Signal transduction by the high-affinity IgE receptor (FcεRI) depends on membrane lipid and protein compartmentalization. Recently published data show that cells treated with 1-heptanol, a cell membrane fluidizer, exhibit changes in membrane properties. However, the functional consequences of 1-heptanol-induced changes on mast cell signaling are unknown. This study shows that short-term exposure to 1-heptanol reduces membrane thermal stability and dysregulates mast cell signaling at multiple levels. Cells treated with 1-heptanol exhibited increased lateral mobility and decreased internalization of the FcεRI. However, this did not affect the initial phosphorylation of the FcεRI-β chain and components of the SYK/LAT1/PLCγ1 signaling pathway after antigen activation. In contrast, 1-heptanol inhibited SAPK/JNK phosphorylation and effector functions such as calcium response, degranulation, and cytokine production. Membrane hyperfluidization induced a heat shock-like response via increased expression of the heat shock protein 70, increased lateral diffusion of ORAI1-mCherry, and unsatisfactory performance of STIM1-ORAI1 coupling, as determined by flow-FRET. Furthermore, 1-heptanol inhibited the antigen-induced production of reactive oxygen species and potentiated stress-induced plasma membrane permeability by interfering with heat shock protein 70 activity. The combined data suggest that 1-heptanol-mediated membrane fluidization does not interfere with the earliest biochemical steps of FcεRI signaling, such as phosphorylation of the FcεRI-β chain and components of the SYK/LAT/PLCγ1 signaling pathway, instead inhibiting the FcεRI internalization and mast cell effector functions, including degranulation and cytokine production.

• MeSH
• Cholesterol MeSH
• Cytokines MeSH
• Heptanol MeSH
• Mast Cells * MeSH
• Signal Transduction * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.

• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Phosphatidylcholines chemistry   metabolism   MeSH
• Ion Channel Gating genetics   MeSH
• Genetic Vectors chemistry   metabolism   MeSH
• HEK293 Cells MeSH
• Protein Interaction Domains and Motifs MeSH
• Protein Conformation, alpha-Helical MeSH
• Protein Conformation, beta-Strand MeSH
• Humans MeSH
• Liposomes chemistry   metabolism   MeSH
• Luminescent Proteins genetics   metabolism   MeSH
• Patch-Clamp Techniques MeSH
• Mutation MeSH
• Neoplasm Proteins chemistry   genetics   metabolism   MeSH
• ORAI1 Protein chemistry   genetics   metabolism   MeSH
• Stromal Interaction Molecule 1 chemistry   genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Genes, Reporter MeSH
• Molecular Dynamics Simulation MeSH
• Amino Acid Substitution MeSH
• Calcium metabolism   MeSH
• Calcium Signaling * MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Ca2+ release-activated Ca2+ (CRAC) channels constitute the major Ca2+ entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca2+-selective Orai channel and the Ca2+-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating. Although the extended transmembrane Orai N-terminal region (Orai1 amino acids 73-91; Orai3 amino acids 48-65) is fully conserved in the Orai1 and Orai3 isoforms, Orai3 tolerates larger N-terminal truncations than Orai1 in retaining store-operated activation. In an attempt to uncover the reason for these isoform-specific structural requirements, we analyzed a series of Orai mutants and chimeras. We discovered that it was not the N termini, but the loop2 regions connecting TM2 and TM3 of Orai1 and Orai3 that featured distinct properties, which explained the different, isoform-specific behavior of Orai N-truncation mutants. Atomic force microscopy studies and MD simulations suggested that the remaining N-terminal portion in the non-functional Orai1 N-truncation mutants formed new, inhibitory interactions with the Orai1-loop2 regions, but not with Orai3-loop2. Such a loop2 swap restored activation of the N-truncation Orai1 mutants. To mimic interactions between the N terminus and loop2 in full-length Orai1 channels, we induced close proximity of the N terminus and loop2 via cysteine cross-linking, which actually caused significant inhibition of STIM1-mediated Orai currents. In aggregate, maintenance of Orai activation required not only the conserved N-terminal region but also permissive communication of the Orai N terminus and loop2 in an isoform-specific manner.

• MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Neoplasm Proteins chemistry   genetics   metabolism   MeSH
• ORAI1 Protein chemistry   genetics   metabolism   MeSH
• Stromal Interaction Molecule 1 chemistry   genetics   metabolism   MeSH
• Protein Domains MeSH
• Protein Structure, Secondary MeSH
• Calcium Channels chemistry   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

STIM1 (stromal interaction molecule 1) and Orai proteins are the essential components of Ca(2+) release-activated Ca(2+) (CRAC) channels. We focused on the role of cholesterol in the regulation of STIM1-mediated Orai1 currents. Chemically induced cholesterol depletion enhanced store-operated Ca(2+) entry (SOCE) and Orai1 currents. Furthermore, cholesterol depletion in mucosal-type mast cells augmented endogenous CRAC currents, which were associated with increased degranulation, a process that requires calcium influx. Single point mutations in the Orai1 amino terminus that would be expected to abolish cholesterol binding enhanced SOCE to a similar extent as did cholesterol depletion. The increase in Orai1 activity in cells expressing these cholesterol-binding-deficient mutants occurred without affecting the amount in the plasma membrane or the coupling of STIM1 to Orai1. We detected cholesterol binding to an Orai1 amino-terminal fragment in vitro and to full-length Orai1 in cells. Thus, our data showed that Orai1 senses the amount of cholesterol in the plasma membrane and that the interaction of Orai1 with cholesterol inhibits its activity, thereby limiting SOCE.

• MeSH
• Biotinylation MeSH
• Point Mutation MeSH
• Cell Membrane metabolism   MeSH
• Cell Line MeSH
• Cholesterol Oxidase metabolism   MeSH
• Cholesterol metabolism   MeSH
• Circular Dichroism MeSH
• Electrophysiological Phenomena MeSH
• Spectrometry, Fluorescence MeSH
• HEK293 Cells MeSH
• Histamine metabolism   MeSH
• Humans MeSH
• Mast Cells metabolism   MeSH
• Mutation MeSH
• Peptides metabolism   MeSH
• Fluorescence Resonance Energy Transfer MeSH
• Signal Transduction MeSH
• Protein Structure, Tertiary MeSH
• Calcium metabolism   MeSH
• Calcium Channels metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH