Fig. 1: Enhanced MLKL channel activity by PI(4,5)P₂ promotes necroptosis and inflammation.

a, b Single-channel current recordings and open probability of MLKL^NT in different lipid compositions (n > 3). c The heatmap shows MLKL^NT currents in various PI(4,5)P₂ concentrations. d Scatterplots of MLKL^NT currents vs. dwell times. e Representative subcellular localization of GFP-PH domain. Cells were treated with or without 5 μM Oxo-M. f The currents of MLKL channel with or without Oxo-M treatment. EC indicate the extracellular solution. g Statistical analysis of MLKL currents (n ≥ 3). h Necroptosis was detected through Annexin V-FITC (AV)/PI staining. L929 cells were treated with 20 ng/mL T and 20 μM Z after PIP5K transfection or ISA treatment (left). Statistical analysis of AV/PI staining (right). i Schematic representation of the residues examined by the solution MLKL^NT structure. j Current traces of MLKL mutants 22A, 34A, and 22A/34A in the presence of different concentrations of PI(4,5)P₂. k Cell viability of MLKL^–/– HeLa cells after co-transfection with PIP5K or PIP3K and MLKL mutant constructs. l The volcano plots for the comparison between proteome patterns of BV2 cells treated with PBS or LPS. m Representative function enrichment analysis of upregulated DEGs. Functional terms were labeled and color-coded with log₁₀ transformed P-value (Fisher’s exact test). n PPI network of proteins shown in Supplementary Fig. S8. Proteins participating in inositol phosphate metabolism are labeled with blue. o Cell viability of BV2 cells after LPS treatment. p, q Potassium efflux levels in LPS-treated BV2 cells and LPS/Q/C-treated BMDM cells (left). Statistical analysis results were shown (right). r Detection of K⁺ efflux level of LPS-treated BV2 cells with or without ISA (left). Statistical analysis results were shown (right). s Enhanced MLKL channel activity by PI(4,5)P₂ promoted necroptosis and inflammation via disturbing ion homeostasis and accelerating potassium efflux.