Abstract

BIII 277 CL {(–)-2R-[2α,3(R*),6α]-3-(2-methoxypropyl)-6,11,11-trimethyl-2,6-methano-1,2,3,4,5,6-hexahydro-3-benzazocin-9-ol hydrochloride} is a novel benzomorphan with neuroprotective and anticonvulsant properties that exhibits high affinity binding to the N-methyl-d-aspartate (NMDA) receptor but, in contrast to other structurally related benzomorphans, low affinity formu opiate and sigma sites. Whole-cell voltage-clamp and single-channel recording were used to study the interaction of BIII 277 CL and its enantiomer BIII 281 CL with native NMDA receptors in cultured hippocampal neurons. BIII 277 CL and BIII 281 CL produced a slow use-dependent block of whole-cell NMDA receptor currents. Once block was established, recovery was slow (<50% in ≥40 min). The steady-state IC₅₀(n_H) values derived from logistic fits to concentration-block isotherms obtained at –60 mV were 5.3 nM (0.67) and 58 nM (1.2), respectively. The benzomorphans had no effect on currents evoked by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and γ-aminobutyric acid but minimally inhibited kainate-evoked currents at high (≥30 μM) concentrations. BIII 277 CL and BIII 281 CL failed to bind and block closed NMDA receptor channels, and the block was occluded by Mg⁺⁺, consistent with an open channel-blocking mechanism. Steady-state block was diminished by depolarization; analysis of the voltage-dependence of block indicated that BIII 281 CL binds within the channel at a site that senses 46% of the transmembrane electric field. Recordings of single NMDA receptor channels in outside-out membrane patches confirmed the slow, persistent blocking action obtained in whole-cell recordings. In addition, at high concentrations, flickering of the unitary currents was observed consistent with a low-affinity channel-blocking action. Taking the present data in conjunction with previously obtained structure-activity information for N-substituted benzomorphans, 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 between modes. The model further proposes that the lower-affinity enantiomer BIII 281 CL binds in modes with one and two but not all three interaction points docked. We conclude that BIII 277 CL and BIII 281 CL are potent and selective, use-dependent (uncompetitive) channel-blocking NMDA receptor antagonists. The substantially higher affinity that BIII 277 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.