In previous studies, requirements and driving forces of high affinity organic anion (35S-BSP) transport by hepatocytes were characterized and a unique Na+-independent rat liver mRNA that encodes an organic anion transport protein (oatp) was functionally cloned. Oatp is the first member of a new family of transport proteins with differing tissue distributions and substrate specificities. It is responsible for a substantial fraction of organic anion transport by the liver. Its distribution in hepatocytes is limited to the basolateral (sinusoidal) plasma membrane, consistent with its role in uptake. Studies of oatp function in HeLa cell transfectants revealed that oatp is an organic anion/HC03 exchanger. Transport activity of oatp in hepatocytes is down-regulated quickly following serine phosphorylation. Although much has been learned regarding the biology of oatp, little is known regarding its functional determinants. The goal of the proposed studies is to elucidate the relationship of oatp structure to its function as a transporter. To accomplish this goal, three specific aims have been proposed. In the first aim, the cellular topology of oatp will be determined utilizing domain-specific antibodies and glycosylation mapping. These studies will test a computer-generated structural model and will be essential for later studies of structure-function relationships.
The second aim will focus on the mechanism by which oatp mediates organic anion transport and will utilize photoaffinity labeling, domain chimerization, and mutagenesis technologies. Functional expression of mutagenized oatp will be evaluated in transiently transfected cells. The third specific aim will determine the molecular mechanism of short-term regulation of oatp transport activity by phosphorylation. The site of phosphorylation will be defined, and the mechanism by which this event influences transport activity will be explored. These studies should provide fundamental important mechanistic information that is applicable to the whole family of oatp-like transporters. Ultimately, this work should permit insight into the mechanism and regulation of organic anion transport in normal liver and in various acquired and inheritable disorders.
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