Structural basis and functional analysis of NMDA receptor regulation by calmodulin

The synaptic plasticity mechanisms that are thought to underlie learning and memory require Ca²⁺ influx mediated by N-methyl-D-aspartate receptors composed of glycine-binding GluN1 and glutamate-binding GluN2 subunits. Calmodulin (CaM) binding to the cytosolic regions in both GluN1 (residues 841-865, called GluN1-C0) and GluN2A (residues 1004-1023, called GluN2A-C0) may be important for Ca²⁺-dependent channel desensitization (CDD). Here, we report NMR, ITC and electrophysiological experiments to probe the structure and functional role of Ca²⁺-bound CaM (Ca²⁺-CaM) binding to both GluN1 and GluN2A subunits. Our ITC studies show that the GluN1-C0 peptide binds to both the N-lobe and C-lobe of Ca²⁺-CaM, whereas the GluN2A-C0 peptide binds to only the Ca²⁺-CaM C-lobe. Our NMR analysis reveals GluN2A residues (W1014 and V1018) interact with exposed hydrophobic residues in the Ca²⁺-CaM C-lobe. The NMR structure of Ca²⁺-CaM bound to the GluN1-C0 peptide indicates the two CaM lobes bind to opposite sides of the GluN1-C0 helix (C-lobe contacts M848, F852, A853 and N-lobe contacts A854, V855, W858). The GluN1 mutant F852E and the GluN2A mutant W1014E both perturbed CaM binding in ITC studies, and also diminished electrophysiologically-measured CDD, suggesting CaM interaction with these residues contributes to CDD. We propose a structural mechanism of CDD wherein channel desensitization is caused by the binding of four CaM per N-methyl-D-aspartate receptor subunit tetramer.