NA+ REABSORPTION AND H+ ION SECRETION RENAL MECHANISMS

总体目标是提供详细的理解和

The overall goal is to provide a detailed understanding and characterization of the amiloride-resistant Na+/H+ isoforms which are of functional importance in renal epithelial tissues. The SPECIFIC AIMS include: A. To define critical functional domains of amiloride-resistant NHE isoforms. cDNA constructs will be stably expressed, and site-directed mutagenesis, truncations, deletions along with cross linking experiments, limited trypsinolysis and protein quantification with Western blots will be used to define the sites of amiloride interation, cation (substrate) binding sites, critical functional groups including histidine and glutamate, and critical cytoplasmic and membrane spanning domains. B. To define the regulation of expression and activity of amiloride-resistant NHE isoforms. Well defined dietary and hormal manipulations (high versus low salt diet, thyroid hormone administration for 5 days, chronic metabolic acidosis) will be used to chronically modulate the level of NHE isoform expression in vivo. Western blots, Northern blots, RNase protection assays, immunoprecipitation and surface labeling will be used to define changes in steady-state mRNA levels and in specific isoform protein expression in response to these in vivo pertubations of NHE expression. C. To identify, clone, stably express and characterize amiloride-resistant NHE isoforms. Commercial cDNA libraries will be screened and clones will be sequenced and analyzed. PCR methods will be used for chromosomal locaiizaton to determine if these are unique isoforms. Partial sequence information will be used for alignment comparisons to describe defined domains of interest with respect to well characterized isoforms. Full length constructs can then be obtained and stably expressed in mouse LAP- cells. Studies of the kinetics (cations, cytosolic pH sensitivity, cooperativity), and inhibitor sensitivity (amiloride analogues, cimetidine) will be carried out. D. To define the tissue specific expression of amiloride-resistant NHE isoforms. Multiple Tissue Northern and Western blots will permit comparisons of relative abundance between a variety of rat and human tissues using isoform- specific cDNA probes and affinity purified antibodies. Immunohistochemistry will define the membrane distribution of specific isoforms in rat cortical tubule preparations using affinity purified antibodies. The studies will provide unique insights into the structure- function relationships of NHE isoforms and define their chronic regulation in specific nephron segments.