Caveolin-3 associates with and affects the function of hyperpolarization-activated cyclic nucleotide-gated channel 4

Targeting of ion channels to caveolae, a subset of lipid rafts, allow cells to respond efficiently to extracellular signals. Hyperpolarization-activated cyclic nucleotide-gated channel (HCN) 4 is a major subunit for the cardiac pacemaker. Caveolin-3 (Cav3), abundantly expressed in muscle cells, is responsible for forming caveolae. P104L, a Cav3 mutant, has a dominant negative effect on wild type (WT) Cav3 and associates with limb-girdle muscular dystrophy and cardiomyopathy. HCN4 was previously shown to localize to lipid rafts, but how caveolae regulate the function of HCN4 is unknown. We hypothesize that Cav3 associates with HCN4 and regulates the function of HCN4 channel. In this study, we applied whole-cell patch clamp analysis, immunostaining, biotinylation, and immunoprecipitation methods to investigate this hypothesis. The immunoprecipitation results indicated an association of HCN4 and Cav3 in the heart and in HEK293 cells. Our immunostaining results showed that HCN4 colocalized with Cav3 but only partially colocalized with P104L in HEK293 cells. Transient expression of Cav3, but not P104L, in HEK 293 cells stably expressing HCN4 caused a 45% increase in HCN4 current (IHCN4) density. Transient expression of P104L caused a two-fold increase in the activation time constant for IHCN4 and shifted the voltage of the steady-state inactivation to a more negative potential. We conclude that HCN4 associates with Cav3 to form a HCN4 macromolecular complex. Our results indicated that disruption of caveolae using P104L alters HCN4 function and could cause a reduction of cardiac pacemaker activity.