The epithelial sodium channel (ENaC) is essential for mediating sodium absorption in several epithelia. Its impaired function leads to severe disorders, including pseudohypoaldosteronism type 1 and respiratory distress. Therefore, pharmacological ENaC activators have potential therapeutic implications. Previously, a small molecule ENaC activator (S3969) was developed. So far, little is known about molecular mechanisms involved in S3969-mediated ENaC stimulation. Here, we identified an S3969-binding site in human ENaC by combining structure-based simulations with molecular biological methods and electrophysiological measurements of ENaC heterologously expressed in oocytes. We confirmed a previous observation that the extracellular loop of β-ENaC is essential for ENaC stimulation by S3969. Molecular dynamics simulations predicted critical residues in the thumb domain of β-ENaC (Arg388, Phe391, and Tyr406) that coordinate S3969 within a binding site localized at the β-γ-subunit interface. Importantly, mutating each of these residues reduced (R388H; R388A) or nearly abolished (F391G; Y406A) the S3969-mediated ENaC activation. Molecular dynamics simulations also suggested that S3969-mediated ENaC stimulation involved a movement of the α5 helix of the thumb domain of β-ENaC away from the palm domain of γ-ENaC. Consistent with this, the introduction of two cysteine residues (βR437C – γS298C) to form a disulfide bridge connecting these two domains prevented ENaC stimulation by S3969 unless the disulfide bond was reduced by DTT. Finally, we demonstrated that S3969 stimulated ENaC endogenously expressed in cultured human airway epithelial cells (H441). These new findings may lead to novel (patho-)physiological and therapeutic concepts for disorders associated with altered ENaC function.

中文翻译：

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小分子激活剂 S3969 通过与通道 β 亚基中的特异性结合袋相互作用来刺激上皮钠通道


上皮钠通道 (ENaC) 对于介导多种上皮细胞的钠吸收至关重要。其功能受损会导致严重疾病，包括 1 型假性醛固酮增多症和呼吸窘迫。因此，药理学 ENaC 激活剂具有潜在的治疗意义。此前，开发了一种小分子ENaC激活剂（S3969）。到目前为止，人们对 S3969 介导的 ENaC 刺激的分子机制知之甚少。在这里，我们通过将基于结构的模拟与分子生物学方法和卵母细胞中异源表达的 ENaC 的电生理学测量相结合，确定了人类 ENaC 中的 S3969 结合位点。我们证实了之前的观察结果，即 β-ENaC 的细胞外环对于 S3969 的 ENaC 刺激至关重要。分子动力学模拟预测了 β-ENaC 拇指结构域（Arg388、Phe391 和 Tyr406）中的关键残基，这些残基在位于 β-γ-亚基界面的结合位点内协调 S3969。重要的是，突变这些残基中的每一个都会减少（R388H；R388A）或几乎消除（F391G；Y406A）S3969介导的ENaC激活。分子动力学模拟还表明，S3969 介导的 ENaC 刺激涉及 β-ENaC 拇指结构域的 α5 螺旋远离 γ-ENaC 手掌结构域的移动。与此一致的是，引入两个半胱氨酸残基（βR437C – γS298C）以形成连接这两个结构域的二硫桥，阻止了 S3969 的 ENaC 刺激，除非二硫键被 DTT 还原。最后，我们证明 S3969 刺激培养的人气道上皮细胞 (H441) 中内源性表达的 ENaC。 这些新发现可能会为与 ENaC 功能改变相关的疾病带来新的（病理）生理学和治疗概念。