Gating of the Orai channel complex involves outer regions surrounding the pore

Ca2+ release-activated Ca2+ (CRAC) channels represent one of the most prominent Ca2+ entry pathway into the cell. They control diverse cellular signaling processes ranging from cell proliferation, gene expression and T-cell activation. Two proteins, the endoplasmic Ca2+ sensor, STIM1, and the Ca2+ selective Orai1 channel, forming a hexameric complex, are sufficient to fully reconstitute CRAC channels. Coupling of STIM1 C-terminus to Orai, via bridging Orai1 N- and C-terminus, is hypothesized to induces a conformational change to the open state. Here, we aim to uncover mechanistic steps how STIM1 activates the Orai channel. Specifically, we will address if STIM1 docking alters i) the orientation of the C-terminal strands and in the following ii) TM helix interactions leading to iii) an open pore conformation. Therefore we will employ a combined approach of patch- clamp, FRET and cysteine crosslinking studies complemented by molecular dynamic (MD) simulations and NMR measurements. Primarily, MD simulations and in vitro experiments together with site-directed mutagenesis aim to elucidate relevant residues and/or intramolecular interactions in TM3/TM4 involved in gating. Our initial experiments revealed indeed that mutation of specific residues in TM2 (H134), TM3 (V181) and TM4 (P245) induce constitutive, Ca2+-selective currents potentially via interactions with neighbouring TM helices. Hence, these amino acids maintain wild- type Orai1 in the closed state and might be directly affected by STIM1 binding to Orai C-terminus thus inducing channel opening. In this context we will investigate if STIM1 coupling alters the orientation of the C-termini and in turn the conformation of outer helical structures by comparing Orai1 wild-type with constitutively active mutants as well as STIM1-activated Orai1. Moreover, as preliminary experiments point to distinct key residues in the intracellular loop2 and the TM3 of Orai1 and Orai3 channels maintaining them in the closed state, we will evaluate our hypothesis that the two channels undergo different conformational changes upon their activation. Hence, we aim to elucidate isoform specific gating mechanisms. In summary, these studies aim at uncovering the molecular mechanism of STIM1-induced conformational changes in Orai1 helices surrounding the pore leading to its open state. Thus, fundamental insight into Orai1 TM reorientations from the closed into the open state will be provided.

Calcium plays a versatile role in the human body as well as in the single cell. Among a variety of Ca2+ entry pathways into the cell, the Ca2+ release-activated Ca2+ (CRAC) channel is best known. It is composed of the ER located Ca2+ sensor STIM and the Ca2+ selective Orai ion channel. Upon depletion of intracellular Ca2+ stores, STIM proteins couple to Orai channels which initiates Ca2+ entry into the cell. This project aimed at the examination to what extent Orai pore opening depends on the pore-surrounding segments of the ion channel complex. Here, we focused especially on the outer transmembrane regions as well as on the cytosolic regions of the Orai complex and their co-regulation with STIM1. The most important results in this project on the STIM/Orai activation machinery can be seen (i) in the identification of functional relevant STIM-Orai-communication sites, (ii) the detection of key sites/ "hot spot" amino acids in the Orai channel, that allow Orai pore opening, as well as (iii) the determination of Orai-subtype-specific regulatory regions. The results were obtained by a complementary approach of experimental and molecular dynamic simulations. In summary, the identified indispensable and modulatory domains within the STIM/Orai complex reveal novel insights in the STIM/Orai activation mechanisms and will additionally provide a basis for specific therapeutic interventions relevant in the treatment of immunological diseases.