Abstract 19025: A Signaling Complex of HCN2/HCN4/Cav3 Resides in Caveolae of Cardiac Cells

Abstract

Macromolecular signaling complexes including ion channels present in the caveolar micro-domains can provide highly specific and localized regulation of cell function. The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are key constituents of the cardiac pacemaker, regulating the rate of diastolic depolarization and cardiac rhythm. HCN4 and HCN2 are the major HCN isoforms found in cardiac tissues. HCN4 is physically associated with Caveolin-3 (Cav3), the “coat protein” for caveolae in cardiac cells. In this report, we dissected the regulation of Cav3 on HCN2. Cardiac HCN2 (60-kDa, HCN2s) or HCN4 was co-localized with Cav3 in mouse LV membrane fractions. A dominant-negative P104L-Cav3 mutation was used to “probe” the association of HCN2/Cav3. HCN2s itself does not encode currents but HCN2s/HCN4 channels generated currents resembled to those recorded from native cells. HEK293 cells expressing full-length-HCN2 (HCN2f)/WTCav3 showed comparable HCN2 density and channel activity to those cells expressing HCN2f/P104L or HCN2 alone. Bioluminescence Resonance Energy Transfer (BRET) assays in conjunction with stable HCN cell lines and lipid raft inhibitors were used to quantify the intra-molecular interactions of each HCN form and Cav3. WTCav3 or P104L was fused to a Renilla Luciferase (Rluc) as the “donor” while each HCN form was fused to YFP as the “acceptor”. All HCN/Cav3 pairs displayed positive BRET ratios, suggesting that HCN and Cav3 were within a 10-100 Å distance to permit energy transfer from RLuc (470-nm) to YFP (530-nm) in the presence of coelenterazine-h. We did not record positive BRET ratios from samples containing no assay substrate. Average BRET ratio data (n=6, p<0.05) suggested that the association for HCN2s/Cav3 is the strongest. The P104L study showed that aa104 is involved in the interactions of HCN2s/Cav3 or HCN4/Cav3 but not HCN2f/Cav3. Our results indicate that Cav3 interacts with HCN4 via HCN2s to form a HCN2s/HCN4/Cav3/caveolae complex, and it may play an important role in cardiac signaling.