Date of Award

5-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Molecular Therapeutics and Cell Signaling

Research Advisor

Alejandro M. Dopico, Ph.D.

Committee

William Earl Armstrong, Ph.D. Charles W. Leffler, Ph.D. Kafait U. Malik, Ph.D. Steven J. Tavalin, Ph.D.

Abstract

Introduction and Rationale: Ethanol (EtOH) at concentrations obtained in circulation during moderate to heavy episodic drinking, such as during binge drinking (30-60 mM) causes cerebral vasoconstriction in many species, including humans. Using rodents as a model to study ethanolinduced cerebral artery constriction, our laboratory demonstrated that ethanol-induced cerebral artery constriction is due to drug-induced reduction of STOCs (Spontaneous Transient Outward Currents) in cerebral artery smooth muscle. In this tissue, STOCs result from the activity of large conductance, calcium-and voltage-gated potassium (BK) channels. Indeed, ethanol (50 mM) decreases the steady-state activity (NPo) of vascular myocyte BK channels leading to an increase in cerebral artery tone. In native tissues, functional BK channels are oligomers of four channel-forming slo1 subunits that are associated with small, accessory subunits (β1-4). β subunits do not form channels themselves but modify BK current phenotype, including its pharmacology. In particular, the vascular smooth muscle-abundant BK β1 subunit is required for ethanol to inhibit cerebral artery myocyte BK channels under physiological conditions of voltage and calcium. In contrast, the neuronally-predominant β4 subunit does not support this ethanol action. The molecular bases of ethanol-mediated inhibition of β1 subunit-containing BK channels and resulting cerebral vasoconstriction remain unknown.

Objective: Identify the BK β1 subunit regions and β1 subunit-dependent channel gating mechanisms underlying ethanol-induced inhibition of cerebral artery smooth muscle BK channel inhibition and eventual cerebral artery constriction.

Methods: Combination of recombinant DNA and other molecular biology in vitro approaches, patch-clamp electrophysiology, allosteric gating modeling, reversible permeabilization of arteries with cDNAs, and artery pressurization techniques.

Results: Ethanol sensitivity of slo1 current is dependent on the channel’s activating ion, i.e., Ca2+ i. Moreover, ethanol-induced modification of activity of slo1 (cbv1) and heteromeric cbv1+β1 BK channels is primarily due to modulation of calcium-driven gating: in particular, increase in Ca2+ i affinity; decrease in allosteric interaction between 1) RCK and voltage sensing domains and 2) RCKs and pore gate domains. Ethanol facilitation of channel inhibition is favored by β1-and β2 but not β3 or β4 subunits. Consistent with the involvement of calciumdependent mechanism, the former two drastically increase the channel’s apparent calcium sensitivity whereas the latter fail to do so. Transmembrane domains of BK-β1 subunit are essential for ethanol-mediated inhibition of β1-containing BK channels. In particular, the second transmembrane domain of β1 subunit is necessary for both inhibition of β1-containing BK channels and cerebral artery constriction evoked by ethanol.

Conclusion: BK β1 subunit TM2 enables ethanol-induced inhibition of β1-containing BK channels and cerebral artery constriction, with drug action on channel activity being dependent on modification of calcium-gating parameters.

DOI

10.21007/etd.cghs.2015.0169

Comments

Two year embargo expires May 2017

Available for download on Thursday, May 30, 2019

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