Work performed during the previous award of this grant tested the hypothesis that P-glycoprotein is the carrier for the renal secretion of organic cations. The experiments indicate that P-glycoprotein does not mediate classical renal organic cation secretion (OCT), i.e., refuting the hypothesis. OCT is characterized by organic cation/proton or organic cation/organic cation-exchange across the apical membrane of proximal tubule cells.
The aim of this application is to clone the cDNA and characterize the protein responsible for OCT. 2'-Deoxytubercidin (dTUB) is a selective substrate for OCT and it will be employed to identify the OCT carrier, as follows. Injection of total poly A+ mRNA into Xenopus oocytes enhances the oocytes' ability to efflux dTUB. The specific mRNA species responsible for the increased efflux will be cloned using this functional expression system. Once cloned and sequenced, biochemical features of the protein will be contrasted to those of other transport proteins. Transfection of the cDNA into MDCK cells (from dog kidney distal tubules) will allow us to study the physiological features of OCT in an epithelium that ordinarily lacks this transport function. The significance of this work is that the OCT has never been purified, hence its biochemical features are currently not known. Nonetheless, OCT is extremely important in that it serves to eliminate environmental and other toxins from our bodies via the kidney.
