2Cr 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, immunoprecipi-tation 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 (xM): 70.3 ?9.8, MG132 (20 xM): 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.
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