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

ATP 敏感钾通道的抑制 ATP 位点和抑制门的化学计量

• 批准号: 9817116
• 负责人: Peter Drain
• 金额: $ 34.5万
• 依托单位: University of Pittsburgh
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9817116&HistoricalAwards=false
• 关键词: Stoichiometry; Inhibitory; ATP; Site; Inhibition

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.

细胞ATP敏感性钾通道（ATP-K通道）因其受细胞内ATP的抑制而得名。 这种抑制在多种动物细胞类型中质膜的电活性与能量代谢的偶联中起着至关重要的作用。 然而，很少有人知道这种耦合的分子机制。 ATP-K通道由四个亚基组成，每个亚基包括一个调节亚基和一个孔形成亚基。 为了理解ATP如何抑制孔的门控打开，理解ATP结合位点相对于门本身结构的数量和位置是至关重要的。 初步研究已经确定了参与ATP结合和门控的氨基酸序列，其映射到孔形成亚基的C-末端结构域。 这些结果将通过构建突变体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存在下如何相互作用。 该方法将结合联合收割机定点诱变和突变通道在青蛙卵母细胞中的表达，其中通道活性可以通过电生理学方法测定。 结果将揭示结合位点和门的亚基排列，以及它们是独立相互作用还是协同作用。 预计由此产生的ATP-K通道分子模型将对其他膜蛋白如何直接受ATP调节从而间接受细胞能量状态调节产生广泛影响。