Journal: bioRxiv
Article Title: NAADP elicits two-pore channel currents by lifting Lsm12-mediated inhibition of PI(3,5)P 2 activation
doi: 10.64898/2026.04.13.718294
Figure Lengend Snippet: (A) Representative time course of TPC2 PM currents at −120 mV during perfusion with 0.3 µM PI(3,5)P 2 . After current activation under control conditions, 0.5 µM Lsm12 was applied to inhibit the current, and subsequent washout of Lsm12 led to recovery. (B, C) Summary of the normalized current amplitudes derived from the maximal (activation) and minimal current (inhibition) levels ( B ) and time constants (τ) ( C ) during the control, Lsm12 wash-on, and wash-off phases from recordings as shown in A ( n = 5). (D) Effects of heat-treated Lsm12 on PI(3,5)P 2 -activated TPC2 PM currents, normalized to currents recorded in the absence of heat-treated Lsm12. (E) Fraction of unbound BODIPY FL PI(3,5)P 2 remaining after incubation of 0.3 µM lipid with 0.05, 0.3, or 1 µM 6×His-tagged Lsm12 ( n = 4), determined from the fluorescence remaining in solution after precipitation of bead-bound Lsm12 with IMAC beads. (F) Representative I–V relationships of constitutively active TPC2 PM carrying the L690A/L694A mutation, recorded under bath conditions alone or after addition of the indicated concentrations of Lsm12 or PI(3,5)P 2 . (G, H) Representative I–V relationships of TPC2 PM currents activated by 0.3 µM ( G ) or 1 µM ( H ) PI(3,5)P 2 in the absence and presence of the indicated concentrations of Lsm12. (I) Concentration-response relationships for inhibition by Lsm12 of TPC2 PM currents activated by 0.3 µM ( n = 10) or 1 µM ( n = 9) PI(3,5)P 2 . (J) PI(3,5)P 2 concentration-response relationships for TPC2 PM currents in the absence and presence of 2 µM Lsm12. (K) Lsm12 concentration-response relationships for TPC2 PM pseudo-WT ( n = 10) and Δ45–50 mutant ( n = 8) channels activated by 0.3 µM PI(3,5)P 2 . Left, modeled structure of Lsm12 highlighting residues 45–50 in stick representation. Summarized data are presented as plots showing mean ± SEM, with or without individual data points, as indicated. ns, not significant ( P > 0.05).
Article Snippet: 20 μl Nuvia IMAC (immobilized metal affinity chromatography) Ni-charged resin (Bio-Rad #7800800) pre-equilibrated with the K + -based solution was added and incubated for 10 min. After centrifugation to pellet the resin, the fluorescence of the unbound BODIPY FL PI(3,5)P 2 in the supernatant was measured by a plate reader.
Techniques: Activation Assay, Control, Derivative Assay, Inhibition, Incubation, Fluorescence, Mutagenesis, Concentration Assay
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