Protons Trap NR1/NR2B NMDA Receptors in a Nonconducting State

Abstract

NMDA receptors are highly expressed in the CNS and are involved in excitatory synaptic transmission, as well as synaptic plasticity. Given that overstimulation of NMDA receptors can cause cell death, it is not surprising that these channels are under tight control by a series of inhibitory extracellular ions, including zinc, magnesium, and H ϩ . We studied the inhibition by extracellular protons of recombinant NMDA receptor NR1/NR2B single-channel and macroscopic responses in transiently transfected human embryonic kidney HEK 293 cells using patch-clamp techniques. We report that proton inhibition proceeds identically in the absence or presence of agonist, which rules out the possibility that protonation inhibits receptors by altering coagonist binding. The response of macroscopic currents in excised patches to rapid jumps in pH was used to estimate the microscopic association and dissociation rates for protons, which were 1.4 ϫ 10 9 M Ϫ1 sec Ϫ1 and 110 -196 sec Ϫ1 , respectively (K d corresponds to pH 7.2). Protons reduce the open probability without altering the time course of desensitization or deactivation. Protons appear to slow at least one time constant describing the intra-activation shut-time histogram and modestly reduce channel open time, which we interpret to reflect a reduction in the overall channel activation rate and possible proton-induced termination of openings. This is consistent with a modest proton-dependent slowing of the macroscopic response rise time. From these data, we propose a physical model of proton inhibition that can describe macroscopic and single-channel properties of NMDA receptor function over a range of pH values.

Glutamate Receptor Ion Channels: Structure, Regulation, and Function

“…Proton inhibition depends on alternative RNA splicing of the GluN1 subunit within the ATD , a region that controls proton sensitivity on its own (Gielen et al, 2009) and through association with Zn 2ϩ (see section VI.B) or ifenprodil (Mott et al, 1998) (see section V.E). Proton inhibition is independent of voltage and ligand binding (Banke et al, 2005). At the single channel level, protons reduce open probability of GluN2B subunit-containing NMDA receptors, with modest effects on open duration and single channel conductance (Traynelis and CullCandy, 1991;Banke et al, 2005).…”

“…Proton inhibition is independent of voltage and ligand binding (Banke et al, 2005). At the single channel level, protons reduce open probability of GluN2B subunit-containing NMDA receptors, with modest effects on open duration and single channel conductance (Traynelis and CullCandy, 1991;Banke et al, 2005). Protons inhibit GluN1/ GluN2A receptors somewhat differently than GluN1/ GluN2B, reducing mean channel open time and open probability Erreger and Traynelis, 2008).…”

“…Unpublished data for GluK2, GluN2A, GluN2C, and GluN2D, from S. M. Dravid, K. M. Vance, and S. F. Traynelis. Data for GluA1 single-channel recordings were from Banke et al (2000), GluK2 single channel recordings were from Zhang et al (2009c), and GluN2B macroscopic current recordings were from Banke et al (2005). 452 both hydrophobic and nonhydrophobic surface regions on the D1 domain (Armstrong and Gouaux, 2000;Sun et al, 2002;Sobolevsky et al, 2009) (see Fig.…”

Control of Assembly and Function of Glutamate Receptors by the Amino-Terminal Domain

“…Protons inhibit all glutamate receptors in a voltage-independent fashion without changing the ionization or EC 50 values of the activating agonist (Christensen and Hida, 1990;Giffard et al, 1990;Tang et al, 1990;Traynelis and CullCandy, 1990;Vyklický et al, 1990;Wu and Christensen, 1996;Ihle and Patneau, 2000;Lei et al, 2001;Mott et al, 2003;Banke et al, 2005). Among NMDA receptors, proton IC 50 for inhibition varies with the GluN2 subunit, with IC 50 values near physiological pH for GluN2A, GluN2B, and GluN2D (7.0 -7.4), leading to the idea that these receptors are under tonic inhibition by physiological levels of protons (Traynelis et al, 1995;Gielen et al, 2009).…”

Regulation of Postsynaptic Ca2+Influx in Hippocampal CA1 Pyramidal Neurons via Extracellular Carbonic Anhydrase

“…An increase in pH e from 7.2 to 7.3 corresponded to an increase in NMDAR currents of ϳ10%, in both the continued presence of agonist (Tang et al, 1990;Traynelis and Cull-Candy, 1990;Vyklicky et al, 1990) and after its rapid removal (Banke et al, 2005). An increase of 0.10 unit pH would be approximately equivalent to the amplification of the recorded alkalosis caused by benzolamide in 0 Mg 2ϩ /PTX media.…”