Fig. 3. Phosphoinositide composition regulates GSDMD pore dynamics in model lipid bilayers.

a PI(4,5)P₂ (PIP2) and PI(3,4,5)P₃ (PIP3) content significantly shortened the time to observe GSDMD protein–membrane interaction compared to baseline phospholipid bilayer. b The dwell time (open duration) of GSDMD pores is similar between PE/PC and PIP2-containing bilayers; PIP3-containing bilayers induced apparently short dwell time but bilayer suffered strong osmotic pressure because pores could not close. c Open probability histogram of single pore events showing distinct responses to phosphoinositide composition. d Histogram of single pore open-close events in different bilayer compositions; either phosphoinositide significantly reduced the current. e Representative traces of single pore recording when PIP2 and its metabolites, PIP3 and diacylglycerol (DAG), were incorporated at the same relative concentrations. General color-shade in this figure: PIP2 (green), DAG (red), or PIP3 (blue). DAG incorporation did not show single pore activity despite detectible protein–membrane interaction, thus is absent from panels (a) to (d). For panels (a), (b), (d), the reported statistical significance was derived from one-way ANOVA with multiple comparisons; “ref” represents the reference; in all cases ****p < 0.0001; ns not significant. In (b), ***p = 0.0002.