Disease-linked mutations alter the stoichiometries of HCN-KCNE2 complexes

Yoann Lussier,Daniel G Bichet,Lena Moeller,Nazzareno D’Avanzo,Rikard Blunck,Oliver Fürst,Marc Leclerc,Eva Fortea,Dimitri Priolo

doi:10.1038/s41598-019-45592-3

Abstract

The four hyperpolarization-activated cylic-nucleotide gated (HCN) channel isoforms and their auxiliary subunit KCNE2 are important in the regulation of peripheral and central neuronal firing and the heartbeat. Disruption of their normal function has been implicated in cardiac arrhythmias, peripheral pain, and epilepsy. However, molecular details of the HCN-KCNE2 complexes are unknown. Using single-molecule subunit counting, we determined that the number of KCNE2 subunits in complex with the pore-forming subunits of human HCN channels differs with each HCN isoform and is dynamic with respect to concentration. These interactions can be altered by KCNE2 gene-variants with functional implications. The results provide an additional consideration necessary to understand heart rhythm, pain, and epileptic disorders.

Highlights

The four mammalian homologs of hyperpolarization activated cyclic-nucleotide gated (HCN1–HCN4) channels represent the molecular correlate of the currents If or Ih in cardiomyocytes and neurons[1,2]
KCNE2-msfGFP fusion protein was co-expressed with hyperpolarization-activated cylic-nucleotide gated (HCN) isoforms in Chinese hamster ovary (CHO-K1) cells in a 1:1 ratio (w:w) and imaged by total-internal reflection (TIRF) microscopy
Fig. 2) were used to account for any KCNE2 that traffics to the plasmalemma or to intracellular organelles within TIRF distance either on its own or in complex with endogenous proteins. This background KCNE2 expression attributed to less than 15% of the analyzed spots and was subtracted from the distribution collected in the presence of HCN channels

Summary

Introduction

The four mammalian homologs of hyperpolarization activated cyclic-nucleotide gated (HCN1–HCN4) channels represent the molecular correlate of the currents If or Ih in cardiomyocytes and neurons[1,2]. The mutation M54T in KCNE2 is genetically linked to sinus bradycardia and reduces Ih density in neonatal rat ventricular myocytes by 80% and slows activation kinetics at physiologically relevant voltages[48]. Targeted deletion of KCNE2 shifts the voltage dependence and alters Ih activation and deactivation kinetics in layer 6 pyramidal neurons, down-regulates HCN1 and HCN2 expression in the brain and results in hyper-susceptibility to the convulsant 4-AP54. These results indicate that HCN-KCNE2 complexes have important physiological implications in both cardiac and neuronal function. We studied how many regulatory KCNE2 subunits interact with the pore forming HCN subunits, and whether the stoichiometry is altered by genetic variations

Methods

Results

Conclusion