Structural Features of the Glutamate Binding Site in Recombinant NR1/NR2A N-Methyl-d-aspartate Receptors Determined by Site-Directed Mutagenesis and Molecular Modeling
We have used site-directed mutagenesis of amino acids located within the S1 and S2 ligand binding domains of the NR2A N -methyl- d -aspartate (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 stu...
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Veröffentlicht in: | Molecular pharmacology 2005-05, Vol.67 (5), p.1470-1484 |
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Zusammenfassung: | We have used site-directed mutagenesis of amino acids located within the S1 and S2 ligand binding domains of the NR2A N -methyl- d -aspartate (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 two electrode voltage clamp. We investigated the effects of mutations in the S1 and S2 regions
on the potencies of the agonists l -glutamate, l -aspartate, ( R , S )-tetrazol-5yl-glycine, and NMDA. Mutation of each of the corresponding residues found in the NR2A receptor subunit, suggested
to be contact residues in the GluR2 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (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 methanethiosulfonate ethylammonium 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. The 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 âcharge conservingâ mutations of two residues
in the binding pocket result in nonfunctional receptor channels and suggest a contributing molecular determinant for why NMDA
is not an agonist at AMPA receptors. |
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ISSN: | 0026-895X 1521-0111 |
DOI: | 10.1124/mol.104.008185 |