come

Structural studies of the 5-HT3 receptor

Abstract

Cys-loop receptors are pentameric ligand-gated ion channels (pLGIC), which play a crucial role in rapid neurotransmission. They are the targets of a legion of drugs (antiemetics, general anesthetics, benzodiazepines, smoke cessation drugs, etc.) and their physiological properties are intensively studied. When pLGICs bind neurotransmitters, they undergo conformational changes, from a resting closed pore state to a transient open pore state; they can also enter a ligand-bound, closed pore, desensitized state. Moreover, the gating properties of pLGICs can be influenced by a variety of compounds (e.g. lipids, competitive inhibitors, allosteric modulators, ions such as Ca 2+ ), which makes them flexible receptors capable of integrating different signals into conformational changes.

In this thesis we focus on structural studies of the mouse serotonin type 3 receptor (m5-HT3R). The first structure of the m5-HT3R, obtained by X-ray crystallography using stabilizing nanobodies, was a closed pore inhibited conformation [START_REF] Hassaine | X-ray structure of the mouse serotonin 5-HT3 receptor[END_REF]. As a follow up, we aimed to obtain structures of the m5-HT3R in other conformations, in order to elucidate its gating mechanism. For this purpose we used both X-ray crystallography and cryo-electron microscopy and thus the whole thesis follows two story-lines.

A general introduction of the pLGIC family is followed by a detailed structural description of the m5-HT3R. In the results section, we present the optimized protocol for the receptor purification, we report that limiting diffraction is a bottleneck in the crystallographic trials and we emphasize limits met using nanobodies for conformational stabilization of the receptor. In the electron microscopy results part we present the optimization of the sample and grid preparation that ultimately permitted data collection. We report four different structures representing distinct functional states of the m5-HT3R: an inhibited tropisetron-bound closed conformation, an open pore state and a putative pre-active state obtained in the presence of serotonin, and finally a closely-related putative pre-active state in the presence of serotonin and of the allosteric modulator TMPPAA. We compare our data with structures of the same receptor obtained by other laboratory.

It was shown for the first time in our work how the antagonist (tropisetron) and the neurotransmitter (serotonin) bind to the full-length m5-HT3R. And our structures deepen the knowledge of the receptor's gating mechanism.

Résumé

Les récepteurs Cys-loop sont des canaux ioniques pentamériques activés par un ligand (pLGIC), qui jouent un rôle essentiel dans la neurotransmission rapide. Ils sont la cible de nombreuses familles de médicaments (antiémétiques, anesthésiques généraux, benzodiazépines, médicaments pour arrêter de fumer, etc.) et leurs propriétés physiologiques sont, en conséquent, très étudiées. Lorsque les pLGIC lient des neurotransmetteurs, ils subissent des modifications conformationnelles, d'un état au repos où le pore est fermé vers un état transitoire ouvert. La liaison de ligand peut également provoquer un état conformationnel fermé et désensibilisé. En outre, les propriétés des pLGIC peuvent être influencées par divers composés (lipides, inhibiteurs compétitifs, modulateurs allostériques, ions tels que Ca 2+ ), ce qui en fait des récepteurs capables d'intégrer différents signaux via des changements de conformation.

Dans cette thèse, nous nous concentrons sur des études structurales du récepteur de la sérotonine de type 3 chez la souris (m5-HT3R). La première structure du m5-HT3R, obtenue par cristallographie aux rayons X, représentait une conformation inhibée à pore fermé, stabilisée par des nanobodies [START_REF] Hassaine | X-ray structure of the mouse serotonin 5-HT3 receptor[END_REF]. Nous avons cherché à obtenir des structures dans d'autres conformations, afin d'élucider son mécanisme moléculaire de fonctionnement. Pour ce faire, nous avons utilisé à la fois la cristallographie aux rayons X et la cryo-microscopie électronique. Les résultats obtenus sont décrits dans 2 chapitres dédiés de la thèse.

Une introduction générale de la famille pLGIC est suivie par une description détaillée de la structure du m5-HT3R. Dans la section résultats, nous présentons le protocole optimisé pour la purification du récepteur, nous expliquons que la mauvaise diffraction est un facteur limitant dans les essais cristallographiques, et nous montrons les freins rencontrés lors de l'utilisation des nanobodies pour la stabilisation conformationnelle du récepteur. Dans les résultats de la microscopie électronique, nous présentons l'optimisation de la préparation de l'échantillon et de la grille qui a finalement permis la collecte de données. Nous décrivons quatre structures différentes représentant des états fonctionnels distincts du m5-HT3R : une conformation inhibée fermée liée au Tropisétron ; un état ouvert et un état pré-actif présumé obtenus en présence de sérotonine ; et enfin un état proche de l'état pré-actif supposé, en présence de sérotonine et du modulateur allostérique TMPPAA. Nous comparons nos données avec les structures du même récepteur obtenues par un autre laboratoire.

Notre travail a montré pour la première fois comment l'antagoniste (Tropisétron) et le neurotransmetteur (sérotonine) se lient au m5-HT3R. Nos structures approfondissent également la connaissance du mécanisme d'action du récepteur.

Table of content

Hugues Nury pour son imense soutien. Pour qu'il a cru en moi meme pendant la period difficile, pour sa incommesurable tranquilité et elegance avec lesquelles il a dirigé mon project. Merci pour toutes les moments inoubliables quand j'avais la possiblité d'appreindre de toi et pour ton patience avec moi. Tu est et tu va rester la plus forte motivation pour ma future carrière scientifique.

I would like to express my sincere gratitude to all committee members: Franck Fieschi, Jean-Michel Jault, Sarah Lummis, Martin Picard and Stéphanie Ravaud for a kind agreement to spend their precious time reading and correcting my manuscript, travelling to the defence and thinking about our research in general. Thank you all! I am deeply grateful to all the current and former members of the laboratory. To Eva Pebay-Peyroula for giving me the chance to join her lab and showing me, that there is a way how to combine science and family and that time can be used super-efficiently, if you are organised. To Stéphanie Ravaud for being my co-supervisor and helping me whenever I needed her. To Jacques Neyton for all his kind permissions regarding the work during weekends and his advices: "c'etait un grand honneur de travailler avec toi!". To Céline Juillan-Binard and Delphine Baud for taking a perfect care about the order, chemicals and instruments, what was making the work in the lab so much easier. To Jonathan Perot for mastering our cell-cultures and protein overexpression and most importantly for his inspiring neatness. To Sonja Minniberger, best master student I have ever met, for her permanent cheerful spirit. To Orso Subrini for his helpful attitude and smart ideas. To Hubert Mayerhofer for showing me, that if you have a reasonable idea it is faster to test if it works rather than to talk about the reasons why it might not work. To Yann Huon de Kermadec for his suppor during my lab and social incorporations. To Florine Dupeux for putting herself out for me. And also to Aleksandra Woźnicka-Misăilă, Georgy Derevyanko and all our stagiaires.

Thank you, all lab members, for being a great team which makes the definition of "lab" being: a place where people are sharing the passion for science in an outstandingly friendly atmosphere. I will miss you all.

I really appreciate the help of our collaborators: Ghérici Hassaine, Lamia Mebarki, Děkuji mé rodině. Rodičům, prarodičům a bráchovi. Můj odchod do zahraničí byl náročným obdobím pro Vás všechny. Nesmírně si vážím Vaší podpory, protože bez ní bych to nikdy nedokázala. Každý z Vás je pro mě jedinečnou inspirací a nekonečným zdrojem k osobnímu rozvoji. Děkuji, že mě tolik milujete. Jsem šťastná, že můžeme být spolu, protože jsou to pro mě jedny z nejdůležitějších chvilek v životě.

At last, but certainly not least, I would like to say "большое спасибо" to my husband who was standing by my side, being my personal and scientific support and to our little daughter Adriana. You are my life drive, please "Stand by me".

A special thank belongs to Dr. Kvido Strisovsky who helped me in my non-standard situation.

5-HT3R

serotonin receptor of type 3

Table of figures

Introduction

When someone is searching for a job, her/his abilities to communicate represent one of the most appreciated skills by human resource recruiters. In biological processes communication also plays a very important role. Unicellular organisms developed the ability to recognise stimuli coming from their surroundings, ways to process the information, and signalling pathways which allow these stimuli to be acted upon (e.g. chemotaxis as a response to a chemical stimulus). Cells in multicellular organisms need to communicate with each other to properly develop into different tissues and organs, fulfil their desired role depending on the cell type, accommodate to external conditions and react to the requirements of the organism. The scope of this thesis is the structural study of a ligand-gated ion channel, which mediates crucial communication, which takes place in excitable cells.

As early as the 19th century, neurons were declared to be the functional building blocks of the nervous system and defined to be independent cells, able to transfer directionally a signal from dendrite towards axones, and the Nobel prize was awarded to Santiago Ramón y Cajal and Camillo Golgi in 1906 for these discoveries. At that time, the propagation of the signal through neurons was not well understood. The development of electrophysiology during the 20th century permitted the mechanism of signal propagation to be elucidated and emphasised the role of ion channels in this process, Nobel prize in Physiology and Medicine awarded to Alan Hodgkin and Andrew Huxley in 1963. Our current molecular understanding of how ion channels operate arises in part from the ability to obtain atomic structures by crystallography or cryo-electron microscopy since the turn of the millennium [START_REF] Doyle | The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and Selectivity[END_REF][START_REF] Liao | Structure of the TRPV1 ion channel determined by electron cryo-microscopy[END_REF].

Action potential

The neuronal signal transfer is divided into two stages: I) propagation along the nerve cell through changes in the membrane potential (electrical signal) and II) the transmission of the signal at the neuron-neuron interface called the synapse, mediated by neurotransmitters (coupling).

The cytoplasmic membrane is the semi-permeable hydrophobic barrier which encloses all living cells. While small hydrophobic molecules can diffuse across membranes, charged molecules such as ions cannot and thus they require specialised proteins to transport them. There are two types of transport across the membrane.

Active transport mediated by pumps and transporters consumes energy to transport molecules against their chemo-electrical gradient. Passive transport occurs along a gradient and is mediated by channels.

Concentrations of ions differ at the interior and exterior side of the plasma membrane. In a typical neuron, there are more Na + ions outside than inside the cell whereas the opposite is true for the K + ions (Fig. 1).The general charge imbalance on different sides of membrane creates electrical field, negative inside, called membrane potential. Polarised neurons have a resting membrane potential of about -70 mV. This polarisation is maintained by sodium/potassium pumps and K + channels. For two K + pumped in 3 Na + ions are pumped out of the nerve cell. If an excitatory stimulus reaches the neuron in the resting state, this results in the opening of ion channels and the passage of sodium ions into the cell. If the stimulus is strong enough, the potential may reach -55 mV and thus overcome a barrier for an all-or-nothing phenomenon. At this threshold, the change of the potential triggers the opening of voltage-gated ion channels, which mediate further depolarisation. This depolarisation is then propagated along the neuronal body to the axon terminal -to the synapse. In synapses, the depolarisation opens calcium channels, yielding a flux of Ca 2+ into the presynaptic terminal. The rise in presynaptic calcium concentration triggers the fusion of intracellular vesicles, filled with neurotransmitters, with the presynaptic membrane and the liberation of neurotransmitters into the synaptic cleft. Ligand-gated ion channels located in the postsynaptic neuronal membrane bind these neurotransmitters, open and let some ions pass through the membrane of the cell. This can result in the depolarisation or hyperpolarisation (depending on the channel selectivity) of the new neuron and the message may eventually be transported through this new neuron as described above. [START_REF] Blaus | Medical gallery of Blausen Medical[END_REF]

The ion channels

Because of their crucial role in the nervous system the ion channels have been intensively studied more than 60 years [START_REF] Hodgkin | A quantitative description of membrane current and its application to conduction and excitation in nerve[END_REF]. Together with the G-protein coupled receptors and kinases, the pore forming units of ion channels are amongst the most abundant genes in the human genome. Almost all ion channels are composed of several subunits and form homomers or heteromers. The existence of different heteromers increases the number of the variants of a certain type of ion channel compared to the number of different subunits encoded by DNA sequences. Differences between the subunits modify the channel properties and thus support their specialisation and plasticity. Functional channels of various oligomeric states exist, from dimeric channels up to hexameric ones. Very often subunits are arranged around a central pseudo-symmetrical axis perpendicular to the plane of the membrane, which coincides with the pore (Fig. 2).

The ion channels do basically two things: they close or open to let the ions pass through the highly hydrophobic core of the membrane. The channel stays in its open or closed state(s) several milliseconds, and the conformational change from the opened to closed gates occurs in the order of a hundred times per second with the flux of 10 6-7 ions/s [START_REF] Gadsby | Ion channels versus ion pumps: the principal difference, in principle[END_REF]. The ion channels are either strictly selective to the certain type of ions (e.g. K + [START_REF] Roux | Ion channels and ion selectivity[END_REF]) or they manifest promiscuity among either cations or anions (e.g. 5-HT3 channel among cations, Na + , K + , Ca 2+ etc. and even some organic cations [START_REF] Yang | Ion permeation through 5-hydroxytryptamine-gated channels in neuroblastoma N18 cells[END_REF]. We distinguish two classes of ion channels: the leaky and gated ion channels. Leaky channels show constitutive opening, and even if they can be regulated by external factors (e.g heat or mechanical stress for TREK channels), they do not directly participate in rapid neurotransmission. The determining characteristic of gated channels is their ability to react to different stimuli coming from their environment, and their rapid response. They all possess a "gate'', which is closed in the resting state and open only once the correct stimulus is detected. Depending on the signal the channels recognise, they can be classified into further subgroups. Voltage-gated ion channels, as the name suggests, react to changes of the membrane potential and this group comprises for example of the K + , Na + and also the Ca 2+ channels. Members of another class of gated ion channels respond to mechanical stimuli. The last group of gated channels are the ligand-gated ion channels. Their opening is dependent on the binding of a chemical compound. This last group comprises of several super-families: the tetrameric glutamate receptors, the TRP channels, the ATP-gated ion channels and the Cys-loop receptors. The Cys-loop receptors are pentameric ligand-gated ion channels and as the serotonin receptor of type 3 (5-HT3R), which is the focus of this thesis, belongs to this family, they are discussed in more detail in the following sections.

Discovery and characteristics of pLGIC

The mechanism behind the chemo-electrical conversion during neurotransmission has been intensively studied since the beginning of the 20th century. The main limiting factors during that period were the available methods and the biological accessibility of the material studied. Key discoveries were made hand in hand with the development of the pharmacological, electrophysiological and biochemical methods.

Emil du Bois-Reymond, known as the father of electrophysiology, was able to detect electrical signals in frog muscle in 1843 and 4 years later, with an improved instrument, he was able to repeat his experiment in humans. In 1868 one of his followers, Julius Bernstein, measured the first action potential across the cellular membrane. In 1902 he formulated the membrane theory further developed by Howard Curtis and Kenneth Cole. After the proposition of ion channel existence in 1952 by Alan Hodgkin and Andrew Huxley, the first direct observation of single functioning ion channels was obtained in the 1970s by the "patch clamp" electrophysiology method invented by Erwin Neher and Bert Sakmann. A breakthrough took place with the injection of brain extracted mRNA into Xenopus laevis oocytes, leading to functional ion channel expression at the cell surface, which could be characterised by electrophysiology [START_REF] Gurdon | Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells[END_REF]. This approach helped to overcome the fact that sequencing and cloning techniques were not available during that period and thus it was not possible to identify and overexpress the protein of interest. Those limiting factors were solved by two biological factors in the case of the nicotinic acetylcholine receptor (nAChR), which made it the first pLGIC discovered and characterised. First, cells of the electric organ from Electrophorus electricus [START_REF] Nachmansohn | Role of acetylcholine in axonal conduction and neuromuscular transmission[END_REF] were shown to be extraordinarily rich in the acetylcholine neurotransmitter sensing receptors. This tissue provided sufficient amounts of working material but it was only of low specificity. The second biological factor that helped to identify the sensing unit was the discovery of a snake venom component, the alpha-bungarotoxin [START_REF] Chang | Isolation of neurotoxins from the venom of bungarus multicinctus and their modes of neuromuscular blocking action[END_REF], which specifically binds to the nAChR receptor and was shown to be a blocker of neurotransmission. A close homologue of this toxin, linked to Sepharose beads, served as the first tool for the affinity purification of receptors. This breakthrough helped to clarify that it is the nAChR and not the acetylcholinesterase that interacts with the toxin. The nAChR was determined to be the bearer of the neurotransmitter response.

In parallel with the advancements being made on the nAChR, the inhibitory neurotransmitters glycine and γ-aminobutyric acid (GABA) were used to identify their receptors, which were both purified in 1982 [START_REF] Pfeiffer | Purification by affinity chromatography of the glycine receptor of rat spinal cord[END_REF][START_REF] Stephenson | Physicochemical characterization of detergent-solubilized gamma-aminobutyric acid and benzodiazepine receptor proteins from bovine brain[END_REF]. Only five years later the DNA regions encoding these two receptors were sequenced [START_REF] Grenningloh | The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors[END_REF][START_REF] Schofield | Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor superfamily[END_REF] and shown to be homologous to the nAChR. The Cys-loop receptor family was further enlarged with the cloning, expression and characterisation of the 5-HT3R [START_REF] Maricq | Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel[END_REF] and the invertebrate glutamate-gated chloride channels (GluCl) [START_REF] Cully | Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans[END_REF]. Prokaryotic homologues were also identified using sequence analysis [START_REF] Tasneem | Identification of the prokaryotic ligand-gated ion channels and their implications for the mechanisms and origins of animal Cys-loop ion channels[END_REF]. Two of them, originating from Gloeobacter violaceus (proton-gated GLIC receptor) and Erwinia chrysanthemi (GABA-gated ELIC receptor) have been extensively studied in structure-function projects aimed at understanding gating and transitions. These two receptors lack the characteristic Cys-loop and thus the alternative name -pentameric ligand-gated ion channels can be considered as more precise. All receptors from this family share a similar architecture but the prokaryotic receptors lack the intracellular domain (ICD).

The pentameric ligand-gated ion channels are classified according to their ion selectivity. The nAChRs and 5-HT3Rs are cation-selective channels, so movement of ions through them causes a depolarisation of the membrane (excitatory postsynaptic potential). Conversely, the glycine receptor (GlyR) and the GABAA receptors (GABAAR) are anion-selective channels and movement of ions through them leads to a hyperpolarisation (inhibitory postsynaptic potential). Both prokaryotic homologues GLIC and ELIC are cation selective.

The variety of the genetically encoded subunits and the ability of the cell to compose pentamers composed of diverse subunits adds to the overall functional plasticity and physiological properties of the receptors. The human genome codes for about 19 different subunits of GABAAR and for 17 different subunits of nAChR. Smaller diversity is observed for the GlyR represented by only 5 different subunits as well as for the 5-HT3R where the five different subunits carry the names from A-E. The phylogenetic relationships between the different subunits of each receptor class are shown in (Fig. 3).

Generally the working mechanism of all the Cys-loop receptors is the same. They open transiently when an agonist binds but after longer exposure to the agonist they switch into the stimuli ignoring state during which the cellular response decreases. This state is called the desensitised state and ions are not able to pass through the channel in this state. After the washout of the agonist the receptor returns into its resting state directly, without the need for reopening. This entire process was first described for the nAChR [START_REF] Katz | A study of the desensitization produced by acetylcholine at the motor end-plate[END_REF]. Since then, its binding specificity and the binding kinetics have been further studied. It is clear now that a variety of molecules might interact with and dissociate from the receptor with different kinetics, which is further influenced by allostery and that this process might be dependent on the receptor conformational state [START_REF] Monod | On the nature of allosteric transitions: A plausible model[END_REF].

The Cys-loop receptors are implicated in multiple diseases, for example in anxiety, epilepsy and Alzheimer's disease. They are targets of clinically used anaesthetics, anti-emetics, barbiturates, steroids and on the biological level also of toxins, alcohol and nicotine [START_REF] Connolly | The Cys-loop superfamily of ligandgated ion channels: the impact of receptor structure on function[END_REF]Lester, 2004;[START_REF] Thompson | The structural basis of function in Cys-loop receptors[END_REF]. I will focus on the detailed functional properties of the 5-HT3R and its structural features compared to the other Cys-loop receptors in the following sections.

The 5-HT3 receptor. Discovery, composition, physiological properties and pharmacology

5-hydroxytryptamine (5-HT) or serotonin is the neurotransmitter biochemically derived from tryptophan. It is mostly produced in the enterochromaffin cells of the gastrointestinal tract but it is also present in the serotonergic nerves in the central nervous system (CNS). There are seven classes of receptors sensing serotonin -the serotonin receptors. The 5-HT3R is the only ion channel out of the 7 different classes of serotonin receptors. The rest of the serotonin receptors are G protein-coupled receptors and they influence physiological processes such as sleep, memory, learning, respiration, mood, anxiety, locomotion and more (the overview of the G-protein coupled serotonin receptors can be found in the review of [START_REF] Pytliak | Serotonin receptors -from molecular biology to clinical applications[END_REF]).

The initial discovery of the 5-HT3 receptor is linked to the determination of the two pharmacologically distinct types of serotonin receptors. The "D" type receptors, which can be inhibited by dibenzyline, mediate the contraction of smooth muscle and the "M" type, which can be inhibited by morphine, cause the depolarisation of the cholinergic neurons in the guinea-pig ileum [START_REF] Gaddum | Two kinds of tryptamine receptor[END_REF]. When the classification and the unique nomenclature was established the "M" type serotonin receptors became the 5-HT3 receptors [START_REF] Bradley | Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine[END_REF] and were further characterised as we know them today [START_REF] Maricq | Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel[END_REF].

5-HT3Rs are located in the peripheral nervous system (PNS) as well as in the CNS [START_REF] Tecott | Nervous system distribution of the serotonin 5-HT3 receptor mRNA[END_REF] both, pre-and post-synaptically. The presence of the 5-HT3R was proven in the peripheral preparations of myenteric plexus [START_REF] Mawe | Peripheral neural serotonin receptors: identification and characterization with specific antagonists and agonists[END_REF], submucous plexus [START_REF] Derkach | 5-HT3 receptors are membrane ion channels[END_REF], nodose ganglion [START_REF] Higashi | 5-Hydroxytryptamine receptors of visceral primary afferent neurones on rabbit nodose ganglia[END_REF] and vagus nerve [START_REF] Azami | The depolarizing action of 5-hydroxytryptamine on rabbit vagal primary afferent and sympathetic neurones and its selective blockade by MDL 72222[END_REF]. After its localisation to the PNS, its existence was also proven in the CNS by mapping of the 5-HT3R activity in the rat brain [START_REF] Kilpatrick | Identification and distribution of 5-HT3 receptors in rat brain using radioligand binding[END_REF]. In the CNS, 5-HT3R is localised to the hippocampus, the amygdala, the area postrema and the nucleus tractus solitarius [START_REF] Koyama | Building a 5-HT3A Receptor Expression Map in the Mouse Brain[END_REF][START_REF] Miquel | Differential subcellular localization of the 5-HT3-As receptor subunit in the rat central nervous system[END_REF] (Fig. 4).

Five subunits A-E of the 5-HT3R have been identified to date. The A subunit was followed by the B subunit discovery and expression four years later [START_REF] Davies | The 5-HT3B subunit is a major determinant of serotonin-receptor function[END_REF]. Three more putative 5-HT3R-encoding genes HTR3C, HTR3D and HTR3E were found in the human brain coding for the C-E subunits [START_REF] Niesler | Cloning, physical mapping and expression analysis of the human 5-HT3 serotonin receptor-like genes HTR3C, HTR3D and HTR3E[END_REF]. Some of the subunits exist in a variety of isoforms. The 5-HT3R A subunit (5-HT3RA) has short and long splice variants differing by 32 amino acids, and other post translational variants are also known for the 5-HT3R B (5-HT3RB) and E subunits. The homopentamer receptors can be made only of A subunits [START_REF] Davies | The 5-HT3B subunit is a major determinant of serotonin-receptor function[END_REF]. None of the subunits B-E forms functional homopentamers and moreover the heteropentamers require the presence of at least one A subunit. The functional properties of the C-E subunits and their presence across species is discussed in detail in the work of Holbrook and co-workers [START_REF] Holbrook | Characterisation of 5-HT 3C, 5-HT 3Dand 5-HT 3Ereceptor subunits: evolution, distribution and function[END_REF] and that of Niesler and co-workers [START_REF] Niesler | Characterization of the Novel Human Serotonin Receptor Subunits 5-HT3C,5-HT3D, and 5-HT3E[END_REF]. The rodents genome codes only for the A and B subunits and interestingly, they never vomit. The absence of the other subunits as a reason of the phenotype is tempting but remains speculative.

The general architecture of all the different subunits is identical. They are composed of the extracellular domain (ECD) formed mostly by beta-sheets. They have four transmembrane helices. Only helix 2 (M2) is the pore forming helix and the rest of the helices protect it from contact with the lipid bilayer. The M3 helix is connected to the M4 helix by the long M3-M4 loop. C-terminal to the M3 there is a peri-membrane short helix (MX) and then the unstructured longer loop connects the MX helix to the cytoplasmic prolongation of the membrane helix 4 (M4) called the MA helix (Fig. 5). The binding site of the receptor is formed by the contact of the two consecutive subunits. The detailed view of the structural features of the 5-HT3 receptors will be provided in section 1.5.2. Hooft et al., 1997). The single channel conductance of native 5-HT3Rs was tissue dependent and ranged between 9 and 17 pS [START_REF] Fletcher | Desperately seeking subunits: are native 5-HT3 receptors really homomeric complexes?[END_REF], whereas it was only in the sub-pS range for 5-HT3A homopentamers expressed in a heterologous system [START_REF] Gill | An electrophysiological investigation of the properties of a murine recombinant 5-HT3 receptor stably expressed in HEK 293 cells[END_REF]. This suggested that multiple forms of the receptor might be present. At first, splice variants of the A subunits were hypothesised to be a source of the heterogeneity but it was shown that the presence variants had only a minor impact on the pharmacological and functional properties of the receptor [START_REF] Hope | Cloning and fnctional expression of an apparent splice variant of the murine 5-HT3 receptor A subunit[END_REF]. In the end, it was a direct comparison between the heterologously expressed 5-HT3RA and the native receptors of the superior cervical ganglion neurons, which provided the evidence for multiple channel forms. Although the pharmacological properties were nearly identical, the conductance of the heterologously expressed receptors was about 0.4-0.6 pS compared to the 9 pS conductivity of the native receptors [START_REF] Hussy | Functional properties of a cloned 5hydroxytryptamine ionotropic receptor subunit: comparison with native mouse receptors[END_REF]. Furthermore, the whole-cell noise analysis of the mureine superior cervical ganglion neuron receptor revealed the single-channel conductance of 3.4 pS. This suggested a co-existence between 9 pS conductance channels and channels with a lower conductance. It implied the presence of at least two different forms of the receptors. The B subunit was discovered and shown to be unable to form a functional homopentamer. The conductivity of the A-B heteromers fitted well with the data obtained from the measurements carried out with the 5-HT3Rs expressed natively [START_REF] Davies | The 5-HT3B subunit is a major determinant of serotonin-receptor function[END_REF] (Fig. 6).

To further explain the effect of the B subunit on the conductance, chimeric receptors swapping domains between the A and B subunits were created [START_REF] Kelley | A cytoplasmic region determines single-channel conductance in 5-HT3 receptors[END_REF]. The conductivity-determining region was shown to be in the cytoplasmic portion of the receptor. The alignment of the sequences of the A and B subunits from different organisms revealed three highly conserved arginine residues (R416, R420, R424) within the cytoplasmic region of the A subunit. When these were mutated to the corresponding residues of the B subunit (R416Q, R420D and R424A) and co-expressed with the WT A subunit, the conductivity increased. Importantly, this conductivity corresponded to the one observed in tissues expressing the A/B heteromers natively. Initially, it was believed that the positive charge of these residues was the main determinant of their role: they would essentially lower the flux of cations exiting the receptor through lateral portals [START_REF] Kelley | A cytoplasmic region determines single-channel conductance in 5-HT3 receptors[END_REF]. Later on, based on the structural prediction of the MA helix and on site directed mutagenesis, it was shown that putative salt bridges could be more important than the actual presence of the charge [START_REF] Kozuska | Impact of intracellular domain flexibility upon properties of activated human 5-HT 3receptors[END_REF]. This was demonstrated by a study where acid residues E414, E418 and D425 were mutated to their amide counterpart (QQN) to break salt bridges made with the other residues. The conductivity of this triple mutant was comparable to that of the initial RRR to QDA mutation and moreover the single mutant E418Q also presented a big conductance. General suggestion that the charged residues in the intracellular domain might play stabilising roles being involved in salt bridges was further supported by mutagenesis of the other charged residues preceding E418.

The RRR residues line the portals of the cytoplasmic vestibule and it was under question whether they determine only the conductance of the receptor or if they also participate in size selectivity towards the permeant ion. By analysis of the ion selectivity of the 5-HT3R mutants missing the M3-M4 loop compared to the selectivity of the WT receptor, it was shown that the RRR triplet is not essential for ion selection [START_REF] Mckinnon | 5-HT3 receptor ion size selectivity is a property of the transmembrane channel, not the cytoplasmic vestibule portals[END_REF].

The channel selectivity filter was determined based on the mutagenesis of the 5-HT3R residues analogous to those influencing the ion selectivity properties of the other Cys-loop receptors [START_REF] Gunthorpe | Conversion of the ion selectivity of the 5-HT(3a) receptor from cationic to anionic reveals a conserved feature of the ligandgated ion channel superfamily[END_REF]. The alignment of anion-selective and cation-selective Cys-loop receptors revealed the conserved presence of positively or negatively charged residues corresponding to the anion-and cation-selectivity, respectively. These rings are located on the extracellular and intracellular ends of the pore. Based on mutagenesis studies results, the lower ring formed by E250 on the intracellular side seems to be the main determinant of the 5-HT3R selectivity [START_REF] Thompson | A single ring of charged amino acids at one end of the pore can control ion selectivity in the 5-HT 3receptor[END_REF]. The 5-HT3R is relatively promiscuous with regard to its selectivity. The selectivity among different cations was predicted based on the property to reverse near 0 mV, suggesting similar permeabilities for Na + and K + cations. The 5-HT3R discriminates between the small monovalent ions (Cs + , K + , Li + < Na + = Rb + ) only weakly [START_REF] Yang | Ion permeation through 5-hydroxytryptamine-gated channels in neuroblastoma N18 cells[END_REF]. Moreover, even some of the organic cations (e.g., ammonium and methylamine) are able to pass through the channel and unsurprisingly their permeability decreases with their increasing size [START_REF] Yang | Ion permeation through 5-hydroxytryptamine-gated channels in neuroblastoma N18 cells[END_REF]. The divalent cations (Ca 2+ , Ba 2+ , Mg 2+ ) also pass through the selectivity filter of the 5-HT3R. The calcium permeability of the receptor varies between the splice variants of the 5-HT3R A subunit, with the shorter variant enabling higher permeability [START_REF] Hargreaves | Ca2+ permeability of cloned and native 5-hydroxytryptamine type 3 receptors[END_REF]. Moreover, the presence of divalent cations modulates the amplitude of the signal and also the duration of the response to the agonist [START_REF] Thompson | Calcium modulation of 5-HT3 receptor binding and function[END_REF]. The absence of calcium cations increases both of these factors. The Ca 2+ inward flux into presynaptic terminals results in exocytosis [START_REF] Rondé | High calcium permeability of serotonin 5-HT3 receptors on presynaptic nerve terminals from rat striatum[END_REF]. As the presynaptic 5-HT3 receptors were shown to mediate GABA release in proportion to the Ca 2+ concentration [START_REF] Turner | Calcium influx through presynaptic 5-HT3 receptors facilitates GABA release in the hippocampus: in vitro slice and synaptosome studies[END_REF], it has been speculated that Ca 2+ permeability was more relevant to presynaptic physiology than to postsynaptic Ca 2+ uptake.

Pharmacology

The 5-HT3Rs are proposed to play a role in the development of multiple diseases and psychological disorders [START_REF] Fakhfouri | 5-HT3 Receptor Antagonists in Neurologic and Neuropsychiatric Disorders: The Iceberg Still Lies beneath the Surface[END_REF][START_REF] Gupta | 5HT3 receptors: Target for new antidepressant drugs[END_REF]. They are the main pharmacological target of drugs marketed for irritable bowel syndrome [START_REF] Tyers | Mechanism of the anti-emetic activity of 5-HT3 receptor antagonists[END_REF] and chemotherapy induced nausea and vomiting (CINV), as well as for post-operative nausea and vomiting [START_REF] Humphrey | Review article: the therapeutic potential of 5-HT3 receptor antagonists in the treatment of irritable bowel syndrome[END_REF]. In both of the aforementioned cases the active drugs are antagonists from the ¨setron¨ family (Fig. 7).

CINV is one of the most dreaded effects of chemotherapy [START_REF] Coates | On the receiving end--patient perception of the side-effects of cancer chemotherapy[END_REF]. It causes discomfort and negatively influences adherence to the chemotherapy treatment regime [START_REF] Bloechl-Daum | Delayed nausea and vomiting continue to reduce patients' quality of life after highly and moderately emetogenic chemotherapy despite antiemetic treatment[END_REF]. Since their discovery in the 1980s, antagonists of 5-HT3R are used and have been further developed as the most potent drugs for the treatment of CINV [START_REF] Sanger | A History of Drug Discovery for Treatment of Nausea and Vomiting and the Implications for Future Research[END_REF]. Currently, together with the NK1 inhibitors and with steroids, they form the three drug CINV treatment regimen [START_REF] Gilmore | Recent advances in antiemetics: new formulations of 5HT3-receptor antagonists[END_REF]. There is a tendency to create universal guidelines for CINV prophylaxis but all attempts are complicated by a lack of understanding of the underlying pathophysiology and by the subjectivity of nausea assessment by patients. Some of the drugs targeting the 5-HT3R such as ondansetron and clozapine have been shown to have a positive effect on schizophrenic symptoms [START_REF] Meltzer | Serotonin receptors : their key role in drugs to treat schizophrenia[END_REF]. Furthermore, mutations in the 5-HT3R were observed in the genomes of patients with bipolar disease and schizophrenia but their impact on pathology has not been directly proven [START_REF] Niesler | Serotonin receptor gene HTR3A variants in schizophrenic and bipolar affective patients[END_REF]. Although further clinical tests are needed it has been concluded that the 5-HT3R antagonists are involved in the treatment of the depression [START_REF] Hewlett | Pilot trial of ondansetron in the treatment of 8 patients with obsessive-compulsive disorder[END_REF]. Indeed, the recently marketed Vortioxetine antidepressant, in addition to its known effects on the serotonin transporter and the 5-HT1 receptors, also potently inhibits the 5-HT3R. However, the role of 5-HT3R inhibition in the clinical effect of Vortioxetine remains undetermined, even if the marketing of the drug insists on its "multimodality" [START_REF] Katona | New generation multi-modal antidepressants: focus on vortioxetine for major depressive disorder[END_REF]. Substance abuse and addiction studied in mice determined that the 5-HT3R is involved in self-administration of ethanol and for cocaine sensitisation [START_REF] Hodge | 5-HT(3A) receptor subunit is required for 5-HT3 antagonist-induced reductions in alcohol drinking[END_REF][START_REF] Hodge | Deletion of the 5-HT(3A)receptor subunit blunts the induction of cocaine sensitization[END_REF]. Importantly, self-administration of ethanol was also shown to be reduced by 5-HT3R antagonists in humans [START_REF] Johnson | Ondansetron reduces the craving of biologically predisposed alcoholics[END_REF]. The 5-HT3R also reacts to anaesthetics from different families including inhalation and intravenous anaesthetics, as well as local anaesthetics [START_REF] Barann | Molecular actions of propofol on human 5-HT3A receptors: enhancement as well as inhibition by closely related phenol derivatives[END_REF][START_REF] Stevens | Modulation of human 5-hydroxytryptamine type 3AB receptors by volatile anesthetics and nalcohols[END_REF][START_REF] Urban | Interactions of anesthetics with their targets: non-specific, specific or both?[END_REF].

Amongst natural compounds acting on the 5-HT3R, morphine (the opioid analgesic from unripe poppy seeds) [START_REF] Gaddum | Two kinds of tryptamine receptor[END_REF], alpha-thujone (the psychotropic component of absinthe from wormwood) [START_REF] Deiml | Alpha-thujone reduces 5-HT3 receptor activity by an effect on the agonist-reduced desensitization[END_REF], and the gingerols and shogaols (the antiemetic compound from the ginger extracts) [START_REF] Abdel-Aziz | Mode of action of gingerols and shogaols on 5-HT3 receptors: binding studies, cation uptake by the receptor channel and contraction of isolated guinea-pig ileum[END_REF]) can be mentioned.

The 5-HT3R structure and its comparison to other known structures of Cys-loop receptors.

Efforts to elucidate the structure of the Cys-loop receptors began with the soluble acetylcholine binding protein, a marine snail protein homologous to the extracellular domain of the Cys-loop receptors. Its X-ray structure was solved in 2001 [START_REF] Brejc | Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors[END_REF] and unveiled the architecture of the extracellular domain of pLGIC and the organization of the orthosteric binding site. The first structure of a full length Cys-loop receptor was the cryo-electron microscopy (cryo-EM) structure of the heteromeric nicotinic acetylcholine receptor extracted from the Torpedo marmorata electric tissue [START_REF] Miyazawa | Structure and gating mechanism of the acetylcholine receptor pore[END_REF][START_REF] Unwin | Refined structure of the nicotinic acetylcholine receptor at 4A resolution[END_REF]. It permitted to understand the receptor global architecture and provided a first structural framework to interpret the accumulated body of functional data on pLGIC. However the resolution remained limited. As a consequence, the register of the amino acid assignment of this structure was later shown to be out by ~1 helix turn beginning with the M1/M2 loop when compared to newly published structures [START_REF] Hibbs | Principles of activation and permeation in an anion-selective Cys-loop receptor[END_REF]. The first full-length structures to be solved by crystallography were of that of pentameric ligand-gated ion channels from prokaryotic organisms, ELIC from Erwinia chrysanthemi [START_REF] Hilf | X-ray structure of a prokaryotic pentameric ligand-gated ion channel[END_REF] and GLIC from Gloeobacter violaceus [START_REF] Bocquet | X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation[END_REF], hypothesised to be in the closed and the open conformation, respectively. Interestingly, neither ELIC nor GLIC possess intracellular domains: they lack the MX helix, the long unstructured loop, and the MA cytoplasmic helix. Thus, their subunits are composed only of the ECD and the 4 membrane helices connected by shorter loops. These structures were followed by the first eukaryotic structure of the Caenorhabditis elegans glutamate-gated chloride channel α, the GluCl channel, where the natural intracellular domain was replaced by the short linker M3-Ala-Gly-Thr-M4 [START_REF] Hibbs | Principles of activation and permeation in an anion-selective Cys-loop receptor[END_REF]. In that case, stabilising antigen-binding fragments (Fab) proteins were used as crystallization chaperones to enhance the pentamer stability and also to help with crystal contacts. In 2014, the structures of the first two mammalian Cys-loop receptors were obtained by X-ray crystallography. These were those of the homomeric GABAA receptor composed of β3 subunits, and with its intracellular domain removed (replaced by the SQPARAA amino acid sequence) [START_REF] Miller | Crystal structure of a human GABAA receptor[END_REF] and of the m5-HT3RA, which included the structured parts of the intracellular domain, the MX and the MA helices [START_REF] Hassaine | X-ray structure of the mouse serotonin 5-HT3 receptor[END_REF]. The latter was obtained in the presence of stabilising nanobodies. The GABAA receptor was supposed to be in the desensitised state and the 5-HT3R was speculated to be in the closed conformation based on the fact that the stabilising nanobody used in the structure is actually acting as an antagonist. Structures of the homomeric glycine receptor in the open, desensitised and closed conformations, obtained by cryo-electron microscopy, enabled direct comparison of the different conformational states [START_REF] Du | Glycine receptor mechanism elucidated by electron cryo-microscopy[END_REF]. The constructs used for that study lacked the intracellular domain, which was replaced by M3-Ala-Gly-Thr-M4. At the same time the X-ray structure of the GlyRα3 was solved, with a resolution up to 3 Å, in the presence of the antagonist strychnine [START_REF] Huang | Crystal structure of human glycine receptor-α3 bound to antagonist strychnine[END_REF]. As in most cases, its intracellular domain was absent and the receptor is supposed to be in the antagonist-bound closed conformation. The only known heteromeric structures to date belong to the nAChR and GABAA family. Thirteen years after the low resolution EM structure of the Torpedo nAChR, the X-ray structure of the recombinant 2α:3β nAChR α4β2 was solved. The structure was obtained in the presence of nicotine bound to the binding site and is believed to be in the desensitised conformation.

After the crystallographic structure of 2α:3β nAChR α4β2, the stoichiometrically distinct nAChR α4β2 3α:2β was solved by cryo-EM [START_REF] Walsh | Structural principles of distinct assemblies of the human α4β2 nicotinic receptor[END_REF]. This enabled a direct comparison between different assemblies and elucidated the principles of the subunit packing mechanism. The cryo-EM structure of the 5-HT3R without ligand (Basak et al., 2018a) revealed only minor differences between the potentially inhibited receptor solved in the presence of VHHs and the apo structure. Two conformationally distinct structures of the GABAA α1β2γ2 receptor [START_REF] Zhu | Structure of a human synaptic GABAA receptor[END_REF] and one additional structure of GABAA α1β1γ2 [START_REF] Phulera | Cryo-EM structure of the benzodiazepine-sensitive α1β1γ2S tri-heteromeric GABAA receptor in complex with GABA[END_REF] were recently solved, all by cryo-EM, showing the organisation of consecutive subunits, revealing the ECD vestibule glycosylations and more importantly illuminating the mode of ligand binding. Interestingly, GABAA α1β1γ2 structure revealed the asymmetry of the TMD subunit organisation around the pore axis and the GABAA α1β2γ2 structure showed the complete gamma subunit collapse into the TMD pore. In contrast to those two structures, the GABAA α1β3γ2 structures [START_REF] Laverty | Cryo-EM structure of the human α1β3γ2 GABAA receptor in a lipid bilayer[END_REF][START_REF] Masiulis | GABAA receptor signalling mechanisms revealed by structural pharmacology[END_REF], solved by cryo-EM as well, presented the symmetrical organisation corresponding to that of the homopentameric GABAA β3 solved by X-ray crystallography. The potential source of dissimilarities might be a use of detergents and engineered constructs (deletion of ICD) in the case of distorted structures in contrast to the use of nanodiscs and full length construct for the symetricaly organised GABAA α1β3γ2 structures. The X-ray structure of the chimeric ELIC(ECD) -GABAA α1(TMD) indicates the differences between the apo and alphaxalone-bound chimeric receptor which is supposed to be desensitised [START_REF] Chen | Structural basis of neurosteroid anesthetic action on GABAA receptors[END_REF]. Finally our recent structural study of the 5-HT3RA receptor mapped the changes that occur during the activation of the receptor by serotonin. These conclusions are supported by the comparison of the apo (Basak et al., 2018a), two open pore structures , two agonist-bound closed pore states [START_REF] Basak | Cryo-EM reveals two distinct serotonin-bound conformations of full-length 5-HT3A receptor[END_REF][START_REF] Polovinkin | Conformational transitions of the serotonin 5-HT3 receptor[END_REF] and one pre-active state [START_REF] Polovinkin | Conformational transitions of the serotonin 5-HT3 receptor[END_REF]. A simplified overview of the pLGIC structures solved up to 2018 is summarised in Fig. 8 and for the sake of completeness table with structures of pLGIC can be found in supplementary data Tab. 1.

The non-physiological membrane domain environment (mostly detergent until last year, and now also nanodiscs) together with the experimental conditions (crystal packing or freezing) often raises questions about the correlation of structures with physiological conformations existing in the cell membrane. Moreover, regarding the Cys-loop receptors, the functional assignments of the captured conformational states might be strenuous. Still the comparison of multiple steady-state structures of the same protein is probably the best tool we have at the moment to understand the molecular mechanism of operation. As with GlyR, more and more receptors are being solved in different conformations and stoichiometries, permitting us to better understand this family.

The overall structure

The 5-HT3R is composed of five subunits arranged in a pseudo-symmetrical manner around the axis formed by the ion permeation pathway. The ECDs are formed by β sheets forming a β-sandwich. Each β-sandwich overlaps with the consecutive one on the outer side of the receptor in a counter-clockwise manner. On the interface of two consecutive ECDs the overlap of the main subunit, the (+) side of the binding pocket, and the complementary subunit, the (-) side of the binding site, create the orthosteric binding site for the neurotransmitter (Fig. 9). The ECDs form multiple contacts with the membrane helices through the loops, which allows signal to be propagated from the bound neurotransmitter to the membrane portion of the receptor (Fig. 10). The membrane helices are organised in concentric rings. The innermost ring is formed by the M2 helices of each subunit which line the pore. These are enclosed by the middle ring, composed of the M1 and M3 helices, and the M4 helices form the peripheral ring (Fig. 11). The position of the M4 helix provides it with a large surface with which to interact with the lipid environment. The MX helices are positioned at the outer side of the M4 helices, reminiscent of clamps that may keep the entire membrane portion together and may stabilize the M4 position. There is no structural information about the unstructured part of the M3-M4 loop. The continuation of the M4 helices protruding into the cytoplasm, the MA helices, are slightly twisted counter-clockwise relative to the ion permeation axis. The M4 ends at the extracellular side, determining the topology of the protein whereby both C-and N-termini are located outside of the cell.

Detailed structural features

Extracellular domain -neurotransmitter binding site

For the Cys-loop receptors, the neurotransmitter binding site was elucidated by the use of the homology, comparing the Cys-loop receptor ECD to the structure of the AChBP [START_REF] Brejc | Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors[END_REF]. The ECD β-sandwiches are formed by the 10 β-strands divided into two main β-sheets. The binding site is formed by "loops" A, B and C from the principal subunit and "loops" D, E, F and G from the complementary subunits [START_REF] Nys | Structural insights into Cys-loop receptor function and ligand recognition[END_REF] (Fig. 9A). It is worth noting that the term "loops" is not strictly correct, because not all of these elements are actually loops. Structurally, only the following elements corresponds to loops: loop A connecting β-strand 4 to β-strand 5 (β4-β5 loop), loop C (β9-β10 loop) and loop F (β8-β9 loop). ¨Loops¨ E, D and G are actually portions of β-strands. Residues that participate in ligand binding in the orthosteric binding site come from "loop" B and are located partially on the β7 strand and partially on the β7-β8 loop. Some of the features of this binding site are well conserved among the Cys-loop receptors, even though their ligands vary. There is a conserved "aromatic box" within the majority of the Cys-loop receptors, located on the interface of some of these conserved elements (Lester, 2004). In the 5-HT3R, the "aromatic box" is defined by aromatic residues W156, Y207, W63, Y126 and F199 from loops B, C, D, E and F, respectively. Some of the binding site residues are conserved across all of the different types of Cys-loop receptors as seen on the sequence alignment in (Fig. 9B). The observation that the loop C is generally more open in the case of the antagonist-bound/closed or resting/closed state, and moves closer to the binding site if an agonist is present and induces the agonist-bound/open conformation or the agonist-bound desensitised/pore closed state, was extrapolated from AChBP to pLGIC. Thus the loop C was commonly used to be a criterion for the determination of the overall receptor functional state. The ligand-bound structures of the pLGIC generally manifest described C loop movement, but it might be very subtle and allows rather comparison of two structures than the interpretation of the structural state by itself. The subunit-subunit rearrangement is a better guideline to the functional state determination [START_REF] Hassaine | X-ray structure of the mouse serotonin 5-HT3 receptor[END_REF]. The structure of the 5-HT 3 R A was solved using the VHH15 nanobody, which binds to the subunit interface from the outside of the binding pocket and actually grasps loop C, bending it towards the outside.

Insights into ligand binding to the 5-HT3R were obtained by comparison with the AChBP solved in the presence of ligands. Mutagenesis of the AChBP binding site to mimic the 5-HT3R binding site led to the creation of the model protein 5-HTBP, which has the binding properties of the 5-HT3R [START_REF] Kesters | Structural basis of ligand recognition in 5-HT3 receptors[END_REF][START_REF] Price | Palonosetron-5-HT3 Receptor Interactions As Shown by a Binding Protein Cocrystal Structure[END_REF].

Based on functional studies of the 5-HT3R binding site mutants, the impact of different amino acids on ligand binding was proposed [START_REF] Beene | Cation-π Interactions in Ligand Recognition by Serotonergic (5-HT 3A) and Nicotinic Acetylcholine Receptors: The Anomalous Binding Properties of Nicotine †[END_REF][START_REF] Spier | The role of tryptophan residues in the 5-Hydroxytryptamine(3) receptor ligand binding domain[END_REF][START_REF] Thompson | Locating an antagonist in the 5-HT3 receptor binding site using modeling and radioligand binding[END_REF]. The majority of the binding studies were done with antagonists of the receptor from the "setron" family, mostly tropisetron and granisetron. The π electrons from the aromatic residues are supposed to interact with the cation provided by the positively-charged ring-embedded nitrogen what was hypothesised for both, agonists [START_REF] Spier | The role of tryptophan residues in the 5-Hydroxytryptamine(3) receptor ligand binding domain[END_REF] and "setron" antagonists. The orientation of the ligands within the binding site was speculative and controversial. Yan and White [START_REF] Yan | Spatial orientation of the antagonist granisetron in the ligand-binding site of the 5-HT3 receptor[END_REF] proposed that the granisetron orients its bicyclic ring to interact with residue W63. Mutagenesis to introduce unnatural amino acids at this position suggested a different interaction mode between granisetron and the W63 position, showing that the cation-π interaction might actually occur via W156 [START_REF] Duffy | Ondansetron and Granisetron Binding Orientation in the 5-HT 3Receptor Determined by Unnatural Amino Acid Mutagenesis[END_REF]. Recently, the binding properties of granisetron and tropisetron, which are closely related, were discussed. Even though the structures of the AChBP solved in the presence of these ligands implies they are orientated in the same way, the extensive mutagenesis of the 5-HT3R binding site and the binding studies of the two agonists implied different properties, suggesting that these two ligands might interact differentially with the 5-HT3R binding site [START_REF] Ruepp | The binding orientations of structurally-related ligands can differ; A cautionary note[END_REF]. Moreover, the binding orientations based on functional studies and modelling, were proposed by the same study.

A very recent structures of the tropisetron bound (see the results presented in section 5.2.2) and granisetron bound m5-HT3R [START_REF] Basak | Molecular mechanism of setron-mediated inhibition of full-length 5-HT3A receptor[END_REF] confirmed the orientation of the ligand in the binding pocket; the amin-carrying bicyclic ring points outwards and the indazole ring mediates the cation-π interaction deeper in the binding pocket.

ECD-TMD interface -binding signal transduction

pLGICs can possess several orthosteric binding sites (5 in the case of the 5-HT3RA) where the binding of a ligand can trigger the channel opening. Conformational changes are often described either as of a ligand-induced selection from the pre-existing conformational states (Monod-Wyman-Changeux model, MWC) or as ligand-induced sequential conformational changes (induced fit model, IF). According to the MWC model all subunits of the existing populations of receptor are in the same conformation and the switch of equilibrium induces the concerted change of all the subunits at once. In contrast, IF model predicts that the binding of ligand changes the conformation of influenced subunits, what increases the binding affinity of the following ligand up to the complete rearrangement of all subunits. In a way the MWC model describes the conformational changes rather at the quaternary structure level and the IF model rather at the tertiary structure. The following paragraphs aim to point to the structurally important and interacting elements (residues, secondary, tertiary and quaternary structures) regardless the ligand-induced cooperativity scheme they follow.

Considering the available structural knowledge of the Cys-loop receptors, a relevant description of the mechanism of signal transduction probably comes from a comparison of the multiple conformations of the GlyR solved by EM [START_REF] Du | Glycine receptor mechanism elucidated by electron cryo-microscopy[END_REF]. This series of structures permits a direct comparison of the interface between the ECD and the TMD among different conformational states within one receptor type. Starting with the binding of a ligand in the binding pocket, the C-loop moves outwards when an antagonist is bound or moves inward (towards the binding site) when an agonist is present. When we consider only the principal (+) subunit , the binding signal might be transduced through movements of the β9 and β10 strands, thus influencing the conformation of loop β8-β9 (F loop) and of the β10/pre-M1 loop region (Fig. 10) at the ECD-TMD interface.

Conformational changes affecting the M1 helix can directly influence the position of the M2 helix. Moreover, the β10/pre-M1 loop region is in contact with the Cys-loop, which in turn interacts with the β1-β2 loop and, most importantly,with the M2-M3 loop. The M2-M3 loop can then directly influence the conformation of the M2 helix. This is also the place where two neighboring subunits meet each other at the ECD-TMD interface. The M2-M3 loop from the (+) subunit interacts with the β8-β9 loop (F loop) and the β10/pre-M1 loop from the neighbouring (-) subuni. In the article reporting the GlyR structures, a direct link between M2 helix rotation and the position of the ECD was described in detail, highlighting residues that play a role in this process [START_REF] Du | Glycine receptor mechanism elucidated by electron cryo-microscopy[END_REF]. The tip of the Cys-loop, which plays the central role in the signal transduction, is very well conserved among the family. The 142-F/YPXD-145 motif in particular is found in all the receptors of the family. Another conserved residue is the arginine located at position 218 in the m5-HT3RA. R218, reported to be responsible for changes in gating properties after ligand binding [START_REF] Hu | Arginine 222 in the Pre-transmembrane Domain 1 of 5-HT3A Receptors Links Agonist Binding to Channel Gating[END_REF] is the key interactor with multiple elements on the ECD-TMD interface. It is sandwiched from the top and bottom by the two aromatic residues W187 of the β8-β9 loop (F loop) and F142 of the Cys-loop. Moreover, R218 is enclosed by multiple negatively charged residues, specifically by D145 of the Cys-loop from one side, by E186 of the β8-β9 loop (F loop) from the other side, and by E53 located on the β1-β2 loop from the front [START_REF] Hassaine | X-ray structure of the mouse serotonin 5-HT3 receptor[END_REF]. Unfortunately, the tip of the M2-M3 loop is not resolved in the 5-HT3R structure and thus hinders the assignment of the interactions of the 4 missing TAIG residues. There is actually an alanine insertion in the mouse construct used in structural studies from our laboratory, mimicking the human M2-M3 loop.

Transmembrane domain -ion pathway through the membrane

The actual pore of the pentameric ligand-gated receptors which is in contact with the passing ions is formed only by the M2 helices. The M2 helices are enclosed by the ring of M1 and M3 helices and are not in direct contact with the M4 helices on the periphery (Fig. 11).

The M2s are oriented more or less perpendicularly to the membrane plane and the pore is defined by the amino acid side chain residues pointing inside to create constrictions parallel to the membrane plane. For the purpose of simplification and better orientation the M2 helix residues of the first nAChR structure to be solved were assigned new numbers, starting with the lowest membrane residue carrying the number 0', up to the residue closest to the extracellular part of the membrane having the number 20' [START_REF] Miyazawa | Structure and gating mechanism of the acetylcholine receptor pore[END_REF]. All structures solved later respect this numbering system and define the 0' residue based on sequence alignment. If some residues, shown to be before the 0' residue, are still within the membrane, they are numbered with negative numbers starting from -1' and going from the 0' residue lower along the M2 helix.

The structures solved to date indicate the existence of multiple constrictions determining the ion pathway. Generally there are two main constriction sites on the M2 helices, one in the middle and the second closer to the intracellular side. The middle position is defined by the highly conserved leucine at the 9' position [START_REF] Alqazzaz | Cys-loop receptor channel blockers also block GLIC[END_REF]. This residue is a geometrical determinant of ion permeability in closed states (Fig. 12) and together with the V13' and I16' residues forms the hydrophobic gates [START_REF] Miyazawa | Structure and gating mechanism of the acetylcholine receptor pore[END_REF]. The V13'S mutation lead to the gain-of-function phenotypes [START_REF] Dang | Probing the role of a conserved M1 proline residue in 5-hydroxytryptamine(3) receptor gating[END_REF]. The L9' mutations to Phe, Tyr or Ala cause the slower desensitisation in contrast to the Thr mutation [START_REF] Yakel | Single amino acid substitution affects desensitization of the 5-hydroxytryptamine type 3 receptor expressed in Xenopus oocytes[END_REF]. Its position differs among the different conformational states of receptors. Even though the relative position of the M2 helices within the receptors in the antagonist-bound/closed and resting/closed conformation vary, the L9' remains the main restriction determinant in all of these structures (5-HT3R, strychnine-GlyRα1 and apo-GluCl). The ion pathway at the P9' position is blocked by the too small radius (~1.4 Å) in the closed GlyRα1 but is widely open (4-5 Å) in the open and desensitised state of the same receptor. In the 5-HT3RA serotonin-bound structure the M2 rotates by 13° in comparison to the apo structure, causing the rotation of L9' away from the pore [START_REF] Basak | Cryo-EM reveals two distinct serotonin-bound conformations of full-length 5-HT3A receptor[END_REF]. Additionally, a tilt was observed at the S2' position, widening the vestibule at the intracellular end.

In the apparently desensitised structures the L9' is not a pore size limiting residue. All the structures of desensitised receptors known to date present the smallest pore radius in the lower part of the M2. For the GlyRα1 in the closed state it is at the proline -2' position [START_REF] Du | Glycine receptor mechanism elucidated by electron cryo-microscopy[END_REF], for the nAChRα4β2 receptor, the channel is tightest at the E-2' position [START_REF] Morales-Perez | X-ray structure of the human α4β2 nicotinic receptor[END_REF], and for GABAA at the A-2' position as well [START_REF] Miller | Crystal structure of a human GABAA receptor[END_REF] (Fig. 12). As mentioned above, these residues are responsible for the selectivity of the ion channels and thus the conserved residues at this position vary according to the channel permeability [START_REF] Thompson | A single ring of charged amino acids at one end of the pore can control ion selectivity in the 5-HT 3receptor[END_REF]. Anion-selective channels usually have uncharged amino acid residues here, and cation-selective channels contain mostly negatively charged glutamic acids at this position.

The X-ray structure of the 5-HT3RA was solved using stabilising nanobodies, which were shown to act as antagonists. The radius formed by the L9' residues is about 4.6 Å, a width that might be too tight for hydrated Na + , which have a radius of about 4.7 Å. This agrees with the suggestion that the structure is in the closed conformation. Moreover, it is supported by the results of MD performed with the recently obtained structures [START_REF] Basak | Cryo-EM reveals two distinct serotonin-bound conformations of full-length 5-HT3A receptor[END_REF] (and our structures presented in the 5.2.2 section). It shows that prediction of the pore de-wetting by MD is a better indicator of the pore permeability rather than the measurement of geometrical constriction. Interestingly, the comparison of the backbone within the structure with the open channels (GLIC and open GluCl) suggests a closer resemblance to these open structures rather than to the closed ELIC and GLIC structures, which led to some speculation questioning if the channel is really closed. The side chain of E-1' was unfortunately not clearly defined in the electron density map and was modeled only as the most plausible rotamer, thus not allowing any further interpretation. The X-ray structure mostly agrees with the apo state receptor solved by cryo-EM, which supports the theory that the X-ray structure is of a closed state and as mentioned above, it is confirmed by the MD.

Different parts of the TMD were proposed to be a predominant location where allosteric modulators bind [START_REF] Sauguet | Crystallographic studies of pharmacological sites in pentameric ligand-gated ion channels[END_REF]. Those are amphipathic molecules including anesthetics, ivermectin, ethanol, as well as lipids/detergents [START_REF] Hibbs | Principles of activation and permeation in an anion-selective Cys-loop receptor[END_REF], [START_REF] Basak | Crystal structure and dynamics of a lipid-induced potential desensitized-state of a pentameric ligandgated channel[END_REF], [START_REF] Nury | X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel[END_REF].

The presence of lipids is a very important factor influencing the functional properties of receptors, mediated by the transmembrane portion of the protein. First it was shown that nAChR solubilised by detergent maintains its ligand-binding capacity but does not allow ion flux [START_REF] Sobel | Large-scale purification of the acetylcholine-receptor protein in its membrane-bound and detergent-extracted forms from Torpedo marmorata electric organ[END_REF], requiring the presence of lipids to help regain its natural functional properties [START_REF] Epstein | Reconstitution of carbamylcholine-dependent sodium ion flux and desensitization of the acetylcholine receptor from Torpedo californica[END_REF]) (Fig. 13). Based on the composition of the membrane naturally surrounding nAChR, tests probing the effects of the addition of cholesterol and of phosphatidic acid (PA, negatively charged head group) were done [START_REF] Baenziger | Effect of membrane lipid composition on the conformational equilibria of the nicotinic acetylcholine receptor[END_REF]. Both of these lipids are able to maintain the coupling between the binding of a ligand and the conformation of the pore. In the presence of phosphatidylcholine lipids only, the nAChR enters a so-called "uncoupled" state that differs from the desensitised state and has lost the capacity to transduce signal from the binding site to the pore [START_REF] Dacosta | Anionic lipids allosterically modulate multiple nicotinic acetylcholine receptor conformational equilibria[END_REF]. When the effect of lipids on the desensitisation process was further studied, the M4 helix, situated at the periphery of the membrane bundle and as such the perfect candidate for lipid interaction, was shown to play a role in this process, at least for nAChRs [START_REF] Dacosta | A distinct mechanism for activating uncoupled nicotinic acetylcholine receptors[END_REF].

Moreover, the GLIC receptor was shown to be sensitive to docosahexaenoic acid (DHA) that induces the desensitised conformation of the receptor [START_REF] Basak | Crystal structure and dynamics of a lipid-induced potential desensitized-state of a pentameric ligandgated channel[END_REF]. DHA binds in between the M4 and M3 helices at the ECD-TMD level, inducing small changes in side chain residues of neighbouring amino acids (among them Phe260 from the M3 and Phe315 from the M4 helix). Another example of the TMD modulators are endogenous neurosteroids which interact with residues T305 (M3 of principal subunit) and Q241+W245 (M1 from consecutive subunit) [START_REF] Laverty | Crystal structures of a GABAA-receptor chimera reveal new endogenous neurosteroid-binding sites[END_REF]. (Baenziger et al., 2017)

The intracellular domain

Although the intracellular domain has been shown to play an important role in conductance determination, in trafficking, in determining life-time kinetics at the synapse and also in protein-protein interactions, structurally it is the least well defined element of the Cys-loop receptors. The prokaryotic homologues lack the intracellular domain completely and the majority of the structures of eukaryotic receptors were solved using mutants where the M3-M4 loop was replaced by short linkers. The M3-M4 loop comprises of the MX peripheral helix, in cation-selective pLGIC, on the intracellular side of the membrane, followed by an unstructured loop and by the MA helix, which becomes the M4 helix at the membrane border (Fig. 5). The only partial structural information available about the M3-M4 region comes from the Torpedo nAChR structure [START_REF] Unwin | Refined structure of the nicotinic acetylcholine receptor at 4A resolution[END_REF] and from the 5-HT3RA structures [START_REF] Hassaine | X-ray structure of the mouse serotonin 5-HT3 receptor[END_REF] where a part of the unstructured domain, the post-M3 loop, and the MX and MA helices are resolved. For the nAChRα4β2 structure, the post-M3 loop and the MX helix were left in the expression construct [START_REF] Morales-Perez | X-ray structure of the human α4β2 nicotinic receptor[END_REF]. However, the loop between the MX helix and the M4 helix is absent in this structure, showing potential flexibility of the unnatural linker. The post-M3 loop is resolved in the very recent GABAA structures but the existence of the MX in anion-selective pLGIC remain unproven because of the poor densities at this level [START_REF] Laverty | Cryo-EM structure of the human α1β3γ2 GABAA receptor in a lipid bilayer[END_REF]Masiulis et al., 2019). Interestingly, the importance of the length of the M3-M4 loop was studied in the 5-HT3R and it was shown that even a loop as short as 2 alanine residues is sufficient to obtain the functional receptor [START_REF] Mckinnon | Length and amino acid sequence of peptides substituted for the 5-HT3A receptor M3M4 loop may affect channel expression and desensitization[END_REF]. Varying the length of the alanine linker resulted in differentially active receptors, suggesting that it is not only the loop length that matters but also the secondary structure of the linker, which may influence proper receptor functioning and expression of the protein [START_REF] Mckinnon | Length and amino acid sequence of peptides substituted for the 5-HT3A receptor M3M4 loop may affect channel expression and desensitization[END_REF].

Functional studies of the 5-HT3R have revealed the importance of the intracellular domain region, particularly that of the residues of the MA helices influencing the single-channel conductance of the receptor [START_REF] Kelley | A cytoplasmic region determines single-channel conductance in 5-HT3 receptors[END_REF]. The residues responsible for the difference in conductivity between the homo-(A subunit only) and heteropentamers (AB receptor) were determined to be R416, R420 and R424 (Fig. 14) as described in detail in the section 1.4.1.

The pathway for ions through the ICD region is still to be elucidated and is speculated not to continue along the pore axis. Cations passing through the 5-HT3R are putatively thought to exit through the lateral portals at the level of the M3-MX loop below which the rings of positively charged arginines are located, whose function might be the repulsion of the positively charged ions or rather the formation of the salt bridges [START_REF] Kozuska | Impact of intracellular domain flexibility upon properties of activated human 5-HT 3receptors[END_REF]. Further down the membrane the MA helices expose hydrophobic residues on the inside of the bundle and this region represents the tightest part of the whole pore vestibule with a minimal width of about 4.2 Å.

Regarding interacting partners of the 5-HT3R, the only known protein binding directly to the ICD is the resistance to inhibitors of cholinesterase type 3 protein (RIC3) [START_REF] Nishtala | Direct interaction of the resistance to inhibitors of cholinesterase type 3 protein with the serotonin receptor type 3A intracellular domain[END_REF], known to be a protein chaperone. Its presence enables the maturation of multiple Cys-loop receptors [START_REF] Halevi | The C. elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors[END_REF][START_REF] Halevi | Conservation within the RIC-3 gene family. Effectors of mammalian nicotinic acetylcholine receptor expression[END_REF][START_REF] Lansdell | RIC-3 Enhances Functional Expression of Multiple Nicotinic Acetylcholine Receptor Subtypes in Mammalian Cells[END_REF][START_REF] Walstab | RIC-3 Exclusively Enhances the Surface Expression of Human Homomeric 5-Hydroxytryptamine Type 3A (5-HT3A) Receptors Despite Direct Interactions with 5-HT3A, -C, -D, and -E Subunits[END_REF].

X-ray crystallography and cryo-electron microscopy of the membrane proteins

During the recent years we have seen a complete revolution in macromolecular structure determination. X-ray crystallography was, for a long time, the main tool supplemented by NMR. However, methodologic development and technical advancement have made it possible to determine the first high-resolution structures of membrane proteins by single-particle cryo-electron microscopy in 2013 [START_REF] Cao | TRPV1 structures in distinct conformations reveal activation mechanisms[END_REF][START_REF] Liao | Structure of the TRPV1 ion channel determined by electron cryo-microscopy[END_REF]. Since then the ratio of membrane protein structures solved by X-ray crystallography compared to cryo-EM has decreased (Fig. 15), with cryo-EM structures representing almost 37% of all membrane protein structures solved during the first four months of 2018 [START_REF] Cheng | Membrane protein structural biology in the era of single particle cryo-EM[END_REF].

Without considering the common difficulties during the overexpression and purification of membrane proteins, both methods have their one specific bottleneck to surpass on the way to atomic resolution structures. X-ray crystallography requires crystal growth, which is generally a very challenging and complex procedure. Moreover, crystal growth of membrane proteins is complicated by the presence of detergent micelles. Solubilised membrane proteins might not be stable enough to form crystals, plus the presence of the micelle may limit the possibility of crystal contact formations. Multiple techniques aiming to solve this problem have been introduced. Among them are the use of antibodies/nanobodies to increase the potential effective surface for crystal contact formation and the insertion of proteins into the lipidic mesophases that may mimic more native-membrane-like conditions.

The cryo-EM method demands the same high-quality protein sample regarding its purity, monodispersity and stability as X-ray crystallography. Crystals are no longer required but free micelles might pose a problem. They increase the noise and make data analysis more complicated (especially for small proteins analysed in combination with large micelle-forming detergents). To eliminate this problem, detergent was replaced by amphipol at least in the early cryo-EM studies of membrane proteins. Nowadays, the structures of membrane proteins are solved using not only amphipols [START_REF] Cao | TRPV1 structures in distinct conformations reveal activation mechanisms[END_REF] but also detergents [START_REF] Du | Glycine receptor mechanism elucidated by electron cryo-microscopy[END_REF] and nanodiscs [START_REF] Efremov | Architecture and conformational switch mechanism of the ryanodine receptor[END_REF][START_REF] Gao | TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action[END_REF]. Another obstacle is the need to have multiple orientations of protein particles in the holes of the frozen grid. Fluorinated detergents can be a helpful tool for membrane proteins [START_REF] Basak | Crystal structure and dynamics of a lipid-induced potential desensitized-state of a pentameric ligandgated channel[END_REF][START_REF] Efremov | Architecture and conformational switch mechanism of the ryanodine receptor[END_REF], but one should keep in mind the fact that those detergents can potentially influence the membrane proteins as has been recently shown for the ryanodine receptor [START_REF] Willegems | Influence of Lipid Mimetics on Gating of Ryanodine Receptor[END_REF].

At the beginning of my PhD the only 5-HT3R structure available was the crystallographic structure of the m5-HT3RA receptor obtained using stabilising nanobodies. We wanted to solve other conformational states of the receptor using X-ray crystallography but the fast development of cryo-EM and the first high-resolution structures of membrane proteins solved via the cryo-EM method encouraged us to start using this method as well. Moreover, the m5-HT3R is an ideal candidate for cryo-EM due to its reasonable size (~250 kDa) and symmetry. The methodology of the X-ray crystallographic and cryo-EM structural studies together with the results and their discussion follows after the Objective of the thesis section.

Objective of the thesis

Due to their important role in neuronal signaling and their potential for pharmacological intervention, many receptors from the pLGIC superfamily are studied extensively, both functionally and structurally. Even though intensive efforts are being made, our knowledge of the signal transduction within the receptor remains limited. The best way to understand the mechanism of the receptor function is the co-interpretation of functional and structural data. Although this thesis focuses on structural studies, all the steps were planned taking into account the functional properties of the receptor known from the literature and complementary functional experiments were carried when needed.

As a follow up of the already described crystallographic structure of the 5-HT3RA receptor solved up to a resolution of 3.5 Å, the aim of my thesis was to try to obtain the structure(s) of an other state(s) and/or potentially obtain a structure with a better resolution, which would allow better structure-function interpretation. In the initial stages, the crystallographic approach was prefered; we quickly realized that the rapidly developing cryo-electron microscopy technique was to become the best tool for the structural studies of our target. Thus, the main goals of my thesis were: I) Crystallography: Use of nanobodies, which were shown to stabilise the receptor and help to create the crystal contacts, for co-crystallisation with the receptor using a classical vapour diffusion method to obtain the receptor locked in (an) other conformation(s).

Use of the lipidic cubic phase to obtain a better resolution structure of the 5-HT3RA.

Co-crystallisation of the receptor with drugs known to improve its stability to obtain better resolution or block the receptor in the open state.

II) Cryo-EM:

To obtain the closed conformation of the receptor as a confirmation of / comparison to the existing crystallographic structure and potentially obtain a structure of the desensitised and/or open state of the receptor.

The m5-HT3R isolation, purification and treatment

The m-5HT3R preparation -materials and methods

DNA construct(s)

Even if I have generated a variety of constructs during my PhD (including 5-HT3 receptor constructs for expression in Leishmania tarentolae cells, ɑ4β2 constructs fused with GFP ), most of the work described here was performed with a single construct encoding for the full-length homomeric receptor. It originates from previous work on the homomeric 5-HT3RA [START_REF] Hassaine | Large scale expression and purification of the mouse 5-HT3 receptor[END_REF] .

The DNA construct for the mouse 5-HT3RA is in a pcDNA5/TO vector, enabling transient transfection of mammalian cells (typically 293F cells), generation of stable inducible cell lines in HEK-Trex cells, and injection into Xenopus laevis oocytes for TEVC functional assays. The insert is composed of a signal sequence, four consecutive Strep tags for purification, a TEV cleavage site and the mature 5-HT3RA sequence (depicted in the Extended Data Figure 1 of our paper [START_REF] Polovinkin | Conformational transitions of the serotonin 5-HT3 receptor[END_REF]).

Cell culture

The adherent, stable, inducible, cell-line HEK 293 5-HT3RA receptor originates from previous work on the homomeric 5-HT3RA receptor [START_REF] Hassaine | Large scale expression and purification of the mouse 5-HT3 receptor[END_REF]. This cell line expresses constitutively the tetracycline repressor, preventing the expression of the target protein in the absence of tetracycline, and it also bears copies of the gene of interest. This cell line was adapted to be used not only for roller bottle expression, which is a tedious and manpower-heavy technique, but also for expression in suspension cultures.

For this purpose we used the adherent stable cell line HEK 293 5-HT3RA. We detached the cells from an 85% confluent 25 mL flask (Dutscher) with 5 mL PBS (Gibco) by gently pipetting up and down and we transferred 1 mL of the cell suspension into a 100 mL Erlenmeyer tube containing 10 mL of FreeStyle media (Gibco) supplemented with 10% of newborn calf serum (Corning). The serum concentration was halved every 3rd day during the passaging of the cells. Cells were plated to the density of 0.8 million cells/mL. At every passage, the amount of antibiotics was increased in a stepwise manner up to the final concentration of 5 μg/mL of Blasticidin and 100 μg/mL of Hygromycin B. Cells were grown while shaking in an orbital shaker at 37 °C, 5% CO 2 and 90 rpm.

Typically, expression was induced by adding 4 mg/L of tetracycline to batches of 2 L of cultures at 2.10 6 cells/mL. The day after induction 4 mM valproic acid was added. Cells were harvested 48-72 h after induction.

Isolation of the 5-HT3RA

I have optimised the purification protocol of the receptor described in [START_REF] Hassaine | X-ray structure of the mouse serotonin 5-HT3 receptor[END_REF] and only the final version is described in the following sections.

Membrane preparation

To avoid contamination by the cytosol debris, I performed a membrane preparation step prior to the solubilisation step.

The collected cells were resuspended in resuspension buffer (10 mM HEPES pH 7.4, 1 mM EDTA and the inhibitor mix CLAPA (1µg/ml Chymostatin,1µg/ml Pepstatin, 1 µg/ml Leupeptin, 1 µg/ml Antipain and 4µg/ml of Aprotinin)) using 10 mL of buffer per 1 g of cells. Cells were kept on ice and lysed using the T25 Ultra Turrax homogeniser by five consecutive 30 s steps with increasing rotation of the homogeniser followed by a 1 min homogenisation at maximal speed. A low speed centrifugation was performed to remove unbroken cells (4000 x g, 20 min, 4 °C). The low-speed supernatant was further spun down using ultracentrifugation (120 000 x g, 1 h, 4 °C). The supernatant was decanted and the pellets containing the membranes were stored at -80 °C.

Solubilisation

Membrane proteins contain hydrophobic parts that are immersed in cellular membranes. To extract this hydrophobic moiety from the membrane and keep it intact and soluble in an aqueous solution, we use amphipathic molecules. These molecules consist of hydrophobic regions which are in contact with the transmembrane hydrophobic part of the protein and of hydrophilic polar group/s which is/are oriented into the solution. The most commonly used surfactants are detergents, which are able to extract the protein from the membrane and maintain it in solution, and amphipols, which are not suitable for the extraction but work well for protein stabilisation in solution [START_REF] Seddon | Membrane proteins, lipids and detergents: not just a soap opera[END_REF]. Detergents are composed of a hydrophobic carbon tail and a polar head. Each detergent has specific properties regarding its harshness, its capability to extract the protein out of the membrane, the level of the native membrane lipids that it is able to remove from around the extracted protein, and its ability to form micelles (the self assembly of single detergent molecules into a supramolecular structure). An important property of a given detergent is its critical micellar concentration (CMC: the concentration border, which once exceeded initiates and favours the formation of the micellar formation over free detergent). For solubilisation of membrane proteins, high detergent concentrations can be necessary (typically 10-100xCMC); but to keep the protein soluble it is generally sufficient to keep detergent levels at at least 1xCMC.

Our membranes were resuspended in the solubilisation buffer (50 mM Tris pH 8, 500 mM NaCl and the inhibitor mix CLAPA) using a 1:25 mass ratio of membranes:buffer. After homogenisation on the magnetic stirrer, 0.15% C 12 E 9 (50xCMC) was used to solubilise the receptor. Solubilisation was performed on the magnetic stirrer for 1.5 h at 4 °C followed by ultracentrifugation (120 000 x g, 1 h, 4 °C ). The supernatant contained the solubilised protein.

Purification of the 5-HT3RA

Affinity chromatography

The Strep tag on the N-terminus of the receptor was used for affinity chromatography on the Strep-Tactin resin (IBA). For a typical purification we used 1.5 mL of the resin/1 g of membranes. We used the gravity-flow set up in columns. The resin was equilibrated with 5x column volumes (CVs) of equilibration buffer (50 mM Tris pH 8, 500 mM NaCl and 0.01% C 12 E 9 ), the solution containing the solubilised protein was loaded once on the column and washed with 5 CVs of washing buffer (50 mM Tris pH 7.4, 125 mM NaCl and 0.01% C 12 E 9 ). The protein was eluted with 3 CVs of the elution buffer (50 mM Tris pH 7.4, 125 mM NaCl and 0.01% C 12 E 9 supplemented with 5 mM d-desthiobiotin). The protein was concentrated using Amicon concentrators (Merck Millipore) with a 100 kDa molecular weight cut-off.

Size exclusion chromatography

Size exclusion chromatography (SEC) on a Superose 6 10/300 GL column (GE Healthcare) was performed after enzymatic treatments (see the following section 3. 1.5.1) to separate the cleavage products or after the amphipol exchange (section 3.1.5.2) to remove the remaining detergent. The column was equilibrated in size exclusion buffer (50 mM Tris pH 7.4). For crystallographic purposes the buffer contained 0.01% C 12 E 9 and 125 mM NaCl. For the cryo-EM experiments, the buffer contained 0.01% C 12 E 9 and only 65 mM NaCl. The SEC performed after amphipol exchange was done using the size exclusion buffer (50 mM Tris pH 7.4, 125 mM NaCl) in the absence of detergent. The 5-HT3RA eluted from the column at around 13.7-14mL and no significant shift was observed for the protein in amphipol compared to the protein in detergent.

Treatement of the 5-HT3RA

Enzymatic treatment

The 5-HT3A contains a partially unstructured intracellular domain, which causes the formation of badly diffracting crystals and we were thus using limited proteolysis by trypsin (0.0125:1 mass ratio of trypsin:protein, 2 h at 30 °C) to cleave this region out. SEC was performed immediately at the end of incubation with trypsin, in order to avoid excessive cleavage which could lead to protein degradation.

On the other hand, this unstructured part seemed to prevent aggregation when the protein was used for cryo-EM and thus only the N-terminal Strep tags were removed when we prepared samples for cryo-EM. They were cleaved by the TEV protease (0.04:1 mass ratio of TEV protease:protein, overnight (ON) at 4 °C).

The 5-HT3RA has 3 N-glycosylation sites. Glycosylations added heterogeneity and were perturbing crystallogenesis. We used PNGase F, which is an amidase that cleaves between the innermost GlcNAc and the asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins. Cleavage was carried out using the 0.1:1 mass ratio of PNGase F:protein (2 h at 4 °C).

Amphipol exchange

The cryoEM structure of TRPV1 [START_REF] Cao | TRPV1 structures in distinct conformations reveal activation mechanisms[END_REF] was a landmark, in the field of membrane protein structures solved by this method. That structure was obtained with a sample solubilized in amphipol, and was the reason why we and others performed experiments in amphipol. It has different properties compared to the detergent. It does not form micelles but wraps the protein around its hydrophobic part, thus once all the proteins are protected by the amphipol molecules, no further free amphipol is needed in the solution. Amphipol is a long polymer composed of a different ratio of the monomers containing polar and nonpolar side chains [START_REF] Tribet | Amphipols: polymers that keep membrane proteins soluble in aqueous solutions[END_REF][START_REF] Zoonens | Amphipols for Each Season[END_REF]. We used commercially available amphipol A8-35 (Anatrace) where the number 35 indicates the percentage of the overall amount of polar monomers present in the polymer. In principle, once amphipol is added to the protein-detergent solution, it starts to compete with the detergent for the hydrophobic parts of the protein. The exchange can only be completed by removal of the detergent from the solution either by dialysis or by the use of polymeric beads. We decided to use the bio-bead method rather than dialysis and we optimised the detergent-amphipol exchange protocol based on the publication explaining the use of the bio-beads [START_REF] Rigaud | Bio-Beads: an efficient strategy for two-dimensional crystallization of membrane proteins[END_REF] for optimal detergent removal and the publication describing the exchange of the TRPV1 receptor from detergent into amphipol [START_REF] Liao | Structure of the TRPV1 ion channel determined by electron cryo-microscopy[END_REF]. Here I present only the final protocol.

First the protein was concentrated to the final concentration of 0.5 mg/mL. Then amphipol A8-35 was added in a 4:1 mass ratio of amphipol:protein and the solution was incubated (3 h, 4 °C, mixing). After the incubation the Bio-Beads (BIO-RAD) were added in a 106:1 mass ratio of Bio-Beads:protein and incubated overnight at 4 °C while mixing, to remove the detergent from the protein-detergent-amphipol solution. Bio-beads with the bound detergent were then removed from the protein-amphipol solution and SEC was performed as described in the section 3.1.4.2.

The m5-HT3R preparation -results

My project started as a follow up of the structure of the m5-HT3RA solved by crystallography. At the beginning I took over the protocols for the protein purification and crystallisation of the receptor [START_REF] Hassaine | X-ray structure of the mouse serotonin 5-HT3 receptor[END_REF] and I tried to optimise them with multiple aspects in mind, specifically aiming to increase the yields, shorten the purification time and lower the costs. The initial protocol comprised of cell homogenisation, membrane separation by ultracentrifugation, protein solubilisation followed by ultracentrifugation, which led to solubilised protein solution. The next step was a filtration which allowed us to proceed with Strep-Tactin affinity chromatography.

Pre-packed Strep-Tactin columns were used and purification was done using the peristaltic pump loading the sample overnight. To improve homogeneity of the sample and to remove potential flexible parts of the protein, deglycosylation was carried out by PNGase F in a ratio of 1 mg : 0.1 mg receptor to deglycosylase (2 h shaking at 37 °C) and limited proteolysis was done by trypsin in a ratio of 1 mg : 0.013 mg receptor to protease (incubated 2 h at 30 °C).

I optimised the solubilisation step, trying to lower the concentration of detergent used and to shorten the incubation time. In contrast to the initially quite high concentration of C 12 E 9 (1%) used for 3 h at 4 °C, I demonstrated that using a concentration of 0.15% for 1.5 h at 4 °C solubilised the protein to the same extent (Fig. 16).

A very time-demanding step was the filtration of solubilised protein solution through 0.22 μm filters. This step was mandatory because of the clogging of the pre-packed Strep-Tactin column filter. We tested the gravity flow set-up and as neither the yield nor the purity of the protein was affected (Fig. 17) we switched to this approach which saved us ~ 2 h of filtration.

A relatively expensive part of sample preparation was the usage of PNGase F. We decided to adapt the protocol from [START_REF] Loo | Using Secretion to Solve a Solubility Problem: High-Yield Expression in Escherichia coli and Purification of the Bacterial Glycoamidase PNGase F[END_REF] to purify PNGase F in-house instead of using the commercially available enzyme. I adapted it and we now express periplasmic PNGase F and purify it using NiNTA and size exclusion chromatography. We verified the activity of in-house purified PNGase F and tested the conditions for its activity. Compared to the initial 2 h incubation at 37 °C we have shown that the same level of deglycosylation can be achieved even when the reaction is incubated at 4 °C. This might prevent protein denaturation caused by increased temperature (Fig, 18).

The isolation, purification and enzymatic treatment of 5-HTRA are carried out over 2 days. Generally, the yields vary from 0.2 -0.3 mg of pure and treated protein per 1 g of membrane fraction after the final size exclusion chromatography, where a nice homogeneous peak corresponding to the pentameric receptor is observed (Fig, 19).

Crystallography

Crystallography -materials and methods

Vapour diffusion trials

All the screening was carried out on the High-Throughput crystallography platform (HTX-lab) at the EMBL in Grenoble.

Manual plates were mostly set up using a protein concentration of ~8 mg/mL. During the optimisation process different ratios of the protein:reservoir drops were used. To slow down the growth kinetics Ali-oil was used to cover the reservoir in the hanging drop set up. Microbatch under-oil crystallography was performed. Different initial concentrations of the protein were used (from 1 mg/mL up to 10 mg/mL). The seeding method used was seeding into the clear drops. Stepwise dehydration and direct dehydration were tested to improve the crystal X-ray diffraction.

The lipidic cubic phase (LCP) trials

The protein was concentrated typically to 40 mg/mL. In one of the trials the VHH15 was used to further stabilise the receptor but its use increased the hydrophilic part pointing out of the lipid bilayer, which is not favourable for the LCP method and thus we used mostly the receptor alone. In collaboration with the platform for high-throughput LCP crystallisation (Valentin Gordeliy's group, currently at EMBL Grenoble), we set up multiple plates. Commercial screens (Cubic phase I and II (QIAGEN) and MemMeso (Molecular Dimensions)) as well as plates based on the optimisation conditions arising from the positive vapour diffusion trials.

The detergent screen with a focus on further use in X-ray crystallography

We re-screened a dozen detergents (C 12 E 9 , C 12 E 10 , DDM, MNG3, OGNG, OG, cymal5, cymal6, cymal7, LDAO, LAPAO, FosC12), measuring the receptor stability upon heating, with the aim of selecting one with a shorter hydrophobic tail forming smaller micelles to allow for the formation of better protein-protein crystal contacts. We followed the intrinsic fluorescence of the protein's tyrosines and tryptophans during thermal denaturation of the receptor. Addition of 9% detergents was followed by 15 min incubation on ice and then the measurement was performed. Typically the measurement was performed in the range of 20-100 °C using 2 °C/min steps. The fluorescence at two wavelengths, 330 and 350 nm was recorded. The 350/330 nm ratio of the two wavelengths was plotted against temperature and represents the unfolding transitions. The data were measured on the Prometheus (NanoTemper).

As a complementary method we used SEC performed after the heat shock. Protein was purified using C 12 E 9 during solubilisation and for stability in solution. Different detergents were added at the 1% concentration and the mixture was incubated 15 min at 55 °C. After ultracentrifugation to remove potential aggregates, the sample was injected onto a Superose 6 column equilibrated with buffer containing 0.01% C 12 E 9 . The same concentration of this detergent was used in the running buffer (running buffer as for protein purification, see 3.1.4.2).

Blue native electrophoresis

Native gels can qualitatively characterise the size of protein complexes. Regarding protein-protein interactions this method can even answer binding stoichiometry questions. We used native electrophoresis for the estimation of the binding ratio in between nanobodies and the 5-HT3A.

All the buffers and procedures were prepared and performed according to the NativePAGE Novex® Bis-Tris Gel System manual (Novex, LifeTec MAN0000557). Briefly, 6% polyacrylamide gels were cast. 10 µg of the purified 5-HT3 were mixed with the 1:2 molar ratio of one receptor subunit:VHH and incubated on ice for 10 minutes. The sample was mixed with the sample buffer (for reference see the manual mentioned above) and 1 µL of 0.4% Coomassie G-250 per 10 µL of mix. Anode and cathode buffers were prepared according to the manual and filled into the electrophoresis system. We loaded the samples onto the gel prior to complete filling of the apparatus with the cathode blue buffer for better visualisation. The electrophoresis was performed either in the cold room or on ice at 150 V for ~2 h. When the run is complete, the blue cathode buffer, migrated through the gel, should start appearing in the normally transparent anode buffer. Extensive destaining of the gel is needed. The gel was first fixed by immersion in 100 mL fixing solution (containing 40% methanol and 10% acetic acid), then microwaved for 45 s and incubated on the shaker for 20 mins. The fixing solution was decanted. The gel was then destained in 8% acetic acid, microwaved for 45 s and incubated until the gel background became transparent.

Crystallography -results

Crystallisation of the m5-HT3RA using nanobodies

VHHs

Camelids possess antibodies made of heavy chains only. Engineered terminal domains of these antibodies are called nanobodies or VHHs. These domains retain their antigen-binding ability, their typical size is ~15 kDa, and they can generally be overexpressed in E. coli. The antigen-binding paratope is made up of 3 complementary binding regions (CDR1-3).

The use of nanobodies has multiple benefits in structural biology [START_REF] Pardon | A general protocol for the generation of Nanobodies for structural biology[END_REF]. They might help to solubilise the protein by increasing its polar surface and they can stabilise the protein, which reduces heterogeneity of the sample. Their use in crystallography can be instrumental in solving the structures of unknown proteins by enabling molecular replacement. Additionally their presence might also help to promote crystal growth by supporting the formation of crystal contacts, as was the case with crystallisation of the m5-HT3RA/VHH15 complex [START_REF] Hassaine | X-ray structure of the mouse serotonin 5-HT3 receptor[END_REF]. After this initial success, our strategy was to try to find another nanobody, which would stabilise the receptor in another conformation.

Nanobodies are obtained following immunisation of the animal (see [START_REF] Pardon | A general protocol for the generation of Nanobodies for structural biology[END_REF] for methods). The protein of interest is injected into an animal and its immune response yields new antibodies. A library of all VHHs is created and those interacting with the protein of interest are selected by phage display and then assayed using ELISA tests with the purified protein. The nanobodies used in our studies were from collaborators in Marseille. The information available to us are the VHH sequences and their ELISA score. For better orientation in our library composed of about 15 nanobodies we sorted them into six groups according to their sequence similarity (Fig. 20).

Co-crystallisation

Following the current trend in crystallography, after the selection of nanobodies from sequentially different groups (one for each group) we used the service of the HTX laboratory on campus to set up commercial screens (generally starting with 6 screens: PACT, PEGs, JCSG and Wizzard I+II).

Protein was purified as described in section 3.1.3-4. After size exclusion chromatography, peak fractions were pooled together and concentrated to ~8 mg/mL. Before crystallisation, the protein was mixed with VHH in a 1:2 molar ratio, (1 monomer of the m5-HT3RA with an excess of 2 VHHs). This mixture was supplemented with 1 mM of Cymal-6. Here the role of Cymal-6 is not to further stabilise the complex, as demonstrated by our thermostability data (Fig, 22). We hypothesise that the positive effect of Cymal-6 during crystallisation is caused by a decrease of the micelle size that promotes protein/protein contacts and results in better crystal packing.

Working towards our goal, we focused on the nanobodies sufficiently distinct from VHH15, hoping that they will stabilise the receptor in another conformation. Unfortunately, even though we have access to biophysical methods to characterise receptor-VHH interactions, we lacked a way to characterise them functionally, which would probably provide the optimal criteria for selection of the crystallisation candidates.

Using 7 different nanobodies we obtained 157 hits, which largely exceeded our expectations. We focused mainly on the conditions which were as different as possible from the conditions under which the structure of receptor in complex with VHH15 was obtained. Manually, we reproduced more than 70 conditions selected to maximise diversity. The majority of them gave crystals. The synchrotron measurements confirmed that these were, apart from very few exceptions, protein crystals with a poor diffraction, ranging from ~30 to ~9 Å at most. We optimised all conditions that yielded crystals but this did not improve their diffraction. Altogether we shot around 550 crystals.

The chemical compositions of the hits obtained were very divergent and we could not observe any clear pattern. Crystals grew at different pHs (5-10), using different organic and inorganic salts and PEGs (from 3350 -10000) (see examples in (Fig. 21)). Changes in chemical composition of the mother liquor (type and amount of ingredients) did not improve the diffraction by our crystals and we focused mainly on physical parameters. From the classical methods used for diffraction improvement we tried: different growth temperatures, seeding, dehydration and growing under oil using microbatch crystallisation. Using recently developed methods we also tried to use CrystalDirect plates, which make it possible to shoot crystals without fishing, directly in the plate. This approach reduces possible manipulation damage but it is compatible only with room-temperature measurements. Except for the micro-batch method, all others listed led to crystals but did not affect diffraction. We collected data of crystals diffracting to 9 Å in the hope of further processing. We were able to determine the space group of crystals grown with two different VHHs but for none of them we were able to get a molecular replacement solution.

Neither the use of the nanobodies at our disposal nor the optimisation of crystallisation conditions led to X-ray diffraction of sufficient quality from any of the many crystals obtained. We thus concluded that larger changes were required and started to re-evaluate the sample preparation protocol. The choice of detergent influences solubility, stability and of course plays an important role during crystallisation. For instance, the crystal quality of the m5-HT3RA/VHH15 complex had been improved when Cymal-6 was used as a supplement to C 12 E 9 , possibly because Cymal-6 reduced the micelle size and thus favoured protein-protein contacts in the crystals. Consequently, we performed a new thermal stability detergent screen using NanoTemper's Prometheus and SEC (see 4.1.3). We observed a correlation between the length of the carbon tail and the protein stability (see below). Although detergents with longer carbon chains were better at stabilising the receptor, some of the shorter tail detergents stood out from the screen. Namely the Cymals and undecyl-maltoside (UDM), the latter being the detergent used for crystallography of the nicotinic α4β2 receptor [START_REF] Morales-Perez | X-ray structure of the human α4β2 nicotinic receptor[END_REF].

Figure 22 depicts thermal denaturation curves for the Cymal series of detergents, where an effect of the carbon tail length was observed, with Tm(Cymal-5) = 52 < ℃ Tm(Cymal-6) =57

< Tm ℃ (Cymal-7) = 60 < Tm ℃ (C 12 E 9 ) = 64 . The comparison of ℃ C 12 E 9 and UDM revealed that UDM might also be a good candidate for crystallographic studies of 5-HT3RA. These detergents stabilise the receptor enough to be used for crystallography, while potentially enabling easier formation of protein-protein contacts.

Because of its similarity to DDM in which the receptor remains active and in view of the recently solved structure of the other Cys-loop receptor in this detergent, we decided to proceed with UDM. Moreover, the size exclusion chromatography profile of the m5-HT3R in presence of UDM as compared to C 12 E 9 looked very encouraging (Fig. 23) Based on our previous experience with the m5-HT3A receptor we set up he PEGs screen and surprisingly observed 5x more promising hits (objects rather than crystals) in the conditions where UDM was used compared to where C 12 E 9 was used, even without the use of nanobodies. At this point, the cryo-EM started to provide early positive results and we decided to focus mainly on it and not to continue with crystallography.

Biochemical characterisation of the nanobodies

From all of the above-mentioned, it is clear that it is not possible to try extensively the combination of all approaches with all different nanobodies and all crystallisation conditions. The difficulties encountered emphasised the need for better biophysical characterisation of our nanobodies.

Looking at other examples of Cys-loop receptors crystallised with chaperones (either Fabs [START_REF] Hibbs | Principles of activation and permeation in an anion-selective Cys-loop receptor[END_REF] or VHHs) we noted that in all successful cases there were 5 chaperones bound per receptor (one per subunit). Thus we sought for a method to determine the binding ratio between the receptor and nanobodies. We tested native electrophoresis for this purpose. The receptor alone and the receptor pre-incubated with VHH15, which binds in a 5 to 1 ratio in the crystal structure, were used as references. Early results indicated clearly the difference between these two samples but the resolution was not good enough. Thus, we turned to BlueNative electrophoresis and replaced the detergent with amphipol. This approach gave us an estimate of the binding ratio (Fig. 24).

For example, VHH4 is very closely related in sequence to VHH15 (97% sequence similarity / 6 point mutations), indicating that it should bind in the same ratio; indeed those two complexes migrate to the same position on the gel. Interestingly, preincubation with nanobody VHH10 does not seem to affect the receptor migration on the BlueNative electrophoresis gel and we were able to confirm the lack of this nanobody in the corresponding crystals by SDS PAGE (data not shown).

Another important parameter influencing the stability of the complex is the binding affinity of the antibody to the receptor. We performed labeled microscale thermophoresis and we were able to demonstrate that this method is not appropriate in our case (extensive trials, results not shown). The conditions of the protein labeling led to m5-HT3R denaturation. The measurement obtained using the non-precipitated fraction of labeled protein gave an uninterpretable binding isotherm. Surface plasmon resonance seems to be a good alternative for assessing protein-protein interactions but, as we know from the trial with VHH15, the single cycle kinetics is the only mode possible, somehow complicating data interpretation.

Finally, we lacked easily accessible functional tests for the elucidation of the nanobody effect on the protein stability. Functional tests would be probably the best indication of stability when looking for the nanobody stabilising the receptor in another conformation than VHH15, which acts as an antagonist. Some of the experiments I performed on the expression optimisation were reported in a case study in Membrane Protein Structure and Function Characterization book [START_REF] Hassaine | Expression, Biochemistry, and Stabilization with Camel Antibodies of Membrane Proteins: Case Study of the Mouse 5-HT3 Receptor[END_REF].

Electron microscopy

Currently, electron microscopy is finding its place next to the other near-atomic resolution structural determination methods, such as X-ray crystallography. It is about 70 years ago when the first biological sample was imaged using EM and since then a huge amount of progress was achieved. The electrons being scattered by the air required the evacuation of the microscope column and thus the sample also needed to be dehydrated, which caused the limited contrast of the images obtained -micrographs. This introduced the need for a contrast increase and that's how negative staining EM appeared [START_REF] Huxley | Preferential staining of nucleic acid-containing structures for electron microscopy[END_REF]. In negative staining, the electron-dense solution, mostly uranyl acetate, surrounds the specimen being observed and increases the contrast. Passing through the imaging of the 2D crystals kept dehydrated in the glucose solution, this led to a resolution of about 7 Å [START_REF] Henderson | Three-dimensional model of purple membrane obtained by electron microscopy[END_REF], but the real breakthrough came with the routine use of sample freezing and the introduction of the cryo-stage into the microscope. That was the beginning of the cryo-EM method as we know it today. Nowadays, the sample in aqueous solution is rapidly frozen by the liquid ethane, allowing formation of the amorphous solid phase instead of crystalline ice, which had resulted in biological sample degradation in the past.

Electron microscopy -material and methods

Negative stain EM of m5-HT3RA

As an initial quality control we performed negative-staining transmission electron microscopy using the electron microscopy platform of IBS. Purified sample (around 1 mg/mL) was deposited on a glow-discharged carbon grid and stained with uranyl acetate pH 7. Images were taken with a Tecnai12 Microscope (FEI) operating at 120 kV, a LaB6 electron source and an Orius 1000 GATAN camera.

Cryo-EM of the m5-HT3RA

During the cryo-EM sample optimisation we performed multiple tests. Samples of different concentrations were used, starting from 0.5 up to 24 mg/mL. We used different commercially available grids. Samples were frozen using a variety of blotting time lengths. Changing the biochemical sample preparation we went back to the optimisation of grid preparation and fine tuned the conditions for all biochemically divergent samples. The typical protein concentration used in amphipol or in detergent was around 10 mg/mL and about 3.5 mg/mL, respectively. Samples were deposited on the glow-discharged carbon grids and frozen by FEI Vitrobot in liquid ethane. Samples were stored under liquid nitrogen until being imaged. Imaging was completed either at our institute on the EM platform using Tecnai G2 Polara cryo-transmission EM with a 300 keV field emission gun (FEG) equipped with a K2 Gatan camera, or in the C-CINA scientific group in Basel on the KRIOS cryo-TEM with a 80-300 keV FEG, Gatan Quantum-LS Energy Filter (GIF) with a Gatan K2 Summit direct electron detector. Particle picking was carried out using Dr. Kai Zhang's Gautomatch and the data treatment was done using Relion software [START_REF] Scheres | RELION: implementation of a Bayesian approach to cryo-EM structure determination[END_REF].

Electron microscopy -results

The Cys-loop receptors are relatively good candidates to be studied by cryo-EM because of their size, their five-fold symmetry and the pre-existence of models (structures solved by X-ray crystallography). The first structure of a Cys-loop receptor ever solved was done by cryo-EM and it was the structure of the nAChR from the electric organ of the Torpedo fish [START_REF] Miyazawa | Structure and gating mechanism of the acetylcholine receptor pore[END_REF]. At the time we started working on the 5-HT3 project, cryo-EM had already been successfully applied to the GlyR [START_REF] Du | Glycine receptor mechanism elucidated by electron cryo-microscopy[END_REF], providing insights into the open, closed and desensitised structures of this receptor.

In my hands, the X-ray crystallization trials did not lead to structures of other conformational states in addition to that already known. Encouraged by the structure of TPRV1, a membrane protein, whose structure was solved at 3.4 Å resolution using cryo-EM [START_REF] Cao | TRPV1 structures in distinct conformations reveal activation mechanisms[END_REF][START_REF] Liao | Structure of the TRPV1 ion channel determined by electron cryo-microscopy[END_REF], we decided to use electron microscopy as a complementary method (and as time passed by, as our main structural method).

Initial trials and optimisation

In our early microscopic trials, a few years back, we used the 5-HT3 receptor solubilised in amphipol. Our/the naive rationale was of a dual nature: the pioneering study of TRPV1 had proven the usefulness of amphipols in structural studies (finally), and microscopists advised us to avoid the presence of detergent during the freezing process. We adapted the exchange of detergent for amphipol as described in section 3.1.5.2. We could obtain a homogeneous pentameric receptor after size-exclusion chromatography (Fig. 25).

Apart from this change in tensio-active, we first kept the protein preparation procedure similar to that for the crystallographic sample, reasoning that if it could crystallise, the protein was of good quality. Thus we used receptor deglycosylated by PNGase F and partially digested by trypsin leading to the removal of the Strep-tag and of the unstructured intracellular region. Negative stain microscopy in the presence of VHH15 nanobodies yielded good-looking micrographs, with side and top orientations of proteins, and almost no aggregation (Fig. 26A) Unexpectedly, when we proceeded to cryo-EM, we observed aggregation of the entire sample caused by the nanobodies, making it impossible to pick single particles (Fig. 26B). The first lesson learnt was thus that a good quality negative stain does not necessarily predict the quality of cryo-EM micrographs. Thus we decided to optimise the conditions directly by cryo-EM.

Early trials with the receptor in amphipol without nanobody present resulted in micrographs containing mostly one orientation of receptors: top views (Fig. 27A). In addition, it seemed that we had an aggregation problem, shown by the presence of 'darker' top views with higher contrast and 'prolonged' side views (Fig. 27B), which we hypothesised were head-to-head or head-to-tail dimers.

We screened different surfactants to solve the orientation and aggregation problems. We tested the more polar amphipol version A8-75 and observed greater aggregation while the orientation problem persisted (Fig. 28).

As fluorinated detergents (F-TAC) had been used to rescue anisotropic orientations of membrane proteins on EM grids, we tested the effect of the addition of F-TAC. The addition of 0.2% F-TAC increased the proportion of side views of the receptor. Because 0.2% roughly correspond to the concentration required to saturate the air/water interface in a grid hole, one hypothesis is that F-TAC impact orientation via surface tension modification, and not via interaction with the protein or amphipols. A side effect of the addition of F-TAC was that fewer particles were present in the holes. We tested grids with a 2-5 nm layer of carbon on top of the holes to increase the number of particles, but this resulted in completely crowded unusable micrographs. We were also concerned that the carbon layer might preclude the acquisition of high-resolution data and decided to instead make changes to the protein concentration. Compensating the lower density of receptor by an increased starting concentration (up to ~20 mg/mL), we collected a dataset with this sample (5-HT3R-Apol-F-TAC) on the G2 Polara cryo-transmission EM with a 300 keV FEG equipped with a K2 Gatan camera but it yielded only a reconstruction of limited resolution, at about 8 Å. We were concerned that conformational flexibility of the receptor might prevent us from reaching a better resolution and we thus collected a second dataset, using a receptor inhibited with 1 mM granisetron. The dataset (5-HT3R-Apol-F-TAC-granisetron) was collected on the Titan Krios microscope in the Center for Cellular Imaging and NanoAnalytics (C-CINA) in Basel. From 421 micrographs, ~125k particles were picked using the low-filtered model of the crystallographic structure. After several rounds of 2D classification, the best looking classes were put together and ~50k particles were used for 3D classification. The best class was further refined, trying the application of different masks, and the final resolution was ~7 Å (Fig. 29).

One of the parameters, which influences the quality of the data collected is the ice thickness. In our case, the ice layer was probably too thick (as evidenced by the difficulty of seeing side views on raw images) to obtain the atomic resolution of the receptor. Apart from the nature of the particles themselves, the composition of the buffer composition also influences ice thickness. The salt concentration, together with the type of tensio-active molecule used, can be crucial. We thus decreased the salt concentration by a factor two but were limited by the relatively high concentration of F-TAC (0.2 %).

We therefore took a step back and returned to the testing of surfactants, focusing on detergents at this time. Specifically, we tried 0.05% DDM, 0.1% MNG3 and 0.01% C 12 E 9 . When using DDM or MNG3, the receptor was purified in C 12 E 9 and we exchanged detergents during the final SEC. According to the SEC profiles, the receptor was perfectly homogenous in the presence of all detergents. Although both alternative detergents, DDM and MNG3, seemed to at least partially solve the orientation problem, they caused significant aggregation of the receptor (Fig. 30).

In the end, the detergent C 12 E 9 was thus shown to be the best choice. It enabled the observation of multiple orientations of protein on the grid and we solved the aggregation problem, which occurred during the freezing step, by omission of the limited digestion (using trypsin) step, which was routinely used for crystallographic studies. Additionally, we retained the disordered intracellular domain of the protein in the hope that its presence will prevent aggregation. To mimic the natural conditions as closely as possible, we used lipid additives (0.01% phosphatidic acid, 0.01% cholesterol hemisuccinate, 0.01% brain phosphatidylcholine, Avanti Polar lipids). After testing a range of grids, we decided to use the Cu-Rh grids, which resulted in good sample quality with the required amount and distribution of particles (Fig. 31). These optimised conditions were a good starting point for performing structural studies.

The gating mechanism of the m5-HT3RA (enclosed publication)

In the last decade, from the appearance of the first ELIC structure (and even before) until today, huge efforts have been made to elucidate the gating mechanism(s) of Cys-loop receptors. The "steady state" structure-to-function interpretation remains the only method available for this purpose. To date, there are only few receptors whose structures have been solved in multiple conformational states, which enables us to understand their complex gating mechanism. Specifically, these are the structures of the WT GLIC receptor, the GluCl receptor, the engineered GlyR structures and the m5-HT3R structures published in parallel. Here I present our recently published data on the 5-HT3R. Four structurally distinct states of 5-HT3RA provide, for the first time, insights into the gating mechanism of the cation-selective pLGIC. To get the most out of the structures obtained, substituted cysteine accessibility mutagenesis (SCAM) and voltage-clamp fluorometry (VCF) were performed to identify the residues lining the channel and involved in local conformational changes. Moreover, in collaboration with F. Dehez and C. Chipot (CNRS, Nancy, France), we performed molecular dynamics simulation studies to further characterise the ion permeation of the channel. Combining the structural and functional studies, we propose a functional interpretation of all of the four structures obtained, widening our knowledge of the gating mechanism (see enclosed publication that follows (pages 77 -98) ).

For the sake of completeness I would like to note that the 5-HT3RA open pore and agonist-activated closed pore structures solved by cryo-EM were published independently by Basak and co-workers (Basak et al., 2018b) and the structural comparison follows the enclosed publication with our results and can be found in the section 5.3.

Electron microscopy -discussion

pLGICs are prominent pharmacological targets, which are studied intensively, as illustrated by the number of structures solved during the last couple of years. However, these structures raise new questions and the detailed functional mechanism still remains shrouded by mystery. A comparison of the gating mechanism of 5-HT3RA with the mechanism elucidated for the other pLGICs (GluCl, GLIC and GlyR) is already discussed in our paper (e.g Extended Data Figures 6 and8). Thus, in this section, I will focus on the direct comparison of the 5-HT3RA structures obtained by our group and independently by the group of Chakrapani, highlighting the main similarities and proposing reasons for potential differences.

In the following text I will keep the nomenclature of structures identical to that used in the original publications. To make it easier for the reader to become oriented in the nomenclature, I will also add the assigned functional state in brackets.

The mouse 5-HT3RA structures solved by cryo-EM were assigned to multiple physiological states, with various levels of confidence in the assignments. There is one apo resting state (Basak et al., 2018a), one inhibited state (T) [START_REF] Polovinkin | Conformational transitions of the serotonin 5-HT3 receptor[END_REF], one potentially pre-activated state plus one structurally closely related state (I1 and I2) [START_REF] Polovinkin | Conformational transitions of the serotonin 5-HT3 receptor[END_REF], a open pore State 1 structure (Basak et al., 2018a) speculated to be non-conducting intermediate or a desensitized state, and two independent open pore structures (State 2 and F respectively) [START_REF] Basak | Cryo-EM reveals two distinct serotonin-bound conformations of full-length 5-HT3A receptor[END_REF][START_REF] Polovinkin | Conformational transitions of the serotonin 5-HT3 receptor[END_REF].

The assignment of the function is indeed more ambiguous for the receptors in the intermediate states where the ECD and ECD-TMD structures resemble the open state but where the pore remains closed. A structural comparison of the intermediate/desensitised structures shows that the I2 state resembles the independently obtained State 1 (agonist-bound non-conducting) to a greater degree, with a root-mean-square deviation (RMSD) of 1.1 Å, than the I1 state (pre-active state), with an RMSD of 1.6 Å (Fig. 32). Drawing conclusions from the RMSD comparison of the two independently obtained structures using different data processing and reaching only limited resolution is tricky. We can speculate that they might be very closely related, but regarding the RMSD differences among the structures of receptors from different families (e.g. comparison of the m-5HT3R to the GABAA subunit with the RMSD of 1.8 Å data shown in the enclosed publication (Extended data Fig 6 ., page 92) the option that they represent distinct physiological states should not be neglected .

Unexpectedly, the biggest surprise comes from the comparison of the two open pore states: the F state and State 2. Unfortunately, the electron density maps are of comparable poor resolution at the level of the ICD in both cases, likely illustrating the ICD flexibility. In contrast to our competitors, we found the building of a structure into this part of the electron density map tricky and thus residues 311-425 were not built in the F state structure. For the purposes of overall structural alignment we use only the shortened version of the State 2 structure cut-down to correspond to the residues resolved in the F state. The overall structural alignment, with an RMSD of 1.9 Å, indicates significant differences. Interestingly, almost the same RMSD (2.0 Å) is observed between the open state F and the inhibited closed state T. Superimposition of the ECDs only shows that the conformation of the F state and of state 2 closely resemble each other with an RMSD of 1.1 Å.

Having a closer look at the binding site, the orientation of the serotonin is the same in both structures, making cation-π interaction with the W156 (Fig. 33). The inward motion of loop C in State 2 seems to be slightly less pronounced compared with the The poorly defined electron density map makes a more detailed comparison impossible also around the conserved Cys-loop region and in close proximity of R218 (important structural features triggering the gating mechanism). Thus it is not possible to draw any conclusions from the flipped side chain of W187 in the State 2 structure compared to all other structures of the receptor solved both by ourselves and by our competitors.

The most striking difference is a different arrangement of the M2 membrane helices, forming the pore, and of their immediate environment, which can be documented by an RMSD of 3.3 Å for the TMD region. Isolated TMD monomers superimpose with an RMSD of 2.1 Å, indicating that the subunit/subunit arrangement accounts for a large part of the differences between the F state and State 2. Accordingly, the interface areas in the TMD, measured using PISA, differ significantly, with ~1100 Å 2 buried for the F state and only ~750 Å 2 for State 2. Comparing the pores, the one of State 2 is generally wider than the one of the F state. This difference is due to the positions of the M2 helices (Fig. 34). The helices start at about the same position on the intracellular site but then they diverge. This results in the ion pathways narrowing at different heights.

Whereas the F state is largely open at the L9' (L260) activation gate, State 2, even though it is widely open, is actually narrowest at the level of this residue (Fig. 35). Compared to the F state, State 2 is significantly wider at the intracellular side of the transmembrane pore creating the vestibule. The pore radius of State 2 resembles that of the GlyR receptor solved by cryo-EM in the presence of glycine (open structure). This widely open pore conformation has brought some controversy [START_REF] Gonzalez-Gutierrez | Chasing the open-state structure of pentameric ligand-gated ion channels[END_REF] because the other open pore structures of pLGIC solved to date are narrower at the intracellular side. The structural studies of the mutated GLIC receptor together with molecular dynamics and electrophysiology studies further support the physiological relevance of the narrower open channel structures [START_REF] Gonzalez-Gutierrez | Chasing the open-state structure of pentameric ligand-gated ion channels[END_REF].

Additionally, State 2 represents a less compact transmembrane helix bundle compared to the F state. The total volume of the State 2 TMD corresponds to 92.17 kÅ 3 (void 27.81 kÅ 3 ; VDW 64.36 kÅ 3 ) with a protein density of 0.698. The F state TMD total volume is 89.03 kÅ 3 (void 26.29 kÅ 3 ; VDW 62.73 kÅ 3 ) with a protein density of 0.705. All data were calculated by ProteinVolume v1.3 [START_REF] Chen | ProteinVolume: calculating molecular van der Waals and void volumes in proteins[END_REF].

Another striking difference between the State 2 structure compared with the other conformational states of the receptor is the movement of the MX helix (Fig. 36A). This peripheral helix located on the intracellular side is in contact with the membrane in the structures of all the other states but is immersed into the membrane in the State 2 structure. This movement is in agreement with the F state that is seen on the alignment of the electron density maps of the F state and the I1 state (Fig. 36B).

The C-terminal end of the MX helix moves ~10 Å along the normal to the membrane. Examples of helices that can enter the membrane exist [START_REF] Entova | Insights into the key determinants of membrane protein topology enable the identification of new monotopic folds[END_REF], and this motion might be part of the operation mechanism of the receptor. It might also be caused by the detergent environment.

The differences between those two open structures raise questions about their assignment to a physiological state. Multiple factors should be kept in mind. The F state and State 2 could potentially correspond to two different conformational states of the open receptor. The opening of the channel is a complex process complicated by the cooperativity of agonists acting at 5 orthosteric binding sites. The existence of multiple open forms has already been proposed for pLGIC [START_REF] Keramidas | An outline of desensitization in pentameric ligand-gated ion channel receptors[END_REF] and the concept was proven to be true for the single-channel study of the ELIC receptor [START_REF] Marabelli | Mechanism of activation of the prokaryotic channel ELIC by propylamine: a single-channel study[END_REF] and for 5-HT3R [START_REF] Corradi | Single-Channel Kinetic Analysis for Activation and Desensitization of Homomeric 5-HT3A Receptors[END_REF]. When comparing the two structures solved independently, it is thus extremely important to consider the [START_REF] Bocquet | X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation[END_REF][START_REF] Chen | Structural basis of neurosteroid anesthetic action on GABAA receptors[END_REF] and suggesting possible regions of the receptor responsible for the lipid-driven modulation. In addition, mutagenesis of the interface between the outermost M4 helix and helices M1/M3 separated the pLGIC superfamily into two groups based on the compactness and tightness of this lipid-exposed region. GLIC, together with GlyR, the GABA receptor and GluCl, have extensive networks of interacting aromatic residues that form a densely packed M4-M1/M3 interface along the entire length of the M4 helix. In contrast, ELIC, the nAChR and the 5-HT3RA have fewer interactions in this region [START_REF] Therien | Pentameric ligand-gated ion channels exhibit distinct transmembrane domain archetypes for folding/expression and function[END_REF]. The Ala mutations of the aliphatic residues have have moderate or no effect as well as mutations of polar residues at the M4 -M1/M3 interface with exception of the M4 -M1 cation-π interactions which mutated abolish the ELIC functioning. Even though the aromatic side chain residues interactions are predominant in both, Ala mutations of single or both aromatic residues impair the GLIC whereas it has a little effect on the ELIC function. Those two groups, represented either by GLIC or by ELIC, should be potentially less or more prone to be influenced by changes in the surrounding lipid environment, respectively. Additionally the group to which the 5-HT3A belongs might undergo conformational changes more readily due to the weaker interactions.

The distorted GABA structures, imaged in detergent, show the gamma subunit collapsing into the pore and assymetric organisation of subunits the two different serotonin receptor structures in the open pore conformation could point to amphipathic molecules as a common denominator. But how does one choose detergents and lipids? Quite often the protein stability is measured prior to structural determination in order to get the most stable sample possible. This method, popularised by the Gouaux lab [START_REF] Gouaux | A Fluorescence-Detection Size-Exclusion Chromatography-Based Thermostability Assay for Membrane Protein Precrystallization Screening[END_REF], has been used for GluCl, the GABA structure, and was probably used even for other structures. However, the generally intuitive idea that conditions, which result in the most thermostable proteins equal conditions where receptors are in a stable physiological state, may not be true in all cases. The artificial lateral pressure potentially created by the amphipathic molecules may result in non physiological tighter packing of the membrane domain helices, thus increasing the intramolecular interaction surface. Specifically, in the presence of an increased level of artificial interactions, the amount of energy needed to break them is higher. Finally, the higher thermal stability of proteins in amphipathic additives might reflect stronger non-native interactions and its maximisation might lead to false positive results. We tested the effect of different detergents on the protein stability and we can conclude that the detergents differ in their ability to stabilise the 5-HT3RA. Furthermore, in our hands the 5-HT3RA manifests the same thermal stability after the addition of lipid master mix (PA, CHS and brain PC; concentrations corresponding to our cryo-EM experiments) as it does in C 12 E 9 (Fig. 38A) (measured by NanoTemper's Prometheus, experiment performed in agreement with the detergent screen described in 4.1.3). Interestingly, the addition of CHS during solubilisation by C 12 E 9 affects the thermostability to a greater extent than addition of CHS just prior to measurement Fig. 38B) .

Conclusion and perspectives

The objectives of my thesis were to obtain structure(s) of other conformational state(s) compared to the 2014 crystal structure and/or increase the resolution of the existing structure of 5-HT3RA (to enable for instance the unambiguous placement of ligands). We have shown that the 5-HT3RA can form crystals in a broad range of conditions (temperature, pH, salts, PEGs) using stabilising nanobodies and Cymal-6 as an additive. We failed to improve the poor diffraction of these crystals with many standard methods (cryoprotection, dehydration of crystals, on-plate measurements). Larger changes need to be implemented to improve the crystal quality. We have shown that changing the detergent might be critical step and we have selected the detergent UDM as a good candidate (section 4.2.1.2).

Starting with no prior experience of electron microscopy, we were able to gradually optimise the sample and the imaging conditions. In the end we obtained four structures of different conformations of the 5-HT3RA by cryo-EM, and could assign them to physiological states and propose a gating mechanism for this receptor compatible with SCAM and VCF experiments, which was also supported by molecular dynamics. The proposed gating mechanism shares some common features (quaternary reorganisation of the ECD, local movements in the ECD-TMD and the upper TMD region, general twist) and possesses some distinct ones compared to the mechanisms inferred for other pLGICs (in contrast to GLIC and GluCl the 5-HT3A and GlyR don't undergo the ECD un-blooming reorganisation, the pore radius of the 5-HT3RA lies in between those of the wider GlyR and the tighter GLIC pore, specific M4 sliding) [START_REF] Polovinkin | Conformational transitions of the serotonin 5-HT3 receptor[END_REF]. Finally, we compared our structures with the structures obtained by Chakrapani's group and noted a puzzling difference in the open pore structures (section 5.3).

The challenging dream to elucidate the gating mechanism of the pLGICs is becoming more and more realistic but together with a better understanding of the gating more questions are raised. Even though the pLGIC share some highly conserved sequences, specifically their overall architecture and general gating apparatus, their actual mechanism seems to differ. To generalise based on what we know about the gating mechanism of one family of receptors is no longer possible and conclusions generalised to the entire superfamily should be made with caution.

Structural studies like ours raise a lot of questions about their physiological relevance (as exemplified by a significant portion of the reviewer's comments to our paper) and they are sometimes still received with scepticism. The purified protein stability, biochemical environment and physical properties used in those studies are indeed not ideal mimics of physiological conditions. Yet it is undeniable that structural studies performed after careful biochemistry deepen our understanding of how membrane proteins function.

A potentially controversial point is the assignment of a structure to a functional state. This assignment can be non-straightforward for all mechanistic proteins, which undergo certain conformational changes during their action. For pLGICs some classical criteria have emerged from the accumulated literature and are used to guide the functional labeling of structures: the position of the loop C, the overall channel radius, the width of the channel at the 9' position, the constriction at the selectivity filters, etc. Fortunately, the field is moving forward with more nuanced definitions of what is a closed/open pore, taking advantage of molecular dynamics. Not every structure to be solved can be easily categorised into the simple three-state model with a resting closed pore, an active open pore and a desensitised closed pore state. The structures of different pLGICs elucidated under a variety of conditions (presence/absence of ligand; use of stabilising nanobodies/Fabs; use of detergents, amphipol or nanodiscs) instead correspond to other classes (most of them known also from electrophysiology) like the apo-resting state, the ligand-bound resting state, or the ligand-bound pre-active state.

In summary, it is clear that the pLGIC field is missing time-resolved structural studies, ideally performed under native conditions. Amongst the methods currently available, this may be achievable using X-ray free electron laser (XFEL) measurements. For this purpose the development of photo-activatable ligands and easy growth of nanocrystal would need to be accomplished first. Both of those aspects, together with very limited availability of XFEL hardware, lower the probability of success using this method.

Considering the "available" methods and instruments, further structural studies by cryo-EM are of great interest. The potential influence of detergent, F-TAC and amphipol on 5-HT3RA could be deduced from structures solved in "membrane-like" conditions, using nanodiscs (or other type of discs). This will require the optimisation of the protocol for protein incorporation into the nanodiscs and possibly even require further rounds of grid preparation optimisation. The structures of the receptor in complex with an allosterically bound ligand (relatively straightforward), of the heteropentamer (requirement for sufficient amounts of the purified heteropentamer, which is more fragile compared to homopentamers) or of the protein with/without ICD in combination with/without the use of calcium in the presence of serotonin (in theory, only the structure without the ICD in the presence of calcium and serotonin is needed to compare with the structure already solved) might bring some insights into the protein pharmacology, functioning and assembly.

Figure 1 |

 1 Figure 1 | A schematic of the resting and the action potential and a detailed view of the electric-to-chemical signal transition at the synapse.......................................................17

Figure 2 |Figure 3 |Figure 4 |Figure 9 |Figure 10 |Figure 13 |Figure 14 |Figure 15 |Figure 16 |Figure 19 |Figure 20 |Figure 21 |Figure 22 |Figure 23 |Figure 26 |Figure 27 |Figure 28 |Figure 30 |Figure 31 |Figure 33 |

 234910131415161920212223262728303133 Figure 2 | Comparison of the architecture of ion channels and membrane gap junction proteins of different oligomeric states.............................................................................18 Figure 3 | Bayesian sequence similarity tree depicting the pLGICs superfamily............21 Figure 4 | The localisation of the 5-HT3R in the neurons of the rat CNS.......................23 Figure 5 | A schematic of the mouse 5-HT3RA subunit.................................................24 Figure 6 | The comparison of the biophysical and pharmacological properties of homo (only A subunit) and heteropentamers (co-expression of A and B subunit)...................25 Figure 7 | The antagonists from the setron family alongside the biological agonist serotonin..........................................................................................................................29 Figure 8 | A chronological overview showing the first discovery of structures of representatives from each pLGIC family potentially by different methods up to the year 2018.................................................................................................................................32 Figure 9 | Structural and sequential features defining the binding site located between two consecutive subunits.................................................................................................34 Figure 10 | Key structural features of signal transduction from the binding site to the membrane domain...........................................................................................................37 Figure 11 | The structural arrangement of the membrane helices and the pore forming M2 helices........................................................................................................................38 Figure 12 | The general comparison of the pore radii forming residues of M2 helix in different pLGIC...............................................................................................................39 Figure 13 | The effect of membrane lipid composition on uncoupling of the nAchRs.. .41 Figure 14 | The intracellular domain conductance-determining features........................43 Figure 15 | Increased use of the cryo-EM method to solve structures of membrane proteins............................................................................................................................44 Figure 16 | The effect of C 12 E 9 detergent concentration and incubation time on the 5-HT3RA solubilisation from membrane fractions.........................................................54 Figure 17 | Comparison of two Strep-Tactin resin based purification setups: gravity flow and pump promoted purification using cartridges...........................................................55

F

  state when an alignment of the ECD is performed. Even though the relative distance (displacement) between the C loops of the apo and the open states solved by Basak et al. are similar to those of the inhibited state T and the open state F it seems that loop C stays further from loop G of the following subunit. It is difficult to judge the possible interaction between residues D202 and R65 in State 2 because of the poor density of the aspartate and the generally low resolution in this region.

Table of figures

 of Figure 35 | The intracellular pore radius of GLIC (open), State 2 and the GlyRα1 (open) are compared..................................................................................................................103 Figure 36 | The pore radius of the F state (pink) compared to that of State 2 (yellow).103 Figure 37 | A comparison of the MX helix position......................................................104 Figure 38 | The thermostability of the 5-HT3A receptor in the presence of lipid additives.........................................................................................................................107

Table 1 |

 1 The summary of the pLGIC structures solved by different methods.........122

Electron microscopy -enclosed publication

Electron microscopy

Supplementary data

Acknowledgements

Acknowledgements

J'aimerai bien exprimer mes remerciments les plus profondes à mon encadrant Acknowledgements Anders A. Jensen, Emmanuelle Neumann, Anders A. Jensen, Solene N. Lefebvre, Pierre-Jean Corringer, Christophe Chipot, Francois Dehez, whose work is stressed in my thesis and in the enclosed publication. Many special thanks go to Guy Schoehn for his patience and for the precious time on microscope which he shared with us.

Abbreviations

Functional properties

The early electrophysiological studies were performed on isolated tissues or on cells expressing the 5-HT3R naturally [START_REF] Hussy | Functional properties of a cloned 5hydroxytryptamine ionotropic receptor subunit: comparison with native mouse receptors[END_REF]. Only after the successful isolation of the gene coding for the A subunit were the homopentamers studied in oocytes (van conditions under which they were obtained.

Therefore, the differences between the two open structures might be for physiological reasons (two distinct open states may actually exist) or due to the experimental conditions used. Firstly, the F state structure was solved in the presence of calcium cations which are known to affect the receptor conductivity. Secondly, pLGIC are known to be sensitive to their lipidic environment and here we are comparing a structure solved in the detergent C 12 E 9 with added lipids (CHS, PA and PC) in the case of the F state and a structure solved in the presence of the same detergent and the addition of fluorinated Fos-choline 8 for State 2. Generally, the non-fluorinated fos-choline detergents are usually efficient in protein solubilisation but they should be used with caution because they might have destabilising and denaturing effect on certain proteins [START_REF] Chipot | Perturbations of Native Membrane Protein Structure in Alkyl Phosphocholine Detergents: A Critical Assessment of NMR and Biophysical Studies[END_REF]. In addition the fluorinated detergents might complicate interpretations since it was shown that they not only interact with ryanodine receptors but also it influences the conformational state of the protein (stabilisation of the closed conformation by the fluorinated octyl maltoside even after the application of strong agonist) through an unknown mechanism [START_REF] Willegems | Influence of Lipid Mimetics on Gating of Ryanodine Receptor[END_REF].

Last but not least, there is an option that one of the structures is too far from biologically existing physiological state, and the result is an artefact.

For a long time, using detergents was the only way to extract membrane proteins from the lipid-bilayer (now, the recently developed SAM/DIBMA copolymers forming "native nanodiscs" may provide an alternative [START_REF] Dörr | Detergentfree isolation, characterization, and functional reconstitution of a tetrameric K +channel: The power of native nanodiscs[END_REF][START_REF] Oluwole | Solubilization of Membrane Proteins into Functional Lipid-Bilayer Nanodiscs Using a Diisobutylene/Maleic Acid Copolymer[END_REF]). Even though the detergent micelles do not reproduce the hallmarks of the lipid-bilayer environment, they remain the traditional choice for protein solubilisation, stabilisation in solution and thus also for structural studies.. Here I would like to sum up the effect of lipids on the pLGICs and how they might influence the structures (and not only those obtained by cryo-EM). The effect of amphipathic molecules, namely lipids and detergents, on the TMD of nAChRs was discussed in section 1.5.2.3 (Fernández Nievas et al., 2008) and the effect of different detergents and lipid additives on the purified 5-HT3R was shown in section 4.2.1.3 and at the end of this section. Interestingly, the 5-HT3R contains the sphingolipid binding motif at the extracellular part of the M1 helix [START_REF] Björkholm | Identification of novel sphingolipid-binding motifs in mammalian membrane proteins[END_REF]. But more generally, amphipathic molecules can have a great impact on the structure and/or function of pLGIC. They interact with the receptors in the allosteric binding site located in the upper and lower part of their TMD. Some lipids, included as additives or even being co-purified, were resolved in the pLGIC structures, suggesting tight interactions with the transmembrane helices [START_REF] Basak | Crystal structure and dynamics of a lipid-induced potential desensitized-state of a pentameric ligandgated channel[END_REF](Basak et al., , 2018a;; Supplementary data 8 Supplementary data This table was generated using data and online software provided by rcsb.org (the PDB; Protein Data Bank). Data generated on 12 August, 2019.