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

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; both and inherited as as mutations in for the hypothesis that GlyR function neurological from the that the of S.G. L. R. Wasmuth J.J. P. O'Connell P. Nat. Genet. PubMed Scopus Google Scholar), B. D. Nat. Genet. PubMed Scopus Google Scholar), and Genet. 3: PubMed Scopus Google all of which carry in a to of the of human the present study, we have characterized the functional properties of three GlyRA1 mutations associated with The R218Q mutation, by members of our G. G. P. A. J. Genet. 2003; PubMed Google Scholar), a residue that is located at the the and and is subunits both and and and and The V260M G. G. G. G. A. J. Genet. PubMed Scopus Google and the Q266H N. L. A. R. L. J. Genet. Google mutations located in the of GlyRA1 subunits but are not believed to in permeation (5Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 424: 949-955Crossref Scopus (1074) Google J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar); are LGIC have used and molecular to the molecular basis of three human and that all three may a of in GlyR gating, with the dramatic associated with the R218Q of were the human GlyRA1 the by with the as P. G. A. L. J. 2002; PubMed Google Scholar). were an of GlyRA1 (wt) and mutant GlyRA1 subunits were expressed of human HEK-293 were in amino acids and in an at with in the were on the were with of the to the with of a encoding the used as a of GlyRA1 were used for the of was of the were were from HEK-293 at a The cell of the was of of G. P. G. L. L. A. 2003; PubMed Scopus Google Scholar). The and and with and with respectively. The was used for and were with the were by of a to a and a within μm of the cell and membrane expression of GlyRA1 subunits in HEK-293 was by of membrane proteins in a cell to the H. J. 2002; PubMed Google Scholar). the were and proteins were isolated by cell lysates with GlyR α subunits in and total lysates were in an an was used as were by a that proteins were not by the were and with an molecular model of a GlyRA1 subunit was a model of the α1 subunit as a PubMed Google A. J.C. Nature. 2003; PubMed Scopus Google 2002; PubMed Scopus Google J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). to in the the GlyRA1 and the and the in the model the in the of as PubMed Google J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The of each GlyR residue were to the of in the with a of and the was with the of were with a of one and a at A. The was by of molecular at and was with the to a of of HE-causing topological of a GlyR α1 the the and the is in the functional consequences of the HE-causing GlyRA1 mutations we HEK-293 with the encoding wtGlyRA1 or R218Q, V260M, or Q266H GlyRA1 and Cl- currents by the expressed channels with the agonist In cells, with concentrations of μm Cl- currents when the cell was at the of currents the Cl- not The of Gly-gated Cl- currents increased with up to concentrations to although the of Gly-gated of Gly-gated currents were to the expressed as a function of and to a of the The for was 26 with a of with those P. Pharmacol. PubMed Scopus Google C.M. A. J. 2003; Full Text Full Text PDF PubMed Scopus Google sensitivity of channels by GlyRA1 subunits HE-causing of the topological of a GlyRA1 subunit (1Breitinger H.G. Becker C.M. ChemBioChem. 2002; 3: 1042-1052Crossref PubMed Scopus (73) Google Scholar). cell to in HEK-293 and R218Q GlyRA1 In this and the the was and the on of the the of for V260M Q266H and R218Q GlyRA1 The the of the to the of the EC50s and for each are is the of in showing elicited by to in wtGlyRA1 and GlyRA1 R218Q have been to of the R218Q mutation on the of channel and in the of is the of in of HEK-293 with R218Q GlyRA1 to the of Gly-gated Cl- currents although the agonist sensitivity of currents was decreased when compared with that of wtGlyRA1 concentrations were to Cl- currents in R218Q analysis that the GlyRA1 channels the R218Q mutation a decrease in sensitivity when compared with wtGlyRA1 channels of with no apparent in the In the Cl- in with the R218Q GlyRA1 was decreased when compared with that of two other GlyRA1 V260M and caused a more in the for the agonist was μm for V260M and 129 μm for Q266H in this no were in the of V260M and Q266H GlyRA1 receptors when compared with wtGlyRA1 the molecular by the R218Q mutation, we the of channel and by with a function the of agonist the of agonist and the agonist in both wtGlyRA1 and R218Q of properties that wtGlyRA1 receptors an increased and a during agonist when compared with GlyRA1 R218Q channels and in the of the agonist and of HE-causing GlyRA1 acids such as β-alanine and taurine behave as agonists at but to be of in mutant as agonists J. Betz H. Pharmacol. PubMed Scopus Google D. B. N. J. Betz H. J. PubMed Scopus Google Scholar). in the Cl- currents elicited by to or in wtGlyRA1 receptors were similar in to those elicited by an and the currents elicited by both and in GlyRA1 the V260M or the Q266H mutation were those by an of in the In GlyRA1 subunits the R218Q mutation, or to Cl- currents In the of the cells, with a to Cl- In the only three in which currents were the was in cells, to Cl- currents and β-alanine sensitivity of GlyRA1 cell to the of Gly, TAU, and at in the cell or mutant GlyRA1 and and in and GlyRA1 as The of the elicited by and wt, V260M, and Q266H) or by and are in and respectively. is the of in of GlyRA1 the R218Q is an antagonist of Gly, which is known to at that the agonist binding on the GlyR (1Breitinger H.G. Becker C.M. ChemBioChem. 2002; 3: 1042-1052Crossref PubMed Scopus (73) Google B. Maksay G. Schemm R. Betz H. Trends Pharmacol. Sci. 2002; 23: 519-527Abstract Full Text Full Text PDF PubMed Scopus (202) Google J. Betz H. Pharmacol. PubMed Scopus Google Scholar). The of is in Cl- currents by channels of wtGlyRA1 subunits were by μm Gly, an agonist to the agonist caused a of the Gly-gated Cl- which were The of Cl- the of antagonist binding to its on the which antagonist concentrations Cl- although the to was not of STR, and in the of the the Cl- the of of the antagonist from its a the was not inhibit the in wtGlyRA1 channels, with the of of and R218Q GlyRA1 channels by cell to in wtGlyRA1 In the with an was with in the with a no was with cell response to in a cell R218Q GlyRA1 was with cell response to μm in a cell wtGlyRA1 cell response to μm in the for with cell response to in the cell R218Q GlyRA1 subunits in or for with The were in at three cells, with comparable GlyRA1 channels the R218Q mutation, the Cl- currents by Gly, with similar to those observed in wtGlyRA1 In similarly to those for wtGlyRA1 channels, a of to the for GlyRA1 R218Q channels was used to Cl- R218Q GlyRA1 subunits, with reduced the response to in the of the of the Cl- elicited by in the of was that of This was similar to that in wtGlyRA1 subunits the of μm in which the of the Cl- elicited by μm in the of was of those in the of of the for by the of D. J. Pharmacol. PubMed Scopus Google D. R. J. Scopus Google were in GlyRA1 R218Q channels to the low of Gly, the of concentrations to of GlyRA1 HE determine the HE-causing mutations membrane of GlyRA1 subunits, in to causing in the functional properties of channels, were in total cell lysates and plasma membrane proteins from HEK-293 with the encoding wtGlyRA1 or mutant The was which an acids by all GlyRA1 subunits were by the of an of an molecular of B. D. Becker C.M. J. PubMed Google Scholar). This was not in from HEK-293 not of this not wtGlyRA1 subunits or GlyRA1 subunits the V260M or the Q266H mutation, both in total lysates and in plasma membrane proteins GlyRA1 R218Q subunits a decrease in the of this both in total lysates and in isolated plasma membrane when compared with In analysis that the the in total lysates and the to in total lysates were and for with wt, V260M, and R218Q GlyRA1 R218Q and the the of the in plasma membrane proteins and the to in total lysates were and for with wt, V260M, and R218Q GlyRA1 R218Q and localization of GlyRA1 subunits HE in total lysates or plasma membrane proteins from HEK-293 with or mutant GlyRA1 are The and in each were with the GlyRA1 subunits or with the of GlyRA1 R218Q the of GlyRs expression agonist sensitivity O. Betz H. J. PubMed Scopus Google D. B. H. P. J. Scopus Google Scholar). In when are expressed at an increased for as as for other agonists such as or the in the present the reduced sensitivity of mutant R218Q receptors have been to a decreased membrane expression of this we decreased membrane expression of wild-type GlyRA1 to the levels of those observed with the R218Q by to the of wtGlyRA1 used to HEK-293 on total lysates and membrane proteins from with concentrations that we a expression of wtGlyRA1 subunits comparable with that of GlyRA1 R218Q mutant subunits when the with of wtGlyRA1 and with and currents not in the apparent sensitivity to when compared with of GlyRA1 receptors of of Gly-gated for the hypothesis that the reduced agonist sensitivity of receptors the R218Q mutation be by a reduced membrane expression of mutant This is supported by the in HEK-293 wtGlyRA1 subunits at a comparable with those of GlyRA1 R218Q subunits the was of each and of each not are similar to those observed in wtGlyRA1 receptor and from those of of the functional consequences of mutations causing human of the and functional determinants of the proteins. In the present study, we have the functional consequences of three GlyRA1 mutations causing The of three is in a molecular model of a GlyRA1 subunit of V260M G. G. G. G. A. J. Genet. PubMed Scopus Google and Q266H N. L. A. R. L. J. Genet. Google Scholar), located in the the R218Q G. G. P. A. J. Genet. 2003; PubMed Google a residue located at the the N and the segment model of a GlyR α1 subunit. the three amino acid at which mutations were are with and are and respectively. β are as and the is as a to transmembrane PubMed Google Scholar). is in to the of the glycine agonist binding an of the the binding and transmembrane acid that may with Arg-218 are in and α are as The of is from the of the binding K. J. Nature. PubMed Scopus Google V260M mutant GlyRA1 receptors a in the for a similar was observed in Q266H GlyRA1 mutant as P. Pharmacol. PubMed Scopus Google Scholar). in agonist sensitivity be caused by in agonist binding or by properties D. J. Pharmacol. PubMed Scopus Google Scholar). The decreased of concentrations of acids and that the agonist binding and channel is in GlyRA1 that two may in the molecular that during the C.M. A. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). are when compared with those by mutations in other the first HE mutation to be identified (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 D. B. N. J. Betz H. J. PubMed Scopus Google with the R218Q G. G. P. A. J. Genet. 2003; PubMed Google were The R218Q mutation caused a decrease in the apparent of at its In channels, concentrations of and were virtually to Cl- in the agonist binding site, but the that Gly-gated currents were similarly antagonized by low concentrations of the antagonist in both and R218Q GlyRA1 channels that at the agonist/antagonist binding be The of and was reduced to in R218Q GlyRA1 channels, suggesting a in the by the R218Q mutation in a residue to the segment of the GlyRA1 subunit. be of and agonist concentrations up to were used in we the that and concentrations have elicited agonist in R218Q GlyRA1 at the both and of wtGlyRA1 channels were those observed in GlyRA1 R218Q channels, with no in are with the hypothesis of a of the R218Q mutation on channel since at agonist the the channel the channel and for receptors A. G. J. PubMed Scopus Google Scholar), that an in the channel the of receptors the from the with the has been to explain the mutation of an residue located the residue to Arg-218 in GlyRA1 receptors in of the LGIC the receptor L. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The of GlyRA1 by the R218Q mutation is supported by our molecular model in which Arg-218 is located a from the agonist binding is to J. Betz H. 1993; PubMed Scopus Google Scholar). In the the of and Arg-218 is in our Arg-218 to other that have been in the (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 D. B. N. J. Betz H. J. PubMed Scopus Google Scholar), and C.M. A. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The function of Arg-218 be from this but are located to Arg-218 and with during the as has been for other within the of the receptor A. J.C. Nature. 2003; PubMed Scopus Google Scholar). is that this residue is subunits both and receptors within the LGIC that is to play a or functional to those mutations that functional in channels that have been the plasma human mutations may channel function by with the of membrane and P. J. Full Text PDF PubMed Scopus Google J. Full Text Full Text PDF PubMed Scopus Google Scholar). that the levels of R218Q GlyRA1 subunit were decreased when compared with wtGlyRA1 subunits, the levels of the V260M or Q266H subunit were as for HE-causing GlyRA1 mutant located in the the transmembrane and B. D. Becker C.M. J. PubMed Google Scholar). in to gating, the R218Q mutation GlyR function by the of receptors the possibly of a decreased increased of the receptor N. A. J. G. Betz H. J. PubMed Scopus Google D. A. J. Kirsch J. 1993; PubMed Scopus Google Scholar). be and at the plasma membrane A. B. N. Betz H. G. J. Full Text Full Text PDF PubMed Scopus Google Scholar). be of to GlyRA1 subunits the R218Q mutation an as a of an increased plasma membrane by the be although in expression GlyR agonist sensitivity O. Betz H. J. PubMed Scopus Google D. B. H. P. J. Scopus Google Scholar), the reduced sensitivity of R218Q mutant receptors to not to be a of the decreased expression of mutant subunits in HEK-293 In agonist sensitivity of was when receptors were expressed at levels comparable with those of the R218Q the that the currents from GlyRA1 R218Q mutant subunits are those from a comparable of wtGlyRA1 subunits to that the R218Q mutation channel or although this to be compared with other GlyRA1 mutations HE-affected the R218Q mutation the dramatic of the in of its effects on the receptor function and the clinical of the disease in the this mutation at one of an of in in to the HE the a of in at of G. G. P. A. J. Genet. 2003; PubMed Google Scholar). This is of in of the that is usually to be in HE (6Zhou L. Chillag K.L. Nigro M.A. Brain Dev. 2002; 24: 669-674Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and no be identified that may explain the of our G. G. P. A. J. Genet. 2003; PubMed Google Scholar). In a similar of a by a severe of HE B. K. Becker C.M. J. Genet. Google Scholar), a decrease of the HE with but during The GlyRA1 mutation in this was a of of the GlyRA1 gene, which in a of This is in with our in the R218Q in which a decrease in of the molecular of the be as a functional GlyRA1 GlyRA1 subunits, in to role in and spinal of muscle in in GlyRs have been characterized in which play a role in and by of with the and the PubMed Scopus Google Scholar), and function be by (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). mechanisms for the of GlyRA1 subunits seem to more in in The mutation in the Genet. 3: PubMed Scopus Google a of GlyR in the central nervous system B. Becker C.M. PubMed Scopus Google Scholar), and all the conclusion, our analysis of the molecular associated with three HE-causing mutations in GlyRA1 subunits a hypothesis for LGIC subunits, in which distinct in agonist and antagonist binding, gating, and (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), and a role for the Arg-218 residue in GlyR This mutation has a dramatic on of GlyR function and is associated with a more severe clinical possibly a role of GlyRA1 subunits in in 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; both and inherited as as mutations in for the hypothesis that GlyR function neurological from the that the of S.G. L. R. Wasmuth J.J. P. O'Connell P. Nat. Genet. PubMed Scopus Google Scholar), B. D. Nat. Genet. PubMed Scopus Google Scholar), and Genet. 3: PubMed Scopus Google all of which carry in a to of the of human the present study, we have characterized the functional properties of three GlyRA1 mutations associated with The R218Q mutation, by members of our G. G. P. A. J. Genet. 2003; PubMed Google Scholar), a residue that is located at the the and and is subunits both and and and and The V260M G. G. G. G. A. J. Genet. PubMed Scopus Google and the Q266H N. L. A. R. L. J. Genet. Google mutations located in the of GlyRA1 subunits but are not believed to in permeation (5Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 424: 949-955Crossref Scopus (1074) Google J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar); are LGIC have used and molecular to the molecular basis of three human and that all three may a of in GlyR gating, with the dramatic associated with the R218Q