Uptake of organic anions such as bilirubin and sulfobromophthalein (BSP) is a fundamental hepatocyte function. Recently, we cloned a unique mRNA encoding a high affinity organic anion transport protein (OATP). Expression of this protein is sufficient to mediate high affinity Cl-- dependent extraction of 35S-BSP from albumin in Xenopus oocytes and transfected HeLa cells. Preliminary studies reveal that presence of this transport protein is limited to liver and kidney, as determined by Northern blot analysis under high stringency conditions. Morphological studies indicate that OATP is limited to the basolateral (sinusoidal) hepatocyte plasma membrane in normal rat liver. The goal of the proposed studies is to better characterize this transport protein and to understand its mechanism of action. The long-range goal of this work is to understand the mechanism of organic anion transport and its relationship to specific proteins in normal liver and in various acquired and inheritable disorders. To accomplish these goals, three specific aims have been proposed. In the first aim, expression and subcellular localization of OATP will be determined by molecular biological, biochemical, and immunological means. Studies will be focused upon modulation of transport function and mechanisms of regulation of transporter expression in normal, developing and regenerating rat liver.
The second aim will focus studies on the synthesis, post-translational modification, and oligomeric state of OATP The relationship of these modifications to transport function will be elucidated by these studies. The third specific aim will examine the mechanism by which OATP mediates organic anion transport. The substrate binding site will be identified by photoaffinity techniques and its function clarified by studies utilizing site-directed mutagenesis. Activation of transport by Cl- and the role of OH- exchange will be studied in cells stably transfected with OATP.
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