MECHANISMS OF G PROTEIN REGULATION OF POTASSIUM CHANNELS

K+通道的异常活性和随后的膜兴奋性变化与神经系统疾病有关。 G蛋白门控内向整流K+通道（GIRK）特别与小鼠的癫痫发作和神经变性有关。 本研究基金的长期目标是阐明G蛋白激活GIRK通道的分子机制。 GIRK通道，最可能在四个亚基（四聚体）的组中，在G蛋白偶联神经递质受体的刺激期间打开。虽然普遍认为G蛋白衍生的G β γ亚基激活GIRK通道，并且GIRK通道的N-和C-末端都结合G β γ，但对G β γ亚基和GIRK通道之间的物理相互作用知之甚少。 将采用电生理学、分子遗传学和生物化学技术的组合来：（1）在天然GIRK通道的背景下定义G β γ结合结构域之间的功能相互作用。 GIRK通道的N-和C-末端的最佳排列将通过生物化学方法确定。 将在非洲爪蟾卵母细胞中表达的基因工程化GIRK多聚体中确定GIRK亚基的临界数量和GIRK亚基的位置，所述GIRK亚基为γ-活化提供专性N-和C-末端。 (2)确定GIRK通道中对γ激活至关重要的共有序列。 通过监测外源性γ-亚基存在下通道活性的变化，在非洲爪蟾卵母细胞中评估遗传改变的GIRK通道中的γ-活化。 将在含有突变序列的亲和力标记融合GIRK蛋白中测量Gbetagamma结合。 将在存在对应于发现结合G β γ的区域的肽片段的情况下测试G β γ通道活性。 (3)鉴定γ亚基和GIRK通道上相互作用的氨基酸对。 将通过生化交联技术鉴定Gbetagamma和GIRK通道中潜在的相互作用氨基酸对。还将探索在受体活化期间G β γ与GIRK的功能性交联。描述这些通道的G蛋白激活所涉及的信号转导机制可能直接影响由于异常膜兴奋性引起的疾病的药物治疗的设计。

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.