Phosphorylation of serine 256 is required for cAMP-dependent regulatory exocytosis of the aquaporin-2 water channel

Abstract

The aquaporin-2 (AQP2) vasopressin water channel is translocated to the apical membrane upon vasopressin stimulation. Phosphorylation of serine 256 of AQP2 by cAMP-dependent protein kinase has been shown, but its relation to vasopressin-regulated translocation has not been elucidated. To address this question, wild type (WT) AQP2 and a mutant with alanine in place of serine 256 of AQP2 (S256A) were expressed in LLC-PK1 cells by electroporation. Measurements by a stopped-flow light-scattering method revealed that the osmotic water permeability (P(f)) of LLC-PK1 cells transfected with WT was 69.6 ± 6.5 μm/s (24.8 ± 2.2 μm/s for mock-transfected), and stimulation by 500 μM 8-(4-chlorophenylthio)-cAMP increased the P(f) by 85 ± 12%. When S256A AQP2 was transfected, the cAMP-dependent increase in the P(f) was only 8 ± 5%. After cAMP stimulation, the increase in surface expression of AQP2 determined by surface biotin labeling was 4 ± 10%, significantly less than that for WT (88 ± 5%). In addition, an in vive [32P]orthophosphate labeling assay demonstrated significant phosphorylation of WT AQP2 and only minimal phosphorylation of S256A AQP2 in LLC-PK1 cells. Our results indicated that serine 256 of AQP2 is necessary for regulatory exocytosis and that cAMP- responsive redistribution of AQP2 may be regulated by phosphorylation of AQP2.