Abstract：
The function of the chloride channel ClC-1 is crucial for the control of muscle excitability. Thus, reduction of ClC-1 functions by CLCN1 mutations leads to myotonia congenita. Many different animal models have contributed to understanding the myotonia pathophysiology. However, these models do not allow in vivo screening of potentially therapeutic drugs, as the zebrafish model does. In this work, we identified and characterized the two zebrafish orthologues (clc-1a and clc-1b) of the ClC-1 channel. Both channels are mostly expressed in the skeletal muscle as revealed by RT-PCR, western blot, and electrophysiological recordings of myotubes, and clc-1a is predominantly expressed in adult stages. Characterization in Xenopus oocytes shows that the zebrafish channels display similar anion selectivity and voltage dependence to their human counterparts. However, they show reduced sensitivity to the inhibitor 9-anthracenecarboxylic acid (9-AC), and acidic pH inverts the voltage dependence of activation. Reduction of clc-1a/b expression hampers spontaneous and mechanically stimulated movement, which could be reverted by expression of human ClC-1 but not by some ClC-1 containing myotonia mutations. Treatment of clc-1-depleted zebrafish with mexiletine, a typical drug used in human myotonia, improves the motor behaviour. Our work extends the repertoire of ClC channels to evolutionary structure-function studies and proposes the zebrafish clcn1 crispant model as a simple tool to find novel therapies for myotonia. KEY POINTS: We have identified two orthologues of ClC-1 in zebrafish (clc-1a and clc-1b) which are mostly expressed in skeletal muscle at different developmental stages. Functional characterization of the activity of these channels reveals many similitudes with their mammalian counterparts, although they are less sensitive to 9-AC and acidic pH inverts their voltage dependence of gating. Reduction of clc-1a/b expression hampers spontaneous and mechanically stimulated movement which could be reverted by expression of human ClC-1. Myotonia-like symptoms caused by clc-1a/b depletion can be reverted by mexiletine, suggesting that this model could be used to find novel therapies for myotonia.
摘要：
氯通道ClC-1的功能对于控制肌肉兴奋性至关重要。因此，CLCN1突变导致ClC-1功能降低导致先天性肌强直。许多不同的动物模型有助于理解肌强直的病理生理学。然而,这些模型不允许在体内筛选潜在的治疗药物，就像斑马鱼模型一样。在这项工作中,我们鉴定并表征了ClC-1通道的两个斑马鱼直向同源物（clc-1a和clc-1b）。通过RT-PCR显示，这两个通道大多在骨骼肌中表达，westernblot,和肌管的电生理记录，clc-1a主要在成年期表达。在非洲爪鱼卵母细胞中的表征表明，斑马鱼通道显示出与其人类对应物相似的阴离子选择性和电压依赖性。然而,它们对抑制剂9-蒽羧酸（9-AC）的敏感性降低，和酸性pH值反转激活的电压依赖性。clc-1a/b表达的减少阻碍了自发和机械刺激的运动，可以通过人ClC-1的表达而不是通过一些含有ClC-1的肌强直突变来恢复。用美西律治疗clc-1耗尽的斑马鱼，一种用于人类肌强直的典型药物，改善电机行为。我们的工作将ClC通道的功能扩展到进化结构功能研究中，并提出了斑马鱼clcn1脆皮模型作为寻找肌强直新疗法的简单工具。关键点：我们已经确定了斑马鱼中ClC-1的两个直系同源物（clc-1a和clc-1b），它们大多在不同发育阶段的骨骼肌中表达。这些通道活性的功能表征揭示了与哺乳动物对应物的许多相似之处，尽管它们对9-AC和酸性pH值较不敏感，但它们对门控的电压依赖性。clc-1a/b表达的减少阻碍了自发和机械刺激的运动，这可以通过人ClC-1的表达来逆转。由clc-1a/b耗竭引起的肌强直样症状可以通过美西律逆转，这表明该模型可用于寻找治疗肌强直的新疗法。
