Isoform- and Species-Specific Proteolysis of Cardiac Pacemaker Channels

Abstract

Proteolysis of cardiac pacemaker channels affects biophysical properties of functional channels. Hyperpolarization-activated channels HCN2 and HCN4 can form homomeric or heteromeric functional pacemaker channels in cardiac ventricles. Employing Western blot and immunoprecipitation techniques with antibodies against N- or C- terminus of HCN2 or HCN4, respectively, we investigated protein expression patterns of endogenous HCN2 and HCN4 in cardiac ventricles of small (mouse, rat) and large (sheep, canine) animals and human. Using an antibody against N-terminus of HCN2, more full length protein at 100kD and less cleaved bands around 50kD were detected in small than in large animals. An additional cleaved band around 60kD was exclusively expressed in human. HCN2 C-terminal antibody could not detect any full length protein in all species tested. A 75kD cleaved band was detected in mice, rat, canine and substantially higher in sheep heart ventricles. A 60kD band was observed in human only. Using an N-terminal HCN4 antibody, the full length protein signals (at 160kD) were present in sheep and canine only. The cleaved bands near 100kD predominated in small animals but absent in large animals. With a C-terminal HCN4 antibody, the full length protein was observed in mice, barely detectable in rat, and clearly present in sheep, canine and human. A cleaved band around 100kD predominated in all animals. A minor cleaved band around 50kD appeared in all tested species except human. Overall, there was less HCN2 and more HCN4 proteolysis in small than in large animal cardiac ventricles. Endogenous myocardial HCN2 and HCN4 underwent intensive proteolysis at both N- and C- termini in an isoform- and species-specific pattern. In conclusion, results obtained from HCN2 and HCN4 protein expression in small animals may not be directly applied to large ones including human.