Channel HCN4 mutation R666Q associated with sporadic arrhythmia decreases channel electrophysiological function and increases protein degradation

Mutations in the hyperpolarization-activated nucleotide-gated channel 4 (HCN4) are known to be associated with arrhythmias in which QT prolongation (delayed ventricular repolarization) is rare. Here, we identified a HCN4 mutation, HCN4-R666Q, in two sporadic arrhythmia patients with sinus bradycardia, QT prolongation and short bursts of ventricular tachycardia. To determine the functional effect of the mutation, we conducted clinical, genetic and functional analyses using whole-cell voltage-clamp, qPCR, western blot, confocal microscopy and co-immunoprecipitation. The mean current density of HEK293T cells transfected with HCN4-R666Q was lower in 24-36 h after transfection, and was much lower in 36-48 h after transfection relative to cells transfected with wild type HCN4. Additionally, we determined that the HCN4-R666Q mutant was more susceptible to ubiquitin-proteasome system-mediated protein degradation than wild type HCN4. This decreased current density for HCN4-R666Q could be partly rescued by treatment with a Proteasome Inhibitor. Therefore, we conclude that HCN4-R666Q had an effect on HCN4 function in two aspects, including decreasing the current density of the channel as a biophysical effect and weakening its protein stability. Our findings provide new insights into the pathogenesis of the HCN4-R666Q mutation.

HCN4; QT prolongation; bradycardia; electrophysiology; short bursts of ventricular tachycardia.