CELLTMCO1内质网中的钙过载激活钙离子通道.pdfVIP

CELL | TMCO1 TMCO1Ca2+Ca2+2016519 CellTMCO1 Is an ER Ca(2+) Load-Activated Ca(2+) Channel (Ca2+) CRAC Fig1. CLAC TMCO1 TMCO1/ CLAC (Ca2+ Load Activated Ca2+) 400-728-0660 1/5 Fig2. TMCO1 Undergoes Oligomerization and Forms Tetramers in Response to ER Ca2+ Overload. (A) Co-immunoprecipitation experiment (coIP) shows TMCO1 forms a complex with itself. The input lanes contained 1/30 of lysates used in coIP reactions. Representative of at least three independent experiments. (B and C) Determination of the TMCO1 stoichiometry via chemical cross-linking. Increasing concentrations of glutaraldehyde (GA) (B) and disuccinimidyl suberate (DSS) (C) were used as indicated. Numbers represents the state of oligomerization: 1, monomer; 2, dimer; 4, tetramer; 8, octamer. Representative of at least three independent experiments. (D) Similar experiments as (C) were performed on HeLa cell lysates prepared from resting cells, cells pre-treated with TG in Ca2+ -free medium (TG+EGTA), and cells pre-treated with 8% ethanol in Ca2+-containing medium (8% ethanol). Representative of at least three independent 400-728-0660 2/5 experiments. (E) D1ER indicated ER Ca2+ concentration manipulated by 8% ethanol in Ca2+ containing medium and by TG in Ca2+ free medium (mean±SEM, n = 9). Each trace line is shown in mean±SEM. (F) Time course of mean FRET ratio changes measured from individual cells expressing CFPand YFP-tagged TMCO1 (green trace line, n = 16) or 146 (purple trace line, n = 6) after application of ethanol and TG. The FRET/CFP ratio before treatment was considered as basal, and FRET changes were represe