The long-term objective of this grant is to understand at a molecular level the structure, function, and regulation of the thiazide diuretic- sensitive Na+:Cl- cotransporter present in the mammalian kidney. the specific objectives of this grant proposal are: (i) to clone the thiazide-sensitive Na+:Cl- cotransporter from rat kidney using a nucleotide probe generated from the cDNA encoding the thiazide-sensitive Na+:Cl- cotransporter that we have recently cloned from the urinary bladder of the winter flounder, Pseudopleuronectes americanus; (ii) to determine the nucleotide and amino acid sequences of the rat renal thiazide-sensitive cotransporter, to develop a topology model the cotransporter, and to compare the nucleotide and amino acid sequences of the Na+:Cl- cotransporter with other sequenced proteins, especially other cotransporter proteins; (iii) to assess the functional characteristics of the cotransporter as expressed in Xenopus laevis oocytes and to compare these characteristics with those of the flounder bladder transporter; (iv) to prepare polyclonal antibodies against the cotransporter which will be used to assess expression of the cotransporter protein; (v) to determine the distribution of the rat Na+:Cl- cotransporter mRNA and protein in both renal and non renal tissues by Northern and Western analyses, PCR of single renal tubules, and immunofluorescence; (vi) to assess whether isoforms of the thiazide- sensitive cotransporter exist in the rat kidney [esp. in the distal tubule and collecting duct] using PCR, and if isoforms can be identified, to determine the structure of these isoforms and to assess their functional characteristics. The results of these proposed studies will provide basic information on which we can begin to model [and the molecular probes on which to assess] the function and regulation of this important renal ion transporting protein at a molecular level.
