Dynamin2, Clathrin, and Lipid Rafts Mediate Endocytosis of the Apical Na/K/2Cl Cotransporter NKCC2 in Thick Ascending Limbs

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.