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

Wei Zhang¹, Edgar Zitron¹², Meike Hömme, Lars Kihm^¶, Christian Morath^¶, Daniel Scherer, Stephan Hegge^||, Dierk Thomas, Claus P. Schmitt, Martin Zeier^¶, Hugo Katus, Christoph Karle¹, , and Vedat Schwenger^¶1

Departments of Cardiology, Paediatrics, ^¶Nephrology, and ^||Parasitology, University Hospital Heidelberg, D-69120 Heidelberg, Germany

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 Thr¹⁵⁷ and Thr²³⁹ (termed AQP1 PKC). 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 Thr¹⁵⁷ or Thr²³⁹ caused a marked attenuation of PKC-dependent current increases, and deletion of both phosphorylation sites in AQP1 PKC channels abolished the effect. In vitro phosphorylation studies with synthesized peptides corresponding to amino acids 154–168 and 236–250 revealed that both Thr¹⁵⁷ and Thr²³⁹ 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 PKC 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.

Received for publication May 10, 1007.

^* This work was supported by Deutsche Forschungsgemeinschaft Project Ka 1714/1-2 (to C. K.) and Forschungsgemeinschaft Project TH 1120/1-1 (to D. T.), University of Heidelberg Postdoctoral Grant of the Medical Faculty (to E. Z.), Deutsche Stiftung für Herzforschung Project F/10/03 (to D. T.), and Deutsche Forschungsgemeinschaft SFB 544 and BMBF-BioFuture Program (to F. F. and S. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Cardiology, Medical University Hospital Heidelberg, Im Neuenheimer Feld 410, D-69120 Heidelberg, Germany. Tel.: 49-6221-568476; Fax: 49-6221-565515; E-mail: edgar.zitron{at}med.uni-heidelberg.de.