Nav1.5 C-Terminal Domains Influence Calcium Regulation of Fast Inactivation Separately from Calmodulin Interaction

The cardiac sodium channel (Nav1.5) has a complex ‘intracellular Ca2+ sensing apparatus’ within its C-terminal domain (CTD) consisting of a partial EF-hand domain (CTD-EF) and a calmodulin (CaM) binding IQ motif (CTD-IQ). There are additional CaM binding motifs within the DIII-DIV linker. Variation in intracellular Ca2+ concentration influences the voltage-dependence of steady-state fast inactivation (SSI) by an unclear mechanism. Here, we mutated 16 key residues implicated in the interactions between CTD-EF and CTD-IQ, CTD-IQ and CaM, or DIII-DIV and CaM and explored their impact on NaV1.5 function and biochemistry. We used quantitative yeast-two-hybrid assays to measure effects of mutations on the interaction between the full length CTD on CaM, and evaluated SSI in high (1 µM free Ca2+) and low [Ca2+]i conditions. Using either BAPTA or HEDTA as chelator, 1 µM free [Ca2+]i was sufficient to shift SSI (elicited by 50 ms prepulses) towards depolarized potentials. We observed that 3 of 5 CTD-IQ mutations (F1912A, A1924T and IQ/AA) strongly reduced the interaction between the CTD and CaM. However, these mutations did not affect the [Ca2+]i effect on SSI suggesting that SSI [Ca2+]i sensitivity does not depend on CaM binding to the CTD-IQ. Unexpectedly, single and combination mutations of the CTD-EF (L1786A, F1791A, Q1807A, L1862A, E1788A-D1790A-D1792A-E1799A, E1804A-D1802A) diminished the CTD-CaM interaction, and some of these mutations (L1786A, E1788A-D1790A-D1792A-E1799A, E1804A-D1802A) also suppressed the [Ca2+]i effect on SSI. Indeed, the only mutations we studied that blunted the [Ca2+]i effect on SSI were within the CTD-EF. These results suggest that the CTD-EF influences NaV1.5 [Ca2+]i sensitivity and mutations in this domain can also alter the interaction of the CTD with CaM, but CaM interaction with the CTD-IQ is not required for the effect of intracellular Ca2+ on inactivation.