CO regulation of Slo1 BKCa potassium channels

一氧化碳（CO）是一种众所周知的有毒气体，但它也可以在我们体内由血红素合成。越来越多的证据表明，一氧化碳起着生理信使的作用。我们之前的合作研究（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都很重要。我们还确定了其与CO功能相互作用所需的特定细胞内通道段。在此基础上，我们建议填补三个最关键的知识空白。首先，一氧化碳激活Slo1 BKCa通道的生理后果是什么？其次，像CO这样的HHDPs在Slo1 BKCa通道中产生了什么构象变化？第三，什么是胞内自由血红素浓度，它是如何随时间变化的？为了评估生理意义，我们将使用从野生型小鼠和基因修饰（敲入）小鼠中分离的海马神经元，这些神经元仅在Slo1 BKCa通道的co依赖性激活特征中受损。为了研究CO等HHDPs诱导的详细构象变化，我们将利用溶液中Slo1蛋白的胞内结构域，并使用色氨酸荧光监测构象动力学。为了估计游离细胞内血红素浓度，我们将开发一种适用于活细胞成像的基于荧光蛋白的游离血红素传感器。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.