The endolysosomal system is essential for cell signaling and physiology. The functions of endocytic vesicles are regulated by a variety of ion channels, including the mucolipin subfamily of transient receptor potential (TRPML) channels, which are localized primarily in endosomes and lysosomes. These channels conduct Ca2+ and Na+ currents from the vesicle lumen to the cytoplasm and are critically involved in membrane trafficking, exocytosis and autophagy. Mutations in TRPML1 cause mucolipidosis type IV (ML IV), a severe lysosomal storage disorder, and mutations in TRPML3 cause deafness and pigmentation defects in mice, underscoring the crucial physiological importance of these channels. The activities of TRPML channels are strongly regulated by endogenous factors such as PIP2, pH, Na+ and Ca2+. The complex regulation in turn controls the physiological functions of these channels. The objective of this project is to elucidate the molecular mechanisms of regulation of TRPML3 by these physiological factors. TRPML3 is regulated by both common and unique mechanisms. Like other TRPMLs, TRPML3 is activated by PI(3, 5)P2 and suppressed by PI(4, 5)P2. However, it is uniquely inhibited by luminal low pH and Na+. This inhibition presumably keeps lysosomal TRPML3 inactive under physiological conditions. Neutralization or damage of lysosomes likely relieves this inhibition and activates TRPML3. We have recently solved cryo-EM structures of full length human TRPML3 in the closed, open and low-pH-inhibited states. These structures reveal a number of unique structural features and suggest new allosteric regulatory mechanisms. We have also uncovered a novel ?Inhibition Memory? that depends on Na+ and amino acid H283. We will build on these exciting findings and determine the structural elements and conformational changes underlying the regulation of TRPML3 by low pH, Na+, PI(3, 5)P2 and PI(4, 5)P2. We will carry out structure-guided mutagenesis studies to test the hypothesis that a luminal pore- loop and H283 are pH sensors and that transmembrane segments S1 and S2 act as allosteric transducers that convert low pH-, Na+-, and PIP2-induced local conformational changes to global conformational changes that either enhance or inhibit channel activity. We will obtain cryo-EM structures of WT and H283A mutant channels in complex with membrane lipids at different pH and with different alkali ions and of WT channels in complex with PI(3, 5)P2 or PI(4, 5)P2 at different pH and Na+ concentrations. These studies will yield rich and deep mechanistic insights into TRPML3 channel regulation and provide new knowledge for the development of therapeutic strategies for ML IV and other endocytic vesicle-related diseases.
TRPML channels are crucial for cellular events such as membrane trafficking, exocytosis and autophagy. Dysfunction of TRPML channels cause human diseases and severe animal phenotypes. Elucidating the molecular mechanisms of regulation of TRPML channels by physiological factors is fundamental for understanding their biological functions and will provide a foundation for studying TRPML channel function, pathogenesis and therapeutic strategies.
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