Isoform-Dependent Cholesterol Regulation of HCN Channels

Abstract

Cholesterol is the major sterol component of all mammalian plasma membranes, and has been shown to regulate numerous ion channels. The hyperpolarization current, Ih, generated by the hyperpolarization-activated cyclic AMP-dependent (HCN) ion channels is a major contributor to the pacemaking in cardiac and neuronal tissue. However, regulation of each human HCN channel isoform (HCN1-4) by membrane lipids, including cholesterol, has not been systematically examined. Cholesterol depletion in CHO-K1 cells by MsCD or Mevastatin or enrichment using a cholesterol-MsCD complex expressing HCN1 or HCN2 channels leads to a reduction in current densities as well as altering the kinetics of activation and deactivation. Immunohistochemistry suggests that there is no change in the distribution of these channels from lipid raft domains to non-raft domains as a result of cholesterol modification. Specific isoform differences between HCN1 and HCN2 could be detected in non-equilibrium state known as hysteresis, examined using ramp protocols. HCN1 demonstrates hysteresis when voltage ramps of 600mV/s are applied, whereas hysteresis is observed in HCN2 channels only at much slower ramp speeds (eg. 150mV/s). However, while the degree of hysteresis is unchanged in HCN1 channels when membrane cholesterol levels are modified, hysteresis in HCN2 channels begins to be apparent at faster ramps speeds (300 mV/s) upon cholesterol depletion. Such isoform dependent differences in cholesterol regulation of HCN channels may contribute to their differential distribution and function in cardiac and neuronal tissue.