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 c ore in various Am-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. I n this regard, Kir6.2 has been shown to physically associate with SUR1 to f orm the pancreatic K-ATP channel. While cardiac K-ATP channel activity can be reconstituted by coexpression of Kir6.2 with a distinct SUR isoform, SUR 2A, no direct proof has been provided for physical association between thes e 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 i n vitro-translated proteins, the Kir6.2-specific antibody coimmunoprecipita ted 38-kDa and 140-kDa proteins corresponding to Kir6.2 and SUR2A, respecti vely. In the absence of 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 aci ds from the carboxy-terminus still coimmunoprecipitated with SUR2A, indicat ing that the distal carboxy-terminus of Kir6.2 is unnecessary for subunit a ssociation. Kir6.2 mutants lacking more proximal carboxy-terminus regions, including the M2 transmembrane domain, failed to immunoprecipitate SUR2A, s uggesting that the proximal carboxy-terminus together with the M2 domain ar e required for channel assembly. These deletion constructs supported cellul ar distribution of Kir6.2. Thus, the present study provides direct evidence for physical association between Kir6.2 and SUR2A, essentially reconstitut ing the cardiac K-ATP channel in vitro. The demonstration of complex format ion between Kir6.2 and SUR2A indicates that the structural basis for channe l function may rely on direct physical interaction of the two subunits. (C) 1999 Academic Press.