STIM1 Human

What is the molecular structure of human STIM1 and its functional domains?

Human STIM1 is a 77.4 kDa protein with multiple functional domains organized for its calcium sensing and signaling roles. The protein structure includes an N-terminal region located in the ER lumen containing two EF-hand Ca²⁺-binding motifs and a sterile alpha motif (SAM); a single transmembrane domain; and a cytosolic C-terminus containing three coiled-coil regions, a channel activation domain (CAD, residues 339-448), a Ser/Pro region, and a Lys-rich domain . This architecture enables STIM1 to function as both a calcium sensor within the ER membrane and an activator of plasma membrane calcium channels .

How does STIM1 activate Orai channels after calcium store depletion?

When Ca²⁺ is depleted from the ER lumen, STIM1 loses its bound Ca²⁺ through its EF-hand motif, which triggers oligomerization and translocation to ER-plasma membrane junctions. At these junctions, STIM1 interacts with and activates the plasma membrane calcium channel Orai1 through its channel activation domain (CAD), enabling store-operated calcium entry (SOCE) . The activation occurs specifically through interaction between the CAD (residues 339-448) within the coiled-coil region of STIM1 and the C-terminus of Orai1 . This process is essential for initiating Ca²⁺ signaling cascades within various cell types .

What experimental evidence supports STIM1's role as a thermosensor?

STIM1 has been identified as a warm sensor through several experimental approaches. Research has demonstrated that STIM1 is directly activated by temperature, as evidenced by the formation of STIM1 multimers after heating . When co-expressed with Orai1, heated STIM1 binds to Orai1 during cooling and opens the channel, resulting in thermally induced calcium influx that is independent of calcium release from the ER . Knockout studies provide physiological evidence, as STIM1 deletion in keratinocytes shifts optimal thermal preference in mice from approximately 32°C to 34°C, affecting temperature selection behavior .

What molecular mechanisms underlie STIM1's thermosensation properties?

The molecular mechanism of STIM1 thermosensation has been elucidated through mutational analyses. The CC1-SOAR region of STIM1 functions as a direct activation domain for temperature sensing, with the transmembrane (TM) region and K domain, but not the EF-SAM domain, being necessary for this process . Both the TM and SOAR domains exhibit similarities and differences between STIM1-mediated thermal sensation and SOCE . The TM23 region (comprising TM2, loop2, and TM3) of Orai1 has been identified as the key domain determining the STIM1/Orai1 thermal response pattern . Importantly, STIM1/Orai1 mediates thermal responses during cooling (heat-off response), while STIM1/Orai3 mediates responses during both heating (heat-on response) and cooling processes .

What are the optimal parameters for designing STIM1 constructs for recombinant protein production?

Successful recombinant STIM1 production requires careful construct design. For C-terminal constructs, those beginning at K246 (corresponding to the first predicted coiled-coil region) yield substantially higher expression levels compared to those starting at Q233 . For N-terminal constructs, truncation beginning from S58 produces the most stable preparations . The table below summarizes expression scores for various hSTIM1 constructs:

Expression score: 4=high, 3=good, 2=low, 1=very low expression

The entire coiled-coil region should be maintained intact, as constructs containing complete coiled-coil domains show higher expression levels than those with partial domains . Specifically, coiled coil 1 appears critical for stability, as confirmed by thermostability shift assays .

What analytical techniques should researchers use to validate STIM1 constructs?

Validation of STIM1 constructs requires multiple complementary techniques. Analytical size exclusion chromatography (ASEC) has proven highly reliable for verifying preparation quality and is typically scalable . Surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and thermostability assays can further validate the integrity and functionality of constructs . For calcium signaling studies, single-cell calcium imaging provides dynamic measurements of STIM1-mediated responses . Statistical analysis should employ unpaired Student's t-tests for comparing two samples or one-way ANOVA for three or more samples, with significance typically denoted at p < 0.05, p < 0.01, and p < 0.001 levels .

How is STIM1 involved in fibrosis and tissue remodeling across different organ systems?

STIM1 plays crucial roles in fibrosis and tissue remodeling in multiple organs. In airway smooth muscle cells, STIM1 catalyzes methacholine-triggered Ca²⁺ oscillations, driving remodeling and hyperresponsiveness . It promotes cell proliferation, migration, and secretion of cytokines and extracellular matrix proteins essential for fibrosis development . In vascular smooth muscle cells, STIM1 and Orai1 mediate the transition from a quiescent to a proliferative state in response to platelet-derived growth factor (PDGF), crucial for vascular repair and remodeling . In kidney mesangial cells, which share similarities with vascular smooth muscle cells, STIM1 expression increases under diabetic conditions, regulating autophagy, cell proliferation, and fibrosis via the PI3K/AKT/mTOR signaling pathway .

What cell-type specific variations exist in STIM1 function and expression?

STIM1 function exhibits notable cell type-specific variations. In airway smooth muscle cells, heightened SOCE activity contributes to greater airway responsiveness, with differences observed between mouse strains (BALB/c vs. C57BL/6) . Enhanced colocalization of Orai1/STIM1 occurs in lung epithelial, interstitial, and luminal immune cells in conditions like asthma and cystic fibrosis . In vascular smooth muscle cells, STIM1 mediates proliferative transitions in response to PDGF . Kidney mesangial cells under diabetic conditions show increased STIM1 expression, with distinct effects on cellular processes . In keratinocytes, STIM1 influences thermal preference behaviors . These variations highlight the importance of considering cell-specific contexts when studying STIM1.

What structural differences explain functional divergence between STIM1 and STIM2?

Despite high sequence homology, STIM1 and STIM2 show significant functional differences. While STIM1 demonstrates temperature sensitivity, STIM2 lacks this capability even when co-expressed with Orai1 . A key difference lies in the K domain, which plays a crucial role in facilitating STIM1 activation and promoting translocation to the plasma membrane, whereas its role in STIM2 is less significant . Experiments with STIM1-K2 (a chimeric construct with STIM2's K domain) rescued part of the thermal response of STIM1-ΔK, suggesting the K domain may be responsible for mediating targeting of STIM1 to the plasma membrane rather than having a direct temperature-sensing role .

How can researchers overcome stability issues when producing recombinant STIM1?

Producing stable recombinant STIM1 presents significant challenges due to its transmembrane nature and oligomerization tendencies. A multi-construct approach has proven effective, with over 200 hSTIM1 constructs tested to identify key domains and boundaries for stable protein production . To overcome stability issues, researchers should: (1) Maintain intact domain boundaries, particularly for coiled-coil regions; (2) Design multiple constructs differing slightly in length (1-2 amino acids) to identify optimal boundaries; (3) Use K246 as a starting point for C-terminal constructs; (4) Include the entire coiled-coil region, especially coiled coil 1, for C-terminal fragment stability; and (5) Employ analytical size exclusion chromatography to verify sample quality before proceeding to functional studies .

What are the best experimental systems for studying STIM1-Orai interactions?

Studying STIM1-Orai interactions requires careful selection of experimental systems. Single-cell calcium imaging combined with confocal analysis provides detailed insights into functional interactions . For thermal response studies, temperature-controlled perfusion systems coupled with calcium imaging enable precise quantification of heat-on and heat-off responses . Creating chimeric proteins by fusing domains of STIM1 with STIM2, or Orai1 with Orai3, helps identify key interaction domains . For in vitro studies, constructs containing the channel activation domain (CAD, residues 339-448) of STIM1 have proven valuable for studying direct interactions with Orai proteins . Both E. coli and S. cerevisiae expression systems have been used for recombinant protein production, with success varying based on specific construct design .