The Mucolipin TRP Ion Channels

描述(由申请人提供)：瞬时受体潜力(TRPML)蛋白的粘脂家族被预测为编码细胞内内小体和溶酶体的离子通道。人类TRPML1的突变会导致IV型粘脂沉积症(ML4)，这是一种幼儿的毁灭性神经退行性疾病。ML4患者表现为运动障碍、智力低下、视网膜变性和缺铁性贫血。带有TRPML3突变的小鼠(Va小鼠)是聋人，表现出旋转行为和色素缺陷。ML4和Va的广谱表型似乎都是内小体/溶酶体功能障碍的某些方面的结果。溶酶体，传统上被认为是生物“垃圾”的末端“回收中心”，最近被发现在多种细胞内信号通路中扮演着不可或缺的角色。然而，由于缺乏可靠的细胞内定位蛋白的功能分析，TRPML蛋白的假定溶酶体功能(S)在很大程度上还不清楚。我们现在已经取得了技术突破，开发了一种膜片钳方法来直接检测分离的晚期内小体/溶酶体中TRPML蛋白的功能。我们发现，TRPML1是一种内向整流(阳离子流出溶酶体)的阳离子通道，同时传导钙和铁。这些发现在分子和电生理学上确定了溶酶体中的第一个钙/铁通道。在ML4患者中发现的突变损害了TRPML1‘S渗透钙和Fe2的能力，其程度与ML4疾病的严重程度密切相关。为了扩大我们的发现，这项拟议研究的目标是应用电生理学、钙离子成像、免疫化学、生物化学和荧光成像等多学科方法来测试我们的中心假设，即TRPML1介导阳离子从内体和溶酶体外流，离子稳态受损是溶酶体功能障碍和ML4表型的基础。我们的第一个目标是确定TRPML1在内溶酶体铁释放中的作用。使用铁成像和铁染色方法，我们将确定在TRPML1缺乏的皮肤成纤维细胞中，内溶酶体释放铁是否受到损害。我们的第二个目标是研究TRPML1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Fe2/Ca2外流。利用细胞死亡分析，我们将研究TRPML1缺陷细胞是否对氧化应激敏感。使用细菌杀灭试验，我们将确定TRPML1缺陷的巨噬细胞是否表现出降低的杀菌活性。我们的第三个目标是研究TRPML1在溶酶体钙信号转导中的作用。与内质网(ER)一样，溶酶体也被认为是某些细胞信号转导过程中的钙释放部位。利用电生理学和钙成像技术，我们将专门测试TRPML1是否被已知的溶酶体钙释放激活剂激活，以及在TRPML1缺陷的成纤维细胞中是否没有诱导的溶酶体钙释放。从长远来看，这些结果应该为铁相关疾病(贫血和铁负荷过载)和退行性疾病(视网膜和神经退行性变)的治疗方法提供临床见解。公共卫生相关性：我们建议的研究结果应该为铁相关疾病(贫血和铁超载)和退行性疾病(视网膜和神经退行性变)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).