Grant: $402,646 - National Institutes of Health - Aug. 4, 2009
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Award Description: Many acid-base transporters contribute to intracellular and extracellular pH regulation in brain. Electrogenic Na/Bicarbonate Cotransporters (NBCs) are particularly important because they alter pH in response to neuronal activity. While the physiologic significance of multiple NBC variants in brain and other tissues is not known, many of these variants exhibit different regulatory profiles. NBCs are regulated by classic second messengers involving protein kinases A and C, as well as interacting proteins. However, the role of other regulatory pathways involving phospholipids for instance has not been elucidated. The long-term objective of this proposal is to identify and characterize the regulatory mechanisms of Na+-coupled bicarbonate transporters (BTs) in an effort to understand the significance of multiple BTs and the molecular basis of transporter function. Patch-clamp techniques and fluorescence imaging will be used to examine phosphatidylinositol 4,5-bisphosphate (PIP2) regulation of pHi and acid extruders (e.g., NBCs, the Na-driven Cl-HCO3 exchanger (NDCBE), and Na-H exchangers (NHEs)) in cultured astrocytes and neurons from rat hippocampus (Aim 1). Intracellular PIP2 levels will be altered in cells by direct perfusion, bath incubation with cell-permeant PIP2, and transfection with phosphoinositide enzymes. Changes in cellular PIP2 will be assessed by mass spectroscopy and a protein-lipid overlay assay. Characteristics to be examined include PIP2 EC50, Na+ and voltage dependencies, and sensitivity to charge screeners and PIP2 blocking antibodies. According to preliminary data, PIP2 stimulates the activity of NBCe1 variants by modulating the regulatory role of their cytoplasmic amino termini (Aim 2). In Aim 2, two-electrode voltage-clamp and macropatch techniques with Xenopus oocytes heterologously expressing truncated and mutant NBCe1 variants will be used to identify and characterize N terminal regions/residues that modulate transporter function and PIP2 sensitivity. PIP2 dose-response curves will be generated. The effect of PIP2 on the biophysical properties (e.g., KM values for transported ions, Vmax values, and current-voltage relationships) of NBCe1 variants will be assessed. The effect of peptides containing identified regulatory regions on NBC function will be assessed. The results from these aims will elucidate PIP2 as a novel regulatory mechanism of NBC activity that likely provides a permissive pH environment for PIP2 targets that modulate neuronal excitability. Characterizing the molecular basis of PIP2¿s stimulation of NBCe1 extends our understanding of the importance of multiple NBCs, and provides mechanistic and structural insight into NBC function. The information will enhance our understanding of acid-base handling by brain cells, particularly in acid-base disturbances associated with ischemia, anoxia/hypoxia, stroke, and reperfusion injury.
Project Description: as defined in the Award Description field
Jobs Summary: Positions created/retained included: Graduate Student Research Associates Faculty Researchers (Total jobs reported: 1)
Project Status: Less Than 50% Completed
This award's data was last updated on Aug. 4, 2009. Help expand these official descriptions using the wiki below.