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• W2103540557 abstract "In this study, we have compared the functional consequences of three mutations (R218Q, V260M, and Q266H) in the α1 subunit of the glycine receptor (GlyRA1) causing hyperekplexia, an inherited neurological channelopathy. In HEK-293 cells, the agonist EC50s for glycine-activated Cl- currents were increased from 26 μm in wtGlyRA1, to 5747, 135, and 129 μm in R218Q, V260M, and Q266H GlyRA1 channels, respectively. Cl- currents elicited by β-alanine and taurine, which behave as agonists at wtGlyRA1, were decreased in V260M and Q266H mutant receptors and virtually abolished in GlyRA1 R218Q receptors. Gly-gated Cl- currents were similarly antagonized by low concentrations of strychnine in both wild-type (wt) and R218Q GlyRA1 channels, suggesting that the Arg-218 residue plays a crucial role in GlyRA1 channel gating, with only minor effects on the agonist/antagonist binding site, a hypothesis supported by our molecular model of the GlyRA1 subunit. The R218Q mutation, but not the V260M or the Q266H mutation, caused a marked decrease of receptor subunit expression both in total cell lysates and in isolated plasma membrane proteins. This decreased expression does not seem to explain the reduced agonist sensitivity of GlyRA1 R218Q channels since no difference in the apparent sensitivity to glycine or taurine was observed when wtGlyRA1 receptors were expressed at levels comparable with those of R218Q mutant receptors. In conclusion, multiple mechanisms may explain the dramatic decrease in GlyR function caused by the R218Q mutation, possibly providing the molecular basis for its association with a more severe clinical phenotype. In this study, we have compared the functional consequences of three mutations (R218Q, V260M, and Q266H) in the α1 subunit of the glycine receptor (GlyRA1) causing hyperekplexia, an inherited neurological channelopathy. In HEK-293 cells, the agonist EC50s for glycine-activated Cl- currents were increased from 26 μm in wtGlyRA1, to 5747, 135, and 129 μm in R218Q, V260M, and Q266H GlyRA1 channels, respectively. Cl- currents elicited by β-alanine and taurine, which behave as agonists at wtGlyRA1, were decreased in V260M and Q266H mutant receptors and virtually abolished in GlyRA1 R218Q receptors. Gly-gated Cl- currents were similarly antagonized by low concentrations of strychnine in both wild-type (wt) and R218Q GlyRA1 channels, suggesting that the Arg-218 residue plays a crucial role in GlyRA1 channel gating, with only minor effects on the agonist/antagonist binding site, a hypothesis supported by our molecular model of the GlyRA1 subunit. The R218Q mutation, but not the V260M or the Q266H mutation, caused a marked decrease of receptor subunit expression both in total cell lysates and in isolated plasma membrane proteins. This decreased expression does not seem to explain the reduced agonist sensitivity of GlyRA1 R218Q channels since no difference in the apparent sensitivity to glycine or taurine was observed when wtGlyRA1 receptors were expressed at levels comparable with those of R218Q mutant receptors. In conclusion, multiple mechanisms may explain the dramatic decrease in GlyR function caused by the R218Q mutation, possibly providing the molecular basis for its association with a more severe clinical phenotype. IntroductionFast inhibitory neurotransmission in the adult mammalian central nervous system is primarily mediated by the amino acids γ-aminobutyric acid (GABA) 1The abbreviations used are: GABA, γ-aminobutyric acid; GlyR, glycine receptor; LGIC, ligand-gated ion channel; HE, hyperekplexia; TAU, taurine; β-ALA, β-alanine; STR, strychnine; wt, wild-type. and Gly, which, by activating Cl--selective ligand-gated ion channels (LGICs), hyperpolarize the membrane of the postsynaptic neuron and inhibit firing (1Breitinger H.G. Becker C.M. ChemBioChem. 2002; 3: 1042-1052Crossref PubMed Scopus (73) Google Scholar). Glycine receptors (GlyRs) are heteropentameric proteins made up of the assembly of two types of homologous subunits: α subunits, which carry the molecular determinants for agonist and antagonist binding, and β subunits, which determine the subsynaptic localization of GlyRs (2Meyer G. Kirsch J. Betz H. Langosch D. Neuron. 1995; 15: 563-572Abstract Full Text PDF PubMed Scopus (348) Google Scholar). In humans, only one gene encoding for β subunits has been identified (3Grenningloh G. Pribilla I. Prior P. Multhaup G. Beyreuther K. Taleb O. Betz H. Neuron. 1990; 4: 963-970Abstract Full Text PDF PubMed Scopus (203) Google Scholar); this gene is widely expressed in the central nervous system at all developmental stages of vertebrates. On the other hand, four genes encoding distinct α subunits (α1–4) are known, each showing a specific regional distribution and ontogenetic profile (4Laube B. Maksay G. Schemm R. Betz H. Trends Pharmacol. Sci. 2002; 23: 519-527Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). In particular, α1 subunits seem to be the predominant isoform expressed postnatally in the spinal cord and brainstem; GlyRs of mature vertebrates are thought to have a 3α12β stoichiometry. The topological organization of the GlyR subunits is believed to be similar to that of other members of the LGIC super-family (5Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 424: 949-955Crossref Scopus (1074) Google Scholar), with both the N and the C termini located extracellularly and four transmembrane segments; the second transmembrane segment (M2) determines permeation properties such as anion selectivity and conductance.GlyRs play a central role in the pathophysiology of hyperekplexia (HE), a rare inherited human neurological disease characterized by muscle stiffness, hyperreflexia, and an exaggerated startle response to sensory stimuli (6Zhou L. Chillag K.L. Nigro M.A. Brain Dev. 2002; 24: 669-674Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The muscle stiffness and hyperreflexia, usually present at birth, decline during the first year of life, although an exaggerated startle response persists during the adult life. The gene associated with hyperekplexia was linked to chromosomal locus 5q33–35 in 1992 (7Ryan S.G. Sherman S.L. Terry J.C. Sparkes R.S. Torres M.C. Mackey R.W. Ann. Neurol. 1992; 31: 663-668Crossref PubMed Scopus (109) Google Scholar); within this region, mutation analysis in HE-affected families proved that the GlyRA1 gene, encoding the α1 subunit of GlyR, was the defective gene in HE (8Shiang R. Ryan S.G. Zhu Y.Z. Hahn A.F. O'Connell P. Wasmuth J.J. Nat. Genet. 1993; 5: 351-358Crossref PubMed Scopus (439) Google Scholar). Several HE-causing mutations are known in the GlyRA1 gene; these include both dominant and recessive inherited mutations, as well as de novo mutations found in sporadic cases. Further support for the hypothesis that abnormal GlyR function causes neurological disturbances derives from the observation that the phenotypes of spasmoid (9Ryan S.G. Buckwalter M.S. Lynch J.W. Handford C.A. Segura L. Shiang R. Wasmuth J.J. Camper S.A. Schofield P. O'Connell P. Nat. Genet. 1994; 7: 131-135Crossref PubMed Scopus (154) Google Scholar), spastic (10Kingsmore S.F. Giros B. Suh D. Bieniarz M. Caron M.G. Seldin M.F. Nat. Genet. 1994; 7: 136-141Crossref PubMed Scopus (190) Google Scholar), and oscillator (11Buckwalter M.S. Cook S.A. Davisson M.T. White W.F. Camper S.A. Hum. Mol. Genet. 1994; 3: 2025-2030Crossref PubMed Scopus (102) Google Scholar) mice, all of which carry genetically determined defects in GlyR-encoding genes, bear a striking resemblance to some of the sensorimotor manifestations of human HE.In the present study, we have characterized the functional properties of three GlyRA1 mutations associated with HE. The R218Q mutation, recently described by members of our group (12Miraglia del Giudice E. Coppola G. Bellini G. Ledaal P. Hertz J.M. Pascotto A. J. Med. Genet. 2003; 40: e71Crossref PubMed Google Scholar), affects a residue that is located at the boundary between the extracellular N-terminal domain and M1 and is highly conserved among subunits forming both anion-selective (GlyR and GABAA,CR) and cation-selective (nicotinic α1–7 and α9, β1–4, γ, δ, ϵ; 5HT-3A and 5HT-3B; some purinergic receptors) LGICs (Supplemental Fig. 1). The V260M (13Miraglia del Giudice E. Coppola G. Bellini G. Cirillo G. Scuccimarra G. Pascotto A. Eur. J. Hum. Genet. 2001; 9: 873-876Crossref PubMed Scopus (25) Google Scholar) and the Q266H (14Milani N. Dalpra L. del Prete A. Zanini R. Larizza L. Am. J. Hum. Genet. 1996; 58: 420-422PubMed Google Scholar) mutations affect residues located in the M2 domain of GlyRA1 subunits but are not believed to participate in permeation (5Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 424: 949-955Crossref Scopus (1074) Google Scholar, 15Bera A.K. Chatav M. Akabas M.H. J. Biol. Chem. 2002; 277: 43002-43010Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar); they are less well conserved among LGIC subunits. We have used pharmacological, biochemical, and molecular modeling techniques to investigate the molecular basis of these three human “channelopathies” and found evidence that all three residues may cause a variable degree of impairment in GlyR gating, with the most dramatic phenotype associated with the R218Q mutation.EXPERIMENTAL PROCEDURESMutagenesis of hGlyRA1 cDNA—Mutations were engineered into the human GlyRA1 cDNA, cloned into the pCIS2 vector, by sequence overlap extension PCR with the Pfu DNA polymerase, as already described (16Castaldo P. Miraglia Del Giudice E. Coppola G. Pascotto A. Annunziato L. Taglialatela M. J. Neurosci. 2002; 22: RC199Crossref PubMed Google Scholar). DNA sequences were verified using an ABI PRISM 310 sequencing apparatus (Applied Biosystem, Foster City, CA).Heterologous Expression of GlyRA1 Subunits—Wild-type (wt) and mutant GlyRA1 subunits were transiently expressed via transfection of human embryonic kidney (HEK-293) cells. HEK-293 cells were grown in minimal essential medium containing 10% fetal bovine serum, nonessential amino acids (0.1 mm), penicillin (50 units/ml), and streptomycin (50 μg/ml), in an humidified atmosphere at 37 °C with 5% CO2 in 100-mm plastic Petri dishes. For electrophysiological experiments, the cells were seeded on glass coverslips (Carolina Biological Supply Company, Burlington, NC). One day after plating, the cells were transfected with 2 μg of the appropriate cDNAs using LipofectAMINE 2000 (Invitrogen), according to the manufacturer's protocol, together with 2 μg of a plasmid encoding the enhanced green fluorescent protein (Clontech) used as a transfection marker. When lower amounts of GlyRA1 cDNA were used for transfection (see Fig. 5), the amount of enhanced green fluorescent protein cDNA was proportionally increased. All of the experiments were performed 1–2 days after transfection.Electrophysiology—Currents were recorded from HEK-293 cells at 20–22 °C using a commercially available amplifier (Axopatch 200A, Axon Instruments, Foster City, CA). The whole cell configuration of the patch clamp technique was adopted using glass micropipettes of 3–5 megaohms of resistance (17Coppola G. Castaldo P. Miraglia del Giudice E. Bellini G. Galasso F. Soldovieri M.V. Anzalone L. Sferro C. Annunziato L. Pascotto A. Tagliatatela M. Neurology. 2003; 61: 131-134Crossref PubMed Scopus (54) Google Scholar). The extracellular and intracellular (pipette) solution contained (in mm): 138 NaCl, 2 CaCl2, 5.4 KCl, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4, with NaOH; and 140 KCl, 2 MgCl2, 10 EGTA, 10 HEPES, 5 Mg-ATP, 0.25 mm cAMP, pH 7.3–7.4, with KOH, respectively. The pCLAMP software (v. 6.0.4, Axon Instruments) was used for data acquisition and analysis. Statistical differences were evaluated with the Student's t test (p < 0.05). Solution exchanges were achieved by means of a cFlow 8 flow controller attached to a cF-8VS 8-valve switching apparatus and a MPRE8 miniature flow outlet positioned within 200 μm of the cell (Cell Microcontrols, Norfolk, VA).Cell Surface Biotinylation and Western Blotting—Plasma membrane expression of GlyRA1 subunits in HEK-293 cells was investigated by surface biotinylation of membrane proteins in intact cells 2 days after transfection using a cell membrane-impermeable reagent (sulfosuccinimidyl-6-(biotinamido) hexanoate; Pierce) according to the manufacturer's protocol (18Wen H. Levitan I.B. J. Neurosci. 2002; 22: 7991-8000Crossref PubMed Google Scholar). Following biotinylation, the cells were lysed, and biotinylated proteins were isolated by reacting cell lysates with ImmunoPure streptavidin beads (Pierce). GlyR α subunits in streptavidin precipitates and total lysates were revealed in Western blots using an anti-GlyRA monoclonal antibody (mAb4a; Alexis Biochemicals, Lausen, Switzerland; dilution 1:4000); an alkaline phosphatase-conjugated sheep anti-mouse IgG (Amersham Biosciences; dilution 1:2000) was used as secondary antibody. Reactive bands were detected by chemiluminescence (SuperSignal, Pierce), using a ChemiDoc station (Bio-Rad). To confirm that intracellular proteins were not labeled by the biotinylation reagent, blots were stripped and reprobed with an anti-α-tubulin antibody (Sigma; dilution 1:5000).Molecular Modeling—A molecular model of a GlyRA1 subunit was built using a previously described model of the GABAAR α1 subunit as a template (19Bertaccini E. Trudell J.R. Int. Rev. Neurobiol. 2001; 48: 141-166Crossref PubMed Google Scholar, 20Kash T.L. Jenkins A. Kelley J.C. Trudell J.R. Harrison N.L. Nature. 2003; 421: 421-425Crossref PubMed Scopus (280) Google Scholar, 21Trudell J.R. Biochim. Biophys. Acta. 2002; 1565: 91-96Crossref PubMed Scopus (46) Google Scholar, 22Trudell J.R. Bertaccini E. Br. J. Anaesth. 2002; 89: 32-40Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We built loops to fill in the gaps between the GlyRA1 sequence and the template sequence and refined the side chains in the model using the auto-rotomer feature in the Biopolymer Module of Insight II (v. 2000.1, Accelrys, San Diego, CA) as described previously (19Bertaccini E. Trudell J.R. Int. Rev. Neurobiol. 2001; 48: 141-166Crossref PubMed Google Scholar, 22Trudell J.R. Bertaccini E. Br. J. Anaesth. 2002; 89: 32-40Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The backbone atoms (C, Cα, N) of each GlyR residue were tethered to the coordinates of corresponding residues in the template with a force constant of 100 kcal/A2, and the structure was optimized with the Discover_3 module of Insight II. Coulombic interactions were calculated with a constant dielectric of one and a cutoff at 9.5 A. The structure was relaxed by performing 5000 2-fs steps of molecular dynamics at 298 K and was then reoptimized with the Discover_3 module to a derivative of 1 kcal/A.RESULTSGlycine Sensitivity of Homomeric GlyRA1s Carrying HE-causing Mutations—The putative topological arrangement of a single GlyR α1 subunit, highlighting the Arg-218, the Val-260, and the Gln-266 residues, is shown in Fig. 1A. To assess the functional consequences of the HE-causing GlyRA1 mutations affecting these residues, we transfected HEK-293 cells with the cDNAs encoding wtGlyRA1 or R218Q, V260M, or Q266H GlyRA1 mutants and recorded Cl- currents carried by the expressed channels upon perfusion with the agonist Gly. In wtGlyRA1-expressing cells, perfusion with concentrations of Gly above 3 μm (0.01–30 mm) activated robust inward Cl- currents when the cell was held at -60 mV (Fig. 1B); the reversal potential of these currents approximated the Cl- equilibrium potential (data not shown), confirming anionic selectivity. The peak amplitude of these Gly-gated Cl- currents increased with Gly concentration up to 300 μm; larger Gly concentrations (3–30 mm) failed to increase Clcurrent peak size, although they accelerated the time course of Gly-gated current desensitization. Peak amplitudes of Gly-gated currents were normalized to the maximum, expressed as a function of Gly concentrations, and fitted to a standard form of the Hill equation (y = ymax ([Gly]nH/[Gly]nH + EC50nH). The EC50 for Gly was 26 ± 4 μm, with a Hill coefficient (nH) of 2.0 ± 0.1 (Fig. 1C). These values concur with those reported previously (23Moorhouse A.J. Jacques P. Barry P.H. Schofield P.R. Mol. Pharmacol. 1999; 55: 386-395Crossref PubMed Scopus (47) Google Scholar, 24Schofield C.M. Jenkins A. Harrison N.L. J. Biol. Chem. 2003; 278: 34079-34083Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar).Fig. 1Glycine sensitivity of homomeric channels formed by GlyRA1 subunits carrying HE-causing mutations. A, schematic representation of the putative topological arrangement of a single GlyRA1 subunit (1Breitinger H.G. Becker C.M. ChemBioChem. 2002; 3: 1042-1052Crossref PubMed Scopus (73) Google Scholar). B, whole cell current responses to Gly in HEK-293 cells expressing wt and R218Q GlyRA1 subunits. In this and the following figures, the holding potential was -60 mV, and the bars on top of the current traces show the duration of drug perfusion. C, Gly doseresponse curves for wt (filled squares), V260M (empty squares), Q266H (filled circles), and R218Q (empty circles) GlyRA1 mutants. The solid lines represent the fits of the experimental data to the form of the Hill equation described under “Results.” EC50s and nH values for each curve are given under “Results.” Each point is the mean ± S.E. of 4–10 determinations in separate cells. D, superimposed traces showing current responses elicited by exposure to 30 mm Gly in wtGlyRA1 and GlyRA1 R218Q channels. Agonist-activated responses have been normalized to facilitate comparison. E, effect of the R218Q mutation on the rate of channel activation, desensitization, and deactivation in the presence of Gly. Each bar is the mean ± S.E. of 6–15 determinations in separate cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Transfection of HEK-293 cells with R218Q GlyRA1 cDNA also led to the appearance of Gly-gated Cl- currents (Fig. 1B), although the agonist sensitivity of these currents was dramatically decreased when compared with that of homomeric wtGlyRA1 channels. Gly concentrations larger than 0.3 mm (1–30 mm) were required to activate Cl- currents in R218Q GlyRA1-transfected cells. Dose-response analysis revealed that the homomeric GlyRA1 channels carrying the R218Q mutation displayed a 200-fold decrease in Gly sensitivity when compared with wtGlyRA1 channels (EC50 of 5747 ± 270 μm), with no apparent change in the Hill coefficient nH (2.3 ± 0.2) (Fig. 1C). In addition, the maximal Cl- current density in cells transfected with the R218Q GlyRA1 cDNA (19.6 ± 8.9 pA/pF; n = 26) was markedly decreased when compared with that of wtGLyRA1-transfected cells (204.7 ± 32.9 pA/pF; n = 20; p < 0.05; see also Fig. 5).The two other HE-associated GlyRA1 mutations, V260M and Q266H, caused a more modest 5-fold increase in the EC50 for the agonist Gly (the EC50 was 135 ± 3 μm for V260M and 129 ± 8 μm for Q266H mutants) (Fig. 1C). Also in this case, no significant differences were detected in the Hill coefficients of V260M (nH = 1.7 ± 0.2) and Q266H (nH = 1.6 ± 0.2) GlyRA1 receptors when compared with wtGlyRA1 receptors.To gain further insight into the molecular defect prompted by the R218Q mutation, we measured the rates of channel activation, desensitization, and deactivation by fitting with a single exponential function the early phases of agonist (30 mm Gly)-induced current activation, the time course of agonist (30 mm Gly)-induced current desensitization, and the current decay upon agonist (3 mm) removal in both wtGlyRA1 and R218Q channels. Comparison of these kinetic properties showed that wtGlyRA1 receptors displayed an increased activation rate and a faster desensitization rate during agonist exposure when compared with GlyRA1 R218Q channels (Fig. 1, D and E), without significant changes in the deactivation rate upon removal of the agonist (Fig. 1E).Taurine and β-Alanine Sensitivity of HE-causing GlyRA1 Mutants—β-amino acids such as β-alanine (β-ALA) and taurine (TAU) behave as agonists at wtGlyR, but they appear to be of lower efficacy than Gly in some mutant receptors, acting as partial agonists (25Schmieden V. Kuhse J. Betz H. Mol. Pharmacol. 1999; 56: 464-472Crossref PubMed Scopus (43) Google Scholar, 26Langosch D. Laube B. Rundstrom N. Schmieden V. Bormann J. Betz H. EMBO J. 1994; 13: 4223-4228Crossref PubMed Scopus (151) Google Scholar). As shown in Fig. 2A, the peak Cl- currents elicited by exposure to 3 mm β-ALA or TAU in wtGlyRA1 receptors were similar in amplitude to those elicited by an identical Gly concentration (Fig. 2, B and C). By contrast, the currents elicited by both β-ALA and TAU in GlyRA1 carrying the V260M or the Q266H mutation were significantly smaller than those triggered by an identical concentration of Gly (3 mm) in the same cells (Fig. 2, A–C). In cells expressing GlyRA1 subunits carrying the R218Q mutation, 3 mm TAU or β-ALA application failed to elicit detectable Cl- currents (Fig. 2A). In the large majority of the R218Q-transfected cells, perfusion with a higher β-ALA concentration (30 mm) also failed to elicit measurable Cl- currents. In the only three cells in which these currents were clearly above background, the Iβ-ALA/IGly ratio was 0.13 ± 0.03 (p < 0.05 versus controls) (Fig. 2B); in R218Q-transfected cells, 30 mm TAU failed to activate measurable Cl- currents (Fig. 2C).Fig. 2Taurine and β-alanine sensitivity of GlyRA1 mutants. A, whole cell current responses to the application of Gly, TAU, and β-ALA (each at 3 mm) in the same cell expressing wt or mutant GlyRA1 subunits. B and C, Iβ-ALA/IGly (B) and ITAU/IGly (C) ratios in wt and GlyRA1 mutants, as indicated. The ratios of the peak current amplitude elicited by 3 mm β-ALA/3 mm Gly and 3 mm TAU/3 mm Gly (for wt, V260M, and Q266H) or by 30 mm β-ALA/30 mm Gly and 30 mm TAU/30 mm Gly (for R218Q) are plotted in B and C, respectively. Each bar is the mean ± S.E. of 3–8 determinations in separate cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Strychnine-induced Blockade of GlyRA1 Channels Carrying the R218Q Mutation—Strychnine (STR) is an antagonist of Gly, which is known to bind at sites that partially overlap the agonist binding site on the GlyR (1Breitinger H.G. Becker C.M. ChemBioChem. 2002; 3: 1042-1052Crossref PubMed Scopus (73) Google Scholar, 4Laube B. Maksay G. Schemm R. Betz H. Trends Pharmacol. Sci. 2002; 23: 519-527Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 25Schmieden V. Kuhse J. Betz H. Mol. Pharmacol. 1999; 56: 464-472Crossref PubMed Scopus (43) Google Scholar). The competitive nature of STR-induced antagonism is shown in Fig. 3A. Cl- currents carried by channels composed of wtGlyRA1 subunits were activated by 30 μm Gly, an agonist concentration close to the EC50. During agonist application, 400 nm STR caused a rapid inhibition of the Gly-gated Cl- currents, which were almost fully abolished. The time course of STR-induced Cl- current inhibition reflects the kinetics of antagonist binding to its site on the receptor, which depend upon antagonist concentration. Lower STR concentrations (100 nm) also inhibited Glygated Cl- currents, although the time required to reach steadystate inhibition was much longer (data not shown). Upon removal of 400 nm STR, and in the continuous presence of the agonist, the Cl- current amplitude recovered slowly, reflecting the kinetics of unbinding of the antagonist from its receptor. When a supramaximal Gly concentration (6 mm, 200-fold higher than the EC50) was applied, 400 nm STR did not inhibit the agonist-induced current in wtGlyRA1 channels, consistent with the competitive nature of STR blockade.Fig. 3Blockade of wt and R218Q GlyRA1 channels by strychnine. A, whole cell current responses to Gly (30 μm, left panel;6mm, right panel) application in cells expressing wtGlyRA1 subunits. In the trace labeled with an empty square, 400 nm STR was co-applied with Gly; in the traces labeled with a filled square, no STR was co-applied with Gly (control). B, whole cell current response to 6 mm Gly application in a cell expressing R218Q GlyRA1 subunits. 400 nm STR was co-applied with Gly. C, left panel, whole cell current response to 30 μm Gly application in a cell expressing wtGlyRA1 subunits. Right panel, whole cell current response to 30 μm Gly in the same cell, after preincubation for 1 min with 400 nm STR. D, whole cell current response to 6 mm Gly application in the same cell expressing R218Q GlyRA1 subunits in control conditions (left panel) or after preincubation for 1 min with 400 nm STR (right panel). The same experiments were performed in at least three additional cells, with comparable results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In GlyRA1 channels carrying the R218Q mutation, 400 nm STR also inhibited the Cl- currents activated by Gly, with kinetics similar to those observed in wtGlyRA1 (Fig. 3B). In these experiments, similarly to those described for wtGlyRA1 channels, a concentration of Gly close to the EC50 (6 mm) for GlyRA1 R218Q channels was used to activate Cl- currents.In cells expressing R218Q GlyRA1 subunits, preincubation with 400 nm STR markedly reduced the response to 6 mm Gly (always in the presence of 400 nm STR) (Fig. 3C); the peak amplitude of the Cl- current elicited by 6 mm Gly in the presence of 400 nm STR was 8.0 ± 1.1% that of control (n = 4). This result was very similar to that obtained in cells expressing wtGlyRA1 subunits using the EC50 concentration of 30 μm (Fig. 3C), in which the peak amplitude of the Cl- current elicited by 30 μm Gly in the presence of 400 nm STR was 5.3 ± 1.7% (n = 5) of those recorded in the absence of STR. Measurements of the true equilibrium dissociation constant for STR by the method of Schild (27Colquhoun D. Br. J. Pharmacol. 1998; 125: 924-947Crossref PubMed Scopus (777) Google Scholar, 28Lewis T.M. Sivilotti L.G. Colquhoun D. Gardiner R.M. Schoepfer R. Rees M. J. Physiol. (Lond.). 1998; 507: 25-40Crossref Scopus (96) Google Scholar) were precluded in GlyRA1 R218Q channels due to the very low potency of Gly, requiring the use of concentrations higher than 10 mm to elicit even small currents.Membrane Localization of GlyRA1 Subunits Carrying HE Mutations—To determine whether the HE-causing mutations modified membrane insertion of GlyRA1 subunits, in addition to causing changes in the functional properties of correctly processed channels, Western blots were performed in total cell lysates and streptavidin-isolated plasma membrane proteins from HEK-293 cells transfected with the cDNAs encoding wtGlyRA1 or mutant GlyRs. The mAb4a monoclonal antibody was used, which recognizes an extracellular epitope (amino acids 96–105) shared by all GlyRA subunits; GlyRA1 subunits were revealed by the appearance of an immunoreactive band of an approximate molecular mass of 48 kDa (29Saul B. Kuner T. Sobetzko D. Brune W. Hanefeld F. Meinck H.M. Becker C.M. J. Neurosci. 1999; 19: 869-877Crossref PubMed Google Scholar). This band was not detected in extracts from untransfected HEK-293 cells (data not shown).The intensity of this 48-kDa band did not show significant differences between cells expressing wtGlyRA1 subunits or GlyRA1 subunits carrying the V260M or the Q266H mutation, both in total lysates (Fig. 4A) and in streptavidin-isolated plasma membrane proteins (Fig. 4B). By contrast, cells expressing GlyRA1 R218Q subunits showed a significant decrease in the intensity of this 48-kDa immunoreactive band, both in total lysates (Fig. 4A) and in isolated plasma membrane fractions (Fig. 4B) when compared with wtGlyRA1-expressing cells. In fact, densitometric analysis revealed that the intensity ratios between the 48-kDa band in total lysates and the band corresponding to α-tubulin (∼55 kDa) in total lysates were 2.61 ± 0.94, 1.65 ± 0.45, 1.88 ± 0.64, and 0.46 ± 0.17 for cells transfected with wt, V260M, Q266H, and R218Q GlyRA1 cDNAs, respectively (n = 4; p < 0.05 between R218Q GlyRA1- and wtGlyRA1-transfected cells). Furthermore, the ratios between the intensity of the GlyRA1-specific band in streptavidin-isolated plasma membrane proteins and the band corresponding to α-tubulin in total lysates were 3.67 ± 1.66, 1.96 ± 0.39, 1.74 ± 0.33, and 0.25 ± 0.16 for cells transfected with wt, V260M, Q266H, and R218Q GlyRA1 cDNAs, respectively (n = 5; p < 0.05 between R218Q GlyRA1- and wtGlyRA1-transfected cells).Fig. 4Membrane localization of GlyRA1 subunits carrying HE mutations. Western blot experiments performed in total lysates (A) or streptavidin-purified biotinylated plasma membrane proteins (B) from HEK-293 cells transfected with wt or mutant GlyRA1 cDNAs are shown. The higher and lower blots in each panel were probed with the mAb4a monoclonal antibody recognizing GlyRA1 subunits or with anti-α-tubulin antibodies, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Decreased Subunit Expression Does Not Explain the Reduced Agonist Sensitivity of GlyRA1 R218Q Channels—In Xenopus oocytes, the level of heterologous GlyRs expression affects agonist sensitivity (30Taleb O. Betz H. EMBO J. 1994; 13: 1318-1324Crossref PubMed Scopus (" @default.
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• W2103540557 title "A Novel Hyperekplexia-causing Mutation in the Pre-transmembrane Segment 1 of the Human Glycine Receptor α1 Subunit Reduces Membrane Expression and Impairs Gating by Agonists" @default.
• W2103540557 cites W148303283 @default.
• W2103540557 cites W149795796 @default.
• W2103540557 cites W163136601 @default.
• W2103540557 cites W166408772 @default.
• W2103540557 cites W1788430178 @default.
• W2103540557 cites W1965996399 @default.
• W2103540557 cites W1971422019 @default.
• W2103540557 cites W1993673269 @default.
• W2103540557 cites W1997505926 @default.
• W2103540557 cites W2002822665 @default.
• W2103540557 cites W2003131372 @default.
• W2103540557 cites W2004368544 @default.
• W2103540557 cites W2005131002 @default.
• W2103540557 cites W2005843451 @default.
• W2103540557 cites W2006428029 @default.
• W2103540557 cites W2012679317 @default.
• W2103540557 cites W2027002330 @default.
• W2103540557 cites W2027315787 @default.
• W2103540557 cites W2027426248 @default.
• W2103540557 cites W2030047965 @default.
• W2103540557 cites W2036791268 @default.
• W2103540557 cites W2039598777 @default.
• W2103540557 cites W2041146982 @default.
• W2103540557 cites W2054690264 @default.
• W2103540557 cites W2057242815 @default.
• W2103540557 cites W2079066138 @default.
• W2103540557 cites W2080207066 @default.
• W2103540557 cites W2090552744 @default.
• W2103540557 cites W2091181395 @default.
• W2103540557 cites W2095947640 @default.
• W2103540557 cites W2107191703 @default.
• W2103540557 cites W2116018623 @default.
• W2103540557 cites W2123122813 @default.
• W2103540557 cites W2140556250 @default.
• W2103540557 cites W2148598721 @default.
• W2103540557 cites W2148619485 @default.
• W2103540557 cites W2152052384 @default.
• W2103540557 cites W2162786423 @default.
• W2103540557 cites W2314222561 @default.
• W2103540557 cites W2333135202 @default.
• W2103540557 cites W2407969353 @default.
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