A Thermodynamic Analysis of Disease-Causing Mutations in the Nav1.5 C-Terminus

The opening of voltage-gated sodium channels (Nav) is responsible for the rapid upstroke of action potentials. A key player during myocardial excitation is the cardiac channel isoform Nav1.5. The general architecture of mammalian Navs is comprised of four homologous domains, containing six transmembrane segments each, and a C-terminal intracellular domain (CTD) carrying an IQ-motif. The individual domains are connected by large intracellular linkers. The linker connecting domain three and four as well as the CTD seem to play a role in channel inactivation which is different from regular channel closing but poses an important function to modulate ion conductance. The rapid inactivation of channels limits the influx of ions and therefore depolarization of the cell per opening signal. In this context the CTD is of particular interest as an interaction partner for regulatory proteins as calmodulin (CaM) as well as hotspot for disease-causing mutations that have a profound influence on channel inactivation. To elucidate the functional effects of disease-causing mutations we expressed mutant channels in Xenopuslaevis oocytes and studied them by two-electrode voltage clamp. To complement the data we analyzed the thermostability of isolated mutant CTDs and performed isothermal titration calorimetry experiments. Isothermal titration calorimetry experiments in the absence and presence of Ca2+ were used to determine binding profiles of individual CaM lobes to WT and mutant CTDs. Our data shows that mutations have distinct effects on the folding stability and ability to bind CaM. Whereas some mutations cause misfolding of the CTD, others selectively affect binding of apoCaM or both apoCaM and Ca2+/CaM, and these changes correlate with the disease phenotype.