Studies on functional sites of organic cation/carnitine transporter OCTN2 (SLC22A5) using a Ser467Cys mutant protein.

Rikiya Ohashi, Ikumi Tamai, Akihiro Inano, Masaki Katsura, Yoshimichi Sai, Jun-ichi Nezu, Akira Tsuji

Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan.

The Journal of pharmacology and experimental therapeutics 2002 Sep

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The organic cation/carnitine transporter OCTN2 mediates transport of carnitine and organic cations in Na(+)-dependent and Na(+)-independent manners, respectively. However, the mechanism of molecular recognition of different substrates has not been clarified yet. We previously found a single amino acid change in OCTN2, Ser467Cys (S467C), in the Japanese population and observed a decreased carnitine transport but unchanged organic cation transport compared with wild type. Therefore, we conducted detailed kinetic and functional analyses of the substrate recognition sites of wild-type and S467C-mutant OCTN2. The K(m) value for carnitine of S467C-mutant was increased about 15-fold over that of the wild type. Mutual inhibition kinetics of carnitine and tetraethylammonium (TEA) were not completely competitive, suggesting that the binding sites are very close to each other, but not identical. Several organic anions such as valproate, as well as organic cations, significantly inhibited carnitine and TEA uptake by OCTN2, and valproate showed Na(+)-dependent inhibition of OCTN2-mediated TEA uptake. The Na(+)-activation kinetics of the S467C mutant was similar to that of the wild type. Furthermore, a significant decrease of the TEA uptake-inhibitory potency of valproate was observed in S467C-mutant OCTN2. These observations suggest that the decrease in affinity of S467C-mutant OCTN2 for carnitine was caused by functional alteration of the anion (carboxyl moiety of carnitine) recognition site located in trans-membrane domain 11, which is closely related to the Na(+)-binding site, on OCTN2 protein. These results demonstrate that OCTN2 has functional sites for carnitine and Na(+) and that the carnitine-binding site is involved, in part, in the recognition of organic cations.

Citation

Rikiya Ohashi, Ikumi Tamai, Akihiro Inano, Masaki Katsura, Yoshimichi Sai, Jun-ichi Nezu, Akira Tsuji. Studies on functional sites of organic cation/carnitine transporter OCTN2 (SLC22A5) using a Ser467Cys mutant protein. The Journal of pharmacology and experimental therapeutics. 2002 Sep;302(3):1286-94