Phosphorylation determines the calmodulin-mediated Ca2+ response and water permeability of AQP0.
In Xenopus oocytes, the water permeability of AQP0 (P(f)) increases with removal of external calcium, an effect that is mediated by cytoplasmic calmodulin (CaM) bound to the C terminus of AQP0. To investigate the effects of serine phosphorylation on CaM-mediated Ca(2+) regulation of P(f), we tested the effects of kinase activation, CaM inhibition, and a series of mutations in the C terminus CaM binding site. Calcium regulation of AQP0 P(f) manifests four distinct phenotypes: Group 1, with high P(f) upon removal of external Ca(2+) (wild-type, S229N, R233A, S235A, S235K, K238A, and R241E); Group 2, with high P(f) in elevated (5 mm) external Ca(2+) (S235D and R241A); Group 3, with high P(f) and no Ca(2+) regulation (S229D, S231N, S231D, S235N, and S235N/I236S); and Group 4, with low P(f) and no Ca(2+) regulation (protein kinase A and protein kinase C activators, S229D/S235D and S235N/I236S). Within each group, we tested whether CaM binding mediates the phenotype, as shown previously for wild-type AQP0. In the presence of calmidazolium, a CaM inhibitor, S235D showed high P(f) and no Ca(2+) regulation, suggesting that S235D still binds CaM. Contrarily, S229D showed a decrease in recruitment of CaM, suggesting that S229D is unable to bind CaM. Taken together, our results suggest a model in which CaM acts as an inhibitor of AQP0 P(f). CaM binding is associated with a low P(f) state, and a lack of CaM binding is associated with a high P(f) state. Pathological conditions of inappropriate phosphorylation or calcium/CaM regulation could induce P(f) changes contributing to the development of a cataract.
Department of Physiology and Biophysics, University of California, Irvine, CA 92697-4561, USA. kkalman@uci.edu
Katalin
K
Kalman
Karin L
KL
Németh-Cahalan
Alexandrine
A
Froger
James E
JE
Hall
D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry / Q000502:physiology*; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D010539:Permeability; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D014867:Water / Q000737:chemistry*; D014982:Xenopus laevis
D002147:Calmodulin; D014867:Water; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium