title: Structural determinants and mechanism of action of a GluN2C-selective NMDA receptor positive allosteric modulator.
abstract: NMDA receptors are tetrameric complexes of GluN1, GluN2A-D, and GluN3A-B subunits and are involved in normal brain function and neurologic disorders. We identified a novel class of stereoselective pyrrolidinone (PYD) positive allosteric modulators for GluN2C-containing NMDA receptors, exemplified by methyl 4-(3-acetyl-4-hydroxy-1-[2-(2-methyl-1H-indol-3-yl)ethyl]-5-oxo-2,5-dihydro-1H-pyrrol-2-yl)benzoate. Here we explore the site and mechanism of action of a prototypical analog, PYD-106, which at 30 μM does not alter responses of NMDA receptors containing GluN2A, GluN2B, and GluN2D and has no effect on AMPA [α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid] and kainate receptors. Coapplication of 50 μM PYD-106 with a maximally effective concentration of glutamate and glycine increases the response of GluN1/GluN2C NMDA receptors in HEK-293 cells to 221% of that obtained in the absence of PYD (taken as 100%). Evaluation of the concentration dependence of this enhancement revealed an EC50 value for PYD of 13 μM. PYD-106 increased opening frequency and open time of single channel currents activated by maximally effective concentrations of agonist but only had modest effects on glutamate and glycine EC50. PYD-106 selectively enhanced the responses of diheteromeric GluN1/GluN2C receptors but not triheteromeric GluN1/GluN2A/GluN2C receptors. Inclusion of residues encoded by GluN1-exon 5 attenuated the effects of PYD. Three GluN2C residues (Arg194, Ser470, Lys470), at which mutagenesis virtually eliminated PYD function, line a cavity at the interface of the ligand binding and the amino terminal domains in a homology model of GluN1/GluN2C built from crystallographic data on GluN1/GluN2B. We propose that this domain interface constitutes a new allosteric modulatory site on the NMDA receptor.
authors.affiliation: Pharmacology Department (A.K., S.A.S., S.F.T.) and Chemistry Department (S.Z., P.B.B., D.C.L., J.P.S.), Emory University, Atlanta, Georgia; Department of Biomedical and Pharmaceutical Sciences, and Center for Biomolecular Structure and Dynamics (K.B.H.), University of Montana, Missoula, Montana; and Cold Spring Harbor Laboratories (E.K., H.F.), Cold Spring Harbor, New York.
authors.forename: Alpa
authors.initials: A
authors.lastname: Khatri
authors.affiliation: Pharmacology Department (A.K., S.A.S., S.F.T.) and Chemistry Department (S.Z., P.B.B., D.C.L., J.P.S.), Emory University, Atlanta, Georgia; Department of Biomedical and Pharmaceutical Sciences, and Center for Biomolecular Structure and Dynamics (K.B.H.), University of Montana, Missoula, Montana; and Cold Spring Harbor Laboratories (E.K., H.F.), Cold Spring Harbor, New York.
authors.forename: Pieter B
authors.initials: PB
authors.lastname: Burger
authors.affiliation: Pharmacology Department (A.K., S.A.S., S.F.T.) and Chemistry Department (S.Z., P.B.B., D.C.L., J.P.S.), Emory University, Atlanta, Georgia; Department of Biomedical and Pharmaceutical Sciences, and Center for Biomolecular Structure and Dynamics (K.B.H.), University of Montana, Missoula, Montana; and Cold Spring Harbor Laboratories (E.K., H.F.), Cold Spring Harbor, New York.
authors.forename: Sharon A
authors.initials: SA
authors.lastname: Swanger
authors.affiliation: Pharmacology Department (A.K., S.A.S., S.F.T.) and Chemistry Department (S.Z., P.B.B., D.C.L., J.P.S.), Emory University, Atlanta, Georgia; Department of Biomedical and Pharmaceutical Sciences, and Center for Biomolecular Structure and Dynamics (K.B.H.), University of Montana, Missoula, Montana; and Cold Spring Harbor Laboratories (E.K., H.F.), Cold Spring Harbor, New York.
authors.forename: Kasper B
authors.initials: KB
authors.lastname: Hansen
authors.affiliation: Pharmacology Department (A.K., S.A.S., S.F.T.) and Chemistry Department (S.Z., P.B.B., D.C.L., J.P.S.), Emory University, Atlanta, Georgia; Department of Biomedical and Pharmaceutical Sciences, and Center for Biomolecular Structure and Dynamics (K.B.H.), University of Montana, Missoula, Montana; and Cold Spring Harbor Laboratories (E.K., H.F.), Cold Spring Harbor, New York.
authors.forename: Sommer
authors.initials: S
authors.lastname: Zimmerman
authors.affiliation: Pharmacology Department (A.K., S.A.S., S.F.T.) and Chemistry Department (S.Z., P.B.B., D.C.L., J.P.S.), Emory University, Atlanta, Georgia; Department of Biomedical and Pharmaceutical Sciences, and Center for Biomolecular Structure and Dynamics (K.B.H.), University of Montana, Missoula, Montana; and Cold Spring Harbor Laboratories (E.K., H.F.), Cold Spring Harbor, New York.
authors.forename: Erkan
authors.initials: E
authors.lastname: Karakas
authors.affiliation: Pharmacology Department (A.K., S.A.S., S.F.T.) and Chemistry Department (S.Z., P.B.B., D.C.L., J.P.S.), Emory University, Atlanta, Georgia; Department of Biomedical and Pharmaceutical Sciences, and Center for Biomolecular Structure and Dynamics (K.B.H.), University of Montana, Missoula, Montana; and Cold Spring Harbor Laboratories (E.K., H.F.), Cold Spring Harbor, New York.
authors.forename: Dennis C
authors.initials: DC
authors.lastname: Liotta
authors.affiliation: Pharmacology Department (A.K., S.A.S., S.F.T.) and Chemistry Department (S.Z., P.B.B., D.C.L., J.P.S.), Emory University, Atlanta, Georgia; Department of Biomedical and Pharmaceutical Sciences, and Center for Biomolecular Structure and Dynamics (K.B.H.), University of Montana, Missoula, Montana; and Cold Spring Harbor Laboratories (E.K., H.F.), Cold Spring Harbor, New York.
authors.forename: Hiro
authors.initials: H
authors.lastname: Furukawa
authors.affiliation: Pharmacology Department (A.K., S.A.S., S.F.T.) and Chemistry Department (S.Z., P.B.B., D.C.L., J.P.S.), Emory University, Atlanta, Georgia; Department of Biomedical and Pharmaceutical Sciences, and Center for Biomolecular Structure and Dynamics (K.B.H.), University of Montana, Missoula, Montana; and Cold Spring Harbor Laboratories (E.K., H.F.), Cold Spring Harbor, New York.
authors.forename: James P
authors.initials: JP
authors.lastname: Snyder
authors.affiliation: Pharmacology Department (A.K., S.A.S., S.F.T.) and Chemistry Department (S.Z., P.B.B., D.C.L., J.P.S.), Emory University, Atlanta, Georgia; Department of Biomedical and Pharmaceutical Sciences, and Center for Biomolecular Structure and Dynamics (K.B.H.), University of Montana, Missoula, Montana; and Cold Spring Harbor Laboratories (E.K., H.F.), Cold Spring Harbor, New York strayne@emory.edu.
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Traynelis
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology*; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D018698:Glutamic Acid / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: D018698:Glutamic Acid; D005998:Glycine; D016194:Receptors, N-Methyl-D-Aspartate; C432823:NR2C NMDA receptor