title: Mutations of the Ca2+-sensing stromal interaction molecule STIM1 regulate Ca2+ influx by altered oligomerization of STIM1 and by destabilization of the Ca2+ channel Orai1.
abstract: A drop of endoplasmic reticulum Ca(2+) concentration triggers its Ca(2+) ssensor protein stromal interaction molecule 1 (STIM1) to oligomerize and accumulate within endoplasmic reticulum-plasma membrane junctions where it activates Orai1 channels, providing store-operated Ca(2+) entry. To elucidate the functional significance of N-glycosylation sites of STIM1, we created different mutations of asparagine-131 and asparagine-171. STIM1 NN/DQ resulted in a strong gain of function. Patch clamp, Total Internal Reflection Fluorescent (TIRF) microscopy, and fluorescence recovery after photobleaching (FRAP) analyses revealed that expression of STIM1 DQ mutants increases the number of active Orai1 channels and the rate of STIM1 translocation to endoplasmic reticulum-plasma membrane junctions with a decrease in current latency. Surprisingly, co-expression of STIM1 DQ decreased Orai1 protein, altering the STIM1:Orai1 stoichiometry. We describe a novel mathematical tool to delineate the effects of altered STIM1 or Orai1 diffusion parameters from stoichiometrical changes. The mutant uncovers a novel mechanism whereby "superactive" STIM1 DQ leads to altered oligomerization rate constants and to degradation of Orai1 with a change in stoichiometry of activator (STIM1) to effector (Orai1) ratio leading to altered Ca(2+) homeostasis.
authors.affiliation: Department of Biophysics, Saarland University, D-66421 Homburg, Germany.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Kilch
authors.forename: Dalia
authors.initials: D
authors.lastname: Alansary
authors.forename: Martin
authors.initials: M
authors.lastname: Peglow
authors.forename: Kathrin
authors.initials: K
authors.lastname: Dörr
authors.forename: Grigori
authors.initials: G
authors.lastname: Rychkov
authors.forename: Heiko
authors.initials: H
authors.lastname: Rieger
authors.forename: Christine
authors.initials: C
authors.lastname: Peinelt
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Niemeyer
mesh_terms: D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D015870:Gene Expression; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D017136:Ion Transport; D019169:Jurkat Cells; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008956:Models, Chemical; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000071740:ORAI1 Protein; D018408:Patch-Clamp Techniques; D055503:Protein Multimerization; D021381:Protein Transport; D000071737:Stromal Interaction Molecule 1; D014162:Transfection
chemical_list: D015220:Calcium Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D002118:Calcium