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 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 (1). NMDA receptors are heteromers containing NR1 subunits, which bind the co-agonist glycine (2, 3), and NR2 subunits, which bind the agonist glutamate (4, 5). 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 (5-8), and that together form a clamshell structure that undergoes a conformational change to enclose the ligand upon binding (6, 9, 10).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 (10) and that desensitization in these receptors results from the dissociation of dimers of the ligand-binding domains of adjacent subunits (11). NMDA receptors exhibit apparent desensitization sensitive to glycine or intracellular Ca 2ϩ (1, 12) 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 receptormediated synaptic events (13-16). 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 (17, 18), as well as to a highly conserved motif (YTANLAAF) in the C-terminal portion of the M3 domain preceding the S2 ligand-binding lobe (19). 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 (17,19), 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 (19). 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 (20). We report here that a residue in the M4 domain of the NR2A subunit exerts a powerful regulatory influence on the desens...

A Site of Alcohol Action in the Fourth Membrane-associated Domain of the N-Methyl-D-aspartate Receptor

“…3 shows plots of power density spectra for NMDA-activated current in cells expressing NR1/ NR2A(M823I) and NR1/NR2A(M823L) mutant receptors. As we have observed previously (23) (14). In addition, a variance analysis of NMDAactivated current indicated that the unitary conductance of the ion channel was not changed in the presence of ethanol in receptors containing NR2A(M823I) or NR2A(M823L) mutant subunits (data not shown).…”

“…To test whether this site might participate in ethanol inhibition of NMDA receptors, we examined ethanol sensitivity of substitution mutants at this site. As we have reported previously, a number of amino acid substitutions at this site yielded nonfunctional receptors (23). All of the functional NR2A(Met 823 ) substitution mutant receptors tested were inhibited by ethanol in a concentration-dependent manner (Fig.…”

“…We previously reported that both the rate and extent of desensitization as well as mean open time differed significantly among the various mutants at NR2A(Met 823 ), indicating an important role for this residue in the regulation of ion channel gating (23). A recent report has also shown a role for the M4 domain in regulating desensitization (29 among the remaining mutants, despite the fact that there were still significant differences among these mutants in both ethanol IC 50 and mean open time.…”

“…We hypothesized that there may be an additional site or sites of alcohol action in one of the membrane-associated domains that participates in the regulation of ion-channel gating and that is accessible from the extracellular environment. We have previously identified a methionine residue in M4 of the NR2A subunit (Met 823 ), which has a predicted location close to the extracellular face of the membrane and which exerts a powerful influence on NMDA receptor ion-channel gating (23). We report here that this residue influences NMDA receptor alcohol sensitivity in a manner that is related both to desensitization of the ion channel and to the molecular volume and hydrophobicity of the substituent amino acid.…”

“…1) (23). To test whether this site might participate in ethanol inhibition of NMDA receptors, we examined ethanol sensitivity of substitution mutants at this site.…”