Identification of an Atypical Calcium-Dependent Calmodulin Binding Site on the C-terminal domain of GluN2A

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 membraneproximal 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 ± 2.4 nM) 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.Keywords: NMDA, Calmodulin, Calcium, GlutamateKeywords: NMDA, Calmodulin, Calcium, Glutamat

Cholinergic ligand-induced affinity changes in Torpedo californica acetylcholine receptor

The binding of cholinergic ligands to Torpedo californica acetylcholine receptor has been studied in vitro by inhibition of the time course of 125I-labeled α-bungarotoxin-receptor complex formation. The extent of inhibition was dependent on the duration of exposure to the ligand, the apparent affinity for ligand increasing with time, and was reversible upon removal of ligand. Ligand concentration, temperature, and Ca2+ ions influenced this effect which is reminiscent of receptor desensitization in vivo. Such effects were observed both for a cholinergic agonist, carbamylcholine, and for an antagonist, bis(3-aminopyridinium)-1,10-decane diiodide. A minimal model is discussed which can account for these effects and for receptor ligand association leading to postsynaptic depolarization

IshmaelJanePharmacyIdentificationAtypicalCalcium(SupplementaryMaterial).pdf

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 membraneproximal 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 ± 2.4 nM) 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.Keywords: NMDA, Calcium, Glutamate, Calmoduli