Two gates mediate NMDAR activity and are under subunit-specific regulation.

Abstract

NMDA receptors (NMDAR) are ionotropic glutamate receptors that mediate fast synaptic transmission and synaptic plasticity. Dysfunction of NMDARs has been found in numerous neurological diseases including Alzheimer's disease, Intellectual disability, epilepsy, autism, and schizophrenia. The NMDARs broad role in neuronal function is reflected in the complexity of its opening and closing kinetics, which can be broadly divided into periods of high opening activity, or clusters, interspersed by periods of no activity, or inter clusters. The initial step in ion channel opening is binding of agonists to the ligand-binding domain (LBD), which generates free energy that propagates to the ion channel to open ‘gate(s)’. NMDARs are heterotetramers composed of the obligate GluN1 and typically some combination of GluN2 subunits. The ion channel pore consists of two transmembrane segments (M1 and M3), a reentrant pore loop (M2) and a peripheral M4. A conserved glycine in the outer most transmembrane helix, the M4 helix, plays important roles in regulating NMDAR function. Here we find that these glycines play different roles in regulating channel opening. The GluN1 glycine mainly regulates single channel events within a cluster or bursts of activity, whereas GluN2A glycine mainly regulates opening into and out of clusters. Molecular dynamic simulations suggest that the GluN1 M4 regulates a ‘gate’ at the M2 loop, whereas the GluN2 M4 (along with GluN2 pre-M1) regulates a gate at the M3 helix bundle crossing. Subsequent functional experiments support this interpretation, leading to the hypothesis that the distinct kinetics of NMDARs are mediated by two gates that are under subunit-specific regulation. These results indicate that the GluN2A subunit regulates early steps in the gating process whereas the GluN1 regulate later steps including transitions form short to long-lived open states.