Ion Channel and Lipid Scramblase Functions of Anoctamins: Roles in Myopathy

 描述（由申请人提供）：Anoctamin-5基因（ANO 5，TMEM 16 E）的隐性突变导致肢带型肌营养不良2L（LGMD 2L）、三好型肌营养不良3（MMD 3）和其他全身性肌病。ANO 5是一个由10个基因组成的超家族的成员，其创始成员（ANO 1和ANO 2）是质膜Ca 2+激活的Cl-通道。由于ANO 5与ANO 1有38%的相同性（54%的相似性），因此人们普遍认为ANO 5是一种Cl-通道，ANO 5肌病是由离子转运缺陷引起的。然而，最近，一些ANO，特别是与ANO 5相似度为75%的ANO 6，具有额外的功能：它们刺激磷脂扰乱（PLS）。PLS是质膜中磷脂不对称性的生理损失，其典型表现为磷脂酰丝氨酸（PtdSer）从其在质膜的细胞质小叶中的位置易位到细胞外小叶。PtdSer在膜中的排列是重要的，原因有二：已知PtdSer充当膜相关蛋白复合物组装的平台，并且是胞内和胞外作用期间膜融合的重要调节剂。该应用测试了ANO 5是磷脂乱序酶和离子通道的假设，然后使用该信息来探索ANO 5相关骨骼肌病理学的机制。ANO 5-肌病和相关的肌病，如dysferlin突变引起的肌病，可以通过修复运动正常产生的膜损伤的机制缺陷来解释。这种损伤通过两个过程愈合：（1）通过组装新的质膜以填充孔来重新密封小损伤，以及（2）肌肉祖干细胞（卫星细胞）的融合以在更严重损伤的部位再生新的肌纤维。我们建议，由ANO 5介导的膜脂重组在这些过程中起着至关重要的作用。有三个具体目标。(1)我们将确定ANO 5是否是一种磷脂乱序酶、一种乱序酶的调节剂和/或一种离子通道。我们将通过膜片钳和PLS通过在过表达ANO 5的HEK细胞和内源性表达ANO 5的肌肉细胞中成像荧光磷脂探针来评估离子通道功能。(2)然后，我们将研究ANO 5介导的PLS在培养的肌管中的细胞机制，并测试离子转运是否起作用。(3)我们将阐明ANO 5在膜修复中的作用，使用表达野生型，破坏，或突变ANO 5的肌管。此外，我们将评估致病性ANO 5变异体的功能，以确定与肌病相关的ANO 5人类变异的功能后果。疾病相关的ANO 5序列变体对离子通道功能、PLS、膜修复和成肌细胞融合的影响将在用这些变体转染的肌管中表征。这项研究有可能开辟一条全新的研究路线，可能导致肌营养不良症的新疗法，特别是由ANO 5功能障碍引起的肌营养不良症，但也可能导致由肌膜脆性或修复缺陷引起的其他类型的肌营养不良症。

 DESCRIPTION (provided by applicant): Recessive mutations in the Anoctamin-5 gene (ANO5, TMEM16E) cause Limb-Girdle Muscular Dystrophy 2L (LGMD2L), Miyoshi Muscular Dystrophy 3 (MMD3), and other generalized myopathies. ANO5 is a member of a 10-gene superfamily, the founding members of which (ANO1 and ANO2) are plasma membrane Ca2+-activated Cl- channels. Because ANO5 is 38% identical (54% similar) to ANO1, it is widely assumed that ANO5 is a Cl- channel and that ANO5 myopathies are explained by defects in ion transport. Recently, however, it has become apparent that some ANOs, notably ANO6 - which is 75% similar to ANO5, have an additional function: they stimulate phospholipid scrambling (PLS). PLS is the physiological loss of phospholipid asymmetry in the plasma membrane, typified by the translocation of phosphatidylserine (PtdSer) from its location in the cytoplasmic leaflet of the plasma membrane to the extracellular leaflet. The arrangement of PtdSer in the membrane is important for two reasons: PtdSer is known to serve as a platform for the assembly of membrane-associated protein complexes and is an important regulator of membrane fusion during endo- and exo- cytosis. This application tests the hypothesis that ANO5 is a phospholipid scramblase and an ion channel and then uses this information to explore the mechanisms of ANO5-associated skeletal muscle pathology. ANO5-myopathies, and related myopathies like ones caused by mutations in dysferlin, are explained by defects in mechanisms that repair membrane injury produced normally by exercise. Such injury is healed by two processes: (1) resealing of small lesions by assembly of new plasma membrane to fill the holes and (2) fusion of muscle progenitor stem cells (satellite cells) to regenerate new muscle fibers at sites of more severe damage. We propose that reorganization of membrane lipids mediated by ANO5 plays a fundamental role in these processes. There are three specific aims. (1) We will determine if ANO5 is a phospholipid scramblase, a regulator of a scramblase, and/or an ion channel. We will evaluate ion channel function by patch clamp and PLS by imaging fluorescent phospholipid probes in both HEK cells overexpressing ANO5 and in muscle cells endogenously expressing ANO5. (2) We will then investigate the cellular mechanisms of ANO5-mediated PLS in cultured myotubes and test whether ion transport plays a role. (3) We will elucidate the role of ANO5 in membrane repair using myotubes expressing wild type, disrupted, or mutant ANO5. Further, we will evaluate the function of pathogenic ANO5 variants to determine the functional consequences of human variations in ANO5 that are linked to myopathy. The effects of disease-associated ANO5 sequence variants on ion channel function, PLS, membrane repair, and myoblast fusion will be characterized in myotubes transfected with these variants. This study has the potential to open a completely novel line of investigation that may lead to new therapies for muscular dystrophies, especially those caused by ANO5 dysfunction, but potentially also other types of muscular dystrophies caused by muscle membrane fragility or defective repair.