The long-term goal of the proposed research is to determine the molecular basis for electrogenic salt absorption by the colon. Understanding the mechanism of colonic ion transport requires (1) the identification and characterization of specific membrane transport proteins and (2) their integration into regulatory schemes which recognize the competing demands imposed on colonic cells by the need to effect wide swings in the rate of transcellular ion flow and nevertheless regulate cell composition within narrow limits. Advances in molecular biology now provide the basis for a systematic approach to the identification of specific membrane proteins which can play an important role in the regulation of cell composition and electrogenic salt absorption: the basolateral Na/H exchanger. Na/H exchangers have been identified in both the apical and basolateral membranes of epithelial cells, but the two forms appear to be functionally distinct. The basolateral Na/H exchanger is modulated by cell volume and has been implicated as a mediator of coordinate regulation of apical and basolateral conductances via cytosolic pH, but its role in electrogenic salt absorption is not well understood. The object of the proposed research is to investigate the physiological role of basolateral Na/H exchange using epithelia which may only express the basolateral form of the antiporter.
Specific aims are: (1) To clone and sequence cDNAs coding for basolateral Na+/H+ exchangers from turtle colon and A6 cells (2) To express the exchangers in oocytes and in antiporter-deficient cells and to compare functional properties with those determined in basolateral membranes. (3) To investigate the modulation by cell volume and aldosterone of (a) the functional expression of Na/H exchange and (b) the temporal and spatial expression of antiporter protein(s). The experiments will utilize two models for electrogenic Na absorption, the turtle colon and the A6 cell line. The turtle colon exhibits high-level expression of a volume- sensitive, basolateral Na/H exchanger which may possess unusual biophysical properties, i.e. a conductive component in the transport cycle. Northern blot analysis of poly A+ MRNA from turtle colon and A6 cells reveals a single band consistent with the size expected from the recently cloned human Na/H antiporter. A6 cells will offer the advantage of a well- defined, easily manipulated cell population which can be used not only for transport studies but also for intracellular pH measurements by means of fluorescent dyes. At each stage of the proposed studies an effort will be made to correlate molecular and functional assays for the exchanger. Functional assays will be designed to compare results obtained with isolated cells with those obtained using intact cell layers in which polarity and differentiated function is more likely to be preserved.
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