Identification of an atypical calcium-dependent calmodulin binding site on the C-terminal domain of GluN2A

Abstract

N-methyl-d-aspartate (NMDA) receptors are calcium-permeable ion channels assembled from four subunits that each have a common membrane topology. The intracellular carboxyl terminal domain (CTD) of each subunit varies in length, is least conserved between subunits, and binds multiple intracellular proteins. We defined a region of interest in the GluN2A CTD, downstream of well-characterized membrane-proximal motifs, that shares only 29% sequence similarity with the equivalent region of GluN2B. GluN2A (amino acids 875–1029) was fused to GST and used as a bait to identify proteins from mouse brain with the potential to bind GluN2A as a function of calcium. Using mass spectrometry we identified calmodulin as a calcium-dependent GluN2A binding partner. Equilibrium fluorescence spectroscopy experiments indicate that Ca2+/calmodulin binds GluN2A with high affinity (5.2±2.4nM) in vitro. Direct interaction of Ca2+/calmodulin with GluN2A was not affected by disruption of classic sequence motifs associated with Ca2+/calmodulin target recognition, but was critically dependent upon Trp-1014. These findings provide new insight into the potential of Ca2+/calmodulin, previously considered a GluN1-binding partner, to influence NMDA receptors by direct association.