Direct M3-M4 Interactions Control NMDA Receptor Gating and Desensitization

Abstract

N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated channels that mediate excitatory neurotransmission in the central nervous system. They are heterotetrameric channels composed of two GluN1 and two GluN2 subunits, each containing four (M1-M4) membrane-embedded helices. Glutamate and glycine binding results in closure of the ligand binding domain and results in opening of the Na+/Ca2+-permeable pore by direct tethering to the gate at the M3 helix. The role of the peripheral M4 helix in modulating channel gating is unclear. To determine if the M4 helix controls gating by direct allosteric interactions with the gate, we performed a cysteine mutagenesis scan and western blot analyses to identify putative regions of direct contact between GluN1 M4 and GluN2A M3. We identified strong crosslinking at GluN2A-F636 with GluN1-G815 and GluN1-L808. We performed alanine mutagenesis on these sites and recorded single-molecule steady-state currents using cell-attached patch clamp. We performed kinetic modelling and used estimated rate constants to execute a double mutant cycle analysis on these recordings to determine the functional impact of these interactions. Each mutant significantly impacted gating open probability (GluN1/GluN2A 0.53 ± 0.10; GluN1G815A/GluN2A 0.32 ± 0.11; GluN1/GluN2AF636A 0.81 ± 0.04; GluN1G815A/GluN2AF636A 0.88 ± 0.04). We found GluN1-G815 made contact with GluN2A-F636 and contributed 1.9 kJ to desensitization. This indicates a functional interaction between the residues that coincides with the physical interaction observed between GluN1-G815 on the M4 helix with GluN2A-F636 on the M3 helix. Our results suggest a model whereby the GluN1 M4 helix makes direct, functionally important contacts with the GluN2A channel gate. This may serve as a mechanism by which intracellular changes in the c-terminal domain can communicate with the channel gate. The GluN1 M4 also houses several disease-associated sites. Mutations that disrupt M3-M4 coupling may be a novel mechanism of disease pathology.