engleski

Characterization of organic cation transporters in zebrafish (Danio rerio)

Uptake transporters represent integral elements in the ADME (Absorption, Distribution, Metabolism, Excretion) processes and toxicological response. They play crucial role in the initial phase of cellular metabolism of xeno- and endobiotics by mediating the uptake of compounds across the cell membrane. The majority of uptake transporters belong to the SLC (Solute Carrier) protein superfamily which is one of the largest known protein superfamilies. Organic cation transporters (OCTs) are members of SLC22A family. There are three human OCT members, OCT1-3. OCT1 and OCT2 are dominantly expressed in liver and kidney, respectively. OCTs are localized in basolateral membranes of blood-tissue barrier cells and they are responsible for polyspecific transport of numerous organic cations. Using phylogenetic and synteny analysis of previously identified two members of zebrafish Octs, we determined orthological relationship with human and other vertebrates Octs. We also determined dominant expression of Oct1 in zebrafish kidney and liver, whereas Oct2 showed lower but ubiquitous expression. Functional characterization was performed using newly identified fluorescent Oct1 substrates, which were used as tool for determination of interactions with various environmentally and physiologically relevant compounds. Initial interaction screen with ASP+ as fluorescent substrate and numerous tested compounds revealed steroid hormones as potent interactors of Oct1, with the highest inhibition constants for progesterone (Ki = 2 µM), androstenedione (5.3 µM) and testosterone (13.2 µM). Among environmental contaminants Oct1 showed high interaction with organotin compounds, especially with dibutyltin and tributyltin chloride with the lowest Ki values of 0.4 and 3.9 µM, respectively. We also modeled tertiary structure Oct1 and performed molecular docking analysis with model substrates and determined interactors, revealing crucial amino acid residues within Oct binding region. In conclusion, this study represents the first functional characterization of organic cation transporters from Slc22 family in fish. The obtained knowledge provides new insight into possible roles of Octs in crucial physiological, defensive and pathological processes in zebrafish, enabling a more reliable use of zebrafish in understanding of OCT/Oct functions in higher vertebrates and human.

organic cation transporters (OCTs) ; zebrafish ; characterization

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