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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AA011560-16
Application #
8494457
Study Section
Special Emphasis Panel (ZRG1-IFCN-A (03))
Program Officer
Orosz, Andras
Project Start
1999-01-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
16
Fiscal Year
2013
Total Cost
$316,379
Indirect Cost
$106,197
Name
University of Tennessee Health Science Center
Department
Pharmacology
Type
Schools of Medicine
DUNS #
941884009
City
Memphis
State
TN
Country
United States
Zip Code
38163
Bukiya, Anna; Dopico, Alejandro M; Leffler, Charles W et al. (2014) Dietary cholesterol protects against alcohol-induced cerebral artery constriction. Alcohol Clin Exp Res 38:1216-26
Bukiya, Anna N; Kuntamallappanavar, Guruprasad; Edwards, Justin et al. (2014) An alcohol-sensing site in the calcium- and voltage-gated, large conductance potassium (BK) channel. Proc Natl Acad Sci U S A 111:9313-8
Kuntamallappanavar, Guruprasad; Toro, Ligia; Dopico, Alex M (2014) Both transmembrane domains of BK ?1 subunits are essential to confer the normal phenotype of ?1-containing BK channels. PLoS One 9:e109306
Ye, Yanping; Jian, Kuihuan; Jaggar, Jonathan H et al. (2014) Type 2 ryanodine receptors are highly sensitive to alcohol. FEBS Lett 588:1659-65
McMillan, Jacob E; Bukiya, Anna N; Terrell, Camisha L et al. (2014) Multi-generational pharmacophore modeling for ligands to the cholane steroid-recognition site in the ?? modulatory subunit of the BKCa channel. J Mol Graph Model 54:174-83
Liu, Jianxi; Bukiya, Anna N; Kuntamallappanavar, Guruprasad et al. (2013) Distinct sensitivity of slo1 channel proteins to ethanol. Mol Pharmacol 83:235-44
Singh, Aditya K; McMillan, Jacob; Bukiya, Anna N et al. (2012) Multiple cholesterol recognition/interaction amino acid consensus (CRAC) motifs in cytosolic C tail of Slo1 subunit determine cholesterol sensitivity of Ca2+- and voltage-gated K+ (BK) channels. J Biol Chem 287:20509-21
Bukiya, Anna N; Vaithianathan, Thirumalini; Kuntamallappanavar, Guruprasad et al. (2011) Smooth muscle cholesterol enables BK *1 subunit-mediated channel inhibition and subsequent vasoconstriction evoked by alcohol. Arterioscler Thromb Vasc Biol 31:2410-23
Bukiya, Anna N; Liu, Jianxi; Dopico, Alejandro M (2009) The BK channel accessory beta1 subunit determines alcohol-induced cerebrovascular constriction. FEBS Lett 583:2779-84