The voltage-gated sodium channel EF-hands form an interaction with the III-IV linker that is disturbed by disease-causing mutations

Voltage-gated sodium channels (Na(V)) are responsible for the rapid depolarization of many excitable cells. They readily inactivate, a process where currents diminish after milliseconds of channel opening. They are also targets for a multitude of disease-causing mutations, many of which have been shown to affect inactivation. A cluster of disease mutations, linked to Long-QT and Brugada syndromes, is located in a C-terminal EF-hand like domain of Na(V)1.5, the predominant cardiac sodium channel isoform. Previous studies have suggested interactions with the III-IV linker, a cytosolic element directly involved in inactivation. Here we validate and map the interaction interface using isothermal titration calorimetry (ITC) and NMR spectroscopy. We investigated the impact of various disease mutations on the stability of the domain, and found that mutations that cause misfolding of the EF-hand domain result in hyperpolarizing shifts in the steady-state inactivation curve. Conversely, mutations in the III-IV linker that disrupt the interaction with the EF-hand domain also result in large hyperpolarization shifts, supporting the interaction between both elements in intact channels. Disrupting the interaction also causes large late currents, pointing to a dual role of the interaction in reducing the population of channels entering inactivation and in stabilizing the inactivated state.