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Functional significance of conserved cysteines in the human organic cation transporter 2
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| Zeitschriftentitel: | American Journal of Physiology-Renal Physiology |
|---|---|
| Personen und Körperschaften: | , , , , , |
| In: | American Journal of Physiology-Renal Physiology, 303, 2012, 2, S. F313-F320 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
American Physiological Society
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| Schlagwörter: |
| author_facet |
Pelis, Ryan M. Dangprapai, Yodying Cheng, Yaofeng Zhang, Xiaohong Terpstra, Jennifer Wright, Stephen H. Pelis, Ryan M. Dangprapai, Yodying Cheng, Yaofeng Zhang, Xiaohong Terpstra, Jennifer Wright, Stephen H. |
|---|---|
| author |
Pelis, Ryan M. Dangprapai, Yodying Cheng, Yaofeng Zhang, Xiaohong Terpstra, Jennifer Wright, Stephen H. |
| spellingShingle |
Pelis, Ryan M. Dangprapai, Yodying Cheng, Yaofeng Zhang, Xiaohong Terpstra, Jennifer Wright, Stephen H. American Journal of Physiology-Renal Physiology Functional significance of conserved cysteines in the human organic cation transporter 2 Physiology |
| author_sort |
pelis, ryan m. |
| spelling |
Pelis, Ryan M. Dangprapai, Yodying Cheng, Yaofeng Zhang, Xiaohong Terpstra, Jennifer Wright, Stephen H. 1931-857X 1522-1466 American Physiological Society Physiology http://dx.doi.org/10.1152/ajprenal.00038.2012 <jats:p>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<jats:sub>t</jats:sub>) 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<jats:sub>2</jats:sub>-biotin did. Mutation of C474 to an alanine or phenylalanine did not affect the K<jats:sub>t</jats:sub>value for MPP. In contrast, the K<jats:sub>t</jats:sub>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 exist within disulfide bridge(s). Each loop cysteine is important for plasma membrane targeting, and their mutation can influence substrate binding. The effect of C474 mutation on TEA transport suggests that it contributes to a TEA binding surface. Given that TEA and MPP are competitive inhibitors, the differential effects of C474 modification on TEA and MPP binding suggest that the binding surfaces for each are distinct, but overlapping in area.</jats:p> Functional significance of conserved cysteines in the human organic cation transporter 2 American Journal of Physiology-Renal Physiology |
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American Journal of Physiology-Renal Physiology |
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| title |
Functional significance of conserved cysteines in the human organic cation transporter 2 |
| title_unstemmed |
Functional significance of conserved cysteines in the human organic cation transporter 2 |
| title_full |
Functional significance of conserved cysteines in the human organic cation transporter 2 |
| title_fullStr |
Functional significance of conserved cysteines in the human organic cation transporter 2 |
| title_full_unstemmed |
Functional significance of conserved cysteines in the human organic cation transporter 2 |
| title_short |
Functional significance of conserved cysteines in the human organic cation transporter 2 |
| title_sort |
functional significance of conserved cysteines in the human organic cation transporter 2 |
| topic |
Physiology |
| url |
http://dx.doi.org/10.1152/ajprenal.00038.2012 |
| publishDate |
2012 |
| physical |
F313-F320 |
| description |
<jats:p>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<jats:sub>t</jats:sub>) 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<jats:sub>2</jats:sub>-biotin did. Mutation of C474 to an alanine or phenylalanine did not affect the K<jats:sub>t</jats:sub>value for MPP. In contrast, the K<jats:sub>t</jats:sub>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 exist within disulfide bridge(s). Each loop cysteine is important for plasma membrane targeting, and their mutation can influence substrate binding. The effect of C474 mutation on TEA transport suggests that it contributes to a TEA binding surface. Given that TEA and MPP are competitive inhibitors, the differential effects of C474 modification on TEA and MPP binding suggest that the binding surfaces for each are distinct, but overlapping in area.</jats:p> |
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| author | Pelis, Ryan M., Dangprapai, Yodying, Cheng, Yaofeng, Zhang, Xiaohong, Terpstra, Jennifer, Wright, Stephen H. |
| author_facet | Pelis, Ryan M., Dangprapai, Yodying, Cheng, Yaofeng, Zhang, Xiaohong, Terpstra, Jennifer, Wright, Stephen H., Pelis, Ryan M., Dangprapai, Yodying, Cheng, Yaofeng, Zhang, Xiaohong, Terpstra, Jennifer, Wright, Stephen H. |
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| description | <jats:p>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<jats:sub>t</jats:sub>) 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<jats:sub>2</jats:sub>-biotin did. Mutation of C474 to an alanine or phenylalanine did not affect the K<jats:sub>t</jats:sub>value for MPP. In contrast, the K<jats:sub>t</jats:sub>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 exist within disulfide bridge(s). Each loop cysteine is important for plasma membrane targeting, and their mutation can influence substrate binding. The effect of C474 mutation on TEA transport suggests that it contributes to a TEA binding surface. Given that TEA and MPP are competitive inhibitors, the differential effects of C474 modification on TEA and MPP binding suggest that the binding surfaces for each are distinct, but overlapping in area.</jats:p> |
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| imprint_str_mv | American Physiological Society, 2012 |
| institution | DE-Zi4, DE-Gla1, DE-15, DE-Pl11, DE-Rs1, DE-14, DE-105, DE-Ch1, DE-L229, DE-D275, DE-Bn3, DE-Brt1, DE-Zwi2, DE-D161 |
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| spelling | Pelis, Ryan M. Dangprapai, Yodying Cheng, Yaofeng Zhang, Xiaohong Terpstra, Jennifer Wright, Stephen H. 1931-857X 1522-1466 American Physiological Society Physiology http://dx.doi.org/10.1152/ajprenal.00038.2012 <jats:p>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<jats:sub>t</jats:sub>) 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<jats:sub>2</jats:sub>-biotin did. Mutation of C474 to an alanine or phenylalanine did not affect the K<jats:sub>t</jats:sub>value for MPP. In contrast, the K<jats:sub>t</jats:sub>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 exist within disulfide bridge(s). Each loop cysteine is important for plasma membrane targeting, and their mutation can influence substrate binding. The effect of C474 mutation on TEA transport suggests that it contributes to a TEA binding surface. Given that TEA and MPP are competitive inhibitors, the differential effects of C474 modification on TEA and MPP binding suggest that the binding surfaces for each are distinct, but overlapping in area.</jats:p> Functional significance of conserved cysteines in the human organic cation transporter 2 American Journal of Physiology-Renal Physiology |
| spellingShingle | Pelis, Ryan M., Dangprapai, Yodying, Cheng, Yaofeng, Zhang, Xiaohong, Terpstra, Jennifer, Wright, Stephen H., American Journal of Physiology-Renal Physiology, Functional significance of conserved cysteines in the human organic cation transporter 2, Physiology |
| title | Functional significance of conserved cysteines in the human organic cation transporter 2 |
| title_full | Functional significance of conserved cysteines in the human organic cation transporter 2 |
| title_fullStr | Functional significance of conserved cysteines in the human organic cation transporter 2 |
| title_full_unstemmed | Functional significance of conserved cysteines in the human organic cation transporter 2 |
| title_short | Functional significance of conserved cysteines in the human organic cation transporter 2 |
| title_sort | functional significance of conserved cysteines in the human organic cation transporter 2 |
| title_unstemmed | Functional significance of conserved cysteines in the human organic cation transporter 2 |
| topic | Physiology |
| url | http://dx.doi.org/10.1152/ajprenal.00038.2012 |