A195V mutation identified in sudden cardiac death decreases membrane localization and activation current density and abolishes cAMP sensitivity of HCN4 channels

Abstract

Abstract Mutation A195V in hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) channel was identified to be genetically associated with sudden unexplained nocturnal death syndrome (SUNDS) and sudden infant death syndrome (SIDS) by us and other group, respectively. However, the underlying mechanism by which this rare variant causes SUNDS and SIDS remains unknow. In this study, HEK293 cells were transfected with wild type or mutant A195V HCN4 plasmid. qPCR, western blot and confocal analysis were conducted to analyze the expression of HCN4 channels. Whole cell clamp was used to analyze the activation current and tail current of HCN4 channels with and without cAMP stimulation. The results showed that A195V decreased the expression and membrane localization of HCN4 channels. Consistent with this, A195V also decreased the activation current density of HCN4 channels under − 140mV to -100mv and under − 50mV to -40mV. The present study was the first to clarify the decreased expression of HCN4 protein and the decreased activation current density and abolished cAMP sensitivity of HCN4 channels caused by A195V, which biophysically confirmed HCN4 A195V as the pathogenic variation for SUNDS and SIDS.