Ethanol actions on slo channels from arteries vs. brain.

Alcohol-induced blackouts (AIB), a form of amnesia linked to dysfunction of the hippocampal CA1 region, often occur upon moderate-heavy, acute ethanol consumption disregarding age, drinking history, sex or education. Despite their high prevalence and detrimental impact on individuals, families and society, biomedical research has largely failed to identify the molecular targets underlying AIB and, more important, to develop pharmacotherapeutic approaches to counteract them. We will cover these gaps in knowledge by departing from all previous work, which focused on AIB-induced disruption of mechanisms operating at the hippocampal neurons themselves, to advance a vascular theory of AIB that is centered on the β1 subunit of ion channels of BK type that is highly expressed in cerebrovascular smooth muscle. Thus, we will test this overarching hypothesis: at concentrations reached in the brain during AIB, specific amino acids (e.g., S160) in BK β1 mediate alcohol-induced BK channel inhibition and, eventually, cerebral artery constriction thus leading to AIB, all these actions being counteracted by selective, novel β1 transmembrane region 2 (TM2)-targeting agents that activate BK channels. We will address three conceptually related, yet independently testable specific aims (SA): SA1 (molecular-cellular resolution) will identify the central role of β1 S160 in mediating ethanol inhibition of BK channels and its reversion by novel β1 TM2-targeting agents whether drug actions are studied using recombinant BK proteins expressed in heterologous, isolated membranes or in the native cell. SA2 (tissue-organ resolution) will identify the role of β1 TM2 specific amino acids in ethanol actions of middle cerebral artery constriction, both in vitro and in vivo, and the reversion of ethanol actions by the β1 TM2-targeting agents. SA3 (organismal resolution) will address the impact of β1 TM2 specific amino acids in a mouse model of AIB and demonstrate AIB reversion by β1 TM2-targeting agents. Testing of the three SAs will be accomplished by combining computational modeling, nano- differential scanning fluorimetry (nanoDSF), point mutagenesis, robotic/manual patch-clamp, electroporation of vascular myocytes and middle cerebral artery with recombinant DNAs, engineered mice (“knockouts” and “knockins”), middle cerebral artery in vitro and in vivo (cranial window) diameter determinations, selective pharmacology, and simultaneous evaluation of CA1 vessels, blood flow and neuron activity in freely behaving mice subject to AIB, all supported by our papers or preliminary data.