N-methyl-D-aspartate (NMDA) receptor channels in cultured CA1 hippocampal neurons were studied using patch-clamp techniques. The purpose of the research was to determine the occupancy of the channel by permeant cations and to determine the influence of charged residues in or near the pore. The concentration dependence of permeability ratios, the mole-fraction dependence of permeability ratios, the concentration dependence of the single-channel conductance, and a single-channel analysis of Mg2+ block all independently indicated that the NMDA receptor behaves as a singly-occupied channel. More precisely, there is one permeant cation at a time occupying the site or sites that are in the narrow region of the pore directly in the permeation pathway. Permeability-ratio measurements in mixtures of monovalent and divalent cations indicated that local charges in or near the pore do not produce a large local surface potential in physiologic solutions. In low ionic strength solutions, a local negative surface potential does influence the ionic environment near the pore, but in normal physiologic solutions the surface potential appears too small to significantly influence ion permeation. The results indicate that the mechanism for the high Ca2+ conductance of the NMDA receptor channel is not the same as for the voltage-dependent Ca2+ channel (VDCC). The VDCC has two high affinity, interacting binding sites that provide high Ca2+ selectivity and conductance. The binding site of the NMDA receptor is of lower affinity. Therefore, the selectivity for Ca2+ is not as high, but the lower affinity of binding provides a faster off rate so that interacting sites are not required for high conductance.

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