A: Basic issues: Our prior work focused on the cloning and mechanistic characterization (stoichiometry and energy coupling) of the basolateral organic anion transporters (OATs), specifically OATs 1 and 3, in particular, focusing on the mechanisms of energy coupling that drives renal secretion of anionic drugs and xenobiotics from the body and the clearance of these agents from the brain and cerebrospinal fluid (CSF). Recently, we have used fold recognition algorithms and the known 3-D structure of a recently crystallized anion exchanger, the glycerol-3-phosphate exchanger (SLC37a2), to model OATs 1 and 3 structure. This approach has enabled us to identify those transmembrane domains (TMD) and amino acid residues involved in substrate recognition and binding. These data have been used to further refine and validate the computational model and will allow us to better understand how these transporters function and explain the differences in substrate specificity between the two OAT isoforms. The first modeling paper on the structure of hOAT1 has been published (JBC, 2006), and two additional manuscripts on hOAT3 structure and a validation study using mutations to test the predictions of the model on xenobiotic transport are nearing completion. We have also assessed the evolution of these critical transporters by comparing the function of related forms from lower vertebrates. This work has shown that the flounder OAT is capable of transporting substrates characteristically handled by either mammalian OAT1 and OAT3. We have begun to examine specific residues that may provide the basis for this marked change in specificity. A second project, focused on determining the mechanisms and energy coupling of additional poorly characterized OATs. We have established an insect cell expression system that permits isolation of membrane vesicles expressing a single OAT, initially hOAT4 - an apically expressed renal drug transporter. Vesicle technology permits investigator control of potential driving forces and isolation of transport events from cellular metabolism. Using this system, we have established that OAT4 functions as an anion exchanger indirectly coupled to sodium via the apical Na/proton exchanger, which in turn is coupled to metabolic energy via the Na,K-ATPase.? B) Modulation of transporter activity: Earlier work focused on identification and characterization of SNPs in hOAT1, demonstrating that naturally occurring SNPs showed significant changes in the affinity of hOAT1 for drugs and xenobiotics. The second aspect of this work has examined regulation of transporter activity. Binding partners that may regulate transporter activity were identified via yeast two-hybid analysis. One of these, PKCz, an atypical PKC isoform, was upon activation (phosphorylation) shown to regulate both OAT3 and OAT1 transport. Thus, activation of PKCz by insulin or epithelial growth factor (EGF) markedly increased transport rate. This stimulatory activity could blocked by pseudosubstate inhibitors of this isoform. Thus, the stimulatory actions of either insulin and EGF on hOAT3 transport was blocked when PKCz activation was prevented. The basis for increased treansport appears to be translocation of of transporter from sub-membrane compartments to the basolateral plasma membrane. Furthermore, translocation is prevented by disruption of microtubles and associated reduction in trafficking. Thus, insulin and EGF appear to control up-regulation of OAT transport via PKCz in a manner analogous to that by which activation of the more typical PKCs physiologically or by phorbal ester treatment leads their down-regulation.? C) Toxicity and disease: This project has taken two approaches, assessment of drug or toxin inhibion of OAT function, leading to decreased elimination and increased retention of OAT subsatrates. These include ochratoxin A, antiviral drugs (e.g., adefovir), and NSAIDs. The second looks at transport as a means of entry for toxic molecules, e.g., mercury via uptake of sulfhydril conjugates, or in complexes with chelating agents.
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