pi(3,5)p 2 (18  (Croda International Plc)

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) Purified Lsm12 protein used throughout this study. Coomassie blue staining of an SDS-PAGE gel shows a single band near 25 kDa, consistent with the predicted molecular mass of 23.8 kDa. (B) Cytosolic application of purified Lsm12 reduced lysosomal TPC2 currents activated by 0.3 µM PI(3,5)P 2 in a concentration-dependent manner. For most Lsm12 concentrations, n = 10; n = 7 for 0.1 µM Lsm12 and n = 8 for the washout condition (“0 again”). (C) Purified Lsm12 (2 µM) markedly reduced endolysosomal TPC1 currents activated by 0.3 µM PI(3,5)P 2 ( n = 7). (D) Purified Lsm12 (2 µM) had little effect on endolysosomal TRPML1 currents activated by 0.3 µM PI(3,5)P 2 ( n = 6). Left panels in B–D: representative current-voltage (I-V) relationships obtained using a voltage ramp from −120 mV to +120 mV, together with schematic illustrations of whole-endolysosome recordings showing the major cations on either side of the membrane, from vacuolin-1-enlarged endolysosomes acutely isolated from HEK293 cells. Right panels in B–D: summary of current amplitudes measured at −120 mV (B, D) or +120 mV (C), showing individual data points and mean ± SEM, normalized to the corresponding current recorded in the absence of Lsm12. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: 500 μl 0.3 μM BODIPY FL PI(3,5)P 2 (Echelon Biosciences #C-35F6) was incubated with designated concentrations (0, 0.05, 0.3, and 1 μM) of 6×His tagged Lsm12 for 10 min in the K + -based solution (145mM KMeSO3, 5mM KCl, and 10mM HEPES (pH 7.4)) that was used for inside-out patch-clamp recordings.

Techniques: Purification, Staining, SDS Page, Concentration Assay, Membrane, Isolation

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: 500 μl 0.3 μM BODIPY FL PI(3,5)P 2 (Echelon Biosciences #C-35F6) was incubated with designated concentrations (0, 0.05, 0.3, and 1 μM) of 6×His tagged Lsm12 for 10 min in the K + -based solution (145mM KMeSO3, 5mM KCl, and 10mM HEPES (pH 7.4)) that was used for inside-out patch-clamp recordings.

Techniques: Activation Assay, Control, Derivative Assay, Inhibition, Incubation, Fluorescence, Mutagenesis, Concentration Assay

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 ) NAADP elicits TPC2 PM currents in the presence of PI(3,5)P 2 and Lsm12. Left : representative time course of TPC2 PM currents recorded at −120 mV during continuous application of 0.3 µM PI(3,5)P 2 , followed by sequential addition of 50 nM Lsm12 alone and then 50 nM Lsm12 together with increasing concentrations of NAADP (10, 100, and 1000 nM), and subsequent withdrawal of NAADP and Lsm12. Middle : summary of current amplitudes measured at −120 mV and normalized to the initial current activated by 0.3 µM PI(3,5)P 2 alone. Right : representative I–V relationships obtained under the corresponding conditions. ( B ) NADP does not elicit TPC2 PM currents in the presence of PI(3,5)P 2 and Lsm12. Current amplitudes were measured at −120 mV and normalized to the initial current activated by 0.3 µM PI(3,5)P 2 alone. ( C ) NAADP fails to induce currents of PI(3,5)P 2 -insensitive mutant TPC2 PM channels. Current amplitudes were measured at −120 mV under the indicated conditions. Path-clamp recordings in (A-C) were performed under the same configuration and ionic conditions. (D) NAADP does not alter the relative Na + /Ca 2+ permeability of PI(3,5)P 2 -activated TPC2 PM currents. Middle: representative I–V relationships under the indicated conditions. Left and right: summaries of normalized current amplitudes at +120 mV and reversal potentials, respectively. For reversal potential analysis, only I–V plots with maximal current amplitudes (+120 mV) between 0.5 and 1 nA were included to minimize error. Throughout this figure, the numbered steps and time points correspond to the color-coded conditions. Unless otherwise specified, the patch-clamp configurations and ionic conditions are illustrated schematically on the left. Bar graphs show individual data points and mean ± SEM. *, p < 0.05; **, p < 0.01; ****, p < 0.0001; ns, p > 0.05.

Article Snippet: 500 μl 0.3 μM BODIPY FL PI(3,5)P 2 (Echelon Biosciences #C-35F6) was incubated with designated concentrations (0, 0.05, 0.3, and 1 μM) of 6×His tagged Lsm12 for 10 min in the K + -based solution (145mM KMeSO3, 5mM KCl, and 10mM HEPES (pH 7.4)) that was used for inside-out patch-clamp recordings.

Techniques: Mutagenesis, Permeability, Patch Clamp

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) Summary of current amplitudes and representative I-V relationships showing that NAADP at 0.1 or 1 µM elicits currents of endolysosomal TPC2 in the presence of 0.3 µM PI(3,5)P 2 and 1 µM Lsm12. ( B ) Representative I–V relationships comparing Lsm12 alone and Lsm12 plus NAADP, showing that NAADP produces minimal activation of TPC2 currents from Lsm12-KO cells in the absence of PI(3,5)P 2 . ( C ) Summary of 1 µM NAADP-induced currents in Lsm12-KO cells supplemented with purified Lsm12 (2 µM), in the presence or absence of 0.3 µM PI(3,5)P 2 . ( D ) Summary of current amplitudes and representative I-V relationships showing that NAADP (1 µM) elicits currents of endolysosomal TPC1 in the presence of 0.3 µM PI(3,5)P 2 and 1 µM Lsm12. Current amplitudes for summary plots were measured at −120 mV in (A) and (C), and at +120 mV in (D). Where indicated, currents were normalized to those recorded in the absence of NAADP and Lsm12 within the same recording. Patch-clamp configurations and ionic conditions were identical for (A–C) and are illustrated schematically to the left of (A) and (D). Bar graphs show individual data points and mean ± SEM. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: 500 μl 0.3 μM BODIPY FL PI(3,5)P 2 (Echelon Biosciences #C-35F6) was incubated with designated concentrations (0, 0.05, 0.3, and 1 μM) of 6×His tagged Lsm12 for 10 min in the K + -based solution (145mM KMeSO3, 5mM KCl, and 10mM HEPES (pH 7.4)) that was used for inside-out patch-clamp recordings.

Techniques: Activation Assay, Purification, Patch Clamp

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) Endogenous PI(3,5)P 2 contributes to NAADP-evoked Ca²⁺ release. Antibodies or lipid-binding “grip” proteins targeting PI(3,5)P 2 or PI(4,5)P2 were co-injected with NAADP into cells. Left: Representative Ca²⁺ indicator traces. Right: Summary of peak NAADP-evoked Ca²⁺ responses. Bar graphs show individual data points and mean ± SEM. ***, p < 0.001. ( B ) Schematic summary of the interplay among PI(3,5)P 2 , Lsm12, and NAADP in TPC regulation. PI(3,5)P 2 activates TPCs, whereas Lsm12 binding to TPCs competitively suppresses PI(3,5)P 2 -activated channels. NAADP binding to Lsm12 relieves this suppression, restoring channel opening in the presence of PI(3,5)P 2 . The interacting sites of the ligand and proteins, as well as the number of Lsm12 molecules depicted in the cartoon, are arbitrary.

Article Snippet: 500 μl 0.3 μM BODIPY FL PI(3,5)P 2 (Echelon Biosciences #C-35F6) was incubated with designated concentrations (0, 0.05, 0.3, and 1 μM) of 6×His tagged Lsm12 for 10 min in the K + -based solution (145mM KMeSO3, 5mM KCl, and 10mM HEPES (pH 7.4)) that was used for inside-out patch-clamp recordings.

Techniques: Binding Assay, Injection