Role Of Altered Membrane Function In Xenobiotic Toxicity

National Institute Of Environmental Health Sciences

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Abstract

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 ready for submission. 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. Kinetic analysis indicates that activation of transport is associated with an increase in maximum velocity, with no change in affinity. This increase in activity following PKCz activation is prevented by disruption of microtubles and associated reduction in trafficking. Together, these results indicate that activation of OAT3 transport requires translocation of of ttransporter form sub-membrane compartments to the basolateral plasma membrane. Finally, downregulation of PKCz activity follows phorbal ester treatment and leads to down-regulation of OAT3 activity and indicates that PKCz plays a pivotal role in controlling the moment to moment activity of this important drug/xenobiotic transporter. C) Basis of Chromium Toxicity: It is known that chromium VI is more toxic in vivo than chromium III. Suggestive evidence indicates that Cr VI may be absorbed more readily than Cr III, resulting in a greater internal dose upon equal exposure. Isolated membrane vesicles were used to examine transport of both forms on the Na-sulfate cotransporter. Cr VI, but not Cr III was an effective competitive inhibitor of this system, indicating that the greater intestinal uptake seen in vivo reflects Cr VI transport on the interstinal Na-sulfate transporter.