Convergent activation of Ca ²⁺ permeability in two-pore channel 2 through distinct molecular routes

Abstract

TPC2 is a pathophysiologically relevant lysosomal ion channel that is activated directly by the phosphoinositide PI(3,5)P 2 and indirectly by the calcium ion (Ca 2+ )–mobilizing molecule NAADP through accessory proteins that associate with the channel. TPC2 toggles between PI(3,5)P 2 -induced, sodium ion (Na + )–selective and NAADP-induced, Ca 2+ –permeable states in response to these cues. To address the molecular basis of polymodal gating and ion-selectivity switching, we investigated the mechanism by which NAADP and its synthetic functional agonist, TPC2-A1-N, induced Ca 2+ release through TPC2 in human cells. Whereas NAADP required the NAADP-binding proteins JPT2 and LSM12 to evoke endogenous calcium ion signals, TPC2-A1-N did not. Residues in TPC2 that bind to PI(3,5)P 2 were required for channel activation by NAADP but not for activation by TPC2-A1-N. The cryptic voltage-sensing region of TPC2 was required for the actions of TPC2-A1-N and PI(3,5)P 2 but not for those of NAADP. These data mechanistically distinguish natural and synthetic agonist action at TPC2 despite convergent effects on Ca 2+ permeability and delineate a route for pharmacologically correcting impaired NAADP-evoked Ca 2+ signals.

Direct measurements of luminal Ca2+with endo-lysosomal GFP-aequorin reveal functional IP3receptors

“…We show that activation of TRPML1 with ML-SA1/PI(3,5)P 2 and TPCs with NAADP produces luminal responses, but the latter were not consistent. Recent studies have shown that NAADP binds to soluble binding proteins to activate TPCs (Gunaratne et al, 2023;Saito et al, 2023). It is possible that loss of these proteins upon permeabilization may account for NAADP insensitivity.…”

Characterisation of ligand gating, ion conduction and the ion selectivity mechanism in the endo-lysosomal ion channel hTPC2

“…Recent research on the NAADP regulation of TPCs has shown that NAADP likely does not directly bind to the TPCs but via binding proteins such as Lsm12 [23] . [24] So far, the selectivity and ligand-gating mechanisms of hTPC2 are insufficiently understood. The selectivity filter (SF) sequences of hTPC2 are 271TAN273 (filter I) and 652VNN654 (filter II) [25] .…”

“…NAADP is a potent and well-known Ca 2+ -mobilizing second messenger, which initiates Ca 2+ release from endo-lysosomal organelles [19] . Recent research on the NAADP regulation of TPCs has shown that NAADP likely does not directly bind to the TPCs but via binding proteins such as Lsm12 [26][18,27] .…”

“…Recent research on the NAADP regulation of TPCs has shown that NAADP likely does not directly bind to the TPCs but via binding proteins such as Lsm12 [23] . [24]…”