Abstract 173: Protein Kinase Cϵ Inhibits Vascular KAtp Channels By Caveolae-dependent Mechanism
Vascular ATP-sensitive K+ (KATP) channels are critical regulators of arterial tone, thus blood flow in response to local metabolic needs. They are important targets for clinically used drugs to treat hypertensive emergency and angina. It is well known that protein kinase C (PKC) activation inhibits KATP channels in vascular smooth muscles. However, the mechanism by which PKC inhibits the channel remains unknown. Since the KATP channel pore-forming subunit Kir6.1 has been localized in caveolar microdomains of arterial smooth muscles, we tested the hypothesis that PKC-induced inhibition of vascular KATP channels is through caveolae-dependent mechanism. The effect of caveolae/caveolin on PKC-mediated inhibition of recombinant KATP channels was investigated in HEK293 cells transfected with Kir6.1 and SUR2B. As expected, whole-cell KATP current density was significantly reduced by activation of PKC with phorbol-12-myristate-13-acetate (PMA, 1μM), but not its inactive congener 4α-PMA (1 μM). The PMA effect was prevented by application of PKCϵ inhibitor peptide Myristoylated PKCϵ V1–2 (Myr PKCϵ V1–2, 200 μM). Interestingly, the inhibitory effect of Myr PKCϵ V1–2 on KATP channels was significantly reduced by disrupting caveolae with cholesterol-depleting agent methyl-β-cyclodextrin (10 mM, 51.9 ± 10.2% vs. 17.2 ± 5.6%, p<0.01, n=5), and further enhanced by overexpression of caveolin-1 (36.8 ± 4.3% vs. 68.3 ± 8.5%, p<0.05, n=5), indicating that integrity of caveolae is essential for PKC-dependent inhibition of KATP channels. Further, Kir6.1 was localized in caveolar fraction by detergent-free sucrose gradient centrifugation. Immunofluorescence images showed that there was substantial colocalization between Kir6.1 and caveolin-1 on the sarcolemmal membrane as punctuate staining pattern. We also show that an antibody against caveolin-1 co-immunoprecipited Kir6.1, suggesting that Kir6.1 and caveolin-1 exist together in a complex. These results indicate that PKC-induced KATP channel inhibition by PMA occurs within caveoar lipid raft microdomains. This study provides first evidence of caveolae-dependent mechanism involved in PKC-mediated KATP channel modulation. Further studies in arterial smooth muscles are under investigation.