Abstract 2325: Caveolae Are Crucial for Receptor-specific Regulation of G Protein-activated Inwardly Rectifying K+ Channel in Mouse Atrial Myocytes
Acetylcholine (ACh) decreases the heart rate by activating G-protein-activated inwardly rectifying K+ (GIRK) channels. In turn, GIRK channels are inhibited by reducing phosphatidyl-inositol 4,5-bisphosphate (PIP2) through Gq protein-coupled receptors (GqPCRs). In previous studies, we found that endothelin-1 (ET-1), but bradykinin (BK) inhibited GIRK currents, even though both of them hydrolyze PIP2 in cardiac myocytes, showing receptor-specificity of GIRK channel regulation. In the present study, we investigated whether caveolae are functioning as signaling microdomain for receptor-specificity in mouse atrial myocytes. Using immunoprecipitation and immunofluorescence analysis, we examined the subcellular localization of GIRK. We found that ACh stimulation (4 min) significantly increased interaction of GIRK with caveolin-3, marker protein for caveolae. In addition, immunoprecipitation study showed that ACh stimulation increased GIRK/ET-1 receptor interaction but decreased GIRK/BK receptor interaction. These data suggested that agonist-mediated organization of signaling complex arises from the activation and translocation of GIRK channel to caveolae. When we disrupted caveolae with methyl-β-cyclodextrin (MβCD, 10 mM), ACh did not increase the interaction of GIRK with caveolin-3. The induction of GIRK/ET-1 receptor signaling complex and the dissociation of GIRK from BK receptors were also disrupted. These data indicated that the integrity of caveolae is important for induction of signaling complex. To determine the functional impact of the caveolae-dependent GIRK/GqPCR signaling complex, the response of GIRK channel to GqPCRs was examined. In control condition, the ET-1 induced inhibition of GIRK was 86.4±1.6% (n=3) and the inhibition of GIRK by BK was minimal (5.3±0.8%, n= 8). In the presence of MβCD, however, both ET-1 and BK caused an inhibition of GIRK currents, and the magnitude varied in a wide range among cells (13.1–95.1% for ET-1 (n=7), 13.0 –98.9% for BK (n=10)), suggesting that caveolae disruption eliminated receptor-specificity. In conclusion, our work indicates that localization of GIRK channels to caveolar macromolecular signaling complex is essential for receptor-specific regulation of GIRK channels.