Mutation of structural determinants lining the N-methyl-D-aspartate receptor channel differentially affects phencyclidine block and spermine potentiation and block

Spermine and other endogenous polyamines potentiate, block and permeate the N-methyl-D-aspartate receptor channel. To identify structural determinants of the N-methyl-D-aspartate channel that mediate spermine's actions, we ge nerated mutant receptors with asparagine (N) to glutamine (Q) or arginine ( R) substitutions in the selectivity filter of the channel. We demonstrate t hat mutation of the three critical asparagines in this domain differentiall y affects block by phencyclidine and both potentiation and block by spermin e. N-to-Q and N-to-R mutations in the N site of the NR1 subunit (N598 in NR 1(011), N619 in NR1(100)) and N-to-Q mutations in the N and N + 1 sites (N5 95 and N596 in NR2A, respectively) of the NR2 subunit (Q/NN, R/NN, N/QN, N/ NQ, Q/QN and Q/NQ receptors) reduced affinity for phencyclidine. The Q/NN r eceptor showed markedly reduced potentiation by spermine, with little or no change in spermine block. The R/NN receptor showed markedly reduced spermi ne potentiation and affinity for spermine at its block site. The N/QN, N/NQ and Q/QN mutant receptors showed somewhat enhanced spermine block, while t he Q/NQ double mutant exhibited significantly more enhanced spermine block. Thus, the asparagine residues critical to Ca2+ permeability and Mg2+ block of N-methyl-D-aspartate channels are also critical to block by spermine an d phencyclidine. To examine the interaction of spermine and phencyclidine within the channel , we performed competition studies. Spermine appeared to compete with phenc yclidine for binding to the receptor; however, blocks by phencyclidine and by spermine were not additive. The findings suggest that spermine can bind to a site in the external vestibule of the channel to impede phencyclidine binding, but allow Na+ influx. (C) 1999 IBRO. Published by Elsevier Science Ltd.