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 i s 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 i s 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 i s 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).
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).
|Cheng, Xiping; Zhang, Xiaoli; Gao, Qiong et al. (2014) The intracellular Ca²? channel MCOLN1 is required for sarcolemma repair to prevent muscular dystrophy. Nat Med 20:1187-92|
|Samie, Mohammad Ali; Xu, Haoxing (2014) Lysosomal exocytosis and lipid storage disorders. J Lipid Res 55:995-1009|
|Bae, Mihyun; Patel, Neha; Xu, Haoxing et al. (2014) Activation of TRPML1 clears intraneuronal A? in preclinical models of HIV infection. J Neurosci 34:11485-503|
|Wang, Wuyang; Zhang, Xiaoli; Gao, Qiong et al. (2014) TRPML1: an ion channel in the lysosome. Handb Exp Pharmacol 222:631-45|
|Sun, Yubing; Yong, Koh Meng Aw; Villa-Diaz, Luis G et al. (2014) Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells. Nat Mater 13:599-604|
|Samie, Mohammad; Wang, Xiang; Zhang, Xiaoli et al. (2013) A TRP channel in the lysosome regulates large particle phagocytosis via focal exocytosis. Dev Cell 26:511-24|
|Li, Xinran; Garrity, Abigail G; Xu, Haoxing (2013) Regulation of membrane trafficking by signalling on endosomal and lysosomal membranes. J Physiol 591:4389-401|
|Yu, Ting; Chung, Chan; Shen, Dongbiao et al. (2012) Ryanodine receptor antagonists adapt NPC1 proteostasis to ameliorate lipid storage in Niemann-Pick type C disease fibroblasts. Hum Mol Genet 21:3205-14|
|Shen, Dongbiao; Wang, Xiang; Li, Xinran et al. (2012) Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release. Nat Commun 3:731|
|Jin, Jie; Wu, Long-Jun; Jun, Janice et al. (2012) The channel kinase, TRPM7, is required for early embryonic development. Proc Natl Acad Sci U S A 109:E225-33|
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