Probing N-methyl-D-aspartate receptor desensitization with the substituted-cysteine accessibility method.
Several forms of macroscopic N-methyl-D-aspartate (NMDA) receptor desensitization affect the amplitude and duration of postsynaptic responses. In addition to its functional significance, desensitization provides one means to examine the conformational coupling of ligand binding to channel gating. Segments flanking the ligand binding domain in the extracellular N terminus of the NMDA receptor NR2 subunit influence the glycine-independent form of desensitization. The NR2A pre-M1 region, the linker between the glutamate binding domain and the channel pore, plays a critical role in desensitization. Thus, we used the substituted-cysteine accessibility method to scan the accessibility of residues in the pre-M1 region and the first transmembrane domain (M1) of NR2A. Cysteine mutants were expressed with NR1 in human embryonic kidney 293 cells and were assayed by whole-cell recording. With activation of the receptor by glutamate and glycine, only a single mutant, V557C, which is located at the beginning of M1, led to irreversible inhibition by the methanethiosulfonate derivative methanethiosulfonate ethyltrimethylammonium (MTSET). The NR2 ligand glutamate was insufficient on its own to induce modification of V557C by MTSET, suggesting that the change in accessibility required channel gating. The rate of MTSET modification of the homologous residue on NR1 (NR1-1a(L562C)/NR2A) was much slower than V557C. We also substituted cysteine in the V557 site of mutant subunits that exhibit either enhanced or reduced desensitization. Modification by MTSET correlated with the degree of desensitization for these subunits, suggesting that V557C is a sensitive detector of desensitization gating.
Thomas
Christopher G
CG
Vollum Institute, Oregon Health and Science University, L474, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA. b
Krupp
Johannes J
JJ
Bagley
Elena E
EE
Bauzon
Reginald
R
Heinemann
Stephen F
SF
Vissel
Bryce
B
Westbrook
Gary L
GL
D002460:Cell Line; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D015640:Ion Channel Gating; D008024:Ligands; D011485:Protein Binding; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism
D008024:Ligands; D016194:Receptors, N-Methyl-D-Aspartate; D003545:Cysteine