Bardou Olivier, Privé Anik, Migneault Francis, Roy-Camille Karl, Dagenais André, Berthiaume Yves, Brochiero Emmanuelle
Centre de recherche, Centre hospitalier de 1'Université de Montréal (CRCHUM) - Hôtel-Dieu, Montral, Quebec, Canada.
Biochim Biophys Acta. 2012 Jul;1818(7):1682-90. doi: 10.1016/j.bbamem.2012.02.025.
Active Na+ absorption by alveolar ENaC is the main driving force of liquid clearance at birth and lung edema resorption in adulthood. We have demonstrated previously that long-term modulation of KvLQT1 and KATP K+ channel activities exerts sustained control in Na+ transport through the regulation of ENaC expression in primary alveolar type II (ATII) cells. The goal of the present study was: 1) to investigate the role of the alpha-ENaC promoter, transfected in the A549 alveolar cell line, in the regulation of ENaC expression by K+ channels, and 2) to determine the physiological impact of K+ channels and ENaC modulation on fluid clearance in ATII cells. KvLQT1 and KATP channels were first identified in A549 cells by PCR and Western blotting. We showed, for the first time, that KvLQT1 activation by R-L3 (applied for 24 h) increased alpha-ENaC expression, similarly to KATP activation by pinacidil. Conversely, pharmacological KvLQT1 and KATP inhibition or silencing with siRNAs down-regulated alpha-ENaC expression. Furthermore, K+ channel blockers significantly decreased alpha-ENaC promoter activity. Our results indicated that this decrease in promoter activity could be mediated, at least in part, by the repressor activity of ERK1/2. Conversely, KvLQT1 and KATP activation dose-dependently enhanced alpha-ENaC promoter activity. Finally, we noted a physiological impact of changes in K+ channel functions on ERK activity, alpha-, beta-, gamma-ENaC subunit expression and fluid absorption through polarized ATII cells. In summary, our results disclose that K+ channels regulate alpha-ENaC expression by controlling its promoter activity and thus affect the alveolar function of fluid clearance.
肺泡上皮钠通道（ENaC）对钠离子的主动重吸收是出生时液体清除以及成年期肺水肿吸收的主要驱动力。我们之前已经证明，长期调节KvLQT1和KATP钾通道活性可通过调控原代II型肺泡上皮（ATII）细胞中ENaC的表达，对钠离子转运产生持续控制作用。本研究的目的是：1）研究转染至A549肺泡细胞系中的α-ENaC启动子在钾通道对ENaC表达调控中的作用；2）确定钾通道和ENaC调节对ATII细胞中液体清除的生理影响。首先通过PCR和蛋白质印迹法在A549细胞中鉴定出KvLQT1和KATP通道。我们首次发现，R-L3（作用24小时）激活KvLQT1可增加α-ENaC的表达，这与吡那地尔激活KATP的效果相似。相反，用药物抑制KvLQT1和KATP或用小干扰RNA（siRNA）使其沉默会下调α-ENaC的表达。此外，钾通道阻滞剂显著降低α-ENaC启动子活性。我们的结果表明，启动子活性的这种降低至少部分可由细胞外信号调节激酶1/2（ERK1/2）的抑制活性介导。相反，KvLQT1和KATP的激活呈剂量依赖性地增强α-ENaC启动子活性。最后，我们注意到钾通道功能变化对ERK活性、α-、β-、γ-ENaC亚基表达以及通过极化的ATII细胞进行液体吸收具有生理影响。总之，我们的结果表明，钾通道通过控制α-ENaC启动子活性来调节其表达，从而影响肺泡的液体清除功能。
