The long term GOAL of this proposal is to identify and study the regulation of ileal Na+/H+ exchangers. Rabbit ileal villus cells have two forms of Na+/H+ exchangers which are distinct in terms of function (transcellular Na absorption vs ph/volume regulation), pharmacology (amiloride sensitivity) and localization (apical vs basolateral). The apical membrane Na+/H+ exchanger is part of neutral NaCl absorption which is inhibited in diarrheal diseases. We have isolated and functionally expressed in fibroblasts deficient in Na+/H+ exchange, a cDNA encoding a rabbit ileal villus basolateral membrane Na*/H* exchanger (NHE) and cloned a partial cDNA encoding a Na+/H+ exchanger related protein (NHERP) which may be the apical membrane Na+/H+ exchanger. Therefore, we propose to obtain a full-length cDNA encoding the NHERP; analyze the tissue distribution of NHERP; and use genomic Southern blot analysis to further confirm that NHE and NHERP are encoded by two separate genes. We will confirm that NHE and NHERP function as Na+/H+ exchangers by transient and stable transfection of the cDNAs into the Na+/H+ exchange deficient fibroblasts; Na+/H+ exchange activity will be determined by demonstration of Na+-dependent recovery from an acid load monitored with fluoresence by the intracellular ph-sensitive dye, BCECF. In contrast to ileal villus cells, crypt cells do not have apical membrane Na+/H+ exchangers. In order to correlate the physiological roles of NHE and NHERP in the ileal epithelium, we will localize the NHE and NHERP messages in cells along the villuscrypt axis by Northern blotting and in situ hybridization using NHE and NHERP cDNAs as probes and localize NHE and NHERP by Western blotting and immunocytochemistry using antibodies raised against NHE and NHERP. The apical membrane Na+/H+ exchanger is regulated by protein kinases. We will establish the functional role of the phosphorylation of NHERP in regulation of Na+/H+ exchange in ileal Na+ absorbing cells by demonstrating that the NHERP is a phosphoprotein and studying the effects of second messenger agonists on its phosphorylation by in vitro and in vivo phosphorylation combined with Western blotting. To provide the basis for identification of the phosphorylation site(s) of the Na+/H+ exchanger, we will identify the phosphoamino acids in NHERP which form in response to elevation in C kinase and A kinase. Then, we will study the structure-funtion relationship of NHE and NHERP by confirming that NHE and NHERP are glycoproteins and identifying ion transporting domains and domains involved in protein kinase regulation of NHE and NHERP by deletion/expression analysis.
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