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). 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. Our work has two main components: A) Basic transporter function and B) Modulation of transporter efficacy. A) Basic issues ? Our prior work has focused on the cloning and mechanistic characterization (stoichiometry and energy coupling) of the basolateral OAT transporters (OATs 1 and 3) that provide the critical 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). This year, 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, focusing on identification of transmembrane domains (TMD) and amino acid residues involved in substrate recognition and binding. These data will be used to further refine and validate the computational model and ultimately to better understand how these transporters function and explain the differences in substrate specificity between the two OAT isoforms. 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. B) Modulation ? 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 looked at the 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 shown to regulate OAT3 and OAT1 transport, markedly increasing transport rate. This stimulatory activity could blocked by pseudosubstate inhibitors of this isoform. Furthermore, the stimulatory actions of both insulin and EGF on hOAT3 transport were blocked when PKCz was inhibited. Thus, these agents may 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.