MECHANISMS OF G PROTEIN REGULATION OF POTASSIUM CHANNELS

K+通道的异常活动和随后膜兴奋性的变化已被认为与神经系统疾病有关。G蛋白门控内向整流钾通道(GIRK)尤其与小鼠癫痫发作和神经变性有关。这项研究拨款的长期目标是阐明GIRK通道G蛋白激活的分子机制。在G蛋白偶联神经递质受体的刺激下，GIRK通道很可能以四个亚单位(四聚体)为一组被打开。虽然人们普遍认为G蛋白来源的Gbetagamma亚基激活GIRK通道，GIRK通道的N端和C端都与Gbetagamma结合，但Gbetagamma亚单位与GIRK通道之间的物理相互作用调控通道活性的研究很少。电生理学、分子遗传学和生化技术的结合将被用来：(1)在天然GIRK通道的背景下定义Gbetagamma结合域之间的功能相互作用。来自GIRK通道的N-和C-末端的最佳排列将通过生物化学来确定。在非洲爪哇卵母细胞中表达的GIRK多聚体中，GIRK亚基的临界数目和为Gbetagamma激活提供专有N末端和C末端的GIRK亚基的位置将被确定。(2)确定GIRK通道中对Gbetagamma激活至关重要的共有序列。在非洲爪哇卵母细胞中，将通过监测在存在外源GIRK亚基的情况下通道活性的变化来评估GIRK通道中的Gbetagamma激活。Gbetagamma结合将在包含突变序列的亲和标记融合GIRK蛋白中进行测量。Gbetagamma通道活性将在存在与发现的Gbetagamma结合区域相对应的多肽片段的情况下进行测试。(3)鉴定Gbetagamma亚基和GIRK通道上相互作用的氨基酸对。Gbetagamma和GIRK通道中潜在的相互作用的氨基酸对将通过生物化学交联技术进行鉴定。还将探索受体激活过程中Gbetagamma与GIRK的功能性交联性。阐明G蛋白激活这些通道的信号转导机制可能直接关系到膜兴奋性异常所致疾病的药物治疗设计。

The aberrant activity of K+ channels and subsequent changes in membrane excitability have been implicated in neurological diseases. G protein-gated inwardly rectifying K+ channels (GIRK) in particular are linked to seizures and neurodegeneration in mice. The long term objective of this research grant is to elucidate the molecular mechanisms underlying G protein activation of GIRK channels. GIRK channels, mostly likely in groups of four subunits (tetramers), are opened during stimulation of G protein-coupled neurotransmitter receptors. While there is general agreement that G protein-derived Gbetagamma subunits activate GIRK channels, and that both the N- and C- termini of GIRK channels bind Gbetagamma, little is known about the physical interaction between Gbetagamma subunits and GIRK channels that governs channel activity. A combination of electrophysiological, molecular genetic, and biochemical techniques will be employed to: (1) Define the functional interactions among the Gbetagamma binding domains in the context of a native GIRK channel. The optimal arrangement of N- and C- termini from GIRK channels will be determined biochemically. The critical number of GIRK subunits and the position of GIRK subunits which donate obligate N- and C-termini for Gbetagamma activation will be determined in genetically engineered GIRK multimers expressed in Xenopus oocytes. (2) Identify the consensus sequences in GIRK channels that are essential for Gbetagamma activation. Gbetagamma activation in genetically altered GIRK channels will be assessed in Xenopus oocytes by monitoring changes in channel activity in the presence of exogenous Gbetagamma subunits. Gbetagamma binding will be measured in affinity-tagged fusion GIRK proteins containing mutant sequences. Gbetagamma channel activity will be tested in the presence of peptide fragments corresponding to regions found to bind Gbetagamma. (3) Identify interacting pairs of amino acids on Gbetagamma subunits and GIRK channels. Potential pairs of interacting amino acids in Gbetagamma and GIRK channels will be identified through biochemical crosslinking techniques. Functional crosslinking of Gbetagamma to GIRK during receptor activation will also be explored. Delineating the signal transduction mechanisms involved in the G-protein activation of these channels may bear directly on design of drug therapies for diseases due to aberrant membrane excitability.