Vitalizing passive STIM1 domains for Orai channel activation

Store-operated Ca2+ entry (SOCE) is essential for many processes ranging from the cellular to the physiological level. Ca2+ entry, initiated by the endoplasmic reticulum (ER) Ca2+ sensor STIM1, regulates various functions including Ca2+ homeostasis, cell growth, proliferation, gene expression and differentiation. Several loss of function mutations of STIM1 result in immune dysfunctions and myopathy. In this project, using electrophysiology, FRET measurements, the new FIRE approach (FRET In a Restricted Environment), and confocal fluorescence imaging we aim to elucidate the molecular processes of STIM1 activation especially focusing on the STIM1 transmembrane domain (TM) and CC1a2. The latter has recently raised some interest, as a mutation therein (R304W) has been associated with the Stormorken syndrome. The impact of the STIM1 TM and CC1a2 domains on the activation state of STIM1 and its coupling to Orai have been only marginally analyzed up to date. Examination of STIM1 TM and CC1a2 mutants with different methods using full length STIM1, conformational sensor constructs and small fragments of STIM1 attached to the ER membrane or cytosolically expressed will help to explore the activating / modulatory role of these two STIM1 domains and the detailed mechanistic steps of STIM1 / Orai activation in the healthy and pathologic system. Public health relevance: The major goal of the presented research is to increase knowledge about the regulatory molecular mechanisms of calcium release activated calcium entry (CRAC). CRAC channels represent one of the major pathways allowing calcium to enter human cells. In case of defective CRAC components like STIM mutants or Orai mutants several pathogenic phenotypes arise: severe combined immunodeficiency, allergy and muscle disorders like tubular aggregate myopathies. Therefore the proposed research is relevant to public health as developing knowledge about key steps in CRAC activation at the molecular level represents an important tool for the generation of therapeutics for the treatment of these diseases.

The so-called Stromal Interaction Molecule1 (STIM1) was characterized in the course of this FWF-funded project. In particular, a specific domain (CC1a2) was investigated and analysed with respect to its modulatory and regulatory functions. STIM1 is part of the Store Operated Calcium Entry (SOCE) into the cell and is essential for a variety of biological processes at both cellular and physiological levels. Calcium influx, triggered by the calcium sensor STIM1 in the endoplasmic reticulum (ER), affects a wide range of functions such as calcium hemoeostasis, cell growth, cell division, gene expression and cell differentiation. Mutations in STIM1 resulting in loss of function result in immune dysfunctions and myopathies. Using biophysical methods such as electrophysiology, FRET measurements, the innovative FIRE system (FRET In a Restricted Environment) and confocal fluorescence microscopy, the molecular processes of STIM1 activation were studied. The CC1a2 domain attracted attention in 2014 because a mutation in this region (R304W), which puts STIM1 in an active state, was associated with Stormorken syndrome in humans. We were able to further investigate and successfully analyze this genetic mutant at the protein level. The activation mechanism of STIM1 in the healthy as well as the diseased (STIM1 R304W Stormorken mutant) system was described on the molecular level and published in the scientific journal "Nature Communications" .