Recruitment of Calmodulin to the Tail of the Voltage-Gated Sodium Channel Nav1.2

Abstract

Voltage-gated sodium channels (NaV) found in excitable cells are responsible for the rising phase of action potentials. These multi-domain transmembrane proteins are regulated by calmodulin (CaM), a highly conserved eukaryotic protein that mediates many calcium-triggered signaling events. Inactivation of sodium channels depends on CaM-mediated feedback during repolarization. In the neuronal sodium channel NaV1.2, CaM binds at least two well-separated sites: an intracellular ''inactivation'' loop between domains DIII and DIV, and an IQ motif [IQRAYRRYLLK] in the cytosolic C-terminal tail. The IQ motif is hypothesized to recruit calcium-free (apo) CaM, making it available to move to the III-IV linker after an influx of calcium. Despite a high degree of sequence identity, the equilibrium constants for CaM binding to nine human NaV IQ motifs span more than 3 orders of magnitude. Apo CaM binds to the NaV1.2 IQ motif with a dissociation constant (Kd) of~6 nM, while the Kd for binding the NaV1.9 IQ motif is~4 mM. Mutational analysis within the IQ motif has not been sufficient to explain the full range of CaM-binding affinities observed for human NaV sequences. Thus, we hypothesized that isoform-specific differences in upstream sequences were making energetically significant contributions to the free energy of binding CaM to NaV1.2. The roles of these residues are being investigated by monitoring CaM binding to biosensors containing mutant sequences of sodium channels bracketed by auto-fluorescent proteins YFP and CFP. Residue-specific information obtained by NMR will provide structural insight into the contributions of residues in the binding interface formed by NaV IQ motif sequences binding to calmodulin from multiple eukaryotes. NIH R01 GM57001.