PI(3,5)P2 controls membrane trafficking by direct activation of mucolipin Ca2+ release channels in the endolysosome

Abstract

Membrane fusion and fi ssion events in intracellular traffi cking are controlled by both intraluminal Ca 2 + release and phosphoinositide (PIP) signalling. However, the molecular identities of the Ca 2 + release channels and the target proteins of PIPs are elusive. In this paper, by direct patch-clamping of the endolysosomal membrane, we report that PI(3,5)P 2 , an endolysosome-specifi c PIP, binds and activates endolysosome-localized mucolipin transient receptor potential (TRPML) channels with specifi city and potency. Both PI(3,5)P 2 -defi cient cells and cells that lack TRPML1 exhibited enlarged endolysosomes / vacuoles and traffi cking defects in the late endocytic pathway. We fi nd that the enlarged vacuole phenotype observed in PI(3,5)P 2 -defi cient mouse fi broblasts is suppressed by overexpression of TRPML1. Notably, this PI(3,5)P 2 -dependent regulation of TRPML1 is evolutionarily conserved. In budding yeast, hyperosmotic stress induces Ca 2 + release from the vacuole. In this study, we show that this release requires both PI(3,5)P 2 production and a yeast functional TRPML homologue. We propose that TRPMLs regulate membrane traffi cking by transducing information regarding PI(3,5)P 2 levels into changes in juxtaorganellar Ca 2 + , thereby triggering membrane fusion / fi ssion events.

A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV

“…2a). We further show that PI(3,5)P 2 , which has been reported recently to activate TRPML channels 14 , has a comparable effect on TRPML1 channel activity as SF-22 when applied at a concentration of 10 mM, respectively (Fig. 2b).…”

“…In recent years, TRPML1 has been characterized using electrophysiological tools and its basic cation channel properties have been investigated [9][10][11][12][13][14][15][16][17][18][19] , its protein-protein interaction network has been explored [20][21][22][23][24] , and knockout mouse models have been generated and investigated [25][26][27][28] . However, effective treatment options for MLIV patients are still missing.…”

Structure of mammalian endolysosomal TRPML1 channel in nanodiscs

“…A luminal linker domain forms a fenestrated canopy atop the channel, providing multiple luminal ion passages to the pore and also creating a negative electrostatic trap – preferably for divalent cations at the luminal entrance. The structure also reveals two equally distributed S4-S5 linker conformations in the closed channel, providing structural implication for the S4-S5 linker-mediated PIP 2 gating mechanism among TRPML channels 7,8 . …”

“…TRPML1 is a Ca 2+ -permeable, non-selective, six-transmembrane (6-TM) tetrameric cation channel, and is believed to be the main lysosomal Ca 2+ release channel important for lysosomal trafficking and signal transduction 15-19 . Like most TRP channels, TRPML1 is ligand-gated and can be activated by the endolysosomal specific lipid, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P 2 ) 7,8 , but is inhibited by the plasma membrane-localized phosphoinositide isoform PI(4,5)P 2 14 (Extended Data Fig. 1).…”

“…2b). The N-terminal poly-basic domain is important for PIP 2 binding 7,14 and its deletion abolishes PI(3,5)P 2 activation of TRPML1 (Extended Data Fig. 1e).…”