TRPML1 channel physiology and pathophysiology

 描述（由申请方提供）：IV型粘脂沉积症（ML IV）是一种重度溶酶体贮积症，其特征为智力和精神发育迟缓、视网膜变性和角膜混浊、缺铁和无氯血症（胃酸水平低）。患有ML IV的儿童通常表现出认知发育迟缓，语言和运动缺陷以及失明，并且通常在语言和运动功能方面达到18个月的最大发育年龄。这种毁灭性的疾病是由编码TRPML 1的基因突变引起的，TRPML 1是TRP离子通道家族的瞬时受体电位粘脂（TRPML）亚家族的成员。TRPML 1通道主要存在于许多细胞类型的细胞内细胞器，特别是晚期内体和溶酶体中。它们是非选择性阳离子通道，可渗透所有单价阳离子。重要的是，TRPML 1通道对Ca 2+和Fe 2+是可渗透的，因此可能对溶酶体中的Ca 2+信号传导至关重要。TRPML 1在溶酶体中的生理功能以及其功能障碍如何导致ML IV在很大程度上尚不清楚。本课题旨在研究TRPML 1的结构、调控、生理功能及致病机制。溶酶体富含脂质，含有高浓度的管腔Ca 2+和Fe 2+，pH值低至约4.5。TRPML 1通道渗透Ca 2+和Fe 2+，并受管腔Ca 2+和pH值的调节。这些特性可能对TRPML 1通道的生理功能至关重要，但其分子和生物物理机制尚不清楚。获得TRPML 1功能重要结构域的高分辨率结构将极大地增强我们对TRPML 1通道生理学和病理生理学的理解。我们已经解决了TRPML 1的前两个跨膜片段之间的~210个氨基酸的接头（称为I-II接头）的晶体结构，该区域是通道功能所必需的。重要的是，该接头含有三个导致ML IV的单个氨基酸突变。结构显示I-II接头形成中心具有孔（称为管腔孔）的四聚体。管腔孔的直径约为14 μ m，并由含有三个天冬氨酸残基和一个推定的丝氨酸脂肪酶位点的管腔孔环内衬，该位点似乎无催化活性。以该晶体结构为蓝本，我们将追求以下具体目标：（1）研究I-II接头在TRPML 1组装和溶酶体靶向中的作用;（2）研究I-II接头ML IV引起的突变对TRPML 1组装、靶向和活性的影响;（3）研究I-II接头在Ca 2+和Fe 2+渗透中的作用以及TRPML 1介导的Ca 2+和Fe 2+信号传导是否对溶酶体生理学至关重要;（4）检验I-II接头对于通过管腔pH调节TRPML 1通道是关键的假设， Ca 2+，这种双重调节对溶酶体生理学很重要。这些研究将产生TRPML 1通道功能的机制见解，阐明ML IV的致病机制，并为这种毁灭性疾病的治疗策略的发展奠定基础。

 DESCRIPTION (provided by applicant): Mucolipidosis type IV (ML IV) is a severe lysosomal storage disorder characterized by mental and psychomotor retardation, retinal degeneration and corneal opacity, iron deficiency, and achlorhydria (low stomach acid level). Children with ML IV often exhibit cognitive retardation, language and motor deficits, and blindness, and typically reach a maximum developmental age of 18 months in language and motor function. This devastating disease is caused by mutations in the gene encoding TRPML1, a member of the transient receptor potential mucolipin (TRPML) subfamily of the TRP family of ion channels. TRPML1 channels are primarily found in intracellular organelles, especially late endosomes and lysosomes, of many cell types. They are non-selective cation channels, permeable to all monovalent cations. Importantly, TRPML1 channels are permeable to Ca2+ and Fe2 and therefore likely critical for Ca2+ signaling in lysosomes. The physiological function of TRPML1 in lysosomes and how its dysfunction leads to ML IV are largely unclear. The objective of this project is to study the structure, regulation, physiological functions and pathogenic mechanisms of TRPML1. Lysosomes are enriched in lipids, contain high concentrations of luminal Ca2+ and Fe2+, and have a low pH of ~4.5. TRPML1 channels permeate both Ca2+ and Fe2+ and are regulated by luminal Ca2+ and pH. These properties may be crucial for the physiological functions of TRPML1 channels, but the molecular and biophysical mechanisms underlying them are unclear. Obtaining high-resolution structures of functionally important domains of TRPML1 would greatly enhance our understanding of TRPML1 channel physiology and pathophysiology. We have solved the crystal structure of a ~210-amino acid linker (named the I-II linker) between the first two transmembrane segments of TRPML1, a region necessary for channel function. Importantly, this linker harbors three single amino acid mutations that cause ML IV. The structure shows that the I-II linker forms a tetramer with a pore (called the luminal pore) in the center. The luminal pore is has a diameter of ~14 Å and is lined by a luminal pore-loop containing three aspartate residues and a putative serine lipase site, which appears catalytically inactive. Using this crystal structure as a blueprint, we will pursue the following specific aims: 1) Studying the role of the I-II linker in TRPML1 assembly and lysosomal targeting; (2) Studying the effect of I-II linker ML IV-causing mutations on TRPML1 assembly, targeting and activity; (3) Investigating the role of the I-II linker in Ca2+ and Fe2+ permeation and whether TRPML1-mediated Ca2+ and Fe2+ signaling is crucial for lysosome physiology; (4) Testing the hypothesis that the I-II linker is critical for the regulation of TRPML1 channels by luminal pH and Ca2+ and that this dual regulation is important for lysosome physiology. These studies will yield mechanistic insights into TRPML1 channel functions, shed light on the pathogenic mechanisms of ML IV, and lay a foundation for the development of treatment strategies for this devastating disease.