Physical association between recombinant cardiac ATP-sensitive K⁺ channel subunits Kir6.2 and SUR2A

The inwardly-rectifying K⁺ channel Kir6.2 serves as a common pore-forming core in various ATP-sensitive K⁺ (K(ATP)) channels, and it is through assembly with sulfonylurea-receptor (SUR) isoforms, which are ATP-binding cassette (ABC) proteins, that tissue-specific channel phenotypes can be generated. In this regard, Kir6.2 has been shown to physically associate with SUR1 to form the pancreatic K(ATP) channel. While cardiac K(ATP) channel activity can be reconstituted by coexpression of Kir6.2 with a distinct SUR isoform, SUR2A, no direct proof has been provided for physical association between these two proteins. Therefore, we tested, by a coimmunoprecipitation procedure in conjunction with an amino-terminal Kir6.2-antibody, physical association between recombinant Kir6.2 and SUR2A. From a mixture of Kir6.2 and SUR2A in vitro-translated proteins, the Kir6.2-specific antibody coimmunoprecipitated 38-kDa and 140-kDa proteins corresponding to Kir6.2 and SUR2A, respectively. In the absence Kir6.2, SUR2A was not precipitated by the anti-Kir6.2 antibody, indicating that the antibody recognized SUR2A only when SUR2A formed a complex with Kir6.2. A Kir6.2 deletion mutant lacking 37 amino acids from the carboxy-terminus still coimmunoprecipitated with SUR2A, indicating that the distal carboxy-terminus of Kir6.2 is unnecessary for subunit association. Kir6.2 mutants lacking more proximal carboxy-terminus regions, including the M2 transmembrane domain, failed to immunoprecipitate SUR2A, suggesting that the proximal carboxy-terminus together with the M2 domain are required for channel assembly. These deletion constructs supported cellular distribution of Kir6.2. Thus, the present study provides direct evidence for physical association between Kir6.2 and SUR2A, essentially reconstituting the cardiac K(ATP) channel in vitro. The demonstration of complex formation between Kir6.2 and SUR2A indicates that the structural basis for channel function may rely on direct physical interaction of the two subunits.