Aquaporin-1 Channel Function Is Positively Regulated by Protein Kinase C
2007; Elsevier BV; Volume: 282; Issue: 29 Linguagem: Inglês
10.1074/jbc.m703858200
ISSN1083-351X
AutoresWei Zhang, Edgar Zitron, Meike Hoömme, Lars Kihm, Christian Morath, D. Scherer, Stephan Hegge, Dierk Thomas, Claus Peter Schmitt, Martin Zeier, Hugo A. Katus, Christoph A. Karle, Vedat Schwenger,
Tópico(s)Magnesium in Health and Disease
ResumoAquaporin-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 ΔPKC). 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 ΔPKC 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 ΔPKC 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. 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 ΔPKC). 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 ΔPKC 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 ΔPKC 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. The aquaporins (AQPs) 3The abbreviations used are: AQP, aquaporin; PKC, protein kinase C; OAG, 1-oleoyl-2-acetyl-sn-glycerol; PMA, phorbol 12-myristate 13-acetate; MOPS, 4-morpholinepropanesulfonic acid; IBMX, isobutylmethylxanthine; CN, cyclic nucleotide; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. 3The abbreviations used are: AQP, aquaporin; PKC, protein kinase C; OAG, 1-oleoyl-2-acetyl-sn-glycerol; PMA, phorbol 12-myristate 13-acetate; MOPS, 4-morpholinepropanesulfonic acid; IBMX, isobutylmethylxanthine; CN, cyclic nucleotide; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. are a family of integral membrane proteins that are widely expressed in bacteria, plants, and animals (1Agre P. King L.S. Yasui M. Guggino W.B. Ottersen O.P. Fujiyoshi Y. Engel A. Nielsen S. J. Physiol. 2002; 542: 3-16Crossref PubMed Scopus (891) Google Scholar). Their main physiological function is rapid transmembrane transport of water driven by osmotic gradients. In addition to water transport, some aquaporins also conduct ion currents. In humans, 11 aquaporins have been found so far (AQP0–AQP10). Of these, AQP1 is the predominant and least specialized subtype. It plays a major role in constitutive water transport through the membranes of several cell types including endothelial cells, red blood cells, and renal proximal tubule cells (1Agre P. King L.S. Yasui M. Guggino W.B. Ottersen O.P. Fujiyoshi Y. Engel A. Nielsen S. J. Physiol. 2002; 542: 3-16Crossref PubMed Scopus (891) Google Scholar). It has been shown that AQP1 may also function as a cyclic nucleotide-gated cation channel that is activated mainly by cGMP and indirectly also by cAMP (2Anthony T.L. Brooks H.L. Boassa D. Leonov S. Yanochko G.M. Regan J.W. Yool A.W. Mol. Pharmacol. 2000; 57: 576-588Crossref PubMed Scopus (144) Google Scholar, 3Yool A.J. Stamer D. Regan J.W. Science. 1996; 273: 1216-1218Crossref PubMed Scopus (141) Google Scholar). Recently, ion currents of native aquaporins were confirmed in choroid plexus epithelium and shown to modulate fluid transport of those cells (4Boassa D. Stamer W.D. Yool A.J. J. Neurosci. 2006; 26: 7811-7819Crossref PubMed Scopus (79) Google Scholar). Because of its significance for determining endothelial water permeability, AQP1 has been found to play a major physiological role in the peritoneal membrane (5Devuyst O. Ni J. Verbavatz J. Biol. Cell. 2005; 97: 667-673Crossref PubMed Scopus (18) Google Scholar). Subsequently, it has been shown to be the molecular correlate of the “ultrasmall pore” responsible for transcellular water permeability during peritoneal dialysis (6Ni J. Verbavatz J.M. Rippe A. Boisde I. Moulin P. Rippe B. Verkman A.S. Devuyst O. Kidney Int. 2006; 69: 1518-1525Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Furthermore, recent reports have demonstrated a previously unexpected role of AQP1 in cell migration (7Sadoun S. Papadopoulos M.C. Hara-Chikuma M. Verkman A.S. Nature. 2005; 434: 786-791Crossref PubMed Scopus (632) Google Scholar, 8Hara-Chikuma M. Verkman A.S. J. Am. Soc. Nephrol. 2006; 17: 39-45Crossref PubMed Scopus (142) Google Scholar, 9Belge H. Devuyst O. Nephrol. Dial. Transplant. 2006; 21: 2069-2071Crossref PubMed Scopus (10) Google Scholar). Endothelial cells lacking AQP1 have impaired cell motility because of reduced formation of lamellipodia, resulting in impaired angiogenesis (7Sadoun S. Papadopoulos M.C. Hara-Chikuma M. Verkman A.S. Nature. 2005; 434: 786-791Crossref PubMed Scopus (632) Google Scholar). Interestingly, AQP1 was also found to be essential for normal migration of renal proximal tubule cells and restitution of renal injury (8Hara-Chikuma M. Verkman A.S. J. Am. Soc. Nephrol. 2006; 17: 39-45Crossref PubMed Scopus (142) Google Scholar, 9Belge H. Devuyst O. Nephrol. Dial. Transplant. 2006; 21: 2069-2071Crossref PubMed Scopus (10) Google Scholar). It is well documented that aquaporin channels may be subject to intense short term regulation by cellular signal cascades, with the most recognized example being the cAMP-dependent protein kinase-dependent regulation of AQP-2 in the kidney collecting duct (1Agre P. King L.S. Yasui M. Guggino W.B. Ottersen O.P. Fujiyoshi Y. Engel A. Nielsen S. J. Physiol. 2002; 542: 3-16Crossref PubMed Scopus (891) Google Scholar, 10Verkman A.S. J. Cell Sci. 2002; 118: 3225-3232Crossref Scopus (481) Google Scholar). Interestingly, however, little is known to date about the short term regulation of AQP1, because this channel was originally considered to be constitutively open (1Agre P. King L.S. Yasui M. Guggino W.B. Ottersen O.P. Fujiyoshi Y. Engel A. Nielsen S. J. Physiol. 2002; 542: 3-16Crossref PubMed Scopus (891) Google Scholar). The role of signaling pathways apart from cyclic nucleotides in the regulation of AQP1 has not been investigated to date. The protein kinase C (PKC) system is a key component of intracellular signaling, and PKC activation is a central pathway downstream of Gq/11 coupled receptors such as adrenergic α1 receptors and muscarinergic M1 receptors (11Rockman H.A. Koch W.J. Lefkowitz R.J. Nature. 2002; 415: 206-212Crossref PubMed Scopus (771) Google Scholar, 12Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2347) Google Scholar). PKC has been shown to be involved in the regulation of structurally diverse membrane proteins, particularly ion channels (13Karle C.A. Zitron E. Zhang W. Wendt-Nordahl G. Kathoöfer S. Thomas D. Gut B. Scholz E. Vahl C.F. Katus H.A. Kiehn J. Circulation. 2002; 106: 1493-1499Crossref PubMed Scopus (61) Google Scholar, 14Kiesecker C. Zitron E. Scherer D. Lueck S. Bloehs R. Scholz E.P. Pirot M. Kathofer S. Thomas D. Kreye V.A. Kiehn J. Borst M.M. Katus H.A. Schoels W. Karle C.A. J. Mol. Med. 2006; 84: 46-56Crossref PubMed Scopus (26) Google Scholar). Recently, it has been demonstrated that PKC mediates the dopamine-dependent down-regulation of renal aquaporin-4 channels (15Zelenina M. Zelenin S. Bondar A.A. Brismar H. Aperia A. Am. J. Physiol. 2002; 283 (–F318): F309Crossref PubMed Scopus (176) Google Scholar, 16Han Z. Wax M.B. Patil R.V. J. Biol. Chem. 1998; 273: 6001-6004Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Notably, a major role of PKC in the regulation of endothelial permeability, angiogenesis, and water transport in the proximal renal tubule has been observed in physiological studies (17Chang Y.S. Munn L.L. Hillsley M.V. Dull R.O. Yuan J. Lakshminarayanan S. Gardner T.W. Jain R.K. Tarbell J.M. Microvasc. Res. 2000; 59: 265-277Crossref PubMed Scopus (110) Google Scholar, 18Siflinger-Birnboim A. Johnson A. Am. J. Physiol. 2003; 284 (–L451): L435Crossref PubMed Scopus (70) Google Scholar, 19Dang L. Seale J.P. Qu X. Clin. Exp. Pharmacol. Physiol. 2005; 32: 771-776Crossref PubMed Scopus (30) Google Scholar, 20Bokhari S.M. Zhou L. Karasek M.A. Paturi S.G. Chaudhuri V. J. Investig. Dermatol. 2006; 126: 460-467Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 21Wang A. Nomura M. Patan S. Ware J.A. Circ. Res. 2002; 90: 609-616Crossref PubMed Scopus (78) Google Scholar, 22Ware J.A. Simons M. Angiogenesis and Cardiovascular Disease. 1999; (, pp. , Oxford University Press, Oxford): 30-59Google Scholar, 23Yao L. Huang D.Y. Pfaff I.L. Nie X. Leitges M. Vallon V. Am. J. Physiol. 2004; 287 (–F304): F299Crossref PubMed Scopus (31) Google Scholar, 24Garcia N.H. Garvin J.L. J. Clin. Investig. 1994; 93: 2572-2577Crossref PubMed Scopus (77) Google Scholar). Here, we show that PKC positively regulates AQP1 channels expressed in Xenopus oocytes. PKC activation induces an increase of both water permeability and ion currents mediated by AQP1. On the molecular level, the effect depends on both consensus PKC phosphorylation sites Thr157 and Thr239 with approximately half of the overall effect being attributable to each site. 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. Ca2+ was omitted from the bath solution to minimize Ca2+-dependent Cl– currents in the oocytes as described by Barish (25Barish M.E. J. Physiol. 1983; 342: 309-325Crossref PubMed Scopus (485) Google Scholar). Current and voltage electrodes were filled with 3 m KCl solution. OAG (1-oleoyl-2-acetyl-sn-glycerol), staurosporine, and chelerythrine (all from Calbiochem) were dissolved in Me2SO to stock solutions of 10 mm and stored at –20 °C. Aliquots of the stock solutions were diluted to the desired concentration with the bath solution on the day of experiments. The maximum concentration of Me2SO in the bath had no effects on the measured currents. All of the measurements were performed at room temperature (20 °C). Electrophysiology and Data Analysis—The two microlelectrode voltage clamp configuration was used to record currents from Xenopus laevis oocytes as published previously (13Karle C.A. Zitron E. Zhang W. Wendt-Nordahl G. Kathoöfer S. Thomas D. Gut B. Scholz E. Vahl C.F. Katus H.A. Kiehn J. Circulation. 2002; 106: 1493-1499Crossref PubMed Scopus (61) Google Scholar). Pclamp software (Axon instruments) was used for generation of the voltage pulse protocols and for data acquisition. The statistical data are presented as the means ± S.E. Statistical significance was evaluated using Student’s t test for pairwise comparison and analysis of variance for comparison of several groups of data. The differences were considered to be significant if the p value was <0.05 and highly significant if the p value was <0.01. In the figures, significance levels are indicated by asterisks (*,p < 0.05; **, p < 0.01; ***, p < 0.001). All of the results were obtained in oocytes from at least three different batches. Analysis of Osmotic Swelling—Water permeability assays at room temperature (20 °C) were initiated with the transfer of AQP1-expressing or control oocytes at time zero from 200 mosm ND96 saline into 100 mm mosm ND96 diluted with distilled water as described by Anthony et al. (2Anthony T.L. Brooks H.L. Boassa D. Leonov S. Yanochko G.M. Regan J.W. Yool A.W. Mol. Pharmacol. 2000; 57: 576-588Crossref PubMed Scopus (144) Google Scholar). After the transfer of the cell, the images were recorded at intervals of 30 s for 5 min. Volume changes were analyzed with ImageJ on the basis of digital images captured every 30 s with a digital Nikon Coolpix 5400 camera on a Nikon SMZ 1500 stereomicroscope. The data were analyzed as proportional change in volume and normalized to the initial volume at time zero. All of the results were obtained in oocytes from at least three different batches. Site-directed Mutagenesis—Sequence analysis revealed two potential PKC phosphorylation residues at positions Thr157 and Thr239. To eliminate PKC-mediated phosphorylation at these positions, the two threonine residues were replaced with alanine. This resulted in the mutated channels T157A and T239A. Through repetitive mutagenesis, both point mutations were introduced into a single clone termed AQP1-ΔPKC. Point mutations were generated with the QuikChange protocol (Stratagene, La Jolla, CA). We used the primers CGTGCTGGCTACTG CCGACCGGAGGCG (forward) and CGCCTCCGGTCGGCAGTAGCCAGCACG (reverse) to introduce mutation T157A, and for mutation T239A, we used the primers CAGCAGTGACCTCGCAGACCGCGTGAAG (forward) and CTTCACGCGGTCTGC GAGGTCACTGCTG (reverse). All of the cDNAs used in this study were verified by complete sequencing (Sequence Laboratories Goöttingen GmBH). Expression of AQP1 Channels in Xenopus Oocytes—The human AQP1 wild type clone was a kind gift from Peter Agre (Baltimore, MD). cRNA was prepared from the corresponding cDNA (AQP1 WT, AQP1-T157A, AQP1-T239A, and AQP1-ΔPKC) with T3 RNA polymerase after linearization with SmaI using the mMESSAGE mMACHINE in vitro transcription kit (Ambion, Austin, TX). AQP1 cRNA of wild type and mutant channels (always at the same concentration of 20 ng/μl) was injected into stage V and VI defolliculated oocytes using a Nanoject automatic injector (Drummond, Broomall, PA). The volume of injected cRNA solution was 46 nl/oocyte. The measurements were performed 2–3 days after expression. Peptide Synthesis—Peptides corresponding to amino acid sequences 154–168 (P154–168, LATTDRRRRDLGGSG, with a single conservative replacement of alanine by glycine at residue 168 to improve solubility) and to 236–250 (P236–250, SDLTDRVKVWTSGEV, with a single conservative replacement of glutamine by glutamate at residue 249 to improve solubility) were obtained from Sigma-Genosys. Homologous peptides containing threonine-to-alanine mutations at residues 157 and 239, respectively, were obtained from the same source and referred to as P154–168(T157A) and P236–250(T239A), respectively. In Vitro Phosphorylation Assays—10 μl (5 μg) of peptide and 25 ng of active PKC (Upstate Cell Signaling Solutions, Lake Placid, NY) were incubated at 30 °C for 15 min in the presence of ADBII, which was composed of 20 mm MOPS, pH 7.2, 25 mm β-glycerol phosphate, 1 mm sodium orthovanadate, 1 mm dithiothreitol, 1 mm CaCl2), 10 μl of lipid activator (1 mg/ml phosphatidylserine, 0.1 mg/ml diacylglycerol, 0.3% Triton X-100, 1 mm dithiothreitol), and 20 μCi of [γ-32P]ATP (GE Healthcare, Munich, Germany) including 15 mm MgCl2 and 200 μm unlabeled ATP in ADBII. The reactions were stopped by adding 4× SDS sample buffer, and the mixture was boiled for 5 min. The peptides were resolved by 20% SDS-polyacrylamide gel electrophoresis. The gels were dried and subjected to autoradiography using Hyperfilm MS film (GE Healthcare, Munich, Germany). The bands were quantified densitometrically with ImageJ on the basis of digitalized images. Oocyte Membrane Isolation and Western Blotting Analysis—Noninjected and AQP1-cRNA-injected (200 ng/μl) Xenopus oocytes were used to isolate crude membrane fractions. 23 oocytes were incubated in 1000 μl of ice-cold hypotonic phosphate buffer (7.5 mm Na2HPO4, 1 mm EDTA, pH 7.5, plus a protease inhibitor mixture tablet (Roche Applied Science)) for 5 min. All of the steps were done at 4 °C. The samples were vortexed and pipetted repeatedly to lyse the oocytes. The yolk and cellular debris were pelleted at 500 × g for 5 min. The membranes were then pelleted at 48,000 × g for 30 min. The membrane pellets were resuspended in 30 μl of 1× SDS protein loading buffer, incubated at 37 °C for 10 min, and electrophoresed on a 12% polyacrylamide SDS gel. Afterward they were electrophoretically transferred onto nitrocellulose filters (Whatman, Dasel, Germany). The blots were blocked for 1 h with TBS-T (10 mm Tris, pH 7.4, 138 mm NaCl, 0.05% Tween 20) containing 3% nonfat dry milk. The blots were then incubated with AQP1 primary antibody (1:1000; Santa Cruz Biotechnology, Heidelberg, Germany) in 3% milk at 4 °C overnight. The blots were washed three times for 15 min with TBS-T and incubated with horseradish peroxidase-conjugated anti-mouse secondary antibody (1:2000) (Cell Signaling Technology, Frankfurt, Germany) for 1 h at room temperature and were washed again three times. After washing, immune complexes were visualized using enhanced chemiluminescence (ECL; GE Healthcare, Munich, Germany). Protein Kinase C Activation Increases Water Permeability of Aquaporin-1 Channels—Human AQP1 channels were expressed heterologously in Xenopus oocytes to allow measurement of water permeability and ion conductance. First, water permeability of the membrane of those cells was assessed by hypotonic swelling experiments according to Anthony et al. (2Anthony T.L. Brooks H.L. Boassa D. Leonov S. Yanochko G.M. Regan J.W. Yool A.W. Mol. Pharmacol. 2000; 57: 576-588Crossref PubMed Scopus (144) Google Scholar). The duration of these experiments had to be limited to 5 min (as in other studies (2Anthony T.L. Brooks H.L. Boassa D. Leonov S. Yanochko G.M. Regan J.W. Yool A.W. Mol. Pharmacol. 2000; 57: 576-588Crossref PubMed Scopus (144) Google Scholar)) to avoid cell destruction as a consequence of the volume increases. To activate PKC in a group of experiments, specific PKC activator OAG (a synthetic and more stable analogue of the physiological activator diacylglycerol) was added to the hypo-osmotic solution at a concentration of 10 μm. No preincubation of cells in OAG was performed. Summary data of the time course of volume change in those cells is plotted in Fig. 1A. Within 5 min, cell volume increased to 131.1 ± 2.6% (n = 8; p < 0.01 in comparison with control experiments without OAG). Without addition of OAG to the hypoosmotic solution, cells expressing AQP1 exhibited a smaller volume increase to 115.3 ± 1.9% (n = 6; Fig. 1A; significantly different from the effect of OAG with p < 0.01). Cells that did not express AQP1 did not show any relevant volume change (100.3 ± 0.3%; n = 7; Fig. 1A; p < 0.001 in comparison with cells expressing AQP1). Regulation of Aquaporin-1 Water Permeability by PKC Is Abolished in AQP1-ΔPKC Channels Lacking Phosphorylation Sites Thr157 and Thr239—Sequence analysis of AQP1 channel amino acid sequence revealed two consensus sites for PKC phosphorylation at Thr157 and Thr239, respectively. To elucidate the functional role of those sites, we modified channel subunits by site-directed mutagenesis. The respective threonin residue was replaced by alanine to abolish phosphorylation at the respective site. Channels lacking both PKC phosphorylation sites (i.e. including mutations T157A and T239A) were termed AQP1-ΔPKC and measured under conditions analogous to those described for wild type channels. The results are summarized in Fig. 1B. After 5 min of exposure to the hypo-osmotic solution, the volume of oocytes expressing AQP1-ΔPKC channels increased to 109.8 ± 1.1% of the respective initial values (Fig. 1B, left column, n = 5). Then experiments were repeated, and OAG (10 μm) was added to the hypo-osmotic solution to activate PKC. In contrast to the effect observed in AQP1 wt channels, volume increase was not enhanced by PKC activation in AQP1-ΔPKC channels. After the observation period of 5 min, the volume of those cells increased to 110.4 ± 0.6% (Fig. 1B, right column, n = 5), i.e. values without significant difference to those observed without application of OAG (p ≫ 0.05). Therefore, we concluded that inactivation of both PKC phosphorylation sites abolishes the positive regulation of AQP1 water permeability by protein kinase C. Protein Kinase C Activation Increases Aquaporin-1 Ion Currents—It has been demonstrated previously that AQP1 channels also conduct ions and that this conductance is activated by an increase in intracellular cyclic nucleotide levels, particularly cGMP and potentially also cAMP (2Anthony T.L. Brooks H.L. Boassa D. Leonov S. Yanochko G.M. Regan J.W. Yool A.W. Mol. Pharmacol. 2000; 57: 576-588Crossref PubMed Scopus (144) Google Scholar, 3Yool A.J. Stamer D. Regan J.W. Science. 1996; 273: 1216-1218Crossref PubMed Scopus (141) Google Scholar). Hence, we examined whether the PKC system also regulates AQP1 ion currents. A standardized voltage protocol was used to elicit AQP1 currents in Xenopus oocytes heterologously expressing these channels. From a holding potential of –30 mV that is close to the reversal potential of AQP1 currents in low K+ solution, voltage steps to potentials from –110 mV to +60 mV (400 ms each) were applied. After recording a measurement under control conditions, phorbol ester PMA (100 nm) was perfused into the bath for 30 min to activate PKC. In contrast to the water permeability measurements, the electrophysiological experiments did not affect the integrity of the cells and could therefore be extended to an observation period of 30 min (instead of 5 min) that allowed a longer observation of the time courses. Measurements were recorded at intervals of 5 min until the end of the observation period. Outward current amplitudes during the step to +60 mV were determined and compared with quantify effects. Typical recordings under control conditions and after 30 min of exposure to PMA are shown in Fig. 2. Under control conditions, the cells merely exhibited small currents (Fig. 2A) with a reversal potential of approximately –30 mV and lack of inward or outward rectification (Fig. 2C). Application of PMA induced a marked increase of currents (Fig. 2B) without affecting reversal potential and rectification (Fig. 2C). During observation of the PMA effect, the currents increased exponentially and reached a plateau after ∼20 min (Fig. 2D). Overall, a relative increase of current amplitudes by 102.5 ± 4.1% was observed (Fig. 2E; n = 10; p < 0.001 in comparison with control experiments). Control experiments were performed with oocytes that did not express AQP1. Those cells exhibited a merely small current increase (Fig. 2, D and E; n = 7; 14.2 ± 4.0%). For additional control experiments, oocytes expressing AQP1 were monitored for 30 min under identical conditions, but without the addition of PMA to the bath solution. Again, only a small current increase was observed in those cells (Fig. 2, D and E; n = 9, 17.1 ± 2.2%). PMA is a well established and highly potent activator of PKC, but it is less specific than OAG. Therefore, we repeated the experiments using OAG instead of PMA to further confirm the role of PKC in the observed effect. Because OAG is less potent than PMA, a higher concentration of OAG had to be applied (10 μm) as already used in previous studies from our laboratory (13Karle C.A. Zitron E. Zhang W. Wendt-Nordahl G. Kathoöfer S. Thomas D. Gut B. Scholz E. Vahl C.F. Katus H.A. Kiehn J. Circulation. 2002; 106: 1493-1499Crossref PubMed Scopus (61) Google Scholar). The experiments were performed as described for PMA. The results are shown in Fig. 3. OAG also induced a marked current increase without affecting biophysical current characteristics (Fig. 3, A–C). Time course of effect was comparable with that seen with PMA (Fig. 3D). Overall, currents increased by 117.9 ± 3.2% (Fig. 3E; n = 12; p < 0.001 in comparison with control experiments). Co-application of PKC inhibitor chelerythrine (10 μm) suppressed the effect almost completely, resulting in a relative current increase by merely 35.6 ± 3.2% (Fig. 3, D and E; n = 9). Control experiments either without exposure to OAG or without expression of AQP1 yielded current increases of 17.1 ± 2.2% (n = 9) and 21.9 ± 3.6% (n = 7), respectively (Fig. 3, D and E). The small current increases in all of the control experiments described in this study were stable and similar effects of statistical significance when compared with the respective initial values (p < 0.01 each). Their relative amplitude was within the range of nonspecific current increases that are commonly observed in the Xenopus oocyte expression system (26Zitron E. Kiesecker C. Luöck S. Kathoöfer S. Thomas D. Kreye V. Kiehn J. Katus H.A. Schoels W. Karle C.A. Cardiovasc. Res. 2004; 63: 520-527Crossref PubMed Scopus (42) Google Scholar, 27Zitron E. Scholz E.P. Owen R. Luck S. Kiesecker C. Thomas D. Kathofer S. Niroomand F. Kiehn J. Kreye V.A. Katus H.A. Schoels W. Karle C.A. Circulation. 2005; 111: 835-838Crossref PubMed Scopus (80) Google Scholar, 28Witchel H.J. Milnes J.T. Mitcheson J.S. Hancox J.C. J. Pharmacol. Toxicol. Methods. 2002; 48: 65-80Crossref PubMed Scopus (109) Google Scholar). Sets of corresponding control experiments are shown in each figure together with the observed effects (Figs. 2, D and E; 3, D and E; 4, D and E; and 5, D and E).FIGURE 4Phosphorylation sites Thr157 and Thr239 are essential for protein kinase C regulation of aquaporin-1 channels. Aquaporin-1 channels exhibit two PKC phosphorylation sites at Thr157 and Thr239, respectively. Mutated AQP1 channels with one inactivated site (AQP1-T157A and AQP1-T239A) or with both sites inactivated (AQP1-ΔPKC) were measured under conditions identical to those described for AQP1 wt channels. PKC was activated by application of OAG (10 μm). AQP1-ΔPKC currents at base line were comparable with those of AQP1 wt and exhibited the same biophysical characteristics. Western blots of cells expressing AQP1 wt and mutant channels demonstrated identical patterns in line with those reported from the literature (2Anthony T.L. Brooks H.L. Boassa D. Leonov S. Yanochko G.M. Regan J.W. Yool A.W. Mol. Pharmacol. 2000; 57: 576-588Crossref PubMed Scopus (144) Google Scholar, 3Yool A.J. Stamer D. Regan J.W. Science. 1996; 273: 1216-1218Crossref PubMed Scopus (141) Google Scholar) (shown as inset in D; results reproduced in two experiments). The position of the 28-kDa band is marked by the black arrow, and the white arrow shows the running direction of the gel. Noninjected oocytes were used as controls. Functionally, however, AQP1 mutants exhibited a markedly attenuated response to OAG (A–C). Time course of measurements is plotted in D. For comparison, the effect observed in wild type (wt) channels is included. Summary data after 30 min are shown in E. Inactivation of either of the two sites reduced PKC effects to approximately half of the values observed in wild type channels with 78.7% in T157A and 78.6% in T239A (n = 9 each; p < 0.001 each). In AQP1-ΔPKC channels lacking both sites, the effect was almost absent, with values approaching those of the control experiments (41%; n = 7; p < 0.05 compared with T157A and T239A). Control experiments performed with all clones yielded results comparable with the control experiments with wild type channels and with those with oocytes that did not express AQP1. In F, results of the in vitro phosphorylation assays of peptides homologous to the phosphorylation sites and the respective mutants are displayed. The autoradiograph is shown in the inset (results were reproduced in two experiments). The relative intensity of the corresponding radiographic band is shown as column graph. Wild type sequence peptides P154–168 (containing Thr157) and P236–250 (containing Thr239) were subject to intense p
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