Grosche Astrid, Sasidharan Anusree, Salathe Matthias, Baumlin Nathalie, Angoli Damiano, Prabhakaran Sreekala, Xu Xiaodong, Vidyasagar Sadasivan
Department of Radiation Oncology, University of Florida, Gainesville, FL, United States.
Department of Internal Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, University of Kansas Medical Center, Kansas City, KS, United States.
Front Pharmacol. 2025 Aug 4;16:1522130. doi: 10.3389/fphar.2025.1522130. eCollection 2025.
In cystic fibrosis (CF), most CFTR mutations cause partial (Class II) or complete (Class I) loss of function. Modulators (VX) can improve CFTR function in Class II mutations but are ineffective for Class I mutations and may cause side effects, resulting in tolerability issues with concerns about long-term safety. Apical anion secretion, essential for maintaining airway surface liquid (ASL) homeostasis, is regulated by CFTR. Alternative anion channels, like ANO1 and SLC26A9, also contribute to ASL homeostasis. Our recent work indicates that specific amino acids can modulate ion channel expression, activity, and trafficking in epithelial cells. We developed a select amino acid formulation (SAA) to enhance anion secretion in primary human bronchial epithelial cells (HBEC) with CF, regardless of mutation.
Transepithelial short-circuit current was measured in wildtype (WT)- and CF-HBEC with various Class I and Class II mutations. Cells were pretreated with DMSO or VX for 24 h before apical exposure to SAA in Ussing chambers. Benzamil-insensitive current was sequentially inhibited to determine the contributions of SLC26A9, CFTR, ANO1, and NKCC1. Cl unidirectional and net fluxes ( Cl) validated chloride secretion. Whole-cell patch-clamp studies determined the current density with SAA in WT- and CF-HBEC. CFTR, SLC26A9, and ANO1 mRNA and protein expression levels were assessed via qPCR and immunofluorescence. ASL volume, ciliary beat frequency (CBF), and mucociliary transport were also assessed.
SAA increased benzamil-insensitive current to 70%-85% of WT cells, and enhanced Cl in both Class I and II mutations. Cl contributed to 72%, 50%, and 39.5% of S9A13-inhibitable current in WT-, F508del-, and G542X/R785X-HBEC, respectively. VX treatment increased current in Class II but did not affect Class I mutations. Increased chloride secretion with SAA was attributed to enhanced activity of SLC26A9 and partial CFTR restoration through elevated mRNA and membrane protein expression. SAA also increased ASL volume and CBF, confirming its effectiveness in Class I mutations.
SAA enhances chloride secretion through SLC26A9 and partial CFTR rescue in Class I and II mutations. These findings suggest SAA functions as a mutation-agnostic therapy to improve anion secretion and clinical symptoms, particularly in Class I mutations.
在囊性纤维化（CF）中，大多数CFTR突变会导致部分（II类）或完全（I类）功能丧失。调节剂（VX）可改善II类突变中的CFTR功能，但对I类突变无效，且可能引起副作用，导致耐受性问题以及对长期安全性的担忧。顶端阴离子分泌对于维持气道表面液体（ASL）稳态至关重要，受CFTR调节。其他阴离子通道，如ANO1和SLC26A9，也有助于ASL稳态。我们最近的研究表明，特定氨基酸可调节上皮细胞中的离子通道表达、活性和转运。我们开发了一种精选氨基酸配方（SAA），以增强患有CF的原代人支气管上皮细胞（HBEC）中的阴离子分泌，无论其为何种突变。
在具有各种I类和II类突变的野生型（WT）-和CF-HBEC中测量跨上皮短路电流。在Ussing小室中，细胞在顶端暴露于SAA之前，先用二甲基亚砜（DMSO）或VX预处理24小时。依次抑制苯扎明不敏感电流，以确定SLC26A9、CFTR、ANO1和NKCC1的贡献。氯离子单向和净通量（Cl）验证了氯化物分泌。全细胞膜片钳研究确定了WT-和CF-HBEC中SAA的电流密度。通过定量聚合酶链反应（qPCR）和免疫荧光评估CFTR、SLC26A9和ANO1的mRNA和蛋白质表达水平。还评估了ASL体积、纤毛摆动频率（CBF）和黏液纤毛运输。
SAA将苯扎明不敏感电流增加至WT细胞的70%-85%，并增强了I类和II类突变中的Cl。在WT-、F508del-和G542X/R785X-HBEC中，Cl分别占S9A13可抑制电流的72%、50%和39.5%。VX处理增加了II类突变中的电流，但不影响I类突变。SAA导致的氯化物分泌增加归因于SLC26A9活性增强以及通过升高mRNA和膜蛋白表达实现的CFTR部分恢复。SAA还增加了ASL体积和CBF，证实了其在I类突变中的有效性。
SAA通过SLC26A9增强氯化物分泌，并在I类和II类突变中部分挽救CFTR。这些发现表明SAA作为一种不依赖突变的疗法，可改善阴离子分泌和临床症状，特别是在I类突变中。
