CAMP-DEPENDENT PHOSPHORYLATION STIMULATES WATER PERMEABILITY OF AQUAPORIN-COLLECTING DUCT WATER CHANNEL PROTEIN EXPRESSED IN XENOPUS OOCYTES

Among water channel proteins (aquaporins), aquaporin-collecting duct ( AQP-CD) is the vasopressin-regulated water channel. Vasopressin causes cAMP production in the renal collecting duct cells, and this is belie ved to lead to exocytic insertion of water channel into the apical mem brane (shuttle hypothesis). AQP-CD contains a consensus sequence for c AMP-dependent protein kinase, residues at positions 253-256 (Arg-Arg-G ln-Ser). To determine the role of this site, Ser-256 was substituted f or Ala, Leu, Thr, Asp, or Glu by site-directed mutagenesis. In Xenopus oocytes injected with wild-type or mutated AQP-CD cRNAs, osmotic wate r permeability (Pf) was 4.8-7.7 times higher than Pf of water-injected oocytes. Incubation with cAMP plus forskolin or direct cAMP injection into the oocytes increased Pf of wildtype, but not mutated, AQP-CD-ex pressing oocytes, whereas the amounts of AQP-CD expression were simila r in wild and mutated types as identified by Western blot analysis. In vitro phosphorylation studies of AQP-CD proteins expressed in oocyte showed that cAMP dependent protein kinase phosphorylated wildtype, but not mutated, AQP-CD proteins. Phosphoamino acid analysis revealed tha t this phosphorylation occurred at the serine residue, Moreover, phosp horylation of AQP-CD protein in intact rat kidney medulla tissues was stimulated by incubation with cAMP. Our data suggest that cAMP stimula tes water permeability of AQP-CD by phosphorylation. This process may contribute to the vasopressin-regulated water permeability of collecti ng duct in addition to the apical insertion of AQP-CD by exocytosis.