Renal elimination of anionic drugs, xenobiotics and toxins are necessary for the survival of mammalian species. This process is mediated by vectorial transport from blood into the urine through the cooperative function of specific transporters in the basolateral and apical membranes of the proximal tubule epithelium. The first step of this process is the extraction of organic anions from the peritubular blood plasma into proximal tubule cells through, in part, the organic anion transporter (OAT) pathway. As a result, OAT pathway is one of the major sites for body drug clearance/detoxification but it is also the site for drug-drug interaction and drug-induced nephrotoxicity. To maximize therapeutic efficacy and minimize toxicity, the transporter structure-function relationships, including drug/inhibitor binding sites, and the regulation of transport mechanisms must be defined. The overall objective of this application is to define the molecular mechanisms underlying drug elimination through the OAT pathway. We have recently isolated a cDNA from mouse kidney which encodes the first member of the OAT family. We have shown that OAT function is down-regulated by two distinct mechanisms: a) activation of PKC (which does not phosphorylate OAT), and b) serine-phosphorylation of OAT by an unknown kinase.
Two specific aims (SA) are therefore proposed. In SA-1, we will identify the functional determinants of OAT through a combination of biochemical, biophysical and genetic engineering approaches. In SA-2, we will identify the short-term regulatory mechanisms of OAT function. We will first identify the site(s) of serine phosphorylation and the kinases which phosphorylate OAT through a combination of mass spectra and site-directed mutagenesis. We will then explore the mechanisms by which kinases/phosphorylationj influence OAT function. The knowledge gained from these studies will be invaluable toward the rational design of novel drugs and inhibitors to optimize drug therapy while avoiding unwanted drug interactions. These studies should also provide fundamental important mechanistic information applicable to the whole family of OAT-like transporters. Ultimately, this work should permit insight into the mechanism and regulation of organic anion transport in normal kidney and in various acquired and inheritable disorders.
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