The Calcium-Calpain-ALIX Axis is the Major Link Between Gasdermin-D Membrane Pores and Terminal Membrane Damage in...

Pyroptosis is an inflammatory form of regulated cell death driven by gasdermin-mediated membrane pore formation. Although gasdermin D (GSDMD) pores are widely regarded as the executioners of pyroptosis, recent studies demonstrate that pore formation is not necessarily lethal because cells can actively repair membrane damage through the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. The molecular mechanism that converts reversible GSDMD pore formation into irreversible membrane rupture and cell death remains unknown. Here, we identify a calcium-calpain-ALIX signaling axis that mechanistically links GSDMD pore formation to catastrophic membrane damage. Using primary macrophages, THP-1 monocytes, and HCT-116 epithelial cells, we show that depletion of the ESCRT adaptor ALG-2-interacting protein X (ALIX) abolishes membrane repair, promotes GSDMD accumulation, and markedly increases susceptibility to pyroptotic death. We further demonstrate that GSDMD pores trigger calcium influx, which induces proteolytic cleavage of ALIX. Preventing calcium influx or chelating intracellular calcium blocks ALIX cleavage, reduces GSDMD accumulation, and markedly improves cell survival. Mechanistically, we identify calpains as the calcium-dependent proteases responsible for ALIX cleavage and establish ALIX as a previously unrecognized calpain substrate. Pharmacologic inhibition or genetic depletion of calpains significantly reduces membrane permeabilization and pyroptotic cell death. Mapping of calpain cleavage sites localizes the major cleavage site within the ALIX V-domain. Importantly, calpain-mediated cleavage disrupts ALIX interaction with the ESCRT-III component CHMP4B, thereby preventing ESCRT assembly and membrane repair. In contrast, calcium depletion or calpain knock down restores CHMP4B recruitment and ESCRT activation Collectively, these findings reveal the first mechanistic pathway linking reversible membrane GSDMD pore formation to irreversible membrane rupture. We propose that GSDMD pore-induced Ca²⁺ influx activates calpains, disables ALIX-dependent ESCRT repair, and drives the transition from repairable membrane injury to terminal pyroptotic lysis. This pathway represents a potential therapeutic target for inflammatory diseases driven by excessive pyroptosis.