Antisense oligonucleotides against the α-subunit of ENaC decrease lung epithelial cation-channel activity
Antisense oligonucleotides against the α-subunit of ENaC decrease lung epithelial cation-channel activity
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DOI：
10.1152/ajplung.1999.276.6.l1046
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发表时间：
1999-06-01
影响因子：
4.9
通讯作者：
Eaton, DC
中科院分区：
文献类型：
--
作者：
Jain, L;Chen, XJ;Eaton, DC
肺上皮细胞对阿米洛利敏感的Na+转运在维持肺泡Nat和水平衡中起关键作用。一般认为Na+转运是由阿米洛利敏感的上皮Nat通道（ENaC）介导的，因为分子生物学研究已经证实了ENaC亚基α、β和γ在肺上皮中的存在。然而，大多数实验室从电生理学研究中报道的主要Na+转运通道是一种非选择性的高电导通道，与其他组织中报道的高选择性、低电导ENaC非常不同。在我们的实验室中，从大鼠肺泡II型（ATII）细胞的原代培养物的顶膜贴片的单通道记录揭示了一个非选择性阳离子通道，电导率为20.6 +/- 1.1 pS，Na+对K+的选择性为0.97 +/- 0.07。该通道被亚微摩尔浓度的阿米洛利抑制。因此，单通道方法观察到的基因产物与克隆的ENaC亚基之间的关系存在一些问题。我们已经采用反义寡核苷酸方法来阻止个别ENaC亚基蛋白质（α，β和γ）的合成，并确定在ATII细胞的顶端膜补丁中观察到的非选择性阳离子通道的密度的亚基表达减少的影响。ATII细胞的反义寡核苷酸治疗抑制每个亚基蛋白的生产，然而，单通道记录显示，只有针对a-亚基的反义寡核苷酸导致非选择性阳离子通道的密度显着降低。单独或一起抑制β-和γ-亚基蛋白不会引起所观察到的通道密度的任何变化。开放概率或其他通道特性没有变化。这些结果支持ENaC的α亚基单独或与β或γ亚基蛋白以外的一些蛋白质组合是肺泡上皮阳离子通道的主要组分的假设。
Amiloride-sensitive Na+ transport by lung epithelia plays a critical role in maintaining alveolar Nat and water balance. It has been generally assumed that Na+ transport is mediated by the amiloride-sensitive epithelial Nat channel (ENaC) because molecular biology studies have confirmed the presence of ENaC subunits alpha, beta, and gamma in lung epithelia. However, the predominant Na+-transporting channel reported from electrophysiological studies by most laboratories is a nonselective, high-conductance channel that is very different from the highly selective, low-conductance ENaC reported in other tissues. In our laboratory, single-channel recordings from apical membrane patches from rat alveolar type II (ATII) cells in primary culture reveal a nonselective cation channel with a conductance of 20.6 +/- 1.1 pS and an Na+-to-K+ selectivity of 0.97 +/- 0.07. This channel is inhibited by submicromolar concentrations of amiloride. Thus there is some question about the relationship between the gene product observed with single-channel methods and the cloned ENaC subunits. We have employed antisense oligonucleotide methods to block the synthesis of individual ENaC subunit proteins (alpha, beta, and gamma) and determined the effect of a reduction in the subunit expression on the density of the nonselective cation channel observed in apical membrane patches on ATII cells. Treatment of ATII cells with antisense oligonucleotides inhibited the production of each subunit protein; however, single-channel recordings showed that only the antisense oligonucleotide targeting the a-subunit resulted in a significant decrease in the density of nonselective cation channels. Inhibition of the beta- and gamma-subunit proteins alone or together did not cause any changes in the observed channel density. There were no changes in open probability or other channel characteristics. These results support the hypothesis that the alpha-subunit of ENaC alone or in combination with some protein other than the beta- or gamma-subunit protein is the major component of lung alveolar epithelial cation channels.