Effects of the sulfhydryl reagent methylmethanethiosulfonate (MMTS) on functions of organic cation transporters (OCTs) were investigated. Currents induced by 10 mM choline [I(max(choline))] in Xenopus laevis oocytes expressing rat OCT1 (rOCT1) were increased four- to ninefold after 30-s incubation with 5 mM MMTS whereas I(max(choline)) by rat OCT2 was 70% decreased. MMTS activated the rOCT1 transporter within the plasma membrane without changing stoichiometry between translocated charge and cation. After modification of oocytes expressing rOCT1 or rOCT2 with MMTS, I(0.5(choline)) values for choline-induced currents were increased. For rOCT1 it was shown that MMTS increased I(0.5) values for different cations by different degrees. Mutagenesis of individual cysteine residues in rOCT1 revealed that modification of cysteine 322 in the large intracellular loop, and of cysteine 451 at the transition of the transmembrane alpha-helix (TMH) 10 to the short intracellular loop between the TMH 10 and 11 is responsible for the observed effects of MMTS. After replacement of cysteine 451 by methionine, the IC(50(choline)) for choline to inhibit MPP uptake by rOCT1 was increased whereas the I(0.5(choline)) value for choline-induced current remained unchanged. At variance, in double mutant Cys322Ser, Cys451Met, I(0.5(choline)) was increased compared with rOCT1 wild-type whereas in the single mutant Cys322Ser I(0.5(choline)) was not changed. The data suggest that modification of rOCT1 at cysteines 322 and 451 leads to an increase in turnover. They indicate that cysteine 451 in rOCT1 interacts with the large intracellular loop and that cysteine 451 in both rOCT1 and rOCT2 is critical for the affinity of choline.

Schmitt BM, Gorbunov D, Schlachtbauer P, Egenberger B, Gorboulev V, Wischmeyer E, Mü ller T, Koepsell H. Charge-tosubstrate ratio during organic cation uptake by rat OCT2 is voltage dependent and altered by exchange of glutamate 448 with glutamine. Am J Physiol Renal Physiol 296: F709 -F722, 2009. First published February 11, 2009 doi:10.1152/ajprenal.90323.2008.-Uptake of substrate and electric charge was measured simultaneously in voltageclamped Xenopus laevis oocytes expressing rat organic cation transporter 2 (rOCT2). At 0 mV, saturating substrate concentrations induced uptake of more positive elementary charges than monovalent organic cations, with charge-to-substrate ratios of 1.5 for guanidinium ϩ , 3.5 for tetraethylammonium ϩ , and 4.0 for 1-methyl-4-phenylpyridinium ϩ . At negative holding potentials, the charge-tosubstrate ratios decreased toward unity. At 0 mV, charge-to-substrate ratios higher than unity were observed at different extracellular pH and after replacement of extracellular Na ϩ , K ϩ , Ca 2ϩ , Mg 2ϩ , and/or Cl Ϫ . Charge-to-substrate ratios were not influenced by intracellular succinate 2Ϫ or glutarate 2Ϫ . The effects of membrane potential and ion substitution strongly suggest that the surplus of transported positive charge is not generated by passive ion permeabilities. Rather, we hypothetize that small cations are taken up together with organic cation substrates whereas the outward reorientation of the empty transporter is electroneutral. Nonselective cotransport of small cations was supported by the three-dimensional structures of rOCT2 in its inward-facing and outward-facing conformations, which we determined by homology modeling based on known corresponding structures of H ϩ -lactose permease of E. coli, and by functional analysis of OCT mutants. In our model, the innermost cavity of the outward-open binding cleft is negatively charged by Glu448 and Asp475, whereas the inward-open innermost cavity is electroneutral, containing Asp379, Asp475, Lys215, and Arg440. Substitution of Glu448 by glutamine reduced the charge-to-TEA ϩ ratio at 0 mV to unity. The observed charge excess associated with organic cation uptake into depolarized cells may contribute to tubular damage in renal failure. organic cation uptake; kidney; stoichiometry; charge translocation; membrane potential ORGANIC CATION TRANSPORTERS (OCTs) of the SLC22 family translocate a wide range of structurally diverse molecules (17, 18). They participate in small intestinal uptake, hepatic excretion and/or renal excretion of various endogenous and exogenous compounds, including choline, monoamine neurotransmitters, coenzymes, and drugs or xenobiotics. The cloning of rat OCT1 (rOCT1) (12) and the subsequent identification of other members of the SLC22 family, including OCT2, which is mainly expressed in the kidney, and OCT3, which has a broad tissue distribution including brain, has led to a much better understanding of these transporters. Particular progress has been made in elucidating their functional characteristics, tiss...