Functional significance of conserved cysteines in the human organic cation transporter 2.

American Journal of Physiology. Renal Physiology
Ryan M PelisStephen H Wright

Abstract

The significance of conserved cysteines in the human organic cation transporter 2 (hOCT2), namely the six cysteines in the long extracellular loop (loop cysteines) and C474 in transmembrane helix 11, was examined. Uptake of tetraethylammonium (TEA) and 1-methyl-4-phenypyridinium (MPP) into Chinese hamster ovary cells was stimulated >20-fold by hOCT2 expression. Both cell surface expression and transport activity were reduced considerably following mutation of individual loop cysteines (C51, C63, C89, C103, and C143), and the C89 and C103 mutants had reduced Michaelis constants (K(t)) for MPP. The loop cysteines were refractory to interaction with thiol-reactive biotinylation reagents, except after pretreatment of intact cells with dithiothreitol or following cell membrane solubilization. Reduction of disulfide bridge(s) did not affect transport, but labeling the resulting free thiols with maleimide-PEO(2)-biotin did. Mutation of C474 to an alanine or phenylalanine did not affect the K(t) value for MPP. In contrast, the K(t) value associated with TEA transport was reduced sevenfold in the C474A mutant, and the C474F mutant failed to transport TEA. This study shows that some but not all of the six extracellular loop cysteines exi...Continue Reading

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Citations

Jul 20, 2012·The Journal of Biological Chemistry·Brigitte EgenbergerHermann Koepsell
Mar 21, 2015·Pharmaceutical Research·Takahiro ShibayamaStephen H Wright
May 28, 2013·The Journal of Pharmacology and Experimental Therapeutics·Mathew BelzerStephen H Wright
Dec 20, 2019·Pharmacological Reviews·Hermann Koepsell
Oct 1, 2019·Drug Metabolism Reviews·Nicolas Brosseau, Dindial Ramotar
Oct 17, 2017·Toxicology in Vitro : an International Journal Published in Association with BIBRA·Jelena DragojevićTvrtko Smital

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