Cystic Fibrosis/Pulmonary Research and Treatment Center, Department of Chemistry, Department of Biochemistry and Biophysics, Departments of Prosthodontics and Pharmacology, Department of Microbiology and Immunology, and Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC 27599.
Proc Natl Acad Sci U S A. 2013 Oct 1;110(40):15973-8. doi: 10.1073/pnas.1311999110. Epub 2013 Sep 16.
The ability to maintain proper airway surface liquid (ASL) volume homeostasis is vital for mucus hydration and clearance, which are essential aspects of the mammalian lung's innate defense system. In cystic fibrosis (CF), one of the most common life-threatening genetic disorders, ASL dehydration leads to mucus accumulation and chronic infection. In normal airways, the secreted protein short palate lung and nasal epithelial clone 1 (SPLUNC1) effectively inhibits epithelial Na(+) channel (ENaC)-dependent Na(+) absorption and preserves ASL volume. In CF airways, it has been hypothesized that increased ENaC-dependent Na(+) absorption contributes to ASL depletion, and hence increased disease. However, this theory is controversial, and the mechanism for abnormal ENaC regulation in CF airways has remained elusive. Here, we show that SPLUNC1 is a pH-sensitive regulator of ENaC and is unable to inhibit ENaC in the acidic CF airway environment. Alkalinization of CF airway cultures prevented CF ASL hyperabsorption, and this effect was abolished when SPLUNC1 was stably knocked down. Accordingly, we resolved the crystal structure of SPLUNC1 to 2.8 Å. Notably, this structure revealed two pH-sensitive salt bridges that, when removed, rendered SPLUNC1 pH-insensitive and able to regulate ASL volume in acidic ASL. Thus, we conclude that ENaC hyperactivity is secondary to reduced CF ASL pH. Together, these data provide molecular insights into the mucosal dehydration associated with a range of pulmonary diseases, including CF, and suggest that future therapy be directed toward alkalinizing the pH of CF airways.
维持适当的气道表面液体 (ASL) 体积平衡的能力对于黏液水合和清除至关重要，这是哺乳动物肺部先天防御系统的重要方面。在囊性纤维化 (CF) 中，这是最常见的危及生命的遗传疾病之一，ASL 脱水导致黏液积聚和慢性感染。在正常气道中，分泌蛋白短腭肺和鼻上皮克隆 1 (SPLUNC1) 有效地抑制上皮钠通道 (ENaC) 依赖性 Na+吸收并维持 ASL 体积。在 CF 气道中，据推测，增加的 ENaC 依赖性 Na+吸收有助于 ASL 耗尽，因此增加了疾病的发生。然而，这一理论存在争议，CF 气道中异常 ENaC 调节的机制仍然难以捉摸。在这里，我们表明 SPLUNC1 是 ENaC 的 pH 敏感调节剂，并且在 CF 气道的酸性环境中无法抑制 ENaC。CF 气道培养物的碱化可防止 CF ASL 过度吸收，而当 SPLUNC1 稳定敲低时，该作用被消除。相应地，我们解析了 SPLUNC1 的 2.8 Å 晶体结构。值得注意的是，该结构揭示了两个 pH 敏感的盐桥，当它们被去除时，SPLUNC1 对 pH 不敏感并且能够在酸性 ASL 中调节 ASL 体积。因此，我们得出结论，ENaC 过度活跃是 CF ASL pH 降低的次要原因。总之，这些数据为与一系列肺部疾病（包括 CF）相关的黏膜脱水提供了分子见解，并表明未来的治疗应针对 CF 气道的碱化。
