Many acid-base transporters contribute to inll'acellular and extracellular pH regul ation in brain. Electroge nic Na/Bicarbonatc Cotransp0l1crs (NBes) arc particu larly imp0l1ant because they al ter pH in response to neuronal activity. While the physiologic significance of multiple NBC vnriants 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 110t been elucida ted. '111e longterm objective of this proposal is to identify and characterize the regulatory mechanisms of Na+-coupled bit:arbonatt! transporters (BTs) in an effort to understand the significance of multiple 13Ts nnd the Illo leculnl'u'lsis of tramipo rler function. Patch-clamp techniques and fluo rcsccnce imaging will he used to exam inc phosphatidylinositol 4,S- hisphosphnte (PIP:!) regulation of pHi and ncid extrudcrs (e.g., NBCs, the Nadrivcn CI-HCO,j exchanger (NDCD E), and )./a-H exchangers (NHEs)) in cult ured astroc)'tes and neurons from rat hippocampus (Aim I). Int racellular P I P ~ levels will be altered in cells by di rect perfusion. b~lth incubation with cell ~ p e r m cant PI P"""""""", and transfection with phosphoinositide enzymes. Changes in cell ular PIP"""""""" will be
Examining the molecular basis of function and PIP2 regulation of Na+-coupled HCO3- transporters such as electrogenic NBCs is necessary to understand how these proteins alter intracellular and extracellular pH in association with neuronal activity under both physiological and pathological conditions. A better molecular understanding of the function of HCO3- transporters will be necessary in future protein-modeling studies. Because changes in ATP levels alter PIP2 levels, the results from this application will contribute to our understanding of pH and associated ion (e.g., Na+ and Ca2+) dysregulation in energy-deficient pathologies such as anoxia/hypoxia, ischemia, stroke, and reperfusion injury.
