Abstract 2695: Role of SUR2A in Coupling ATP-sensitive K+ Channels to Caveolin-3
We have recently shown that ATP-sensitive potassium (KATP) channels in cardiac myocytes are mostly localized in caveolae. The present study was designed to explore molecular compositions involved in the interaction between cardiac KATP channels and caveolins. Co-immunoprecipitation experiments were performed in both adult cardiac myocytes and COS-7 cells transfected with either HA-tagged wild-type (Octameric Kir6.2/SUR2A) or mutant KATP channel (tetrameric Kir6.2C4A), with or without caveolin-3 or caveolin-1. It is known that unlike Kir6.2, Kir6.2C4A (mutation of ER retention signal LRKR) is able to express on the cell surface without SUR. Our data 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 mutant Kir6.2C4A/caveolin-3, Kir6.2C4A was detected with anti-HA antibody 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. In contrast, caveolin-1 did not co-immunoprecipitate Kir6.2 or Kir6.2C4A in cells transfected with Kir6.2/SUR2A/caveolin-1 or Kir6.2C4A/caveolin-1. When the caveolin-3 scaffolding domain peptide (10 μM, SDP), corresponding to amino acid residues 55–74 of caveolin-3, was incubated with cell lysates before and throughout immunoprecipitation with anti-caveolin-3 antibody, the peptide prevented the co-immunoprecipitation of caveolin-3 and Kir6.2 but did not block the co-immunoprecipitation of caveolin-3 and Kir6.2C4A. Similar finding was observed in the adult rat cardiac myocytes where the caveolin-3 SDP eliminated the interaction between endogenous KATP channels and caveolin-3. These results indicate that SUR2A subunit plays an important role in coupling cardiac KATP channels to caveolin-3, possibly via caveolin-3 scaffolding domain. This interaction may serve to anchor or regulate KATP channels in caveolae.
This research has received full or partial funding support from the American Heart Association, National Center.