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 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.

Public Health Relevance

TRPML1 channels have unique properties and play unique and important roles in lysosomes. Mutations of the TRPML1 gene cause mucolipidosis type IV (ML IV), a severe human lysosomal storage disorder. Children with ML IV often have cognitive retardation, motor and language deficits, and blindness. Studying the structure, function, regulation, physiology and pathophysiology of TRPML1 channels will shed light on their physiological roles within lysosomes and lead to a better understanding of the pathogenesis of ML IV, facilitating the development of new therapeutic strategies against this disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM085234-05
Application #
8880595
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Nie, Zhongzhen
Project Start
2009-05-01
Project End
2019-02-28
Budget Start
2015-05-01
Budget End
2016-02-29
Support Year
5
Fiscal Year
2015
Total Cost
$331,805
Indirect Cost
$118,672
Name
Columbia University (N.Y.)
Department
Biology
Type
Other Domestic Higher Education
DUNS #
049179401
City
New York
State
NY
Country
United States
Zip Code
10027
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