Intracellular Mg2+ interacts with structural determinants of the narrow constriction contributed by the NR1-subunit in the NMDA receptor channel

N -methyl-D-aspartate (NMDA) receptor channels are blocked by intracellular Mg 2+ in a voltage-dependent manner. Amino acid residues positioned at or near the narrow constriction that interact with intracellular Mg 2+ were identified in recombinant NR1-NR2A channels expressed in Xenopus oocytes or human embryonic kidney (HEK) 293 cells. In the absence of extracellular Ca 2+ , the block of wild-type channels by intracellular Mg 2+ measured using macroscopic currents showed a voltage dependence (δ) of around 0.38 and a voltage-independent affinity for the channel of 4 mM. These parameters were independent of the Mg 2+ concentration (0.05-10 mM), and were indistinguishable from those found for the reduction of single channel amplitudes under the same ionic conditions. Under biionic conditions with high intracellular Mg 2+ and K + extracellularly, Mg 2+ was weakly permeant. Mg 2+ efflux, however, attenuated the block only at positive potentials (> +80 mV). Substitutions of the N-site asparagine in the NR1-subunit altered intracellular Mg 2+ block over physiological membrane potentials (+10 to +50 mV). Substitution of glycine, glutamine or serine attenuated the extent of block whereas the negatively charged aspartate enhanced it, consistent with the side chain of the native asparagine at this position contributing to a blocking site for intracellular Mg 2+ . Substitutions of the N-site or N + 1 site asparagine in the NR2A-subunit, which form a blocking site for extracellular Mg 2+ , also altered the block by intracellular Mg 2+ . However, for the NR2A-subunit N-site asparagine, the block was reduced but only at non-physiological high potentials (> +70 mV). The NR2A-subunit N + 1 site asparagine, which together with the NR1-subunit N-site asparagine forms the narrow constriction of the channel, also contributed to a blocking site for intracellular Mg 2+ . However, it did so to a lesser extent than the NR1-subunit N-site and in a manner different from its contribution to a blocking site for extracellular Mg 2+ . It is concluded that intracellular Mg 2+ interacts with residues that form the narrow constriction in the NMDA receptor channel with the N-site asparagine of the NR1-subunit representing the dominant blocking site. Thus, intracellular Mg 2+ interacts with different asparagine residues at the narrow constriction than extracellular Mg 2+ , although the two blocking sites are positioned very close to each other.