Aquaporin-1 Channel Function Is Positively Regulated by Protein Kinase C

Wei Zhang,Edgar Zitron,Meike Hoömme,Lars Kihm,Christian Morath,Daniel Scherer,Stephan Hegge,Dierk Thomas,Claus P Schmitt,Martin Zeier,Hugo Katus,Christoph Karle,Vedat Schwenger

doi:10.1074/jbc.m703858200

Wei Zhang, Edgar Zitron + Show 11 more

Open Access

https://doi.org/10.1074/jbc.m703858200

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Abstract

Aquaporin-1 (AQP1) channels contribute to osmotically induced water transport in several organs including the kidney and serosal membranes such as the peritoneum and the pleura. In addition, AQP1 channels have been shown to conduct cationic currents upon stimulation by cyclic nucleotides. To date, the short term regulation of AQP1 function by other major intracellular signaling pathways has not been studied. In the present study, we therefore investigated the regulation of AQP1 by protein kinase C. AQP1 wild type channels were expressed in Xenopus oocytes. Water permeability was assessed by hypotonic challenges. Activation of protein kinase C (PKC) by 1-oleoyl-2-acetyl-sn-glycerol (OAG) induced a marked increase of AQP1-dependent water permeability. This regulation was abolished in mutated AQP1 channels lacking both consensus PKC phosphorylation sites Thr(157) and Thr(239) (termed AQP1 DeltaPKC). AQP1 cationic currents measured with double-electrode voltage clamp were markedly increased after pharmacological activation of PKC by either OAG or phorbol 12-myristate 13-acetate. Deletion of either Thr(157) or Thr(239) caused a marked attenuation of PKC-dependent current increases, and deletion of both phosphorylation sites in AQP1 DeltaPKC channels abolished the effect. In vitro phosphorylation studies with synthesized peptides corresponding to amino acids 154-168 and 236-250 revealed that both Thr(157) and Thr(239) are phosphorylated by PKC. Upon stimulation by cyclic nucleotides, AQP1 wild type currents exhibited a strong activation. This regulation was not affected after deletion of PKC phosphorylation sites in AQP1 DeltaPKC channels. In conclusion, this is the first study to show that PKC positively regulates both water permeability and ionic conductance of AQP1 channels. This new pathway of AQP1 regulation is independent of the previously described cyclic nucleotide pathway and may contribute to the PKC stimulation of AQP1-modulated processes such as endothelial permeability, angiogenesis, and urine concentration.

Highlights

We provide data demonstrating that this new pathway of AQP1 regulation is independent of the previously described cyclic nucleotide pathway
Molecular Basis of AQP1 Regulation by Protein Kinase C—A parallel effect of protein kinase C on ion currents and water permeability mediated by aquaporin-1 channels was observed
There is an interaction between those two signaling pathways. measurements of water permeability are limited by the AQP1-⌬PKC channels lacking functional PKC mechanical robustness of the cells that do not tolerate excessive phosphorylation sites were expressed in Xenopus oocytes, and volume increases

Summary

EXPERIMENTAL PROCEDURES

Solutions and Drug Administration—Two microelectrode voltage clamp measurements of Xenopus oocytes were performed in isotonic NaCl saline containing (in mM) 100 NaCl, 2 KCl, 4.3 MgCl, and 5 HEPES, pH 7.3. The respective threonin residue was membrane pellets were resuspended in 30 ␮l of 1ϫ SDS protein replaced by alanine to abolish phosphorylation at the respective loading buffer, incubated at 37 °C for 10 min, and electrophore- site. The blots were incubated sure to the hypo-osmotic solution, the volume of oocytes with AQP1 primary antibody (1:1000; Santa Cruz Biotechnol- expressing AQP1-⌬PKC channels increased to 109.8 Ϯ 1.1% of ogy, Heidelberg, Germany) in 3% milk at 4 °C overnight.

RESULTS

DISCUSSION