Evidence of cysteine pairing in the long extracellular loop of human organic cation transporter 2 (hOCT2)

OCT2 assists in the renal elimination of many toxic organic cations. The hOCT2 sequence contains thirteen cysteine residues, and of these, six occur in the long extracellular loop (“loop” cysteines; between helices 1 and 2). The conservation across species of loop cysteines suggests they are important for protein structure/function. Previously we showed that the loop cysteines of hOCT2 are inaccessible to maleimide‐PEO 2 ‐biotin (MB), leading to speculation that they occur in pairs through disulfide bridges. To test this, a mutant of hOCT2 containing only the six loop cysteines (other 7 cysteines mutated to alanine) was created. The wild‐type (WT) and mutant transporter displayed similar TEA transport activity when stably expressed in CHO cells. Whereas WT hOCT2 could be precipitated with MB (biotinylation followed by Western blotting) precipitation of the mutant transporter required pre‐treatment with 10 mM dithiothreitol (DTT). DTT treatment had no effect on the kinetics (maximal rate and affinity) of TEA transport by WT hOCT2. These data are consistent with an association through a disulfide bridge of at least one pair of cysteines in the long extracellular loop of hOCT2. (DK58251)