Charge-to-substrate ratio during organic cation uptake by rat OCT2 is voltage dependent and altered by exchange of glutamate 448 with glutamine.
Uptake of substrate and electric charge was measured simultaneously in voltage-clamped 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(+), 3.5 for tetraethylammonium(+), and 4.0 for 1-methyl-4-phenylpyridinium(+). At negative holding potentials, the charge-to-substrate 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(+), K(+), Ca(2+), Mg(2+), and/or Cl(-). Charge-to-substrate ratios were not influenced by intracellular succinate(2-) or glutarate(2-). 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(+)-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(+) 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.
Schmitt
Bernhard M
BM
Institute of Anatomy and Cell Biology, Koellikerstr. 6, 97070 Würzburg, Germany.
Gorbunov
Dmitry
D
Schlachtbauer
Peter
P
Egenberger
Brigitte
B
Gorboulev
Valentin
V
Wischmeyer
Erhard
E
Müller
Thomas
T
Koepsell
Hermann
H
D000818:Animals; D001665:Binding Sites; D002412:Cations; D003998:Dicarboxylic Acids / Q000378:metabolism; D005942:Gluconates / Q000378:metabolism; D018698:Glutamic Acid; D005973:Glutamine; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D019344:Lactic Acid / Q000378:metabolism; D008564:Membrane Potentials; D008958:Models, Molecular; D009154:Mutation; D009865:Oocytes; D027701:Organic Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000075102:Organic Cation Transporter 2; D010539:Permeability; D011487:Protein Conformation; D051381:Rats; D013329:Structure-Activity Relationship; D019789:Tetraethylammonium / Q000378:metabolism; D014982:Xenopus laevis
D002412:Cations; D003998:Dicarboxylic Acids; D005942:Gluconates; D027701:Organic Cation Transport Proteins; D000075102:Organic Cation Transporter 2; C100590:Slc22a2 protein, rat; D005973:Glutamine; D019344:Lactic Acid; D018698:Glutamic Acid; D019789:Tetraethylammonium; C030691:gluconic acid