NMDA channel gating is influenced by a tryptophan residue in the M2 domain but calcium permeation is not altered.
N-Methyl-D-aspartate (NMDA) receptors are susceptible to open-channel block by dizolcipine (MK-801), ketamine and Mg(2+) and are permeable to Ca(2+). It is thought that a tryptophan residue in the second membrane-associated domain (M2) may form part of the binding site for open-channel blockers and contribute to Ca(2+) permeability. We tested this hypothesis using recombinant wild-type and mutant NMDA receptors expressed in HEK-293 cells. The tryptophan was mutated to a leucine (W-5L) in both the NMDAR1 and NMDAR2A subunits. MK-801 and ketamine progressively inhibited currents evoked by glutamate, and the rate of inhibition was increased by the W-5L mutation. An increase in open channel probability accounted for the acceleration. Fluctuation analysis of the glutamate-evoked current revealed that the NMDAR1 W-5L mutation increased channel mean open time, providing further evidence for an alteration in gating. However, the equilibrium affinities of Mg(2+) and ketamine were largely unaffected by the W-5L mutation, and Ca(2+) permeability was not decreased. Therefore, the M2 tryptophan residue of the NMDA channel is not involved in Ca(2+) permeation or the binding of open-channel blockers, but plays an important role in channel gating.
Buck
D P
DP
John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia.
Howitt
S M
SM
Clements
J D
JD
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D016291:Dizocilpine Maleate / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating; D007649:Ketamine / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010539:Permeability; D017434:Protein Structure, Tertiary; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; D007649:Ketamine; D016291:Dizocilpine Maleate; D014364:Tryptophan; D008274:Magnesium; D002118:Calcium