Modulation of voltage- and Ca2+-gated potassium channels (BK) by acute ethanol exposure is involved in several physiological processes known to be altered during alcohol intoxication. In some cases, ethanol action in the body requires drug-mediated BK activation, while in others ethanol inhibits BK to modify tissue function. The long- term goal of our research is to pinpoint the molecular mechanisms and targets that determine differential ethanol responses of BK and the contribution of such modulation to acute ethanol actions in the body. This goal will help to address a long-standing enigma, that is, the mechanism of ethanol action on ion channels, and will lead to rational therapeutic interventions in alcohol intoxication. We recently showed that ethanol actions on BK result from a basic interaction among the channel-forming (slo) subunit, the BK natural ligand (Ca2+) and the drug, yet several other elements, such as posttranslational modification of slo, BK accessory subunits (?) and the lipid environment around the channel complex are able to fine-tune the final ethanol effect. We also showed that ethanol at concentrations obtained in circulation during binge drinking and known to increase the risk for stroke, causes cerebrovascular constriction by reducing cerebral artery myocyte BK currents. However, the mechanisms and molecular targets of ethanol action on cerebral vessels remain unknown. Cerebral artery myocyte BK result from the tight association of slo and ?1, the latter controlling BK Ca2+ sensitivity and coupling to ryanodine receptors (RyR). RyR generates sparks, a local Ca2+ signal that activates BK. The central hypothesis of this proposal is that ?1, by controlling slo Ca2+ sensitivity and BK-RyR coupling, is the key element that leads to ethanol inhibition of BK current and, thus, cerebral artery constriction. We will test 3 specific aims (A). In A1, we will use rat and mouse models (including ?1 K/O mice), determination of ethanol action on cerebral artery tone, in vitro electrophysiology, and pharmacology to test whether ethanol-induced arterial constriction and reduction of BK current in native cells require ?1. In A2, we will use mutated ?s, single channel recordings and kinetic modeling to pinpoint subunit domain and mechanism by which ?1 enables ethanol direct inhibition of BK. In A3, we will use patch-clamp and lipid bilayer electrophysiology, selective antibodies, confocal Ca2+ imaging and pharmacology to determine whether ethanol inhibits RyR and, thus, decreased BK function. At the end of the project period, we expect to have identified both molecular target and mechanism leading to ethanol- induced cerebrovascular constriction, the fundamental element in cerebrovascular disease linked to alcohol intoxication.

Public Health Relevance

Binge drinking is the predominant form of alcohol abuse in the US. Binge drinking may lead to cerebral artery constriction, vasospasm and stroke, all consequences of ethanol- induced contraction of cerebral artery smooth muscle. We identified that this ethanol action is mediated by smooth muscle ion channels of the BK type, which are complex heterooligomeric proteins. This proposal will identify the protein subunit site and molecular mechanism leading to ethanol inhibition of BK current and cerebral artery constriction. The proposal will bring critical information to design rational pharmacotherapeutic interventions in cerebrovascular disease associated with alcohol intoxication.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (ZRG1-IFCN-A (03))
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Orosz, Andras
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University of Tennessee Health Science Center
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North, Kelsey; Bisen, Shivantika; Dopico, Alex M et al. (2018) Tyrosine 450 in the Voltage- and Calcium-Gated Potassium Channel of Large Conductance Channel Pore-Forming (slo1) Subunit Mediates Cholesterol Protection against Alcohol-Induced Constriction of Cerebral Arteries. J Pharmacol Exp Ther 367:234-244
Bukiya, Anna N; Dopico, Alex M (2018) Fetal Cerebral Circulation as Target of Maternal Alcohol Consumption. Alcohol Clin Exp Res 42:1006-1018
Kuntamallappanavar, Guruprasad; Bisen, Shivantika; Bukiya, Anna N et al. (2017) Differential distribution and functional impact of BK channel beta1 subunits across mesenteric, coronary, and different cerebral arteries of the rat. Pflugers Arch 469:263-277
Simakova, Maria N; Bisen, Shivantika; Dopico, Alex M et al. (2017) Statin therapy exacerbates alcohol-induced constriction of cerebral arteries via modulation of ethanol-induced BK channel inhibition in vascular smooth muscle. Biochem Pharmacol 145:81-93
Dopico, A M; Bukiya, A N; Kuntamallappanavar, G et al. (2016) Modulation of BK Channels by Ethanol. Int Rev Neurobiol 128:239-79
Bisen, Shivantika; Seleverstov, Olga; Belani, Jitendra et al. (2016) Distinct mechanisms underlying cholesterol protection against alcohol-induced BK channel inhibition and resulting vasoconstriction. Biochim Biophys Acta 1861:1756-1766
Kuntamallappanavar, Guruprasad; Dopico, Alex M (2016) Alcohol modulation of BK channel gating depends on ? subunit composition. J Gen Physiol 148:419-440
Chang, Jennifer; Fedinec, Alexander L; Kuntamallappanavar, Guruprasad et al. (2016) Endothelial Nitric Oxide Mediates Caffeine Antagonism of Alcohol-Induced Cerebral Artery Constriction. J Pharmacol Exp Ther 356:106-15
Tan, Xing-Lin; Xue, Yue-Qiang; Ma, Tao et al. (2015) Partial eNOS deficiency causes spontaneous thrombotic cerebral infarction, amyloid angiopathy and cognitive impairment. Mol Neurodegener 10:24
Bukiya, Anna N; McMillan, Jacob; Liu, Jianxi et al. (2014) Activation of calcium- and voltage-gated potassium channels of large conductance by leukotriene B4. J Biol Chem 289:35314-25

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