Specificity of Calmodulin Recognition of Human Voltage-Gated Sodium Channels

Abstract

Voltage-dependent sodium channels (Nav1.X) control the rising phase of action potentials in excitable cells. Mutational pathologies include epilepsy, Long QT syndrome, familial autism, and pain insensitivity. This family of ion channels is regulated by calmodulin (CaM), a small, essential eukaryotic calcium sensor that contains two highly homologous domains whose affinities for calcium differ by an order of magnitude. CaM is known to recognize at least two regions of NaV: the highly conserved inactivation gate between transmembrane domains III and IV, and an IQ motif, located in the intracellular C-terminus of the Nav. The IQ motif, which may serve as a CaM “sink”, holds CaM available to quickly re-associate with the inactivation gate upon calcium binding. High-resolution structures of apo CaM bound to the IQ motifs of NaV1.2, 1.5 and 1.6 show many similarities. However, the free energies of CaM binding to these motifs are markedly different. In our studies of CaM binding to the IQ motif of NaV1.2, the C-domain of both apo (calcium-free) and calcium-saturated CaM bind and calcium binding to CaM lowers its affinity for the IQ motif. To better understand calcium-mediated feedback control, we conducted thermodynamic analyses of CaM binding to the IQ motif sequences representing all 9 members of the human Nav family and the consensus inactivation gate. CaM binding was detected by monitoring the loss of FRET intensity of a biosensor containing the IQ motif sandwiched between two YFP and CFP. The nine sodium channels split into two classes based on their binding affinity for apo CaM at the IQ motif. We are exploring the roles of individual residues to determine the positions that are necessary and sufficient to confer preferential binding of apo or calcium-saturated CaM. Support: NIH R01 GM57001.