Defense Date

2009

Document Type

Thesis

Degree Name

Master of Science

Department

Physiology

First Advisor

Diomedes Logothetis

Abstract

Phosphatidylinositol bisphosphate (PIP2) directly regulates electrophysiological activity in a diverse family of ion channels whether the effect is stimulatory or inhibitory. Much has been unveiled about the apparent affinity and modulatory function of PIP2 using a chemically modified dioctanoyl PIP2 (diC8), a membrane delimited cytosolic co-factor in inside-out macropatch experiments. Yet, the scarcity of molecular tools that permit fine external control in whole-cell systems has precluded future studies from probing the physiological role of PIP2 in cells in the presence of a fully intact cytoplasm. Here we introduce light as an external control for PIP2 through photocaging of diC8, and test its activation of Kir2.3 (IRK3), an inwardly rectifying ion channel that has previously shown to possess moderate binding affinity to PIP2, in excised, inside-out macropatches. Our experiments revealed that photocaged-diC8 and irradiated photocaged-diC8 have significantly different activation kinetics than the fully active diC8. Surprisingly, the activation of caged-diC8 by UV irradiation attenuated Kir2.3 activity, while the inactivated diC8 (caged-diC8) resulted in similar magnitude of channel activity compared to the currents elicited by unmodified diC8. Interestingly, we also show that application of both activated (irradiated) and inactive (caged) diC8 in macropatches generated highly fluctuating ion channel activity.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

August 2009

Included in

Physiology Commons

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