Interplay of Nav1.5 Disease-Causing Mutations and Phosphorylation Revealed by Protein Semi-Synthesis and Molecular Dynamics Simulations

Abstract

Activation of the voltage-gated sodium channel Nav1.5 initiates the cardiac action potential and numerous Nav1.5 mutations are known to cause severe and potentially life-threatening arrhythmias. These are typically caused by changes to channel function, such as altered inactivation. Additionally, it is becoming increasingly recognised that Nav1.5 undergoes heavy post-translational modification in vivo. However, conventional approaches are unable to reliably mimic post-translational modification, such as phosphorylation. This has prevented investigation of a potential functional interplay with patient mutations. Here, we overcome this limitation by using protein semi-synthesis of Nav1.5 in live cells in combination with molecular dynamics simulations. Indeed, we introduce stable phosphorylation mimics on both WT and two different pathogenic long-QT mutant channel backgrounds and decipher functional and pharmacological effects with unique precision. Our data demonstrate that the altered steady-state inactivation profile caused by phosphorylation of Y1495 is due to a destabilization of the inactivation particle in its binding site. Surprisingly, we find that unlike the ΔK1500 mutant, the Q1476R mutation does not itself change channel inactivation properties, but that phosphorylation of this mutant results in a greatly increased right-shift in steady-state inactivation compared to WT (20 mV vs 10 mV). Additionally, free energy calculations suggest that unbinding of the inactivation particle is energetically favored when Y1495 is phosphorylated, and this effect is again exacerbated in the presence of the Q1476R mutation. Lastly, we show that both phosphorylation and patient mutations can impact Nav1.5 sensitivity towards the clinically used anti-arrhythmic drug quinidine, but not flecainide. In short, functional effects can be exacerbated by phosphorylation in the presence of human patient mutations in Nav1.5. This has implications for the interpretation of mutational phenotypes, as well as possibly the design of future drug regimens.