NA/ION REABSORPTION AND H/ION SECRETION RENAL MECHANISMS

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The long term objective of this proposal is to characterize the ion transport processes in apical and basolateral membranes of rental epithelial cells, with a specific focus on the factors which account for long term regulation of these transporters at the cellular and subcellular level. A specific hypothesis to be tested in the present application is that two related forms of the Na/H antiporter are expressed in the apical and basolateral membranes of renal epithelial cells. While similar in structure, and perhaps even the produce of the same gene, the two forms have district functional differences which may be related to distinct structural domains of the protein. In addition, we hypothesize that there are critical groups (e.., histidine, lysine glutamate) in a hydrophobic region of the amiloride-binding site which regulate the maximal transport rate of the Na/H antiporter and interact specifically with 5-N substituted amiloride analogues. The specific aims for the current proposal are: 1) To define the functional and physical distinctions between the apical and basolateral forms of the human Na/H antiporter. 2) To clone, sequence and express the apical and basolateral forms of the human NA/H antiporter. 3)To examine the role of transcriptional regulation, antiporter to acid-base perturbations and other long term "regulators" of antiporter activity. 4) To define the functional domains of the apical and basolateral form of the a/H antiporter, and relate these findings to the functional characteristics of the two forms as determined under specific aim #1. 5) To determine if a "charge relay" mechanism is involved in the transepithelial transport of sodium an protons by the apical and basolateral forms of the Na/H antiporter. These studies will provide basic insights into the function and regulation of the apical and basolateral forms of the Na/H antiporter, their roles in the maintenance of intracellular pH and, in polarized epithelial systems, in the vectorial transport of protons and sodium. These experiments will use affinity-purifed antibodies to the Na/H antiporter to immunopurify the apical form of the exchanger for human renal BBMV, and the basolateral form from human placental membrane vesicles. The specific amino acid sequences obtained from the purified proteins and peptide will be compared to sequences obtained for cDNA libraries constructed from human renal cortex, and human placenta. Transcriptional regulation of the Na/H antiporter(s) activity will be studied in cultured cells which express apical and/or basolateral exchangers. Site specific mutagenesis will be undertake to examine the molecular details of the active site of the various forms of the Na/H antiporter. In vitro expression systems frog oocytes, transection assays) will be used to assess the structure-function aspects of the Na/H antiporter and its polarized expression.