TRPML1 channel physiology and pathophysiology

 描述(申请人提供)：IV型粘脂沉积症(ML IV)是一种严重的溶酶体储存障碍，特征是智力和精神运动迟缓、视网膜变性和角膜混浊、缺铁和酸中毒(低胃酸水平)。患有ML IV的儿童通常表现出认知迟缓、语言和运动障碍以及失明，通常在语言和运动功能方面的最高发育年龄为18个月。这种毁灭性的疾病是由编码TRPML1的基因突变引起的，TRPML1是Trp家族离子通道的瞬时受体潜在粘蛋白(TRPML)亚家族的成员。TRPML1通道主要存在于多种细胞类型的胞内细胞器中，尤其是晚期内小体和溶酶体。它们是非选择性的阳离子通道，可被所有单价阳离子渗透。重要的是，TRPML1通道对Ca~(2+)和Fe~(2+)具有通透性，因此可能对溶酶体内的Ca~(2+)信号转导起关键作用。TRPML1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。本课题的目的是研究TRPML1的结构、调控、生理功能及其致病机制。溶酶体富含脂质，含有高浓度的钙离子和铁离子，pH值低至~4.5。TRPML1通道对钙离子和铁离子均有渗透作用，并受钙离子和pH的调节。这些特性可能对TRPML1通道的生理功能至关重要，但其背后的分子和生物物理机制尚不清楚。获得TRPML1重要功能结构域的高分辨率结构将极大地加深我们对TRPML1通道生理学和病理生理学的理解。我们已经解决了TRPML1前两个跨膜片段之间的~210个氨基酸连接体(称为I-II连接体)的晶体结构，这是通道功能所必需的区域。重要的是，这个连接子含有三个导致ML IV的单一氨基酸突变。结构表明，I-II连接子形成一个四聚体，中心有一个孔(称为腔孔)。管腔孔的直径为~14ä，内侧有一个管腔孔环，其中包含三个天冬氨酸残基和一个可能的丝氨酸脂肪酶位点，似乎没有催化活性。以此晶体结构为蓝图，我们将追求以下具体目标：1)研究I-II连接子在TRPML1组装和溶酶体靶向中的作用；(2)研究I-II连接子ML IV突变对TRPML1组装、靶向性和活性的影响；(3)研究I-II连接子在钙和铁渗透中的作用以及TRPML1介导的钙和Fe2+信号转导是否对溶酶体生理至关重要；(4)验证I-II连接子对管腔pH和Fe2+调节TRPML1通道至关重要的假设 这一双重调节对溶酶体生理非常重要。这些研究将从机制上深入了解TRPML1通道的功能，阐明ML IV的致病机制，并为开发这种毁灭性疾病的治疗策略奠定基础。

 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.