The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family. The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family. Hydrogen peroxide (H2O2) 2The abbreviations used are: H2O2, hydrogen peroxide; AQP, aquaporin; CM-H2DCFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester; ROS, reactive oxygen species; TIP, tonoplast intrinsic protein; wt, wild type; MES, 4-morpholineethanesulfonic acid. belongs to the group of reactive oxygen species (ROS). ROS are generated in a number of key metabolic processes in cells like the electron transport chain in the inner mitochondrial membrane (1Møller I.M. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 561-591Crossref PubMed Scopus (1337) Google Scholar) and, specific for plants, the chloroplast thylakoid membrane (2Foyer C.H. Noctor G. Physiol. Plant. 2003; 119: 355-364Crossref Scopus (1061) Google Scholar). Because ROS can potentially damage proteins, lipids, and nucleic acids, cells have a number of ROS-scavenging systems that are able to remove these molecules and to maintain a relatively low and constant ROS concentration (3Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine.3rd Ed. Oxford University Press, New York1999: 105-245Google Scholar). However, ROS are also intermediates in various signal transduction pathways and have been shown to initiate responses to various stresses and disorders (for recent reviews, see Refs. 4Ardanaz N. Pagano P.J. Exp. Biol. Med. (Maywood). 2006; 231: 237-251Crossref PubMed Scopus (198) Google Scholar and 5Cai H. Cardiovasc. Res. 2005; 6: 26-36Crossref Scopus (469) Google Scholar). Arabidopsis mutants lacking an NADPH oxidase were not able to respond adequately to potassium deficiency (6Shin R. Schachtman D.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8827-8832Crossref PubMed Scopus (460) Google Scholar) and were impaired in stomatal closure (7Bright J. Desikan R. Hancock J.T. Weir I.S. Neill S.J. Plant J. 2006; 45: 113-122Crossref PubMed Scopus (834) Google Scholar), providing genetic evidence for a role of NADPH oxidase in signaling. ROS are interconvertible molecules including singlet oxygen, superoxide, hydroxyl radical, and H2O2.H2O2 has a distinctive set of features compared with other ROS. (i) It is not charged, (ii) it is not a radical, (iii) it possesses an intermediate oxidation number, (iv) it is relatively stable under physiological conditions, and (v) catalase can disproportionate it into water and molecular oxygen without the expense of reduction equivalents. Although substantial progress has been made regarding the formation and scavenging of ROS, little is known about their transport from the site of origin to the place of action or detoxification. Recently three studies from mammalian systems have provided evidence that H2O2, in addition to the well studied role in intracellular signaling, is also used as an intercellular signal molecule (8Pletjushkina O.Y. Fetisova E.K. Lyamzaev K.G. Ivanova O.Y. Dom-nina L.V. Vyssokikh M.Y. Pustovidko A.V. Alexeevski A.V. Alexeevski D.A. Vasiliev J.M. Murphy M.P. Chernyak B.V. Skulachev V.P. Biochemistry (Mosc.). 2006; 71: 60-67Crossref PubMed Scopus (35) Google Scholar, 9Pelle E. Mammone T. Maes D. Frenkel K. J. Investig. 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Expression of aquaporins in yeast secretory vesicles lowered the activation energy for diffusion of water from between 46 and 55 to 17 kJ mol-1 (11Coury L.A. Hiller M. Mathai J.C Jones E.W. Zeidel M.L. Brodsky J.L. J. Bacteriol. 1999; 181: 4437-4440Crossref PubMed Google Scholar). H2O2 has a permanent dipole moment of 2.26 × 10-18 electrostatic unit (12Ardon M. Oxygen. W. A. Benjamin Inc., New York1965: 82Google Scholar), very similar to that of water (1.85 × 10-18 electrostatic unit). Consequently simple passive diffusion of H2O2 across the lipid bilayer should be limited as for water. Recent studies have demonstrated that some membranes are indeed poorly permeable to H2O2 (13Seaver L.C. Imlay A.J. J. Bacteriol. 2001; 183: 7182-7189Crossref PubMed Scopus (361) Google Scholar, 14Branco M.R. Marhino H. Cyrne L. Antunes F. J. Biol. Chem. 2004; 279: 6501-6506Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 15Makino N. Sasaki K. Hashida K. Sakakura Y. Biochim. Biophys. 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The yeast used all aquaporin strains were on with various of H2O2. was at the of addition of H2O2 decreased growth and survival of the different yeast strains to various wt, and were able to in the presence of H2O2, growth of and was at H2O2 of H2O2 the growth was by for at to the growth similar growth responses were with and without of the not with the of aquaporins not the sensitivity of yeast cells toward of H2O2 in the from are shown as an However, of and growth and cell survival on H2O2 The was not of and growth of all yeast strains was at a concentration of H2O2 compared with the The was by and Although for the with was able to on to H2O2, growth of with was at H2O2. of growth and cell survival to the wild was by addition of H2O2 at very similar is known to a role in K. A. J. H. H. J. L. J.M. S. 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Gutteridge J.M.C. Free Radicals in Biology and Medicine.3rd Ed. Oxford University Press, New York1999: 105-245Google Scholar). the can into yeast the the cells and it to oxidation by ROS. yeast cells were five and fluorescence was all yeast the cells with were made permeable with and the were to H2O2. all an in fluorescence was not the of H2O2 in the of the yeast cells with either or a in fluorescence of H2O2 to or yeast cells fluorescence and However, addition of H2O2 to yeast cells and fluorescence The was for not cells were from the assay by fluorescence could be addition of H2O2 to the assay However, with assay yeast cells with and the in fluorescence addition of H2O2 as in of the cells by of the assay of also the fluorescence signal from the assay this that the in fluorescence was not to of the fluorescent from the cells and was by of H2O2 into the the of fluorescence the first addition of H2O2. Expression of to a in fluorescence of The a reduction in the activation energy for the diffusion of H2O2 from to about kJ mol-1 of yeast cells for with the fluorescence addition of H2O2 and an in of with an fluorescence signal addition of and H2O2 as the in the of not the reduction of the fluorescence addition of in was not to a of on the fluorescence in of yeast and is with the cells and not from of the from yeast cells to the yeast with or were in the assay and by were in assay and both the and the cells were to fluorescence addition of ions and can with H2O2 to hydroxyl could a similar H2O2 was with or and used in the fluorescence with either or ions to a with H2O2 with or ions not in an in fluorescence to yeast cells However, H2O2 with was able to an in fluorescence of cells with as used in and to a fluorescence The results that not with H2O2 at (for both H2O2 and and that a of cells with is needed to of The of H2O2 by by cells with either or were with CM-H2DCFDA, and to was as a with an within the of cells compared with R. 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