Calmodulin Regulation of the Neuronal Voltage-Dependent Sodium Channel

Abstract

Calmodulin (CaM) is an essential eukaryotic calcium sensor comprised of two homologous domains (N, C). Ca2+ binding to CaM changes its conformation and determines how CaM recognizes and regulates target proteins such as the neuronal voltage-dependent sodium channel (Nav1.2) which is essential for the generation and propagation of action potentials. Nav1.2 is a multimer with one pore-forming α-subunit and one or more β-subunits. CaM binds to an IQ-motif (IQxxxBGxxxB, B=K,R) of Nav1.2 that is near the C-terminus of the α-subunit. Prior thermodynamic studies showed that this IQ peptide (Nav1.2IQp, KRKQEEVSAIVIQRAYRRYLLKQKVKK) selectively lowers the Ca2+-binding affinity sites in the C-domain of CaM, without affecting the N-domain (Theoharis et al, Biochemistry 2008). This selective decrease correlates with Nav1.2IQp having a higher affinity for apo CaM than for calcium-saturated CaM. Structural studies of complexes of CaM bound to target peptides or proteins demonstrated that the 4-helix bundle of the CaM C-domain adopts an “open” conformation when Ca2+-saturated. There is only one high-resolution structure (2IX7) of apo CaM bound to an IQ motif; it shows the C-domain having a “semi-open” conformation. To understand the Ca2+-dependent conformational switching CaM when regulating Nav1.2, we applied heteronuclear NMR methods. Amide exchange, hnNOE, and chemical shift perturbation experiments revealed residue-specific changes consistent with a “semi-open” conformation of the apo C-domain of CaM when bound to Nav1.2IQp. NMR experiments are complete and analysis is underway to determine the solution structure of the apo C-domain of CaM bound to Nav1.2IQp. Understanding the interface between CaM and the IQ-motif of the channel will result in a more complete model of how CaM regulates Nav1.2 function at low physiological [Ca2+] in neuronal tissues. NIH GM57001