Constitutive endocytosis of NKCC2 in thick ascending limbs is dynamin dependent

Abstract

The Na/K/2Cl cotransporter (NKCC2) mediates NaCl absorption by the epithelial cells of the thick ascending limb (TAL). Trafficking into (exocytosis) and out (endocytosis) of the apical membrane regulates surface NKCC2 levels. Dynamin‐2 is a ubiquitous protein that mediates endocytosis in many epithelial cells. It is not known whether NKCC2 undergoes constitutive endocytosis and whether dynamin‐2 is involved. We hypothesized that constitutive endocytosis of NKCC2 is mediated by dynamin‐2. We measured surface NKCC2 and the rate of NKCC2 endocytosis by biotinylation and Western blot. First, we studied whether dynamin‐2 is expressed in TAL. We found that dynamin‐2 is expressed in rat medullary TAL suspensions as a single band at the predicted molecular weight of 100 kDa. In control TALs we observed constitutive NKCC2 endocytosis over 30 min that averaged 10.1 ± 0.8 a.u/min. We tested whether the dynamin inhibitor (Dynasore) inhibits constitutive NKCC2 endocytosis. In the presence of Dynasore (100 μM), the rate of constitutive NKCC2 endocytosis averaged 2.8 ± 0.6 a.u/min ( n = 6, p < 0.01 vs control), a 72% inhibition. Acute (20 min) blockade of endocytosis with Dynasore increased surface NKCC2 by 66 ± 26% (p<0.05). The total pool of NKCC2 was not changed by Dynasore. We concluded that constitutive NKCC2 endocytosis is mediated by dynamin‐2. These are the first data showing NKCC2 endocytosis and a role for dynamin in TALs.