Ethanol Actions on Slo Channels from Arteries vs. Brain

Overview

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, biological sex, gender 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 ß1 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 brain during AIB, specific amino acids (e.g., S160) in BK ß1 mediate alcohol-induced BK channel inhibition and, eventually, cerebral artery constriction thus leading to AIB, all these actions being counteracted by selective, novel ß1 transmembrane region 2 (TM2)-targeting agent(s) 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 ß1 S160 in mediating ethanol inhibition of BK channels and its reversion by novel ß1 TM2-targeting agent(s) 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 ß1 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 ß1 TM2-targeting agent(s). SA3 (organismal resolution) will address the impact of ß1 TM2 S160 in a mouse model of AIB and demonstrate AIB reversion by ß1 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.