Abstract 1873: ATP-sensitive K+ Channels are Regulated by Caveolin-Enriched Microdomains in Cardiac Myocytes
ATP-sensitive potassium (KATP) channels in the heart are critical regulators of cellular excitability and action potentials during ischemia. However, little is known about subcellular localization of these channels and their regulation. The present study was designed to assess the hypothesis that KATP channels in cardiac myocytes are localized in caveolin-enriched microdomains and regulated by caveolae/caveolin. Both adult and neonatal rat cardiomyocytes were used. Subcellular fractionation by density gradient centrifugation, Western blotting, co-immunoprecipitation and immunofluorescence confocal microscopy were employed in combination with whole-cell voltage clamp recordings and siRNA gene silencing. We detected that the majority of KATP channel subunits Kir6.2/SUR2A on the plasma membrane of cardiac myocytes were localized in low buoyant density fractions by density gradient centrifugation followed by Western blotting. In contrast, clathrin heavy-chain was detected across a broad range of the gradient fractions. Measurement of cholesterol levels within each fraction showed cholesterol to be enriched in cavelin-rich fractions. Immunofluorescence confocal microscopy revealed extensive colocalization of KATP channel subunits Kir6.2/SUR2A and caveolin-3 along the plasma membrane. Co-immunoprecipitation of cardiac myocytes showed significant association of Kir6.2 and caveolin-3. Furthermore, whole-cell voltage clamp studies suggested that adenosine A1 receptor-mediated activation of KATP channels was largely eliminated by disrupting caveolae with methyl-β-cyclodextrin (MβCD) in adult rat cardiomyocytes (35.4 ± 13.8 vs. 7.5 ± 4.3 pA/pF, control vs. MβCD, p<0.05) or by small interfering RNA targeting caveolin-3 in neonatal rat cardiomyocytes (24.1 ± 4.0 vs. 1.5 ± 0.6 pA/pF, control siRNA vs. caveolin-3 siRNA, p<0.01), whereas pinacidil-induced KATP activation was not altered. In conclusion, we demonstrate that KATP channels are localized to caveolin-enriched microdomains. This microdomain association is essential for adenosine receptor-mediated regulation of KATP channels in cardiac myocytes.
This research has received full or partial funding support from the American Heart Association, AHA National Center.