Regulation of calcium-activated potassium channels by lipid messengers

描述(申请人提供)：大电导钙和电压门控K+(Slo1BK)通道发挥着多种生理和病理生理作用，其变构门控机制受到多种细胞信号通路的调节。越来越多的证据表明，某些脂质可能是信号分子。我们建议揭示两种脂质信使--磷脂酰肌醇4，5-二磷酸(PIP2)和二十二碳六烯酸(DHA)--一种富含油性鱼类的omega-3长链多不饱和脂肪酸--调节sO1BK通道的生物物理和物理化学机制。PIP2对Slo1 BK通道的作用最近有报道，但其作用机制尚不清楚。我们将通过进行彻底的机械电生理测量来填补这一关键的知识空白。此外，我们将确定对PIP2的作用重要的辅助β亚基的结构决定因素。我们的电生理测量将与分离和纯化的Slo1门环蛋白的色氨酸荧光测量相补充。DHA是一种新出现的脂质信使，其作用于slo1通道的生物物理和物理化学机制也将用类似的电生理和荧光方法进行研究。这一研究结果有望为PIP和DHA在Slo1通道变构门控机制中的作用提供明确的机制，并建立Slo1通道是omega-3脂肪酸受体的新范式。DHA对slo1 BK通道的调节可能是omega-3长链脂肪酸促进健康的基础。

DESCRIPTION (provided by applicant): Large-conductance Ca2+- and voltage-gated K+ (Slo1 BK) channels play numerous physiological and pathophysiological roles and their allosteric gating mechanism is subject to modulation by a variety of cellular signaling pathways. Increasing evidence suggests that certain lipids may serve as signaling molecules. We propose to reveal the biophysical and physicochemical mechanisms of modulation of Slo1 BK channels by two lipid messengers, phosphatidylinositol 4,5-bisphosphate (PIP2) and docosahexaenoic acid (DHA), an omega-3 long-chain polyunsaturated fatty acid enriched in oily fish. The effect of PIP2 on the Slo1 BK channel was reported recently but the mechanism is only poorly known. We will fill this critical knowledge gap by performing thorough mechanistic electrophysiological measurements. Furthermore, we will identify the structural determinants of the auxiliary beta subunit important for the action of PIP2. Our electrophysiological measurements will be complemented with measurements of tryptophan fluorescence of the isolate and purified Slo1 gating ring protein. The biophysical and physicochemical mechanisms of the action of DHA, an emerging lipid messenger, on the Slo1 channel will be also investigated similarly using the electrophysiological and fluorescence methods. The research outcome is expected to provide definitive mechanisms of the PIP and DHA actions on the allosteric gating mechanism of the Slo1 channel and establish the novel paradigm that the Slo1 channel is an omega-3 fatty acid receptor. The modulation of the Slo1 BK channel by DHA may underlie the health-promoting effects of omega-3 long-chain fatty acids.