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Date of Award

2014

Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science

Department

Biochemistry & Molecular Biol.

First Advisor

Dr. Linda M. Boland

Abstract

Characterization and investigation of the physiological properties of a novel tandem pore domain potassium channel (K2P) cloned from the marine sponge, Amphimedon queenslandica was accomplished through the use of a Xenopus oocyte expression system. Previous research has determined that alkaline pH and arachidonic acid (AA) are necessary for the activation this AqK2P channel and that the C-terminus is one of the crucial regions for AA and stretch activation. We propose to investigate the role of the C- terminus by designing a mutant AqK2P channel containing an HA-Epitope tag in an extracellular facing loop that would allow for determination of the surface expression of the channel while still maintaining its function. The HA-AqK2P F278, D112 and A98 constructs were successfully created, with their expression at the surface of the cell membrane confirmed through chemiluminescence assays, and functionality preserved through measurable current detected in electrophysiology recordings. Future directions involve designing C-terminal mutants of the HA-AqK2P channels to investigate the role of the C-terminus in trafficking and expression at the cell membrane in order to support or reject our hypotheses that the resulting loss of signal and current in the C-terminus truncation mutations of the AqK2P channel is due to a low expression of these channels at the membrane, attributable to a protein trafficking defect, or due to a loss of pH and lipid sensitivity.

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