The Mucolipin TRP Ion Channels

描述（由申请人提供）：瞬时受体电位（TRPML）蛋白的粘磷脂家族被预测为细胞内核内体和溶酶体的离子通道编码。人类TRPML1突变导致IV型粘脂病（ML4），这是幼儿中一种毁灭性的神经退行性疾病。ML4患者表现为运动缺陷、智力迟钝、视网膜变性和缺铁性贫血。TRPML3基因突变的小鼠（变异型waddler， Va小鼠）耳聋，表现出绕圈行为和色素沉着缺陷。ML4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。溶酶体，传统上被认为是生物“垃圾”的终端“回收中心”，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，由于缺乏对这些细胞内定位蛋白的可靠功能测定，TRPML蛋白的推测溶酶体功能一直不清楚。我们现在已经取得了技术上的突破，开发了一种膜片钳法来直接测量分离的晚期内核体/溶酶体中TRPML蛋白的功能。我们发现TRPML1是一个向内整流（阳离子流出溶酶体）阳离子通道，传导Ca2+和Fe2+。这些发现在分子和电生理学上鉴定了溶酶体中的第一个Ca2+/Fe2+通道。在ML4患者中发现的突变损害了TRPML1渗透Ca2+和Fe2+的能力，其程度与ML4疾病的严重程度密切相关。为了扩大我们的研究结果，本研究的目标是应用多学科方法，使用电生理学、Ca2+成像、免疫化学、生物化学和荧光成像来验证我们的中心假设，即TRPML1介导内体和溶酶体的阳离子外溢，以及溶酶体功能障碍和ML4表型的离子稳态受损。我们的第一个目标是确定TRPML1在内溶酶体铁释放中的作用。利用铁成像和铁染色方法，我们将确定在trpml1缺失的皮肤成纤维细胞中，内切溶酶体的铁释放是否受损。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+外排。通过细胞死亡试验，我们将研究trpml1缺陷细胞是否易受氧化应激的影响。使用细菌杀灭试验，我们将确定trpml1缺陷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TRPML1在溶酶体Ca2+信号传导中的作用。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Ca2+释放位点。利用电生理学和Ca2+成像，我们将专门测试TRPML1是否被已知的溶酶体Ca2+释放激活剂激活，以及诱导的溶酶体Ca2+释放是否在TRPML1缺陷成纤维细胞中缺失。从长远来看，该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（贫血和铁超载）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).