Date of Award

1-1-2020

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Biological Sciences

Content Description

1 online resource (viii, 72 pages) : illustrations (some color)

Dissertation/Thesis Chair

Haijun Chen

Committee Members

Gregory Lnenicka, Sho-ya Wang

Keywords

Potassium channel, SGY1528, Triton X-100, TWIK, Yeast, Potassium channels, Yeast fungi

Subject Categories

Biology

Abstract

Two-pore domain potassium (K2P) channels conduct leak or background K+ currents, which primarily maintain resting membrane potential and regulate cellular excitability. Among the TWIK subfamily of K2P channels, TWIK-1 (K2P1) and TWIK-2 (K2P6) channels are characterized as weak inwardly rectifying K+ channels, whereas TWIK-3 (K2P7) channels do not produce detectable currents in heterologous expression systems. Although K2P channels were first identified in the 1990s, only a few K2P-specific pharmacological agents are available. In this study, I developed a yeast-based rescue system to screen the effects of pharmacological agents on TWIK channels. First, I employed K+-uptake-deficient SGY1528 yeasts that K+ transporters Trk1 and Trk2 are genetically deleted. SGY1528 yeast cannot grow in agar plates or culture medium with low extracellular K+ concentrations ([K+]o). Heterologous expression of TWIK-1, TWIK-2, or TWIK-3 channels rescued growth of SGY1528 yeast, supporting that TWIK channels produce K+ uptake in SGY1528 yeasts. Second, we studied the effects of pharmacological agents on TWIK channels expressed in SGY1528 yeasts by monitoring growth curves of these yeasts in the absence and presence of pharmacological agents. As Triton X-100 has been previously reported to inhibit some K+ channels with either inward or outward currents, it is examined here for its potential inhibition on TWIK channels. Application of 0.5% Triton X-100 caused a ring-shaped inhibition zone of SGY1528 yeasts expressing TWIK-1, TWIK-2, or TWIK-3 channels on agar plates with low [K+]o in a dose-dependent manner. Application of Triton X-100 also inhibited growth of TWIK-expressing SGY1528 yeast in culture medium with low [K+]o. In contrast, application of Ba2+ or quinine, which are not potent inhibitors of TWIK channels, had no effect on growths of TWIK-expressing SGY1528 yeasts. Therefore, I constructed a yeast-based system that can be potentially used for high-throughput screening effects of chemical compounds on TWIK channels.

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