engleski

Functional characterization of MATE (SLC47A) and OCT (SLC22A) transporters in zebrafish (Danio rerio)

In this paper we present the first data on identification, molecular characterization and understanding of potential (eco)toxicological relevance of two important classes of polyspecific uptake and efflux membrane transport proteins: organic cation transporters (OCTs, SLC22A family) and multidrug and toxin extrusion proteins (MATEs, SLC47A family). OCTs have important role in uptake and distribution of cationic drugs and toxins in mammals. MATEs, unlike ABC transporters which are ATP dependent, use the proton gradient as the driving force for substrate efflux and are known to excrete cationic endogenous substances and xenobiotics. Using zebrafish (Danio rerio) as a model organism we performed phylogenetic and synteny analyses, followed by qRT-PCR determinations of tissue distribution and sex differences in mRNA expression of related genes. Two zebrafish OCT co-orthologs and 6 MATE orthologs were identified and their syntenic relations determined. High expression of Oct2 was determined in testes and moderate in kidney, and Oct1 showed very high expression in kidney, and high expression in liver and testes of male zebrafish. Unlike mammalian MATEs, all six zebrafish MATEs are quite ubiquitously expressed. In order to determine the substrate specificity of identified OCTs and MATEs, we transiently expressed corresponding transporters in HEK293 cells and performed initial transport activity assays using a series of model fluorescent probes: 4- (4-dimethylaminostyryl)-N-methylpyridinium (ASP+), ethidium bromide, amiloride, berberine, 4', 6-diamidino-2-phenylindole (DAPI) and rhodamine 123. Finally, using developed functional assays with ASP+ as selected fluorescent probe for OCTs, and DAPI for MATEs, respectively, we tested a series of potential physiological substrates along with selected set of environmentally relevant contaminants on their interaction with zebrafish transporters. Taken together our results imply that: (1) zebrafish OCT1 probably has a pivotal physiological role in uptake of hormones in testes and their clearance from blood through kidney and liver, (2) OCT1 may have an important defensive role in fish metabolic response to organotines ; and (3) zebrafish MATEs appears to have important functions both in fish physiology and in elimination of xenobiotics through liver and kidney.

organic cation transporters (OCTs) ; multidrug and toxin extrusion proteins (MATEs) ; zebrafish ; functional characterization

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