We have recently shown that ATP-sensitive potassium (K-ATP) channels in the heart are localized in the caveolae of cardiac myocytes and regulated by caveolae-related signaling. However, little is known about the role of caveolins, signature proteins of caveolae, in cardiac K-ATP channel function. The present study was designed to explore the potential functional interaction between caveolin-3 and K-ATP channels. The cardiac K-ATP channel subunits Kir6.2 and SUR2A were transiently transfected in HEK293T cells with or without co-transfection of caveolin-3 or caveolin-1. Our data demonstrated that the recombinant K-ATP channel activity in HEK293T cells was inhibited by expression of caveolin-3, but not caveolin-1. The application of caveolin-3 scaffolding domain peptide, corresponding to amino acid residues 5574 of caveolin-3, blocked the inhibitory effect of caveolin-3 on K-ATP channels. However, the same peptide did not have any significant effect on K-ATP channels in HEK293T cells without caveolin-3 expression. We further confirmed that K-ATP channels co-immunoprecipitated with caveolin-3 but not caveolin-1. The association of K-ATP channels with caveolin-3 was largely prevented by caveolin-3 scaffolding domain peptide. Our results indicate that caveolin-3 negatively regulates Kir6.2/SUR2A channel function. (C) 2009 Elsevier Inc. All rights reserved.