Diversity of NR1/NR2B Receptor Gating Kinetics

Abstract

NMDA receptors are heteromeric glutamate-activated ion channels composed of NR1- and NR2-subunits. Controlled expression of four NR2-isoforms (A - D) results in receptors with distinct gating properties, contributing to the diversity of excitatory post-synaptic currents. Additionally, the NR1/NR2A-isoform can itself respond with distinct kinetics due to modal gating. To investigate whether the NR1/NR2B-isoform also gates with modal kinetics, we recorded steady-state single-channel activity from cell-attached patches of HEK293-cells transfected with NR1 and NR2B, in the continuous presence of saturating agonists. Single-channel records (n=37) revealed a variety of gating patterns illustrated by a 25-fold range in measured equilibrium open probability: range, 0.02 - 0.49; Po (mean ± s.e.m.) = 0.20 ± 0.02. This diversity reflects mainly differences in mean closure durations per file: range, 6 - 200 ms; mean closed time = 43 ± 8 ms; with less spread for mean open durations: range, 1.8 - 10.5 ms; mean open time = 4.8 ± 0.3 ms. Kinetic analyses revealed that each record had 2-4 open and at least 5 closed components in the respective interval duration distributions. As with NR1/2A-receptors, in all NR1/2B-records we observed sporadic gating changes due to sudden changes in mean open durations, indicative of modal behavior. We identified three gating regimes, each having at least two open time components: a ubiquitous brief component (0.27 ± 0.01 ms) and at least one of three longer components (tau-L = 2.5 ± 0.1 ms; tau-M = 5.0 ± 0.2 ms; or tau-H = 10.0 ± 1.0 ms). In contrast to NR1/2A-channel behavior where modal gating allowed characterization of all observed channels, for the NR1/2B-receptor we also observed gating patterns which differ in mean duration of closures. These data and analyses reveal the variety of mechanisms generating the previously observed diversity of macroscopic NR1/2B-responses.