Evans Blue is not a suitable inhibitor of the epithelial sodium channel δ-subunit

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.