In arterial myocytes, activation of Ca2+-gated K+ (BK) channels limits Ca2+ influx and, thus, leads to vasodilation. In most cells, BK channels consist of channel-forming (1) and accessory (2) subunits. The 21 subtype is highly expressed in vascular myocytes and barely found in other cells, and serves as a key element to limit vascular myocyte contraction. Thus, BK 21 emerges as an ideal target to develop novel vasodilators. Several steroids activate BK channels, yet their mechanism of action remains unclear. Steroids target BK channels of variant subunit combinations, which questions whether steroids activate BK via specific docking on the channel or, rather, secondarily to nonspecific perturbation of membrane lipids. We recently found that cholane steroids, such as lithocholate (LC), selectively activate 21-containing BK, causing dilation of resistance -size arteries. Notably, other 2 subtypes (2-4) failed to provide LC sensitivity to BK channels, suggesting the existence of a cholane steroid-recognizing region in 21. Using chimeric 2s and computer dynamics, we identified two candidate sites in 21 for steroid recognition. In this proposal, we will use computer dynamics, organic synthesis, point mutagenesis and patch-clamp to identify the actual site and chemical forces involved in cholane steroid docking on BK 21. We designed selected LC nonsteroid analogs (NSA) that will be used to define the structural features of the docking site. Supported by preliminary data, NSA that dock onto this site more effectively than LC will be probed on mutated BK to determine NSA efficacy as channel activators. Once ligand docking site and efficacy are determined (Aim 1), we will combine voltage- and current-clamp methods, single channel kinetic modeling and confocal microscopy in studies that will go from native channels in isolated membranes to integrative approaches that will assess the interplay among BK current, membrane potential and local Ca2+ signals in intact myocytes. These studies will identify the mechanism of action by which LC and NSA docking on BK 21 leads to increased BK current and thus, depressed myocyte contractility (Aim 2). Finally, using pressurized, cannulated arteries, a cranial window, and evaluation of mesenteric artery diameter changes in vivo, we will determine the contribution of activation of 21-containing BK channels to dilation of resistance-size, small arteries, addressing any possible role of local Ca2+ and smooth muscle membrane potential in ligand action (Aim 3).
For Aims 2 &3 we will take advantage of the BK 21 K/O mouse model. The proposal will bring fundamental new information on steroid site and mechanisms of action on 21- containing BK channels and eventual dilation of resistance-size arteries. Translational potential resides on developing novel SA vasodilators that act via selective targeting of BK 21, independently of endothelium, and devoid of steroid actions. Given the high prevalence of disease requiring acute dilation of both mesenteric and cerebral arteries, we focus on 3rd-4th branches of mesenteric artery and resistance-size, middle cerebral artery.

Public Health Relevance

The importance of cell membrane-initiated signaling by physiological steroids is increasingly recognized, yet the molecular site and mechanisms of steroid action on membrane proteins, including ion channels, often remain unknown. Combining theoretical (single channel kinetic modeling, computational dynamics) and experimental in vitro and in vivo rodent models, we will identify the molecular site in BK channel 21 subunit and mechanism of action by which cholane steroids and nonsteroid analogs (NSA) activate smooth muscle BK channels and, thus, dilate resistance-size arteries. Results will be critical to design novel, steroid side effect-free NSA to treat prevailing human diseases that require acute vasodilation of both systemic and cerebral arteries, independently of intact endothelial function.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL104631-02
Application #
8080805
Study Section
Special Emphasis Panel (ZRG1-CVRS-F (03))
Program Officer
OH, Youngsuk
Project Start
2010-06-01
Project End
2015-11-30
Budget Start
2011-06-01
Budget End
2012-11-30
Support Year
2
Fiscal Year
2011
Total Cost
$387,087
Indirect Cost
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
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Bukiya, Anna N; Dopico, Alejandro M (2017) Common structural features of cholesterol binding sites in crystallized soluble proteins. J Lipid Res 58:1044-1054
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
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
Bukiya, Anna N; Osborn, Catherine V; Kuntamallappanavar, Guruprasad et al. (2015) Cholesterol increases the open probability of cardiac KACh currents. Biochim Biophys Acta 1848:2406-13
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
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
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
Ye, Yanping; Jian, Kuihuan; Jaggar, Jonathan H et al. (2014) Type 2 ryanodine receptors are highly sensitive to alcohol. FEBS Lett 588:1659-65

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