Date of Award

6-1-2017

Document Type

Thesis and Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

School of Biological Sciences

First Advisor

Craig Gatto

Abstract

This work focuses on the elucidation of the mechanism of passive proton import through the Na,K-ATPase. This enzyme uses the energy in ATP hydrolysis to exchange three intracellular Na+ for two extracellular K+ to maintain ion gradients within the cell, and while in the absence of physiological external Na+ and K+, the phosphorylated externally open (E2P) conformation passively imports protons, generating an inward current (IH). Chapter one reports on the effects of intracellular cations and nucleotides to shift the Na,K-ATPase into the E2P conformation. We identified that a combination of either internal Na+ and ATP or K+ and Pi. In chapter two, we investigated the effects of extracellular inhibitors on the ability for H+ to enter through the E2P conformation. We used two different known extracellular tetrapropylammonium (TPA) and ethylenediamine (EDA) in order to block extracellular access at different expected depths within the Na,K-ATPase. Using a combination of electrophysiological and biochemical techniques, the results showed that TPA inhibits the Na,K-ATPase as well as IH near the extracellular surface as previously demonstrated. EDA on the other hand, inhibited at or near the shared sites, induced dephosphorylation and accentuate IH at acidic pH 6.0. The appendix of this work shows that while perpetually pseudo-phosphorylated by BeF3- the Na,K-ATPase, Na+ and K+ are still able to interact in the phosphorylated conformation.

Comments

Imported from ProQuest Stanley_ilstu_0092E_11039.pdf

Page Count

127

Available for download on Wednesday, September 12, 2018

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