MECHANISMS OF G PROTEIN REGULATION OF POTASSIUM CHANNELS

K+通道的异常活动和随后的膜兴奋性变化与神经系统疾病有关。G蛋白门控的内向纠偏K+通道（GIRK）尤其与小鼠癫痫发作和神经变性有关。这项研究的长期目标是阐明G蛋白激活GIRK通道的分子机制。在G蛋白偶联的神经递质受体的刺激下，GIRK通道大多以四个亚基（四聚体）为组打开。虽然人们普遍认为G蛋白衍生的Gbetagamma亚基激活了GIRK通道，并且GIRK通道的N端和C端都与Gbetagamma结合，但关于Gbetagamma亚基与GIRK通道之间控制通道活性的物理相互作用知之甚少。电生理学、分子遗传学和生物化学技术的结合将被用于：(1)定义在天然GIRK通道背景下Gbetagamma结合域之间的功能相互作用。来自GIRK通道的N-和C-末端的最佳排列将由生物化学决定。在非洲爪蟾卵母细胞中表达的基因工程GIRK多聚体中，将确定用于Gbetagamma激活的GIRK亚基的临界数量和提供专性N-和c -末端的GIRK亚基的位置。(2)确定对伽马激活至关重要的GIRK通道的一致序列。通过监测外源性伽马射线亚基存在时通道活性的变化，将评估遗传改变的非洲爪蟾卵母细胞中的伽马射线激活。β - γ结合将在含有突变序列的亲和标记融合的GIRK蛋白中进行测量。伽马通道活性将在与发现的伽马结合区域相对应的肽片段存在的情况下进行测试。(3)确定伽马亚基和GIRK通道上相互作用的氨基酸对。通过生物化学交联技术将确定Gbetagamma和GIRK通道中潜在的相互作用氨基酸对。在受体激活过程中，我们还将探讨伽马与GIRK的功能性交联。描述这些通道中g蛋白激活的信号转导机制可能直接影响到由于异常膜兴奋性引起的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.