Regulation of cerebral blood flow is necessary for survival as the brain requires a large amount of circulating oxygen and nutrients. Resistance-size cerebral arteries manage constant blood flow to the brain by myogenic autoregulation mechanisms. Abnormal cholesterol levels trigger dysregulation of resistance-size cerebral arteries via the calcium- and voltage-gated potassium channel of large conductance (BK), contributing to common cerebrovascular pathologies such as stroke, cognitive deficits including some forms of dementia, and the disruption of cerebral artery function by recreational alcohol. Cholesterol inhibition of the BK channel alters contractility of vascular smooth muscle impacting cerebral artery diameter, and dysregulates delivery of oxygen and nutrients throughout the brain. While cholesterol diminishes BK channel activity, the molecular mechanism(s) by which this occurs are currently unknown. Cholesterol recognition/interaction amino acid consensus (CRAC) motifs are potential binding sites for cholesterol, and ten are found throughout the BK channel amino acid sequence. The cytosolic tail domain contains seven of these ten CRAC motifs, and it has been demonstrated that cholesterol modulates BK currents by one or more of these cytosolic tail domain CRAC motifs. My goal is to determine the molecular mechanisms that govern cholesterol regulation of the BK channel by interacting with certain cytosolic tail domain CRAC motif(s), and to define the impact of this regulation on cerebral artery diameter. This proposal addresses two main aims: Aim 1 will determine the structural basis and gating mechanisms that lead to cholesterol-induced hindering of BK function through cholesterol direct interactions with the BK channel-forming slo1 subunit. The hypothesis that cholesterol modulates BK currents via interaction with specific CRACs will be addressed by electrophysiology and binding experiments. I will also identify which BK gating parameter(s) are altered upon cholesterol interaction. 1.1. I will first determine the contribution of distinct CRAC motifs to cholesterol binding and the consequent inhibition of homomeric slo1 channel activity. 1.2. Next, I will determine the critical physicochemical features of distinct CRAC motifs that allow for modulation of the channel’s cholesterol sensitivity. 1.3. Finally, I will identify the cholesterol-sensitive gating parameters that lead to cholesterol-induced hindering of slo1 channel activity. Aim 2 will address the physiological and pharmacological consequences of cholesterol-slo1 interactions via CRAC4 motif as an example on native BKs in cerebral artery smooth muscle and cerebral artery diameter. 2.1. I will determine the effects of cholesterol interactions with CRAC4 in native BKs in arterial myocytes under physiological conditions. 2.2. I will also determine the consequences of cholesterol regulation of BK currents via slo1 CRAC4 on artery diameter. This proposal will for the first time develop a unifying scheme that explains the actions of cholesterol on BK channel function and cerebral artery diameter at both molecular and cellular levels based on direct binding of the sterol to the BK channel-forming slo1 subunit and/or allosteric regulation secondary to the sterol interaction.

The goal of this proposal is to determine the molecular mechanisms that govern cholesterol regulation of the protein termed calcium- and voltage-gated potassium channel of large conductance (BK), and the impact of this regulation on blood vessel diameter in the brain. We will for the first time develop a unifying scheme that explains the actions of cholesterol on the function of BK-forming protein and artery function in the brain at both molecular and cellular levels based on direct binding of cholesterol to the BK channel-forming subunit and/or allosteric regulation secondary to cholesterol interaction with third parties. Considering that cholesterol is prevalent in modern Western diets and has implications for multiple pathological conditions (atherosclerosis, stroke, cognitive deficits including some forms of dementia), the data obtained from this proposal will have high relevance for deepening our understanding of fundamental mechanisms behind these pathologies.