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

We studied the effects of two mutations of the extracellular loop of the alpha-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) alpha-, beta-, gamma-rENaC cRNA (8.3 ng/subunit) resulted 48-72 h later in inward Na+ currents (-5.5 +/- 0.8 muA; means +/- SE at -100 mV; n = 21), which were completely inhibited by amiloride. Oocytes injected with either alpha(Y279A)- or alpha(Y283A)- and beta-, gamma-rENaC cRNAs had significantly lower Na+ currents. Furthermore, alpha(Y279A)-, beta-, gamma-rENaC-injected oocytes had a higher K-i for amiloride (0.54 +/- 0.97 vs. 0.10 +/- 0.04 muM; P < 0.01). Exposure of oocytes to SIN-1 (1 mM) for 5 min decreased both total N+ and amiloride-sensitive currents across wt and alpha(Y279A)- but not alpha(Y283A)-, beta-, gamma-rENaC. Furthermore, exposure to SIN-1 increased the K-i for amiloride across wt but not alpha(Y279A)-, beta-, gamma-rENaC-injected oocytes. These data indicate that both tyrosines are important for proper ENaC function and their oxidative modifications contribute to altered ENaC function.