β Subunits Functionally Differentiate Human Kv4.3 Potassium Channel Splice Variants.

Geoffrey W Abbott

doi:10.3389/fphys.2017.00066

Abstract

The human ventricular cardiomyocyte transient outward K+ current (Ito) mediates the initial phase of myocyte repolarization and its disruption is implicated in Brugada Syndrome and heart failure (HF). Human cardiac Ito is generated primarily by two Kv4.3 splice variants (Kv4.3L and Kv4.3S, diverging only by a C-terminal, S6-proximal, 19-residue stretch unique to Kv4.3L), which are differentially remodeled in HF, but considered functionally alike at baseline. Kv4.3 is regulated in human heart by β subunits including KChIP2b and KCNEs, but their effects were previously assumed to be Kv4.3 isoform-independent. Here, this assumption was tested experimentally using two-electrode voltage-clamp analysis of human subunits co-expressed in Xenopus laevis oocytes. Unexpectedly, Kv4.3L-KChIP2b channels exhibited up to 8-fold lower current augmentation, 40% slower inactivation, and 5 mV-shifted steady-state inactivation compared to Kv4.3S-KChIP2b. A synthetic peptide mimicking the 19-residue stretch diminished these differences, reinforcing the importance of this segment in mediating Kv4.3 regulation by KChIP2b. KCNE subunits induced further functional divergence, including a 7-fold increase in Kv4.3S-KCNE4-KChIP2b current compared to Kv4.3L-KCNE4-KChIP2b. The discovery of β-subunit-dependent functional divergence in human Kv4.3 splice variants suggests a C-terminal signaling hub is crucial to governing β-subunit effects upon Kv4.3, and demonstrates the potential significance of differential Kv4.3 gene-splicing and β subunit expression in myocyte physiology and pathobiology.

Highlights

Human voltage-gated potassium (Kv) channels are generated by a gene family expressing 40 different α subunits, producing a wide range of current properties and diversity of conductances, gating kinetics and voltage dependence
It is demonstrated that KChIP2b and KCNEs unexpectedly introduce a number of striking differences in baseline function between human Kv4.3L and Kv4.3S
Xenopus laevis Oocyte Channel cRNA and Peptide Injections cRNA transcripts encoding hKv4.2, hKv4.3L, hKv4.3S, and hKChIP2b were generated by in vitro transcription (T7 polymerase mMessage mMachine kit, Thermo Fisher Scientific) from cDNA sub-cloned into plasmids

Summary

INTRODUCTION

Human voltage-gated potassium (Kv) channels are generated by a gene family expressing 40 different α subunits, producing a wide range of current properties and diversity of conductances, gating kinetics and voltage dependence. The KCNEs ( termed MinK-related peptides, or MiRPs) are singletransmembrane-domain subunits that co-assemble with Kv α subunits to dictate fundamental properties including channel α subunit composition, forward trafficking, endocytosis, ion selectivity, conductance, gating kinetics, voltage dependence, and the effects of regulation by other proteins (Abbott, 2015, 2016a,b). KChIPs are cytosolic proteins that modulate multiple aspects of Kv channel biology, including trafficking, current density, gating kinetics, and voltage dependence (An et al, 2000; Rhodes et al, 2004). It is demonstrated that KChIP2b and KCNEs unexpectedly introduce a number of striking differences in baseline function between human Kv4.3L and Kv4.3S These data both identify a 19-residue C-terminal stretch important for Kv4.3 regulation by β subunits, and suggest the functional and pathophysiological significance of differential remodeling of Kv4.3 splice variants in HF

MATERIALS AND METHODS

Findings

DISCUSSION