The role of Orai N-terminus and loop 2 in the coupling with STIM1

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 amino acids particularly within the N-terminus and loop 2 of Orai essential for its activation by STIM1. Initial focus will be laid on the role of the conserved, membrane-adjacent region within Orai1 N-terminus that is involved, besides Orai C-terminus, in the interaction/gating with STIM1. (i) Key residues therein will be functionally identified by the effect of Orai1 N-terminal truncations as well as point mutants on the current activation. A decrease or lack of current activation might be caused by an impeded overall interaction with STIM1 and/or a gating defect. (ii) Potential impairment of STIM1 interaction with the mutated Orai1 N-terminus will be specifically resolved by a novel approach based on the constitutively active Orai1 V102A/C mutant. The Ca2+ selectivity of this mutant can be recovered as long as Orai1 N-terminal interactions with STIM1 occur providing a precise read-out parameter for specific detection of STIM1 interaction with mutated Orai1 N-terminal strands. (iii) To differentiate between impairment of the gating machinery or the N-terminal/STIM1 interactions, N-terminal Orai1 mutants will be examined for a potential recovery of current activation or Ca2+ selectivity when swapping loop2 and/or C-terminus of Orai3. This chimeric approach is based on our finding that Orai1 channels with identical N-terminal truncations as Orai3 channels already lack current activation pointing to additional structural elements promoting Orai3/STIM1 interaction/gating. (iv) In a complementary approach we will address the individual impact of Orai cytosolic strands to the overall interaction with STIM1 by our novel FIRE assay. Orai key domains or residues therein identified in the functional experiments above will be examined for their potential to interact in a 2D environment with STIM1 fragments mimicking the interaction of membrane-localized STIM1/Orai. Moreover, this approach will be utilized to identify new domains within STIM1 C-terminus targeting Orai N-terminus and loop 2. The identified domains generally involved in the interaction will additionally provide a basis for specific interference with the Orai/STIM coupling 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 aimed at identifying amino acids particularly within the N-terminus and loop 2 of Orai essential for its activation by STIM1. Initial focus has been laid on the role of the conserved, membrane-adjacent region within Orai1 N-terminus that is involved, besides Orai C-terminus, in the interaction/gating with STIM1. The most important results in this project on the STIM/Orai activation machinery can be seen in the resolution of (i) hot-spot residues within the conserved N-terminal Orai domain essential to the activation and gating of Orai channels, (ii) the three dimensional structure of a complex of interacting STIM1-Orai1 C-termini, and the understanding of the activation state of STIM1 by (iii) a coiled-coil clamp mechanism and by (iv) a missense disease-related mutation. By the development of two novel techniques for the characterisation of functional STIM/Orai interactions of of their fragments, we further increased the general technique repertoire for studying protein-protein interactions. In summary, the identified domains involved in and controlling the interaction as well as activation of the STIM/Orai system will additionally provide a basis for specific therapeutic interventions relevant in the treatment of immunological diseases.