Localization of Pacemaker Channels in Lipid Rafts Regulates Channel Kinetics

Lipid rafts are discrete membrane subdomains rich in sphingolipids and cholesterol. In ventricular myocytes a function of caveolae, a type of lipid rafts, is to concentrate in close proximity several proteins of the β-adrenergic transduction pathway. We have investigated the subcellular localization of HCN4 channels expressed in HEK cells and studied the effects of such localization on the properties of pacemaker channels in HEK and rabbit sinoatrial (SAN) cells. We used a discontinuous sucrose gradient and Western blot analysis to detect HCN4 proteins in HEK and in SAN cells, and found that HCN4 proteins localize to low-density membrane fractions together with flotillin (HEK) or caveolin-3 (SAN), structural proteins of caveolae. Lipid raft disruption by cell incubation with methyl-β-cyclodextrin (MβCD) impaired specific HCN4 localization. It also shifted the midpoint of activation of the HCN4 current in HEK cells and of If in SAN cells to the positive direction by 11.9 and 10.4 mV, respectively. These la...