Lorenz E, Terzic A
Department of Internal Medicine, Mayo Clinic, Mayo Foundation, Rochester, Minnesota 55905, USA.
J Mol Cell Cardiol. 1999 Feb;31(2):425-34. doi: 10.1006/jmcc.1998.0876.
The inwardly-rectifying K+ channel Kir6.2 serves as a common pore-forming core in various ATP-sensitive K+ (KATP) 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 KATP channel. While cardiac KATP 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 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 acids from the carboxyterminus 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 carboxyterminus 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 KATP 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.
内向整流钾通道Kir6.2是各种ATP敏感性钾（KATP）通道中常见的孔形成核心，通过与磺脲类受体（SUR）亚型组装（SUR亚型是ATP结合盒（ABC）蛋白），可产生组织特异性通道表型。在这方面，已证明Kir6.2与SUR1物理结合形成胰腺KATP通道。虽然通过将Kir6.2与不同的SUR亚型SUR2A共表达可重建心脏KATP通道活性，但尚未提供这两种蛋白之间物理结合的直接证据。因此，我们通过免疫共沉淀程序结合氨基末端Kir6.2抗体，检测重组Kir6.2与SUR2A之间的物理结合。在体外翻译的Kir6.2和SUR2A蛋白混合物中，Kir6.2特异性抗体共免疫沉淀出分别对应于Kir6.2和SUR2A的38 kDa和140 kDa蛋白。在没有Kir6.2的情况下，抗Kir6.2抗体不会沉淀SUR2A，这表明该抗体仅在SUR2A与Kir6.2形成复合物时才识别SUR2A。一个从羧基末端缺失37个氨基酸的Kir6.2缺失突变体仍能与SUR2A共免疫沉淀，这表明Kir6.2的远端羧基末端对于亚基结合并非必需。缺失更多近端羧基末端区域（包括M2跨膜结构域）的Kir6.2突变体无法免疫沉淀SUR2A，这表明近端羧基末端与M2结构域对于通道组装是必需的。这些缺失构建体支持了Kir6.2的细胞分布。因此，本研究为Kir6.2与SUR2A之间的物理结合提供了直接证据，基本在体外重建了心脏KATP通道。Kir6.2与SUR2A之间复合物形成的证明表明通道功能的结构基础可能依赖于两个亚基的直接物理相互作用。
