Stoichiometry of the Inhibitory ATP Site and Inhibition Gate of the ATP Sensitive Potassium Channel

细胞的ATP敏感性钾通道(ATP-K通道)因其受细胞内ATP的抑制而得名。在多种动物细胞中，这种抑制在质膜的电活动与能量代谢的偶联中起着至关重要的作用。然而，人们对这种偶联的分子机制知之甚少。ATP-K通道由四个亚基组成，每个亚基是一个调节亚基和一个成孔亚基。为了了解ATP是如何抑制门控的孔道开放的，了解ATP结合位点的数量和位置相对于门本身的结构是至关重要的。初步研究已经确定了与ATP结合和门控有关的氨基酸序列，这些序列映射到成孔亚基的C-末端结构域。这些结果将通过构建突变亚基和野生型亚基的多聚体融合蛋白来扩展，以分析结合位点和门的数量以及它们在ATP存在时是如何相互作用的。该方法将结合定点突变和青蛙卵母细胞中突变通道的表达，其中通道活性可以通过电生理方法进行分析。结果将揭示结合位点和门的亚基排列，以及它们是独立作用还是协同作用。可以预期，由此得到的ATP-K通道的分子模型将对其他膜蛋白如何直接由ATP调节，从而间接由细胞的能量状态调节具有广泛的意义。

ATP-sensitive potassium channels (ATP-K channels) of cells are so-named because of their inhibition by intracellular ATP. This inhibition plays a crucial role in the coupling of electrical activity of the plasma membrane to energy metabolism in a variety of animal cell types. However, little is known about the molecular mechanism of this coupling. The ATP-K channel is formed from four subunits each of a regulatory subunit, and a pore-forming subunit. To understand how ATP inhibits the gated opening of the pore, it is fundamental to understand the number and location of ATP binding sites relative to the structure of the gate itself. Preliminary studies have defined amino acid sequences involved in ATP binding and gating which map to the C-terminal domain of the pore-forming subunit. These results will be extended by constructing multimeric fusion proteins of mutant and wild-type subunits to analyze the number of binding sites and gates and how they interact in the presence of ATP. The approach will combine site-directed mutagenesis and expression of mutant channels in frog oocytes where channel activity can be assayed by electrophysiological methods. The results will reveal the subunit arrangement of both binding sites and gates and whether they interact independently or cooperatively. It is anticipated that the resulting molecular model for the ATP-K channel will have broad implications for how other membrane proteins may be regulated directly by ATP and hence indirectly by the energy state of the cell.