CO regulation of Slo1 BKCa potassium channels

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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.