All ETDs from UAB

Advisory Committee Chair

Mark O Bevensee

Advisory Committee Members

Dale J Benos

Inyeong Choi

Lori L McMahon

Harald W Sontheimer

Document Type

Dissertation

Date of Award

2009

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Na-Coupled Bicarbonate Transporters (NCBTs) are members of the bicarbonate transporter superfamily that play important roles in regulating intracellular pH (pHi) and extracellular pH (pHo) in the central nervous system. Electrogenic Na/bicarbonate Cotransporter 1 (NBCe1) is an NCBT that is expressed in different mammalian tissues including the brain. NBCe1 has three splice variants - NBCe1-A, -B and -C - that differ in the amino and carboxy termini. We have first performed a systematic characterization of the localization profiles of the three NBCe1 splice variants at mRNA and protein levels in rat brain. In these studies, we have used anti-sense probes and C-terminal antibodies that distinguish between the different NBCe1 splice variants. Using in situ hybridization technique, we have found that NBCe1-A is absent in rat brain whereas NBCe1-B and NBCe1-C are the brain-specific variants. Our results from immunohistochemistry were confirmed by those from immunoelectron microscopy demonstrating intracellular expression of NBCe1-A/B in neurons, and plasma membrane expression of NBCe1-C in glial cells more so than the surrounding neurons. Therefore, NBCe1 splice variants have different expression profiles in rat brain. Next, we examined whether the electrogenic NBC expressed in neurons is functional. We performed electrophysiology experiments in cultured rat hippocampal neurons bathed in either 5% CO2/24mM HCO3- or 20% CO2/96mM HCO3- using two different assays in whole-cell and perforated-patch modes. Based on the results from both assays, we found a population of rat hippocampal neurons that exhibited functional electrogenic NBC activity. At the immunohistochemistry level, using C-terminal antibodies to NBCe1-A/B and -C we found that all neurons in culture expressed NBCe1-A/B and -C variants. Also, performing double labeling studies on neurons using antibodies to NBCe1-A/B and -C and an antibody to glutamic acid decarboxylase 67 (marker of inhibitory neurons), we found that NBCe1-A/B and -C were expressed in both excitatory and inhibitory neurons. Therefore, the heterogeneity in NBC function was not due to differences in NBCe1 expression.

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