Molecular Determinants and stoichiometry of STIM1/Orai activation

A rapid change in cytosolic calcium levels is one of the main mechanisms to stimulate cellular processes including secretion, contraction and gene activation. Store-operated Ca2+ channels represent a widespread route for Ca2+ entry into cells, among which the Ca2+ release-activated Ca2+ (CRAC) channel is a prototypic one. Its key components, the luminal Ca2+ sensor STIM1 and the three highly Ca2+ selective Orai channels couple to each other upon depletion of endoplasmic reticulum (ER) stores thereby activating Ca2+ entry. The project utilizes a combined approach of electrophysiology, confocal FRET and single molecule fluorescence microscopy together with molecular biology and biochemistry techniques to provide a detailed insight into the composition of the active STIM1/Orai complex. Initially I will aim to identify the role of an N-terminal polybasic cluster in Orai in the activation of this ion channel. Another main part of my project represents the elucidation of the stoichiometry of STIM1 proteins and intact Orai subunits that are required for a functional STIM1/Orai complex. Moreover I will investigate if Orai proteins form stable tetramers or if STIM1 induces oligomerization of Orai dimers upon store-depletion. Furthermore, I will aim to uncover essential amino acids in the STIM1 homomerization domain. Finally, dominant negative fragments will be identified that interfere with Orai/CRAC stimulation. In summary, I will provide a collective overview on the molecular prerequisites finally allowing Orai activation.