NMDA Channel Gating is Influenced by a Tryptophan Residue in the M2 Domain but Calcium Permeation Is Not Altered

Abstract

N-Methyl-D-aspartate (NMDA) receptors are susceptible to open-channel block by dizolcipine (MK-801), ketamine and Mg2+ and are permeable to Ca2+. 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 Ca2+ 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 Mg2+ and ketamine were largely unaffected by the W-5L mutation, and Ca2+ permeability was not decreased. Therefore, the M2 tryptophan residue of the NMDA channel is not involved in Ca2+ permeation or the binding of open-channel blockers, but plays an important role in channel gating.