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. Our work this year has three main components: A) Basic transporter function, B) Modulation of transporter efficacy, and C) Toxicity/Therapy. 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). We have demonstrated that while OAT1 and OAT3 both play important roles in xenobiotic elimination by the kidney, only OAT3 contributes significantly to elimination of drugs and toxins from the cerebrospinal fluid. In addition, 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. For both OATs, TMD 5, 7, 10, and 11 surround the binding pocket. Charged and aromatic amino acids lining this putative pocket have now been mutated and shown to alter transport following Xenopus oocyte expression. Constructs are currently being expressed in mammalian cell lines to assess the impact of these mutations on trafficking of the mutant proteins to the cell membrane. Initial data indicates that defective trafficking is not respoonsible for the observed decrease in transport. This 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 begun to assess 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 ? We have looked at two aspects of the differences in transporter function between individuals. First, we have searched for polymorphic variants of human OAT1, identifying 20 SNPs. Of these 9 are in exons and 2 lead to amino acid changes in the transporter. Initial analysis focused on the non-synonymous changes (R50H and K525I). These SNPs did not lead to changes in the selectivity of the transporter, but the R50H did increase its affinity for several drugs. The second aspect of this work was directed toward the regulation of transporter activity. Using yeast two-hybrid analysis to determine binding partners that may regulate transporter activity, we have identified several proteins involved in cell signaling, intracellular trafficking, structural or chaperone proteins, or metabolism that interact with the C-terminal of OAT3. One of these, 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 theisoform. Furthermore, the stimulatory actions of both insulin and EGF were blocked when this PKC was inhibited. Thus, these agents may control up-regulation of OAT transport via the atypical PKC in a manner parallel to that by which activation of the more typical PKCs physiologically or by phorbal ester treatment leads their down-regulation. C) Toxicity/Therapy ? The OATs and the parallel system for organic cations, the OCTs, also play important roles in governing the retention and toxicity of a variety of environmental agents and drugs. We have shown that Oat1 plays an important role in protection against mercury and methy-mercury accumulation via secretion of various metal conjugates, particularly metal conjugates with organic sulfates. Studies using the cloned OATs were also successful in identifying the potential hazards associated with the use of the natural sweetener, stevioside, as a sugar substitute. Although stevioside was not transported by any of the OATs, its aglycone metabolite, steviol, was transported and was a potent inhibitor of OATs 1 and 3 (Ki?s 1-10 uM), and a less potent inhibitor of OATs 2 and 4. Thus, metabolism to steviol poses a risk for interactions with the elimination of drugs taken concurrently with this natural product. Finally, we have looked at the role of OCT transporters in the intestinal absorption of two H2-receptor antagonists, famotidine and ranitidine. The data indicate that only OCT1 is both well expressed in intestine, demonstrates a significant affinity for these drugs. Thus, OCT1 is the likely mediator of intestinal absorption of this drug. In addition, both drugs were potent inhibitors of OCTS 1,2, and 3. Thus, they have significant potential for drug-drug interactions via inhibition of the OCTs, particularly in kidney and liver.