Mutations in the extracellular loop of α-rENaC alter sensitivity to amiloride and reactive species

Abstract

Matalon. Mutations in the extracellular loop of ␣-rENaC alter sensitivity to amiloride and reactive species. Am J Physiol Renal Physiol 286: F1202-F1208, 2004. First published February 17, 2004 10.1152/ajprenal. 00352.2003.-We studied the effects of two mutations of the extracellular loop of the ␣-subunit of the (ENaC) on amiloride-sensitive current in Xenopus laevis oocytes and the inhibition of this current by 3-morpholinosydnonimine (SIN-1). Injection of oocytes with wild-type (wt) ␣-,␤-,␥-rENaC cRNA (8.3 ng/subunit) resulted 48 -72 h later in inward Na ϩ currents (Ϫ5.5 Ϯ 0.8 A; means Ϯ SE at Ϫ100 mV; n ϭ 21), which were completely inhibited by amiloride. Oocytes injected with either ␣ Y279A-or ␣Y283A-and ␤-,␥-rENaC cRNAs had significantly lower Na ϩ currents. Furthermore, ␣Y279A-,␤-,␥-rENaC-injected oocytes had a higher K i for amiloride (0.54 Ϯ 0.97 vs. 0.10 Ϯ 0.04 M; P Ͻ 0.01). Exposure of oocytes to SIN-1 (1 mM) for 5 min decreased both total Na ϩ and amiloride-sensitive currents across wt and ␣Y279A-but not ␣ Y283A-,␤-,␥-rENaC. Furthermore, exposure to SIN-1 increased the Ki for amiloride across wt but not ␣Y279A-,␤-,␥-rENaC-injected oocytes. These data indicate that both tyrosines are important for proper ENaC function and their oxidative modifications contribute to altered ENaC function.

Inhibition of Lung Fluid Clearance and Epithelial Na+ Channels by Chlorine, Hypochlorous Acid, and Chloramines

“…Reactive intermediates also inhibit vectorial Na ϩ transport across ATII cells by decreasing the activity of epithelial Na ϩ channels via post-translational modifications of either ENaC per se or of structural proteins (such as actin and fondrin) that are necessary for proper action of ENaC (6,74). Substitution of a single tyrosine in the extracellular loop of ␣ENaC (Tyr-283) with alanine prevented the peroxynitrite-induced decrease of amiloride-sensitive currents (75). ENaC was also inhibited by sulfhydryl-reactive agents added in the cytoplasm, and reducing agents reversed the rundown activity of ENaC in excised patches (76).…”

Reactive Species Mediate Inhibition of Alveolar Type II Sodium Transport during Mycoplasma Infection

“…Furthermore, NO donors added to the apical compartments of Ussing chambers containing rat ATII cell monolayers inhibited Na ϩ transport (11) through cGMP-independent mechanisms. Excessive production of reactive oxygen species may result in post-translational modification of ENaC proteins through nitration or oxidation of limited domains or specific key amino acid residues (13,18,48,49). Likewise, reactive oxygen species may directly alter channel protein activity (11) or cytoskeletal structures, which are required for proper Na ϩ channel function (50).…”

“…Activated neutrophils also secrete myeloperoxidase (MPO) into the alveolar space, which catalyzes the reaction of nitrite, hydrogen peroxide, and chloride to produce new intermediates capable of nitrating, oxidizing, and chlorinating proteins (17); these events have been shown to decrease ENaC function (18). To determine the impact of RONS and MPO on ion transport during M. pulmonis infection in vivo, we measured alveolar fluid clearance (AFC) in infected and uninfected mice lacking MPO (MPO Ϫ/Ϫ ).…”

ENaCs and ASICs as therapeutic targets

“…In contrast, superoxide seems to enhance ENaC activity, and protects against the negative influence of RONS (114,169,431). The potential underlying mechanisms for the negative effects of peroxynitrite include posttranslational modification of vulnerable tyrosine and/or cysteine residues in the channel, although little direct evidence has been obtained to support this (81). The effects of superoxide are most likely due to modulation of NADPH oxidase, and scavengers/inhibitors of superoxide resulted in an acute decrease in channel P o (379).…”