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

 描述(由申请人提供)：Anoctamin-5基因(ANO5，TMEM16E)的隐性突变会导致肢体-Girdle肌营养不良症2L(LGMD2L)、Miyoshi肌营养不良症3(MMD3)和其他全身性肌肉疾病。ANO5是一个10基因超家族的成员，其创始成员(ANO1和ANO2)是质膜钙激活的氯离子通道。因为ANO5与ANO1有38%的同源性(54%相似)，所以人们普遍认为ANO5是一个氯离子通道，ANO5的肌病是由离子转运缺陷来解释的。然而，最近，一些ANO变得明显，特别是ANO6-它与ANO5有75%的相似之处-有一个额外的功能：它们刺激磷脂扰乱(PLS)。PL是细胞膜磷脂不对称性的生理性丧失，表现为磷脂酰丝氨酸(PtdSer)从质膜内的细胞质小叶向细胞外小叶的移位。PtdSer在膜上的排列很重要，有两个原因：PtdSer被认为是膜相关蛋白复合体组装的平台，是细胞内外膜融合的重要调节因子。这项应用测试了ANO5是磷脂扰乱酶和离子通道的假设，然后利用这一信息来探索ANO5相关骨骼肌病理的机制。ANO5-肌病，以及相关的肌病，如由deferlin突变引起的肌病，可以通过修复正常运动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)通过组装新的质膜来填充孔洞来重新封闭小的损伤，以及(2)融合肌肉前体干细胞(卫星细胞)以在更严重的损伤部位再生新的肌肉纤维。我们认为ANO5介导的膜脂重组在这些过程中起着基础性的作用。有三个具体目标。(1)我们将确定ANO5是否是磷脂扰乱酶、扰乱酶的调节因子和/或离子通道。我们将通过膜片钳和偏最小二乘法对过度表达ANO5的HEK细胞和内源性表达ANO5的肌肉细胞中的荧光磷脂探针成像来评估离子通道功能。(2)我们将研究ANO5在培养的肌管中介导的偏最小二乘法的细胞机制，并测试离子转运是否起作用。(3)我们将阐明ANO5在表达野生型、破碎型或突变型ANO5的肌管膜修复中的作用。此外，我们将评估致病ANO5变异的功能，以确定与肌病相关的人类ANO5变异的功能后果。疾病相关的ANO5序列变异对离子通道功能、偏最小二乘法、膜修复和成肌细胞融合的影响将在转染这些变异的肌管中得到表征。这项研究有可能开启一条全新的研究路线，可能导致治疗肌肉营养不良的新疗法，特别是那些由ANO5功能障碍引起的肌肉营养不良，但也可能包括由肌膜脆性或修复缺陷引起的其他类型的肌肉营养不良。

 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.