CO regulation of Slo1 BKCa potassium channels

一氧化碳（CO）是一种众所周知的有毒气体，但它也是在我们自己的身体从血红素合成。越来越多的证据表明，CO作为生理信使发挥作用。我们之前的合作成果（P3和P4）表明，CO直接刺激大电导Ca2+和电压门控K+（Slo1 BKCa）通道，这在包括神经和肌肉兴奋性在内的许多功能中很重要。我们还确定了一个特定的细胞内段的通道所需的功能与CO的相互作用。建立在这个坚实的知识基础上，我们建议填补三个最关键的知识空白。首先，CO特异性激活Slo1 BKCa通道的生理后果是什么？第二，HHDP如CO在Slo1 BKCa通道中产生什么样的构象变化？第三，什么是游离的细胞内血红素浓度，它如何随时间变化？为了评估生理意义，我们将使用从野生型小鼠和仅在Slo 1 BKCa通道的CO依赖性激活特征中受损的基因修饰（敲入）小鼠中分离的海马神经元。为了研究HHDP如CO诱导的详细构象变化，我们将利用Slo1蛋白在溶液中的胞内结构域，并使用色氨酸荧光监测构象动力学。为了估计细胞内游离血红素浓度，我们将开发一种基于荧光蛋白的游离血红素传感器，适用于活细胞成像。预期的研究成果将在多个组织层面推进HHDP信号领域。

Carbon monoxide (CO) is a well-known toxic gas but it is also synthesized in our own body from heme. Increasing evidence suggests that CO functions as a physiological messenger. Our previous collaborative effort (P3 and P4) demonstrated that CO directly stimulates the large-conductance Ca2+- and voltage-gated K+ (Slo1 BKCa) channel important in numerous functions including nerve and muscle excitability. We also identified a specific intracellular segment of the channel required for its functional interaction with CO. Building on this solid knowledge base, we propose to fill three of the most critical knowledge gaps. First, what are the physiological consequences of activation of Slo1 BKCa channels specifically by CO? Second, what conformational changes do HHDPs such as CO produce in the Slo1 BKCa channel? Third, what is the free intracellular heme concentration and how does it change with time? To assess the physiological significance, we will use hippocampal neurons isolated from wild-type mice and genetically modified (knock-in) mice impaired only in the CO-dependent activation feature of the Slo1 BKCa channel. To study the detailed conformational changes induced by HHDPs such as CO, we will utilize the intracellular domain of the Slo1 protein in solution and monitor the conformational dynamics using tryptophan fluorescence. To estimate the free intracellular heme concentration, we will develop a fluorescent protein-based free heme sensor suitable for live cell imaging. The anticipated research outcomes will advance the field of HHDP signaling at multiple levels of organization.