ABSTRACT
Calcium (Ca2+ ) signaling controls T-cell activation and functions. Ca2+ concentrations are locally detected and controlled by Ca2+ -sensors (STIM1 and 2 detecting the depletion from ER stores channels) and Ca2+ -channels (ORAI1-3 in the cell membrane and VDAC1 in the outer mitochondrial membrane). We first validated and titrated antibodies to assess the expression of these Ca2+ -sensors and -channels in human and murine cells, and further devised a 18-antibodies mass cytometry panel to characterize their expression in primary murine lymphocyte subsets.
Subject(s)
Calcium Channels/isolation & purification , Flow Cytometry/methods , Gene Expression Regulation/genetics , Animals , Calcium Channels/genetics , Cell Membrane/genetics , Cell Membrane/metabolism , Humans , Lymphocyte Subsets/immunology , Lymphocyte Subsets/metabolism , Mice , Mitochondrial Membranes/metabolism , ORAI1 Protein/genetics , ORAI1 Protein/isolation & purification , Stromal Interaction Molecule 1/genetics , Stromal Interaction Molecule 1/isolation & purification , Stromal Interaction Molecule 2/genetics , Stromal Interaction Molecule 2/isolation & purification , Voltage-Dependent Anion Channel 1/geneticsABSTRACT
We report a new NMR-scale purification procedure for two recombinant wild type fragments of the stromal interaction molecule 1 (STIM1). This protein acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol accumulating at ER - plasma membrane (PM) junctions upon calcium store depletion ultimately leading to activation of the Orai/CRAC channel. The functionally relevant cytosolic part of STIM1 consists of three coiled coil domains, which are mainly involved in intra- and inter-molecular homomeric interactions as well as coupling to and gating of CRAC channels. The optimized one-step rapid purification procedure for two 15N,13C isotope-labeled cytosolic coiled coil fragments, which avoids the problems of previous approaches. The high yields of soluble well folded 15N,13C isotope-labeled cytosolic coiled coil fragments followed by detergent screening provide for initial NMR characterization of these domains. The longer 30.5â¯kDa fragment represents the largest STIM1 wild type fragment that has been recombinantly prepared and characterized in solution without need for mutation or refolding.