Abstract 16943: Dissecting the Molecular Interaction of Caveolin3 and the Regulatory Subunit (SUR2) of Cardiac KATP Channels

Abstract

Macromolecular signaling complexes including ion channels present in caveolar microdomains can provide highly specific and localized regulation of cell function. Prior studies have shown that Caveolin3 (Cav3) may interact with a Kir6.2 pore of the cardiac ATP-sensitive potassium channels (KATP). In this report, we studied the molecular interaction of Cav3 and SUR2, the regulatory subunit of KATP. Cav3 and SUR2 were detected in the same caveolar fractions isolated from mouse left ventricles. A P104L-Cav3 mutation was used to probe Cav3-SUR2 trafficking and molecular interaction in COS1 cells. Immunostaining data showed that SUR2 co-localized with Cav3, however, P104L caused retention of SUR2-specific fluorescent signals intracellularly. A quantitative Bioluminescence Resonance Energy Transfer (BRET) assay using coelenterazine-h as substrate was established to study Cav3-SUR2 interaction. WT or P104L-Cav3 was fused to a Renilla Luciferase (Rluc) while SUR2 was fused to YFP. Both WTCav3/SUR2 (0.19±0.04, n=10) and P104LCav3/SUR2 (0.17±0.02, n=10, P=0.05) pairs displayed positive BRET ratios, suggesting that the two proteins were within a 10-100 Å distance to permit energy transfer from RLuc (470-nm) to YFP (530-nm). We did not record positive BRET ratios from negative control samples containing no substrate. Our data showed that Cav3 could directly bind to SUR2, and this interaction was not significantly affected by the P104L mutation, indicating that aa104 is probably not a binding site for the two proteins. To determine which portion of SUR2 binds to Cav3, 3 expression clones harboring SUR2 transmembrane domain TMD0, TMD1 or TMD2 were made. BRET results showed that Cav3 preferably binds to TMD0 (0.19±0.01, n=5) or TMD1 (0.15±0.03, n=5) over TMD2 (0.02±0.01, n=5). Co-immunoprecipitation results confirmed our BRET data. Mouse neonatal myocytes infected with adenoviral particles expressing P104LCav3 displayed a significantly prolonged action potential (APD90, 20.5±4, n=6) than WTCav3-expressing cells (9.3±4, n=4, p<0.05), which suggested that Cav3 levels affected membrane electrical properties. Together with other data, a Cav3-SUR2-Kir6.2 complex that resides in the caveolar microdomains may play an important role in cardiac signaling.