STIM Proteins Cluster Orai1 Channels and Modulate Receptor-Mediated Calcium Signals

Abstract

Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels plays an indispensable role in receptor-evoked Ca2+ signaling. The two main CRAC channel facilitators are the PM Orai channels, and the ER Ca2+ sensors, STIM1 and STIM2. The molecular mechanism of STIM1/Orai1 coupling remains unsolved. Our recent work provided insight into the coupling mechanism between the Orai1 activating domain of STIM1 (SOAR) and the Orai1 C-terminus. Using rationally-designed mutational screens of the purported Orai1 interaction site within SOAR, we determined that mutating residue F394 to histidine completely prevents binding of full-length STIM1. We developed concatenated SOAR dimers containing one or two F394H mutations, and demonstrated that only a single functional subunit within a SOAR dimer is necessary for Orai1 binding. Current studies reveal that concatenated SOAR heterodimers (SH-S) are unable to form Orai1 clusters, whereas cells expressing wild-type dimers (S-S) retain this ability. The recent discovery of the splice variant of STIM2 (STIM2.1), which has an abrogated binding capacity similar to STIM1-F394H, provided us with another tool to investigate STIM/Orai1 coupling and its physiological role in receptor-evoked Ca2+ signals. Using concatenated heterodimers (SH-S or S2.1-S), we observed profound effects on dimer mobility and generation of ICRAC as compared to wild-type SOAR1 homodimers (S-S), in cells stably expressing Orai1. The physiological role of clustering Orai1 channels is suggested from co-expressing full length STIM1 and STIM2.1. We observe profoundly altered oscillatory responses in HEK cells induced by physiological levels of CCh. Our studies suggest that clustering may affect the ability for Orai1 channels to generate fully functional Ca2+ entry microdomains within ER-PM junctions. This provides strong evidence supporting the “unimolecular model” of STIM1-Orai1 coupling, which appears to be critical for channel clustering, ICRAC generation, and maintenance of Ca2+ oscillations.