Wellhauser Leigh, Kim Chiaw Patrick, Pasyk Stan, Li Canhui, Ramjeesingh Mohabir, Bear Christine E
Research Institute, Hospital for Sick Children and Departments of Biochemistry and Physiology, University of Toronto, Canada.
Mol Pharmacol. 2009 Jun;75(6):1430-8. doi: 10.1124/mol.109.055608. Epub 2009 Apr 1.
The deletion of Phe-508 (DeltaPhe508) constitutes the most prevalent of a number of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that cause cystic fibrosis (CF). This mutation leads to CFTR misfolding and retention in the endoplasmic reticulum, as well as impaired channel activity. The biosynthetic defect can be partially overcome by small-molecule "correctors"; once at the cell surface, small-molecule "potentiators" enhance the channel activity of DeltaPhe508-CFTR. Certain compounds, such as VRT-532, exhibit both corrector and potentiator functions. In the current studies, we confirmed that the inherent chloride channel activity of DeltaPhe508-CFTR (after biosynthetic rescue) is potentiated in studies of intact cells and membrane vesicles. It is noteworthy that we showed that the ATPase activity of the purified and reconstituted mutant protein is directly modulated by binding of VRT-532 [4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol] ATP turnover by reconstituted DeltaPhe508-CFTR is decreased by VRT-532 treatment, an effect that may account for the increase in channel open time induced by this compound. To determine whether the modification of DeltaPhe508-CFTR function caused by direct VRT-532 binding is associated with structural changes, we evaluated the effect of VRT-532 binding on the protease susceptibility of the major mutant. We found that binding of VRT-532 to DeltaPhe508-CFTR led to a minor but significant decrease in the trypsin susceptibility of the full-length mutant protein and a fragment encompassing the second half of the protein. These findings suggest that direct binding of this small molecule induces and/or stabilizes a structure that promotes the channel open state and may underlie its efficacy as a corrector of DeltaPhe508-CFTR.
苯丙氨酸508缺失（ΔPhe508）是囊性纤维化跨膜传导调节因子（CFTR）中导致囊性纤维化（CF）的多种突变中最常见的一种。这种突变导致CFTR错误折叠并滞留在内质网中，同时通道活性受损。小分子“校正剂”可部分克服这种生物合成缺陷；一旦到达细胞表面，小分子“增强剂”可增强ΔPhe508-CFTR的通道活性。某些化合物，如VRT-532，兼具校正剂和增强剂功能。在当前研究中，我们证实在完整细胞和膜囊泡研究中，ΔPhe508-CFTR（生物合成挽救后）的固有氯离子通道活性得到增强。值得注意的是，我们发现纯化并重组的突变蛋白的ATP酶活性可通过VRT-532[4-甲基-2-(5-苯基-1H-吡唑-3-基)-苯酚]的结合直接调节，VRT-532处理可降低重组ΔPhe508-CFTR的ATP周转，这种效应可能解释了该化合物诱导的通道开放时间增加。为了确定由VRT-532直接结合引起的ΔPhe508-CFTR功能改变是否与结构变化相关，我们评估了VRT-532结合对主要突变体蛋白酶敏感性的影响。我们发现VRT-532与ΔPhe508-CFTR的结合导致全长突变蛋白及包含该蛋白后半部分的片段对胰蛋白酶的敏感性轻微但显著降低。这些发现表明，这种小分子的直接结合诱导和/或稳定了促进通道开放状态的结构，这可能是其作为ΔPhe508-CFTR校正剂发挥功效的基础。
