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Abstract

The high flux rate of Ca²⁺ through NMDA receptor (NMDAR) channels is critical for their biological function and may depend on a Ca²⁺ binding site in the extracellular vestibule. We screened substitutions of hydrophilic residues exposed in the vestibule and identified a cluster of charged residues and a proline, the DRPEER motif, positioned C terminal to M3, that is unique to the NR1 subunit. Charge neutralization or conversion of residues in DRPEER altered fractional Ca²⁺ currents in a manner consistent with its forming a binding site for Ca²⁺. Similarly, in a mutant channel in which all of the negative charges are neutralized (ARPAAR), the block by extracellular Ca²⁺ of single-channel current amplitudes is attenuated. In these same channels, the block by extracellular Mg²⁺is unaffected. DRPEER is located extracellularly, and its contribution to Ca²⁺ influx is distinct from that of the narrow constriction. We conclude that key residues in DRPEER, acting as an external binding site for Ca²⁺, along with a conserved asparagine in the M3 segment proper, contribute to the high fractional Ca²⁺ currents in these channels under physiological conditions. Therefore, these domains represent critical molecular determinants of NMDAR function in synaptic physiology.