Date of Graduation

2014

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

Gregory M Dick

Committee Co-Chair

Robert W Brock

Committee Member

Gregory M Dick

Committee Member

John M Hollander

Committee Member

Mark Olfert

Committee Member

William F Wonderlin

Abstract

The following documents our attempt at answering a fundamental question in vascular smooth muscle: Which K+ channels control membrane potential, and thus, vascular tone? A number of groups have approached this question and great strides have been made; however, uncertainty remains as to which specific K+ channel(s) underlies this physiologically important current. Previous studies using nonselective pharmacological inhibitors of voltage-dependent K+ (KV) channels, particularly delayed rectifier K+ (KDR) channels, indicate membrane depolarization, Ca2+ influx, and vasoconstriction. Importantly, however, there are more than 40 genes known to encode KV channel subunits. We hypothesize that KDR channels composed of KV 1 subunits are responsible for the polarizing current of arterial smooth muscle. Recently, diphenyl phosphine oxide-1 (DPO-1) has been suggested as a specific KV1.5 inhibitor. Therefore, we used DPO-1 as a pharmacological tool to investigate the role of KV1.5 subunits in the KDR channels of arterial smooth muscle. In Chapter 2, we tested the specificity of DPO-1 for KV1.5 using KV1.5 knockout (KO) mice. Whole-cell patch clamp recordings revealed reduced current and less percent block by DPO-1 in aortic smooth muscle cells from KO mice. Additionally, in Chapter 2, we determined that resistance vessels from rat brain and skeletal muscle contain a DPO-1-sensitive KDR current, constrict in response to DPO-1, and these KDR channels control reactivity. In Chapter 3, we investigated the mechanism of block by DPO-1 of KDR channels in porcine coronary smooth muscle cells and found that it was largely similar to that known for cloned KV1.5 channels. Together this work suggests that KV1.5 is a major contributor to the KDR current observed in arterial smooth muscle across a number of species and in a variety of arterial vessel types. Furthermore, these DPO-1-sensitive KDR channels regulate vascular tone, buffer vasoconstriction, and control reactivity. An additional interest of our group, and incorporated into the body of this document as Chapter 4, is the effects of the xenoestrogenic endocrine disruptor bisphenol A (BPA). We have previously shown that BPA activates large conductance voltage- and Ca2+-activated K+ (BK) channels and here we build on this knowledge by examining which side of the cell membrane BPA mediates the activity of BK. This was done by formation of a membrane impermeable BPA-monosulfate (BPA-MS) and subsequent electrophysiological experiments determined that BPA may have both intracellular and extracellular binding sites on the beta1 auxiliary subunit. Collectively, this dissertation is an investigation of the physiology and pharmacology of K+ channels with respect to an underlying interest in vascular smooth muscle physiology.

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