David Schnell

Andrea Strasser

Dr Roland Seifert
email: seifert.roland@mh-hannover.de

Comparison of the pharmacological properties of human and rat histamine H 3 -receptors

Keywords: active receptor state, histamine H 3 -receptor, imoproxifan, molecular dynamics simulations, Sf9 insect cells

Abbreviations used: GPCR, G protein-coupled receptor; GTPS, guanosine 5'-[thio]triphosphate; h, human; r, rat; β 2 AR, β 2 -adrenoceptor; H 3 R, histamine H 3 -receptor, H 4 R, histamine H 4 -receptor; TM, transmembrane domain; JNJ-7753707, (4-fluorophenyl)(1methyl-2-an-1H-imidazol-5-yl)methanone; FUB181, 4-(3-(3-(4-chlorophenyl)propoxy)propyl)-1H-imidazole; A-304121, (R)-2-amino-1-(4-(3-(4-(cyclopropanecarbonyl)phenoxy)

A c c e p t e d M a n u s c r i p t

INTRODUCTION

Histamine (HA) exhibits its biological effects through the activation of four different G protein-coupled receptors (GPCRs). The histamine H 1 -receptor (H 1 R) is associated with inflammatory and allergic reactions, e. g. it increases vascular permeability and NO production [START_REF] Hill | International Union of Pharmacology. XIII. Classification of histamine receptors[END_REF]. The histamine H 2 -receptor (H 2 R) regulates gastric acid production, but also shows a positive inotropic effect on the heart [START_REF] Hill | International Union of Pharmacology. XIII. Classification of histamine receptors[END_REF]. The histamine H 3 -receptor (H 3 R) is a presynaptic auto-and heteroreceptor, regulating the release of HA and various other neurotransmitters in the nervous system, and is involved in important physiological processes like the sleep-wake cycle, eating behaviour and cognition [START_REF] Leurs | The histamine H 3 receptor: from gene cloning to H 3 receptor drugs[END_REF]. The histamine H 4receptor (H 4 R) mediates inflammatory and immunological processes, e. g. chemotaxis of eosinophils, mast cells and dendritic cells, but it is also present on neurons mediating HAinduced itching [START_REF] Leurs | Molecular and biochemical pharmacology of the histamine H 4 receptor[END_REF][START_REF] Zampeli | The role of histamine H 4 receptor in immune and inflammatory disorders[END_REF]. H 1 R and H 2 R antagonists have been used as therapeutics for decades, H 3 R and H 4 R are still explored and promising new drug targets [START_REF] Tiligada | Histamine H 3 and H 4 receptors as novel drug targets[END_REF].

The H 3 R was pharmacologically identified in the early 1980s, but cloned almost 20 years later in 1999 as an orphan GPCR [START_REF] Leurs | The histamine H 3 receptor: from gene cloning to H 3 receptor drugs[END_REF]. The reason for this delay was that it only shares ~20% homology to the H 1 R and H 2 R. The complex gene structure of the human H 3 R (hH 3 R) gives rise to many possible splice variants. To date about 20 hH 3 R splice variants are known [START_REF] Bongers | Molecular aspects of the histamine H 3 receptor[END_REF], but their function still remains elusive. The H 3 R displays high constitutive, i. e. ligandindependent, activity in many experimental systems [START_REF] Arrang | Constitutive activity of the histamine H 3 receptor[END_REF]. The H 3 R is one of the very few GPCRs for which constitutive activity has also been demonstrated in vivo [START_REF] Morisset | High constitutive activity of native H 3 receptors regulates histamine neurons in brain[END_REF].

For the H 3 R it has also been shown that species-differences exist [START_REF] Ireland-Denny | Species-related pharmacological heterogeneity of histamine H 3 receptors[END_REF][START_REF] Wulff | Characteristics of recombinantly expressed rat and human histamine H 3 receptors[END_REF]. Fig. 1 shows the amino acid sequences of hH 3 R and rH 3 R. Although the H 3 R sequence has a high degree of similarity among species, differences located in key regions of the receptor protein account for differences in antagonist affinity [START_REF] Yao | Molecular modeling and pharmacological analysis of species-related histamine H 3 receptor heterogeneity[END_REF][START_REF] Ligneau | Distinct pharmacology of rat and human histamine H 3 receptors: role of two amino acids in the third transmembrane domain[END_REF]. Additionally, splice variants differ in composition and expression pattern between species, and there are potential differences in signal transduction processes between either tissues and/or species [START_REF] Hancock | Genetic and pharmacological aspects of histamine H 3 receptor heterogeneity[END_REF]. Nevertheless, there are still unresolved questions about species differences of the full-length and un- In the present study, we systematically compared the pharmacological properties of hH 3 R and rH 3 R. Fig. 2 shows the structures of the compounds studied, all of them being imidazole-containing ligands. We co-expressed hH 3 R and rH 3 R in Sf9 cells together with mammalian G proteins in a defined stoichiometry, determined the affinity of ligands in radioligand binding studies, and their potency and efficacy in steady-state GTPase assays.

The baculovirus/Sf9 cell system is very suitable for the analysis of G i /G o -coupled receptors and in particular constitutively active receptors, because in Sf9 cells no endogenous G i /G oproteins or GPCRs with constitutive activity are present. The controlled expression of receptor and G proteins in Sf9 cell membranes represents more the physiological situation than, for example, the construction of GPCR-Gα fusion proteins, because fusion proteins do not exist physiologically and the mobility of the G proteins is not restricted in the coexpression system. Moreover, the use of very proximal read-outs, like radioligand binding or steady-state GTPase assays prevent possible bias in later steps of the signal transduction cascade.

Many studies of ligand-receptor interactions come to a point where structural information on the atomic level is needed to explain experimental results. In the case of GPCRs, this is a very challenging and time-consuming process, and at the end only snapshots of static ligand-receptor complexes are resolved [START_REF] Kobilka | New G-protein-coupled receptor crystal structures: insights and limitations[END_REF]. However, more and more high-resolution crystal structures of inactive-and active-state GPCR-ligand complexes are becoming available and can be used to generate better homology models [START_REF] Rasmussen | Crystal structure of the human β 2 -adrenergic G-protein-coupled receptor[END_REF][START_REF] Jaakola | The 2.6 Å crystal structure of a human A 2A adenosine receptor bound to an antagonist[END_REF][START_REF] Park | Crystal structure of the ligandfree G-protein-coupled receptor opsin[END_REF].

Several molecular modelling studies with regard to ligand-receptor interaction at H 3 R are found in literature [START_REF] Yao | Molecular modeling and pharmacological analysis of species-related histamine H 3 receptor heterogeneity[END_REF][START_REF] Stark | Different antagonist binding properties of human and rat histamine H 3 receptors[END_REF][START_REF] Schlegel | Generation of a homology model of the human histamine H 3 -receptor for ligand docking and pharmacophore-based screening[END_REF][START_REF] Rai | Modeling G protein-coupled receptors for structure-based drug discovery using low-frequency normal modes for refinement of homology models: Application to H 3 antagonists[END_REF]. Since active-state models for H 3 Rs do not yet exist, we generated and used those models to explain the pharmacological species differences at hH 3 R and rH 3 R on the basis of experimental data.

A c c e p t e d M a n u s c r i p t 5

MATERIALS AND METHODS

Materials.

The cDNAs of the hH 3 R and rH 3 R were kindly provided by Dr. T. Lovenberg (Johnson & Johnson Pharmaceutical R&D, San Diego, CA, USA). An alignment of the corresponding amino acid sequences is given in Fig. 1. Anti-hH 3 R Ig and anti-rH 3 R Ig were from Bio-Trend (Cologne, Germany). The antibody recognizing both species homologs was from GeneWay (San Diego, CA, USA). All other antibodies, purified G proteins, reagents for molecular biology, recombinant baculoviruses encoding mammalian G protein subunits, and the sources of test compounds were described before [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF]. Chemical structures of H 3 R ligands are depicted in Fig. 2. Stock solutions (10 mM) of all H 3 R ligands described in this paper were prepared in distilled water and stored at -20°C. [γ-32 P]GTP was synthesized as described [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF]. Unlabeled nucleotides were from Roche (Indianapolis, IN, USA) and all other reagents were of the highest purity available and from standard suppliers.

Construction of FLAG epitope-and hexahistidine

SDS-PAGE and immunoblot analysis.

The protocols for virus amplification, protein expression and western blot analysis were described before [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF]. Proteins transferred to nitrocellulose membranes were reacted with anti-hH 3 R (N-term) (1:1000), anti-rH 3 R (C-term) (1:1000) and anti-H 3 R (i3) (1:1000) Igs.

[ 35 S]GTPS saturation binding assay.

Experiments were performed in analogy to the assay described in Schnell et al. [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF].

Membranes were thawed and sedimented by a 10-min centrifugation at 4°C and 15,000g to remove residual endogenous guanine nucleotides as far as possible. Membranes were resuspended in binding buffer (12.5 S]GTPγS by filtration through GF/C filters, followed by three washes with 2 ml of binding buffer (4°C). Filter-bound radioactivity was determined by liquid scintillation counting.

The experimental conditions chosen ensured that not more than 10 % of the total amount of radioactivity added to binding tubes was bound to filters. The maximum number of Gα i/orelated GTPγS binding sites in membranes expressing H 3 Rs plus Gα-subunits plus β 1 γ 2 was corrected by the binding determined in parallel in membranes expressing H 3 Rs plus β 1 γ 2 alone. These reference membranes were always prepared under exactly the same conditions as the other ones. To ensure the same viral load in the reference membranes, Sf9 cells were infected with baculoviruses encoding H 3 Rs, β 1 γ 2 and virus encoding no recombinant protein at all. In this manner, only the number of functionally intact and heterologously expressed mammalian Gα i/o -subunits was quantitated. Experiments were performed in analogy to the assay described in Schnell et al. [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF]. Briefly, membranes were thawed, sedimented and resuspended in 10 mM Tris/HCl, pH 7.4. Assay tubes contained Sf9 membranes (10 -20 μg of protein/tube), 5.0 mM MgCl 2 , 0.1 mM EDTA, 0.1 mM ATP, 100 nM GTP, 0.1 mM adenylyl imidodiphosphate, 1.2 mM creatine phosphate, 1 μg of creatine kinase, and 0.2% (w/v) bovine serum albumin in 50 mM Tris/HCl, pH 7.4, and H 3 R ligands at various concentrations. Reaction mixtures (80 μl) were incubated for 2 min at 25°C before the addition of 20 μl of [γ-32 P]GTP (0.1 μCi/tube). All stock and work dilutions of [γ-32 P]GTP were prepared in 20 mM Tris/HCl, pH 7.4. Reactions were conducted for 20 min at 25°C. Reactions were terminated by the addition of 900 μl of slurry consisting of 5% (w/v) activated charcoal and 50 mM NaH 2 PO 4 , pH 2.0. Charcoal absorbs nucleotides but not P i . Charcoal-quenched reaction mixtures were centrifuged for 7 min at room temperature at 15,000g. Six hundred microliters of the supernatant fluid of reaction mixtures were removed, and 32 P i was determined by liquid scintillation counting. Enzyme activities were corrected for spontaneous degradation of [γ-32 P]GTP. Spontaneous [γ-32 P]GTP degradation was determined in tubes containing all of the above described components plus a very high concentration of unlabeled GTP (1 mM) that, by competition with [γ-32 P]GTP, prevents [γ-32 P]GTP hydrolysis by enzymatic activities present in Sf9 membranes. Spontaneous [γ-32 P]GTP degradation was <1% of the total amount of radioactivity added using 20 mM Tris/HCl, pH 7.4, as solvent for [γ-32 P]GTP. The experimental conditions chosen ensured that not more than 10% of the total amount of [γ-32 P]GTP added was converted to 32 P i .

Radioligand binding assays.

Experiments were performed in analogy to the assay described in Schnell et al. [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF].

Membranes were thawed and sedimented by a 10-min centrifugation at 4°C and 15,000g and resuspended in binding buffer (12.5 mM MgCl 2 , 1 mM EDTA, and 75 mM Tris-HCl, pH 7.4), to remove residual endogenous guanine nucleotides as much as possible. In Filter-bound radioactivity was determined by liquid scintillation counting. The experimental conditions chosen ensured that not more than 10 % of the total amount of radioactivity added to binding tubes was bound to filters.

Construction of inactive and active models of hH 3 R and rH 3 R.

Based on the crystal structure of the human β 2 -adrenergic receptor [START_REF] Rasmussen | Crystal structure of the human β 2 -adrenergic G-protein-coupled receptor[END_REF][START_REF] Cherezov | High-resolution crystal structure of an engineered human β 2 -adrenergic G proteincoupled receptor[END_REF], a homology model of the inactive hH 3 R and rH 3 R were generated. Based on the active state model of guinea pig H 1 R (gpH 1 R) [START_REF] Strasser | Analysis of the activation mechanism of the guinea-pig Histamine H 1 -receptor[END_REF][START_REF] Strasser | Ligand-specific contribution of the N terminus and E2-loop to pharmacological properties of the histamine H 1 -receptor[END_REF], an active model of hH 3 R was constructed by homology modelling. All models were refined and energetically minimized with SYBYL 7.0 (Tripos, St.

Louis, MO, USA), as described [START_REF] Igel | Synthesis and structureactivity relationships of cyanoguanidine-type and structurally related histamine H 4 receptor agonists[END_REF]. Imoproxifan was docked manually into the binding pocket of the active hH 3 R and the inactive rH 3 R. Thereby, previous results of similar compounds were taken into account [START_REF] Stark | Different antagonist binding properties of human and rat histamine H 3 receptors[END_REF][START_REF] Schlegel | Generation of a homology model of the human histamine H 3 -receptor for ligand docking and pharmacophore-based screening[END_REF]. The resulting structures were embedded in a simulation box, including lipid bilayer, water, sodium and chlorine ions, as described [START_REF] Strasser | Pharmacological profile of histaprodifens at four recombinant histamine H 1 -receptor species isoforms[END_REF].

Subsequently, molecular dynamic simulations with GROMACS 3.3.1 [START_REF] Van Der Spoel | GROMACS: fast, flexible, and free[END_REF] were performed, using a simulation protocol, previously described [START_REF] Strasser | Pharmacological profile of histaprodifens at four recombinant histamine H 1 -receptor species isoforms[END_REF]. Molecular biology was planned with GCK 2.5 (Textco BioSoftware, West Lebanon, NH, USA). Ligand structures were illustrated using ChemDraw Ultra 8.0 (CambridgeSoft, Cambridge, MA, USA). The sequence alignment was performed using ClustalX (2.0), which is a windows interface based on the Clustal W algorithm [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF]. Protein was determined using the DC protein assay kit (Bio-Rad, Hercules, CA, USA). [ 3 H]Dihydroalprenolol was obtained from Perkin Elmer (Boston, MA, USA) and protein quantification via western blot performed as described in Schnell et al. [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF]. All analyses of experimental data were performed with the Prism 5 program (GraphPad Software, San Diego, CA, USA).

RESULTS

Western blot analysis of hH 3 R and rH 3 R expressed in Sf9 insect cell membranes.

Membranes of Sf9 cells expressing hH 3 R or rH 3 R plus mammalian G proteins were prepared and analyzed via immunoblot. It has to be mentioned, that membranes co-expressing rH 3 R plus different mammalian G proteins were prepared in parallel and under exactly the same conditions as the membranes expressing hH 3 R [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF]. Thus, the comparison of hH 3 R and rH 3 R pharmacology in this system is not based on historical data but direct. Both hH 3 R and rH 3 R bands were doublets, probably representing differently glycosylated forms (Fig. 3). H 3 R species homologs presumably exhibit similar glycosylation patterns since the putative Nglycosylation site for the H 3 R (Asn11) is fully conserved within their sequences (Fig. 1). The H 3 R species homologs could be clearly discriminated by anti-hH 3 R Ig, raised against an 18 amino acid peptide within the extracellular N-terminus of the hH 3 R, and anti-rH 3 R Ig, raised against an 18 amino acid peptide within the cytoplasmatic C-terminus of the rH 3 R (Figs. 3A and3B). Additionally, anti-H 3 R (i3) Ig was used to confirm the above mentioned results (Fig. 3C). This antibody was raised against a peptide sequence within the third intracellular loop (i3) of the hH 3 R, but turned out to be not species-selective. Again, all H 3 R bands occurred as doublets at ~49 kDa. However, there were some additional bands at lower molecular weight, which are presumably non-specific, since they also appeared at the control lane loaded with A c c e p t e d M a n u s c r i p t 10 uninfected Sf9 cell membranes. Thus, our data indicate that hH 3 R and rH 3 R were equally well and properly expressed in Sf9 cells. In analogy to our recent publication [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF], the rH 3 R was also co-expressed with different mammalian G proteins (Gα i1 , Gα i2 , Gα i3 or Gα o1 , and β 1 γ 2 dimers, respectively) to analyze the coupling profile. All proteins were properly detected by different selective antibodies (Fig. 3). Moreover, we also quantified the expression levels of receptors and G proteins by immunoblot, using hβ 2 AR or purified G protein subunits as standards (Fig. 3D and3H, I). The results of these studies are summarized in Table 1.

Quantitative analysis of receptor-to-G protein stoichiometries.

Protein quantification via western blot is semi-quantitative and does not discriminate between functional and non-functional proteins. Therefore, we directly used a recently described combination of antagonist [³H]JNJ-7753707-and [ 35 S]GTPγS-saturation binding [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF] and calculated the functional GPCR/Gα protein ratios (Table 1). Similar to the membranes expressing hH 3 R, we detected an excess of mammalian G proteins in the case of rH 3 R, confirming the previously reported results [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF]. Thus, G protein expression level is not limiting in this experimental system, too.

hH 3 R and rH 3 R coupling to different Gα-subunits.

The G protein coupling profile of rH 3 R (Table 2) was also investigated as for the hH 3 R [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF].

Briefly, receptor-dependent [γ-32 P]GTP hydrolysis of different Gα-subunits was measured under steady-state conditions. GTPase activities were determined in parallel under basal conditions, maximal stimulation with the physiological (and full) agonist histamine (10 µM) and a saturating concentration of the inverse agonist thioperamide (10 µM) in Sf9 cell membranes co-expressing the rH 3 R and different G proteins.

Like hH 3 R, rH 3 R coupled efficiently to all co-expressed mammalian Gα i/o -subunits (Gα i1 , Gα i2 , Gα i3 or Gα o1 , and β 1 γ 2 dimers, respectively), as was evident by the high basal GTPase activities and the large absolute stimulatory and inhibitory effects of histamine and thioperamide, respectively (Table 2). Also, the relative stimulatory effects of histamine and A c c e p t e d M a n u s c r i p t 11 the relative inhibitory effects of thioperamide based on total ligand-regulated GTPase activity were similar for each of the four systems studied, indicating that the constitutive activity of rH 3 R was comparable and not substantially influenced by the type of G protein (Table 2). The constitutive activity of rH 3 R coupled to cognate G i /G o -proteins was rather high and comparable to the constitutive activity of hH 3 R, rendering the two systems suitable for an analysis of species-specific ligand effects, without possible bias due to differences in basal activity between membranes.

Ligand potencies and efficacies in the steady-state GTPase assay at rH 3 R compared to hH 3 R co-expressed with different Gα-subunits.

Next, we examined a series of imidazole-based ligands in Sf9 cell membranes expressing rH 3 R and different Gα i/o -proteins in the steady-state GTPase assay. The data (Table 3) were then compared with the results for hH 3 R [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF] (Fig. 4 A andB). The endogenous agonist histamine (1) and the standard H 3 R ligands N  -methylhistamine ( 2) and (R)-methylhistamine (3) were full agonists and equally potent in all membranes. There were essentially no species-differences. The highly potent standard H 3 R agonist imetit (4) was almost a full agonist at rH 3 R, too. Interestingly, proxifan (5) was again a strong partial agonist in all systems, independent of the G protein subtype co-expressed, corroborating the notion that this ligand does not show functional selectivity at H 3 Rs [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF]. In contrast to hH 3 R, impentamine (6) was a strong and more potent partial agonist at rH 3 R in all experimental settings. Strikingly, imoproxifan [START_REF] Arrang | Constitutive activity of the histamine H 3 receptor[END_REF] was an almost full agonist at hH 3 R (Fig. 5A), but an inverse agonist at rH 3 R (Fig. 5B). The type of G protein subunit did not change the pharmacological profile of imoproxifan (Table 3). The inverse agonists ciproxifan (8) and thioperamide [START_REF] Wulff | Characteristics of recombinantly expressed rat and human histamine H 3 receptors[END_REF] were more potent but less efficacious at rH 3 R than at hH 3 R and again, the G protein subtype caused no changes in their profiles. Clobenpropit (9) was neither speciesspecific nor did the G protein subtype change its pharmacology. Moreover, there is also a strong linear correlation between potencies and efficacies of imidazole-based ligands at membranes expressing rH 3 R and different Gα i/o -subunits, as was found for the hH 3 R (Fig. 6;

A c c e p t e d M a n u s c r i p t

Table 3). Thus, the pharmacological profile of the rH 3 R is also very similar under the various experimental conditions and, like at hH 3 R, ligand-specific receptor conformations leading to coupling differences do not exist for the compounds investigated [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF].

Collectively, these results confirm the findings regarding the relative stimulatory and inhibitory effects of histamine and thioperamide, respectively (Table 2), based on total ligandregulated GTPase activity and are indicative for similar constitutive activity of hH 3 R and rH 3 R under all experimental conditions. If there had been differences in constitutive activity between hH 3 R and rH 3 R, then systematic changes in the potencies of full agonists as well as potencies and efficacies of partial agonists and inverse agonists would have occurred. This, however, was not the case. In contrast, the behaviour of ligands, e. g. impentamine or imoproxifan, at one H 3 R species homolog often opposed each other, against every expectation. Thus, these ligand effects are solely species-specific and not due to differences in constitutive activity of hH 3 R and rH 3 R.

[ 3 H]NAMH binding studies at hH 3 R and rH 3 R.

Since the type of G protein co-expressed did not change the pharmacology of ligands at both hH 3 R and rH 3 R in the steady-state GTPase assay, we performed radioligand binding experiments for a further characterization only at membranes expressing hH 3 R or rH 3 R plus Gα i2 plus β 1 γ 2 dimers.

At first, we addressed the formation of a high-affinity ternary complex between the agonist [ 3 H]NAMH, the hH 3 R or rH 3 R and nucleotide-free G protein in saturation binding experiments (Fig. 5 C andD). The K d of [ 3 H]NAMH at hH 3 R was 0.62 ± 0.21 nM, and the B max was 0.62 ± 0.02 pmol/mg (n = 3). At rH 3 R, the K d value was 1.37 ± 0.36 nM, and the B max was 0.48 ± 0.03 pmol/mg (n = 3). Interestingly, binding of 2), (R)--methylhistamine (3), imetit (4) and proxifan (5) had essentially the same affinities at hH 3 R and rH 3 R. Impentamine [START_REF] Bongers | Molecular aspects of the histamine H 3 receptor[END_REF], imoproxifan (7) (Fig. 5 E andF), ciproxifan [START_REF] Morisset | High constitutive activity of native H 3 receptors regulates histamine neurons in brain[END_REF] and thioperamide (10) (Fig. 5 E andF) bound with higher affinity to rH 3 R. Clobenpropit (9) also bound with similar affinity to both receptors. The pharmacological profiles, determined in [ 3 H]NAMH competition binding (Table 4) and steady-state GTPase assays (Table 3), compared with the literature, were very similar [START_REF] Lim | Evaluation of histamine H 1 -, H 2 -, and H 3 -receptor ligands at the human histamine H 4 receptor: identification of 4-methylhistamine as the first potent and selective H 4 receptor agonist[END_REF][START_REF] Bongers | An 80-amino acid deletion in the third intracellular loop of a naturally occurring human histamine H 3 isoform confers pharmacological differences and constitutive activity[END_REF]. The pK i and pEC 50 values determined at hH 3 R correlated well, suggesting a direct signal transfer in the Sf9 cell system (Fig. 7). However, at rH 3 R, the correlation coefficient was rather low, due to an extraordinary high affinity of impentamine [START_REF] Bongers | Molecular aspects of the histamine H 3 receptor[END_REF]. Interestingly, the pK i values of imoproxifan [START_REF] Arrang | Constitutive activity of the histamine H 3 receptor[END_REF] were significantly lower than the corresponding pEC 50 values determined in the GTPase assay (t test, p < 0.05).

Binding mode of imoproxifan at hH 3 R and rH 3 R.

To understand the molecular basis for the unique behaviour of imoproxifan, we performed molecular modelling studies with hH 3 R and rH 3 R. The binding modes of imoproxifan at active hH 3 R and inactive rH 3 R, representing the most favoured ligand-receptor-complexes, are presented in Fig. 8.

Imoproxifan is bound to hH 3 R and rH 3 R in E-configuration, representing the transisomer of the oxime-moiety. The E-configuration was also determined to be the favoured one by crystallographic studies [START_REF] Sasse | New histamine H 3receptor ligands of the proxifan series: imoproxifan and other selective antagonists with high oral in vivo potency[END_REF]. The electrostatic surface potential of the amino acids with the ligand in the binding pocket is shown (Fig. 8, A andB). In hH 3 R and rH 3 R, the positively charged terminal imidazole moiety of imoproxifan interacts with the highly conserved Asp 3.32 (Fig. 8, A and B, black arrow). However, there are large differences in electrostatic surface between TM V and TM III (Fig. 8, A andB, yellow, dotted line). In this region, the electrostatic surface potential is positive in hH 3 R due to the NH moiety of Trp 6.48 , The analoguous region in rH 3 R shows a slightly negative potential due to the OH moiety of Thr 6.52 . The consequence is a different orientation, and thus a different hydrogen bond networking of the oxime moiety The reason for the differences in electrostatic surface potential between hH 3 R and rH 3 R are explained by the amino acid difference at position 3.37 between hH 3 R and rH 3 R. In hH 3 R, Glu 5.46 can electrostatically interact with Thr 3.37 (Fig. 8,C, yellow, dotted line). Thus, Glu 5.46 points towards Thr 3.37 and away from the binding pocket. Consequently, the electrostatic potential surface in this region is neutral and a small pocket for the methyl moiety of imoproxifan is build. In contrast, in rH 3 R, Thr 3.37 is exchanged into Ala 3.37 . Thus, an electrostatic interaction between Glu 5.46 and the amino acid side chain in position 3.37 is no longer possible. Instead, the modelling studies revealed an electrostatic interaction of Glu 5.46 and Tyr 3.33 in rH 3 R (Fig. 8,D, yellow, dotted line). Consequently, the negatively charged side chain of Glu 5.46 points partially toward the binding pocket, resulting in an aromatic contact between Tyr 3.33 and the imoproxifan.

A second species-difference between hH 3 R and rH 3 R near to the binding pocket is located at position 3.40. There is an alanine in hH 3 R, but a bulkier valine in rH 3 R (Fig. 8,C and D, green, dotted line). It is likely that this amino acid difference also directs the oxime moiety of imoproxifan into a distinct orientation. Since Ala 3.40 is not as bulky as Val 3.40 , there is more space for the oxime moiety to point downward in direction to 3.40 in hH 3 R, than in rH 3 R.

Additionally, Trp 6.48 is shown in its active conformation at hH 3 R (Fig. 8,C, blue arrow) and in its inactive conformation in rH 3 R (Fig. 8,D, blue arrow). Trp 6.48 is part of a highly conserved motif among GPCRs, thought to function as a toggle-switch during receptor activation, as is evident due to structural and biophysical studies [START_REF] Ahuja | Multiple switches in G protein-coupled receptor activation[END_REF]. Trp 6.48 horizontal to the membrane surface stabilizes the active state of a GPCR. Trp 6.48 vertical to the membrane surface stabilizes the inactive state of a GPCR. As consequence of the different amino acids at position 3.37 and 3.40 between hH 3 R and rH 3 R, the oxime moiety of imoproxifan can establish a hydrogen bond interaction to Trp 6.48 in its active conformation, thus stabilizing the active conformation of hH 3 R (Fig. 8,E, yellow, dotted line). This interaction was not found in A c c e p t e d M a n u s c r i p t rH 3 R. Here, the hydrogen of the oxime moiety interacts electrostatically with a negatively charged surface established by Thr 6.52 (Fig. 8, F, yellow, dotted line). In rH 3 R, the methyl group of imoproxifan is located in a small pocket established by Val 3.40 and Trp 6.48 in its inactive conformation.

Collectively, the different binding modes of imoproxifan in hH 3 R and rH 3 R presumably lead to differences in efficacies due to a different orientation of the oxime moiety and thus, stabilization of Trp 6.48 either horizontal or vertical to the membrane surface.

In contrast to imoproxifan [START_REF] Arrang | Constitutive activity of the histamine H 3 receptor[END_REF], ciproxifan (8) (Fig. 4B) was found to act as an inverse agonist at hH 3 R as well as at rH 3 R. This observation is quite interesting, since both compounds only differ in the substitution pattern at the phenyl moiety (Fig. 2). To obtain more insight into these pharmacological differences, we docked ciproxifan (8) into the binding pocket of the active conformation of hH 3 R and performed a minimization with regard to potential energy. An overlay with the corresponding imoproxifan -hH 3 Rcomplex showed, that the cyclopropyl moiety of ciproxifan [START_REF] Morisset | High constitutive activity of native H 3 receptors regulates histamine neurons in brain[END_REF], which is more space-filling than the methyl moiety of imoproxifan [START_REF] Arrang | Constitutive activity of the histamine H 3 receptor[END_REF], lead to a shift of the neighbouring Glu 5.46 away from the binding pocket. Subsequently, the hydrogen bond between Glu 5.46 and Thr 3.37 is broken, which may lead to a destabilization of the entire ligand-receptor complex. In contrast, ciproxifan (8) could be docked very well into the binding pocket of the inactive hH 3 R. The resulting binding mode is similar to that of imoproxifan [START_REF] Arrang | Constitutive activity of the histamine H 3 receptor[END_REF] at the inactive rH 3 R. Since there is the smaller alanine in position 3.40 at hH 3 R (compared to valine at rH 3 R), the more space-filling cycloproyl moiety fits optimally into this small pocket at hH 3 R. However, the exchange of the oxime moiety into a carbonyl moiety in ciproxifan (8) leads to the loss of one hydrogen bond between ligand and receptor, as found for imoproxifan (Fig. 8F).

DISCUSSION

Ligand pharmacology at hH 3 R and rH 3 R is species-dependent (Fig. 4) [START_REF] Lovenberg | Cloning of rat histamine H 3 receptor reveals distinct species pharmacological profiles[END_REF][START_REF] Esbenshade | Pharmacological classification of histamine H 3 receptor agents across species is attributable to transmembrane 3 sequence differences[END_REF].

Unexpectedly, the species-differences can even span from agonism to inverse agonism in the case of imoproxifan [START_REF] Esbenshade | Pharmacological classification of histamine H 3 receptor agents across species is attributable to transmembrane 3 sequence differences[END_REF]. In this study, we unraveled the underlying molecular A c c e p t e d M a n u s c r i p t 16 mechanism of this reversal in efficacy. In steady-state GTPase assays, imoproxifan was an inverse agonist at rH 3 R, but almost full agonist at hH 3 R. Competition binding studies with [ 3 H]NAMH confirmed that the effect was receptor-mediated. Both hH 3 R and rH 3 R were expressed at similar levels and defined receptor-to-G protein stoichiometries. The basal activity in the two systems was comparable, as indicated by the similar inhibitory effects of the standard inverse agonist thioperamide. Thus, the unexpected behaviour of imoproxifan can only be due to species-specific differences in ligand recognition and receptor activation.

Previous modelling studies described the binding mode of FUB181, a compound, similar to imoproxifan [START_REF] Schlegel | Generation of a homology model of the human histamine H 3 -receptor for ligand docking and pharmacophore-based screening[END_REF]. The orientation of imoproxifan in the binding pocket of H 3 R, determined in the present study, is similar to these previous findings. In another study, it was suggested, that the Ala 3.40 Val amino acid difference between hH 3 R and rH 3 R is responsible for the observed species-differences in antagonist pharmacology [START_REF] Yao | Molecular modeling and pharmacological analysis of species-related histamine H 3 receptor heterogeneity[END_REF]. It was pointed out that thioperamide or compound A-304121 are in closer contact to Val 3.40 in rH 3 R, than to Ala 3.40 in hH 3 R. We could reproduce this finding because the affinity and potency of thioperamide was higher at rH 3 R than hH 3 R in our experimental system, too. Our molecular modelling studies further revealed that both amino acid differences in TM III, at position 3.37 and 3.40, are responsible for the differences in pharmacology of imoproxifan between hH 3 R and rH 3 R. The oxime moiety points downward in direction of Ala 3.40 in hH 3 R. Thus, the polar oxime moiety is able to establish a hydrogen bond interaction with Trp 6.48 in its active conformation. In contrast, in rH 3 R, the oxime moiety points toward TM V. Additionally, the methyl moiety of imoproxifan fits optimally into a small pocket between the bulky Val 3.40 and Trp 6.48 in its inactive conformation. The highly conserved Trp 6.48 is suggested to act as a switch for receptor activation within biogenic amine receptors. Trp 6.48 oriented horizontally to the membrane surface is thought to stabilize the active state of a receptor, while Trp 6.48 oriented vertically to the membrane surface stabilizes the inactive state of a receptor. Since the hydrogen bond interaction between the oxime moiety and Trp 6.48 stabilizes Trp 6.48 in its active conformation in hH 3 R, the partial agonism of imoproxifan is explained on a molecular level. This hydrogen bond-supported stabilization of Trp 6.48 in its active conformation is not possible in rH 3 R. Here, in contrast to hH 3 R, the methyl group near Val 3.40 and Trp 6.48 stabilizes Trp 6.48 in its inactive conformation due to a hydrophobic interaction. Thus, the modelling studies provide an explanation for the inverse agonism of imoproxifan at rH 3 R on a molecular level, too. Additionally, the molecular modelling studies could be used to explain the switch from partial agonism of imoproxifan [START_REF] Arrang | Constitutive activity of the histamine H 3 receptor[END_REF] to inverse agonism of ciproxifan (8) at hH 3 R. The modelling studies suggest that the main reason for this is the more space-filling cycloproyl moiety of ciproxifan ( 8), compared to the methyl moiety in imoproxifan [START_REF] Arrang | Constitutive activity of the histamine H 3 receptor[END_REF]. This leads to a loss of hydrogen bonding between Glu 5.46 and Thr 3.37 . Furthermore, a hydrogen bond between the carbonyl moiety and the NH moiety of Trp 6.48 , stabilizing the active conformation of hH 3 R, could not be established.

Interestingly, the pK i values imoproxifan [START_REF] Arrang | Constitutive activity of the histamine H 3 receptor[END_REF] at hH 3 R and rH 3 R were significantly higher than their pEC 50 values (t test, p < 0.05). These results suggest that hH 3 R and rH 3 R can exist in a state of low partial agonist/inverse agonist affinity that interacts efficiently with G proteins. Another study, analyzing the hH 3 R expressed in SK-N-MC cells by [ 3 H]NAMH competition binding and CRE-β-galactosidase reporter gene assays, revealed similar disparities [START_REF] Lim | Evaluation of histamine H 1 -, H 2 -, and H 3 -receptor ligands at the human histamine H 4 receptor: identification of 4-methylhistamine as the first potent and selective H 4 receptor agonist[END_REF]. Similar results were also obtained when the human formyl peptide receptor, coupled to various G i -proteins, was studied in Sf9 cell membranes [START_REF] Wenzel-Seifert | Quantitative analysis of formyl peptide receptor coupling to Gα i1 , Gα i2 , and Gα i3[END_REF]. In this study, the K d of the agonist radioligand [ 3 H]fMLP was ~100-fold lower than the EC 50 determined in GTPase experiments. Interestingly, at hH 3 R expressed in Sf9 cells the low affinity state stabilized by imoproxifan [START_REF] Arrang | Constitutive activity of the histamine H 3 receptor[END_REF] leads to an activation of G proteins, whereas in rH 3 R, the low affinity state inhibited the activation of G proteins.

The G protein coupling profile of hH 3 R and rH 3 R was similar, too (Table 2; in [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF]). An important fact is that like for hH 3 R [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF], at rH 3 R no evidence for functional selectivity was observed (Table 3). However, in another study, evidence for functional selectivity at H 3 R could be obtained [START_REF] Krueger | G protein-dependent pharmacology of histamine H 3 receptor ligands: evidence for heterogeneous active state receptor conformations[END_REF]. Possible reasons for those discrepancies were already previously discussed [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF]. Both hH 3 R and rH 3 R coupled effectively with G i /G o -proteins in Sf9 cell membranes, as was shown by GTPγS-insensitive ternary complex formation, using In line with the data for hH 3 R and rH 3 R, high-affinity agonist binding to the structurally closely related histamine H 4 -receptor is also largely GTPS-insensitive [START_REF] Schneider | High constitutive activity and a G-proteinindependent high-affinity state of the human histamine H 4 -receptor[END_REF]. The H 4 -receptor possesses very high constitutive activity as well. Similar constitutive activity renders the system well suited for the analysis of species-specific ligand effects, since differences in constitutive activity between GPCRs can alter their pharmacological profiles and lead to a further complication of data interpretation [START_REF] Preuss | Constitutive activity and ligand selectivity of human, guinea pig, rat, and canine histamine H 2 receptors[END_REF][START_REF] Preuss | Mutations of Cys-17 and Ala-271 in the human histamine H 2 receptor determine the species selectivity of guanidine-type agonists and increase constitutive activity[END_REF].

The pharmacology of all histamine receptors (H x Rs) is species-dependent. This is especially true for H x R agonists. At the H 1 R, several classes of bulky ligands exhibit species differences [START_REF] Seifert | Multiple differences in agonist and antagonist pharmacology between human and guinea pig histamine H 1 -receptor[END_REF]. Some of them show unique behaviours, like epimeric members of the ergoline family or chiral histaprodifens, switching from silent antagonism to partial agonism depending on the species studied [START_REF] Strasser | Pharmacological profile of histaprodifens at four recombinant histamine H 1 -receptor species isoforms[END_REF][START_REF] Pertz | Striking differences of action of lisuride stereoisomers at histamine H 1 receptors[END_REF]. Detailed molecular studies dissected some of the underlying mechanisms [START_REF] Strasser | Ligand-specific contribution of the N terminus and E2-loop to pharmacological properties of the histamine H 1 -receptor[END_REF][START_REF] Strasser | Molecular basis for the selective interaction of synthetic agonists with the human histamine H 1 -receptor compared with the guinea pig H 1 -receptor[END_REF]. At the H 2 R, bulky agonists like the long-chained impromidine-and arpromidine-derived guanidines or N G -acylated imidazolylpropylguanidines (AIPGs), are more potent and efficacious at the gpH 2 R than at the hH 2 R [START_REF] Wenzel-Seifert | Quantitative analysis of formyl peptide receptor coupling to Gα i1 , Gα i2 , and Gα i3[END_REF][START_REF] Kelley | Distinct interaction of human and guinea pig histamine H 2 -receptor with guanidine-type agonists[END_REF]. Metiamide was identified to be an inverse agonist at the hH 2 R, gpH 2 R and rH 2 R, but a weak partial agonist at the cH 2 R [START_REF] Preuss | Constitutive activity and ligand selectivity of human, guinea pig, rat, and canine histamine H 2 receptors[END_REF]. At the H 4 R, which has the lowest sequence similarity between species, studies focusing on ligand-receptor interactions of agonists are beginning to emerge [START_REF] Igel | Synthesis and structureactivity relationships of cyanoguanidine-type and structurally related histamine H 4 receptor agonists[END_REF][START_REF] Lim | Phenylalanine 169 in the second extracellular loop of the human histamine H 4 receptor is responsible for the difference in agonist binding between human and mouse H 4 receptors[END_REF]. However, the species-differences of imoproxifan at hH 3 R and rH 3 R described in this study represent the most substantial differences in pharmacology among H X Rs identified so far. This is particularly compelling in view of the fact that hH 3 R and rH 3 R sequences display a high degree of homology and only two amino acid residues cause the disparities.

In conclusion, we have shown that hH 3 R and rH 3 R expressed in Sf9 cells both similarly couple to defined G i /G o -protein heterotrimers and display similar constitutive activities. We show species-differences in pharmacological properties of imoproxifan and offer an explanation on the molecular basis for these differences. Most importantly, we to the Ballesteros/Weinstein nomenclature. The most conserved residue in each TM is numbered as X.50, where X is the number of the respective TM domain [START_REF] Ballesteros | Integrated methods for the construction of three dimensional models and computational probing of structure-funtion relations in G-protein coupled receptors[END_REF]. Amino acids shown in white with black shading represent a putative glycosylation site of the H 3 R. Amino acids in frame represent putative interaction sites of HA with the H 3 R [START_REF] Uveges | The role of transmembrane helix 5 in agonist binding to the human histamine H 3 receptor[END_REF][START_REF] Yao | Molecular modeling and pharmacological analysis of species-related histamine H 3 receptor heterogeneity[END_REF]. were used as standard to assess the expression levels of the rH 3 R in different membrane preparations with anti-FLAG Ig. In E, the same membranes were reacted with anti-His6 Ig. In A c c e p t e d M a n u s c r i p t F, the membranes were reacted with anti-Gα i-common Ig. In G, the membranes were reacted with anti-G β-common Ig. In H, 0.5, 1.0, 1.5 and 2.0 µg of a membrane expressing the rH 3 R + Gα i2 + β 1 γ 2 was analyzed in order to quantify the Gα-subunits, using 2, 4, 7.5, 15 and 30 pmol of purified Gα i2 as standard. In I, 0.5, 1.0, 1.5 and 2.0 µg of a corresponding membrane of the same batch expressing rH 3 R + Gα o1 + β 1 γ 2 was analyzed to quantify the Gα-subunits, using 2, 4, 7.5, 15 and 30 pmol of purified Gα o2 an almost identical splice variant of Gα o1 as standard. A, electrostatic potential surface in the binding pocket of active hH 3 R with imoproxifan in its binding conformation. B, electrostatic potential surface in the binding pocket of the inactive rH 3 R with imoproxifan in its binding conformation. A and B, yellow dotted circle: the electrostatic potential is rather positive at hH 3 R due to the NH moiety of Trp 6.48 , but slightly negatively charged in rH 3 R due to the OH moiety of Thr 6.52 . The consequence is a different orientation of the ligands oxime moiety. C, conformation of amino acids in the imoproxifan bound state of active hH 3 R. D, conformation of amino acids in the imoproxifan bound state of inactive rH 3 R. C and D, yellow dotted circle: important differences in side chain conformation of Glu 5.46 between hH 3 R and rH 3 R. At hH 3 R, Glu 5.46 interacts with Thr 3.37 and points away from the binding pocket. In rH 3 R, Thr 3.37 is exchanged to Ala 3.37 . Thus, the interaction between Glu 5.46 and the amino acid in position 3.37 is no longer possible and Glu 5.46 interacts with Tyr 3.33 . Green dotted circle: in position 3.40, there is a small Ala in hH 3 R, but the more bulky Val at rH 3 R. It is suggested that this species difference is also be important for the different orientations of the oxime moiety between hH 3 R and rH 3 R. E, interaction between imoproxifan and hH 3 R; F, interaction between imoproxifan and rH 3 R. E and F, yellow dotted circle: at hH 3 R, the oxime moiety of imoproxifan points downwards and stabilizes the highly conserved Trp 6.48 in its active conformation by a hydrogen bond; at rH 3 R, the oxime moiety of imoproxifan points upwards to TM V and interacts with Thr The quantification of receptors and G proteins via immunoblot was performed as described [START_REF] Schnell | No evidence for functional selectivity of proxifan at the human histamine H 3 -receptor coupled to defined G i /G o protein heterotrimers[END_REF].

[³H]JNJ-7753707 saturation bindings were performed as described under Methods.

A c c e p t e d M a n u s c r i p t 4 spliced H 3

 43 Rs (445 amino acids), especially regarding the detailed molecular mechanisms involved in ligand-receptor interactions.

  [ 3 H]JNJ-7753707 (= [ 3 H]RWJ-422475) (30 Ci/mmol) was kindly donated from Dr. P. Bonaventure (Johnson & Johnson Pharmaceutical R&D, San Diego, CA, USA). [ 3 H]N α -methylhistamine (74-85 Ci/mmol) and [ 35 S]GTPS (1100 Ci/mmol) were obtained from Perkin Elmer (Boston, MA, USA).

  -tagged cDNAs for hH 3 R and rH 3 R. The cDNA for the tagged rH 3 R protein was generated by sequential overlap-extension PCR in analogy to the procedure described recently for hH 3 R [23]. In the case of rH 3 R, the sense primer RAT HRH3-F-(5´-GAC GAT GAT GAC GCC ATG GAG CGC GCG CCG CC-3´) consisted of 15 bp of the 3'-end of SF and the first 17 bp of the 5'-end of the rH 3 R. The antisense primer RAT HRH3-RV (5´-GA TCC TCT AGA TTA GTG ATG GTG ATG ATG GTG CTT CCA GCA CTG CTC -3´) consisted of 15 bp of the C-terminus of the rH 3 R, and encoded a hexahistidine tag, the stop codon, and a Xba I site. As template, a plasmid (pCIneo) containing the sequence of rH 3 R was used.

A c c e p t e d M a n u s c r i p t 6 2. 3 .

 63 Generation of recombinant baculoviruses, cell culture and membrane preparation,

  mM MgCl 2 , 1 mM EDTA, and 75 mM Tris-HCl, pH 7.4), supplemented with 0.05% (m/v) BSA. Each tube (total volume of 250 or 500 μl) contained 10 -20 μg of membrane protein. Tubes contained 0.2 -2 nM [ 35 S]GTPγS plus unlabeled GTPγS to give the desired final ligand concentrations (0.2 -50 nM). Neither GDP nor H 3 R ligands were included in assays. Non-specific binding was determined in the presence of 100 μM unlabeled GTPγS and amounted to less than 1% of total binding. Incubations were conducted for 90 minutes at 25°C and shaking at 250 rpm. Bound [ 35 S]GTPγS was separated from free [

A c c e p t e d M a n u s c r i p t 7 2. 5 .

 75 Steady-state GTPase activity assay.

  [ 3 H]NAMH binding assays, each tube (total volume, 250 or 500 µl) contained 10 to 50 µg of protein.

A c c e p t e d M a n u s c r i p t 8

 8 Non-specific binding was determined in the presence of [3 H]NAMH at various concentrations plus 10 µM THIO and amounted to ~10% of total binding at saturating concentrations (10 nM). Incubations were conducted for 60 min at RT and shaking at 250 rpm. Saturation binding experiments were carried out using 0.3 to 10 nM [ 3 H]NAMH in the presence or absence of 10 µM GTPγS. In competition binding experiments, tubes contained 1 nM [ 3 H]NAMH and unlabeled ligands at various concentrations. Bound [ 3 H]NAMH was separated from free [ 3 H]NAMH by filtration through GF/C filters pretreated with 0.3% (m/v) polyethyleneimine, followed by three washes with 2 ml of binding buffer (4°C). [ 3 H]JNJ-7753707 (= [ 3 H]RWJ-422475) binding experiments were performed using the same procedure as described above for [ 3 H]NAMH. With [ 3 H]JNJ-7753707 as radioligand, nonspecific binding was about 20-30% of total binding at saturating concentrations (10 nM).

  [ 3 H]NAMH was only slightly GTPγS-sensitive in both cases. The K d values of [ 3 H]NAMH in the presence of GTPγS (10 µM) were about 2-fold lower, but the B max values did not change significantly (Fig. 5 C and D).

  A c c e p t e d M a n u s c r i p t 13 In competition binding experiments (Fig. 4 C), histamine (1) (Fig. 5 E and F), N methylhistamine (

A

  c c e p t e d M a n u s c r i p t 14 of imoproxifan. In hH 3 R, the methyl moiety points into direction of TM V, whereas in rH 3 R, the methyl moiety points downward.

[ 3 H

 3 ]NAMH as radioligand, and steady-state [γ-32 P]GTP hydrolysis. The similarly small shifts of the [ 3 H]NAMH saturation binding curves at hH 3 R and rH 3 R by GTPγS indicate a similarly strong interaction of both receptors with the G protein and are in line with the high constitutive activity of the H 3 Rs. Thus, the results confirm the data of the GTPase experiments.

AFig. 1 .

 1 FIGURE CAPTIONS

Fig. 2 .Fig. 3 .

 23 Fig. 2. Structures of imidazole-containing H 3 R-ligands: full agonists 1-3, partial agonists 4-6, imoproxifan 7, and antagonists/inverse agonists 8-10.

Fig. 4 .

 4 Fig. 4. Comparison of the pharmacological properties of hH 3 R and rH 3 R. A, Comparison of the potencies of ligands in the GTPase assay. Data for hH 3 R were taken from Ref. 23, data for rH 3 R were taken from Table 3. B, Comparison of the efficacies of ligands in the GTPase assay. Data for hH 3 R were taken from Ref. 23, data for rH 3 R were taken from Table 3. C, Comparison of the affinities of ligands in the [ 3 H]NAMH competition binding assay. Data

Fig. 5 .

 5 Fig. 5. Comparison of the effects of histamine, imoproxifan and thioperamide in

Fig. 6 .

 6 Fig. 6. Correlation of potency and efficacy of ligands at the rH 3 R in the presence of different co-expressed Gα i/o -proteins. Data shown in Table 3 were analyzed by linear regression. Numbers designate individual ligands decoded in Fig. 2. In A, C and E, the

Fig. 7 .

 7 Fig. 7. Correlation of affinity and potency of ligands at the hH 3 R and rH 3 R. Data shown were analyzed by linear regression. Numbers designate individual ligands decoded in Fig. 2.In A, the affinities and potencies of ligands at membranes co-expressing the hH 3 R, Gα i2 and β 1 γ 2 dimers were correlated. A, r 2 = 0.83; slope = 0.94 ± 0.15. In B, the affinities and potencies of ligands at membranes co-expressing the rH 3 R, Gα i2 and β 1 γ 2 dimers were correlated. B, r 2 = 0.50; slope = 0.83 ± 0.29. The dotted lines indicate the 95% confidence

Fig. 8 .

 8 Fig. 8. Binding mode of imoproxifan at the active hH 3 R and inactive rH 3 R.

[ 35 S

 35 ]GTPγS saturation bindings were performed, using Sf9 cell membranes from the same batch of preparation. Reaction mixtures contained membranes (10 -20 µg of protein), 0.2 -2 nM of [ 35 S]GTPγS, and unlabeled GTPγS to give the desired final ligand concentrations for saturation (0.2 -50 nM). GDP or additional H 3 R ligands were not present in the reaction mixtures. Data were analyzed by nonlinear regression and were best fitted to hyperbolic onesite saturation isotherms. The maximal number of GTPγS binding sites in membranes expressing rH 3 R plus Gα i2 plus β 1 γ 2 was corrected by the binding determined in rH 3 R plus β 1 γ 2 . By this way, the number of functionally intact and heterologously expressed G protein subunits was quantified. Data shown are the means ± S. E. M. of 3 independent experiments performed in triplicate. Receptor-to-G protein ratios were calculated, using the B max values determined for the different membrane preparations.

A c c e p t e d M a n u s c r i p t 30

 30

A c c e p t e d M a n u s c r i p t

A c c e p t e d M a n u s c r i p t 32 Steady

 32 -state GTPase activity in Sf9 membranes expressing rH 3 R, different Gα i/o subunits and β 1 γ 2 was determined as described under Materials and Methods. Reaction mixtures contained ligands at concentrations from 0.1 nM to 10 µM as appropriate to generate saturated concentration/response curves. Data were analyzed by nonlinear regression and were best fit to sigmoid concentration/response curves. Typical basal GTPase activities ranged between 1.0 and 4.0 pmol * mg -1 * min -1 , and the maximal stimulatory effect of histamine (10 µM) amounted to ~40 to ~90% above basal. The efficacy (E max ) of histamine was determined by nonlinear regression and was set to 1.00. The E max values of other agonists and inverse agonists were referred to this value. Data shown are the means ± S. E. M. of three to four experiments performed in duplicates each. Statistical analysis was performed using one-way ANOVA, followed by Dunnet's multiple comparison test using the values determined at hH 3 R, Gα i2 and β 1 γ 2 as a reference. Significant differences to the membrane expressing Gα i2 are shown following comparison with other Gα i/o subunits. (no symbol: not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001). Additionally, data shown were correlated and analyzed by linear regression. The potencies and efficacies of ligands at membranes co-expressing the rH 3 R, Gα i1 , Gα i3 or Gα o1 , and β 1 γ 2 dimers, respectively, were correlated with values determined at the reference membrane expressing Gα i2 . The correlation coefficients (r 2 ) and the slopes are presented at the bottom of the table.

of 0. 1

 1 nM to 10 µM as appropriate to generate saturated competition curves. Data were analyzed by nonlinear regression and were best fit to one-site (monophasic) competition curves. Data shown are the means ± S. E. M. of three to five independent experiments performed in duplicate at 3 different membrane preparations. Additionally, data shown were correlated and analyzed by linear regression. The affinities and potencies of ligands at membranes co-expressing the hH 3 R or rH 3 R plus Gα i2 plus β 1 γ 2 dimers, respectively, were correlated. The correlation coefficients (r 2 ) and the slopes of all tested ligands are presented at the bottom of the table.

  Fig.

1

 1

Figure 1 Page 35 of 42 A c c e p t e d M a n u s c r i p t

 142 Figure 1

Figure 2 A

 2 Figure 2

Figure 3 A

 3 Figure 3

Figure 4 Page 38 of 42 A c c e p t e d M a n u s c r i p t Fig. 5 hH 3 R

 44253 Figure 4

Figure 6 Page

 6 Figure 6

Figure 7

 7 Figure 7

  35

Table 1 : Quantification of rH 3 R-to-G protein ratios via western blot, [³H]JNJ-7753707- and [ 35 S]GTPγS-saturation binding.

 1 6.52 .

	TABLES
			B max ± S. E. M. (pmol × mg -1 )
	membrane immunoblot: anti-FLAG Ig anti-Gα Igs [³H]JNJ-7753707 [ 35 S]GTPγS R : G ratio	+ Gα i1 ~1.5 -2.5 n. d. 0.67 ± 0.03 3.40 ± 0.80 ~1 : 5	rH 3 R + β 1 γ 2 ~1.5 -2.5 + Gα i2 ~1.5 -2.5 + Gα i3 ~50 -100 n. d. 0.77 ± 0.02 1.05 ± 0.04 4.43 ± 0.53 2.52 ± 0.37 a n u s c r i p t + Gα o1 ~1.5 -2.5 ~50 -100 1.25 ± 0.04 8.19 ± 1.27 M ~1 : 6 ~1 : 2 ~1 : 7
	A c c e p t e d
			29

Table 2 : Analysis of rH 3 R/G protein coupling -GTPase activities in Sf9 membranes expressing rH 3 R and different Gα i/o -proteins.

 2

	GTPase activity		rH 3 R + β 1 γ 2
	± S. E. M.	+ Gα i1	+ Gα i2	+ Gα i3	+ Gα o1
	basal (pmol × mg -1 × min -1 )	1.22 ± 0.21	1.75 ± 0.22	1.12 ± 0.04	4.29 ± 0.12
	+ ago. (pmol × mg -1 × min -1 )	2.01 ± 0.26	3.17 ± 0.37	2.13 ± 0.14	5.95 ± 0.14
	Δ ago. (pmol × mg -1 × min -1 )	0.79 ± 0.07	1.42 ± 0.15	1.01 ± 0.10	1.66 ± 0.09
	Agonist stimulation (% of basal)	67.44 ± 0.09 81.78 ± 2.32 89.38 ± 5.14 38.78 ± 2.38
	+ inv. ago. (pmol × mg -1 × min -1 )	0.71 ± 0.10	0.85 ± 0.12	0.59 ± 0.03	2.86 ± 0.14
	Δ inv. ago. (pmol × mg -1 × min -1 )	0.51 ± 0.13	0.90 ± 0.10	0.53 ± 0.02	1.43 ± 0.13
	Inverse agonist
	inhibition	40.32 ± 5.93 51.66 ± 0.96 47.4 ± 0.82 33.29 ± 2.88
	(% of basal)
	Steady-state GTPase experiments were performed as described in Methods. Reaction
	mixtures contained 0.1 µCi [γ-32 P]GTP and 100 nM unlabeled GTP in the presence of solvent
	(basal), 10 µM HA (+ ago.) or 10 µM THIO (+ inv. ago.). Data shown are the means ± S. E.
	M. of three to four independent experiments for each membrane preparation performed in
	duplicates. The absolute agonist-stimulation (Δ ago.) and inverse agonist-inhibition (Δ inv.
	ago.) of GTP hydrolysis, as well as the relative agonist-stimulation and inverse agonist-
	inhibition of GTP hydrolysis (% of basal), were calculated.

Table 3 : Ligand potencies and efficacies in the GTPase assay at Sf9 cell membranes expressing the rH 3 R and different G proteins. rH

 3 3 R + Gα i1 + β 1 γ 2 rH 3 R + Gα i2 + β 1 γ 2 rH 3 R + Gα i3 + β 1 γ 2 rH 3 R + Gα o1 + β 1 γ 2

		pEC 50		pEC 50		pEC 50		pEC 50
			E max ± S. E. M.		E max ± S. E. M.		E max ± S. E. M.		E max ± S. E. M.
		± S. E. M.		± S. E. M.		± S. E. M.		± S. E. M.
	HA	7.64 ± 0.07*	1.00	7.94 ± 0.05	1.00	7.88 ± 0.06	1.00	7.72 ± 0.05	1.00
	NAMH	8.30 ± 0.23	0.91 ± 0.10	8.98 ± 0.12	1.06 ± 0.07	9.11 ± 0.30	1.16 ± 0.21	8.88 ± 0.18	1.10 ± 0.11
	RAMH	8.06 ± 0.17	0.88 ± 7.14	8.54 ± 0.10	0.91 ± 0.05	8.50 ± 0.08	0.90 ± 0.03	8.21 ± 0.10	0.84 ± 0.04
	IME	9.86 ± 0.19	0.95 ± 0.10	9.76 ± 0.13	0.89 ± 0.06	10.14 ± 0.36	1.39 ± 0.30	9.78 ± 0.12	0.99 ± 0.07
	PRO	8.12 ± 0.22	0.48 ± 0.05	8.52 ± 0.12	0.66 ± 0.04	8.42 ± 0.10	0.64 ± 0.04	8.19 ± 0.16	0.66 ± 0.05
	IMP	8.33 ± 0.15	0.72 ± 0.05	8.94 ± 0.20	0.91 ± 0.10	8.99 ± 0.26	0.95 ± 0.14	8.76 ± 0.12	0.84 ± 0.06
	IMO	9.03 ± 0.26	-0.61 ± 0.08	9.03 ± 0.13	-0.61 ± 0.04	9.05 ± 0.23	-0.46 ± 0.05	8.93 ± 0.16	-0.96 ± 0.08**
	CIP	8.71 ± 0.27	-0.81 ± 0.10	8.64 ± 0.10	-0.75 ± 0.03	8.64 ± 0.15	-0.61 ± 0.04	8.58 ± 0.16	-1.01 ± 0.07
	CLOB	9.04 ± 0.26	-0.43 ± 0.05	8.95 ± 0.16	-0.44 ± 0.03	8.77 ± 0.13	-0.41 ± 0.02	8.68 ± 0.11	-0.69 ± 0.03**
	THIO	7.82 ± 0.24	-0.66 ± 0.07	7.66 ± 0.12	-0.62 ± 0.03	7.61 ± 0.13	-0.50 ± 0.03	7.64 ± 0.08	-0.99 ± 0.03***
	r 2	0.77	0.99	1.00	1.00	0.97	0.96	0.96	0.996
	slope	0.99 ± 0.19	0.97 ± 0.04	1.00	1.00	1.17 ± 0.07	0.99 ± 0.07	1.05 ± 0.07	1.20 ± 0.03

Table 4 : [³H]NAMH competition bindings in Sf9 membranes expressing hH 3 R or rH 3 R in combination with Gα i2 and β 1 γ 2 .

 4 Experiments were performed as described under Methods. Reaction mixtures contained Sf9 membranes (10 -50 µg of protein), 1 nM [³H]NAMH, and unlabeled ligands at concentrations

		pK i ± S. E. M.
		hH 3 R + Gα i2 + β 1 γ 2	rH 3 R + Gα i2 + β 1 γ 2
	HA	8.20 ± 0.04	7.89 ± 0.07
	NAMH	9.22 ± 0.03	8.70 ± 0.09
	RAMH	8.91 ± 0.07	8.62 ± 0.07
	IME	9.20 ± 0.05	9.38 ± 0.08
	PRO	7.87 ± 0.07	8.08 ± 0.10
	IMP	8.84 ± 0.06	10.11 ± 0.05
	IMO	6.92 ± 0.06	8.47 ± 0.09
	CIP	7.03 ± 0.12	8.87 ± 0.08
	CLOB	9.34 ± 0.06	9.11 ± 0.06
	THIO	7.34 ± 0.04	7.94 ± 0.04
	r 2 (pK i /pEC 50 )	0.83	0.50
	slope (pK i /pEC 50 )	0.94 ± 0.15	0.83 ± 0.29

ACKNOWLEDGEMENTS

We would like to thank Mrs. G. Wilberg and Mrs. A. Seefeld for their excellent technical assistance. Thanks are also due to the reviewers for their helpful suggestions. This work was supported by the Research Training Program (Graduiertenkolleg) [GRK760] "Medicinal Chemistry: Molecular Recognition -Ligand-Receptor Interactions" of the German Research Foundation (DFG). The authors are affiliated with COST Action [BM0806].

This paper documents substantial pharmacological differences between human and rat histamine H 3 -receptor. Most strikingly, in human H 3 -receptor, imoproxifan stabilizes an active conformation. In rat H 3 -receptor, imoproxifan stabilizes an inactive conformation.

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*Graphical Abstract