先天性肌强直症(MC)是一种遗传性罕见疾病,其特征是收缩后肌肉松弛受损,导致肌肉僵硬.它是由骨骼肌氯化物通道ClC-1中的功能丧失突变引起的,这对于稳定静息膜电位和动作电位的复极化阶段很重要。多亏了体外功能研究,ClC-1突变改变氯离子流入细胞的分子机制已经部分阐明,将它们分类为“门控缺陷”或“表达缺陷”突变。迄今为止,由于没有直接的ClC-1激活剂,因此MC的治疗仅是姑息性的。理想的药物应该是在门控缺陷突变的情况下能够纠正ClC-1的生物物理缺陷的药物,或者是能够在质膜上恢复ClC-1蛋白表达的药物,用于运输缺陷的药物。在这项研究中,我们测试了尼氟灭酸(NFA)的能力,一种商业非甾体抗炎药,作为贩运缺陷型MC突变体的药理伴侣(A531V,V947E)。在HEK293细胞中表达野生型(WT)或MC突变体ClC-1通道,并在NFA孵育前后用膜片钳技术记录全细胞氯电流。进行膜生物素化测定和蛋白质印迹以支持电生理结果。由于ClC-1总蛋白水平和质膜上的通道表达减少,A531V和V947E突变导致氯化物电流密度降低。用50µMNFA处理A531V和V947E转染的细胞恢复了氯电流,达到与WT相似的水平。此外,电压依赖性没有观察到显著差异,这表明NFA增加了蛋白质膜的表达而不改变ClC-1的功能。的确,生化实验证实,NFA孵育后V947E总蛋白表达及其质膜分布恢复,达到与WT相似的蛋白质水平。因此,在贩运缺陷型ClC-1通道突变中使用NFA作为药物伴侣可能是治疗MC的良好策略.由于这种药物的良好安全性,我们的研究可能很容易为旨在验证携带特定ClC-1通道突变的部分患者NFA的抗强直性活性的确证人类试验研究开辟道路.
Myotonia congenita (MC) is an inherited rare disease characterized by impaired muscle relaxation after contraction, resulting in muscle stiffness. It is caused by loss-of-function mutations in the skeletal muscle chloride channel ClC-1, important for the stabilization of resting membrane potential and for the repolarization phase of action potentials. Thanks to in vitro functional studies, the molecular mechanisms by which ClC-1 mutations alter chloride ion influx into the cell have been in part clarified, classifying them in \"gating-defective\" or \"expression-defective\" mutations. To date, the treatment of MC is only palliative because no direct ClC-1 activator is available. An ideal drug should be one which is able to correct biophysical defects of ClC-1 in the case of gating-defective mutations or a drug capable to recover ClC-1 protein expression on the plasma membrane for trafficking-defective ones. In this study, we tested the ability of niflumic acid (NFA), a commercial nonsteroidal anti-inflammatory drug, to act as a pharmacological chaperone on trafficking-defective MC mutants (A531V, V947E). Wild-type (WT) or MC mutant ClC-1 channels were expressed in HEK293 cells and whole-cell chloride currents were recorded with the patch-clamp technique before and after NFA incubation. Membrane biotinylation assays and western blot were performed to support electrophysiological results. A531V and V947E mutations caused a decrease in chloride current density due to a reduction of ClC-1 total protein level and channel expression on the plasma membrane. The treatment of A531V and V947E-transfected cells with 50 µM NFA restored chloride currents, reaching levels similar to those of WT. Furthermore, no significant difference was observed in voltage dependence, suggesting that NFA increased protein membrane expression without altering the function of ClC-1. Indeed, biochemical experiments confirmed that V947E total protein expression and its plasma membrane distribution were recovered after NFA incubation, reaching protein levels similar to WT. Thus, the use of NFA as a pharmacological chaperone in trafficking defective ClC-1 channel mutations could represent a good strategy in the treatment of MC. Because of the favorable safety profile of this drug, our study may easily open the way for confirmatory human pilot studies aimed at verifying the antimyotonic activity of NFA in selected patients carrying specific ClC-1 channel mutations.