The Orai activation machinery

Among diverse Ca2+ entry pathways into the cell, the Ca2+ release-activated Ca2+ (CRAC) channel is best characterized. Its key components include the ER located Ca2+ sensor STIM1 and the three Ca2+ selective Orai channels. Upon store-depletion STIM1 proteins multimerize and redistribute into punctae close to the plasma membrane. Coupling of STIM1 to Orai proteins involves their cytosolic C-termini and culminates in activation of CRAC currents. This project aims at identifying N- as well as C-terminal Orai domains essential for its activation. Initial focus will be laid on Orai3 that allows for activation by STIM1 or 2-APB in an attempt to pinpoint essential regions for either of these stimuli. STIM1-relevant regions of Orai3 will be analogously examined on Orai1. Moreover, N- and C- terminal chimera of Orai1/3 will be utilized to test for a co-operation of these cytosolic strands for Orai activation. In a complementary approach we will address conformational coupling aspects of the Orai activation machinery via a novel STIM1-derived conformational sensor. Conformational rearrangements of a double-labeled STIM1 C- terminal fragment measured upon its coupling to a variety of Orai channel constructs will be related to their function ranging from inactive to fully active. The results obtained by this project will reveal key determinants within Orai N- or C-terminus that are essential for Orai activation, particularly via STIM1. The identified regions might be utilized as dominant negative species to interfere with Orai activation and thus represent potential drug targets. The developed STIM1 conformational sensors are interesting tools not only to relate Orai function to STIM1 C-terminal conformational rearrangements, but additionally represent reporter sensors for potential drug evaluation at the level of STIM1. Thus, both topics will provide a basis for novel approaches to interfere with the STIM1/Orai activation machinery.

Among diverse Ca2+ entry pathways into the cell, the Ca2+ release-activated Ca2+ (CRAC) channel is best characterized. Its key components include the ER located Ca2+ sensor STIM1 and the three Ca2+ selective Orai channels. Upon store-depletion STIM1 proteins multimerize and redistribute into punctae close to the plasma membrane. Coupling of STIM1 to Orai proteins involves their cytosolic C-termini and culminates in activation of CRAC currents. One goal of this project was to identify both N- as well as C-terminal Orai domains, which are essential for this activation. Furthermore, visualization of conformational changes during this activation process was approached. On the latter point, we designed a STIM1-derived conformational sensor, which allowed for the first time to pursue, upon binding of STIM1 to Orai1, a "switch" from a closed to an open conformation. Furthermore, it was possible to simulate the physiologically active conformation of STIM1 by introducing point mutations, which additionally enabled us to identify domains participating in the STIM1 conformational switch. Insights into the domains involved in Orai activation were obtained in particular on the basis of Orai3 and its activation by STIM1 as well as 2-APB. We could show that the N-terminal domain conserved among Orai proteins is essential for the gating of the Orai channel, and it involves distinct stretches for the activation by STIM1 and 2-APB. Furthermore, we succeeded in characterizing the STIM/Orai interaction site by resolving the three-dimensional structure of STIM1 and Orai C-termini in complex. With regard to the use of Orai/STIM as a potential target for immune-regulating drugs, we characterized the molecular action of novel CRAC channel inhibitors. With the results achieved in this project, we have identified key determinants within the Orai N- and C-termini that are essential for the Orai activation, in particular by STIM1. The developed STIM1 conformational sensor is an interesting tool to relate Orai activation to STIM1 C-terminal re-arrangements, and it additionally allows for evaluation of novel STIM1- targeting drugs.