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Role for SUR2A in coupling cardiac K(ATP) channels to caveolin-3.

Wei Sun, Keli Hu

Cell. Physiol. Biochem., 2010 , 25, 409-18

ATP-sensitive K+ channels in the heart are localized in the caveolae. However, little is known about the molecular mechanism underlying the caveolar targeting of those ion channels. The present study was designed to explore the molecular compositions involved in the interaction between cardiac K(ATP) channels and caveolin-3. The HA-tagged wild-type (Kir6.2/SUR2A) or mutant K(ATP) channel (Kir6.2C4A or Kir6.2Delta36) subunits were transiently transfected into COS-7 cells with or without caveolin-3. Both Kir6.2C4A and Kir6.2Delta36 are able to form tetrameric Kir6.2 channels on the cell membrane without SUR subunit. We demonstrated that caveolin-3 co-immunopreciptiated Kir6.2 in COS-7 cells transfected with Kir6.2/SUR2A/caveolin-3, whereas Kir6.2 was not detected in the caveolin-3 immunoprecipitates in cells transfected with either caveolin-3 or Kir6.2/SUR2A alone. In cells transfected with Kir6.2C4A/caveolin-3, Kir6.2C4A was detected with anti-HA antibody but at a significantly lower level in the caveolin-3 immunoprecipitates when compared with Kir6.2 detected in cells transfected with the Kir6.2/SUR2A/caveolin-3. Kir6.2C4A was not co-immunoprecipitated with caveolin-3 in cells transfected with caveolin-3 or Kir6.2C4A alone. The application of caveolin-3 scaffolding domain peptide, corresponding to amino acid residues 55-74 of caveolin-3, largely blocked the co-immunoprecipitation of caveolin-3 and Kir6.2/SUR2A octameric channels but did not prevent the co-immunoprecipitation of caveolin-3 and the Kir6.2 tetrameric channels. Disrupting caveolae with methyl-beta-cyclodextrin significantly attenuated association of tetrameric Kir6.2 channels with caveolin-3. Immunofluorescence microscopy revealed that a higher percentage of cells showed significant colocalization of caveolin-3 with Kir6.2 than colocalization of caveolin-3 with Kir6.2C4A or Kir6.2Delta36. We further confirmed that in adult rat cardiac myocytes the association of endogenous octameric K(ATP) channels with caveolin-3 was largely prevented by caveolin-3 scaffolding domain peptide but not control peptide. We concluded that SUR2A is important for coupling cardiac K(ATP) channels to caveolin-3, possibly through the caveolin-3 scaffolding domain.