Received for publication October 20, 2004.
Revised January 9, 2005.
Accepted for publication February 8, 2005.

Structural features of the glutamate binding site in recombinant NR1/NR2A N-methyl-D-aspartate receptors determined by site-directed mutagenesis and molecular modeling

Abstract

We have used site-directed mutagenesis of amino acids located within the S1 and S2 ligand binding domains of the NR2A NMDA receptor subunit to explore the nature of ligand binding. Wild-type or mutated NR1/NR2A NMDA receptors were expressed in Xenopus laevis oocytes and studied using TEVC. We investigated the effects of mutations in the S1 and S2 regions on the potencies of the agonists L-glutamate, L-aspartate, RS-tetrazol-5-yl glycine and NMDA. Mutation of each of the corresponding residues found in the NR2A receptor subunit, suggested to be contact residues in the GluR2 AMPA receptor subunit, caused a rightward shift in the concentration-response curve for each agonist examined. None of the mutations examined altered the efficacy of glutamate as assessed by MTSEA potentiation of agonist-evoked currents. In addition none of the mutations altered the potency of glycine. Homology modeling and molecular dynamics were used to evaluate molecular details of ligand binding of both wild-type and mutant receptors, as well as to explore potential explanations for agonist selectivity between glutamate receptor subtypes. Our modeling studies support our interpretation of the mutagenesis data and indicate a similar binding strategy for L-glutamate and NMDA when they occupy the binding site in NMDA receptors as has been proposed for glutamate binding to the GluR2 AMPA receptor subunit. Furthermore, we offer an explanation as to why 'charge conserving' mutations of two residues in the binding pocket result in non-functional receptor-channels and suggest a contributing molecular determinant of why NMDA is not an agonist at AMPA receptors.

Key words: Glutamate, Molecular dynamics, Structure-activity relationships and modeling, Func. analysis receptor/ion channel mutants, Mutagenesis/Chimeric approaches

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