A Site in the Fourth Membrane-associated Domain of the N-Methyl-d-aspartate Receptor Regulates Desensitization and Ion Channel Gating

Abstract

The N-methyl-d-aspartate (NMDA) receptor has four membrane-associated domains, three of which are membrane-spanning (M1, M3, and M4) and one of which is a re-entrant pore loop (M2). The M1–M3 domains have been demonstrated to influence the function of the ion channel, but a similar role for the M4 domain has not been reported. We have identified a methionine residue (Met823) in the M4 domain of the NR2A subunit that regulates desensitization and ion channel gating. A tryptophan substitution at this site did not alter the EC50 for glycine or the peak NMDA EC50 but decreased the steady-state NMDA EC50 and markedly increased apparent desensitization, mean open time, and peak current density. Results of rapid solution exchange experiments revealed that changes in microscopic desensitization rates and closing rates could account for the changes in macroscopic desensitization, steady-state NMDA EC50, and current density. Other amino acid substitutions at this site could increase or decrease the rate of desensitization and mean open time of the ion channel. Both mean open time and desensitization were dependent primarily upon the hydrophobic character of the amino acid at the position. These results demonstrate an important role for hydrophobic interactions at Met823 in regulation of NMDA receptor function. The N-methyl-d-aspartate (NMDA) receptor has four membrane-associated domains, three of which are membrane-spanning (M1, M3, and M4) and one of which is a re-entrant pore loop (M2). The M1–M3 domains have been demonstrated to influence the function of the ion channel, but a similar role for the M4 domain has not been reported. We have identified a methionine residue (Met823) in the M4 domain of the NR2A subunit that regulates desensitization and ion channel gating. A tryptophan substitution at this site did not alter the EC50 for glycine or the peak NMDA EC50 but decreased the steady-state NMDA EC50 and markedly increased apparent desensitization, mean open time, and peak current density. Results of rapid solution exchange experiments revealed that changes in microscopic desensitization rates and closing rates could account for the changes in macroscopic desensitization, steady-state NMDA EC50, and current density. Other amino acid substitutions at this site could increase or decrease the rate of desensitization and mean open time of the ion channel. Both mean open time and desensitization were dependent primarily upon the hydrophobic character of the amino acid at the position. These results demonstrate an important role for hydrophobic interactions at Met823 in regulation of NMDA receptor function. N-methyl-d-aspartate human embryonic kidney 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid analysis of variance The N-methyl-d-aspartate (NMDA)1 receptor is a subtype of ionotropic glutamate receptor that plays essential roles in neuronal development, synaptic plasticity, and several types of neurological disorders (1Dingledine R. Borges K. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar). NMDA receptors are heteromers containing NR1 subunits, which bind the co-agonist glycine (2Lynch D.R. Anegawa N.J. Verdoorn T. Pritchett D.B. Mol. Pharmacol. 1994; 45: 540-545PubMed Google Scholar, 3Kuryatov A. Laube B. Betz H. Kuhse J. Neuron. 1994; 12: 1291-1300Abstract Full Text PDF PubMed Scopus (339) Google Scholar), and NR2 subunits, which bind the agonist glutamate (4Laube B. Hirai H. Sturgess M. Betz H. Kuhse J. Neuron. 1997; 18: 493-503Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, 5Anson L.C. Chen P.E. Wyllie D.A. Colquhoun D. Schoepfer R. J. Neurosci. 1998; 18: 581-589Crossref PubMed Google Scholar). The agonist-binding domains of all ionotropic glutamate receptor subunits consist of two lobes (S1 and S2) that are formed by the region of the extracellular N-terminal domain preceding the first membrane-associated (M) domain and the loop between the M3 and M4 domain, respectively (5Anson L.C. Chen P.E. Wyllie D.A. Colquhoun D. Schoepfer R. J. Neurosci. 1998; 18: 581-589Crossref PubMed Google Scholar, 6Stern-Bach Y. Bettler B. Hartley M. Sheppard P.O. O'Hara P.J. Heinemann S.F. Neuron. 1994; 13: 1345-1357Abstract Full Text PDF PubMed Scopus (398) Google Scholar, 7Wafford K.A. Kathoria M. Bain C.J. Marshall G., Le Bourdellès B. Kemp J.A. Whiting P.J. Mol. Pharmacol. 1995; 47: 374-380PubMed Google Scholar, 8Hirai H. Kirsch J. Laube B. Betz H. Kuhse J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6031-6036Crossref PubMed Scopus (197) Google Scholar), and that together form a clamshell structure that undergoes a conformational change to enclose the ligand upon binding (6Stern-Bach Y. Bettler B. Hartley M. Sheppard P.O. O'Hara P.J. Heinemann S.F. Neuron. 1994; 13: 1345-1357Abstract Full Text PDF PubMed Scopus (398) Google Scholar, 9Armstrong N. Sun Y. Chen G.Q. Gouaux E. Nature. 1998; 395: 913-917Crossref PubMed Scopus (611) Google Scholar, 10Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar). Observations from x-ray crystallographic studies on non-NMDA glutamate receptor constructs suggest that the degree of binding domain closure induced by a particular agonist appears to determine both the degree of receptor activation and the extent of desensitization produced by that agonist (10Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar) and that desensitization in these receptors results from the dissociation of dimers of the ligand-binding domains of adjacent subunits (11Sun Y. Olson R. Horning M. Armstrong N. Mayer M. Gouaux E. Nature. 2002; 417: 245-253Crossref PubMed Scopus (603) Google Scholar). NMDA receptors exhibit apparent desensitization sensitive to glycine or intracellular Ca2+ (1Dingledine R. Borges K. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar, 12McBain C.J. Mayer M.L. Physiol. Rev. 1994; 74: 723-760Crossref PubMed Scopus (0) Google Scholar) in addition to true glycine-insensitive desensitization (subsequently referred to simply as desensitization). NMDA receptor desensitization is physiologically relevant, as it can influence the amplitude, duration, and following frequency of NMDA receptor-mediated synaptic events (13Tong G. Shepherd D. Jahr C.E. Science. 1995; 267: 1510-1512Crossref PubMed Scopus (364) Google Scholar, 14Jones M.V. Westbrook G.L. Trends Neurosci. 1996; 19: 96-101Abstract Full Text PDF PubMed Scopus (410) Google Scholar, 15Lester R.A.J. Jahr C.E. J. Neurosci. 1992; 12: 635-643Crossref PubMed Google Scholar, 16Lieberman D.N. Mody I. Nature. 1994; 369: 235-239Crossref PubMed Scopus (440) Google Scholar). Determinants of NMDA receptor desensitization have been localized to two regions in or near the S1 ligand-binding site in the N-terminal domain, one that shows homology to leucine/isoleucine/valine-binding proteins and one located in the region immediately preceding the M1 domain (17Krupp J.J. Vissel B. Heinemann S.F. Westbrook G.L. Neuron. 1998; 20: 317-327Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 18Villarroel A. Regalado M.P. Lerma J. Neuron. 1998; 20: 329-339Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), as well as to a highly conserved motif (YTANLAAF) in the C-terminal portion of the M3 domain preceding the S2 ligand-binding lobe (19Kohda K. Wang Y. Yuzaki M. Nat. Neurosci. 2000; 3: 315-322Crossref PubMed Scopus (182) Google Scholar). The pre-M1 and M3 domains have been suggested to be involved in transducing the conformational changes induced by agonist binding into those responsible for ion channel gating (17Krupp J.J. Vissel B. Heinemann S.F. Westbrook G.L. Neuron. 1998; 20: 317-327Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 19Kohda K. Wang Y. Yuzaki M. Nat. Neurosci. 2000; 3: 315-322Crossref PubMed Scopus (182) Google Scholar), but the nature of the conformational changes and molecular determinants underlying NMDA receptor ion channel gating remain unclear. The YTANLAAF motif in M3 has been shown to play an important role in the regulation of ion channel gating, as a point mutation in this region of the NR1 subunit increases mean open time in receptors formed from coexpression with NR2A subunits (19Kohda K. Wang Y. Yuzaki M. Nat. Neurosci. 2000; 3: 315-322Crossref PubMed Scopus (182) Google Scholar). Mutations at a tryptophan residue in the M2 domain of the NR1 or NR2A subunit have also been reported to subtly affect mean open time and opening frequency (20Buck D.P. Howitt S.M. Clements J.D. Biophys. J. 2000; 79: 2454-2462Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). We report here that a residue in the M4 domain of the NR2A subunit exerts a powerful regulatory influence on the desensitization and gating of the NMDA receptor ion channel. Site-directed mutagenesis in plasmids containing NR1-1a and NR2A subunit cDNA (Drs. D. R. Lynch, University of Pennsylvania; and D. M. Lovinger, Vanderbilt University) was performed using the QuikChange kit (Stratagene, La Jolla, CA), and all mutations were verified by DNA sequencing. Human embryonic kidney (HEK) 293 cells were seeded in 35-mm dishes, allowed to grow to 70–95% confluence, and transfected with cDNA for the wild-type or mutant NR1 and NR2A subunits and green fluorescent protein at a 2:2:1 ratio, respectively, using LipofectAMINE 2000 or calcium phosphate (both from Invitrogen). The culture medium during and after the transfection step contained 100 μmketamine and 200 acid to to were in experiments after and were performed as by Chen T. J. Neurosci. 1999; 19: PubMed Google Scholar) and and J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), 293 cells were with and with the in A and in for at were and with A containing 100 glycine for at and in and for were with in for at The was at for at and the was with in B. were to and protein acid protein and the was with at and the were to were to and for in with containing were with in with at with the were with as the for were by and using and was performed at using an or were formed using with of and of were by cells were the of the after a to increase the of the solution in these were from and of were with and of following were at were for and for and at on a using a and were at in extracellular medium containing and the was to using and the was to using The solution in contained and the was to using and the was to using from the solution contained and in extracellular of and were in extracellular medium and to cells using a rapid solution exchange the by the and The time for solution exchange in cells was which was using from to in the of of amino were using following using the for amino acid and were reported Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. J. Mol. PubMed Scopus Google Scholar, N. R. J. PubMed Scopus Google Scholar). were using the A. P.J. D. J. Physiol. PubMed Google Scholar), which of from by the in of an for the of of the that results from these to be reported for and are those by the to and are and respectively, and is the analysis of of were were and the were with a function of the form shown in is the at is the frequency and is the were from the current and variance of the were using in were from the is the and is the of the to the region of a current from were using the A. J. Physiol. PubMed Scopus Google Scholar), at which a These were at to of and using similar results were using and a were to time using and were to using was to for as is for NMDA receptors Colquhoun D. J. Physiol. 1992; PubMed Scopus Google Scholar). in rapid solution exchange experiments were to a in which A and the and and of the channel, this and are the agonist binding and and are the channel opening and closing and are the rates for into and from the from rapid solution exchange experiments were to the using the CA), to the in in which NMDA receptor subunit have been B. Chen N. T. Y. Nat. Neurosci. 2002; PubMed Scopus Google Scholar), J.D. Westbrook G.L. Neuron. Full Text PDF PubMed Scopus Google with to the of the mean open time from analysis and for receptors containing NR2A and subunits, and with all rates allowed to current were using the with all in the to from to the of variance and analysis were performed using the was to determine following are reported as the The results of a have demonstrated that in the M4 domain of the NMDA receptor NR1 subunit are not to the of the ion channel B. T. Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of these to as the and hydrophobic amino be the substitution to interactions J. Physiol. 1999; PubMed Scopus Google Scholar), produced or change in receptor function by apparent not with the of the which was methionine residue is conserved in NMDA receptor NR1 and NR2 subunits tryptophan substitution of the residue in the NR2A subunit a on receptor function. A current by of NMDA in receptors of wild-type subunits and subunits is the mutant a degree of apparent desensitization at all NMDA The mutation did not alter the EC50 of NMDA for activation of peak current but the EC50 of NMDA for activation of steady-state current the for glycine was not by the mutant The mutation did not alter the EC50 of glycine for activation of peak current or steady-state current mutation the EC50 for steady-state not by of NMDA and glycine in 293 cells or The time and current for to all for NMDA the of in 293 cells wild-type or mutant are the of four to and the are to for glycine the of in 293 cells or are the of three to and the are to the mutation the apparent desensitization of the We the of the mutation on the desensitization of current by of NMDA in cells using a solution exchange the of cells wild-type NR2A or mutant subunits, desensitization of current following of NMDA two The time for the was not by NMDA in wild-type or mutant receptors and did not between wild-type and mutant receptors of desensitization was to the of Traynelis S.F. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, K. T. C.J. P.J. Traynelis S.F. Nat. Neurosci. PubMed Scopus Google Scholar), as it was in the of not the time for the was dependent upon NMDA in receptors containing both wild-type and mutant subunits and was in receptors with wild-type receptors determine the mutation could alter ion channel gating in addition to desensitization, in from cells wild-type or mutant receptors at was not by the mutation for wild-type in from cells wild-type receptors open time that could be with three with time of and time of in cells receptors four with time of and The first three time in containing receptors did not from the in cells wild-type receptors the of the in a mean open time of which was increased with the of in wild-type were in experiments using analysis of current by NMDA at of revealed that the of the ion channel was not by the mutation for mutant and wild-type of to of current mean open of for wild-type receptors and for mutant receptors in the shown from two cells in the mean open time of NMDA receptors containing the mutant was increased with that of wild-type receptors the in analysis the open time in and the of the open time these the in analysis to primarily the of the two time These well with the of the and open time in analysis and for mutant and wild-type The for wild-type or mutant receptors in exchange experiments could be to a for rate from the of the to the that the mutation increased the rate of into the for mutant and wild-type and decreased the rate of from this for mutant and wild-type but did not alter the rates of ion channel opening for mutant and wild-type or agonist for mutant and wild-type of from the using the from to the that the can account not for the increase in macroscopic desensitization produced by the but also for the increase in peak current and decrease in steady-state current in cells the mutant subunit increase in peak current was not to an increase in receptor as the mutation did not alter receptor as in experiments an to the of the at the NR2A Met823 site responsible for the gating of the ion channel, and a of substitution at this of highly and or glycine at this site did not was for the a of the mutant subunits in the EC50 for NMDA activation of steady-state the steady-state current ratio, and the mean open time the NMDA activation of peak current and the were The to this was the which a of and an increased EC50 for NMDA activation of peak current with wild-type The mean open time of receptors with that of wild-type receptors which could account for an apparent in to the of channel opening events to the time of the analysis of the results with the of NR2A Met823 revealed a between the steady-state current and the amino acid but not molecular or and the mean open time of the of was to as well as to but not to molecular or and was an apparent an between steady-state current and mean open time in the NR2A Met823 these were not The for the steady-state NMDA EC50 highly with steady-state current was between the for the peak NMDA EC50 and steady-state current of NMDA receptors containing NR2A substitution open from for the wild-type receptors from for the wild-type receptors from for the wild-type receptors from for the wild-type receptors from for the wild-type receptors from for the wild-type receptors from for the wild-type receptors from for the wild-type receptors from for the wild-type receptors from for the wild-type receptors from for the wild-type receptors from for the wild-type receptors from for the wild-type receptors from for the wild-type receptors from for the wild-type receptors from for the wild-type receptors from for the wild-type receptors and steady-state and peak current are the open were for steady-state current by or NMDA in the of of or at this from for the wild-type receptors in a between NMDA receptor desensitization and mean open time or NMDA The of current by NMDA and glycine the mean open time of by and glycine or the EC50 for NMDA activation of peak or steady-state current and steady-state and peak current are the open were for steady-state current by or NMDA in the of of or at this this mutation of a highly conserved methionine residue (Met823) in the NR2A subunit of the NMDA receptor to tryptophan increased the rate of macroscopic desensitization as well as the mean open time of the ion channel. wild-type the extent of desensitization at and the time of desensitization at were similar to those similar and (17Krupp J.J. Vissel B. Heinemann S.F. Westbrook G.L. Neuron. 1998; 20: 317-327Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, K. T. C.J. P.J. Traynelis S.F. Nat. Neurosci. PubMed Scopus Google Scholar, C.J. G.L. J. 1999; PubMed Scopus Google Scholar). The of the two of apparent desensitization was primarily to the of in the extracellular as was in the reported for apparent desensitization to K. T. C.J. P.J. Traynelis S.F. Nat. Neurosci. PubMed Scopus Google Scholar), and as it was not in the of the of desensitization was also not by the the of desensitization was markedly by the A in the rates of into and from the in receptors containing the mutant was also the were to a it is that the mutation have a this have been as an of desensitization as a change in the rate of an residue in the M4 domain appears to be in the of the ion channel into and of the NR2A Met823 regulates the rate of ion channel as was in analysis experiments in which the mutation increased the mean open time of the ion channel. Results from that this increase in mean open time in receptors containing the mutant was to the of an open time not in wild-type Results of studies a role for mean open time and desensitization of NMDA receptors in the regulation of synaptic in the of NMDA receptors can changes in mean open time that affect the of NMDA receptor-mediated synaptic events R. Science. 1997; PubMed Scopus Google D.N. Mody I. Nat. Neurosci. 1999; PubMed Scopus Google Scholar), and as that decrease NMDA receptor mean open time PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar) the of the NMDA receptor of G. J. Neurosci. PubMed Google Scholar). NMDA receptor desensitization can influence the and following frequency as well as the of synaptic events (13Tong G. Shepherd D. Jahr C.E. Science. 1995; 267: 1510-1512Crossref PubMed Scopus (364) Google Scholar, 14Jones M.V. Westbrook G.L. Trends Neurosci. 1996; 19: 96-101Abstract Full Text PDF PubMed Scopus (410) Google Scholar, 15Lester R.A.J. Jahr C.E. J. Neurosci. 1992; 12: 635-643Crossref PubMed Google Scholar, 16Lieberman D.N. Mody I. Nature. 1994; 369: 235-239Crossref PubMed Scopus (440) Google Scholar). the time of desensitization by the mutation in this is to that of the glutamate in the synaptic J.D. R.A.J. G. Jahr C.E. Westbrook G.L. Science. 1992; PubMed Scopus Google Scholar), this of desensitization in the regulation of NMDA receptor function. NMDA receptor channel gating the dissociation of glutamate R.A.J. Jahr C.E. J. Neurosci. 1992; 12: 635-643Crossref PubMed Google Scholar, R.A.J. Clements J.D. Westbrook G.L. Jahr C.E. Nature. PubMed Scopus Google Scholar), with the that the time for of NMDA receptor-mediated synaptic can be in the R.A.J. Clements J.D. Westbrook G.L. Jahr C.E. Nature. PubMed Scopus Google Scholar, S. J. Physiol. PubMed Scopus Google Scholar). time for from desensitization S. Wang Wang D.R. J. 1998; 79: PubMed Scopus Google Scholar). The of the of NMDA receptor desensitization to regulation of synaptic be to increase with receptor as during which could a of the receptors to in the The a between desensitization and open time in the NR2A Met823 in this in the the channel is the it is to the to with the molecular of desensitization for non-NMDA glutamate receptors (11Sun Y. Olson R. Horning M. Armstrong N. Mayer M. Gouaux E. Nature. 2002; 417: 245-253Crossref PubMed Scopus (603) Google Scholar). The of a between steady-state current and mean open time in the that as desensitization and open time at this Mutations at NR2A Met823 did not influence the NMDA EC50 for peak with the of the in which it is that the of ion channel opening in this mutant was responsible for the increase in the NMDA EC50 for activation of peak current to a influence of ion channel gating on agonist binding D. J. Pharmacol. 1998; PubMed Scopus Google Scholar). to the results for the NMDA EC50 for peak of NR2A Met823 mutations decreased the NMDA EC50 for steady-state current The decreased steady-state EC50 were to the increased desensitization in these mutant as were highly with steady-state current and were also by the not this is the increase in apparent for a degree of desensitization from an increase in the of agonist on receptors B. S. J. Physiol. PubMed Scopus Google Scholar), as has been in acid A receptors M.V. Westbrook G.L. Neuron. 1995; Full Text PDF PubMed Scopus Google Scholar), from an increase in the of the binding site for is also with the of between peak and steady-state NMDA in the the of between NMDA EC50 for peak current and steady-state current and the of a change in the agonist rate using The results of using a of of amino were with an important influence of the of the at NR2A Met823 upon both mean open time and hydrophobic interactions between NR2A Met823 and a residue or region of the protein the ion channel in the and The that the molecular of the did not influence in a that the regulation by the residue at this site on ion channel gating not a as appears to be the for similar in acid A and glycine receptors S. M.P. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, A. N. A. Mol. Pharmacol. 1999; PubMed Scopus Google Scholar, T. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. E. A. A. A. A. J. Neurosci. Google Scholar). The of the of an for molecular at this at which desensitization is The of an between ion channel open time and desensitization in the is with a influence of this site on the rates for the and of the ion channel. is that these be to of the amino acid at the which are not well by as molecular and of the A the of in the region of M4 of the NR1 subunit B. T. Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) did not report in ion channel function following substitution at NR1 the site to NR2A the substitution of a tryptophan at this in tryptophan is the and hydrophobic amino the of function of this mutant NR1 subunit is to an of this to the increase in molecular or with the that tryptophan substitutions at the adjacent in the NR1 subunit did not alter receptor that this residue influence ion channel not in a to that of site in the NR2A Results of studies on the NR1 subunit B. T. Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Neuron. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) suggest that of the N-terminal of the region identified as M4 is located of the NR2A Met823 be located to the extracellular of the be upon in the of the M4 that the of the M4 domain that the is in NR2A Met823 is one or of the membrane-associated domains, the changes in the residue at this could influence ion channel function in a that is with a but to for a the results of this demonstrate that this site the of the ion channel gating in the pre-M1 and M3 domains also influence ion channel gating and desensitization and be involved in transducing agonist binding into ion channel gating (17Krupp J.J. Vissel B. Heinemann S.F. Westbrook G.L. Neuron. 1998; 20: 317-327Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 19Kohda K. Wang Y. Yuzaki M. Nat. Neurosci. 2000; 3: 315-322Crossref PubMed Scopus (182) Google Scholar). non-NMDA glutamate agonist binding appears to a of the S2 ligand-binding domain to S1 (11Sun Y. Olson R. Horning M. Armstrong N. Mayer M. Gouaux E. Nature. 2002; 417: 245-253Crossref PubMed Scopus (603) Google Scholar), which a on the M3 domain that results in ion channel results from of dimers of the S1 domains, the on the M3 domain and the channel to (11Sun Y. Olson R. Horning M. Armstrong N. Mayer M. Gouaux E. Nature. 2002; 417: 245-253Crossref PubMed Scopus (603) Google Scholar). similar conformational changes ion channel gating and desensitization in NMDA the results of the that NR2A Met823 ion channel gating hydrophobic interactions with an adjacent site in M3 or membrane-associated We and for and and and for on the