MLKL forms cation channels

Abstract

The mixed lineage kinase domain-like (MLKL) protein is a key factor in tumor necrosis factor-induced necroptosis. Recent studies on necroptosis execution revealed a commitment role of MLKL in membrane disruption. However, our knowledge of how MLKL functions on membrane remains very limited. Here we demonstrate that MLKL forms cation channels that are permeable preferentially to Mg2+ rather than Ca2+ in the presence of Na+ and K+. Moreover, the N-terminal domain containing six helices (H1-H6) is sufficient to form channels. Using the substituted cysteine accessibility method, we further determine that helix H1, H2, H3, H5 and H6 are transmembrane segments, while H4 is located in the cytoplasm. Finally, MLKL-induced membrane depolarization and cell death exhibit a positive correlation to its channel activity. The Mg2+-preferred permeability and five transmembrane segment topology distinguish MLKL from previously identified Mg2+-permeable channels and thus establish MLKL as a novel class of cation channels.

The MLKL Channel in Necroptosis Is an Octamer Formed by Tetramers in a Dyadic Process

“…We showed previously (26) that translocation of the artificial MLKL N-terminal domain oligomers to the plasma membrane led to ion influx and is sufficient to induce necroptosis; Hildebrand et al (35) and Dondelinger et al (27) showed that overexpression of the MLKL N-terminal four-␣-helix boundary is enough to trigger necroptosis; Wang et al (28) and Dondelinger et al (27) showed that the MLKL N-terminal four-␣-helix boundary could bind to negatively charged phospholipids and cardiolipin and disrupted the membrane integrity in the liposome. A recent work by Xia et al demonstrated the cation channel activity of MLKL in the liposome, supporting the idea that ion influx plays a role in necroptotic cell death (31). Our data demonstrate that MLKL forms a 180°rotational symmetry complex.…”

“…We propose that ␣-helices 1, 2, 3, and 5 but not ␣-helix 6 of MLKL are the transmembrane fragments because this is the only possibility, given the fact that the N-and C-terminal ends are at the inner side of the plasma membrane. The fact that ␣-helix 6 was detected as a transmembrane fragment in the study of Xia et al is likely because they used the N-terminal 178 amino acids of MLKL in the SCAM experiment (31). To have the C-terminal kinase-like domain of MLKL in its position of the octamer, ␣-helix 6 has to be unleashed to allow opening of the ␣-helix bundle, as suggested by published structural studies of MLKL (32,36), and thus, ␣-helix 6 is not involved in penetration of the plasma membrane.…”

“…Using a substituted cysteine accessibility mutagenesis (SCAM) strategy, Xia et al showed that ␣-helices 1, 2, 3, 5, and 6 of MLKL are transmembrane fragments in the liposome (31). We also tried to apply SCAM in our culture cell system but failed.…”

“…The affinity of MLKL to selected phospholipids in the membrane plays a role in MLKL translocation to the plasma membrane (26)(27)(28)(29). The cation channel activity of MLKL was recently shown using liposomes (31), supporting the role of ion influx in causing the high intracellular osmotic pressure that leads to necrosis (26,29).…”

“…MLKL is a well-known effector of necroptosis and is activated downstream of RIP3 (24)(25)(26)(27)(28)(29)(31)(32)(33)(34). Although its monomer structure has been solved and a number of amino acids or clusters of amino acids were revealed to be important for MLKL oligomerization and targeting to the plasma membrane (27,32,(34)(35)(36), the details of its oligomerization and oligomer structure are still largely unknown.…”