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Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity
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| Zeitschriftentitel: | American Journal of Physiology-Renal Physiology |
|---|---|
| Personen und Körperschaften: | , , , |
| In: | American Journal of Physiology-Renal Physiology, 292, 2007, 5, S. F1583-F1591 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
American Physiological Society
|
| Schlagwörter: |
| author_facet |
Pelis, Ryan M. Dangprapai, Yodying Wunz, Theresa M. Wright, Stephen H. Pelis, Ryan M. Dangprapai, Yodying Wunz, Theresa M. Wright, Stephen H. |
|---|---|
| author |
Pelis, Ryan M. Dangprapai, Yodying Wunz, Theresa M. Wright, Stephen H. |
| spellingShingle |
Pelis, Ryan M. Dangprapai, Yodying Wunz, Theresa M. Wright, Stephen H. American Journal of Physiology-Renal Physiology Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity Physiology |
| author_sort |
pelis, ryan m. |
| spelling |
Pelis, Ryan M. Dangprapai, Yodying Wunz, Theresa M. Wright, Stephen H. 1931-857X 1522-1466 American Physiological Society Physiology http://dx.doi.org/10.1152/ajprenal.00496.2006 <jats:p>Human organic cation transporter 2 (hOCT2) is essential for the renal tubular secretion of many toxic organic cations. Previously, of the cysteines (C437, C451, C470, and C474) that occur within transmembrane helices that comprise the hydrophilic cleft (proposed site of substrate binding), only C474 was accessible to maleimide-PEO<jats:sub>2</jats:sub>-biotin (hydrophilic thiol-reactive reagent), and covalent modification of this residue caused lower transport rates (Pelis RM, Zhang X, Dangprapai Y, Wright SH, J Biol Chem 281: 35272–35280, 2006). Thus it was hypothesized that the environmental contaminant Hg<jats:sup>2+</jats:sup>(as HgCl<jats:sub>2</jats:sub>) would interact with C474 to reduce hOCT2-mediated transport. Uptake of [<jats:sup>3</jats:sup>H]tetraethylammonium (TEA) into Chinese hamster ovary cells stably expressing hOCT2 was reduced in a concentration-dependent manner by HgCl<jats:sub>2</jats:sub>, with an IC<jats:sub>50</jats:sub>of 3.9 ± 0.11 μM. Treatment with 10 μM HgCl<jats:sub>2</jats:sub>caused a sixfold reduction in the maximal rate of TEA transport but did not alter the affinity of hOCT2 for TEA. To determine which cysteines interact with Hg<jats:sup>2+</jats:sup>, a mutant with all four cleft cysteines converted to alanines (quadruple mutant), and four variants of this mutant, each with an individual cysteine restored, were created. The quadruple mutant was less sensitive to HgCl<jats:sub>2</jats:sub>than wild-type, whereas the C451- and C474-containing mutants were more sensitive than the quadruple mutant. Consistent with the HgCl<jats:sub>2</jats:sub>effect on transport, MTSEA-biotin only interacted with C451 and C474. These data indicate that C451 and C474 of hOCT2 reside in the aqueous milieu of the cleft and that interaction of Hg<jats:sup>2+</jats:sup>with these residues causes reduced TEA transport activity.</jats:p> Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity American Journal of Physiology-Renal Physiology |
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10.1152/ajprenal.00496.2006 |
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American Journal of Physiology-Renal Physiology |
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| title |
Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity |
| title_unstemmed |
Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity |
| title_full |
Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity |
| title_fullStr |
Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity |
| title_full_unstemmed |
Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity |
| title_short |
Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity |
| title_sort |
inorganic mercury interacts with cysteine residues (c451 and c474) of hoct2 to reduce its transport activity |
| topic |
Physiology |
| url |
http://dx.doi.org/10.1152/ajprenal.00496.2006 |
| publishDate |
2007 |
| physical |
F1583-F1591 |
| description |
<jats:p>Human organic cation transporter 2 (hOCT2) is essential for the renal tubular secretion of many toxic organic cations. Previously, of the cysteines (C437, C451, C470, and C474) that occur within transmembrane helices that comprise the hydrophilic cleft (proposed site of substrate binding), only C474 was accessible to maleimide-PEO<jats:sub>2</jats:sub>-biotin (hydrophilic thiol-reactive reagent), and covalent modification of this residue caused lower transport rates (Pelis RM, Zhang X, Dangprapai Y, Wright SH, J Biol Chem 281: 35272–35280, 2006). Thus it was hypothesized that the environmental contaminant Hg<jats:sup>2+</jats:sup>(as HgCl<jats:sub>2</jats:sub>) would interact with C474 to reduce hOCT2-mediated transport. Uptake of [<jats:sup>3</jats:sup>H]tetraethylammonium (TEA) into Chinese hamster ovary cells stably expressing hOCT2 was reduced in a concentration-dependent manner by HgCl<jats:sub>2</jats:sub>, with an IC<jats:sub>50</jats:sub>of 3.9 ± 0.11 μM. Treatment with 10 μM HgCl<jats:sub>2</jats:sub>caused a sixfold reduction in the maximal rate of TEA transport but did not alter the affinity of hOCT2 for TEA. To determine which cysteines interact with Hg<jats:sup>2+</jats:sup>, a mutant with all four cleft cysteines converted to alanines (quadruple mutant), and four variants of this mutant, each with an individual cysteine restored, were created. The quadruple mutant was less sensitive to HgCl<jats:sub>2</jats:sub>than wild-type, whereas the C451- and C474-containing mutants were more sensitive than the quadruple mutant. Consistent with the HgCl<jats:sub>2</jats:sub>effect on transport, MTSEA-biotin only interacted with C451 and C474. These data indicate that C451 and C474 of hOCT2 reside in the aqueous milieu of the cleft and that interaction of Hg<jats:sup>2+</jats:sup>with these residues causes reduced TEA transport activity.</jats:p> |
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| author | Pelis, Ryan M., Dangprapai, Yodying, Wunz, Theresa M., Wright, Stephen H. |
| author_facet | Pelis, Ryan M., Dangprapai, Yodying, Wunz, Theresa M., Wright, Stephen H., Pelis, Ryan M., Dangprapai, Yodying, Wunz, Theresa M., Wright, Stephen H. |
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| description | <jats:p>Human organic cation transporter 2 (hOCT2) is essential for the renal tubular secretion of many toxic organic cations. Previously, of the cysteines (C437, C451, C470, and C474) that occur within transmembrane helices that comprise the hydrophilic cleft (proposed site of substrate binding), only C474 was accessible to maleimide-PEO<jats:sub>2</jats:sub>-biotin (hydrophilic thiol-reactive reagent), and covalent modification of this residue caused lower transport rates (Pelis RM, Zhang X, Dangprapai Y, Wright SH, J Biol Chem 281: 35272–35280, 2006). Thus it was hypothesized that the environmental contaminant Hg<jats:sup>2+</jats:sup>(as HgCl<jats:sub>2</jats:sub>) would interact with C474 to reduce hOCT2-mediated transport. Uptake of [<jats:sup>3</jats:sup>H]tetraethylammonium (TEA) into Chinese hamster ovary cells stably expressing hOCT2 was reduced in a concentration-dependent manner by HgCl<jats:sub>2</jats:sub>, with an IC<jats:sub>50</jats:sub>of 3.9 ± 0.11 μM. Treatment with 10 μM HgCl<jats:sub>2</jats:sub>caused a sixfold reduction in the maximal rate of TEA transport but did not alter the affinity of hOCT2 for TEA. To determine which cysteines interact with Hg<jats:sup>2+</jats:sup>, a mutant with all four cleft cysteines converted to alanines (quadruple mutant), and four variants of this mutant, each with an individual cysteine restored, were created. The quadruple mutant was less sensitive to HgCl<jats:sub>2</jats:sub>than wild-type, whereas the C451- and C474-containing mutants were more sensitive than the quadruple mutant. Consistent with the HgCl<jats:sub>2</jats:sub>effect on transport, MTSEA-biotin only interacted with C451 and C474. These data indicate that C451 and C474 of hOCT2 reside in the aqueous milieu of the cleft and that interaction of Hg<jats:sup>2+</jats:sup>with these residues causes reduced TEA transport activity.</jats:p> |
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| imprint | American Physiological Society, 2007 |
| imprint_str_mv | American Physiological Society, 2007 |
| institution | DE-Pl11, DE-Rs1, DE-105, DE-14, DE-Ch1, DE-L229, DE-D275, DE-Bn3, DE-Brt1, DE-Zwi2, DE-D161, DE-Gla1, DE-Zi4, DE-15 |
| issn | 1931-857X, 1522-1466 |
| issn_str_mv | 1931-857X, 1522-1466 |
| language | English |
| last_indexed | 2024-03-01T15:30:23.564Z |
| match_str | pelis2007inorganicmercuryinteractswithcysteineresiduesc451andc474ofhoct2toreduceitstransportactivity |
| mega_collection | American Physiological Society (CrossRef) |
| physical | F1583-F1591 |
| publishDate | 2007 |
| publishDateSort | 2007 |
| publisher | American Physiological Society |
| record_format | ai |
| recordtype | ai |
| series | American Journal of Physiology-Renal Physiology |
| source_id | 49 |
| spelling | Pelis, Ryan M. Dangprapai, Yodying Wunz, Theresa M. Wright, Stephen H. 1931-857X 1522-1466 American Physiological Society Physiology http://dx.doi.org/10.1152/ajprenal.00496.2006 <jats:p>Human organic cation transporter 2 (hOCT2) is essential for the renal tubular secretion of many toxic organic cations. Previously, of the cysteines (C437, C451, C470, and C474) that occur within transmembrane helices that comprise the hydrophilic cleft (proposed site of substrate binding), only C474 was accessible to maleimide-PEO<jats:sub>2</jats:sub>-biotin (hydrophilic thiol-reactive reagent), and covalent modification of this residue caused lower transport rates (Pelis RM, Zhang X, Dangprapai Y, Wright SH, J Biol Chem 281: 35272–35280, 2006). Thus it was hypothesized that the environmental contaminant Hg<jats:sup>2+</jats:sup>(as HgCl<jats:sub>2</jats:sub>) would interact with C474 to reduce hOCT2-mediated transport. Uptake of [<jats:sup>3</jats:sup>H]tetraethylammonium (TEA) into Chinese hamster ovary cells stably expressing hOCT2 was reduced in a concentration-dependent manner by HgCl<jats:sub>2</jats:sub>, with an IC<jats:sub>50</jats:sub>of 3.9 ± 0.11 μM. Treatment with 10 μM HgCl<jats:sub>2</jats:sub>caused a sixfold reduction in the maximal rate of TEA transport but did not alter the affinity of hOCT2 for TEA. To determine which cysteines interact with Hg<jats:sup>2+</jats:sup>, a mutant with all four cleft cysteines converted to alanines (quadruple mutant), and four variants of this mutant, each with an individual cysteine restored, were created. The quadruple mutant was less sensitive to HgCl<jats:sub>2</jats:sub>than wild-type, whereas the C451- and C474-containing mutants were more sensitive than the quadruple mutant. Consistent with the HgCl<jats:sub>2</jats:sub>effect on transport, MTSEA-biotin only interacted with C451 and C474. These data indicate that C451 and C474 of hOCT2 reside in the aqueous milieu of the cleft and that interaction of Hg<jats:sup>2+</jats:sup>with these residues causes reduced TEA transport activity.</jats:p> Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity American Journal of Physiology-Renal Physiology |
| spellingShingle | Pelis, Ryan M., Dangprapai, Yodying, Wunz, Theresa M., Wright, Stephen H., American Journal of Physiology-Renal Physiology, Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity, Physiology |
| title | Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity |
| title_full | Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity |
| title_fullStr | Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity |
| title_full_unstemmed | Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity |
| title_short | Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity |
| title_sort | inorganic mercury interacts with cysteine residues (c451 and c474) of hoct2 to reduce its transport activity |
| title_unstemmed | Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity |
| topic | Physiology |
| url | http://dx.doi.org/10.1152/ajprenal.00496.2006 |