Molecular interactions of the Na_V1.5 C-terminal domain: CaM sequestered the IQ motif from the CTD

Voltage-gated sodium channels (Na_V) are important for life. Alterations to the synchronized timing of ion conduction can create life-threatening conditions. How Na_V conduction responds to changes in intracellular Ca²⁺ concentration has been the subject of extensive investigation. Crystal structures of the cardiac Na_V (Na_V1.5) cytosolic components were reported as trimeric complexes that are restructured in the presence of Ca²⁺. These results formed the basis for a gating model where two Na_V1.5 molecules interact to alter function in response to Ca²⁺ concentration. Here, we investigated the binding site surface of these trimeric interactions in solution. Nuclear magnetic resonance spectroscopy demonstrated that these trimeric complexes do not form in solution. Analysis of the available structural data indicated that the Na_V1.5 IQ motif can only accommodate interaction with one protein at a time. Our nuclear magnetic resonance spectroscopy data were consistent with the Na_V1.5 C-terminal domain (CTD) and the Ca²⁺- sensing protein calmodulin (CaM) engaging the same binding site surface of the Na_V1.5 IQ motif. Titrations of IQ motif peptide into a 1:1 mixture of ¹⁵N CTD: CaM sample revealed the Na_V1.5 channel is biophysically distinct from neuronal Na_V1.2 as Ca²⁺ enhanced CaM's ability to sequester the Na_V1.5 IQ motif from the Na_V1.5 CTD. Stopped-flow kinetic measurements quantified Ca²⁺ release rates from the CaM-IQ and CaM-inactivation gate (IGATE) complexes to provide insight into complex lifetimes. Our work advanced understanding the molecular machinery that underlies Na_V1.5 gating (CTD-IGATE and CTD-IQ motif interactions) and provided insight into the structural details of CaM-facilitated Na_V1.5 modification (CaM-IQ motif and CaM-IGATE interactions).