Popescu, G. K. | bioRxiv Science

bioRxiv2020

Contributions by N-terminal Domains to NMDA Receptor Currents

To investigate the role of the N-terminal domains (NTDs) in NMDA receptor signaling we used kinetic analyses of one-channel currents and compared the reaction mechanism of recombinant wild-type GluN1/GluN2A and GluN1/GluN2B receptors with those observed for NDT-lacking receptors. We found that truncated receptors maintained the fundamental gating mechanism characteristic of NMDA receptors, which includes a multi-state activation sequence, desensitization steps, and mode transitions. This result establishes that none of the functionally-defined NMDA receptor activation events require the NTD. Notably, receptors that lacked the entire NTD layer retained isoform-specific kinetics. Together with previous reports, these results demonstrate that the entire gating machinery of NMDA receptors resides within a core domain that contains the ligand-binding and the channel-forming transmembrane domains, whereas the NTD and C-terminal layers serve modulatory functions, exclusively.

biophysics↗

bioRxiv2020

Cross-subunit Interactions that Stabilize Open States Mediate Gating in NMDA Receptors

NMDA receptors are excitatory channels with critical functions in the physiology of central synapses. Their activation reaction proceeds as a series of kinetically distinguishable, reversible steps, whose structural bases are of current interest. Very likely, the earliest steps in the activation reaction include glutamate binding to and compression of the ligand-binding domain. Later, three short linkers transduce this movement to open the gate by mechanical coupling with transmembrane helices. Here, we used double-mutant cycle analyses to demonstrate that a direct chemical interaction between GluN1-I642 (on M3) and GluN2A-L550 (on L1-M1) stabilizes receptors after they have opened, and therefore represents one of the structural changes that occur late in the activation reaction. This native interaction extends the current decay, and its absence predicts deficits in charge transfer by GluN1-I642L, a pathogenic human variant. SIGNIFICANCE STATEMENTNMDA receptors are glutamatergic channels whose activations control the strength of excitatory synapses in the central nervous system. Agonist binding initiates a complex activation reaction that consists of a stepwise sequence of reversible isomerizations. In addition to previously identified steps in this series, which include agonist-induced closure of the ligand-binding lobes, and the subsequent mechanical pulling by the ligand-binding domain on the gate-forming transmembrane helix, we identify a new cross-subunit interaction, which stabilizes open receptors and slows the rate of the current decay. Naturally occurring NMDA receptor variants lacking this interaction are pathogenic.

biophysics↗