The intracellular Ca ²⁺ channel TRPML3 is a PI3P effector that regulates autophagosome biogenesis

Abstract

Autophagy is a multiple fusion event, initiating with autophagosome formation and culminating with fusion with endo-lysosomes in a Ca 2+ -dependent manner. The source of Ca 2+ and the molecular mechanism by which Ca 2+ is provided for this process are not known. The intracellular Ca 2+ permeable channel transient receptor potential mucolipin 3 (TRPML3) localizes in the autophagosome and interacts with the mammalian autophagy-related protein 8 (ATG8) homolog GATE16. Here, we show that lipid-regulated TRPML3 is the Ca 2+ release channel in the phagophore that provides the Ca 2+ necessary for autophagy progress. We generated a TRPML3-GCaMP6 fusion protein as a targeted reporter of TRPML3 compartment localization and channel function. Notably, TRPML3-GCaMP6 localized in the phagophores, the level of which increased in response to nutrient starvation. Importantly, phosphatidylinositol-3-phosphate (PI3P), an essential lipid for autophagosome formation, is a selective regulator of TRPML3. TRPML3 interacted with PI3P, which is a direct activator of TRPML3 current and Ca 2+ release from the phagophore, to promote and increase autophagy. Inhibition of TRPML3 suppressed autophagy even in the presence of excess PI3P, while activation of TRPML3 reversed the autophagy inhibition caused by blocking PI3P. Moreover, disruption of the TRPML3–PI3P interaction abolished both TRPML3 activation by PI3P and the increase in autophagy. Taken together, these results reveal that TRPML3 is a downstream effector of PI3P and a key regulator of autophagy. Activation of TRPML3 by PI3P is the critical step providing Ca 2+ from the phagophore for the fusion process, which is essential for autophagosome biogenesis.

TRPML3/BK complex promotes autophagy and bacterial clearance by providing a positive feedback regulation of mTOR via PI3P

“…Although our data clearly show that TRPML3/BK regulates autophagy, it remains unclear whether TRPML3/BK control autophagy induction, autophagy maturation, and/or autolysosomal degradation. To differentiate these possibilities, we adopted an autophagic flux assay by measuring LC3-II levels in the presence of bafilomycin A1 which inhibits autolysosomal degradation as well as the fusion of lysosomes with autophagosomes ( 2 , 42 ). We found that TRPML3 overexpression increased autophagic flux, and this was eliminated by Paxilline ( Fig.…”

“…TRPML3 is a Ca 2+ /Na + release channel, residing in both endosomal and lysosomal (endolysosomal) membranes. While TRPML1 is only activated by PI3,5P2, a PIP specifically localized in late endosomes and lysosomes ( 1 ), TRPML3 is activated by both PI3P, a PIP present in the phagophore and early endosomes, and PI3,5P2 ( 1 , 2 ). TRPML3 activity can also be enhanced by replacing luminal Na + with K + ( 3 – 6 ) and by increasing luminal pH ( 4 , 5 , 7 – 9 ).…”

“…Bacterial infection inhibits mTOR, leading to autophagy induction, which provides an innate immune mechanism for intracellular bacterial clearance via lysosome degradation ( 17 – 19 ). While there are multiple links between TRPML1 and autophagy regulation ( 20 – 24 ), the role of TRPML3 in autophagy remained less understood ( 11 , 25 – 27 ) until a recent work suggested that TRPML3 is recruited to phagophores where it is activated by PI3P to facilitate autophagy induction ( 2 ).…”

Coordinating activation of endo‐lysosomal two‐pore channels and TRP mucolipins

“…PI(3)P binds to both cytosolic N-terminus and the large luminal loop. Interestingly, TRPML3 is activated by cytosolic but not luminal PI(3)P, but when combined, robust currents are observed (Kim et al, 2022) (Fig. 3).…”

“…TRPML1 and TRPML3 were not mechanosensitive. TRPML3 is a selective target of PI(3)P (Kim et al., 2022; Xu et al., 2023). PI(3)P binds to both cytosolic N‐terminus and the large luminal loop.…”