Molecular motions that shape the cardiac action potential: Insights from voltage clamp fluorometry

Abstract

Very recently, voltage-clamp fluorometry (VCF) protocols have been developed to observe the membrane proteins responsible for carrying the ventricular ionic currents that form the action potential (AP), including those carried by the cardiac Na þ channel, Na V 1.5, the L-type Ca 2þ channel, Ca V 1.2, the Na þ /K þ ATPase, and the rapid and slow components of the delayed rectifier, K V 11.1 and K V 7.1. This development is significant, because VCF enables simultaneous observation of ionic current kinetics with conformational changes occurring within specific channel domains. The ability gained from VCF, to connect nanoscale molecular movement to ion channel function has revealed how the voltage-sensing domains (VSDs) control ion flux through channel pores, mechanisms of post-translational regulation and the molecular pathology of inherited mutations. In the future, we expect that this data will be of great use for the creation of multi-scale computational AP models that explicitly represent ion channel conformations, connecting molecular, cell and tissue electrophysiology. Here, we review the VCF protocol, recent results, and discuss potential future developments, including potential use of these experimental findings to create novel computational models.

Mechanisms and models of cardiac sodium channel inactivation

“…Because computational models of channels are readily incorporated into multiscale cell and tissue models, 136,138 this approach could be used to identify how inactivation should be targeted therapeutically to stabilize excitation at the tissue level to prevent arrhythmia. As additional structural data emerges, including the Cryo-EM structure of a mammalian Na V channel, 139 conformational data from fluorescence experiments, 140 and even NMR data showing channel dynamics, 141 these models will only improve, provided that the computational approaches needed to incorporate this type of information into the models continue to be developed in parallel.…”

Mechanisms of noncovalent β subunit regulation of NaV channel gating

“…For many years, this protocol has been applied to study the skeletal muscle isoform Na V 1.4, and it has provided great insight into the VSD roles in determining activation and inactivation gating kinetics (Cha et al, 1999; Chanda and Bezanilla, 2002 ; Silva and Goldstein, 2013a , b ), the mechanisms of local anesthetic regulation of the VSDs ( Muroi and Chanda, 2009 ; Arcisio-Miranda et al, 2010 ), and details of how toxins pathologically affect VSD activation ( Campos et al, 2007 , 2008 ). We have recently broadened this approach by developing VCF constructs to track VSD conformations of all four domains in the cardiac paralog, Na V 1.5 ( Varga et al, 2015 ; Zhu et al, 2016 ), whose ionic current modulation by the β subunit in oocytes mirrors the mammalian cell phenotype.…”

Regulation of Na+ channel inactivation by the DIII and DIV voltage-sensing domains

“…We have recently developed VCF protocols for hNa V 1.5 and observed that the DIII-VSD was uniquely immobilized by prolonged inactivation-inducing pulses (Varga et al, 2015; Zhu et al, 2016). Here, we apply these methods to observe the VSD effects of inactivation-deficient mutations within the DIII–DIV linker, the DIII S4–S5 linker, and the DIV S4–S5 linker to discover how they interact to regulate Na V channel inactivation.…”