title: Evans Blue is not a suitable inhibitor of the epithelial sodium channel δ-subunit.
abstract: The Epithelial Sodium Channel (ENaC) is a heterotrimeric ion channel which can be either formed by assembly of its α-, β- and γ-subunits or, alternatively, its δ-, β- and γ-subunits. The physiological function of αβγ-ENaC is well established, but the function of δβγ-ENaC remains elusive. The azo-dye Evans Blue (EvB) has been routinely used to discriminate between the two channel isoforms by decreasing transmembrane currents and amiloride-sensitive current fractions of δβγ-ENaC expressing Xenopus oocytes. Even though these results could be reproduced, it was found by precipitation experiments and spectroscopic methods that the cationic amiloride and the anionic EvB directly interact in solution, forming a strong complex. Thereby a large amount of pharmacologically available amiloride is removed from physiological buffer solutions and the effective amiloride concentration is reduced. This interaction did not occur in the presence of albumin. In microelectrode recordings, EvB was able to abrogate the block of δβγ-ENaC by amiloride or its derivative benzamil. In sum, EvB reduces amiloride-sensitive ion current fractions in electrophysiological experiments. This is not a result of a specific inhibition of δβγ-ENaC but rather represents a pharmacological artefact. EvB should therefore not be used as an inhibitor of δ-ENaC.
authors.affiliation: Institute for Animal Physiology, Justus-Liebig-University, Heinrich-Buff-Ring 26, D-35392 Giessen, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Perniss
authors.affiliation: Institute for Animal Physiology, Justus-Liebig-University, Heinrich-Buff-Ring 26, D-35392 Giessen, Germany.
authors.forename: Annemarie
authors.initials: A
authors.lastname: Wolf
authors.affiliation: Institute for Animal Physiology, Justus-Liebig-University, Heinrich-Buff-Ring 26, D-35392 Giessen, Germany.
authors.forename: Lukas
authors.initials: L
authors.lastname: Wichmann
authors.affiliation: Department of Chemistry, Stanford University, 337 Campus Drive, Stanford, CA 94305-4401337, USA; Institute for Radiochemistry and Molecular Imaging, University Clinic Cologne, Kerpener Strasse 62, 50931 Cologne, Germany. Electronic address: mschoenb@stanford.edu.
authors.forename: Matthias
authors.initials: M
authors.lastname: Schönberger
authors.affiliation: Institute for Animal Physiology, Justus-Liebig-University, Heinrich-Buff-Ring 26, D-35392 Giessen, Germany. Electronic address: mike.althaus@bio.uni-giessen.de.
authors.forename: Mike
authors.initials: M
authors.lastname: Althaus
mesh_terms: D000418:Albumins / Q000378:metabolism; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D004396:Coloring Agents / Q000494:pharmacology; D062686:Epithelial Sodium Channel Blockers / Q000494:pharmacology*; D053503:Epithelial Sodium Channels / Q000187:drug effects* / Q000378:metabolism; D005070:Evans Blue / Q000494:pharmacology*; D005260:Female; D006801:Humans; D009865:Oocytes / Q000378:metabolism; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D012996:Solutions; D014982:Xenopus laevis
chemical_list: D000418:Albumins; D002021:Buffers; D004396:Coloring Agents; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels; D011994:Recombinant Proteins; C507051:SCNN1D protein, human; D012996:Solutions; C013407:benzamil; D005070:Evans Blue; D000584:Amiloride
keywords: Amiloride; Delta-ENaC; ENaC; Epithelial sodium channel; Evans blue