Necroptosis and pyroptosis are two forms of programmed cell death with a common feature of plasma membrane rupture. Here we studied the morphology and mechanism of pyroptosis in comparison with necroptosis. Different from necroptosis, pyroptosis undergoes membrane blebbing and produces apoptotic body-like cell protrusions (termed pyroptotic bodies) prior to plasma membrane rupture. The rupture in necroptosis is explosion-like, whereas in pyroptosis it leads to flattening of cells. It is known that the execution of necroptosis is mediated by mixed lineage kinase domain-like (MLKL) oligomers in the plasma membrane, whereas gasdermin-D (GSDMD) mediates pyroptosis after its cleavage by caspase-1 or caspase-11. We show that N-terminal fragment of GSDMD (GSDMD-N) generated by caspase cleavage also forms oligomer and migrates to the plasma membrane to kill cells. Both MLKL and GSDMD-N are lipophilic and the N-terminal sequences of both proteins are important for their oligomerization and plasma membrane translocation. Unlike MLKL which forms channels on the plasma membrane that induces influx of selected ions which osmotically swell the cells to burst, GSDMD-N forms non-selective pores and does not rely on increased osmolarity to disrupt cells. Our study reveals the pore-forming activity of GSDMD and channel-forming activity of MLKL determine different ways of plasma membrane rupture in pyroptosis and necroptosis.

/pdf/pyroptosis-is-driven-by-non-selective-gasdermin-d-pore-and-2kw23biea0.pdf

Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis

The interaction of Staphylococcus aureus alpha-toxin with planar lipid membranes results in the formation of ionic channels whose conductance can be directly measured in voltage-clamp experiments. Single-channel conductance depends linearly on the solution conductivity suggesting that the pores are filled with aqueous solution; a rough diameter of 11.4 +/- 0.4 A can be estimated for the pore. The conductance depends asymmetrically on voltage and it is slightly anion selective at pH 7.0, which implies that the channels are asymmetrically oriented into the bilayer and that ion motion is restricted at least in a region of the pore. The pores are usually open in a KCl solution but undergo a dose- and voltage-dependent inactivation in the presence of di- and trivalent cations, which is mediated by open-closed fluctuations at the single-channel level. Hill plots indicate that each channel can bind two to three inactivating cations. The inhibiting efficiency follows the sequence Zn2+ greater than Tb3+ greater than Ca2+ greater than Mg2+ greater than Ba2+, suggesting that carboxyl groups of the protein may be involved in the binding step. A voltage-gated inactivation mechanism is proposed which involves the binding of two polyvalent cations to the channel, one in the open and one in the closed configuration, and which can explain voltage, dose and time dependence of the inactivation.

Ionic channels formed by Staphylococcus aureus alpha-toxin: Voltage-dependent inhibition by divalent and trivalent cations

Membrane damage by hemolytic viruses, toxins, complement, and other cytotoxic agents. A common mechanism blocked by divalent cations.

Physiological roles of zinc in the plasma membrane of mammalian cells

We have previously demonstrated (M. Stubbs, Z. M. Bhujwalla, G. M. Tozer, L. M. Rodrigues, R. J. Maxwell, R. Morgan, F. A. Howe, and J. R. Griffiths, NMR Biomed., 5: 351, 1992) that the intracellular pH (pHi) of several rat tumors is higher (>pH 7.0) than that of the tumor extracellular fluid (pHe), in contrast to normal tissues ( e.g., liver) in which pHi is lower than pHe. In this paper we confirm a pHe of 6.8 ± 0.07 (SEM) in Morris hepatoma 9618a by an independent method and report the tissue content of other ions by both 31P magnetic resonance spectroscopy and by conventional analysis in hepatomas and livers in rats. Compared with liver, tissue Na+ was 2-fold higher and tissue K+ was lower. Tissue Ca2+ was 8-fold higher (7.4 ± 4.3 µmol/g wet weight) and tissue Pi was 2-fold higher (8.5 ± 1.3 µmol/g wet weight) suggesting the presence of insoluble calcium phosphate. Cl- was unchanged (∼40 µmol/g wet weight), whereas HCO-3 was lower in the hepatoma (12.4 ± 0.83 compared to 15.5 ± 0.76 µmol/g wet weight). Total tissue Mg2+ was similar in both tissues, but free [Mg2+] (calculated by two different methods) was ∼5-fold lower in the hepatoma. The ATP values were 3.5-fold and [NAD]/[NADH] 9-fold lower in the hepatoma. The results are compatible with the hypothesis that the chronic partial hypoxia of tumor tissue involves changes in the linked equilibria of many ions and metabolites and may help explain such pathologies as calcification.

Metabolic Consequences of a Reversed pH Gradient in Rat Tumors

The concentration of Na+and K+ and the pH in the cytoplasm of Lettre cells was measured by monitoring the net flux of H+, Na+, or K+ across the plasma membrane which had been rendered permeable to these ions by the action of Sendal virus. Ion flux was measured directly by analysis of cell composition, or indirectly by observing the change in membrane potential of cells treated with a specific ionophore. Cytoplasmic concentrations of cations were obtained by establishing the concentration of the cation in the medium at which addition of Sendai virus causes no change in cytoplasmic cation content. The value of Lettre-cell pH was confirmed by direct measurement employing 3tp nuclear magnetic resonance, and the values of Na+ and K+ concentration were confirmed by analysis of cell cation and water content. Lettre cells suspended at 32°C in Hepes-buffered saline at pH 7.3 maintain a cytosolic pH of 7.0 and contain 30 mM Na+ and 80 mM K+.

A novel method for measuring intracellular pH and potassium concentration

The interaction of pore-forming agents, such as Sendai virus, influenza virus (at pH 5 3), activated complement,Staphylococcus aureus α-toxin, melittin and polylysine, with the surface membrane of cells has been studied. In each case the following changes are initiated: collapse of membrane potential, leakage of ions, and leakage of phosphorylated metabolites. The changes can be inihibited by extracellular Ca2+ at physiological concentration; Mg2+ is less effective, and Zn2+ is more effective, than Ca2+ Ca2+ appears to act at a stage subsequent to the binding of pore-forming agent to cells. It is concluded that divalent cations are able to protect cells against the damaging effects of certain viruses, toxins or the components of activated complement in a manner that is worthy of further investigation.

Ca2+ and the interaction of pore-formers with membranes

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A novel method for measuring intracellular pH and potassium concentration

Ca2+ and the interaction of pore-formers with membranes