Abstract
The Ca2+-binding protein calmodulin has emerged as a pivotal player in tuning Na+ channel function, although its impact in vivo remains to be resolved. Here, we identify the role of calmodulin and the NaV1.5 interactome in regulating late Na+ current in cardiomyocytes. We created transgenic mice with cardiac-specific expression of human NaV1.5 channels with alanine substitutions for the IQ motif (IQ/AA). The mutations rendered the channels incapable of binding calmodulin to the C-terminus. The IQ/AA transgenic mice exhibited normal ventricular repolarization without arrhythmias and an absence of increased late Na+ current. In comparison, transgenic mice expressing a lidocaine-resistant (F1759A) human NaV1.5 demonstrated increased late Na+ current and prolonged repolarization in cardiomyocytes, with spontaneous arrhythmias. To determine regulatory factors that prevent late Na+ current for the IQ/AA mutant channel, we considered fibroblast growth factor homologous factors (FHFs), which are within the NaV1.5 proteomic subdomain shown by proximity labeling in transgenic mice expressing NaV1.5 conjugated to ascorbate peroxidase. We found that FGF13 diminished late current of the IQ/AA but not F1759A mutant cardiomyocytes, suggesting that endogenous FHFs may serve to prevent late Na+ current in mouse cardiomyocytes. Leveraging endogenous mechanisms may furnish an alternative avenue for developing novel pharmacology that selectively blunts late Na+ current.
Highlights
Voltage-gated NaV channels initiate action potentials in excitable tissue and are fundamental to defining cellular excitability
We found that the NaV1.5 IQ/AA mutant channels in cardiomyocytes did not appreciably increase late Na+ current, hinting at the presence of endogenous protective mechanisms
To determine regulatory factors that prevent late Na+ current for IQ/AA mutant ch, we considered fibroblast growth factor homologous factors (FHFs), which are within the NaV1.5 proteomic subdomain in the heart, can bind to the C-terminal domain of NaV1.5, and have been implicated in the regulation of late Na+ current [11, 12, 19,20,21]
Summary
Voltage-gated NaV channels initiate action potentials in excitable tissue and are fundamental to defining cellular excitability. Voltage-gated Na+ channels consist of 4 transmembrane domains, each containing 6 transmembrane α-helices, connected by intracellular linkers, and intracellular N-terminal and C-terminal domains (Figure 1A). Voltage sensor movement drives channel openings, while ensuing channel inactivation depends on the allosteric blockade of ion conduction triggered by the interaction of the hydrophobic IFM motif in the III–IV linker with a hydrophobic pocket between domains III and IV [1]. Impaired inactivation leads to late inward Na+ current that can cause cardiac arrhythmias or dilated cardiomyopathy [2, 3]. Increased inward late Na+ influx underlies SCN5A-mediated long QT syndrome type 3 (LQT3) [4]. Increased late Na+ current is sufficient to cause structural and electrical remodeling in the atria of mice, leading to spontaneous atrial fibrillation [5]. Determining how changes in Na+ channel structure [7] and interactome cause functional alterations in channel properties that beget cardiac disease is critical to devise new therapies
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