Single‐channel analysis of an NMDA receptor possessing a mutation in the region of the glutamate binding site

Abstract

Recombinant NR1a/NR2A(T671A) N‐methyl‐D‐aspartate (NMDA) receptor‐channels, which carry a point mutation in the putative glutamate binding site that reduces glutamate potency by around 1000‐fold, have been expressed in Xenopus laevis oocytes and their single‐channel properties examined using patch‐clamp recording techniques. Shut time distributions of channel activity were fitted with a mixture of five exponential components. The first three components in each distribution were considered to occur within a channel activation as they exhibited little or no dependence on agonist concentration. Bursts of single‐channel openings were defined by a critical gap length with a mean of 5.65 ± 0.37 ms. Shut intervals with a duration longer than this value were considered to occur between separate bursts of channel openings. Distributions of the lengths of bursts of openings were fitted with a mixture of four exponential components. The longest two components carried the majority of the charge transfer in the channel recordings and had means of 7.71 ± 1.1 and 37.7 ± 4.3 ms. The overall probability of a channel being open during a burst was high (mean 0.92 ± 0.01). Brief concentration jumps (1 ms) of 10 mm glutamate were applied to outside‐out patches so that a comparison between the macroscopic current relaxation and steady‐state single‐channel activity evoked by glutamate could be made. The decay of such macroscopic currents was fitted with a single exponential component with a mean of 32.0 ± 3.53 ms. The good agreement between macroscopic current decay following brief agonist exposure and the value for the slowest component of the burst length distribution suggests that the bursts of openings that we identified in steady‐state recordings represent individual activations of recombinant NR1a/NR2A(T671A) NMDA receptor‐channels. A new way of displaying geometric distributions is suggested, and the utility of a modified definition of the ‘probability of being open within a burst’ is discussed. The single‐channel data that we present in this paper support further the idea that the point mutation T671A in the NR2A NMDA receptor subunit affects mainly the ability of glutamate to remain bound to these channels.

Structural Features of the Glutamate Binding Site in Recombinant NR1/NR2AN-Methyl-d-aspartate Receptors Determined by Site-Directed Mutagenesis and Molecular Modeling

“…Previous studies have characterized the properties of glutamate-activated NR1/NR2A(T671A) receptors and the homologous mutation in NR2D-containing NMDA receptors at both the whole-cell and single-channel level (Anson et al, 1998(Anson et al, , 2000Chen et al, 2004). In the present study, we find that the potencies for all four agonists were decreased by this mutation, with effects on glutamate potency being the largest (848-fold; Table 1).…”

“…Several structure-function studies of ionotropic glutamate receptors and related kainate binding proteins have identified residues located in the S1 and S2 binding domains that, when mutated, lead to a reduction in the ability of the ligand to remain bound to its binding site (Kuryatov et al, 1994;Kuusinen et al, 1995;Paas et al, 1996;Laube et al, 1997Laube et al, , 2004Anson et al, 1998Anson et al, , 2000Wo et al, 1999;Kalbaugh et al, 2004; for review, see Erreger et al, 2004). This study is the first to describe systematically the effects of mutating each of the proposed contact residues in the ligand binding Fig.…”

Deactivation kinetics of acid-sensing ion channel 1a are strongly pH-sensitive

“…This surprising effect is conserved in rat ASIC1a, human ASIC1a (hASIC1a), and chicken ASIC1a (cASIC1a), but is not seen in rat ASIC1a/2a heteromers. From a biophysical perspective, our findings are unexpected because, classically, deactivation kinetics are independent of agonist concentration (32)(33)(34)(35)(36). In addition, these findings are physiologically intriguing because they indicate that synaptic ASIC1a could shift between very fast AMPA-like kinetics and slower NMDA-like kinetics depending on the recent history of the synapse.…”

Equilibrium Constants for (R)-[(S)-1-(4-Bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic Acid (NVP-AAM077) Acting at Recombinant NR1/NR2A and NR1/NR2BN-Methyl-d-aspartate Receptors: Implications for Studies of Synaptic Transmission

“…Notwithstanding, this study is the first to report equilibrium constants for NVP-AAM077 action at NR1/NR2A and NR1/NR2B NMDA receptors and to demonstrate that its action is indeed competitive in nature and is surmountable by increasing the concentration of glutamate used to activate receptors. In addition, our results show that at least one of the residues (Thr671) that interacts directly with glutamate in the ligand binding domain (for review, see Chen and Wyllie, 2006; also see Anson et al, 1998Anson et al, , 2000Chen et al, 2004Chen et al, , 2005Furukawa et al, 2005) also influences NVP-AAM077 binding. The NR2A(T671A) mutation has previously been shown to reduce D-AP5 potency (Anson et al, 1998), suggesting that this residue plays an important role in competitive antagonist binding as well as agonist binding in NR2A NMDA receptor subunits.…”