Perniss_2015_Biochem.Biophys.Res.Commun_466_468

The Epithelial Sodium Channel (ENaC) is a heterotrimeric ion channel which can be either formed by assembly of its alpha-, beta- and gamma-subunits or, alternatively, its delta-, beta- and gamma-subunits. The physiological function of alphabetagamma-ENaC is well established, but the function of deltabetagamma-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 deltabetagamma-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 deltabetagamma-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 deltabetagamma-ENaC but rather represents a pharmacological artefact. EvB should therefore not be used as an inhibitor of delta-ENaC.