The Cardiac Channel NaV1.5 Inactivation is Modulated by its C-Terminal EF-hand Domain

Abstract

The cardiac isoform of voltage-gated sodium channels Nav1.5 is responsible for the rapid upstroke of the action potential after excitation. The underlying gene of the alpha-subunit SCN5A is mostly expressed in the myocardium although some expression in other tissue has been reported. Auxiliary proteins like calmodulin have been identified to modify channel gating. Mutations in SCN5A and calmodulin have been linked to several arrhythmias, including Brugada syndrome and Long-QT syndrome type 3. The channel architecture is organized in four homologous transmembrane repeats and a C-terminal EF-hand domain. After channel opening Nav1.5 undergoes inactivation within milliseconds which leads to a non-conducting state of the channel. The cytoplasmic DIII-IV linker is known as inactivation gate. It contains the hydrophobic IFM motif which has been shown to stabilize the inactivated state. A previous study suggested a direct interaction of the C-terminal part of the DIII-IV linker and the C-terminal EF-hand domain of the channel stabilizing the inactivation gate in the inactivated state. Here, we present an alternative model where the EF-hand destabilizes the inactivated state through binding to the DIII-IV linker. Isothermal titration calorimetry was utilized to characterize binding of WT and mutant inactivation gate peptides to the EF-hand domain. NMR spectroscopy enabled us to map the exact interaction site on the EF-hand domain. Two-electrode voltage clamp measurements in Xenopus laevis oocytes provided data on inactivation gate mutants and EF-hand disease mutants in the context of full length channel. Mutations that abolished EF-hand binding lead to a profound left-shift of steady-state inactivation, suggesting a role of the EF-hand domain in destabilizing the inactivated state.