Contributions of Carboxy-Terminal Domains to NMDA Receptor Gating

Abstract

The intracellularly located carboxy terminal domain (CTD) of NMDA receptors represents almost half of these receptors’ mass; it interacts with a wide variety of regulatory proteins; and it serves to control receptor targeting and function. Calcium-activated proteins such as calmodulin, actinin and calcineurin, bind to or modify residues on CTDs and results in run-down, inactivation or desensitization of macroscopic responses. The mechanisms by which these kinetic changes occur are unknown. As a prelude into investigations of calcium-dependent modulation mechanism, we first asked how do CTDs of GluN1 and GluN2A subunits contribute to the receptor's gating reaction. We used cell-attached single-channel recordings, kinetic analyses and modeling to characterize the stationary gating kinetics of NMDA receptors lacking CTDs: GluN1(K838Stop) and/or GluN2(K844Stop). We found that compared to wild-type receptors (Po, .53 ± 0.03; MOT, 6.0 ± 0.6 ms; MCT, 5.4 ± 0.7; n = 17) receptors lacking CTD of GluN1 subunits had similar open probabilities but with slightly longer openings and closures (Po, .5±0.05 , MOT, 8.5 ± 1.2 ms, MCT, 11.0 ±2.4 ms; n = 9). In contrast, receptors lacking the CTD of GluN2A subunits were ∼2.5-fold less active (Po, .2±0.06; n = 9), with slightly longer openings (MOT, 10.7±1.3 ms,) and ∼17-fold longer closures. Receptors completely devoid of CTD, were much less active then either of the single-subunit CTD deletions, mainly due to substantially longer closures (Po, 0.1 ± 0.03, MOT, 6.5±1.4 ms, MCT, 175.2±53.4; n=8). These results demonstrate that the CTDs of both the GluN1 and GluN2A subunits contribute to normal gating kinetics, with the CTD of GluN2A receptors playing a major role in maintaining high open probabilities.