Charged Residues at the 2′ Position of Human GABAC ρ1 Receptors Invert Ion Selectivity and Influence Open State Probability
2004; Elsevier BV; Volume: 279; Issue: 52 Linguagem: Inglês
10.1074/jbc.m410625200
ISSN1083-351X
AutoresJane E. Carland, Andrew J. Moorhouse, Peter H. Barry, Graham A.R. Johnston, Mary Chebib,
Tópico(s)Ion channel regulation and function
ResumoThe ability of members of the nicotinicoid superfamily of ligand-gated ion channels to selectively conduct anions or cations is critical to their function within the central nervous system. Recent work has demonstrated that residues at the intracellular end of the second transmembrane domain, between the –3′ and 2′ positions, form the ion selectivity filter of these receptors. In this study, the proline residue at the 2′ position (Pro-2′) at the intracellular end of the second transmembrane domain of the γ-aminobutyric acid type C ρ1 subunit was mutated to glutamate (ρ1P2′E) and arginine (ρ1P2′R). Dilution potential experiments indicated that the charge selectivity of the ρ1P2′E receptor channels had been inverted, with the channels now becoming predominantly cation selective, indicating the ability of negatively charged residues at this 2′ position to control charge selectivity. The mutation was also seen to have significantly decreased agonist potency and intrinsic efficacy. In contrast, the ρ1P2′R receptor channels were anion-selective but were now found to be constitutively open with high holding currents (inhibited by low γ-aminobutyric acid doses and the competitive antagonist, 1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid alone) and increased agonist activity. Hill coefficients of both mutants were decreased, but competitive antagonist studies indicated that their binding sites were not significantly affected. The ability of members of the nicotinicoid superfamily of ligand-gated ion channels to selectively conduct anions or cations is critical to their function within the central nervous system. Recent work has demonstrated that residues at the intracellular end of the second transmembrane domain, between the –3′ and 2′ positions, form the ion selectivity filter of these receptors. In this study, the proline residue at the 2′ position (Pro-2′) at the intracellular end of the second transmembrane domain of the γ-aminobutyric acid type C ρ1 subunit was mutated to glutamate (ρ1P2′E) and arginine (ρ1P2′R). Dilution potential experiments indicated that the charge selectivity of the ρ1P2′E receptor channels had been inverted, with the channels now becoming predominantly cation selective, indicating the ability of negatively charged residues at this 2′ position to control charge selectivity. The mutation was also seen to have significantly decreased agonist potency and intrinsic efficacy. In contrast, the ρ1P2′R receptor channels were anion-selective but were now found to be constitutively open with high holding currents (inhibited by low γ-aminobutyric acid doses and the competitive antagonist, 1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid alone) and increased agonist activity. Hill coefficients of both mutants were decreased, but competitive antagonist studies indicated that their binding sites were not significantly affected. The vertebrate members of the Cys loop or nicotinicoid superfamily of ligand-gated ion-channel (N-LGIC) 1The abbreviations used are: N-LGIC, nicotinicoid superfamily of ligand-gated ion channel; nAChR, nicotinic acetylcholine receptor; GlyR, glycine receptor; GABAAR, γ-aminobutyric acid receptor subtype A; GABACR, GABA receptor subtype C; M2, second transmembrane; I4AA, imidazole-4-acetic acid; THIP, 4,5,6,7-tetrahydroisoxazole2.0+/– pyridine-3-ol; TACA, trans-4-aminocrotonic acid; CACA, cis-4-aminocrotonic acid; (+/–)-TAMP, trans-2-(+/–)-(aminomethyl)cyclopropanoic acid; +/–-CAMP, cis-2-(+/–)-(aminomethyl)cyclopropanoic acid; TPMPA, 1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid. 1The abbreviations used are: N-LGIC, nicotinicoid superfamily of ligand-gated ion channel; nAChR, nicotinic acetylcholine receptor; GlyR, glycine receptor; GABAAR, γ-aminobutyric acid receptor subtype A; GABACR, GABA receptor subtype C; M2, second transmembrane; I4AA, imidazole-4-acetic acid; THIP, 4,5,6,7-tetrahydroisoxazole2.0+/– pyridine-3-ol; TACA, trans-4-aminocrotonic acid; CACA, cis-4-aminocrotonic acid; (+/–)-TAMP, trans-2-(+/–)-(aminomethyl)cyclopropanoic acid; +/–-CAMP, cis-2-(+/–)-(aminomethyl)cyclopropanoic acid; TPMPA, 1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid. receptors consist of nicotinic acetylcholine receptor (nAChR), strychnine-sensitive glycine receptor (GlyR), serotonin type 3 receptor, the A and C subtypes of the γ-aminobutyric acid receptors (GABAA/CR), and a recently discovered zinc-activated channel (1Chebib M. Johnston G.A.R. J. Med. Chem. 2000; 43: 1427-1447Crossref PubMed Scopus (301) Google Scholar, 2Cully D.F. Vassilatis D.K. Liu K.K. Paress P.S. Van der Ploeg L.H.T. Schaeffer J.M. Arena J.P. Nature. 1994; 371: 707-711Crossref PubMed Scopus (574) Google Scholar, 3Davies P.A. Wang W. Hales T.G. Kirness E.F. J. Biol. Chem. 2003; 278: 712-717Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 4Lester H.A. Dibas M.I. Dahan D. Leite J.F. Dougherty D.A. Trends Neurosci. 2004; 27: 329-336Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Members of this family are formed by a pentameric arrangement of subunits around a central ion channel pore, which is lined by residues within the second transmembrane (M2) domain of each subunit (5Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-955Crossref PubMed Scopus (1075) Google Scholar). Although these receptors display a high degree of sequence and structural homology, they differ in the nature of the ions conducted. For example, nACh receptors are selectively permeable to monovalent and divalent cations, whereas GlyR, GABAAR, and GABACRs predominantly conduct anions, typically chloride. This difference critically underpins the role of different LGICs in mediating excitatory or inhibitory transmission at synapses. The proximity of residues within the M2 domain to the ion permeation pathway, and the channel gate(s), suggests that they have a considerable influence on channel gating and ion conduction, and this has been confirmed by a number of mutagenesis studies (6Imoto K. Busch C. Sakmann B. Mishina M. Konno T. Nakai J. Bujo H. Mori Y. Fukuda K. Numa S. Nature. 1988; 335: 645-648Crossref PubMed Scopus (605) Google Scholar, 7Corringer P-J. Le Novère N. Changeux J.-P Annu. Rev. Pharmacol. Toxicol. 2000; 40: 431-458Crossref PubMed Scopus (706) Google Scholar, 8Keramidas A. Moorhouse A.J. Schofield P.R. Barry P.H. Prog. Biophys. Mol. Biol. 2004; 86: 161-204Crossref PubMed Scopus (160) Google Scholar, 9Galzi J-L. Devillers-Thiéry A. Hussy N. Betrand S. Changeux J.-P. Bertrand D. Nature. 1992; 359: 500-505Crossref PubMed Scopus (342) Google Scholar). Electron microscopic pictures of the closed nAChR reveal that hydrophobic residues around 9′–14′ form a girdle to occlude ion permeation (5Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-955Crossref PubMed Scopus (1075) Google Scholar). In the desensitized state, the structure of a putative "desensitization gate" has not been definitively resolved but may involve a more extensive number of M2 residues (10Auerbach A. Akk G. J. Gen. Physiol. 1998; 112: 181-197Crossref PubMed Scopus (143) Google Scholar, 11Wilson G.G. Karlin A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1241-1248Crossref PubMed Scopus (132) Google Scholar). In the open state of the channel, the pore tapers to a constriction at the intracellular end. In the nAChR a threonine at the 2′ position forms the narrowest part of the pore, and mutations to this position markedly alter permeation properties (12Unwin N. Proc. R. Soc. Lond. B Biol. Sci. 2000; 355: 1813-1829Crossref Scopus (104) Google Scholar). Mutations to other residues within this constricted intracellular end have also been shown to have marked effects on ion permeation and seem to be particularly important for determining ion selectivity (6Imoto K. Busch C. Sakmann B. Mishina M. Konno T. Nakai J. Bujo H. Mori Y. Fukuda K. Numa S. Nature. 1988; 335: 645-648Crossref PubMed Scopus (605) Google Scholar, 8Keramidas A. Moorhouse A.J. Schofield P.R. Barry P.H. Prog. Biophys. Mol. Biol. 2004; 86: 161-204Crossref PubMed Scopus (160) Google Scholar). Even from early work investigating the ion selectivity of N-LGIC receptors, it was suggested that a critical role was played by rings of charged residues at the extracellular and intracellular ends of the M2 domain, the charged side chains being thought to favor the permeation of ions of the opposite charge (6Imoto K. Busch C. Sakmann B. Mishina M. Konno T. Nakai J. Bujo H. Mori Y. Fukuda K. Numa S. Nature. 1988; 335: 645-648Crossref PubMed Scopus (605) Google Scholar, 13Dani J.A. Biophys. J. 1986; 49: 607-618Abstract Full Text PDF PubMed Scopus (131) Google Scholar). Imoto et al. (6Imoto K. Busch C. Sakmann B. Mishina M. Konno T. Nakai J. Bujo H. Mori Y. Fukuda K. Numa S. Nature. 1988; 335: 645-648Crossref PubMed Scopus (605) Google Scholar) had also shown that the changes in charge of the intermediate ring in the intracellular end of the M2 domain had the greatest effect on ion conductance in the AChR channel. More recently, mutations to other residues, especially of a charged nature, within this constricted intracellular end of the M2 domain have also been shown to have marked effects on ion permeation and seem to be particularly important for determining ion charge selectivity (8Keramidas A. Moorhouse A.J. Schofield P.R. Barry P.H. Prog. Biophys. Mol. Biol. 2004; 86: 161-204Crossref PubMed Scopus (160) Google Scholar). Bertrand and colleagues (9Galzi J-L. Devillers-Thiéry A. Hussy N. Betrand S. Changeux J.-P. Bertrand D. Nature. 1992; 359: 500-505Crossref PubMed Scopus (342) Google Scholar) were the first to invert the ion selectivity of a N-LGIC receptor, converting the α7 nAChR to an anion conducting receptor by mutating two residues (E-1′A and V13′T) and introducing a proline residue at the intracellular end of the channel pore (Pro-–2′). Complementary mutations successfully inverted the ion selectivity of the GlyR, serotonin type 3 receptor A, and GABAAR (14Gunthorpe M.J. Lummis S.C.R. J. Biol. Chem. 2001; 276: 10977-10983Abstract Full Text Full Text PDF Scopus (115) Google Scholar, 15Jensen M.L. Timmermann D.B. Johansen T.H. Schousboe A. Varming T. Ahring P.K. J. Biol. Chem. 2002; 277: 41438-41447Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 16Keramidas A. Moorhouse A.J. French C.R. Schofield P.R. Barry P.H. Biophys. J. 2000; 78: 247-259Abstract Full Text Full Text PDF Scopus (101) Google Scholar), with the extension of this work demonstrating that double and even single mutations at critical positions (0′, –1′) within this region are sufficient to invert ion selectivity (15Jensen M.L. Timmermann D.B. Johansen T.H. Schousboe A. Varming T. Ahring P.K. J. Biol. Chem. 2002; 277: 41438-41447Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 17Keramidas A. Moorhouse A.J. Pierce K.D. Schofield P.R. Barry P.H. J. Gen. Physiol. 2002; 119: 393-410Crossref PubMed Scopus (80) Google Scholar, 18Moorhouse A.J. Keramidas A. Zaykin A. Schofield P.R. Barry P.H. J. Gen. Physiol. 2002; 119: 411-425Crossref PubMed Scopus (36) Google Scholar, 19Wotring V.E. Miller T.S. Weiss D.S. J. Physiol. 2003; 2003: 527-540Crossref Scopus (50) Google Scholar). These studies indicate that the ion selectivity filter is located at the intracellular end of the channel corresponding to the narrowest region of the channel between the –3′ and 2′ positions and that N-LGIC receptors share common physical characteristics in these regions. The current study has focused on the role of the proline residue at the 2′ position (Pro-2′) in the M2 domain of GABACRs. This proline is highly conserved across the species from which the ρ1 subunit has been cloned, suggesting its importance for normal receptor function. It is located at the extracellular edge of the constricted region and there is a high degree of sequence variation at this position between the N-LGIC receptors. We have recently shown that mutations to this residue altered the relative actions of a series of agonists and partial agonists at recombinant ρ1 GABACRs (20Carland J.E. Moore A.M. Hanrahan J.R. Mewett K.N. Johnston G.A.R. Chebib M. Neuropharmacology. 2004; 46: 770-781Crossref PubMed Scopus (19) Google Scholar). We interpreted these effects in terms of mutations to 2′ changing the gating equilibrium between the closed and open states of the channel and found a correlation with side-chain hydrophobicity (using mutations to alanine, glycine, serine, and phenylalanine). In the current study, we investigated the impact of charged residues at the 2′ position of the human GABAC ρ1 subunit in the M2 domain on the pharmacology and ion selectivity of the human ρ1 GABACR (see Fig. 1). Production of Mutant Human GABAC ρ1 Subunits—Mutation of the native proline at the 2′ position of the human GABAC ρ1 subunit in the M2 domain to arginine (ρ1P2′R) and glutamate (ρ1P2′E) was achieved using the QuikChange site-directed mutagenesis kit (Stratagene). All mutations were verified by DNA sequencing (Australian Genome Research Facility). Plasmids containing mutant and wild-type inserts were linearized with Xba-I, and mRNA was synthesized using the T7 mMESSAGE mMACHINE kit (Ambion, Austin, TX). Expression of Wild-type and Mutant ρ1 GABACRs in Xenopus Oocytes—Oocytes were harvested from Xenopus laevis as described previously (21Chebib M. Mewett K.N. Johnston G.A.R. Eur. J. Pharmacol. 1998; 357: 227-234Crossref PubMed Scopus (59) Google Scholar). Stage V-VI oocytes were injected with mRNA (10 ng/50 nl) and then stored at 18 °C in ND96 solution (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 5 mm HEPES, pH 7.5) supplemented with 2.5 mm sodium pyruvate, 0.5 mm theophylline, and 50 μgml–1 gentamycin. Recordings of receptor activity were obtained after 2–8 days by a two-electrode voltage clamp (see next section). Oocytes were voltage-clamped at –60 mV and continually perfused with ND96 solution at room temperature. Known concentrations of compounds tested were dissolved in ND96 and applied until maximum responses were obtained. The oocyte was then washed for 3–10 min, which was sufficient for the complete recovery of the control response to GABA. In the case of antagonists, a complete GABA concentration response curve was obtained under control conditions and then again in the presence of a known concentration of antagonist. This enabled the determination of single point estimates from individual cells. All compounds were tested on oocytes from at least three harvests. Studies of wild-type and mutant receptors were conducted in parallel. Electrophysiology and Solutions—Recordings of receptor currents and current-voltage data were obtained with a two-electrode voltage clamp using a Digidata 1200, a Geneclamp 500 amplifier, and pClamp 8 for Windows (Axon Instruments Inc., Foster City, CA), together with a MacLab 2e recorder (AD Instruments, Sydney, NSW) and Chart version 3.6.3 program, as further described previously (21Chebib M. Mewett K.N. Johnston G.A.R. Eur. J. Pharmacol. 1998; 357: 227-234Crossref PubMed Scopus (59) Google Scholar). Microelectrodes, filled with 3 m KCl were used for both the current and voltage intracellular electrodes, with the external bath reference electrode being a Ag/AgCl electrode, which needed correction for changes in Cl– activity when appropriate. Steady-state current measurements were obtained in response to (100 ms) voltage steps from –30 mV to potentials between –60 mV and 80 mV, in 10-mV increments. The current-voltage relationships for the GABA (100 μm)-evoked current were obtained after the subtraction of control responses to the same voltage protocol recorded in the absence of GABA. Current-voltage measurements were undertaken for each receptor in the presence of three solutions with decreasing levels of extracellular NaCl, OR2 (a control 100% NaCl, 82.5 mm NaCl, 2 mm KCl, 1 mm MgCl2·6H2O, 5 mm HEPES (with 2.5 mm NaOH)) and then in two OR2-like solutions 0.5OR2 (a 50% NaCl solution in which 82.5 mm NaCl was replaced with 41.25 mm sodium chloride and 78 mm sucrose) and 0.25OR2 (a 25% NaCl solution in which 82.5 mm NaCl was replaced with 20.6 mm NaCl and 112.5 mm sucrose). The concentration of sucrose required to maintain solution osmolarity was obtained from the CRC Handbook of Chemistry and Physics (22Wolf A.V. Brown M.G. Prentiss P.G. Weast R.C. Astle M.J. CRC Handbook of Chemistry and Physics. Chemical Rubber Publishing Co., Florida1980: D261-D270Google Scholar). GABA, imidazole-4-acetic acid (I4AA), 4,5,6,7-tetrahydroisoxazole[5,4-C]pyridine-3-ol (THIP), and muscimol were obtained from Sigma Chemical Co. Trans-4-aminocrotonic acid (TACA), cis-4-aminocrotonic acid (CACA), trans-2-(+/–)-(aminomethyl)cyclopropanoic acid ((+/–)-TAMP), cis-2-(+/–)-(aminomethyl)cyclopropanoic acid ((+/–)-CAMP), and (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA) were prepared as previously reported (23Hanrahan J.R. Mewett K.N. Chebib M. Burden P.M. Johnston G.A.R. J. Chem. Soc. Perkins Trans. 1. 2001; 19: 2389-2392Crossref Scopus (28) Google Scholar, 24Duke R.K. Chebib M. Allan R.D. Mewett K.N. Johnston G.A.R. J. Neurochem. 2000; 75: 2602-2610Crossref PubMed Scopus (54) Google Scholar). General Analysis of Data—All current responses were normalized to the maximum GABA-activated current recorded in the same cell and expressed as a percentage of this maximum. This was then plotted as a function of agonist concentration using Kaleidagraph 3.0. This data was fitted by least squares to the equation I = IM[A]nH/(EC50nH + [A]nH), where I is the response to a known concentration of agonist, IM is the maximum current obtained, [A] is the agonist concentration, EC50 is the concentration of agonist that activates 50% of receptors and nH is the Hill coefficient. The apparent binding constant for antagonists (KB) was estimated using the Schild equation KB = ({A}/{A*} – 1) – [Ant], where {A} is the EC50 of agonist in the presence of antagonist, {A*} is the EC50 of agonist in the absence of antagonist, and [Ant] is the concentration of antagonist. This model assumes simple competitive antagonism. Dilution Potential Fitting—Current-voltage relationships were plotted using Clampfit, and reversal potentials were directly read from linear or polynomial fits to these data and averaged. Reversal potentials obtained in the three extracellular solutions (OR2, 0.5OR2, and 0.25OR2) were corrected for liquid junction potentials and changes in Ag/AgCl electrode potentials, because of changes in Cl– activity when the extracellular solutions were diluted, using the MS Windows version of JPCalc (25Barry P.H. J. Neurosci. Methods. 1994; 51: 107-116Crossref PubMed Scopus (545) Google Scholar). SigmaPlot 5 was used to fit reversal potentials (Erev) to a modified version of the Goldman-Hodgkin-Katz equation (26Hodgkin A.L. Katz B. J. Physiol. 1949; 108: 37-77Crossref PubMed Scopus (1806) Google Scholar, 27Goldman D.E. J. Gen. Physiol. 1943; 27: 37-60Crossref PubMed Scopus (1667) Google Scholar) Erev = (RT/F) ln ((aNao + βaKo + αaCli)/(aNai + βaKi + αaClo)). Where aNa, aK, and aCl represent the ionic activities of Na+, K+, and Cl–, respectively, α = PCl/PNa and β = PK/PNa, and superscripts o and i refer to external and internal solution values, respectively. Because of the large changes in ionic strength of the external solutions, it was necessary to use ion activities (a) rather than ion concentrations (C) in all data fitting calculations. For the NaCl concentrations in the three different OR2 solutions (87, 45, and 25 mm) mean activity coefficients (γ, where a = γC) of 0.793, 0.837, and 0.873 at 25 °C were interpolated from Table I of Appendix 8.9 in Robinson and Stokes (28Robinson R.A. Stokes R.H. Activity Coefficients of Electrolytes at 25 °C. Electrolyte Solutions, Butterworths, London1965Google Scholar). Activities of each of the ions is listed in Table I. Intracellular ion concentrations were assumed to be the same as those measured by Barish (29Barish M.E. J. Physiol. 1983; 342: 309-325Crossref PubMed Scopus (489) Google Scholar), using intracellular ion-selective microelectrodes, with [Cl]i = 33 mm, [Na]i = 6mm, and [K]i = 92 mm. The [Cl]i value was close to the value of 35 mm estimated by Wotring et al. (19Wotring V.E. Miller T.S. Weiss D.S. J. Physiol. 2003; 2003: 527-540Crossref Scopus (50) Google Scholar).Table IExternal concentrations and activities of sodium, chloride, and potassium ionsSolutionNa+Cl-K+Activity coefficient (γ)aThe activity coefficients were obtained by extrapolation from Table I of Appendix 8.9 in Robinson and Stokes (28) for Cl- concentrations of 87, 45, and 25 mm, respectively.mmCjobCjo is the external concentration of an ion.OR28586.520.7930.5OR243.7545.2520.8370.25OR223.124.620.873ajocajo is the external activity of an ion.OR267.468.61.60.5OR236.637.91.70.25OR220.221.51.7a The activity coefficients were obtained by extrapolation from Table I of Appendix 8.9 in Robinson and Stokes (28Robinson R.A. Stokes R.H. Activity Coefficients of Electrolytes at 25 °C. Electrolyte Solutions, Butterworths, London1965Google Scholar) for Cl- concentrations of 87, 45, and 25 mm, respectively.b Cjo is the external concentration of an ion.c ajo is the external activity of an ion. Open table in a new tab Relative permeabilities were calculated from reversal potential data for each individual experiment and then averaged for all the experiments. A one-way analysis of variance, using Tukey's multiple comparison test, was performed to determine the statistical significance of the results, where 0.05 was treated as the point of significance. ρ1P2′R Receptors Have an Increased Basal Holding Current—The expression of wild-type and mutant GABAC ρ1 subunit mRNA in Xenopus oocytes produced functional GABA-activated receptors. However, when clamped at –60 mV the holding current for cells expressing ρ1P2′R receptors (–704 ± 76 nA, n = 46) was significantly (p < 0.001) greater than that in cells expressing wild-type (–40 ± 15 nA, n = 22) and ρ1P2′E receptors (–38 ± 7 nA, n = 26). This holding current was inhibited by low doses of GABA (Fig. 2c) and by TPMPA alone (Fig. 3, IC50 = 2.55 ± 0.76 μm, n = 4). This indicates that the ρ1P2′R receptors allow some ion permeation even in the absence of the agonist. In these receptors, in response to application of maximal GABA concentrations, inward currents were observed at –60 mV that were much larger than the holding currents (Fig. 2c) suggesting that the proportion of time that these receptors are spontaneously open is much smaller than channel openings induced by ligand binding. Overall, the whole-cell currents measured for wild-type and mutant receptors varied, ranging between 100 and 3000 nA for wild-type receptors, 100 and 400 nA for ρ1P2′E receptors, and 100 and 600 nA for ρ1P2′R receptors.Fig. 3a, TPMPA (300 μm, duration indicated by closed bar) inhibits the basal holding current of ρ1P2′R receptors expressed in Xenopus oocytes in the absence of GABA. GABA (100 μm, duration indicated by hatched bar) produces a maximal response at ρ1P2′R receptors. b, the holding current is inhibited by TPMPA in a dose-dependent manner. Data are the mean ± S.E. (n = 4–24 oocytes) obtained from at least three harvests.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Potency and Efficacy of Full and Partial Agonists at Wild-type and Mutated Receptors—The potencies and efficacies of the full agonists, GABA, TACA, and (+/–)-CAMP differed at mutant receptors compared with the wild-type receptors (Table II and Fig. 4). The potency of GABA (p < 0.001, n = 24) and (+/–)-CAMP (p < 0.01, n = 5) was significantly increased at the ρ1P2′R receptors, whereas in contrast, all three compounds were significantly less potent at the ρ1P2′E receptors (p < 0.001, n = 4–15). The intrinsic efficacy of these full agonists tested was maintained at the mutant receptors, with the exception of a significant increase for TACA (p < 0.001, n = 3) and a significant ∼50% decrease for (+/–)-CAMP at ρ1P2′E receptors (p < 0.001, n = 4).Table IIEffects of agonists, partial agonists and antagonists at wild-type and mutant ρ1 GABAC receptors expressed in X. laevis oocytesWild-typer1P2′Er1P2′REC50aEC50 is the concentration required to obtain 50% activation. Data are the mean ± S.E. (n = 3-24 oocytes) obtained over a minimum of three harvests.nHbnH is the Hill coefficient which is a measure of the cooperativity of agonists. Data are the mean ± S.E. (n = 3-24 oocytes) obtained over a minimum of three harvests.IMcIM is the intrinsic efficacy of the agonist and is measured as a percentage of the maximum response of GABA, which is designated as 100%. Data are the mean ± S.E. (n = 3-24 oocytes) obtained over a minimum of three harvests.EC50aEC50 is the concentration required to obtain 50% activation. Data are the mean ± S.E. (n = 3-24 oocytes) obtained over a minimum of three harvests.nHbnH is the Hill coefficient which is a measure of the cooperativity of agonists. Data are the mean ± S.E. (n = 3-24 oocytes) obtained over a minimum of three harvests.IMcIM is the intrinsic efficacy of the agonist and is measured as a percentage of the maximum response of GABA, which is designated as 100%. Data are the mean ± S.E. (n = 3-24 oocytes) obtained over a minimum of three harvests.EC50aEC50 is the concentration required to obtain 50% activation. Data are the mean ± S.E. (n = 3-24 oocytes) obtained over a minimum of three harvests.nHbnH is the Hill coefficient which is a measure of the cooperativity of agonists. Data are the mean ± S.E. (n = 3-24 oocytes) obtained over a minimum of three harvests.IMcIM is the intrinsic efficacy of the agonist and is measured as a percentage of the maximum response of GABA, which is designated as 100%. Data are the mean ± S.E. (n = 3-24 oocytes) obtained over a minimum of three harvests.μm%μm%μm%Agonist or partial agonistsGABA1.1 ± 0.12.1 ± 0.11004.9 ± 0.3gp < 0.001 compared to wild-type.1.3 ± 0.1gp < 0.001 compared to wild-type.1000.2 ± 0.03gp < 0.001 compared to wild-type.1.2 ± 0.1gp < 0.001 compared to wild-type.100TACA0.40 ± 0.022.5 ± 0.292 ± 23.0 ± 0.4gp < 0.001 compared to wild-type.1.2 ± 0.1111 ± 3gp < 0.001 compared to wild-type.0.2 ± 0.031.0 ± 0.196 ± 3CACA40.8 ± 6.72.0 ± 0.376 ± 4118.2 ± 7.7gp < 0.001 compared to wild-type.1.2 ± 0.00333 ± 1gp < 0.001 compared to wild-type.8.1 ± 1.2fp < 0.01 compared to wild-type.1.2 ± 0.282 ± 3(+/-)-TAMP17.9 ± 1.01.5 ± 0.235 ± 310.1 ± 0.7gp < 0.001 compared to wild-type.1.0 ± 0.128 ± 21.2 ± 0.1gp < 0.001 compared to wild-type.1.0 ± 0.0469 ± 5gp < 0.001 compared to wild-type.(+/-)-CAMP40.8 ± 11.92.3 ± 0.3107 ± 292.6 ± 2.2gp < 0.001 compared to wild-type.1.2 ± 0.155 ± 3gp < 0.001 compared to wild-type.9.0 ± 1.2fp < 0.01 compared to wild-type.1.1 ± 0.196 ± 2fp < 0.01 compared to wild-type.Muscimol1.43 ± 0.151.6 ± 0.179 ± 45.8 ± 0.7gp < 0.001 compared to wild-type.1.0 ± 0.0438 ± 1gp < 0.001 compared to wild-type.0.2 ± 0.1fp < 0.01 compared to wild-type.1.0 ± 0.190 ± 3ep < 0.05 compared to wild-type.I4AA8.6 ± 1.01.2 ± 0.18 ± 15.9 ± 0.71.1 ± 0.113 ± 20.4 ± 0.1gp < 0.001 compared to wild-type.1.1 ± 0.157 ± 5gp < 0.001 compared to wild-type.Antagonists (KB)dKB is the estimated binding constant of the antagonist. Data are the mean ± S.E. values (n = 4-8 oocytes) obtained over a minimum of three harvests.TPMPA2.3 ± 0.22.6 ± 0.31.9 ± 0.6I4AA1.85 ± 0.351.9 ± 0.50.5 ± 0.2fp < 0.01 compared to wild-type.THIP30.3 ± 1.822.8 ± 5.721.2 ± 2.4a EC50 is the concentration required to obtain 50% activation. Data are the mean ± S.E. (n = 3-24 oocytes) obtained over a minimum of three harvests.b nH is the Hill coefficient which is a measure of the cooperativity of agonists. Data are the mean ± S.E. (n = 3-24 oocytes) obtained over a minimum of three harvests.c IM is the intrinsic efficacy of the agonist and is measured as a percentage of the maximum response of GABA, which is designated as 100%. Data are the mean ± S.E. (n = 3-24 oocytes) obtained over a minimum of three harvests.d KB is the estimated binding constant of the antagonist. Data are the mean ± S.E. values (n = 4-8 oocytes) obtained over a minimum of three harvests.e p < 0.05 compared to wild-type.f p < 0.01 compared to wild-type.g p < 0.001 compared to wild-type. Open table in a new tab A similar pattern of changes were observed for the partial agonists muscimol, CACA, (+/–)-TAMP, and I4AA (Table II and Fig. 5). The potency of muscimol was increased by ∼7-fold at the ρ1P2′R receptors (p < 0.01, n = 5) and decreased by 4-fold at the ρ1P2′E receptors (p < 0.001, n = 3). Similarly, CACA was 5-fold more potent at the ρ1P2′R receptors (p < 0.01, n = 5) compared with wild-type, with a 3-fold decrease in potency being observed at ρ1P2′E receptors (p < 0.001, n = 3). The potency of (+/–)-TAMP and I4AA was also increased at the ρ1P2′R receptors, by 15- and 21-fold, respectively (p < 0.001, n = 5–9). Similarly, (+/–)-TAMP and I4AA were also more potent at ρ1P2′E receptors compared with wild-type, this increase was significant for (+/–)-TAMP (p < 0.001, n = 4). The intrinsic efficacy of the partial agonists was, in general, decreased at the ρ1P2′E receptors and increased at the ρ1P2′R receptors (Table II), with the greatest change observed being the 7-fold increase in the measured intrinsic efficacy of I4AA at the ρ1P2′R receptors (p < 0.001, n = 9). The Hill coefficients (nH) for activation of wild-type receptors ranged from 1.2 to 2.5 for the different full and partial agonists (Table II). The mean nH for activation of both mutant receptors seemed to be generally decreased compared with the wild-type, with values ranging from 1.0 t
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