Sure Florian, Einsiedel Jürgen, Gmeiner Peter, Duchstein Patrick, Zahn Dirk, Korbmacher Christoph, Ilyaskin Alexandr V
Institute of Cellular and Molecular Physiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
Department of Chemistry and Pharmacy, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
J Biol Chem. 2024 Apr;300(4):105785. doi: 10.1016/j.jbc.2024.105785. Epub 2024 Feb 23.
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 Xenopus laevis 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.
上皮钠通道（ENaC）对于介导多种上皮组织中的钠吸收至关重要。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，ENaC药理学激活剂具有潜在的治疗意义。此前，已开发出一种小分子ENaC激活剂（S3969）。到目前为止，对于S3969介导的ENaC刺激所涉及的分子机制知之甚少。在此，我们通过将基于结构的模拟与分子生物学方法以及在非洲爪蟾卵母细胞中异源表达的ENaC的电生理测量相结合，确定了人ENaC中的一个S3969结合位点。我们证实了先前的一项观察结果，即β - ENaC的细胞外环对于S3969刺激ENaC至关重要。分子动力学模拟预测了β - ENaC拇指结构域中的关键残基（Arg388、Phe391和Tyr406），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降低（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键会阻止S3969对ENaC的刺激，除非二硫键被二硫苏糖醇（DTT）还原。最后，我们证明了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 - ）生理和治疗概念。
