Cui Y, Giblin J P, Clapp L H, Tinker A
Centre for Clinical Pharmacology, Department of Medicine, University College London, The Rayne Institute, 5 University Street, London WC1E 6JJ, United Kingdom.
Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):729-34. doi: 10.1073/pnas.98.2.729. Epub 2001 Jan 2.
ATP-sensitive potassium channels are an octomeric complex of four pore-forming subunits of the Kir 6.0 family and four sulfonylurea receptors. The Kir 6.0 family consists of two known members, Kir 6.1 and Kir 6.2, with distinct functional properties. The tetrameric structure of the pore-forming domain leads to the possibility that mixed heteromultimers may form. In this study, we examine this by using biochemical and electrophysiological techniques after heterologous expression of these subunits in HEK293 cells. After the coexpression of Kir 6.1 and Kir 6.2, Kir 6.1 can be coimmunoprecipitated with isoform-specific Kir 6.2 antisera and vice versa. Coexpression of SUR2B and Kir 6.2 with Kir 6.1 dominant negatives at a 1:1 expression ratio and vice versa led to a potent suppression of current. Kir 6.1, and Kir 6.2 dominant negative mutants were without effect on an inwardly rectifying potassium channel from a different family, Kir 2.1. Single-channel analysis, after coexpression of SUR2B, Kir 6.1, and Kir 6.2, revealed the existence of five distinct populations with differing single-channel current amplitudes. All channel populations were inhibited by glibenclamide. A dimeric Kir 6.1-Kir 6.2 construct expressed with SUR2B had a single-channel conductance intermediate between that of either Kir 6.2 or Kir 6.1 expressed with SUR2B. In conclusion, Kir 6.1 and Kir 6.2 readily coassemble to produce functional channels, and such phenomena may contribute to the diversity of nucleotide-regulated potassium currents seen in native tissues.
ATP敏感性钾通道是由四个Kir 6.0家族的成孔亚基和四个磺脲类受体组成的八聚体复合物。Kir 6.0家族由两个已知成员Kir 6.1和Kir 6.2组成，它们具有不同的功能特性。成孔结构域的四聚体结构导致可能形成混合异源多聚体。在本研究中，我们通过在HEK293细胞中异源表达这些亚基后使用生化和电生理技术来研究这一问题。在共表达Kir 6.1和Kir 6.2后，Kir 6.1可以与同种型特异性的Kir 6.2抗血清共免疫沉淀，反之亦然。以1:1的表达比例共表达SUR2B和Kir 6.2与Kir 6.1显性阴性突变体，反之亦然，导致电流的有效抑制。Kir 6.1和Kir 6.2显性阴性突变体对来自不同家族的内向整流钾通道Kir 2.1没有影响。在共表达SUR2B、Kir 6.1和Kir 6.2后进行的单通道分析显示存在五个具有不同单通道电流幅度的不同群体。所有通道群体均被格列本脲抑制。与SUR2B一起表达的二聚体Kir 6.1-Kir 6.2构建体的单通道电导介于与SUR2B一起表达的Kir 6.2或Kir 6.1的单通道电导之间。总之，Kir 6.1和Kir 6.2很容易共同组装产生功能性通道，这种现象可能有助于解释在天然组织中观察到的核苷酸调节钾电流的多样性。
