The Mucolipin TRP Ion Channels

描述（由申请方提供）：预期瞬时受体电位（TRPML）蛋白的粘脂家族编码细胞内内体和溶酶体的离子通道。人类TRPML 1突变导致IV型粘脂沉积症（ML 4），这是一种在幼儿中具有破坏性的神经退行性疾病。ML 4患者表现出运动缺陷、智力迟钝、视网膜变性和缺铁性贫血。TRPML 3突变的小鼠（varitint-waddler，Va小鼠）是耳聋的，表现出盘旋行为和色素沉着缺陷。ML 4和Va的广谱表型似乎是由内体/溶酶体功能障碍的某些方面引起的。传统上被认为是生物“垃圾”的终端“回收中心”的溶酶体，最近被发现在多种细胞内信号通路中起着不可或缺的作用。然而，TRPML蛋白的推定溶酶体功能一直不清楚，主要是由于缺乏对这些细胞内定位蛋白的可靠功能测定。我们现在已经通过开发膜片钳方法直接测量分离的晚期内体/溶酶体中TRPML蛋白的功能取得了技术突破。我们发现，TRPML 1是一个内向整流（阳离子流出溶酶体）的阳离子通道传导Ca 2+和Fe 2+。这些发现在分子和电生理学上鉴定了溶酶体中的第一个Ca 2 +/Fe 2+通道。在ML 4患者中发现的突变损害TRPML 1渗透Ca 2+和Fe 2+的能力，其程度与ML 4疾病的严重程度密切相关。为了扩大我们的研究结果，提出的研究的目标是应用多学科的方法，使用电生理学，Ca 2+成像，免疫化学，生物化学和荧光成像来测试我们的中心假设，即TRPML 1介导阳离子从内体和溶酶体流出，受损的离子稳态是溶酶体功能障碍和ML 4表型的基础。我们的第一个目标是确定TRPML 1在内溶酶体铁释放中的作用。使用铁成像和铁染色方法，我们将确定是否从内溶酶体铁释放TRPML 1缺陷的皮肤成纤维细胞受损。我们的第二个目的是研究TRPML 1在溶酶体介导的细胞生物学功能中的作用，这些功能已被证明涉及溶酶体的Fe 2 +/Ca 2+流出。使用细胞死亡测定，我们将研究TRPML 1缺陷细胞是否对氧化应激敏感。使用细菌杀伤试验，我们将确定TRPML 1缺陷的巨噬细胞是否表现出降低的杀菌活性。我们的第三个目的是研究TRPML 1在溶酶体Ca 2+信号转导中的作用。与内质网（ER）一样，溶酶体也被认为是某些细胞信号传导过程中的Ca 2+释放位点。使用电生理学和Ca 2+成像，我们将专门测试TRPML 1是否被已知的溶酶体Ca 2+释放激活剂激活，以及TRPML 1缺陷成纤维细胞中是否不存在诱导的溶酶体Ca 2+释放。从长远来看，这些结果应该为铁相关疾病（贫血和铁过载）和退行性疾病（视网膜和神经变性）的治疗方法提供临床见解。公共卫生相关性：我们提出的研究结果应该为铁相关疾病（贫血和铁过载）和退行性疾病（视网膜和神经变性）的治疗方法提供临床见解。

DESCRIPTION (provided by applicant): The mucolipin family of Transient Receptor Potential (TRPML) proteins is predicted to encode ion channels of intracellular endosomes and lysosomes. Mutations of human TRPML1 cause type IV mucolipidosis (ML4), a devastating neurodegenerative disease in young children. ML4 patients exhibit motor defects, mental retardation, retinal degeneration, and iron-deficiency anemia. Mice with mutations in TRPML3 (the varitint-waddler, Va mice) are deaf and exhibit circling behavior and pigmentation defects. The broad-spectrum phenotypes of both ML4 and Va appear to result from certain aspects of endosomal/lysosomal dysfunction. Lysosomes, traditionally believed to be the terminal "recycle center" for biological "garbage", have recently been revealed to play indispensable roles in multiple intracellular signaling pathways. The putative lysosomal function(s) of TRPML proteins, however, has been unclear largely due to the lack of a reliable functional assay for these intracellularly-localized proteins. We have now made a technical breakthrough by developing a patch-clamp method to directly measure the functions of TRPML proteins in the isolated late endosome/lysosome. We found that TRPML1 is an inwardly-rectifying (cations flowing out of the lysosome) cation channel conducting both Ca2+ and Fe2+. These findings molecularly and electrophysiologically identified the first Ca2+/Fe2+ channel in the lysosome. Mutations found in ML4 patients impair TRPML1's ability to permeate Ca2+ and Fe2+ at degrees that correlate well with the severity of the ML4 disease. To expand our findings, the goal of the proposed research is to apply a multidisciplinary approach using electrophysiology, Ca2+ imaging, immunochemistry, biochemistry, and fluorescence imaging to test our central hypotheses that TRPML1 mediates cation efflux from endosomes and lysosomes and that impaired ion homeostasis underlies lysosomal dysfunction and ML4 phenotypes. Our first aim is to determine the role of TRPML1 in endolysosomal iron release. Using iron imaging and iron staining methods, we will determine whether iron release from endo-lysosomes is impaired in TRPML1-deficient skin fibroblasts. Our second aim is to investigate the roles of TRPML1 in the lysosome-mediated cell biological functions that have been shown to involve Fe2+/Ca2+ efflux from lysosomes. Using cell death assays, we will investigate whether TRPML1-deficient cells are susceptible to oxidative stress. Using bacteria killing assays, we will determine whether TRPML1-deficient macrophages exhibit reduced bactericidal activity. Our third aim is to investigate the role of TRPML1 in lysosomal Ca2+ signaling. Like the endoplasmic reticulum (ER), lysosomes are also believed to be the Ca2+ release sites during certain cellular signaling. Using electrophysiology and Ca2+ imaging, we will specifically test whether TRPML1 is activated by known lysosome Ca2+-release activators, and whether the induced lysosomal Ca2+ release is absent in TRPML1-deficient fibroblasts. In the long term, the results should provide clinical insights into therapeutic approaches for both iron-related disorders (anemia and iron overload) and degenerative diseases (retinal and neural degeneration). PUBLIC HEALTH RELEVANCE: The outcome of our proposed research should provide clinical insights into therapeutic approaches for both iron-related disorders (anemia and iron overload) and degenerative diseases (retinal and neural degeneration).