Stationary Gating of GluN1/GluN2B Receptors in Intact Membrane Patches

Abstract

NMDA receptors are heteromeric glutamate-gated channels composed of GluN1 and GluN2 subunits. Receptor isoforms that differ in their GluN2-subunit type (A-D) are expressed differentially throughout the central nervous system and have distinct kinetic properties in recombinant systems. How specific receptor isoforms contribute to the functions generally attributed to NMDA receptors remains unknown, due in part to the incomplete functional characterization of individual receptor types and unclear molecular composition of native receptors. We examined the stationary gating kinetics of individual rat recombinant GluN1/GluN2B receptors in cell-attached patches of transiently transfected HEK293 cells and used kinetic analyses and modeling to describe the full range of this receptor's gating behaviors. We found that, like GluN1/GluN2A receptors, GluN1/GluN2B receptors have three gating modes that are distinguishable by their mean open durations. However, for GluN1/GluN2B receptors, the modes also differed markedly in their mean closed durations and thus generated a broader range of open probabilities. We also found that regardless of gating mode, glutamate dissociation occurred approximately 4-fold more slowly (k(-) = 15 s(-1)) compared to that observed in GluN1/GluN2A receptors. On the basis of these results, we suggest that slow glutamate dissociation and modal gating underlie the long heterogeneous activations of GluN1/GluN2B receptors.

One-channel Cell-attached Patch-clamp Recording

“…Recordings from early ( Figure 8A, 5 days in vitro) culture more closely resemble kinetics described for GluN1/GluN2B receptors and recordings from late ( Figure 8B, 27 days in vitro) cultures more closely resemble kinetics described for GluN1/ GluN2A receptors (Figures 3A and 5A). This observation is consistent with patterns of NMDA receptor expression isoforms and with careful characterizations of currents recorded from cell attached one-channel patches in hippocampal cultures 16 . The pipette was attached at the soma of a neuron that was dissociated from the prefrontal cortex of a rat embryo and was maintained in culture for 5 days (A) or 27 days (B).…”

“…Under these conditions, and when applying a pipette holding potential of +100 mV (leading to an approximate membrane potential of -120 mV), single recombinant GluN1/GluN2A and GluN1/GluN2B are open with high probability (P o , 0.3-0.5) to one relatively high conductance level (> 50 pS) (Figures 3A and 5B) [15][16][17] . When recording, current noise spikes, baseline drifts, or periods of excessive noise may manifest (Figures 4A-C), however as mentioned, these may be corrected if brief and minor.…”

Ifenprodil Effects on GluN2B-Containing Glutamate Receptors

“…1B) (Amico-Ruvio and Popescu, 2010). Both data sets included only records that originated from a single active channel, and we processed and analyzed all records in an identical manner (Kussius et al, 2009;Amico-Ruvio and Popescu, 2010).…”

“…All methods were similar to those described in detail previously (Kussius et al, 2009;Amico-Ruvio and Popescu, 2010). In brief, rat GluN1-1a (NR1-1a, U08261) and GluN2B (NR2B, NM012574) clones were expressed from pcDNA3.1(ϩ) in human embryonic kidney 293 cells together with GFP.…”

“…Essentially, the activation of 2B receptors consists of rapid agonist binding, slow channel gating, even slower channel desensitization, and occasional gatingmode changes. Single-channel measurements showed that after binding glutamate and before populating open states, (glycine-bound) receptors transition through at least three kinetically resolvable preopen states; occasionally, receptors escape this active gating cycle by entering desensitized states, and on a minutes time scale they can also change the gating mode (Banke and Traynelis, 2003;Amico-Ruvio and Popescu, 2010). With this model in mind, allosteric ligands may inhibit NMDA receptors by causing preopen or desensitized events to become longer, open events to become shorter, low-activity gating modes to become more prevalent, or any combination of the above mechanisms.…”