Abstract
Voltage-gated sodium channels (NaV) 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 NaV1.5, 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.
Highlights
Mammalian voltage-gated sodium channels (NaVs) are large plasma membrane proteins that selectively conduct Na+ ions down their electrochemical gradient[1]
NaV1.5 is the target for multiple mutations linked to Long-QT type 3 (LQT3) and Brugada Syndromes[20,21,22], several of which are in the EF-hand region (Fig. 1)
Tailoring the exact voltage-dependence of this curve can dictate the amount of available sodium channels
Summary
Mammalian voltage-gated sodium channels (NaVs) are large plasma membrane proteins that selectively conduct Na+ ions down their electrochemical gradient[1]. The mammalian α-subunit can associate with one or more of four different β-subunit isoforms[4] These form single-transmembrane helices, with an extracellular immunoglobulin-like domain[3,5,6,7] and a short cytosolic tail. Several high-resolution structures have been described for the C-terminal portion of mammalian NaVs8–14 (Fig. 1b). These show an EF-hand domain after the last transmembrane segment, followed by an IQ motif. Despite the presence of high Ca2+ concentrations in conditions used for multiple crystallography studies, no Ca2+ has been observed to bind to it This is in contrast with previous experiments suggesting direct binding of Ca2+ to the EF-hand domain[15]. Several functional experiments have hinted at a role for the C-terminal region in inactivation[18,19]
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