All ETDs from UAB

Advisory Committee Chair

Mark O Bevensee

Advisory Committee Members

Walter F Boron

Kevin L Kirk

Robin Aj Lester

Jacques I Wadiche

Document Type

Dissertation

Date of Award

2014

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The electrogenic Na/bicarbonate cotransporter (NBCe1) is an acid/base regulator that is also involved in coordinating epithelial ion transport. Splice variants of NBCe1 differ at their cytosolic amino- (N-) and/or carboxy- (C-) termini. These different cytosolic termini impart differential regulation for each variant. For example, the N-terminus of NBCe1-A is autostimulatory, whereas the N-terminus of NBCe1-B and -C is autoinhibitory. We examine the regulatory role of PIP2 for NBCe1 splice variants. In the first study of this dissertation, we characterize the effect of increasing PIP2 on the activity of NBCe1-A, -B, and -C expressed in Xenopus laevis oocytes. Injecting PIP2 stimulated NBCe1-B and -C current by ~150% as monitored by the two-electrode voltage-clamp technique. The majority of this stimulation required PIP2 hydrolysis to IP3 and ER Ca2+ stores, and was mediated by a staurosporine-sensitive kinase. The second study focuses on the effect of PIP2 itself on the activity of NBCe1 expressed in oocytes. The two-electrode voltage-clamp technique was used to control the activation of a co-expressed voltage sensitive phosphatase (VSP), that dephosphorylates PIP2, and to monitor associated changes in the current of co-expressed NBCe1 variants. VSP activation by depolarizing an oocyte to +60 mV for 10 s inhibited NBCe1-B and -C by ~35%. When VSP was subsequently inactivated by repolarization to -60 mV, NBCe1 currents slowly recovered to baseline levels. Both NBCe1 currents and PIP2 levels were simultaneously monitored by the two-electrode voltage-clamp technique and confocal imaging of a PIP2-binding pleckstrin homology conjugated to green fluorescent protein, respectively. The slow NBCe1 current recovery mirrored PIP2 replenishment at the membrane. NBCe1 inhibition was not observed in the same experimental protocols with a catalytically dead VSP. These combined studies reveal that PIP2 can regulate NBCe1 activity by a dual mechanism that involves both PIP2 itself and the classic IP3/Ca2+ pathway. This dissertation provides insight into how acid-base regulation can be tightly coupled to receptors that hydrolyze PIP2, such as Gq-coupled receptors. Because PIP2 is a ubiquitous signaling epicenter, the findings may have implications across many types of tissue.

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