REGULATION OF G PROTEIN-GATED K+ CHANNEL FUNCTION

本研究旨在阐明调节心率和突触传递的G蛋白门控K+通道(KACH通道/GIRK)的激活和调节机制。最近的研究表明，KACH通道与膜上的磷脂酰肌醇-4，5-二磷酸(PIP2)的相互作用是至关重要的，因为这允许其他门控分子(β-γ，Na+)发挥作用。Betagamma是最有效的门控分子，因为它能产生最大的激活。我们最近发现在心房和大脑的细胞质中存在一种抑制脂质物质，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物(切除的斑块)的情况下，KACH通道转变为具有长时间开放的“高开放概率”模式。在抑制物(细胞质)的存在下，KACH通道的活性要低得多，只显示出短暂的开放，类似于在细胞贴附的斑块(完整细胞)中观察到的KACH通道状态。利用电生理学和分子生物学方法，我们建议通过选择性地将内源性抑制物从细胞中移除来鉴定内源性抑制物并研究其在KACH通道功能中的生理作用(特定目标编号1)。利用嵌合通道(GIRK/IRK)和PIP2抗体，我们将研究其抑制KACH通道活性的分子基础。这将通过测试这一假设来实现，即该抑制剂阻止PIP2-KACH通道相互作用，从而降低门控分子(如Betagamma和Na+(特定目标编号2))的有效性。由于细胞质抑制剂引起的Kach通道动力学变化与快速脱敏过程中观察到的变化相似，我们将检验假定的抑制剂介导激动剂诱导的Kach电流(特定空气数3)的快速脱敏的假设。这些研究将揭示新的信号通路参与激动剂诱导的KACH通道的激活和脱敏，并有助于更好地理解心率的控制。

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.