Bing-qing XIA,Sui FANG,Xue-qin CHEN,Hong HU,Pei-yuan CHEN,Hua-yi WANG,Zhao-bing GAO

（1.State Key Laboratory of Drug Research,Shanghai Institute of Materia Medica,Chinese Academy of Sciences,Shanghai 201203,China;2.School of Life Science and Technology,Shanghai Tech University,Shanghai 200031,China）

T3-5

MLKL forms cation channels

Bing-qing XIA1,Sui FANG1,Xue-qin CHEN1,Hong HU2,Pei-yuan CHEN1,Hua-yi WANG2,Zhao-bing GAO1

（1.State Key Laboratory of Drug Research,Shanghai Institute of Materia Medica,Chinese Academy of Sciences,Shanghai 201203,China;2.School of Life Science and Technology,Shanghai Tech University,Shanghai 200031,China）

OBJECTIVETo investigate how MLKL functions on the membrane and explore its electrophysiological characters and structure.METHODSThe full-length human MLKL were expressed in SF21 cells and purified using glutathione-sepharose affinity chromatography.The currents of purified MLKL proteins were recorded in avoltage-clamp mode using a Warner BC-535 bilayer clamp amplifier.The currents were digitized using pCLAMP 10.2 software.HEK293 cells were cultured and transfected with MLKL plasmid.Cell viability was examined using the CellTiter-Glo Luminescent Cell Viability Assay kit.RESULTMLKL forms cation channels that are permeable preferentially to Mg2+rather than Ca2+in the presence of Na+and K+.Moreover,each MLKL monomer contains five transmembrane helices:H1,H2,H3,H5 and H6 of the N-terminal domain which is sufficient to form channels.Finally,MLKL-induced membrane depolarization and cell death exhibit a positive correlation to its channel activity.

MLKL;magnesium channel;bilayer lipid membrane;cation channel;necroptosis

The project supported by State Key Program of Basic Research of China(2013CB910604);National Natural Science Foundation of China(61327014,61175103,61433017 and31571427);and the External Cooperation Program of BIC,ChineseAcademy of Sciences(1536631KYSB20130003)

Zhao-bing GAO,Tel:(021)20239067,E-mail：zbgao@simm.ac.cn;Hua-yi WANG,Tel:(021)20776023,E-mail:wanghuayi@shanghaitech.edu.cn