| Autor: |
Ares GR; Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Wayne State University, Detroit, Michigan 48202, USA., Ortiz PA |
| Jazyk: |
angličtina |
| Zdroj: |
The Journal of biological chemistry [J Biol Chem] 2012 Nov 02; Vol. 287 (45), pp. 37824-34. Date of Electronic Publication: 2012 Sep 12. |
| DOI: |
10.1074/jbc.M112.386425 |
| Abstrakt: |
Steady-state surface levels of the apical Na/K/2Cl cotransporter NKCC2 regulate NaCl reabsorption by epithelial cells of the renal thick ascending limb (THAL). We reported that constitutive endocytosis of NKCC2 controls NaCl absorption in native THALs; however, the pathways involved in NKCC2 endocytosis are unknown. We hypothesized that NKCC2 endocytosis at the apical surface depends on dynamin-2 and clathrin. Measurements of steady-state surface NKCC2 and the rate of NKCC2 endocytosis in freshly isolated rat THALs showed that inhibition of endogenous dynamin-2 with dynasore blunted NKCC2 endocytosis by 56 ± 11% and increased steady-state surface NKCC2 by 67 ± 27% (p < 0.05). Expression of the dominant negative Dyn2K44A in THALs slowed the rate of NKCC2 endocytosis by 38 ± 8% and increased steady-state surface NKCC2 by 37 ± 8%, without changing total NKCC2 expression. Inhibition of clathrin-mediated endocytosis with chlorpromazine blunted NKCC2 endocytosis by 54 ± 6%, while preventing clathrin from interacting with synaptojanin also blunted NKCC2 endocytosis by 52 ± 5%. Disruption of lipid rafts blunted NKCC2 endocytosis by 39 ± 4% and silencing caveolin-1 by 29 ± 4%. Simultaneous inhibition of clathrin- and lipid raft-mediated endocytosis completely blocked NKCC2 internalization. We concluded that dynamin-2, clathrin, and lipid rafts mediate NKCC2 endocytosis and maintain steady-state apical surface NKCC2 in native THALs. These are the first data identifying the endocytic pathway for apical NKCC2 endocytosis. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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