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, B ) Representative I–V relationships and corresponding summaries of TPC2 PM currents activated by 40 µM (A) or 10 µM (B) TPC2-A1-P. Currents were recorded in the inside-out excised patch configuration in the absence or presence of 2 µM Lsm12 or 1 µM NAADP, as indicated. For summary plots, current amplitudes were measured at −120 mV and normalized to those recorded in the absence of Lsm12. Conditions are color-coded as indicated. ( C ) Representative I–V relationships of TPC2 PM currents activated by extracellularly applied TPC2-A1-P at the indicated concentrations and recorded in the cell-attached configuration from WT, Lsm12-KO, and Lsm12-reexpressing HEK293 cells. ( D ) Summary of TPC2 PM current amplitudes measured at −120 mV, shown as absolute amplitudes (left) or normalized to currents elicited by 40 µM TPC2-A1-P in the same recording (right). ( E ) Immunoblot of lysates from WT, Lsm12-KO, and Lsm12-reexpressing HEK293 cells probed with an anti-Lsm12 antibody. For Lsm12 reexpression, a C-terminal FLAG- and 6×His-tagged construct was used. Cell types are color-coded as indicated in (C–E). Bar graphs show individual data points and mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, p > 0.05.

Article Snippet: The TPC2 or TPC1 channel currents were elicited by perfusion of PtdIns( , )P2 diC8 (Echelon Biosciences #P-3058), TPC2-A1-P (MedChemExpress #HY-131615 or Sigma-Aldrich #SML3700), or TPC2-A1-N (MedChemExpress #HY-131614 or Sigma-Aldrich #SML3562) on the cytosolic side in inside-out recordings and whole endolysosomal recordings, or of TPC2-A1-P on the extracellular side in cell-attached recordings.

Techniques: Western Blot, Construct

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: Lsm12 knockout enhances sensitivity of TPC2-expressing cells to TPC2-A1-P–induced Ca 2+ elevation Left: Representative time courses of Ca 2+ indicator signals in response to 10 µM TPC2-A1-P in WT and Lsm12-KO HEK293 cells, with or without TPC2 expression. Right : Summary of peak Ca 2+ responses (ΔF/F₀) evoked by the indicated concentrations of TPC2-A1-P in WT and Lsm12-KO HEK293 cells, with or without TPC2 expression. Bar graphs show individual data points and mean ± SEM. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, p > 0.05.

Article Snippet: The TPC2 or TPC1 channel currents were elicited by perfusion of PtdIns( , )P2 diC8 (Echelon Biosciences #P-3058), TPC2-A1-P (MedChemExpress #HY-131615 or Sigma-Aldrich #SML3700), or TPC2-A1-N (MedChemExpress #HY-131614 or Sigma-Aldrich #SML3562) on the cytosolic side in inside-out recordings and whole endolysosomal recordings, or of TPC2-A1-P on the extracellular side in cell-attached recordings.

Techniques: Knock-Out, Expressing

Journal: Acta pharmacologica Sinica

Article Title: Tetrandrine augments melanoma cell immunogenicity via dual inhibition of autophagic flux and proteasomal activity enhancing MHC-I presentation.

doi: 10.1038/s41401-025-01507-9

Figure Lengend Snippet: Fig. 8 Mechanistic illustration of tetrandrine-mediated enhance- ment of melanoma cell recognition and killing by CD8+ T cells through the inhibition of autophagy and proteasomal activity. The diagram illustrates how MHC-I molecules in melanoma cells can be degraded through both autophagy and proteasomal pathways, leading to a reduction in surface MHC-I molecules and facilitating immune escape of the melanoma cells. Tetrandrine disrupts this degradation process by concurrently inhibiting late- stage autophagic flux, through lysosomal acidification disrup- tion, and suppressing proteasomal activity. This dual inhibition prevents MHC-I degradation, thereby increasing MHC-I-mediated antigen presentation on the surface of melanoma cells. The elevated antigen presentation enhances CD8+ T cell recognition and cytotoxicity against melanoma cells. Further mechanistic exploration revealed that tetrandrine exerts its effects by blocking the lysosomal calcium efflux channel TPC2, leading to elevated lysosomal calcium levels and reduced cytosolic calcium concentrations. This calcium imbalance inhibits lysosomal acidification and suppresses cytoplasmic proteasomal activity, collectively contributing to reduced MHC-I degradation. The schematic emphasizes tetrandrine’s pivotal role in modulating both autophagic and proteasomal pathways, ultimately enhan- cing the immunogenicity of melanoma cells and increasing their susceptibility to CD8+ T cell-mediated cytotoxicity. Tet tetrandrine.

Article Snippet: The TPC2 agonist TPC2-A1-N (Cat. No. HY-131614) was obtained from MedChemExpress (Monmouth, NJ, USA).

Techniques: Inhibition, Activity Assay, Immunopeptidomics, Blocking Assay