Interactions of STIM and ORAI

The identification of the molecular basis of Ca2+ release activated channels (CRAC) is only starting to emerge. Recently, knock-down strategies identified the stromal interaction protein 1 (STIM1) and ORAI1 as essential parts in the CRAC complex. STIM1 is an ER-located Ca2+-sensor, and ORAI1, consisting of four transmembrane regions, turned out to be all or part of the CRAC pore. In this project we will focus especially on the identification of intra- and intermolecular interactions of STIM1 and ORAI1 in the standard expression system HEK293 cells in order to characterize the minimal key domains mediating their assembly and coupling. For this, we will use a combined approach of several techniques including electrophysiology, Ca2+ imaging, confocal FRET as well as TIRF microscopy together with molecular biology and biochemical methods. For detailed mapping of relevant interaction sites we will additionally employ the peptide array SPOT technology. Furthermore X-ray crystallography will be applied to potentially interacting fragments of STIM1 and ORAI1 to elucidate their 3-diemensional structure, both alone and in a complex. Our studies will be extended to ORAI2 and ORAI3 to compare the major interaction domains with those of ORAI1 probably revealing explanations for different functions. Key domains mediating or inhibiting STIM1/ORAI activation will be probed for their potential interference with endogenous CRAC in rat basophilic leukemia (RBL) cells representing a model system where STIM1 and ORAI1 seem to manifest the molecular origin of CRAC. These results will provide a basis to specifically manipulate endogenous CRAC currents of native cells in an attempt to elucidate therapeutical targets in the long term.

The identification of the molecular basis of store-operated or Ca2+ release activated channels (SOC or CRAC) is only starting to emerge. While members of the transient receptor potential (TRPs) ion channels family (TRPs) including the canonical TRPs (TRPCs) and Ca2+ selective vanilloid TRPV6 are suggested as channel components, in the past two years knock-down strategies identified the stromal interaction protein 1 (STIM1) and Orai1 as essential parts in the CRAC complex. STIM1 is an ER-located Ca2+-sensor, and Orai1, consisting of four transmembrane regions, turned out to be all or part of the CRAC pore. In this study we have focused especially on the identification of intra- and intermolecular interactions of STIM and Orai in the standard expression system HEK293 cells to analyze the key domains mediating their assembly and function. For this, we have used a combined approach of several techniques including electrophysiology, Ca2+ imaging, confocal FRET microscopy together with molecular biology and biochemical methods. Significant results have been obtained regarding (i) the minimal domain of STIM1 required for interaction with Orai, (ii) the domains within STIM1 and Orai that mediate their interaction, (iii) a modulatory domain that mediates fast inactivation of Orai channels, (iv) the heteromeric assembly of Orai1/Orai3 proteins affecting the Ca2+- selectivity and (v) the stochiometry of an Orai1 protein assembly in dependence of activation by STIM1. Identification of key domains mediating STIM/Orai activation has provided a basis to specifically manipulate endogenous CRAC/SOC currents of native cells. Rat basophilic leukemia (RBL) have been employed as model systems to probe STIM/Orai fragments or mutants for a potential interference with endogenous CRAC currents.