REGULATION OF G PROTEIN-GATED K+ CHANNEL FUNCTION

G 蛋白门控 K 通道功能的调节

• 批准号: 6638426
• 负责人: Donghee Kim
• 金额: $ 23.4万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6638426
• 关键词: G protein; electrophysiology; fatty acid binding protein; free fatty acids; guanine nucleotide binding protein; guanosine triphosphate; immunologic assay /test; inhibitor /antagonist; intermolecular interaction; laboratory rat; long chain fatty acid; membrane activity; membrane permeability; membrane potentials; nucleoside analog; phosphatidylinositols; phospholipase A2; potassium channel; protein structure function; radiotracer; tissue /cell culture

The goal of our research is to elucidate the mechanism of activation and modulation of the G protein- gated K+ channel (KACh channel/GIRK) which regulates heart rate and synaptic transmission. Recent studies have shown that interaction of the KACh channel with phosphatidylinositol-4,5-bisphosphate (PIP2) in the membrane is critical, as this allows other gating molecules (beta gamma, Na+) to work. Betagamma is the most effective gating molecule as it produces the greatest activation. We recently discovered the existence of an inhibitory lipid substance in the cytoplasm of atria and brain that plays a crucial role in KACh channel function. In the absence of the endogenous lipid inhibitor (excised patch), the KACh channel shifts to a "high open probability" mode with long- lived openings. In the presence of the inhibitor (cytoplasm), the KACh channel activity is much lower showing only short-lived openings, similar to the KACh channel state observed in cell-attached patches (intact cells). Using electrophysiological and molecular biological methods, we propose to identify the endogenous inhibitor and study its physiological role in KACh channel function by selectively removing it from the cell (specific aim number 1). Using chimeric channels (GIRK/IRK) and PIP2 antibody, we will study the molecular basis for the inhibitory effect on the KACh channel activity. This will be done by testing the hypothesis that the inhibitor blocks PIP2-KACh channel interaction and thus reduces the effectiveness of gating molecules such as betagamma and Na+ (specific aim number 2). As changes in KACh channel kinetics produced by the cytoplasmic inhibitor are similar to those observed during the fast desensitization, we will test the hypothesis that the putative inhibitor mediates the fast desensitization of the agonist-induced KACh current (specific air number 3). These studies should reveal novel signaling pathways involved in agonist-induced activation and desensitization of the KACh channel, and help better understand the control of heart rate.

我们研究的目标是阐明 G 蛋白门控 K+ 通道（KACh 通道/GIRK）的激活和调节机制，该通道调节心率和突触传递。 最近的研究表明，KACh 通道与膜中磷脂酰肌醇 4,5-二磷酸 (PIP2) 的相互作用至关重要，因为这使得其他门控分子（β、γ、Na+）发挥作用。 Betagamma 是最有效的门控分子，因为它产生最大的激活。 我们最近发现心房和大脑细胞质中存在一种抑制性脂质物质，它在 KACh 通道功能中起着至关重要的作用。 在缺乏内源性脂质抑制剂（切除的斑块）的情况下，KACh 通道转变为具有长寿命开放的“高开放概率”模式。 在存在抑制剂（细胞质）的情况下，KACh 通道活性低得多，仅显示短暂的开口，类似于在细胞附着斑块（完整细胞）中观察到的 KACh 通道状态。 我们建议使用电生理学和分子生物学方法来鉴定内源性抑制剂，并通过选择性地将其从细胞中去除来研究其在 KACh 通道功能中的生理作用（具体目标 1）。 我们将使用嵌合通道 (GIRK/IRK) 和 PIP2 抗体，研究对 KACh 通道活性抑制作用的分子基础。 这将通过测试以下假设来完成：抑制剂阻断 PIP2-KACh 通道相互作用，从而降低门控分子（如 βamma 和 Na+）的有效性（具体目标编号 2）。由于细胞质抑制剂产生的 KACh 通道动力学变化与快速脱敏过程中观察到的相似，我们将检验假定抑制剂介导激动剂诱导的 KACh 电流（特定空气数 3）快速脱敏的假设。这些研究应该揭示参与激动剂诱导的 KACh 通道激活和脱敏的新信号通路，并有助于更好地了解心率的控制。