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

Anoctamins 的离子通道和脂质扰乱酶功能：在肌病中的作用

基本信息

批准号：
9027618
负责人：
H. CRISS HARTZELL
金额：
$ 34.1万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-17 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9027618
关键词：
Attention Binding Blood Coagulation Disorders Blood Platelets Cell membrane Cells Chemicals Coupled DYSF gene Data Defect Disease Dysplasia Dystonia Epithelial Exercise Exhibits Exocytosis Failure Functional disorder Genes Goals Healed Human Image Injury Investigation Ion Channel Ion Transport Ions Knock-out Lead Lesion Life Ligands Limb-Girdle Muscular Dystrophies Link Lipids Location Maintenance Measures Mediating Membrane Membrane Fusion Membrane Lipids Membrane Protein Traffic Membrane Proteins Molecular Monomeric GTP-Binding Proteins Mus Muscle Muscle Cells Muscle Fibers Muscular Dystrophies Mutation Myoblasts Myoepithelial cell Myopathy Natural regeneration Pathology Phosphatidylserines Phospholipids Physiological Play Process Protein Analysis Proteins Proteomics Regulation Role Scott syndrome Signal Transduction Site Skeletal Muscle Smooth Muscle Myocytes Specificity Spinocerebellar Ataxias Stem cells Structure Testing Variant annexin A5 anoctamin 5 base cell growth regulation cellular imaging extracellular healing human disease inhibitor/antagonist insight member mutant novel overexpression patch clamp phospholipid scramblase progenitor protein complex protein function public health relevance repaired satellite cell trafficking

项目摘要

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.

描述（由申请人提供）：Anoctamin-5 基因（ANO5、TMEM16E）的隐性突变导致肢带型肌营养不良症 2L（LGMD2L）、三好肌营养不良症 3（MMD3）和 ANO5 的其他全身性肌病的成员。 10基因超家族，其创始成员（ANO1和ANO2）是血浆由于 ANO5 与 ANO1 有 38% 相同（54% 相似），因此人们普遍认为 ANO5 是一种 Cl- 通道，并且最近认为 ANO5 肌病是由离子转运缺陷来解释的。很明显，一些 ANO，尤其是 ANO6（与 ANO5 相似 75%）具有附加功能：它们刺激磷脂加扰 (PLS)。质膜中磷脂不对称性的生理损失，以磷脂酰丝氨酸 (PtdSer) 从质膜细胞质小叶中的位置易位到细胞外小叶为代表。 PtdSer 在膜中的排列很重要，原因有二：众所周知，它是膜相关蛋白复合物组装的平台，并且是胞吞和胞吐过程中膜融合的重要调节因子。该应用测试了 ANO5 是一种磷脂扰乱酶和离子通道的假设，然后利用该信息来探索 ANO5 相关骨骼肌病理学的机制，以及相关肌病（如由 Dysferlin 突变引起的肌病）的解释。修复通常由运动产生的膜损伤的机制存在缺陷，这种损伤可以通过两个过程来治愈：（1）通过组装新的质膜来填充孔洞并重新封闭小损伤。 (2) 肌肉祖细胞（卫星细胞）融合，在更严重损伤的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发挥着重要作用 1。）我们将确定 ANO5 是否是磷脂扰乱酶、扰乱酶的调节剂和/或离子通道。我们将通过膜片钳和 PLS 通过荧光成像来评估离子通道功能。过表达 ANO5 的 HEK 细胞和内源表达 ANO5 的肌肉细胞中的磷脂探针 (2) 然后我们将研究培养的肌管中 ANO5 介导的 PLS 的细胞机制并测试离子转运是否发挥作用 (3) 我们将阐明。使用表达野生型、破坏型或突变型 ANO5 的肌管，ANO5 在膜修复中的作用此外，我们将评估致病性 ANO5 变体的功能以确定其功能。与肌病相关的人类 ANO5 变异的后果将在转染这些变异的肌管中得到表征。开启了一条全新的研究路线，可能会带来针对肌营养不良症的新疗法，尤其是由 ANO5 功能障碍引起的肌营养不良症，但也可能针对由肌膜脆性或修复缺陷引起的其他类型的肌营养不良症。