Bertrand Carol A, Mitra Shalini, Mishra Sanjay K, Wang Xiaohui, Zhao Yu, Pilewski Joseph M, Madden Dean R, Frizzell Raymond A
Departments of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania;
Departments of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Am J Physiol Lung Cell Mol Physiol. 2017 Jun 1;312(6):L912-L925. doi: 10.1152/ajplung.00178.2016. Epub 2017 Mar 30.
Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9's interaction with CAL. Mutation of SLC26A9's PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9.
阴离子转运体SLC26A家族的几个成员与囊性纤维化跨膜传导调节因子（CFTR）相互作用，形成复合物，其中CFTR和SLC26A的功能相互调节。这些相互作用被认为是由PDZ支架相互作用促进的。在气道上皮细胞中，CFTR已被证明受NHERF - 1正向调节，受CAL负向调节。然而，尚不清楚哪些PDZ结构域蛋白与SLC26A9相互作用，SLC26A9是在气道上皮细胞中发现的SLC26A家族成员。我们之前已经表明，来自非囊性纤维化供体的原代人支气管上皮细胞（HBE）表现出由SLC26A9介导的组成性阴离子分泌。然而，囊性纤维化供体的HBE中不存在组成性阴离子分泌。我们研究了SLC26A9组成性活性的变化是否可归因于CFTR转运的丧失，以及PDZ相互作用发挥了什么作用。与共表达SLC26A9和野生型CFTR的细胞相比，共表达SLC26A9与转运突变体F508del CFTR的HEK293细胞的组成性电流显著降低。我们发现，当与F508del CFTR共表达时，SLC26A9表现出复杂的糖基化，但它在质膜上的表达降低。SLC26A9与NHERF - 1和CAL都相互作用，并且与野生型CFTR共表达时，它与两者的相互作用都显著增加。然而，与F508del CFTR共表达仅增加了SLC26A9与CAL的相互作用。SLC26A9的PDZ基序突变减少了与CAL的这种关联，并恢复了其组成性活性。用校正剂VX - 809纠正囊性纤维化HBE中异常的F508del CFTR转运也恢复了SLC26A9的活性。我们得出结论，当SLC26A9与F508del CFTR共表达时，其转运缺陷导致SLC26A9在细胞内以对PDZ基序敏感的方式滞留。
