Generation of a selective 5-HT3B subunit-recognising polyclonal antibody; identification of immunoreactive cells in rat hippocampus.

The present study generated a polyclonal antibody (AP86/3) that recognises a peptide sequence of the h5-HT(3B) receptor subunit. Western blot analysis of homogenates prepared from cell lines expressing either homomeric (h5-HT(3A)) or heteromeric (h5-HT(3A/3B)) receptors, as well as immunocytochemical studies with the same cell lines, indicated that AP86/3 recognised, selectively, the 5-HT(3B) subunit. Immunohistochemical labelling was also apparent in cells in the rat hippocampus that displayed the distribution and morphology of interneurones.

Pharmacological comparison of human homomeric 5-HT3A receptors versus heteromeric 5-HT3A/3B receptors.

The present study determined the detailed pharmacological profile of heterologously expressed human (h) homomeric 5-HT3A receptors in direct comparison to heteromeric h5-HT3A/3B receptors. The very minor differences in their respective pharmacological profiles indicates that the 5-HT3B receptor subunit alters, predominantly, the biophysical rather than the pharmacological properties of the 5-HT3 receptor.

Molecular determinants of (+)-tubocurarine binding at recombinant 5-hydroxytryptamine3A receptor subunits.

The 5-hydroxytryptamine type 3 (5-HT3) receptor is a transmitter-gated ion channel mediating neuronal excitation. The receptor native to neurons, or as a homopentameric assembly of 5-HT3A receptor subunits, displays a species-dependent pharmacology exemplified by a 1800-fold difference in the potency of (+)-tubocurarine [(+)-Tc] as an antagonist of the current response mediated by mouse and human receptor orthologs. Here, we attempt to identify amino acid residues involved in binding (+)-Tc by use of chimeric and mutant 5-HT3A subunits of mouse and human expressed in Xenopus laevis oocytes. Replacement of the entire extracellular N-terminal domain of the mouse 5-HT3A (m5-HT3A) subunit by that of the human ortholog and vice versa exchanged the differential potency of (+)-Tc, demonstrating the ligand binding site to be contained wholly within this region. Mutagenesis of multiple amino acid residues within a putative binding domain that exchanged nonconserved residues between mouse and human receptors shifted the apparent affinity of (+)-Tc in a reciprocal manner. The magnitude of the shift increased with the number of residues (3, 5, or 7) exchanged, with septuple mutations of m5-HT3A and human 5-HT3A subunits producing a 161-fold decrease and 53-fold increase in the apparent affinity of (+)-Tc, respectively. The effect of point mutations was generally modest, the exception being m5-HT3A D206E, which produced a 9-fold decrease in apparent affinity. We conclude that multiple amino acids within a binding loop of human and mouse 5-HT3A subunits influence the potency of (+)-Tc.

The effect of a transmembrane amino acid on etomidate sensitivity of an invertebrate GABA receptor.

1. The gamma-aminobutyric acid (GABA)-modulatory and GABA-mimetic actions of etomidate at mammalian GABA(A) receptors are favoured by beta2- or beta3- versus beta1-subunit containing receptors, a selectivity which resides with a single transmembrane amino acid (beta2 N290, beta3 N289, beta1 S290). Here, we have utilized the Xenopus laevis oocyte expression system in conjunction with the two-point voltage clamp technique to determine the influence of the equivalent amino acid (M314) on the actions of this anaesthetic at an etomidate-insensitive invertebrate GABA receptor (Rdl) of Drosophila melanogaster. 2. Complementary RNA-injected oocytes expressing the wild type Rdl GABA receptor and voltage-clamped at -60 mV responded to bath applied GABA with a concentration-dependent inward current response and a calculated EC50 for GABA of 20+/-0.4 microM. Receptors in which the transmembrane methionine residue (M314) had been exchanged for an asparagine (RdlM314N) or a serine (RdlM314S) also exhibited a concentration-dependent inward current response to GABA, but in both cases with a reduced EC50 of 4.8+/-0.2 microM. 3. Utilizing the appropriate GABA EC10, etomidate (300 microM) had little effect on the agonist-evoked current of the wild type Rdl receptor. By contrast, at RdlM314N receptors, etomidate produced a clear concentration-dependent enhancement of GABA-evoked currents with a calculated EC50 of 64+/-3 microM and an Emax of 68+/-2% (of the maximum response to GABA). 4. The actions of etomidate at RdlM314N receptors exhibited an enantioselectivity common to that found for mammalian receptors, with 100 microM R-(+)-etomidate and S-(-)-etomidate enhancing the current induced by GABA (EC10) to 52+/-6% and 12+/-1% of the GABA maximum respectively. 5. The effects of this mutation were selective for etomidate as the GABA-modulatory actions of 1 mM pentobarbitone at wild type Rdl (49+/-4% of the GABA maximum) and RdlM314N receptors (53+/-2% of the GABA maximum) were similar. Additionally, the modest potentiation of GABA produced by the anaesthetic neurosteroid 5alpha-pregnan-3alpha-ol-20-one (Rdl = 25+/-4% of the GABA maximum) was not altered by this mutation (RdlM314N = 18+/-3% of the GABA maximum). 6. Etomidate acting at beta1 (S290)-containing mammalian GABA(A) receptors is known to produce only a modest GABA-modulatory effect. Similarly, etomidate acting at RdlM314S receptors produced an enhancement of GABA but the magnitude of the effect was reduced compared to RdlM314N receptors. 7. Etomidate acting at human alpha6beta3gamma2L receptors is known to produce a large enhancement of GABA-evoked currents and at higher concentrations this anaesthetic directly activates the GABA(A) receptor complex. Mutation of the human beta3 subunit asparagine to methionine (beta3 N289M found in the equivalent position in Rdl completely inhibited both the GABA-modulatory and GABA-mimetic action of etomidate (10-300 microM) acting at alpha6beta3 N289Mgamma2L receptors. 8. It was concluded that, although invertebrate and mammalian proteins exhibit limited sequence homology, allosteric modification of their function by etomidate can be influenced in a complementary manner by a single amino acid substitution. The results are discussed in relation to whether this amino acid contributes to the anaesthetic binding site, or is essential for transduction. Furthermore, this study provides a clear example of the specificity of anaesthetic action.

Ion permeation and conduction in a human recombinant 5-HT3 receptor subunit (h5-HT3A).

1. A human recombinant homo-oligomeric 5-HT3 receptor (h5-HT3A) expressed in a human embryonic kidney cell line (HEK 293) was characterized using the whole-cell recording configuration of the patch clamp technique. 2. 5-HT evoked transient inward currents (EC50 = 3.4 microM; Hill coefficient = 1.8) that were blocked by the 5-HT3 receptor antagonist ondansetron (IC50 = 103 pM) and by the non-selective agents metoclopramide (IC50 = 69 nM), cocaine (IC50 = 459 nM) and (+)-tubocurarine (IC50 = 2.8 microM). 3. 5-HT-induced currents rectified inwardly and reversed in sign (E5-HT) at a potential of -2.2 mV. N-Methyl-D-glucamine was finitely permeant. Permeability ratios PNa/PCs and PNMDG/PCs were 0.90 and 0.083, respectively. 4. Permeability towards divalent cations was assessed from measurements of E5-HT in media where Ca2+ and Mg2+ replaced Na+. PCa/PCs and PMg/PCs were calculated to be 1.00 and 0.61, respectively. 5. Single channel chord conductance (gamma) estimated from fluctuation analysis of macroscopic currents increased with membrane hyperpolarization from 243 fS at -40 mV to 742 fS at -100 mV. 6. Reducing [Ca2+]o from 2 to 0.1 mM caused an increase in the whole-cell current evoked by 5-HT. A concomitant reduction in [Mg2+]o produced further potentiation. Fluctuation analysis indicates that a voltage-independent augmentation of gamma contributes to this phenomenon. 7. The data indicate that homo-oligomeric receptors composed of h5-HT3A subunits form inwardly rectifying cation-selective ion channels of low conductance that are permeable to Ca2+ and Mg2+.

Characterization of a human 5-hydroxytryptamine3 receptor type A (h5-HT3R-AS) subunit stably expressed in HEK 293 cells.

1. A cloned cDNA encoding a human 5-hydroxytryptamine3 receptor type A subunit (h5-HT3R-As) was transfected into human embryonic kidney (HEK 293) cells maintained in cell culture and a stable cell line expressing a high density of the recombinant receptor was selected. 2. Membrane homogenates prepared from transfected, but not untransfected, cells exhibited a homogeneous and saturable population (Bmax = 4.49 +/- 0.46 pmol mg-1 protein) of sites that bound the radiolabelled 5-HT3 receptor antagonist, [3H]-granisetron with high affinity (pKD = 8.87 +/- 0.08). Kinetic studies (at 37 degrees C) revealed rapid association (kappa +1 4.76 +/- 0.3 x 10(8) M-1 min-1) and dissociation (kappa -1 = 0.21 +/- 0.003 min-1) of the radioligand. 3. Selective and non-selective 5-HT3 receptor ligands competed for [3H]-granisetron binding with a rank order of potency (granisetron > ondansetron > meta-chlorophenylbiguanide > 5-HT > 2-methyl-5-HT > metoclopramide > > phenylbiguanide > cocaine > (+)-tubocurarine) identical to that established for 5-HT3 receptors endogenous to the human CNS. 4. In electrophysiological recordings performed on transfected cells, voltage-clamped at a holding potential of -60 mV, locally applied 5-HT (10 microM) evoked transient inward current responses that reversed in sign at a potential of -1.0 +/- 1.1 mV. Such responses were antagonized in a reversible manner by granisetron (1 nM). 5. The construction of a stable cell line expressing a high density of recombinant human 5-HT3 receptors which display appropriate pharmacology and function will assist in the further characterization of this receptor subtype and the exploration of species differences in 5-HT3 receptor pharmacology.

Cloning and functional expression of a human 5-hydroxytryptamine type 3AS receptor subunit.

A human 5-hydroxytryptamine receptor type 3AS (5-HT3R-AS) subunit has been cloned from an amygdala cDNA library. We report the nucleotide and predicted amino acid sequence of the human subunit, which possesses 85% and 84% amino acid sequence identity with mouse and rat 5-HT3R-AS subunits, respectively. Acting on Xenopus laevis oocytes injected with RNA transcripts of the clone, 5-HT and selective 5-HT3 receptor agonists elicited inwardly directed current responses that displayed desensitization. Such currents were blocked in a concentration-dependent manner by selective and nonselective 5-HT3 receptor antagonists but were unaffected by compounds acting at G protein-linked 5-HT receptors. A quantitative comparison of the pharmacological profiles of human and mouse recombinant 5-HT3R-AS receptor complexes revealed differences in the potencies of some antagonist or agonist compounds tested, the most dramatic example being (+)-tubocurarine, which demonstrated an approximately 1800-fold discrepancy in antagonist potency. In view of the small number of sequence substitutions that occur between the human and mouse homologues of the 5-HT3R-AS in the extracellularly located aminoterminal domain, compounds such as (+)-tubocurarine, in conjunction with site-directed mutagenesis, may prove to be valuable in locating amino acid residues that contribute to the ligand binding site(s) of the 5-HT3 receptor. Also, when methodological differences are taken into account, the present study suggests that a homo-oligomeric assembly of human 5-HT3R-AS subunits can account for the distinctive ligand binding properties of human 5-HT3 receptors established in postmortem brain tissue.

Evidence that the atypical 5-HT3 receptor ligand, [3H]-BRL46470, labels additional 5-HT3 binding sites compared to [3H]-granisetron.

1. The radioligand binding characteristics of the 3H-derivative of the novel 5-HT3 receptor antagonist BRL46470 were investigated and directly compared to the well characterized 5-HT3 receptor radioligand [3H]-granisetron, in tissue homogenates prepared from rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen. 2. In rat cerebral cortex/hippocampus, rat ileum, NG108-15 cell and HEK-5-HT3As cell homogenates, [3H]-BRL46470 bound with high affinity (Kd (nM): 1.57 +/- 0.18, 2.49 +/- 0.30, 1.84 +/- 0.27, 3.46 +/- 0.36, respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 102 +/- 16, 44 +/- 4, 968 +/- 32 and 2055 +/- 105, respectively; mean +/- s.e. mean, n = 3-4) but failed to display specific binding in human putamen homogenates. 3. In the same homogenates of rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen as used for the [3H]-BRL46470 studies, [3H]-granisetron also bound with high affinity (Kd (nM): 1.55 +/- 0.61, 2.31 +/- 0.44, 1.89 +/- 0.36, 2.03 +/- 0.42 and 6.46 +/- 2.58 respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 39 +/- 4, 20 +/- 2, 521 +/- 47, 870 +/- 69 and 18 +/- 2, respectively; mean +/- s.e. mean, n = 3-4). 4. Competition studies with a range of structurally different 5-HT3 receptor ligands indicated that in both rat cerebral cortex/hippocampus and rat ileum homogenates, [3H]-BRL46470 binding exhibited a pharmacological profile consistent with the labelling the 5-HT3 receptor with compounds competing with Hill coefficients close to unity.5 In HEK-5-HT3As cell homogenates, [3H]-BRL46470 and [3H]-granisetron associated rapidly((3.84+/-0.4)106 M-1S-1 and (5.85+/-0.2)106 M-1S-1, respectively, mean+/-s.e.mean, n=3-4) in an apparently monophasic manner. Following the establishment of equilibrium, both [3H]-BRL46470 and [3H]-granisetron at a saturating concentration ([3H]-BRL46470 approximately 16 nM; [3H]-granisetron approximately 18 nM) and at a sub-Kd concentration (approximately 1 nm for both radioligands)dissociated biphasically in HEK-5-HT3As cell homogenates (saturating concentration; [3H]-BRL464704.05 x 10-3+/-2.53 x I0-3 s-1 and 5.83 x 10-5+0.91 x I0-5 s-1; [3H]-granisetron 3.20 x 10-3+ 1.70 x IO-3 s-1 and18.58 x 10-5 +/- 4.19 x I0-5 s-1: sub-Kd concentration; [3H]-BRL46470 2.47 x 10-3+/- 1.18 x 10-3 s-1 and 9.30x 10-5+/-2.59x 10-5 S-1; [3H]-granisetron 65.91 x 10-3+/-22.14x I0-3 s-1 and 49.96x 10-5+/-12.26x 10-5s- 1 mean+/- s.e.mean, n = 4-8) when induced by a 300 fold dilution in ice-cold Tris/Krebs.6 In conclusion, the present study provides evidence that [3H]-BRL46470 specifically labels the 5-HT3receptor in rat cerebral cortex/hippocampus, rat ileum, NG108-15 cell and HEK-5-HT3As cell homogenates, but fails to label the 5-HT3 receptor expressed in human putamen. Whilst the pharmacological profile of the site labelled by [3H]-BRL46470 is directly comparable to that labelled by [3H]-granisetron, [3H]-BRL46470 consistently labelled approximately twice the density of sites compared to [3H]-granisetron in the same tissue homogenates prepared from rat cortex/hippocampus, ratileum, NG108-15 cells and HEK-5-HT3As cells.

The interaction of trichloroethanol with murine recombinant 5-HT3 receptors.

1. The effects of ethanol, chloral hydrate and trichloroethanol upon the 5-HT3 receptor have been investigated by use of electrophysiological techniques applied to recombinant 5-HT3 receptor subunits (5-HT3R-A or 5-HT3R-As) expressed in Xenopus laevis oocytes. Additionally, the influence of trichloroethanol upon the specific binding of [3H]-granisetron to membrane preparations of HEK 293 cells stably transfected with the murine 5-HT3R-As subunit and 5-HT3 receptors endogenous to NG 108-15 cell membranes was assessed. 2. Ethanol (30-300 mM), chloral hydrate (1-30 mM) and trichloroethanol (0.3-10 mM), produced a reversible, concentration-dependent, enhancement of 5-HT-mediated currents recorded from oocytes expressing either the 5-HT3R-A, or the 5-HT3R-As subunit. 3. Trichloroethanol (5 mM) produced a parallel leftward shift of the 5-HT concentration-response curve, reducing the EC50 for 5-HT from 1 +/- 0.04 microM (n = 4) to 0.5 +/- 0.01 microM (n = 4) for oocytes expressing the 5-HT3R-A. A similar shift, from 2.1 +/- 0.05 microM (n = 11) to 1.3 +/- 0.1 microM (n = 4), was observed in oocytes expressing the 5-HT3R-As subunit. Trichloroethanol (5 mM) had little or no effect upon the maximum current produced by 5-HT for either recombinant receptor. 4. Trichloroethanol (5 mM) similarly reduced the EC50 for 2-methyl-5-HT from 13 +/- 0.4 microM (n = 4) to 4.6 +/- 0.2 microM (n = 4) and from 15 +/- 2 microM (n = 4) to 5 +/- 0.4 microM (n = 4) for oocytes expressing the 5-HT3R-A and 5-HT3R-As subunit respectively. Additionally, trichloroethanol (5 mM) produced a clear enhancement of the maximal current to 2-methyl-5-HT (expressed as a percentage of the maximal current to 5-HT) from 63 +/- 0.7% (n = 4) to 101 +/- 1.6% (n = 4) and from 9 +/- 0.2% (n = 4) to 74 +/- 2% (n = 4) for oocytes expressing the 5-HT3R-A and 5-HT3R-As subunit respectively. 5. Trichloroethanol (2.5 mM) had no effect upon the Kd, or Bmax, of specific [3H]-granisetron binding to membrane homogenates of NG 108-15 cells or HEK 293 cells. Similarly, competition for [3H]-granisetron binding by the 5-HT3 receptor antagonists ondansetron and tropisetron was unaffected. However, competition for [3H]-granisetron binding by the 5-HT3 receptor agonists, 5-HT, 2-methyl-5-HT and phenylbiguanide was enhanced by trichloroethanol (2.5 mM). 6 Unexpectedly, the competition for [3H]-granisetron binding by the 5-HT3 receptor antagonist,quipazine, was enhanced by 2.5 mM trichloroethanol. Quipazine (1 nM-0.3 microM) antagonized 5-HT evoked currents recorded from oocytes expressing the 5-HT3R-A subunit with an IC50 of 18 +/- 3 nM(n = 4). Additionally, quipazine (30 nM-0.3 microM) produced a small inward current which was greatly enhanced by 5 mM trichloroethanol and antagonized by 100 nM ondansetron. Collectively, these observations suggest that quipazine may act as a partial agonist.7. The demonstration that a recombinant homo-oligomeric receptor, expressed in a foreign membrane,retains a sensitivity to alcohols, together with the sequencing of alcohol-insensitive 5-HT3 receptor subunits, may lead to a better definition of the alcohol binding site(s).

Pharmacological characterization of the apparent splice variants of the murine 5-HT3 R-A subunit expressed in Xenopus laevis oocytes.

The actions of 5-hydroxytryptamine3 (5-HT3) receptor agonists and antagonists have been determined on the recombinant murine 5-HT3 R-A and an apparent splice variant of this subunit, termed 5-HT3 R-AS. When expressed in Xenopus laevis oocytes, both forms of the subunit functioned as a homo-oligomeric complex and exhibited inward current responses to bath applied 5-HT. Analysis of the 5-HT concentration-response curve obtained with either homo-oligomer gave Hill coefficients greater than two, suggesting positive co-operativity within the receptor complex. The rank order of potency of a range of 5-HT3 receptor agonists [m-chlorophenylbiguanide > 5-HT > 2-methyl-5-HT (2-Me-5-HT) > or = phenylbiguanide] was identical for both subunits. Indeed, with the exception of 2-Me-5-HT, for the agonists tested there was little difference across the subunits in either their potency, or the maximal current response that they elicited relative to 5-HT. Although 2-Me-5-HT exhibited a similar potency for both subunits, the maximal response evoked by this agonist at the 5-HT3 R-AS subunit was much reduced when compared to the 5-HT3 R-A subunit. The 5-HT-induced current mediated by either form of the subunit was inhibited by the 5-HT3 receptor selective antagonists BRL 46470, granisetron and ondansetron and the non-selective antagonists (+)-tubocurarine, metoclopramide and cocaine in a reversible and concentration-dependent manner. These antagonists did not discriminate between the subunits and their potencies were similar to those reported previously for 5-HT3 receptors native to murine neuronal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and functional expression of an apparent splice variant of the murine 5-HT3 receptor A subunit.

The polymerase chain reaction has been employed to isolate a cDNA encoding a functional 5-HT3 receptor subunit from the murine neuroblastoma cell line N1E-115. Overall, the amino acid sequence predicted from this clone demonstrates a 98% homology with the 5-HT3 receptor A subunit cloned from NCB-20 hybridoma cells. A deletion of 6 amino acid residues located within the putative large intracellular loop, which may result from alternative splicing, represents the principal difference between the two clones. Upon expression in Xenopus oocytes, the homo-oligomeric receptor displayed pharmacological properties which define it as a functional 5-HT3 receptor.