Functional cross-talk between phosphorylation and disease-causing mutations in the cardiac sodium channel Na _v 1.5

Abstract

The voltage-gated sodium channel Nav1.5 initiates the cardiac action potential. Alterations of its activation and inactivation properties due to mutations can cause severe, life-threatening arrhythmias. Yet despite intensive research efforts, many functional aspects of this cardiac channel remain poorly understood. For instance, Nav1.5 undergoes extensive posttranslational modification in vivo, but the functional significance of these modifications is largely unexplored, especially under pathological conditions. This is because most conventional approaches are unable to insert metabolically stable posttranslational modification mimics, thus preventing a precise elucidation of the contribution by these modifications to channel function. Here, we overcome this limitation by using protein semisynthesis of Nav1.5 in live cells and carry out complementary molecular dynamics simulations. We introduce metabolically stable phosphorylation mimics on both wild-type (WT) and two pathogenic long-QT mutant channel backgrounds and decipher functional and pharmacological effects with unique precision. We elucidate the mechanism by which phosphorylation of Y1495 impairs steady-state inactivation in WT Nav1.5. Surprisingly, we find that while the Q1476R patient mutation does not affect inactivation on its own, it enhances the impairment of steady-state inactivation caused by phosphorylation of Y1495 through enhanced unbinding of the inactivation particle. We also show that both phosphorylation and patient mutations can impact Nav1.5 sensitivity toward the clinically used antiarrhythmic drugs quinidine and ranolazine, but not flecainide. The data highlight that functional effects of Nav1.5 phosphorylation can be dramatically amplified by patient mutations. Our work is thus likely to have implications for the interpretation of mutational phenotypes and the design of future drug regimens.

The dispensability of 14-3-3 proteins for the regulation of human cardiac sodium channel Nav1.5

“…Another limitation of heterologously expressed proteins is the difficulty in controlling post-translational modifications, such as phosphorylation, that impact Na v 1.5 function and 14-3-3 signaling. A recent study reported a successful semisynthetic approach to stabilize Na v 1.5 phosphorylation in vitro 29 . Thus, it would be interesting to test difopein-mediated regulation under stably phosphorylated Na v 1.5.…”

Modulation of IKs channel–PIP2 interaction by PRMT1 plays a critical role in the control of cardiac repolarization

“…Despite the clear association between heart failure and arrhythmias, widely applicable treatments to simultaneously treat both diseases have been difficult to develop. Rather than targeting and modulating the ion channels themselves, targeting the posttranslational modifications of ion channels, which have more subtle effects on excitability, might prove to be an interesting alternative to treat arrhythmias (Galleano et al, 2021). Therefore, the benefits of PRMT1 on CaMKII and I Ks in cardiomyocytes open the possibility of using the PRMT1 signaling pathway as a novel therapeutic target for treating both heart failure and arrhythmias.…”

Structural Modeling of Peptide Toxin - Ion Channel Interactions using RosettaDock

“…In addition, both structural models and native structures represent only snapshots of the full spectrum of protein dynamics. Using static ion channel structures as inputs for molecular dynamics simulations can provide a broader perspective of the dynamics underlying the ion channel gating and modulation (Flood et al, 2019; Galleano et al, 2021; Nguyen et al, 2019; Rovšnik et al, 2021; Suma et al, 2020). Finally, structure-guided computational protein design has achieved significant milestones in recent years (Anishchenko et al, 2021; Cao et al, 2020, 2022; Sahtoe et al, 2022; Silva et al, 2019), and these approaches can be applied to engineer ion channels (Xu et al, 2020; Yang et al, 2016) or design novel ion channel modulators (Xu et al, 2021).…”