Role Of Altered Membrane Function In Xenobiotic Toxicity

Environmental Health Sciences

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Abstract

A fundamental tenet of pharmacology is that the effects of a drug or toxicant are determined by the duration and concentration of exposure at critical target sites. Our bodies use two primary strategies to limit such exposure, a) metabolism to less toxic forms and b) excretion from the body as a whole, or from specific sites, e.g., the brain. The transport proteins mediating excretory and barrier function are the focus of our work, with primary emphasis on kidney and choroid plexus (a main site of the blood-cerebrospinal fluid (CSF) barrier). In addition, we examine the contribution of normal or altered membrane function in the development of target organ toxicity. A particularly important component of this work has been identification of the carrier proteins contributing to these processes, cloning the transporters, determining their subcellular distribution, and defining the driving forces and mechanisms that mediate xenobiotic transport. Thus, some years ago, we were able to show that renal organic anion (OA) secretion was mediated by indirect coupling to the out>in sodium gradient via basolateral OA exchange for intracellular dicarboxylate (DC), specifically alpha-ketoglutarate. We subsequently cloned the first transport protein responsible for this function (rOAT1) from rat kidney and demonstrated that it was indeed an OA/DC exchanger. Several additional OATs have now been cloned, and we have recently proven that one of these, OAT3, is a basolateral OA/DC exchanger like OAT1. Interestingly, two of the others, OATs 2 and 4, appear to use a different mechanism. We are attempting to characterize their driving forces and mechanism. Parallel studies were conducted examining the molecular basis for the other main xenobiotic exporting system, the organic cation (OC) secretory system. We have shown that OCT2 is the main basolateral transporter in human kidney and that it is a facilitated diffusion type carrier, driven by the inside negative membrane potential of the proximal epithelial cell. In parallel with these renal studies, we have also investigated the contribution of these cloned transporters to the blood-CSF barrier. OATs 1-4 were all shown to be present in the choroid plexus, as well as OCT2, OCT3, OCT-N1 and OCT-N2. We have established that OAT3 is the predominant organic anion transporter expressed and that the polarity of transporter expression was reversed relative to the kidney, i.e., it is expressed in the apical membrane where it mediates transport anionic xenobiotics from the CSF toward the blood for subsequent elimination by kidney or liver. OCT2 is also apically expressed, where it plays an important role in choline homeostasis. Additional studies using expressed transporters in various cell lines were used to assess the role of OA transport in the etiology of the pronounced proximal tubular damage caused by methyl- and inorganic mercury, and by the fungal toxin, ochratoxin A. In each case, toxicity was shown to be augmented in OAT1 expressing cells and could be markedly attenuated by treatment with OAT1 inhibitors. Current work focuses on determination of the sites of specific functions (e.g., substrate binding and regulatory sites and regions responsible for protein protein interactions) within the structure of the transporters proteins; regulation of transporter expression; splice variants and SNPs in these drug transporters; mechanistic studies on OATs 2 and 4; and in a new initiative, on the mechanism and driving forces of the OATP family of transporters.