N-METHYL-D-ASPARTATE RECEPTOR-CHANNEL BLOCK BY THE ENANTIOMERIC 6,7-BENZOMORPHANS BIII-277-CL AND BIII-281-CL

BIII 277 CL {(-)-2R-[2 alpha,3(R),6 ,6-methano-1,2,3,4,5,6-hexahydro- 3-benzazocin-9-ol hydrochloride) is a novel benzomorphan with neuropro tective and anticonvulsant properties that exhibits high affinity bind ing to the N-methyl-D-aspartate (NMDA) receptor but, in contrast to ot her structurally related benzomorphans, low affinity for mu opiate and sigma sites. Whole-cell voltage-clamp and single-channel recording we re used to study the interaction of BIII 277 CL and its enantiomer BII I 281 CL with native NMDA receptors in cultured hippocampal neurons. B ill 277 CL and Bill 281 CL produced a slow use-dependent block of whol e-cell NMDA receptor currents. Once block was established, recovery wa s slow (<50% in greater than or equal to 40 min). The steady-state IC5 0 (n(H) values derived from logistic fits to concentration-block isoth erms obtained at -60 mV were 5.3 nM (0.67) and 58 nM (1.2), respective ly. The benzomorphans had no effect on currents evoked by pha-amino-3- hydroxy-5-methyl-4-isoxazolepropionate and gamma-aminobutyric acid but minimally inhibited kainate-evoked currents at high (greater than or equal to 30 mu M) concentrations. Bill 277 CL and Bill 281 CL failed t o bind and block closed NMDA receptor channels, and the block was occl uded by Mg++ consistent with an open channel-blocking mechanism. Stead y-state block was diminished by depolarization; analysis of the voltag e-dependence of block indicated that BIII 281 CL binds within the chan nel at a site that senses 46% of the transmembrane electric field. Rec ordings of single NMDA receptor channels in outside-out membrane patch es confirmed the slow, persistent blocking action obtained in whole-ce ll recordings. In addition, at high concentrations, flickering of the unitary currents was observed consistent with a low-affinity channel-b locking action. Taking the present data in conjunction with previously obtained structure-activity information for N-substituted benzomorpha ns, a three-mode-blocking model was developed in which there are three interaction sites for binding of the high-affinity ligand BIII 277 CL . In this model, the drug can bind in one of three modes by docking at one, two or all three interaction points but cannot transition betwee n modes. The model further proposes that the lower-affinity enantiomer Bill 281 CL binds in modes with one and two but not all three interac tion points docked. We conclude that Bill 277 CL and Bill 281 CL are p otent and selective, use-dependent (uncompetitive) channel-blocking NM DA receptor antagonists. The substantially higher affinity that BIII 2 77 CL exhibits for the NMDA receptor in comparison with its enantiomer and other benzomorphans appears to be due to stabilization of binding at three sites within the channel.