Molecular determinants of coordinated proton and zinc inhibition of N- methyl- d -aspartate NR1/NR2A receptors

Abstract

Modulation of the N-methyl-D-aspartate (NMDA)-selective glutamate receptors by extracellular protons and Zn 2؉ may play important roles during ischemia in the brain and during seizures. Recombinant NR1͞ NR2A receptors exhibit a much higher apparent affinity for voltageindependent Zn 2؉ inhibition than receptors with other subunit combinations. Here, we show that the mechanism of this apparent high-affinity, voltage-independent Zn 2؉ inhibition for NR2A-containing receptors results from the enhancement of proton inhibition. We also show that the N-terminal leucine͞isoleucine͞valine binding protein (LIVBP)-like domain of the NR2A subunit contains critical determinants of the apparent high-affinity, voltage-independent Zn 2؉ inhibition. Mutations H42A, H44G, or H128A greatly increase the Zn 2؉ IC50 (by up to Ϸ700-fold) with no effect on the potencies of glutamate and glycine or on voltage-dependent block by Mg 2؉ . Furthermore, the amino acid residue substitution H128A, which mediates the largest effect on the apparent high-affinity Zn 2؉ inhibition among all histidine substitutions we tested, is also critical to the pH-dependency of Zn 2؉ inhibition. Our data revealed a unique interaction between two important extracellular modulators of NMDA receptors.G lutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the central nervous system. Although overactivation of the N-methyl-D-aspartate (NMDA)-selective glutamate receptors can trigger neurodegeneration in neuropathological conditions such as stroke, NMDA receptor function is regulated under normal conditions by several extracellular ions (Mg 2ϩ , H ϩ , and Zn 2ϩ ), which exert strong tonic inhibition in a subunit selective manner (1). Inhibition by extracellular protons is particularly relevant for the neuropathological consequences of occlusive stroke because acidification of the extracellular environment during ischemia has been hypothesized to inhibit NMDA receptor overactivation by extrasynaptic glutamate that accumulates following metabolic failure (2, 3). The proton inhibition of NMDA receptors could delay their contribution to subsequent neuronal death until the pH gradients are restored, and this delay may provide a therapeutic window for postinsult treatment with NMDA receptor antagonists. Acidification of the extracellular space during electrographic seizure may also contribute to seizure termination through inhibition of NMDA receptor function (4, 5). Because of these potentially important aspects of the pH sensitivity of the NMDA receptor, we have sought to understand the structural basis by which the receptor might control its regulation by extracellular protons.In the central nervous system, the extracellular Zn 2ϩ concentration has been shown to vary under normal brain function as well as neuropathological conditions (6, 7). In addition, there are large amounts of chelatable Zn 2ϩ in the glutamatergic terminals of hippocampus (8-10), which are released in a Ca 2ϩ -dependent manner (for review, see ref. 6). ...

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Channels Lacking the N-terminal Domain

“…Glycine-independent desensitization of NMDA receptors was assigned to two regions in the NR2A subunit, the X domain and an area within the S1 segment (13,17). Moreover, participation of the N-terminal domain in the regulation of NMDA receptor channel function by allosteric modulators, including Zn 2ϩ ions (14,16), ifenprodil (8,15,16), spermine (8), protons (25), and redox agents (26), has been demonstrated. In some cases, this modulation has been shown to involve an direct allosteric interaction between the N-terminal domain and the agonist-binding domain (16).…”

“…First, this domain is implicated as a determinant in the assembly of oligomeric channels (10 -12). Second, the X domain may mediate the allosteric transitions involved in the channel activation, desensitization, or modulation by ions and drugs as has been observed for NMDA receptors (8,(13)(14)(15)(16)(17). Third, the X domain may provide docking sites for extracellular proteins, which serve to cluster the receptors or stabilize their localization.…”

Protons Trap NR1/NR2B NMDA Receptors in a Nonconducting State

“…The finding that protons appear to shift receptors to an inactive state also carries structural implications and suggests that other forms of allosteric modulation could proceed through creation of briefly occupied inactive states [e.g., calcineurin effects on GluR1 (Banke et al, 2000)]. Given that a number of regulators, such as polyamines, extracellular Zn 2ϩ , ifenprodil, and alternative splicing, all appear to alter NMDA receptor function by shifting the pKa of the proton sensor (Traynelis et al, 1995;Mott et al, 1998;Choi and Lipton, 1999;Low et al, 2000), our findings have widespread implications for NMDA receptor function.…”

“…Allosteric regulators such as polyamines acting at NR1/NR2B NMDA receptors (Traynelis et al, 1995), ifenprodil acting at NR1/NR2B (Mott et al, 1998), and zinc acting at NR1/NR2A (Choi and Lipton, 1999;Low et al, 2000;Paoletti et al, 2000 have been proposed to exert their actions through modification of tonic inhibition by physiological pH. Thus, proton-sensitive gating could be a downstream feature of receptor activation that is regulated by exogenous molecules and therapeutic agents.…”

Constitutive Activation of the N-Methyl-d-aspartate Receptor via Cleft-spanning Disulfide Bonds

“…Barium was used as the divalent cation to minimize secondary activation of calcium-activated Cl Ϫ currents (25). EDTA was included to chelate trace amounts of the soft metal divalent cations Cd 2ϩ and Zn 2ϩ , which have been reported to contaminate buffer solutions and inhibit the NMDA receptor by binding to a high affinity site (26,27). EDTA also removes a zinc-dependent component of desensitization (28).…”