Activated by phosphorylation, mixed lineage kinase domain-like protein (MLKL) was found to translocate to the plasma membrane, where MLKL interacts with phosphatidylinositol phosphates (PIPs) via a patch of positively charged amino acids at the surface of a four-helical bundle domain (4HBD) located in its N-terminal region (Dondelinger Y et al. 2014; Wang H et al. 2014; Hildebrand JM et al. 2014; Su L et al. 2014; Quarato G et al. 2016). Interfering with the formation of PI(5)P or PI(4,5)P2 using PIP binders such as PIKfyve (P5i) efficiently inhibited TNF-induced necroptosis in both mouse L929 and the human FADD-null Jurkat cells (Dondelinger Y et al. 2014). In vitro liposome experiments revealed that MLKL induces leakage of PIP- or cardiolipin-containing liposomes suggesting that MLKL may have pore-forming capacities to mediate cell death by membrane's permeabilizing (Dondelinger Y et al. 2014; Wang H et al. 2014; Tanzer MC et al. 2016; Petrie EJ et al. 2018). Liposome permeabilization assays demonstrated that the N-terminal 4HB domain of MLKL compromised membrane integrity, and was more effective on liposomes whose composition resembled that of plasma membranes than on those mimicking mitochondrial membranes (Tanzer MC et al. 2016). One study has proposed the 4HB domain might reorganize in membranes to assemble into ion channels (Xia B et al. 2016); however, this remains to be fully explored in cellular contexts and structurally. Other studies implicated MLKL in engaging mitochondrial membranes to provoke mitochondrial fission or promote ion channel activity (Cai Z et al. 2014), although subsequent studies have discounted these possibilities (Murphy JM et al. 2013; Tait SW et al. 2013; Moujalled DM et al. 2014; Wang H et al. 2014; reviewd by Murphy JM 2020). Based on studies showing that the 4HB domain can permeabilize membranes in vitro (Dondelinger Y et al. 2014; Su L et al. 2014; Wang H et al. 2014; Tanzer MC et al. 2016; Petrie EJ et al. 2018), it is thought that MLKL kills cells via direct action on the plasma membrane (Murphy JM 2020).

Even in the Cai NCB 2013 paper this was not true of all cell types tested, despite this being written into folklore.

In terms of “pores” - this is a term that implies some sort of ordered structure. I prefer membrane perturbations or disruption as a term. If you look at Samson Nat Comm 2020, you can see these membrane structures are irregular in size and form. They also argue against channels as the destination for MLKL. Instead pMLKL coalesces with tight junction proteins in HT29 cells, although whether this is because of the membrane topology at this site or because of other accessory proteins being localized to these junctions, remains unknown.

Also Zargarian Plos Biol 2017. However, the idea of necroptotic bubbles was challenged in Samson Nat Comm 2020 because the bubbles could come and go at sites pMLKL did not accumulate and where membrane damage (“blowout”) did not occur

Various studies showed that the endosomal sorting complexes required for transport (ESCRT) pathway can remove phosphorylated MLKL-containing membrane vesicles from cells undergoing necroptosis, thereby attenuating the cell death process (Gong YN et al. 2017; Yoon S et al. 2017; Fan W et al. 2019). The ESCRT-associated proteins, programmed cell death 6-interacting protein (PDCD6IP or ALG-2-interacting protein X, ALIX) and syntenin-1 (SDCBP), were found to antagonize MLKL-mediated plasma membrane alteration (Fan W et al. 2019). In addition, flotillin-mediated endocytosis was proposed to suppress necroptosis by removing MLKL from the plasma membrane and redirecting it for lysosomal degradation (Fan W et al. 2019).

Studies in human cell lines suggest that upon induction of necroptosis MLKL shifts to the plasma membrane and membranous organelles such as mitochondria, lysosome, endosome and ER (Wang H et al. 2014), but it is trafficking via a Golgi-microtubule-actin-dependent mechanism that facilitates plasma membrane translocation, where membrane disruption causes death (Samson AL et al. 2020).


Based on studies showing that the 4HB domain can permeabilize membranes in vitro (Dondelinger et al. 2014; Su et al. 2014; Wang et al. 2014; Tanzer et al. 2016; Petrie et al. 2018), it is thought that MLKL kills cells via direct action on the plasma membrane.