cGMP induces degradation of NKCC2 in the thick ascending limb via the ubiquitin-proteasomal system.

The thick ascending limb of Henle's loop (TAL) reabsorbs NaCl via the apical Na-K-2Cl cotransporter (NKCC2). NKCC2 activity is regulated by surface NKCC2 levels. The second messenger cGMP decreases NKCC2 activity by decreasing surface NKCC2 levels. We found that surface NKCC2 undergoes constitutive degradation. Therefore, we hypothesized that cGMP decreases NKCC2 levels by increasing NKCC2 ubiquitination and proteasomal degradation. We measured surface NKCC2 levels by biotinylation of surface proteins, immunoprecipitation of NKCC2, and ubiquitin in TALs. First, we found that inhibition of proteasomal degradation blunts the cGMP-dependent decrease in surface NKCC2 levels [vehicle: 100%, db-cGMP (500 µM): 70.3 ± 9.8%, MG132 (20 µM): 97.7 ± 5.0%, and db-cGMP + MG132: 103.3 ± 3.4%, n = 5, P < 0.05]. We then found that cGMP decreased the internalized NKCC2 pool and that this effect was prevented by inhibition of the proteasome but not the lysosome. Finally, we found that NKCC2 is constitutively ubiquitinated in TALs and that cGMP enhances the rate of NKCC2 ubiquitination [vehicle: 59 ± 14% and db-cGMP (500 µM): 111 ± 25%, n = 5, P < 0.05]. We conclude that NKCC2 is constitutively ubiquitinated and that cGMP stimulates NKCC2 ubiquitination and proteasomal degradation. Our data suggest that the cGMP-induced NKCC2 ubiquitination and degradation may contribute to the cGMP-induced decrease of the NKCC2-dependent NaCl reabsorption in TALs.