Richard Dufour
email: richard.dufour@orange.com

Géraldine Damnati
email: geraldine.damnati@orange.com

Delphine Charlet
email: delphine.charlet@orange.com

Frédéric Béchet
email: frederic.bechet@lif.univ-mrs.fr

Automatic transcription error recovery for Person Name Recognition

Keywords: transcription error detection, transcription error correction, person name entity recognition

Person Name Recognition from transcriptions of TV shows spoken content is a crucial step towards multimedia document indexing. Recognizing Person Names implies the combination of three main modules: Automatic Speech Recognition, Named-Entity Recognition and Entity Linking to associate the recognized surface form to a normalized Person Name. The three modules are potentially error prone. Hence, beyond each module's intrinsic complexity, the Person Names issue suffers from the highly dynamic evolution of vocabularies and occurrence contexts that are correlated to various dimensions (such as actuality, topic of the show…). This paper focuses on the first module and proposes an approach to recover from transcription errors made on Person Names. An error correction method is applied on the textual ASR output and we show that it is all the more efficient that it is coupled with a specific error region detection system. Experiments on the French REPERE database show that Person Names transcription can be efficiently corrected while preserving the overall transcription quality and thus increasing the performance of the whole Person Name Recognition process.

Introduction

Person Name Recognition (PNR) is a particular case of Named Entity recognition which is particularly important from an applicative point of view. Indexing documents by the mentioned persons is relevant in itself but recognizing Person Names is also a preliminary step for further tasks such as Person Identification in documents. Person Identification is under the scope of the French REPERE1 evaluation program for multi-modal person recognition in video documents. This program addresses both supervised and unsupervised identification, the latter requiring that no biometric model should be used and identification should be performed only by analysing the document itself. In this case, useful information regarding persons' identity can be found in the video through the detection and recognition of person names in overlaid texts and in the audio channel through the detection and recognition of person names in the spoken content. This paper addresses the second issue and proposes an enhanced process towards Person Name Recognition from automatic transcription of speech in TV contents.

A fully automatic PNR system is evaluated consisting of three modules: Automatic Speech recognition (ASR), Named Entity Recognition (NER) and Person Entity Linking (PEL).

From a sequence of transcribed words "la campagne de Hollande (Hollande's campaign)", the NER module is in charge of localizing "Hollande" and assessing that it is a Person Entity (and not the French word for Netherlands) while the latter module allows the localized Person Entity to be associated to a normalized reference form (here François_HOLLANDE).

The three modules are potentially error prone. Hence, beyond each module's intrinsic complexity, the Person Names issue suffers from the highly dynamic evolution of vocabularies and occurrence contexts that are correlated to various dimensions (such as actuality, topic of the show, period when processing archives…). Even for the most efficient Speech-to-Text engines, the transcription of PN is by essence problematic. Some approaches have been designed to handle specific spoken term detection using sophisticated vocabulary adaptation techniques [START_REF] Lecorvé | An unsupervised Web-based topic language model adaptation method[END_REF] or post-processing in multiple outputs (word lattices or subword unit lattices for hybrid approaches [START_REF] Rastrow | A new method for OOV detection using hybrid word/fragment system[END_REF]). From a large scale applicative point of view, specifically tuning a system for a given content is not always feasible. In this perspective, we propose an alternative approach consisting of post-processing textual output provided by a state-of-the art industrial generic Speech to Text engine in order to recover from potential errors on PN.

The objective is to improve PN transcription without degrading the overall transcription quality. In fact, higher level Spoken Language Understanding processes (e.g. prediction of mentioned persons' presence in the show [START_REF] Béchet | Detecting person presence in TV shows with linguistic and structural features[END_REF], named identification of speakers [START_REF] Jousse | Automatic named identification of speakers using diarization and ASR systems[END_REF]) need to analyse the context in which PN occur. Hence, in order to exploit PNR it is essential to maintain high-quality transcription for the surrounding words.

Section 2 gives an overview of our Person Name Recognition system, presenting each of the three above mentioned modules. Section 3 presents the transcription error recovery approach and evaluations are provided in section 4.

Person Name Recognition system

Automatic Speech Recognition

Automatic transcription is performed with an industrial Speech to Text (STT) engine: the VoxSigma speech recognizer V3.5 from Vocapia Research, based on LIMSI technology [START_REF] Gauvain | The LIMSI Broadcast News Transcription System[END_REF]. This is a generic engine dedicated to Broadcast contents. This software provides speaker segmentation and speech recognition along with word-level confidence measures based on posterior probability for each recognized word. For genericity purpose and due to applicative constraints, we have chosen to directly exploit the one-best automatic transcription provided by this on-the-shelf STT engine. Hence we propose to improve the transcription of Person Names by only relying on the textual form of the STT one-best output, without any re-decoding process. The error recovery approach is detailed in section 3.

ISCA Archive

Named Entity Recognition

The Named Entity Recognition (NER) system used in this study is LIA_NE [START_REF] Béchet | Unsupervised knowledge acquisition for extracting named entities from speech[END_REF]. This system is based on a machine learning approach that includes two components: a generative HMMbased process used to predict Part-Of-Speech tags as well as semantic labels for each input word; a discriminative CRF-based process that takes as features the words as well as the syntactic and semantic labels given by the HMM in order to determine the span and the type of each entity detected.

The main advantage of a machine learning approach for NER when dealing with speech input is the ability to train the statistical model on a corpus that matches the output of ASR systems (no punctuation and unreliable capital letters). In LIA_NE the HMM and CRF models have been trained on the ESTER Broadcast News training corpus from which all punctuation and capitalization have been removed. This leads to increase the robustness of the system towards ASR errors [START_REF] Béchet | Unsupervised knowledge acquisition for extracting named entities from speech[END_REF].

Person Entity Linking

The goal of the Person Entity Linking module is to associate a unique identifier to each PN detected in a text, regardless of the way the name is expressed. For example, in the news corpus used to build this module, the current wife of the French president Sarkozy can be found under 6 forms: Carla Bruni-Sarkozy, Carla Bruni, Mme Bruni-Sarkozy, Carla Sarkozy, Carla Bruni Sarkozy, Mme Sarkozy. The PEL module is in charge of translating each of these forms into the normalized identifier: Carla_BRUNI-SARKOZY. Previous works on entity linking have focused on solving the linking ambiguities by using the context of the name occurrence in the document, either with heuristics or machine learning approaches [START_REF] Stern | A Joint Named Entity Recognition and Entity Linking System[END_REF]. We are dealing here with the opposite problem: given a potential list of persons that are likely to occur in TV shows, we want to predict all the different forms that can be used to express their names.

In usual frameworks, a closed list is chosen so as to be consistent with the ASR lexicon. Since we propose an approach to correct person names, we are not limited by the ASR lexicon. Hence, we have relaxed this constraint by considering a very large dictionary of person names, much larger that the one used by the ASR decoder. Each person name in this dictionary is associated to a normalized form. When there is an ambiguity (for example: Mme Sarkozy can refer to several persons) the most frequent normalized form is chosen. We built the person name dictionary used in this study with the following process:

• a corpus of newswire collected over the period 2004-2011: 9.2M PN entities have been detected with LIA_NE corresponding to 811K different forms, further reduced to 220K by removing PN occurring less than 4 times • a clustering method has been applied to this 220K list by associating forms sharing common substrings and occurring in the same newswire • finally a set of 110K clusters has been obtained, each of them representing several forms of a person name; the normalized form chosen is the most frequent sequence firstname+lastname in the cluster. The error recovery strategy consists in: first detecting Person Names error regions and then searching for Person Names in error regions on the basis of their phonetic representation.

Error recovery approach

Error detection and characterization

In previous work [START_REF] Lecorvé | An unsupervised Web-based topic language model adaptation method[END_REF], we have proposed an approach for error region detection and characterization in LVCSR transcriptions. It is a well-known phenomenon that LVCSR errors tend to appear in consecutive error regions. Beyond the classical confidence measure approach that estimates the probability of each word to be correct, we consider error detection as a segmentation task. Furthermore, simply detecting error regions is not enough for general purpose and it is important to characterize them. We have proposed to characterize error region with respect to the nature of the words that yielded the error region. Characterization is done along four error classes: Person Names, Other Proper Nouns, Homophones, Others. Driven by applicative considerations, we believe that this approach is more suitable to define relevant strategies in order whether to ignore errors (e.g. Homophones) or to try to correct them.

In this paper we combine two of the approaches described in [START_REF] Lecorvé | An unsupervised Web-based topic language model adaptation method[END_REF] and we focus on the detection of Person Names error regions.

Sequential approach

The segmentation and characterization steps are applied sequentially. Error detection is simply performed thanks to a threshold on the ASR confidence measures. Segmentation is performed by gathering consecutive errors to form an error region. Then a supervised classifier (icsiboost1 ) is applied on error regions with various features representing the region itself (word bigrams, POS trigram and syntactic chunks2 , number of words, average number of syllables per words) and its context (quadrigram on the 5 previous words, duration and average confidence measure of the speaker turn).

Integrated approach

Error regions have specific properties depending on their nature (various average length for instance) suggesting to use integrated approaches in order to simultaneously segment and categorize error regions. This is achieved thanks to Conditional Random Fields (CRF) with an IUO underlying model (Inside, Unique, Outside) and the following feature set: word bigrams POS tags and syntactic chunks, confidence measure and duration of current, previous and next word.

Error correction

Given an automatically detected error region, the error correction process consists in searching in a Person Name dictionary for the entity whose phonetic representation is the most similar to the phonetic sequence associated to the recognized words. This approach was originally studied in the context of open-vocabulary spoken term detection [START_REF] Dubois | Using textual information from LVCSR transcripts for phonetic-based spoken term detection[END_REF]. In the general case, such an approach can be efficient in terms of spoken term detection recall but has to be carefully used in order to avoid low precision rates. In this paper, we show that it can be a relevant approach for searching PN provided that the search step is guided by a preliminary error detection step.

Constitution of the PN database

In order to be consistent with the further Person Named Entity detection and Linking stages, the dictionary is extracted from the one described in section 2.3 and is composed of the 20K most frequent PN. Each entry is associated to its phonetic representation thanks to the Orange Labs grapheme to phoneme converter (G2P). We keep pronunciation variants for each entry, leading to a total amount of 135K phonetic sequences dictionary.

Phonetic search

Given a detected error region E={w 1 ,…,w n } of n consecutive words, correction is achieved along the following process:

•

The sequence of n words is transformed into a sequence of phonemes, preserving the inter-word coarticulation phenomena (which are particularly usual in French with the liaison phenomenon). The G2P providing phonetic variants, we keep the longest phoneme sequence as our phonetic search space Φ={φ 1 ,…, φ k } .

•

Phonetic search is performed thanks to dynamic programming through the phoneme sequence Φ. It looks for optimal alignment (i.e. with minimum distance) between the sequences of phonemes of the PN database entries and any sub-sequences within Φ. The alignment distance is defined as the sum of the costs of the operations (phoneme pairs substitution, deletion and insertion) involved in the alignment. It is then normalized by the number of operations. The insertion, deletion and substitution costs are derived from a pre-computed phoneme confusion matrix.

•

The result of the phonetic search step is an n-best list of potential Person Names matching with a sub-sequence of phonemes from Φ and ranked according to their normalized distance to the matched sub-sequence.

•

The previous matching scores do not reflect length constraints. It is then possible that a very short name matches perfectly a subsequence of one or two phonemes from the original Φ sequence. In order to alleviate this problem, a re-ranking process is applied to include coverage constraints. It allows favoring the detection of a longer name with a higher distance provided that this distance ranges in a fixed delta from the one-best distance.

•

Finally, it can happen that several Person Names are mentioned in a single PN error region. The previous step is iterated as long as non-overlapping hypotheses can be produced from the n-best list.

Implementation

Using a phoneme confusion matrix allows taking into account the fact that some phonemes are more likely to be omitted or inserted than others and that some phoneme pairs are more likely to be confused than others. It is computed maximising the likelihood of the alignment between a reference phoneme sequence obtained from manual transcriptions and the one obtained from LVCSR output on the same training corpus. This learning step is performed iteratively by the EM algorithm.

In practise, it can happen that only the last name yields an error region while the first name is correctly transcribed (eg. a common first name and an unknown last name). In order to increase the precision of the search process, we systematically append the error region with the previously transcribed word when this word is tagged as a first name by the POS.

The next section shows how this error correction approach, coupled with a suitable error detection process can improve the overall PNR process.

Experiments

Database description

The error region detection and characterization models have been trained on a 14 hours TV Broadcast News shows corpus composed of 38 shows from 7 French generalist channels [START_REF] Dufour | Automatic error region detection and characterization in LVCSR transcriptions of TV news shows[END_REF]. The phoneme confusion matrix, for our 33 phonemes set, has been trained on the same corpus: 521k reference phonemes were aligned with 555k hypothesized phonemes.

PNR experiments are run on a corpus of TV shows from two French channels (2 shows from BFMTV and 5 shows from LCP, none of them are part of the training corpus) provided by the French REPERE evaluation program. The corpus is characterized by a strong variety in terms of topics and types of shows (debates, extracts of parliament allocutions, reports, news…). A development corpus (DEV) is used to tune the error recovery strategy and a test corpus (TEST) is used to assess to overall PNR performance. DEV and TEST both consist of 3 hours of speech from about 30 extracts of shows. PN entities have been manually annotated and associated with a normalized linked form. Our entity linking approach can associate a last name to a reference form but does not perform contextual reference resolution (isolated first name are not linked to a reference form). As a consequence, we discard from the database persons that are only mentioned by their first name.

DEV

Impact of error recovery strategies

In order to quantify the impact of the error recovery strategy on the overall PNR task, we measure PNR recall and PNR precision. PNR recall is the number of normalized Person Names that are correctly recognized, over the total amount of reference normalized PN. PNR precision is the equivalent value over the total amount of detected normalized PN. In Figure 1, PNR recall is plotted against PNR precision by varying the threshold on the tolerated distance between the original phoneme sequence and the hypothesized names phoneme sequences. The top curve (ref) corresponds to the optimal case where PN error regions are manually segmented. It illustrates the interesting potential of the proposed correction method and provides Oracle PNR performance with the whole process including our correction paradigm. Starting with a 62.0% PNR recall and a 87.6% PNR precision, the curve shows that it is possible to reach an optimal 70.2% PNR recall with an improved 89.4% PNR precision.

Systematically searching PN in automatic transcriptions would necessarily yield poor performance. Contrastive experiments were achieved for the sake of comparison and leaded to around 30% absolute drop in PNR precision even with a very strict tolerance on the distance confirming the need for an error detection strategy in order to guide the correction process. The three other curves presented in Figure 1 correspond to three different error region detection strategies. As a baseline, we directly used the word level Confidence Measure (CM) provided by the ASR engine. If several consecutive words have a CM below a given threshold (see [START_REF] Dufour | Automatic error region detection and characterization in LVCSR transcriptions of TV news shows[END_REF] for threshold optimisation) they are considered as an error region. Without any further selection process, this strategy (illustrated by the CM curve) eventually allows increasing the PNR recall but with a significant drop in PNR precision.

Additionally using a CRF to detect error regions ("CM+CRF" through a simple OR fusion) improves the quality of the overall process. In fact, it can happen that a misrecognized PN generates a succession of several erroneous words among which one can be a short word with a high CM. Simply applying a threshold results in multiple error regions, thus preventing the correction process to be applied on the relevant phonetic support. Due to the rich modelling provided by the structural and contextual features, CRFs are more likely to detect a single error region, even if all recognized words do not have a CM below the threshold. As a result, PNR recall is higher for the CM+CRF curve. However, the PNR precision is still degraded.

The last experiment illustrates the importance of the error region characterization step. Following the method described in section 3.1, we only apply the correction strategy to those error regions that are automatically characterized as error regions generated by misrecognized person names. The "CM+CRF PN only" curve shows that the PNR recall can be increased to a certain extent without any loss in PNR precision (following the Oracle curve).

In order to validate the approach on the TEST corpus, operating points have been chosen in order to optimise the Fmeasure for the "ref" and "CM+CRF PN only" curves. The second column in Table 2 confirms the potential of the error correction approach when applied on manually segmented error regions and the third column confirms the performance of the fully automatic approach with an increasing recall and precision for equivalent overall word error rate (w.e.r.). When focusing on the speaker turns where a correction has been applied, the w.e.r. decreases from 23.1% to 22.1% for the manual error segmentation approach and it decreases from 18.3% to 18.0% for the fully automatic approach.

TEST

Conclusion

We have proposed an error recovery approach dedicated to errors on Person Names (PN). It has been integrated in a complete Person Name Recognition (PNR) task (including ASR, Named Entity Recognition and Person Entity Linking) and evaluated on a TV shows corpus available from the REPERE evaluation program. The error correction approach, consisting of searching for PN within the phonetic representation of wrongly recognised words, proved to be very promising when applied to the true (manually detected) PN error regions. When coupled with an automatic PN error region detection system, we were able to increase the overall PNR recall without degrading PNR precision nor the overall word error rate. Further improvements in the difficult error region detection and characterization task should yield even better results.

Figure 1 :

 1 Figure 1: Person Name Recognition Recall and Precision for various error region detection strategies.

  likely to result in transcribed words that are phonetically close to the original name. It can be a simple substitution (Karim examen instead of Karim Benzema, Marc librement instead of Marc Lièvremont) or a sequence of erroneous short words (ou ma Thurman instead of Uma Thurman or Calais les valses instead of Cadel Evans). In this paper, we propose to recover from such errors by comparing the phonetic representation of recognized words to a PN dictionary.

Transcription errors on Person Names, be them caused by Out of Vocabulary (OOV) words, by unseen context or by any possible cause, are

Table 2 :

 2 Performances on the TEST corpus

		No	Manual	Automatic
		correction	error reg.	error reg.
	PNR Recall	62.1	68.6	63.3
	PNR Precision	80.7	82.6	81.0
	overall w.e.r.	24.4 %	24.1 %	24.3 %

funded by the ANR agency, http://www.defi-repere.fr

http://code.google.com/p/icsiboost

http://pageperso.lif.univ-mrs.fr/~frederic.bechet/download.html


Emmanuel Bourinet

Gerald W Zamponi
email: zamponi@ucalgary.ca

Block of voltage-gated calcium channels by peptide toxins

Keywords:

Venoms from various predatory species, such as fish hunting molluscs scorpions, snakes and arachnids contain a large spectrum of toxins that include blockers of voltage-gated calcium channels. These peptide blockers act by two principal manners -physical occlusion of the pore and prevention of activation gating. Many of the calcium channel-blocking peptides have evolved to tightly occupy their binding pocket on the principal pore forming subunit of the channel, often rendering block poorly reversible. Moreover, several of the best characterized blocking peptides have developed a high degree of channel subtype selectivity. Here we give an overview of different types of calcium channel-blocking toxins, their mechanism of action, channel subtype specificity, and potential use as therapeutic agents.

Overview of voltage-gated calcium channels

Voltage-gated calcium channels are the major source of depolarization-evoked calcium entry into excitable cells of brain, heart, and muscle [START_REF] Zamponi | The physiology, pharmacology and pathology of voltage-gated calcium channels and their therapeutic potential[END_REF]. This in turn supports many critical physiological functions that range from muscle contraction, to the release of neurotransmitters and calciumdependent gene transcription, among many others. The mammalian genome encodes as many as ten different genes that lead to different types of Cav subunits, the principal pore forming subunit that forms the core of the calcium permeable ion channel [START_REF] Simms | Neuronal voltage gated calcium channels: structure, function and dysfunction[END_REF]. They have been grouped into three major classes (Cav1, Cav2 and Cav3) which in turn correspond to different types of calcium currents that have been identified in native tissues (for review, see [START_REF] Simms | Neuronal voltage gated calcium channels: structure, function and dysfunction[END_REF]. The Cav1 family encodes four different types of L-type channels. Among the Cav2 family, Cav2.1, Cav2.2 and Cav2.3 correspond, respectively, to P/Qtype, N-type and R-type currents. The Cav3 family represents three different types of T-type calcium channels (also known as low-voltage activated channels due to their hyperpolarized voltage range of activation) [START_REF] Catterall | International Union of Pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels[END_REF]. Members of the Cav3 family are thought to be monomers whereas Cav1 and Cav2 channels are multimeric complexes that also include a cytoplasmic Cavb and an extracellular Cava2d subunit [START_REF] Simms | Neuronal voltage gated calcium channels: structure, function and dysfunction[END_REF][START_REF] Catterall | International Union of Pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels[END_REF]. These ancillary subunits serve primarily to regulate calcium channel trafficking and function, but have also been shown to alter the pharmacological characteristics of the channels [START_REF] Dolphin | Voltage-gated calcium channels and their auxiliary subunits: physiology and pathophysiology and pharmacology[END_REF]. All Cav subunits share a common transmembrane topology of four homologous transmembrane domains that each contain six membrane spanning helices plus a reentrant p-loop motif that forms the pore of the channel and imparts calcium selectivity [START_REF] Catterall | International Union of Pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels[END_REF]. The fourth transmembrane helix in each domain contains a positively charged amino acid residue in every third position and forms the voltage sensor of the channel [START_REF] Catterall | Ion channel voltage sensors: structure, function, and pathophysiology[END_REF]. The major domains are connected by large cytoplasmic regions, in addition to being flanked by intracellular N-and C-terminal regions. The domain I-II linker is the locus for Cavb subunit interactions [START_REF] Pragnell | Calcium channel beta-subunit binds to a conserved motif in the I-II cytoplasmic linker of the alpha1-subunit[END_REF]. The recent breakthrough resolution of the Cav1.1 channel structure at a nearly atomic level brings us a step closer to the understanding of the interface between the Cav subunit and ancillary subunits [START_REF] Wu | Structure of the voltagegated calcium channel Cav1.1 complex[END_REF][START_REF] Wu | Structure of the voltage-gated calcium channel Cav1.1 at 3.6 A resolution[END_REF]. Notably these studies revealed that Cava2d has a docking site formed by extracellular segments of domains I, II, and III, all organized with other extracellular loops into a dome structure above the selectivity filter. Whether this organization is common to other Cav subtypes remains to be determined.

The Cav subunit is the target for pharmacological agents that block (and in some cases enhance) calcium channel activity [START_REF] Zamponi | The physiology, pharmacology and pathology of voltage-gated calcium channels and their therapeutic potential[END_REF]. This is relevant in the context of therapeutics, as calcium channel inhibitors have been used to treat disorders such as hypertension, pain and epilepsy (for review, see [START_REF] Khosravani | Voltage-gated calcium channels and the idiopathic generalized epilepsies[END_REF][START_REF] Waxman | Regulating excitability of peripheral afferents: emerging ion channel targets[END_REF][START_REF] Bourinet | Calcium permeable ion channels in pain signaling[END_REF][START_REF] Zamponi | The physiology, pharmacology and pathology of voltage-gated calcium channels and their therapeutic potential[END_REF][START_REF] Zamponi | Targeting voltage-gated calcium channels in neurological and psychiatric diseases[END_REF]. Calcium channel blockers include small inorganic ions such as cadmium which act by occluding the permeation pathway [START_REF] Lansman | Blockade of current through single calcium channels by Cd2þ, Mg2þ, and Ca2þ. Voltage and concentration dependence of calcium entry into the pore[END_REF], small organic molecules such as dihydropyridines which have been used as a tool to identify native L-type calcium currents [START_REF] Randall | Pharmacological dissection of multiple types of Ca2þ channel currents in rat cerebellar granule neurons[END_REF], and larger peptide-based toxins that are isolated from a wide variety of venomous animal species. Here, we provide an overview of different peptide toxins that act on various members of the calcium channel superfamily, and touch on their therapeutic potential.

u-conotoxins

The family of u-conotoxins is derived from the venoms of a variety of different marine molluscs that use their venom to hunt fish. They are typically between twenty and thirty amino acids in size and display a rigid backbone structure that is spatially constrained by the formation of disulfide bonds formed between six conserved cysteine residues [START_REF] Olivera | Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using u-conotoxin from Conus magus venom[END_REF][START_REF] Olivera | Conotoxins[END_REF][START_REF] Lewis | Conus venom peptide pharmacology[END_REF]. Blocking affinity and channel subtype selectivity vary with amino acid sequence in the various loops between the cysteine bonds. In general, u-conotoxins act by physically occluding the pore of the channel, thus preventing calcium influx. In many cases, binding is very tight, leading to slow dissociation rates and hence poorly reversible block (for example, [START_REF] Mintz | Ptype calcium channels blocked by the spider toxin u-Aga-IVA[END_REF][START_REF] Boland | u-Conotoxin block of N-type calcium channels in frog and rat sympathetic neurons[END_REF][START_REF] Ellinor | Structural determinants of the blockade of N-type calcium channels by a peptide neurotoxin[END_REF].

One of the defining characteristics of Cav2.2 (N-type) calcium channels is their inhibition by u-conotoxin GVIA, a 27 amino acid peptide isolated from the fish hunting mollusc Conus geographus [START_REF] Olivera | Calcium channel diversity and neurotransmitter release: the u-conotoxins and u-agatoxins[END_REF][START_REF] Mccleskey | u-conotoxin: direct and persistent blockade of specific types of calcium channels in neurons but not muscle[END_REF]. GVIA block is exquisitely selective for Cav2.2 channels and virtually irreversible [START_REF] Boland | u-Conotoxin block of N-type calcium channels in frog and rat sympathetic neurons[END_REF][START_REF] Mcdonough | Interactions among toxins that inhibit N-type and P-type calcium channels[END_REF], however, strong membrane hyperpolarization has been shown to accelerate the dissociation of this peptide from the channel [START_REF] Stocker | Preferential interaction of omega-conotoxins with inactivated N-Type Ca2þ channels[END_REF][START_REF] Feng | Determinants of inhibition of transiently expressed voltage-gated calcium channels by u-conotoxins GVIA and MVIIA[END_REF]. Structure-function analysis based on chimeric calcium channel constructs has revealed that GVIA interacts with the outer vestibule of the pore comprised of the extracellular domain III S5eS6 (p-loop) region [START_REF] Ellinor | Structural determinants of the blockade of N-type calcium channels by a peptide neurotoxin[END_REF]. Moreover, mutations in this region (especially a Glycine residue at position 1326) have been shown to affect not only the blocking rate constant, but also to have the capability to render the block reversible [START_REF] Feng | Residue Gly1326 of the N-type calcium channel alpha 1B subunit controls reversibility of u-conotoxin GVIA and MVIIA block[END_REF]. These data fit with a structural homology model of Cav2.2 based on the structure of bacterial sodium channels [START_REF] Lewis | Conus venom peptide pharmacology[END_REF]. This picture is likely to be refined in light of the recent structural data of the Cav1.1 channel in complex with a Cava2d subunit that is docked near the top of the channel pore [START_REF] Wu | Structure of the voltage-gated calcium channel Cav1.1 at 3.6 A resolution[END_REF]. Such structural information may offer some mechanistic insights into why the Cava2d subunit affects blocking and unblocking rate constants for certain types of u-conotoxins [START_REF] Mould | The a2d auxiliary subunit reduces affinity of uconotoxins for recombinant N-type calcium channels[END_REF]. The venom of Conus geographus also contains other related u-conotoxins, such as GVIB, GVIC, GVIIA, and GVIIA, however they are not as well characterized at the electrophysiological level as GVIA [START_REF] Olivera | Calcium channel diversity and neurotransmitter release: the u-conotoxins and u-agatoxins[END_REF].

A number of calcium channel blocking peptides have been isolated from the venom of Conus magus, another fish hunting snail. This includes u-conotoxins MVIIA, MVIIB, MVIIC, and MVIID (Hillyard, 1992;[START_REF] Olivera | Calcium channel diversity and neurotransmitter release: the u-conotoxins and u-agatoxins[END_REF], which for the most part preferentially target Cav2.2 channels [START_REF] Olivera | Peptide neurotoxins from fish-hunting cone snails[END_REF][START_REF] Monje | A new Conus peptide ligand for Ca channel subtypes[END_REF][START_REF] Fox | Irreversible and reversible blockade of IMR32 calcium channel currents by synthetic MVIIA and iodinated MVIIC u-conopeptides[END_REF]. However, MVIIC also blocks Cav2.1 calcium channels and its blocking effects are reversible [START_REF] Woppmann | Calcium channel subtypes in rat brain: biochemical characterization of the high-affinity receptors for uconopeptides SNX-230 (synthetic MVIIC), SNX-183 (SVIB), and SNX-111 (MVIIA)[END_REF][START_REF] Grantham | u-conotoxin MVIIC reversibly inhibits a human N-type calcium channel and calcium influx into chick synaptosomes[END_REF], underscoring the point that small changes in amino acid composition between MVIIA and MVIIC are sufficient to alter channel subtype selectivity. The mode of action of these peptides is similar to that of GIVA, and they compete for a common interaction site on the channel [START_REF] Olivera | Calcium channel diversity and neurotransmitter release: the u-conotoxins and u-agatoxins[END_REF], although subtle differences in the way these toxins interact with the channel have been suggested [START_REF] Woppmann | Calcium channel subtypes in rat brain: biochemical characterization of the high-affinity receptors for uconopeptides SNX-230 (synthetic MVIIC), SNX-183 (SVIB), and SNX-111 (MVIIA)[END_REF].

MVIIA has received particular attention, as this peptide can be synthesized such that it retains its native conformation [START_REF] Xiao | Synthetic omega-conopeptides applied to the site of nerve injury suppress neuropathic pains in rats[END_REF]. This has allowed the exploration of this peptide as a therapeutic agent for pain, based on the underlying principle that the Cav2.2 channel plays a major role in the transmission of pain signals in the spinal dorsal horn [START_REF] Bourinet | Calcium permeable ion channels in pain signaling[END_REF]. Intrathecal delivery of MVIIA (a.k.a. ziconotide or Prialt) mediates analgesic effects in both animals and humans. However, although Prialt is approved for treating intractable cancer pain in humans it has a narrow therapeutic window and the potential for causing severe CNS side effects [START_REF] Antanassoff | Ziconotide, a new N-Type calcium channel blocker, administered intrathecally for acute postoperative pain[END_REF][START_REF] Penn | Adverse effects associated with the intrathecal administration of ziconotide[END_REF][START_REF] Miljanich | Ziconotide: neuronal calcium channel blocker for treating severe chronic pain[END_REF][START_REF] Staats | Intrathecal ziconotide in the treatment of refractory pain in patients with cancer or AIDS: a randomized controlled trial[END_REF][START_REF] Thompson | Treatment challenges and complications with Ziconotide monotherapy in established pump patients[END_REF][START_REF] Wallace | Intrathecal Ziconotide in the treatment of chronic nonmalignant pain: a randomized, double-blind, placebo-controlled clinical trial[END_REF][START_REF] Ver | An open-label, multicenter study of the safety and efficacy of intrathecal ziconotide for severe chronic pain when delivered via an external pump[END_REF]. The toxicity of MVIIA has recently been attributed to a methionine residue at position 12 of the toxin molecule, which is known to dock to a hydrophobic binding pocket comprised of residues I300, F302, L305 of the Cav2.2 subunit [START_REF] Wang | Molecular basis of toxicity of N-type calcium channel inhibitor MVIIA[END_REF].

A number of additional u-conotoxins have been identified in the venoms of Conus fulman and Conus catus and shown to mediate potent inhibition of Cav2.2 channels and to exhibit analgesic effects. This includes u-conotoxins FVIA (a reversible N-type channel blocker; [START_REF] Lee | Analgesic effect of highly reversible omega-conotoxin FVIA on N type Ca2þ channels[END_REF], as well as CVIB [START_REF] Motin | Omega-conotoxin CVIB differentially inhibits native and recombinant N-and P/Q-type calcium channels[END_REF], CVID [START_REF] Lewis | Novel u-conotoxins from Conus catus discriminate among neuronal calcium channel subtypes[END_REF][START_REF] Scott | Actions of intrathecal u-conotoxins CVID, GVIA, MVIIA, and morphine in acute and neuropathic pain in the rat[END_REF][START_REF] Adams | Omega-conotoxin CVID inhibits a pharmacologically distinct voltage-sensitive calcium channel associated with transmitter release from preganglionic nerve terminals[END_REF], CVIE and CVIF [START_REF] Berecki | Analgesic (omega)-conotoxins CVIE and CVIF selectively and voltagedependently block recombinant and native N-type calcium channels[END_REF]. Interestingly, CVID and CVIE (but not CVIF) mediate analgesia in mice even after systemic administration [START_REF] Sadeghi | Novel u-conotoxins from C. catus reverse signs of mouse inflammatory pain after systemic administration[END_REF]. Of further note, CVID has been advanced to human clinical trials where a larger therapeutic window was observed compared with Prialt [START_REF] Schroeder | N-type calcium channel blockers: novel therapeutics for the treatment of pain[END_REF], however, this peptide did to our knowledge not advance beyond phase II. Nonetheless, the larger therapeutic window has been attributed to the fact that CVID shows greater selectivity for Cav2.2 channels over Cav2.1.

The u-conotoxins SIA, SVIA and SVIB have been isolated from Conus striatus venom. Although they are pore blockers, it has been suggested that their sites of action are distinct from those of GVIA [START_REF] Ramilo | Novel aand u-conotoxins from Conus striatus venom[END_REF]. Moreover, similar to MVIIC, SVIB reversibly blocks Cav2.2 and Cav2.1 channels [START_REF] Woppmann | Calcium channel subtypes in rat brain: biochemical characterization of the high-affinity receptors for uconopeptides SNX-230 (synthetic MVIIC), SNX-183 (SVIB), and SNX-111 (MVIIA)[END_REF][START_REF] Nielsen | A consensus structure for u-conotoxins with different selectivities for voltage-sensitive calcium channel subtypes: comparison of MVIIA, SVIB and SNX-202[END_REF].

The venoms of Conus Victoriae and Conus regis, respectively, contain a-conotoxins Vc1.1 and Rg1A. These peptides were classified as a-conotoxins in line with their action on nicotinic acetylcholine receptors [START_REF] Clark | The synthesis, structural characterization, and receptor specificity of the alpha-conotoxin Vc1.1[END_REF][START_REF] Nevin | Are alpha9alpha10 nicotinic acetylcholine receptors a pain target for alpha-conotoxins?[END_REF]. Subsequently, potent inhibition by these toxins of Cav2.2 calcium channels was reported. However, both Vc1.1 and Rg1A do not act directly on the channel, but instead by activating GABA B receptors [START_REF] Callaghan | Analgesic alpha-conotoxins Vc1.1 and Rg1A inhibit N-type calcium channels in rat sensory neurons via GABAB receptor activation[END_REF][START_REF] Callaghan | Analgesic alpha-conotoxins Vc1.1 and RgIA inhibit N-type calcium channels in sensory neurons of alpha9 nicotinic receptor knockout mice[END_REF][START_REF] Cuny | gamma-Aminobutyric acid type B (GABAB) receptor expression is needed for inhibition of N-type (Cav2.2) calcium channels by analgesic alpha-conotoxins[END_REF][START_REF] Huynh | Novel mechanism of voltagegated N-type (Cav2.2) calcium channel inhibition revealed through a-conotoxin Vc1.1 activation of the GABA(B) receptor[END_REF]. This is thought to occur by interference with the receptor dimer ectodomain interface [START_REF] Adams | Mechanisms of conotoxin inhibition of N-type (Ca(v) 2.2) calcium channels[END_REF], which then triggers inhibition of Cav2.2 channels via a G protein pathway. By doing so, the Vc1.1 peptide produces analgesia in rodent models of neuropathic and gastrointestinal pain, and reduces the excitability of human dorsal root ganglion neurons [START_REF] Klimis | A novel mechanism of inhibition of high-voltage activated calcium channels by a-conotoxins contributes to relief of nerve injury-induced neuropathic pain[END_REF][START_REF] Castro | a-Conotoxin Vc1.1 inhibits human dorsal root ganglion neuroexcitability and mouse colonic nociception via GABAB receptors[END_REF]. Vc1.1 is being explored as a possible pain therapeutic for humans, and this has been facilitated through the generation of a cyclized version of the peptide that remarkably allows oral delivery of Vc1.1 [START_REF] Carstens | Engineering of conotoxins for the treatment of pain[END_REF].

While the majority of u-conotoxins appear to target Cav2.2 and Cav2.1 calcium channels, there are some examples of L-type calcium channel blocking u-conotoxins. For example, u-conotoxin TxVII from Conus textile blocks L-type calcium channels from a freshwater pond snail, Lymnaea stagnalis [START_REF] Fainzilber | A novel hydrophobic omega-conotoxin blocks molluscan dihydropyridine-sensitive calcium channels[END_REF]. Glacontryphan-M, an unusual peptide isolated from the venom of Conus marmoreus contains g-carboxyglutamyl (Gla) residues in positions 2 and 4 that bestow calcium binding ability onto this toxin. It has been shown to block L-type currents in mouse pancreatic beta cells [START_REF] Hansson | The first gamma-carboxyglutamic acid-containing contryphan. A selective L-type calcium ion channel blocker isolated from the venom of Conus marmoreus[END_REF]. Block, however, was found to be incomplete with ~35% inhibition at saturating concentrations. Substitution of the Gla residues with glutamate abolished block, as did removal of extracellular calcium.

u-Agatoxins

The venom of the American funnel-web spider Agenelopsis aperta contains a number of different neurotoxins, including the calcium channel blocking uagatoxins [START_REF] Adams | Toxityping rat brain calcium channels with u-toxins from spider and cone snail venoms[END_REF][START_REF] Olivera | Calcium channel diversity and neurotransmitter release: the u-conotoxins and u-agatoxins[END_REF]. Among these, u-agatoxin IVA has been used as an experimental tool to identify and distinguish native P-and Q-type currents [START_REF] Adams | Toxityping rat brain calcium channels with u-toxins from spider and cone snail venoms[END_REF][START_REF] Llinas | Blocking and isolation of a calcium channel from neurons in mammals and cephalopods utilizing a toxin fraction (FTX) from funnel-web spider poison[END_REF][START_REF] Turner | Calcium channels coupled to glutamate release identified by u-Aga-IVA[END_REF], with P-type currents showing an approximately ten-fold greater sensitivity to this toxin. Both P-type and Q-type currents are supported by different variants of Cav2.1 channels (i.e., different splice isoforms), and differential assembly with different types of ancillary Cavb subunits [START_REF] Bourinet | Splicing of a1A subunit gene generates phenotypic variants of P-and Q-type calcium channels[END_REF][START_REF] Mermelstein | Properties of Q-type calcium channels in neostriatal and cortical neurons are correlated with beta subunit expression[END_REF], with alternate splicing of an asparagine-proline motif in the domain IV S3eS4 linker greatly affecting blocking affinity [START_REF] Bourinet | Splicing of a1A subunit gene generates phenotypic variants of P-and Q-type calcium channels[END_REF]. This region is implicated further in toxin block by a study that examined the role of a specific glutamate residue that appears to be critical for toxin block (Winterfield and Swartz, 2000). u-agatoxin IVA is somewhat larger than the family of uconotoxins (i.e., 48 amino acids), but also has constrained backbone structure through formation of as many as four disulfide bonds [START_REF] Olivera | Calcium channel diversity and neurotransmitter release: the u-conotoxins and u-agatoxins[END_REF].

Unlike the u-conotoxins, u-agatoxin IVA does not cause pore block, but instead acts as a gating inhibitor (Fig. 1). Binding of the toxin antagonizes the activation of the channel by preventing the movement of the voltage sensor domains [START_REF] Mintz | Ptype calcium channels blocked by the spider toxin u-Aga-IVA[END_REF]McDonough et al., 1997a;[START_REF] Bourinet | Splicing of a1A subunit gene generates phenotypic variants of P-and Q-type calcium channels[END_REF], which fits the notion that toxin binding occurs at a region adjacent to the voltage sensor in domain IV. This inhibition can be overcome by application of strong depolarizing voltage pulses [START_REF] Mintz | Ptype calcium channels blocked by the spider toxin u-Aga-IVA[END_REF]. The voltage-induced reversibility of blocking action is also reflected in a massive (i.e., many tens of millivolts) shift in the apparent halfactivation potential of the channel that is and likely due to dislodging of the toxin from its binding site by virtue of outward voltage sensor movement [START_REF] Mintz | Ptype calcium channels blocked by the spider toxin u-Aga-IVA[END_REF]. Although u-agatoxin IVA is selective for Cav2.1channels, higher concentrations of this toxin also have the propensity to affect other members of the Cav2 channel family, including Cav2.2 [START_REF] Sidach | Low-affinity blockade of neuronal N-type Ca channels by the spider toxin u-agatoxin-IVA[END_REF] and Cav2.3 [START_REF] Williams | Structure and functional characterization of neuronal a1E calcium channel subtypes[END_REF].

A much larger member of the u-agatoxin family is u-agatoxin IIIA [START_REF] Venema | Antagonism of synaptosomal calcium channels by subtypes of omega-agatoxins[END_REF][START_REF] Olivera | Calcium channel diversity and neurotransmitter release: the u-conotoxins and u-agatoxins[END_REF]. It is nearly double in size compared to IVA (78 amino acid residues) and contains as many as 12 cysteine residues that are capable of six disulfide bonds [START_REF] Olivera | Calcium channel diversity and neurotransmitter release: the u-conotoxins and u-agatoxins[END_REF]. This toxin has poor selectivity among the family of high-voltage activated channels, blocking L-type, P/Qtype and N-type channels, whereas T-type calcium channels appear to be spared [START_REF] Mintz | Inhibition of N-and L-type Ca2þ channels by the spider venom toxin u-Aga-IIIA[END_REF][START_REF] Mintz | Block of Ca channels in rat central neurons by the spider toxin u-Aga-IIIA[END_REF]. This toxin acts as a pore blocker, however, a residual current remains even at saturating concentrations. In Cav2.2 channels, this residual current is insensitive to u-conotoxin GVIA, indicating that u-agatoxin IIIA may act via incomplete pore occlusion [START_REF] Mintz | Block of Ca channels in rat central neurons by the spider toxin u-Aga-IIIA[END_REF]. In contrast, in P-type channels, u-agatoxin IVA blocks this residual current, consistent with the idea that u-agatoxin IVA does not require access to the pore of the channel [START_REF] Mintz | Block of Ca channels in rat central neurons by the spider toxin u-Aga-IIIA[END_REF].

The American funnel web spider also produces two other uagatoxins, namely u-agatoxin IA and IIA. The former appears to be specific for insect calcium channels, whereas the latter blocks both mammalian and invertebrate channels [START_REF] Olivera | Calcium channel diversity and neurotransmitter release: the u-conotoxins and u-agatoxins[END_REF]. The two toxins differ in structure, with u-agatoxin IA being a heterodimer with five disulfide bonds [START_REF] Santos | Heterodimeric structure of the spider toxin u-agatoxin IA revealed by precursor analysis and mass spectrometry[END_REF], and u-agatoxin IIA displaying only three disulfide bonds.

Other toxins from spiders and scorpions

The venom of the tarantula Grammostola spatulata contains a calcium channel blocking peptide termed u-grammotoxin SIA. This toxin acts on Cav2.1, Cav2.2 and Cav2.3 channels with micromolar affinity [START_REF] Lampe | Isolation and pharmacological characterization of u-grammotoxin SIA, a novel peptide inhibitor of neuronal voltage-sensitive calcium channel responses[END_REF][START_REF] Piser | u-Grammotoxin blocks action-potential-induced Ca2þ influx and whole-cell Ca2þ current in rat dorsal-root ganglion neurons[END_REF][START_REF] Piser | u-grammotoxin SIA blocks multiple, voltage-gated, Ca2þ channel subtypes in cultured rat hippocampal neurons[END_REF]McDonough et al., 1997b;[START_REF] Turner | Characterization of presynaptic calcium channels with u-conotoxin MVIIC and u-grammotoxin SIA: role for a resistant calcium channel type in neurosecretion[END_REF]. u-grammotoxin SIA is a gating modifier whose action is reminiscent of that of u-agatoxin IVA (McDonough et al., 1997a), such that it produces a massive shift in the half-activation potential of the channel. Interestingly, the effects of u-grammotoxin SIA and u-agatoxin IVA on Cav2.2 channels are additive, such that the apparent half activation voltage of the channels is shifted by as much as þ150 mV when both toxins are present (McDonough et al., 1997b). This finding implies that the two toxins, although both acting by preventing voltage-sensor movement, target distinct sites or domains of the Cav2.2 subunit.

Binding studies reveal that u-conotoxin GVIA cannot displace ugrammotoxin SIA from its binding site [START_REF] Lampe | Isolation and pharmacological characterization of u-grammotoxin SIA, a novel peptide inhibitor of neuronal voltage-sensitive calcium channel responses[END_REF], consistent with the notion that the latter is not a pore blocker.

Huwenotoxin-1 is a 33 amino acid peptide isolated from the spider Selenocosmia huwena. It has been shown to bock N-type currents in NG108-15 cells with an affinity of approximately 100 nM [START_REF] Peng | The effect of Huwentoxin-I on Ca(2þ) channels in differentiated NG108-15 cells, a patch-clamp study[END_REF]. L-type calcium channels were also shown to be targeted by this peptide, but with much lower affinity. Low-voltage activated channels were not affected [START_REF] Peng | The effect of Huwentoxin-I on Ca(2þ) channels in differentiated NG108-15 cells, a patch-clamp study[END_REF]. Based on its folding pattern, Huwenotoxin-1 likely acts though a pore blocking mechanism similar to that of u-conotoxin MVIIA.

Venom from the tarantula Hysterocrates gigas contains a calcium channel blocking peptide termed SNX-482 which is a potent inhibitor of Cav2.3 calcium channels [START_REF] Newcomb | Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas[END_REF]. Like u-grammotoxin SIA and u-agatoxin IVA, this toxin triggers a shift in half activation potential of Cav2.3 channels by approximately 70 mV, indicating that it too is an activation gating modifier. This peptide also blocks Cav1.2 calcium channels, albeit incompletely [START_REF] Bourinet | Interaction of SNX482 with domains III and IV inhibits activation gating of a1E (Cav2.3) calcium channels[END_REF]. Although its precise site of action on Cav2.3 channels is unknown, block appears to involve domains III and IV of the channel as revealed by chimeras between Cav2.3 and Cav1.2. These findings indicate that despite their high affinity for calcium channels, peptides trapping voltage-sensing domains have promiscuous effects on distinct channels. This has been well documented for voltage-gated sodium channels [START_REF] Bosmans | Targeting voltage sensors in sodium channels with spider toxins[END_REF]. Along these lines, SNX482 was recently shown to potently affect Kv4.2 and 4.3 potassium channels, suggesting that caution is warranted in the interpretation of neurophysiological data obtained with this peptide [START_REF] Kimm | Inhibition of A-type potassium current by the peptide toxin SNX-482[END_REF]. Nonetheless, SNX-482 was demonstrated to have interesting effects on the descending pathways controlling opiate analgesia and on reducing morphine tolerance to pain, an effect that is mirrored in the Cav2.3 KO mice [START_REF] Yokoyama | Blocking the R-type (Cav2.3) Ca2þ channel enhanced morphine analgesia and reduced morphine tolerance[END_REF].

SNX-325 is a 31 amino acid long peptide found in the venom of the spider Segestria florentina that contains 6 cysteine residues [START_REF] Newcomb | SNX-325, a novel calcium antagonist from the spider Segestria florentina[END_REF]. It preferentially blocks Cav2.2 calcium channels, but can target other members of the high-voltage activated calcium channel family at higher concentrations. Since it is able to displace u-conotoxin MVIIA from its binding site [START_REF] Newcomb | SNX-325, a novel calcium antagonist from the spider Segestria florentina[END_REF] this toxin likely acts as a pore blocker.

The venom of the scorpion Parabuthus transvaalicus contains kurtoxin, a 63 amino acid peptide that was the first published example of a toxin acting on a member of the T-type calcium channel family [START_REF] Chuang | Inhibition of Ttype voltage-gated calcium channels by a new scorpion toxin[END_REF]. However, subsequent studies have that this peptide also targets members of the Cav1 and Cav2 channel families [START_REF] Sidach | Kurtoxin, a gating modifier of neuronal high-and low-threshold Ca channels[END_REF], in addition to voltage-gated sodium channels [START_REF] Zhu | Actions of kurtoxin on tetrodotoxin-sensitive voltage-gated Naþ currents, NaV1.6, in murine vas deferens myocytes[END_REF]. At least for Cav3 channels, kurtoxin acts as a gating modifier [START_REF] Sidach | Kurtoxin, a gating modifier of neuronal high-and low-threshold Ca channels[END_REF]. The solution structure of kurtoxin reveals striking similarities to a-scorpion toxins, but somewhat different surface properties that may explain why this toxin acts on Cav3 channels in addition to sodium channels [START_REF] Lee | Solution structure of kurtoxin: a gating modifier selective for Cav3 voltage-gated Ca(2+) channels[END_REF].

Finally, protoxins I and II, found in the venom of the Tarantula Thrixoppelma pruriens, are potent sodium channel blockers [START_REF] Middleton | Two tarantula peptides inhibit activation of multiple sodium channels[END_REF][START_REF] Schmalhofer | ProTx-II, a selective inhibitor of NaV1.7 sodium channels, blocks action potential propagation in nociceptors[END_REF][START_REF] Xiao | The tarantula toxins ProTx-II and huwentoxin-IV differentially interact with human Nav1.7 voltage sensors to inhibit channel activation and inactivation[END_REF], however, they have also been shown to potently block Cav3 calcium channels [START_REF] Edgerton | Inhibition of the activation pathway of the T-type calcium channel Ca(V)3.1 by ProTxII[END_REF][START_REF] Bladen | Block of T-type calcium channels by Protoxins I and II[END_REF]. Protoxin I preferentially blocks Cav3.1 over Cav3.3 channels with about one order of magnitude higher affinity [START_REF] Ohkubo | Tarantula toxin ProTx-I differentiates between human T-type voltage-gated Ca2þ channels Cav3.1 and Cav3.2[END_REF][START_REF] Bladen | Block of T-type calcium channels by Protoxins I and II[END_REF]. This toxin appears to interact with residues in the domain IV region with some contribution from sites located in domain II of the Cav3.1 channels. In contrast, Cav3.2 channels are blocked with about 100-fold lower affinity. There appears to be some conservation in the binding sites of this toxin on Cav3 and Nav channels [START_REF] Bladen | Block of T-type calcium channels by Protoxins I and II[END_REF]. In contrast, protoxin II acts preferentially on Cav3.2 calcium channels and causes a curious shift in the half-inactivation potential of the channel [START_REF] Bladen | Block of T-type calcium channels by Protoxins I and II[END_REF] that remains to be further explored. Recent structure function experiments in which the voltage sensing domains of Cav3.1 channels were transferred individually onto a Kv2.1 backbone have revealed that protoxin II may preferentially target the voltage sensor in domain III of Cav3 channels [START_REF] Salari | Molecular interactions between Tarantula toxins and low-voltage-activated calcium channels[END_REF].

Additional toxins have been isolated from the Parabuthus transvaalicus scorpion (i.e., KLI and KLII) and the tarantula Theraphosidae (PsPTx3). The former two peptides block T-type channels and sodium channels [START_REF] Olamendi-Portugal | Two new scorpion toxins that target voltage-gated Ca2þ and Naþ channels[END_REF], whereas the latter preferentially targets T-type calcium channels, with some selectivity for Cav3.2 (E. Bourinet, unpublished observations, patent WO 2010081971 A1).

In summary, a number of calcium channel blocking peptides from various arachnids have been shown to block voltage-gated calcium channels. Many of these act as gating modifiers, however, the evolutionary significance of gating block as opposed to pore occlusion in peptide toxins from spiders versus cone snails remains to be determined.

Calcium channel blocking toxins from snakes

Black mamba (Dendroaspis polylepis) venom contains calciseptine, a 60 amino acid peptide (de [START_REF] De Weille | Calciseptine, a peptide isolated from black mamba venom, is a specific blocker of the L-type calcium channel[END_REF]. This peptide has been shown to selectively block L-type calcium channels from brain and heart [START_REF] De Weille | Calciseptine, a peptide isolated from black mamba venom, is a specific blocker of the L-type calcium channel[END_REF], and was subsequently shown to compete with dihydropyridine binding to synaptosomes [START_REF] Yasuda | Calciseptine binding to a 1,4-dihydropyridine recognition site of the L-type calcium channel of rat synaptosomal membranes[END_REF]. Interestingly, this peptide appears to have an agonist function on Cav1.1 channels in skeletal muscle [START_REF] García | Calciseptine, a Ca2þ channel blocker, has agonist actions on L-type Ca2þ currents of frog and mammalian skeletal muscle[END_REF]. FS2 is a related 60 amino acid peptide, also from black mamba venom, that differs from calciseptine in three amino acids. Like calciseptine, it potently blocks L-type calcium channels in smooth muscle [START_REF] Yasuda | FS2. a mamba venom toxin, is a specific blocker of the L-type calcium channels[END_REF].

Calcicludine is a 60 amino acid peptide isolated from the venom of the green mamba Dendroaspis angusticeps. It has been shown to block high-voltage activated calcium channels in cerebellar granule cells with high affinity [START_REF] Schweitz | Calcicludine, a venom peptide of the Kunitz-type protease inhibitor family, is a potent blocker of high-threshold Ca2þ channels with a high affinity for L-type channels in cerebellar granule neurons[END_REF], with L-type channels being most effectively inhibited. The effects of this toxin were also studied on heterologously expressed channels [START_REF] Stotz | Block of voltage-dependent calcium channel by the green mamba toxin calcicludine[END_REF], revealing several interesting features. First, block of Cav1.2, Cav2.1, Cav2.2 and Cav2.3 channels was found to be irreversible and incomplete at saturating concentrations, with Cav1.2 channels exhibiting the greatest degree of inhibition. Second, for Cav2 channels, the toxin induced a small hyperpolarizing shift in halfactivation potential, leading to a current enhancement at negative membrane potentials. Finally, experiments with chimeric calcium channels revealed that multiple membrane domains are involved in toxin block. Calcicludine binding was subsequently shown to mediate allosteric coupling to the dihydropyridine binding site [START_REF] Wang | Calcicludine binding to the outer pore of Ltype calcium channels is allosterically coupled to dihydropyridine binding[END_REF] in line with experiments involving calciseptine [START_REF] Yasuda | Calciseptine binding to a 1,4-dihydropyridine recognition site of the L-type calcium channel of rat synaptosomal membranes[END_REF].

Altogether, mamba venom contains at least three related peptide toxins that remarkable all are 60 amino acids in length, and all act by inhibiting L-type channel activity.

How useful are calcium channel peptide toxins as therapeutics?

Peptide toxins isolated from predatory species such as cone snails have long been explored as possible therapeutics, and is exemplified by the fact that entire research conferences have been dedicated to this subject (for example, Venoms to Drugs). With regard to calcium channel blocking peptides, most effort has been focused on their utility as potential analgesics. This is due to the fact that these peptides, which do not normally cross the blood brain barrier and can thus typically not be administered systemically, can be delivered to key sites of action in the pain pathway intrathecally. The only clinically approved calcium channel blocking peptide so far is Prialt (u-conotoxin MVIIA) [START_REF] Staats | Intrathecal ziconotide in the treatment of refractory pain in patients with cancer or AIDS: a randomized controlled trial[END_REF]). However, it can hardly be described as a blockbuster drug, and both the need for intrathecal delivery via an implanted mini pump and the limited therapeutic index is severely limiting [START_REF] Ver | An open-label, multicenter study of the safety and efficacy of intrathecal ziconotide for severe chronic pain when delivered via an external pump[END_REF]. Modification of the peptide structure into cyclized versions may provide a means for oral delivery, and thus the potential for much wider application for clinical use in humans. However, although there is now proof of concept with Vc1.1, producing such orally available peptides without loss of specificity and affinity remains challenging. Nonetheless, in addition to having been established as important and selective tools for isolation of native calcium currents [START_REF] Randall | Pharmacological dissection of multiple types of Ca2þ channel currents in rat cerebellar granule neurons[END_REF], peptide toxins retain promise as possible therapeutic agents, as either drugs, or templates for small organic mimetics. The resolution of the Cav1.1 structure may help advance the discovery of such peptide mimetics.

Fig. 1 .

 1 Fig.1. Comparison of action of pore blockers and gating modifiers. A. Pore blockers such as u-conotoxin MVIIA act by binding to amino acid residues in the outer vestibule of the channel pore, thus physically occluding the permeation pathway. Hence, at sub-saturating concentrations, there is a reduction of whole cell conductance across the entire spectrum of voltages without an apparent shift in the voltage-dependence of activation (bottom panel). B, Gating modifiers such as u-agatoxin IVA bind to a site located outside of the permeation pathway that is coupled to the activation gating machinery. Toxin binding thus prevents channel opening, and hence calcium entry through the channel. Strong membrane depolarizations can overcome this effect, allowing full channel opening at very depolarized membrane potentials. This is reflected in a rightward shift of the activation curve of the channel (bottom panel).

Please cite this article in press as: Bourinet, E., Zamponi, G.W., Block of voltage-gated calcium channels by peptide toxins, Neuropharmacology (2016), http://dx.doi.org/10.1016/j.neuropharm.2016.10.016

Acknowledgments

Work in the Zamponi laboratory is supported by a Foundation grant from the Canadian Institutes of Health Research, and a Canada Research Chairs award. The Bourinet laboratory is supported by grants from the Fondation pour la Recherche M edicale and the Agence Nationale de la Recherche.