Chen Lan, Fuller Catherine M, Kleyman Thomas R, Matalon Sadis
Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 901 19th St. S, BMR II, Rm. 224, Birmingham, AL 35205-3703, USA.
Am J Physiol Renal Physiol. 2004 Jun;286(6):F1202-8. doi: 10.1152/ajprenal.00352.2003. Epub 2004 Feb 17.
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 microA; 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 microM; P < 0.01). Exposure of oocytes to SIN-1 (1 mM) for 5 min decreased both total Na(+) 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.
我们研究了上皮钠通道（ENaC）α亚基细胞外环的两种突变对非洲爪蟾卵母细胞中氨氯地平敏感电流的影响，以及3 - 吗啉代亚硝基胍（SIN - 1）对该电流的抑制作用。向卵母细胞注射野生型（wt）α - 、β - 、γ - rENaC的cRNA（8.3 ng/亚基），48 - 72小时后产生内向Na⁺电流（-5.5±0.8 μA；-100 mV时的平均值±标准误；n = 21），该电流被氨氯地平完全抑制。注射α（Y279A）- 或α（Y283A）- 以及β - 、γ - rENaC的cRNA的卵母细胞具有明显更低的Na⁺电流。此外，注射α（Y279A）- 、β - 、γ - rENaC的卵母细胞对氨氯地平的抑制常数（K₁）更高（0.54±0.97对0.10±0.04 μM；P < 0.01）。将卵母细胞暴露于SIN - 1（1 mM）5分钟，可降低野生型和α（Y279A）- 但不降低α（Y283A）- 、β - 、γ - rENaC的总Na⁺电流和氨氯地平敏感电流。此外，暴露于SIN - 1会增加野生型但不会增加注射α（Y279A）- 、β - 、γ - rENaC的卵母细胞对氨氯地平的K₁。这些数据表明，这两个酪氨酸对于ENaC的正常功能都很重要，并且它们的氧化修饰会导致ENaC功能改变。
