Inside-out Ca2+ signalling prompted by STIM1 conformational switch

Abstract

Store-operated Ca2+ entry mediated by STIM1 and ORAI1 constitutes one of the major Ca2+ entry routes in mammalian cells. The molecular choreography of STIM1-ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca2+ store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution toward the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca2+ signaling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. Based on these findings we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.

Molecular mechanisms underlying inhibition of STIM1-Orai1-mediated Ca2+ entry induced by 2-aminoethoxydiphenyl borate

“…Protocols for the loading of Fura-2 AM were similar to those described before (13): HEK293 cells were first kept in the imaging solution with 1 mM CaCl 2 and 2 μM Fura-2 AM for 30 min. Next, cells were kept in Fura-2 AM free imaging solution with 1 mM CaCl 2 for another 30 min.…”

The STIM-Orai Pathway: Conformational Coupling Between STIM and Orai in the Activation of Store-Operated Ca2+ Entry

“…Transient ER luminal Ca 2+ depletion results in a major STIM protein conformational change whereby the luminal N-termini of the dimeric proteins become more closely associated. This conformational change induces the C-terminal cytosolic portion of the dimeric STIM protein to unfold and extend into the cytosol and be able to associate with the PM in ER-PM junctions (Ma et al 2015). The C-termini of the dimeric STIM proteins are highly conserved between the two STIM isoforms particularly in a region termed the STIM-Orai activating region (SOAR; 344–442 in STIM1) (Yuan et al 2009).…”

“…The exposed SOAR domain in the activated STIM1 protein is free to bind Orai1 at the PM. Binding to Orai1 channels causes transient trapping and activation of the channels within ER-PM junctions (Zhou et al 2013; Yang et al 2012; Ma et al 2015). Ca 2+ brought in through Orai1 trapped in ER-PM junctions potentiates calcineurin-induced NFAT dephosphorylation, which regulates transcription of a large number of genes in many different cell types (Kar and Parekh 2015; Zhou et al 2015a).…”

Optogenetic Immunomodulation: Shedding Light on Antitumor Immunity

“…In addition to its best-established role in driving T lymphocyte activation, intracellular Ca 2+ mobilization promotes inflammasome activation in macrophages, DC maturation, and antigen presentation [17–19]. By mimicking a conformational switch in STIM1 [18, 20] and thus obviating the need for physiological stimulus or store depletion to elicit Ca 2+ entry, He et al developed an Opto-CRAC system (Figure 1b) to photo-trigger Ca 2+ influx and faithfully phenocopy hallmark Ca 2+ -dependent responses in immune cells following light stimulation [21, 22]. When introduced into therapeutic DCs in a mouse model of melanoma, engineered Opto-CRAC channels function as “photoactivatable adjuvants” to enhance DC maturation and antigen presentation (step 1; Figure 1a), thereby promoting T cell priming and activation (step 2) to facilitate the killing of melanoma and its metastases at distal sites [21].…”