{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T02:16:57Z","timestamp":1760149017895,"version":"build-2065373602"},"reference-count":41,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2023,6,28]],"date-time":"2023-06-28T00:00:00Z","timestamp":1687910400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["Be2253\/8-2"],"award-info":[{"award-number":["Be2253\/8-2"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>(1) Background: Several members of the ubiquitous aquaporin family, AQP, of water and neutral solute channels carry a cysteine residue in the selectivity filter region. Traditionally, toxic mercury-containing compounds are used to bind to the cysteine as covalent AQP inhibitors for physiological studies or analysis of structure\u2013function relationships. (2) Methods: We tested thiol-reactive methylthiosulfonate reagents, MTS, as alternative Cys modifiers for AQP inhibition. Three MTS reagents transferring S-alkyl moieties of increasing size, i.e., S-methyl, S-n-propyl, and S-benzyl, were used with yeast-expressed water-selective AQP1 and the aquaglyceroporin AQP9. Respective Cys-to-Ala variants and mouse erythrocytes that naturally express AQP1 and AQP9 served as controls. (3) Results: Both wildtype AQP isoforms were inhibited by the Cys modifiers in a size-dependent manner, whereas the Cys-to-Ala-variants exhibited resistance. Sub-millimolar concentrations and incubation times in the minute range were sufficient. The modifications were reversible by treatment with the thiol reagents acetylcysteine, ACC, and dithiothreitol, DTT. (4) Conclusions: MTS reagents represent a valid alternative of low toxicity for the inhibition of mercurial-sensitive AQPs.<\/jats:p>","DOI":"10.3390\/cells12131742","type":"journal-article","created":{"date-parts":[[2023,6,29]],"date-time":"2023-06-29T00:49:47Z","timestamp":1687999787000},"page":"1742","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Methylthiosulfonate-Based Cysteine Modifiers as Alternative Inhibitors of Mercurial-Sensitive Aquaporins"],"prefix":"10.3390","volume":"12","author":[{"given":"Katrin","family":"Jeuken","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Gutenbergstr. 76, 24118 Kiel, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emmi","family":"Jaeger","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Gutenbergstr. 76, 24118 Kiel, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emily","family":"Matthews","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Gutenbergstr. 76, 24118 Kiel, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5912-6626","authenticated-orcid":false,"given":"Eric","family":"Beitz","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Gutenbergstr. 76, 24118 Kiel, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,6,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1016\/j.jmb.2007.02.070","article-title":"Structural basis of aquaporin inhibition by mercury","volume":"368","author":"Savage","year":"2007","journal-title":"J. Mol. Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1002\/jcp.1040830316","article-title":"Effect of PCMBS on water transfer across biological membranes","volume":"83","author":"Naccache","year":"1974","journal-title":"J. Cell. Physiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1126\/science.256.5055.385","article-title":"Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein","volume":"256","author":"Preston","year":"1992","journal-title":"Science"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1007\/978-981-19-7415-1_6","article-title":"Evolutionary overview of aquaporin superfamily","volume":"1398","author":"Ishibashi","year":"2023","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"624","DOI":"10.1038\/42512","article-title":"The three-dimensional structure of aquaporin-1","volume":"387","author":"Walz","year":"1997","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"481","DOI":"10.1126\/science.290.5491.481","article-title":"Structure of a glycerol-conducting channel and the basis for its selectivity","volume":"290","author":"Fu","year":"2000","journal-title":"Science"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2413","DOI":"10.1007\/s00018-007-7163-2","article-title":"Aquaporins with selectivity for unconventional permeants","volume":"64","author":"Wu","year":"2007","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2188","DOI":"10.1038\/emboj.2009.182","article-title":"Concerted action of two cation filters in the aquaporin water channel","volume":"28","author":"Wu","year":"2009","journal-title":"EMBO J."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Geistlinger, K., Schmidt, J.D.R., and Beitz, E. (2022). Lactic acid permeability of aquaporin-9 enables cytoplasmic lactate accumulation via an ion trap. Life, 12.","DOI":"10.3390\/life12010120"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1073\/pnas.0507225103","article-title":"Point mutations in the aromatic\/arginine region in aquaporin 1 allow passage of urea, glycerol, ammonia, and protons","volume":"103","author":"Beitz","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/S0021-9258(18)54108-9","article-title":"The mercury-sensitive residue at cysteine 189 in the CHIP28 water channel","volume":"268","author":"Preston","year":"1993","journal-title":"J. Biol. Chem."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1080\/15216549900201333","article-title":"Cloning and functional expression of rAOP9L a new member of aquaporin family from rat liver","volume":"47","author":"Ko","year":"1999","journal-title":"IUBMB Life"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"872","DOI":"10.1038\/414872a","article-title":"Structural basis of water-specific transport through the AQP1 water channel","volume":"414","author":"Sui","year":"2001","journal-title":"Nature"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1038\/nrd4226","article-title":"Aquaporins: Important but elusive drug targets","volume":"13","author":"Verkman","year":"2014","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1016\/j.pharmthera.2015.08.002","article-title":"Challenges and achievements in the therapeutic modulation of aquaporin functionality","volume":"155","author":"Beitz","year":"2015","journal-title":"Pharmacol. Ther."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1086","DOI":"10.1002\/cmdc.201300107","article-title":"Aquaporin inhibition by gold(III) compounds: New insights","volume":"8","author":"Martins","year":"2013","journal-title":"ChemMedChem"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Florio, M., Engfors, A., Gena, P., Larsson, J., Massaro, A., Timpka, S., Reimer, M.K., Kjellbom, P., Beitz, E., and Johanson, U. (2022). Characterization of the aquaporin-9 inhibitor RG100204 in vitro and in db\/db mice. Cells, 11.","DOI":"10.3390\/cells11193118"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"613","DOI":"10.1002\/jcp.1041640320","article-title":"Evidence for the presence of a Hg-inhibitable water-permeability pathway and aquaporin 1 in A5 salivary epithelial cells","volume":"164","author":"Lazowski","year":"1995","journal-title":"J. Cell. Physiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"5171","DOI":"10.1074\/jbc.271.9.5171","article-title":"Structure of aquaporin-2 vasopressin water channel","volume":"271","author":"Bai","year":"1996","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"538","DOI":"10.1021\/bi9520038","article-title":"Selected cysteine point mutations confer mercurial sensitivity to the mercurial-insensitive water channel MIWC\/AQP-4","volume":"35","author":"Shi","year":"1996","journal-title":"Biochemistry"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1016\/S0167-4781(00)00268-2","article-title":"Functional characterization of a water channel of the nematode Caenorhabditis elegans","volume":"1517","author":"Kuwahara","year":"2000","journal-title":"Biochim. Biophys. Acta (BBA) Gene Struct. Expr."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"39873","DOI":"10.1074\/jbc.M207008200","article-title":"Characterization of aquaporin-6 as a nitrate channel in mammalian cells. Requirement of pore-lining residue threonine 63","volume":"277","author":"Ikeda","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1153","DOI":"10.1073\/pnas.0307295101","article-title":"Molecular dissection of water and glycerol permeability of the aquaglyceroporin from Plasmodium falciparum by mutational analysis","volume":"101","author":"Beitz","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"7487","DOI":"10.1074\/jbc.M111.329102","article-title":"Functional characterization of a novel aquaporin from Dictyostelium discoideum amoebae implies a unique gating mechanism","volume":"287","author":"Kai","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1038\/ncb2656","article-title":"Intracellular lumen extension requires ERM-1-dependent apical membrane expansion and AQP-8-mediated flux","volume":"15","author":"Khan","year":"2013","journal-title":"Nat. Cell Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"118569","DOI":"10.1016\/j.bbamcr.2019.118569","article-title":"Aquaporin-1 plays a key role in erythropoietin-induced endothelial cell migration","volume":"1867","author":"Maltaneri","year":"2020","journal-title":"Biochim. Biophys. Acta (BBA)-Mol. Cell Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"877","DOI":"10.1007\/s13280-023-01831-6","article-title":"Our evolved understanding of the human health risks of mercury","volume":"52","author":"Basu","year":"2023","journal-title":"AMBIO"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"228","DOI":"10.3109\/09687688.2014.960493","article-title":"The arginine-facing amino acid residue of the rat aquaporin 1 constriction determines solute selectivity according to its size and lipophilicity","volume":"31","author":"Krenc","year":"2014","journal-title":"Mol. Membr. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"9358","DOI":"10.1074\/jbc.M117.782516","article-title":"Electrostatic attraction of weak monoacid anions increases probability for protonation and passage through aquaporins","volume":"292","author":"Rothert","year":"2017","journal-title":"J. Biol. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"464","DOI":"10.1080\/09687680701342669","article-title":"The conserved sequence NXX[S\/T]HX[S\/T]QDXXXT of the lactate\/pyruvate:H(+) symporter subfamily defines the function of the substrate translocation pathway","volume":"24","author":"Paiva","year":"2007","journal-title":"Mol. Membr. Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1038\/nprot.2007.13","article-title":"High-efficiency yeast transformation using the LiAc\/SS carrier DNA\/PEG method","volume":"2","author":"Gietz","year":"2007","journal-title":"Nat. Protoc."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"15634","DOI":"10.1016\/S0021-9258(19)37635-5","article-title":"Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules","volume":"263","author":"Denker","year":"1988","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"8407","DOI":"10.1074\/jbc.273.14.8407","article-title":"Evidence for the presence of aquaporin-3 in human red blood cells","volume":"273","author":"Roudier","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"12560","DOI":"10.1073\/pnas.0705313104","article-title":"Aquaporin 9 is the major pathway for glycerol uptake by mouse erythrocytes, with implications for malarial virulence","volume":"104","author":"Liu","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"667","DOI":"10.1289\/ehp.1307864","article-title":"Mercury exposure and health impacts among individuals in the artisanal and small-scale gold mining community: A comprehensive review","volume":"122","author":"Gibb","year":"2014","journal-title":"Environ. Health Perspect."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1016\/0076-6879(77)47042-3","article-title":"Novel sulfhydryl reagents","volume":"47","author":"Kenyon","year":"1977","journal-title":"Methods Enzymol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"6052","DOI":"10.1111\/febs.16024","article-title":"Cysteine 159 delineates a hinge region of the alternating access monocarboxylate transporter 1 and is targeted by cysteine-modifying inhibitors","volume":"288","author":"Geistlinger","year":"2021","journal-title":"FEBS J."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"4874","DOI":"10.1074\/jbc.M110683200","article-title":"A single, bi-functional aquaglyceroporin in blood-stage Plasmodium falciparum malaria parasites","volume":"277","author":"Hansen","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"797","DOI":"10.1016\/0028-3908(96)00129-3","article-title":"On the use of thiol-modifying agents to determine channel topology","volume":"35","author":"Holmgren","year":"1996","journal-title":"Neuropharmacology"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1479","DOI":"10.1021\/bi962256g","article-title":"External cysteine residues in the serotonin transporter","volume":"36","author":"Chen","year":"1997","journal-title":"Biochemistry"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1124\/mol.111.073692","article-title":"The activity of human aquaporin 1 as a cGMP-gated cation channel is regulated by tyrosine phosphorylation in the carboxyl-terminal domain","volume":"81","author":"Campbell","year":"2012","journal-title":"Mol. Pharmacol."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/12\/13\/1742\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:03:07Z","timestamp":1760126587000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/12\/13\/1742"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,6,28]]},"references-count":41,"journal-issue":{"issue":"13","published-online":{"date-parts":[[2023,7]]}},"alternative-id":["cells12131742"],"URL":"https:\/\/doi.org\/10.3390\/cells12131742","relation":{},"ISSN":["2073-4409"],"issn-type":[{"type":"electronic","value":"2073-4409"}],"subject":[],"published":{"date-parts":[[2023,6,28]]}}}