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Calmodulin Binding to the Polybasic C-Termini of STIM Proteins Involved in Store-Operated Calcium Entry †
Abstract
Translocation of STIM1 and STIM2 from the endoplasmic reticulum to the plasma membrane is a key step in store-operated calcium entry in the cell. We show by isothermal titration calorimetry that calmodulin binds in a calcium-dependent manner to the polybasic C-termini of STIM1 and STIM2, a region critical for their translocation to the plasma membrane ( K D < or = 1 microM in calcium). HSQC NMR spectroscopy shows this interaction is in the fast exchange regime. By binding STIM1 and STIM2, calmodulin may regulate store refilling, thereby ensuring the maintenance of its own action in intracellular signaling.
... The interactions among CRAC2A and STIM1 or Orai1 involve the coiled-coil domain, as well as the proline/lysine rich segment of the former, and considering Orai1, map to the N-terminal domain with K85 and K87 serving as critical interaction sites (Figure 1) [21]. It is worth mentioning that the sites of CRACR2A-STIM1 and CRACR2A-Orai1 interaction overlap with these described to bind calmodulin (CaM) in the presence of Ca 2+ , explicitly including hOrai1 and the lysin-rich domain of hSTIM1 [21,83]. CRAC2A associations with both basic components of the CRAC channel complex are associated with a stringent dependence on the activation state, as these are promoted successively to store depletion yet exclusively under Ca 2+ -free conditions, while otherwise strong binding between the mentioned domains of STIM1/Orai1 and CRACR2A was reported to vanquish if exposed to buffer solutions supplemented with 2 mM Ca 2+ [21]. ...
... This is explained by the fact that the side chains of those residues homologous to W76 and Y80 of human Orai1 proteins that emerged as critical for CaM to associate, reside within the central pathway of ionpermeation, putatively precluding binding of calmodulin for steric reasons [18,[140][141][142]. Calmodulin in its Ca 2+ -complexed form was found to associate with the poly-basic domain of STIM1 as well, relying in particular on the residues L390 and F391 of activated STIM1 [83,143,144]. This led to alternative concepts of a role of CaM in slow inactivation (SCDI)rather than FCDI, whereby binding of Ca 2+ -saturated calmodulin was proposed to manipulate the ability of STIM1 to couple to Orai1 and/or the propensity to engage homomeric interactions. ...
... Indeed, Li and colleagues reported that overexpression of wildtype calmodulin rather than Ca 2+ -insensitive variants was capable of lessening the level of STIM1-STIM1 association upon store depletion. Considering the residence of 390 LF 391 in the immediate vicinity of the coupling interface of STIM1 and Orai1, couplings among the CRAC channel and fragments of STIM1 were reported to be sensitive to the presence of Ca 2+ calmodulin alike, yet awareness is needed that activation of Orai1 channels by STIM1 itself seems to be sensitive to sequence alterations at these two loci as well [83,143]. In any case, interactions of the aforementioned hydrophobic dipeptide and Ca 2+ -bound calmodulin were recently reproduced by Bhardwaj et al., in addition to detecting thereto unseen associations of the Ca 2+ -bound sensor protein with juxtaposed L374 and V375 residues of hSTIM1. ...
The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca2+ from the endoplasmic reticulum (ER), is critical for Ca2+ homeostasis and active signal transduction in a plethora of cell types. Spurred by the long-sought decryption of the molecular nature of the CRAC channel, considerable scientific effort has been devoted to gaining insights into functional and structural mechanisms underlying this signalling cascade. Key players in CRAC channel function are the Stromal interaction molecule 1 (STIM1) and Orai1. STIM1 proteins span through the membrane of the ER, are competent in sensing luminal Ca2+ concentration, and in turn, are responsible for relaying the signal of Ca2+ store-depletion to pore-forming Orai1 proteins in the plasma membrane. A direct interaction of STIM1 and Orai1 allows for the re-entry of Ca2+ from the extracellular space. Although much is already known about the structure, function, and interaction of STIM1 and Orai1, there is growing evidence that CRAC under physiological conditions is dependent on additional proteins to function properly. Several auxiliary proteins have been shown to regulate CRAC channel activity by means of direct interactions with STIM1 and/or Orai1, promoting or hindering Ca2+ influx in a mechanistically diverse manner. Various proteins have also been identified to exert a modulatory role on the CRAC signalling cascade although inherently lacking an affinity for both STIM1 and Orai1. Apart from ubiquitously expressed representatives, a subset of such regulatory mechanisms seems to allow for a cell-type-specific control of CRAC channel function, considering the rather restricted expression patterns of the specific proteins. Given the high functional and clinical relevance of both generic and cell-type-specific interacting networks, the following review shall provide a comprehensive summary of regulators of the multilayered CRAC channel signalling cascade. It also includes proteins expressed in a narrow spectrum of cells and tissues that are often disregarded in other reviews of similar topics.
... Since STIM1 and STIM2 comprise several predicted Ca 2 + /CaM-binding sites in their CTDs [20], cytosolic Ca 2 + may also contribute directly to their regulation. One of the Ca 2 + /CaMbinding sites overlaps with the lipid-binding K-rich domain of STIM1 and STIM2 and isothermal titration calorimetry with STIM1 and STIM2 peptides revealed K d 's for Ca 2 + /CaM of 0.8 and 0.9 μM, respectively [21]. This suggests that binding of Ca 2 + /CaM may compete with lipid binding and ER-PM contact formation but the mechanism of this interaction remains unknown. ...
... Together with a faster activation of STIM2 in response to the depletion of Ca 2 + from the ER lumen [15], these features may contribute on the one hand to high sensitivity of STIM2 as Ca 2 + sensor, but on the other hand they bear the risk of an overactivation of STIM2 and constitutively high cytosolic Ca 2 + levels. In this context, the previously described Ca 2 + -dependent binding of CaM to the Krich domains of STIM1 and STIM2 point towards an additional regulation of ER-PM contact formation via competing PI(4,5)P 2 and Ca 2 + /CaM-binding sites [21]. ...
... In contrast to the isothermal titration calorimetry results of Bauer et al. (2008), reduced monomeric GFP-STIM2K showed 8.4-fold higher binding to Ca 2 + /CaM beads than GFP-STIM1K ( Figure 3B). In order to determine whether formation of an amphipathic α-helix in STIM2 K-rich domain enhances Ca 2 + /CaM binding, we mutated both prolines in GFP-STIM1K and tested binding. ...
Ca2+ homeostasis and signalling rely on physical contacts between Ca2+ sensors in the endoplasmic reticulum (ER) and Ca2+ channels in the plasma membrane (PM). STIM1 and STIM2 Ca2+ sensors oligomerize upon Ca2+ depletion in the ER lumen, contact phosphoinositides at the PM via their cytosolic lysine (K)-rich domains, and activate Ca2+ channels. Differential sensitivities of STIM1 and STIM2 towards ER luminal Ca2+ have been studied but responses towards elevated cytosolic Ca2+ concentration and the mechanism of lipid binding remain unclear. We found that tetramerization of the STIM1 K-rich domain is necessary and sufficient for binding to PI(4,5)P2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation. In contrast, dimerization of STIM2 K-rich domain was sufficient for lipid binding. Further, the K-rich domain of STIM2, but not of STIM1, forms an amphipathic α-helix. These distinct features of the STIM2 K-rich domain cause an increased affinity for PI(4,5)P2, consistent with the lower activation threshold of STIM2 and a function as regulator of basal Ca2+ levels. Concomitant with higher affinity for PM lipids, binding of Calmodulin inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PI(4,5)P2 concentration-dependent manner. Therefore, we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER-PM contacts via Calmodulin binding.
... To identify the ER retention signal in STIM1, I first focused on its lipid-binding domain, the K-rich domain. The K-rich domain can associate with Ca 2+ /Calmodulin (Bauer et al, 2008;Bhardwaj et al, 2013) and phospholipids in the PM during the formation of ER-PM contacts Park et al, 2009;Walsh et al, 2010). Therefore, I examined whether the K-rich domain plays a role in retention of STIM1 in the ER. ...
... Apart from binding to PI(4,5)P 2 via the K-rich domain, STIM1 also interacts directly (Bauer et al, 2008;Bhardwaj et al, 2013), which may lead to conformational changes resulting in exposure of RXR retention signals or to interference with recruitment of STIM1 to ER exit sites. Other factors such as physical interaction between STIM1 and the cytosolic Ca 2+ sensor CRACR2A, which ! ))! stabilizes CRAC channels in T cells, at ER-PM contact sites (Srikanth et al, 2010) and yet unknown components could regulate the trafficking and stability of STIM1 at the PM (Ercan et al, 2012). ...
... Local [Ca 2+ ] c may regulate the interaction between the K-rich domain of STIM1 and lipids in the PM, which would influence the distribution of STIM1(McLaughlin & Murray, 2005). In addition to PI(4,5)P 2 , the K-rich domain of STIM1 can also bind to Ca 2+ /CaM in presence of 1 mM CaCl 2 , suggesting that fully-Ca 2+ saturated CaM binds to K-rich domain of STIM1 at locally high [Ca 2+ ](Bauer et al, 2008;Bhardwaj et al, 2013). This binding to Ca 2+ /CaM via its K-rich domain would allow STIM1 to sense changes in local [Ca 2+ ] c indirectly.In addition to the K-rich domain, STIM1 contains at least two more Ca 2+ /CaM binding sites(Ercan, 2011). ...
The coupling of endoplasmic reticulum (ER) and plasma membrane (PM) is crucial for
calcium (Ca2+) homeostasis. STIM1 and STIM2 are type I membrane proteins of the ER and
function as Ca2+ sensors in a process known as store-operated calcium entry (SOCE). They
sense a drop in luminal Ca2+ concentration and undergo conformational changes and
oligomerization. The active oligomerized STIM proteins translocate to ER-PM contact sites,
where they bind to phosphoinositides (PIPs) at the inner leaflet of the PM via their lysine (K)-
rich domains and activate Orai1, a pore-forming Ca2+ release-activated Ca2+ (CRAC)
channel subunit in the PM.
I found that STIM2, but not STIM1, contains a di-lysine ER-retention signal. This
signal restricts the function of STIM2 as Ca2+ sensor to the ER while STIM1 can reach the
PM via the classical secretary pathway. The intracellular distribution of STIM1 is regulated in
a cell-cycle-dependent manner with cell surface expression of STIM1 during mitosis.
Efficient retention of STIM1 in the ER during interphase depends on its K-rich domain and a
di-arginine ER retention signal. SOCE enhances ER retention, suggesting that trafficking of
STIM1 is regulated and this regulation contributes to STIM1’s role as multifunctional
component in Ca2+-signaling.
In contrast to mitotic cells, interphase cells retain most of their STIM1 intracellularly.
Under resting condition, the ER-resident STIMs are preferentially located in PI(4,5)P2
containing preexisting ER-PM contact sites, which are expanded upon ER Ca2+ depletion.
The lipid-binding, K-rich domains are required to localize STIM proteins in preexisting ERPM
contact sites. Moreover, STIM2 recruits ER more efficiently to the PM. This is consistent
with the fact that STIM2 has higher lipid-binding affinity and lower activation threshold than
STIM1 and that STIM2 functions as a regulator of basal Ca2+ homeostasis.
Finally, I studied the role of microtubules in ER-PM contact site formation. I observed
that STIM1 aligns along microtubules. Alignment of STIM proteins with microtubules is a
conserved process. In addition to accumulation of STIM1 at microtubule plus ends, STIM1
moves along microtubules in an EB-1-independent manner. I identified two EB-1-
independent microtubule-binding sites located within the C-terminus of STIM1 and found
that oligomerization increases the EB-1-independent microtubule-binding affinity of STIM1.
However, the physiological function of this EB1-independent microtubule binding activity
remains elusive.
... The polybasic domains of STIM1 and STIM2 have both been shown to interact directly with calmodulin (CaM) in a Ca 2+ dependent manner using isothermal titration calorimetry (Bauer et al., 2008). It was proposed that when cytoplasmic Ca 2+ levels increase, CaM associates with Ca 2+ thereby increasing its affinity for STIM1 and STIM2. ...
... It was proposed that when cytoplasmic Ca 2+ levels increase, CaM associates with Ca 2+ thereby increasing its affinity for STIM1 and STIM2. The interaction with CaM and the STIM proteins may result in the destabilisation and dissociation of STIM1 from plasma membrane Orai1 and act as an effective switch to terminate SOC influx when cytosolic Ca 2+ levels are high (Bauer et al., 2008). However, an interaction between full length STIM1 and CaM has not been demonstrated. ...
... Another protein shown to bind to STIM1 includes the microtubule plus end tracking protein, EB1, although this interaction seems to be required for remodelling the ER and was not shown to be required for SOCE (Grigoriev et al., 2008). There is also evidence to suggest that calmodulin binds to STIM1 (Bauer et al., 2008) and Orai1 ) and may be involved in the Ca 2+ -dependent inactivation of SOCE . One of the main aims of this study was to identify a novel interacting partner for STIM1 and/or Orai1 which may be involved in the regulation of SOCE. ...
... Dysfunctions in Ca 2+ /CaM based regulatory mechanisms are linked to several human diseases [49][50][51][52]. The key components of the CRAC machinery, STIM and Orai channel proteins have been shown to interact with Ca 2+ /CaM [16,46,[53][54][55][56][57][58][59][60]. However, Ca 2+ /CaM, STIM1 and Orai1 interaction sites and the underlying regulatory mechanism of SCDI still remain unclear. ...
... Yet, it was shown that these Orai1 residues enable conformational changes within the pore, leading to CRAC channel inactivation without the requirement of Ca 2+ /CaM [59]. STIM1 K-rich domain binds Ca 2+ /CaM and is required for the STIM1-SARAF interaction as well as for SARAF-mediated SCDI of I CRAC [43] [16,54]. Deletion of the K-rich domain of STIM1 led to only a small decrease in Ca 2+ /CaM binding, suggesting that there are likely additional Ca 2+ /CaM binding motifs in STIM1 [16]. ...
Background/aims:
Store-operated Ca2+ entry (SOCE) through plasma membrane Ca2+ channel Orai1 is essential for many cellular processes. SOCE, activated by ER Ca2+ store-depletion, relies on the gating function of STIM1 Orai1-activating region SOAR of the ER-anchored Ca2+-sensing protein STIM1. Electrophysiologically, SOCE is characterized as Ca2+ release-activated Ca2+ current (ICRAC). A major regulatory mechanism that prevents deleterious Ca2+ overload is the slow Ca2+-dependent inactivation (SCDI) of ICRAC. Several studies have suggested a role of Ca2+/calmodulin (Ca2+/CaM) in triggering SCDI. However, a direct contribution of STIM1 in regulating Ca2+/CaM-mediated SCDI of ICRAC is as yet unclear.
Methods:
The Ca2+/CaM binding to STIM1 was tested by pulling down recombinant GFP-tagged human STIM1 C-terminal fragments on CaM sepharose beads. STIM1 was knocked out by CRISPR/Cas9 technique in HEK293 cells stably overexpressing human Orai1. Store-operated Ca2+ influx was measured using Fluorometric Imaging Plate Reader and whole-cell patch clamp in cells transfected with STIM1 CaM binding mutants. The involvement of Ca2+/CaM in SCDI was investigated by including recombinant human CaM in patch pipette in electrophysiology.
Results:
Here we identified residues Leu374/Val375 (H1) and Leu390/Phe391 (H2) within SOAR that serve as hydrophobic anchor sites for Ca2+/CaM binding. The bifunctional H2 site is critical for both Orai1 activation and Ca2+/CaM binding. Single residue mutations of Phe391 to less hydrophobic residues significantly diminished SOCE and ICRAC, independent of Ca2+/CaM. Hence, the role of H2 residues in Ca2+/CaM-mediated SCDI cannot be precisely evaluated. In contrast, the H1 site controls exclusively Ca2+/CaM binding and subsequently SCDI, but not Orai1 activation. V375A but not V375W substitution eliminated SCDI of ICRAC caused by Ca2+/CaM, proving a direct role of STIM1 in coordinating SCDI.
Conclusion:
Taken together, we propose a mechanistic model, wherein binding of Ca2+/CaM to STIM1 hydrophobic anchor residues, H1 and H2, triggers SCDI by disrupting the functional interaction between STIM1 and Orai1. Our findings reveal how STIM1, Orai1, and Ca2+/CaM are functionally coordinated to control ICRAC.
... Interestingly, one study suggests that ER refilling by itself may not be sufficient for reversing the SOCE process and may require Ca 2ϩ entry through Orai1 into the cytosol in addition to the rebinding of luminal Ca 2ϩ to the STIM1 EF hand (344) (see also Ref. 219). The Ca 2ϩ sensor for this process is not known, but SARAF (274), CRACR2A (364), and CaM bound to the PBD (17) or other parts of STIM1 (111) are possible candidates. ...
... An increasing number of STIM-or Orai-binding proteins have been isolated through tandem affinity purification, mass spectrometry/proteomics, and RNAi screens. These include STIM1-binding proteins such as CRACR2A (364), P100 (412), junctate (363), Golli (97,98,401), POST (180), SARAF (274), the ER oxidoreductase ERp57 (305), calnexin (330), and CaM (17,266). Orai-binding proteins include CaM (257), CRACR2A (364), POST (180), and SPCA2 (99). ...
Store-operated cofor calcium signaling in virtually all metozoan cells and serve a wide variety of functions ranging from gene expression, motility, and secretion to tissue and organ development and the immune response. SOCs are activated by the depletion of Ca2+ from the endoplasmic reticulum (ER), triggered physiologically through stimulation of a diverse set of surface receptors. Over 15 years after the first characterization of SOCs through electrophysiology, the identification of the STIM proteins as ER Ca2+ sensors and the Orai proteins as store-operated channels has enabled rapid progress in understanding the unique mechanism of store-operate calcium entry (SOCE). Depletion of Ca2+ from the ER causes STIM to accumulate at ER-plasma membrane (PM) junctions where it traps and activates Orai channels diffusing in the closely apposed PM. Mutagenesis studies combined with recent structural insights about STIM and Orai proteins are now beginning to reveal the molecular underpinnings of these choreographic events. This review describes the major experimental advances underlying our current understanding of how ER Ca2+ depletion is coupled to the activation of SOCs. Particular emphasis is placed on the molecular mechanisms of STIM and Orai activation, Orai channel properties, modulation of STIM and Orai function, pharmacological inhibitors of SOCE, and the functions of STIM and Orai in physiology and disease.
... Despite the intriguing variations, the function of STIMs and CaM are not totally mutually exclusive. CaM can bind to the cytosolic domains of both STIM1 and STIM2 and may play a role in targeting of STIMs to ER-PM junctions or in disassembling STIMs from Orai proteins during inactivation [216]. The binding sites on STIM1 and STIM2 are located at the far C-terminus of the molecules (i.e. ...
... The binding sites on STIM1 and STIM2 are located at the far C-terminus of the molecules (i.e. residues 667e685 and residues 730e746, respectively) [216] and each contain potential hydrophobic anchor residues. Additionally, CaM has been shown to bind to the N-terminal gating helix of Orai1 and is associated with Ca 2þ -dependent inactivation of SOCE [217]. ...
The calcium (Ca(2+)) ion is a universal signalling messenger which plays vital physiological roles in all eukaryotes. To decode highly regulated intracellular Ca(2+) signals, cells have evolved a number of sensor proteins that are ideally adapted to respond to a specific range of Ca(2+) levels. Among many such proteins, calmodulin (CaM) is a multi-functional cytoplasmic Ca(2+) sensor with a remarkable ability to interact with and regulate a plethora of structurally diverse target proteins. CaM achieves this 'multi-talented' functionality through two EF-hand domains, each with an independent capacity to bind targets, and an adaptable flexible linker. By contrast, stromal interaction molecule-1 and -2 (STIMs) have evolved for a specific role in endoplasmic reticulum (ER) Ca(2+) sensing using EF-hand machinery analogous to CaM; however, whereas CaM structurally adjusts to dissimilar binding partners, STIMs use the EF-hand machinery to self-regulate the stability of the Ca(2+) sensing domain. The molecular mechanisms underlying the Ca(2+)-dependent signal transduction by CaM and STIMs have revealed a remarkable repertoire of actions and underscore the flexibility of nature in molecular evolution and adaption to discrete Ca(2+) levels. Recent genomic sequencing efforts have uncovered a number of disease-associated mutations in both CaM and STIM1. This article aims to highlight the most recent key structural and functional findings in the CaM and STIM fields, and discusses how these two Ca(2+) sensor proteins execute their biological functions.
Copyright © 2015. Published by Elsevier Inc.
... Moreover, in addition to binding PM-lipids, isotermal calorimetry and nuclear magnetic resonance studies revealed that peptides of STIM1 and STIM2 K-rich domains bind to Ca 2+ /CaM (127). This finding is really important, since it is well known that the Ca 2+ sensor CaM interacts with many proteins such as phosphatase calcineurin, cytoskeletal proteins and ion transporters and regulates their functions (reviewed in (128)). ...
... Moreover, independently, it was shown that the K-rich region of both STIM1 and STIM2 are capable of binding Ca 2+ /CaM in vitro (127). I was therefore interested in whether the K-rich region contribute to the ER-retention albeit having no KKXX signal. ...
Das endoplasmatische Retikulum (ER) ist in seiner Morphologie verschiedenartig. Es setzt sich aus plattenförmigen und tubulären Strukturen zusammen. Die verschiedenen Domänen des ERs erfüllen vielfache Funktionen u.a. in der kotranslationalen Proteintranslokation, der Lipidsynthese, der Qualtitätskontrolle und dem Protein- und Lipidtransport zu unterschiedlichen Organellen. Das ER steht in engem Kontakt mit anderen Organellen, um Überleben und Wachstum einer Zelle zu gewährleisten. Zusätzlich bildet das ER Kontakte mit der Plasmamembran (PM) aus. An diesen Kontakten finden Lipidtransfer und Kopplung von Ca2+-Signalen statt. Eine Komponente des kortikalen ERs in der Bäcker-Hefe ist das integrale Membranprotein Ist2. Die Sortierung von Ist2 in das kortikale ER erfolgt mit Hilfe seines kortikalen Sortierungssignals (CSS), das an Lipide der PM bindet. Da bisher nicht bekannt war, ob Ist2 im kortikalen ER verbleibt oder durch einen unkonventionellen Weg zur Plasmamembran gelangt, habe ich die Lokalisation von Ist2 in mammalischen Zellen untersucht. Meine Ergebnisse demonstrieren, dass Ist2 das ER nicht verläßt und dort periphere Domänen ausbildet, die in enger Nachbarschaft zur PM liegen. Um die Merkmale dieser peripheren ER-Strukturen weiter zu analysieren, habe ich mammalische Typ1-Membranproteine mit dem CSSIst2 markiert und deren Lokalisation untersucht. Durch die Interaktion des CSSIst2 mit Lipiden der PM wurden alle getesteten Chimären zu den peripheren ER-Strukturen rekrutiert. Weiterhin konnte ich zeigen, daß diese peripheren ER-Strukturen statisch und mit dem restlichen ER verbunden sind. Neben dem Hefeprotein Ist2 sind die mammalischen Proteine STIM1 und STIM2 ebenfalls in der Lage, ER-PM-Kontaktstellen zu bilden. STIM-Proteine haben ihre Funktion in der Signalverstärkung während des Speicher-abhängigen Ca2+-Eintritts. Sie erkennen Ca2+-Konzentrationen durch ihre N-terminalen EF-Hand-Domänen. Nach Ca2+-Depletion des ERs multimerisieren sie und formen ER-PM-Kontaktstellen, an denen sie mit dem Ca2+-Kanal der PM, Orai1, interagieren und diesen aktivieren. Darüber hinaus habe ich den molekularen Mechanismus der Ausbildung von ER-PM-Kontaktstellen aufgedeckt. Durch in vitro Liposomen-Bindungsstudien habe ich gezeigt, dass die C-Termini von STIM1 und STIM2 mittels ihrer Lysin (K)-reichen Domänen an Lipide der PM binden. Diese Ergebnisse verdeutlichen, daß die Ausbildung der ER-PM-Kontaktstellen von der Expression einen integralen membranenprotein mit einem PM-Lipid-Bindungssignal abhängig ist. Da für das STIM1-Protein bereits eine Lokalisation an der Zelloberfläche demonstriert worden war, habe ich seine Retention im ER untersucht. Dabei konnte ich zwei Mechanismen identifizieren. Der erste Mechanismus beruht auf dem Zurückhalten des STIM1-Proteins im ER über mehrere Di-Arginin ER-Retentionssignale. Der zweite Mechanismus ist abhängig von der cytosolischen Ca2+-Konzentrationen. Meine Ergebnisse zeigen, dass die Depletion von cytosolischem Ca2+ den Transport von STIM1 an die Zelloberfläche fördert, wo STIM1 Orai1 aktiviert. Ausgehend von meinen Daten erkennt STIM1 indirekt das cytosolische Ca2+ durch seine K-reiche Domäne, die Ca2+ im Komplex mit Calmodulin bindet. Somit integriert STIM1 Ca2+-Signale im ER und im Cytosol.
... Calmodulin is an essential protein present in all eukaryotic cells. It transmits the signal of increased cytoplasmic Ca 2+ concentration to the activation or attenuation of several hundred enzymes, transporters and other target proteins [20][21][22][23][24][25][26][27][28]. Calmodulin is thereby involved in the regulation of higher order functions such as memory and learning, cell motility and growth, and immune response [25,26,28]. ...
... It transmits the signal of increased cytoplasmic Ca 2+ concentration to the activation or attenuation of several hundred enzymes, transporters and other target proteins [20][21][22][23][24][25][26][27][28]. Calmodulin is thereby involved in the regulation of higher order functions such as memory and learning, cell motility and growth, and immune response [25,26,28]. One of the most striking aspects of calmodulin is its ability to blend promiscuity with specificity in target binding. ...
This study explores the influence of long range intra-protein electrostatic interactions on the conformation of calmodulin in solution. Ensemble Förster resonance energy transfer (FRET) is measured for calmodulin with a fluorophore pair incorporated specifically with a donor at residue 17 and an acceptor at position 117. This construct was generated by a combination of solid phase peptide synthesis, cloning, expression and native chemical ligation. This labelling method has not previously been used with calmodulin and represents a convenient method for ensuring the explicit positioning of the fluorophores. The ensemble FRET experiments reveal significant electrostatic repulsion between the globular domains in the calcium free protein. At low salt, calmodulin has a relatively extended conformation and the distance between the domains is further increased by denaturation, by heat, or by non-ionic denaturants. The repulsion between domains is screened by salt and is also diminished by calcium binding, which changes the protein net charge from -23 to -15. Compared to the calcium-free form at low salt,, the inter-domain distance in the calcium bound form has, on average, decreased by 25%. The conformation of the calcium form is insensitive to salt screening. These results imply that when the two globular domains of calmodulin interact with target, there is no significant free energy penalty due to electrostatic interactions. © 2013 The Authors Journal compilation © 2013 FEBS.
... Physiologically, a small increase in cytosolic Ca 2+ is sufficient to active CaM and CaMKII, which then provides feedback regulation on Ca 2+ signal. For instance, activation of CaM upon Ca 2+ rise acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid SOCE inactivation in many types of cells [16,34,36], playing as a down-regulator of SOCE. In contrast, CaMKII activation usually enhances Ca 2+ signaling; such as promoting ryanodine receptor and L-type Ca 2+ channel activation in cardiomyocytes [19][20][21], as well as enhancing SOCE in non-excitable cells [4,31,35]. ...
... Moreover, CaMKII can be auto-activated in pathological situation [17,37]. Therefore, the key players, CaM and CaMKII, in Ca 2+ /CaM/CaMKII pathway can regulate Ca 2+ signaling independently through CaMKII auto-activation or cooperatively through internal Ca 2+ increase and act on different target proteins and pathways [17,[34][35][36][37], rendering much complicated and fine-tuned Ca 2+ activities to fit various biological needs in cellular function. ...
Background/aims:
Upon Ca2+ store depletion, stromal interaction molecule 1 (STIM1) oligomerizes, redistributes near plasmalemma to interact with Ca2+ selective channel-forming subunit (Orai1) and initiates store-operated Ca2+ entry (SOCE). Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a regulator of SOCE, but how CaMKII regulates SOCE remains obscure.
Methods:
Using Fura2, confocal microscopy, co-immunoprecipitation, specific blocker and overexpression/knockdown approaches, we evaluated STIM1 aggregation and its interaction with Orai1, and SOCE upon Ca2+ store depletion in thapsigargin (TG) treated HEK293 and HeLa cells.
Results:
Overexpression of CaMKIIδ enhanced TG-induced STIM1 co-localization and interaction with Orai1 as well as SOCE. In contrast, CaMKIIδ knockdown and a specific inhibitor of CaMKII suppressed them. In addition, overexpression or knockdown of CaMKIIδ in TG treated cells exhibited increased or reduced STIM1 clustering and plasmalemma redistribution, respectively.
Conclusion:
CaMKII up-regulates SOCE by increasing STIM1 aggregation and interaction with Orai1. This study provides an additional insight into SOCE regulation and a potential mechanism for CaMKII involvement in some pathological situations through crosstalk with SOCE.
... In order to identify the signals in STIM1 that are responsible for ER retention, we focused on the Krich domain. This domain interacts with Ca 2+ /Calmodulin (CaM) (42) and binds to PM lipids during the formation of ER-PM contacts (20,21,(24)(25)(26). Moreover, it has been identified as a regulator of TRPC Ca 2+ channels (43). ...
... In STIM1, the K-rich domain functions in regulation of its trafficking. This could be achieved by interaction with phosphoinositides (20,21,24,25) at the PM or by Ca 2+ /CaM (42). ...
STIM1 is a core component of the store-operated Ca²⁺-entry channel involved in Ca²⁺-signaling with an important role in the activation of immune cells and many other cell types. In response to cell activation, STIM1 protein senses low Ca²⁺ concentration in the lumen of the endoplasmic reticulum (ER) and activates the channel protein Orai1 in the plasma membrane by direct physical contact. The related protein STIM2 functions similar but its physiological role is less well defined. We found that STIM2, but not STIM1, contains a di-lysine ER-retention signal. This restricts the function of STIM2 as Ca²⁺ sensor to the ER while STIM1 can reach the plasma membrane. The intracellular distribution of STIM1 is regulated in a cell-cycle-dependent manner with cell surface expression of STIM1 during mitosis. Efficient retention of STIM1 in the ER during interphase depends on its lysine-rich domain and a di-arginine ER retention signal. Store-operated Ca²⁺-entry enhanced ER retention, suggesting that trafficking of STIM1 is regulated and this regulation contributes to STIM1s role as multifunctional component in Ca²⁺-signaling.
... Thus, mitochondria cannot independently regulate channel activity [160]. It is therefore reasonable to assume that the mitochondrial-dependent Ca 2+ microdomains that are formed around the channels may regulate the function of Ca 2+ binding proteins such as calmodulin, which binds to the polybasic domain of STIM [210], and CRACR2A, which participates in the formation and stability of STIM-ORAI1 complex [211]. Both proteins may thus help mitochondria influence CRAC channel activity. ...
During T cell activation, the engagement of a T cell with an antigen-presenting cell (APC) results in rapid cytoskeletal rearrangements and a dramatic increase of intracellular calcium (Ca2+) concentration, downstream to T cell antigen receptor (TCR) ligation. These events facilitate the organization of an immunological synapse (IS), which supports the redistribution of receptors, signaling molecules and organelles towards the T cell-APC interface to induce downstream signaling events, ultimately supporting T cell effector functions. Thus, Ca2+ signaling and cytoskeleton rearrangements are essential for T cell activation and T cell-dependent immune response. Rapid release of Ca2+ from intracellular stores, e.g. the endoplasmic reticulum (ER), triggers the opening of Ca2+ release-activated Ca2+ (CRAC) channels, residing in the plasma membrane. These channels facilitate a sustained influx of extracellular Ca2+ across the plasma membrane in a process termed store-operated Ca2+ entry (SOCE). Because CRAC channels are themselves inhibited by Ca2+ ions, additional factors are suggested to enable the sustained Ca2+ influx required for T cell function. Among these factors, we focus here on the contribution of the actin and microtubule cytoskeleton. The TCR-mediated increase in intracellular Ca2+ evokes a rapid cytoskeleton-dependent polarization, which involves actin cytoskeleton rearrangements and microtubule-organizing center (MTOC) reorientation. Here, we review the molecular mechanisms of Ca2+ flux and cytoskeletal rearrangements, and further describe the way by which the cytoskeletal networks feedback to Ca2+ signaling by controlling the spatial and temporal distribution of Ca2+ sources and sinks, modulating TCR-dependent Ca2+ signals, which are required for an appropriate T cell response. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Herve.
... Many Ca 2+ transport systems in the cell are directly regulated by CaM (Fig. 1), counting for example the plasma membrane Ca 2+ -ATPase (reviewed in Refs. [35,42,43]), diverse Ca 2+ channels such as IP 3 receptors [48], ryanodine receptors [49], Orai/CRAC (calcium release-activated calcium) channels [50,51] and its regulator stromal-interacting molecule 1 (STIM1) [52], voltage-gated Ca 2+ channels (reviewed in Ref. [53]), TRPM2 [54] and other transient receptor potential (TRP) channels (reviewed in Ref. [55]), as well as gap junction channels and hemichannels (reviewed in Ref. [56]). Furthermore, the hyperpolarizing Ca 2+dependent K + channels, that favor Ca 2+ entry via hyperpolarizationsensitive channels, are also CaM-dependent (reviewed in Ref. [57]). ...
Calmodulin (CaM) is a ubiquitous Ca(2+) receptor protein mediating a large number of signaling processes in all eukaryotic cells. CaM plays a central role in regulating a myriad of cellular functions via interaction with multiple target proteins. This review focuses on the action of CaM and CaM-dependent signaling systems in the control of vertebrate cell proliferation, programmed cell death and autophagy. The significance of CaM and interconnected CaM-regulated systems for the physiology of cancer cells including tumor stem cells, and processes required for tumor progression such as growth, tumor-associated angiogenesis and metastasis are highlighted. Furthermore, the potential targeting of CaM-dependent signaling processes for therapeutic use is discussed.
... The protein concentrations were spectrophotometrically determined at 280 nm with the extinction coefficients 1490 M −1 cm −1 for CB, 67 14600 M −1 cm −1 for scMN, 68 and 3200 M −1 cm −1 for calmodulin. 69 To retain CB in the apo form, 200 μM EDTA was added to all buffers. ...
The aggregation of amyloid β peptides (Aβ) into amyloid fibrils is implicated in the pathology of Alzheimer's disease. In light of the increasing number of proteins reported to retard Aβ fibril formation, we investigated the influence of small hydrophilic model proteins of different charge on Aβ aggregation kinetics and their interaction with Aβ. We followed the amyloid fibril formation of Aβ40 and Aβ42 using thioflavin T fluorescence in the presence of six charge variants of calbindin D9k and single-chain monellin. The formation of fibrils was verified with transmission electron microscopy. We observe retardation of the aggregation process from proteins with net charge +8, +2, -2 and -4, whereas no effect is observed for proteins with net charge of -6 and -8. The single-chain monellin mutant with the highest net charge, scMN+8, has the largest retarding effect on the amyloid fibril formation process, which is noticeably delayed at as low as a 0.01:1 scMN+8 to Aβ40 molar ratio. scMN+8 is also the mutant with the fastest association to Aβ40 as detected by surface plasmon resonance, although all retarding variants of calbindin D9k and single-chain monellin bind to Aβ40.
... As STIM1 was postulated to control the gating of TRPC1, it is attractive to hypothesize that the truncation in STIM1 is causally related to the exaggerated response of SNS in SHRSP through abnormal regulation of TRPC1. In addition, Bauer et al. showed that calmodulin bound to the polybasic C-terminal of STIM1 in a calcium-dependent manner [25]. This implied that the lack of the C-terminal residues of STIM1 affected the calmodulin-dependent regulation of STIM1 as well. ...
The stroke-prone spontaneously hypertensive rat (SHRSP) is known to have exaggerated sympathetic nerve activity to various types of stress, which might contribute to the pathogenesis of severe hypertension and stroke observed in this strain. Previously, by using a congenic strain (called SPwch1.72) constructed between SHRSP and the normotensive Wistar-Kyoto rat (WKY), we showed that a 1.8-Mbp fragment on chromosome 1 (Chr1) of SHRSP harbored the responsible gene(s) for the exaggerated sympathetic response to stress. To further narrow down the candidate region, in this study, another congenic strain (SPwch1.71) harboring a smaller fragment on Chr1 including two functional candidate genes, Phox2a and Ship2, was generated. Sympathetic response to cold and restraint stress was compared among SHRSP, SPwch1.71, SPwch1.72 and WKY by three different methods (urinary norepinephrine excretion, blood pressure measurement by the telemetry system and the power spectral analysis on heart rate variability). The results indicated that the response in SPwch1.71 did not significantly differ from that in SHRSP, excluding Phox2a and Ship2 from the candidate genes. As the stress response in SPwch1.72 was significantly less than that in SHRSP, it was concluded that the 1.2-Mbp congenic region covered by SPwch1.72 (and not by SPwch1.71) was responsible for the sympathetic stress response. The sequence analysis of 12 potential candidate genes in this region in WKY/Izm and SHRSP/Izm identified a nonsense mutation in the stromal interaction molecule 1 (Stim1) gene of SHRSP/Izm which was shared among 4 substrains of SHRSP. A western blot analysis confirmed a truncated form of STIM1 in SHRSP/Izm. In addition, the analysis revealed that the protein level of STIM1 in the brainstem of SHRSP/Izm was significantly lower when compared with WKY/Izm. Our results suggested that Stim1 is a strong candidate gene responsible for the exaggerated sympathetic response to stress in SHRSP.
... Although the exact mechanism remains unclear, one possibility is that the binding of Ca 2+ -CAM to the Orai1 N-terminus displaces STIM1 from its binding site at this location, thereby destabilizing activation gating and reducing CRAC channel activity. Interestingly, calmodulin was also shown to bind to STIM1 and STIM2 in a Ca 2+ -dependent manner and may exert completely unrelated mode of modulation via STIM 87 . Golli, an alternative splice variant of the myelin basic protein is reported to negatively regulate SOCE 88 . ...
... Worth mentioning, the effects of CaM on TRPC channels required calcium (Fig. 5C). CaM has been shown to interact with the polybasic domain from STIM1 and STIM2, resulting in reduced refilling of the ER [53]. However, since in our study we used only the SOAR fragment from STIM1 (which is soluble and not attached to the ER), any effect of CaM on ER refilling that may be reflected in changes on TRPC channel PO is unlikely. ...
Depletion of intracellular calcium ion stores initiates a rapid cascade of events culminating with the activation of the so-called Store-Operated Channels (SOC) at the plasma membrane. Calcium influx via SOC is essential in the initiation of calcium-dependent intracellular signaling and for the refilling of internal calcium stores, ensuring the regeneration of the signaling cascade. In spite of the significance of this evolutionary conserved mechanism, the molecular identity of SOC has been the center of a heated controversy spanning over the last 20 years. Initial studies positioned some members of the transient receptor potential canonical (TRPC) channel superfamily of channels (with the more robust evidence pointing to TRPC1) as a putative SOC. Recent evidence indicates that Stromal Interacting Molecule 1 (STIM1) activates some members from the TRPC family of channels. However, the exact subunit composition of TRPC channels remains undetermined to this date. To identify the subunit composition of STIM1-activated TRPC channels, we developed novel method, which combines single channel electrophysiological measurements based on the patch clamp technique with single molecule fluorescence imaging. We termed this method Single ion Channel Single Molecule Detection technique (SC-SMD). Using SC-SMD method, we have obtained direct evidence of the subunit composition of TRPC channels activated by STIM1. Furthermore, our electrophysiological-imaging SC-SMD method provides evidence at the molecular level of the mechanism by which STIM1 and calmodulin antagonize to modulate TRPC channel activity.
... Interestingly, CaM also binds to the polybasic tail of STIM1 and STIM2 in a Ca 2 +-dependent manner [65], suggesting that the local cytosolic Ca 2 + elevations are sensed by CaM, which then binds to the STIM1 Cterminus and/or to the N-terminus of Orai1 to induce the rapid inactivation of SOCE channels. Mitochondria were proposed to minimize the slow Ca 2 +-dependent inactivation by sequestering the Ca 2 + ions entering across SOCE channels [51,66,67]. However, given the proximity between the cortical ER and the PM, the notion that mitochondria can have an impact on the local Ca 2 + concentration at the channel mouth has been challenged, and measurements with Ca 2 +-sensitive probes anchored on the outer mitochondrial membrane revealed that subplasmalemmal mitochondria are not exposed to high Ca 2 + microdomains [68]. ...
The SOCE (store-operated Ca2+ entry) pathway is a central component of cell signalling that links the Ca2+-filling state of the ER (endoplasmic reticulum) to the activation of Ca2+-permeable channels at the PM (plasma membrane). SOCE channels maintain a high free Ca2+ concentration within the ER lumen required for the proper processing and folding of proteins, and fuel the long-term cellular Ca2+ signals that drive gene expression in immune cells. SOCE is initiated by the oligomerization on the membrane of the ER of STIMs (stromal interaction molecules) whose luminal EF-hand domain switches from globular to an extended conformation as soon as the free Ca2+ concentration within the ER lumen ([Ca2+]ER) decreases below basal levels of ~500 μM. The conformational changes induced by the unbinding of Ca2+ from the STIM1 luminal domain promote the formation of higher-order STIM1 oligomers that move towards the PM and exposes activating domains in STIM1 cytosolic tail that bind to Ca2+ channels of the Orai family at the PM and induce their activation. Both SOCE and STIM1 oligomerization are reversible events, but whether restoring normal [Ca2+]ER levels is sufficient to initiate the deoligomerization of STIM1 and to control the termination of SOCE is not known. The translocation of STIM1 towards the PM involves the formation of specialized compartments derived from the ER that we have characterized at the ultrastructural level and termed the pre-cortical ER, the cortical ER and the thin cortical ER. Pre-cortical ER structures are thin ER tubules enriched in STIM1 extending along microtubules and located deep inside cells. The cortical ER is located in the cell periphery in very close proximity (8-11 nm) to the plasma membrane. The thin cortical ER consists of thinner sections of the cortical ER enriched in STIM1 and devoid of chaperones that appear to be specialized ER compartments dedicated to Ca2+ signalling.
... Additionally, several studies have provided evidence for involvement of the C-terminal polylysine sequence of STIM1 in other interactions. This polylysine sequence has been identified as a direct binding partner for canonical transient receptor potential (TRP) channels (Zeng et al., 2008), as a structural determinant of the inwardly rectifying character of I CRAC (Yuan et al., 2009) and as a binding site for calmodulin (Bauer et al., 2008). ...
We have previously shown that PIP5KIβ and PIP5KIγ generate functionally distinct pools of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] important for antigen-stimulated Ca(2+) entry in mast cells. In the present study, we find that association of the endoplasmic reticulum (ER) Ca(2+) sensor, STIM1, and the store-operated Ca(2+) channel, Orai1, stimulated by thapsigargin-mediated ER store depletion, is enhanced by overexpression of PIP5KIβ and inhibited by overexpression of PIP5KIγ. These different PIP5KI isoforms cause differential enhancement of PtdIns(4,5)P(2) in detergent-resistant membrane (DRM) fractions, which comprise ordered lipid regions, and detergent-solubilized membrane (DSM) fractions, which comprise disordered lipid regions. Consistent with these results, the inositol 5-phosphatase L10-Inp54p, which is targeted to ordered lipids, decreases PtdIns(4,5)P(2) in the DRM fraction and inhibits thapsigargin-stimulated STIM1-Orai1 association and store-operated Ca(2+) entry, whereas the inositol 5-phosphatase S15-Inp54p, which is targeted to disordered lipids, decreases PtdIns(4,5)P(2) in the DSM fraction and enhances STIM1-Orai1 association. Removal of either the STIM1 C-terminal polylysine sequence (amino acids 677-685) or an N-terminal polyarginine sequence in Orai1 (amino acids 28-33) eliminates this differential sensitivity of STIM1-Orai1 association to PtdIns(4,5)P(2) in the distinctive membrane domains. Our results are consistent with a model of PtdIns(4,5)P(2) balance, in which store-depletion-stimulated STIM1-Orai1 association is positively regulated by the ordered lipid pool of PtdIns(4,5)P(2) and negatively regulated by PtdIns(4,5)P(2) in disordered lipid domains.
... 71 It should also be noted that Ca 2+ -CaM can bind to the polybasic tails of both STIM1 (i.e., residues 667-685) and STIM2 (i.e., residues 730-746) with K d ~1μM; moreover, this affinity is reduced by 2 orders of magnitude in the absence of Ca 2+ . 73 Therefore, CaM may also downregulate SOCE via binding to STIMs in a Ca 2+ -dependent manner after localized cytosolic Ca 2+ level increases, thereby inhibiting and/ or disrupting ER-PM targeting of STIM1 molecules which is dependent on the polybasic stretches of STIMs. 38,56,59,74,75 ...
Store-operated calcium (Ca (2+)) entry is the process by which molecules located on the endo/sarcoplasmic reticulum (ER/SR) respond to decreased luminal Ca (2+) levels by signaling Ca (2+) release activated Ca (2+) channels (CRAC) channels to open on the plasma membrane (PM). This activation of PM CRAC channels provides a sustained cytosolic Ca (2+) elevation associated with myriad physiological processes. The identities of the molecules which mediate SOCE include stromal interaction molecules (STIMs), functioning as the ER/SR luminal Ca (2+) sensors, and Orai proteins, forming the PM CRAC channels. This review examines the current available high-resolution structural information on these CRAC molecular components with particular focus on the solution structures of the luminal STIM Ca (2+) sensing domains, the crystal structures of cytosolic STIM fragments, a closed Orai hexameric crystal structure and a structure of an Orai1 N-terminal fragment in complex with calmodulin. The accessible structural data are discussed in terms of potential mechanisms of action and cohesiveness with functional observations.
... Our comparison of STIM2.1 and STIM2.2 CAD domain binding to CaM using SPR shows that both domains bind to Ca 2 þ-CaM with a much higher affinity than the C-terminal site (10 and 40 nM versus B1 mM 36,37 ). STIM2.1 has an increased affinity and an altered CaM-binding motif likely generated in part by the specific aa sequence (IQ). ...
Cellular homeostasis relies upon precise regulation of Ca2+ concentration. Stromal interaction molecule (STIM) proteins regulate store-operated calcium entry (SOCE) by sensing Ca2+ concentration in the ER and forming oligomers to trigger Ca2+ entry through plasma membrane-localized Orai1 channels. Here we characterize a STIM2 splice variant, STIM2.1, which retains an additional exon within the region encoding the channel-activating domain. Expression of STIM2.1 is ubiquitous but its abundance relative to the more common STIM2.2 variant is dependent upon cell type and highest in naive T cells. STIM2.1 knockdown increases SOCE in naive CD4+ T cells, whereas knockdown of STIM2.2 decreases SOCE. Conversely, overexpression of STIM2.1, but not STIM2.2, decreases SOCE, indicating its inhibitory role. STIM2.1 interaction with Orai1 is impaired and prevents Orai1 activation, but STIM2.1 shows increased affinity towards calmodulin. Our results imply STIM2.1 as an additional player tuning Orai1 activation in vivo.
... Calmodulin has been reported to interact with TRP proteins, including Drosophila TRPL [52] and mammalian TRPC1 [35]. More recently, calmodulin was found to interact with the polybasic Ctermini of STIM1 [53] and with a membrane-proximal N-terminal domain of Orai1 [36] in a Ca 2+ dependent manner. Different experimental manoeuvres have demonstrated that calmodulin is involved in Ca 2+ -dependent inactivation of SOCE, including expression of TRPC1 or Orai1 mutants lacking the ability to bind calmodulin, which prevented Ca 2+ -dependent inactivation of SOCE in human salivary gland cells [35], or I CRAC in HEK-293 cells [36], respectively, or over-expression of a calmodulin inhibitor peptide and a Ca 2+ -insensitive calmodulin mutant, which results in reduced inactivation of I SOC in liver cells [54]. ...
Store-operated Ca(2+) entry (SOCE) is a major pathway for Ca(2+) influx in non-excitable cells. Recent studies favour a conformational coupling mechanism between the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 and Ca(2+) permeable channels in the plasma membrane to explain SOCE. Previous studies have reported a role for the cytoskeleton modulating the activation of SOCE; therefore, here we have investigated whether the interaction between STIM1 and the Ca(2+) permeable channels is modulated by the actin or microtubular network. In HEK-293 cells, treatment with the microtubular disrupter colchicine enhanced both the activation of SOCE and the association between STIM1 and Orai1 or TRPC1 induced by thapsigargin (TG). Conversely, stabilization of the microtubules by paclitaxel attenuated TG-evoked activation of SOCE and the interaction between STIM1 and the Ca(2+) channels Orai1 and TRPC1, altogether suggesting that the microtubules act as a negative regulator of SOCE. Stabilization of the cortical actin filament layer results in inhibition of TG-evoked both association between STIM1, Orai1 and TRPC1 and SOCE. Interestingly, disruption of the actin filament network by cytochalasin D did not significantly modify TG-evoked association between STIM1 and Orai1 or TRPC1 but enhanced TG-stimulated SOCE. Finally, inhibition of calmodulin by calmidazolium enhances TG-evoked SOCE and disruption of the actin cytoskeleton results in inhibition of TG-evoked association of calmodulin with Orai1 and TRPC1. Thus, we demonstrate that the cytoskeleton plays an essential role in the regulation of SOCE through the modulation of the interaction between their main molecular components.
... A recent study raised the possibility that an ER-resident membrane protein, TMEM110, may cooperate with STIM2 to regulate long-term maintenance of ER-plasma membrane junctions and their short-term physiological remodeling during storedependent Ca 2+ signaling (Quintana et al. 2015). Binding of Ca 2+ /CaM to the K-rich domain of STIM2 contributes to regulation of STIM2-mediated Ca 2+ influx (Bauer et al. 2008). ...
Tight control of basal cytosolic Ca2+ concentration is essential for cell survival and to fine-tune Ca2+-dependent cell functions. A way to control this basal cytosolic Ca2+ concentration is to regulate membrane Ca2+ channels including store-operated Ca2+ channels and secondary messenger-operated channels linked to G-protein-coupled or tyrosine kinase receptor activation. Orai, with or without its reticular STIM partner and Transient Receptor Potential (TRP) proteins, were considered to be the main Ca2+ channels involved. It is well accepted that, in response to cell stimulation, opening of these Ca2+ channels contributes to Ca2+ entry and the transient increase in cytosolic Ca2+ concentration involved in intracellular signaling. However, in various experimental conditions, Ca2+ entry and/or Ca2+ currents can be recorded at rest, without application of any experimental stimulation. This led to the proposition that some plasma membrane Ca2+ channels are already open/activated in basal condition, contributing therefore to constitutive Ca2+ entry. This article focuses on direct and indirect observations supporting constitutive activity of channels belonging to the Orai and TRP families and on the mechanisms underlying their basal/constitutive activities.
... When Ca 2þ is not bound, however, STIM1 and STIM2 bind to CaM with much lower affinity (K d of 55 and 150 mM, respectively). 87 Although the functional significance of CaM-binding remains uncertain, recent evidence suggests that binding of Ca 2þ /CaM to STIM2 competitively inhibits interactions of the STIM2 K-rich domain with liposomes and PIP 2 in the PM. 59 This interaction may prevent Ca 2þ overload by reducing STIM2mediated ER-PM contacts when [Ca 2þ ] c is elevated, and consequently diminish the store-operated Ca 2þ current. ...
Ca ²⁺ release from the endoplasmic reticulum is an important component of Ca ²⁺ signal transduction that controls numerous physiological processes in eukaryotic cells. Release of Ca ²⁺ from the endoplasmic reticulum is coupled to the activation of store-operated Ca ²⁺ entry into cells. Store-operated Ca ²⁺ entry provides Ca ²⁺ for replenishing depleted endoplasmic reticulum Ca ²⁺ stores and a Ca ²⁺ signal that regulates Ca ²⁺ -dependent intracellular biochemical events. Central to connecting discharge of endoplasmic reticulum Ca ²⁺ stores following G protein-coupled receptor activation with the induction of store-operated Ca ²⁺ entry are stromal interaction molecules (STIM1 and STIM2). These highly homologous endoplasmic reticulum transmembrane proteins function as sensors of the Ca ²⁺ concentration within the endoplasmic reticulum lumen and activators of Ca ²⁺ release-activated Ca ²⁺ channels. Emerging evidence indicates that in addition to their role in Ca ²⁺ release-activated Ca ²⁺ channel gating and store-operated Ca ²⁺ entry, STIM1 and STIM2 regulate other cellular signaling events. Recent studies have shown that disruption of STIM expression and function is associated with the pathogenesis of several diseases including autoimmune disorders, cancer, cardiovascular disease, and myopathies. Here, we provide an overview of the latest developments in the molecular physiology and pathophysiology of STIM1 and STIM2.
Impact statement
Intracellular Ca ²⁺ signaling is a fundamentally important regulator of cell physiology. Recent studies have revealed that Ca ²⁺ -binding stromal interaction molecules (Stim1 and Stim2) expressed in the membrane of the endoplasmic reticulum (ER) are essential components of eukaryote Ca ²⁺ signal transduction that control the activity of ion channels and other signaling effectors present in the plasma membrane. This review summarizes the most recent information on the molecular physiology and pathophysiology of stromal interaction molecules. We anticipate that the work presented in our review will provide new insights into molecular interactions that participate in interorganelle signaling crosstalk, cell function, and the pathogenesis of human diseases.
... This region (667-685) did not contain any known CaM-binding site and consists of several basic and hydrophobic residues. ITC experiments in which the polybasic peptide was titrated into CaM revealed a strong binding affinity between these two molecules (1 μM) 54 . Second, through pull-down and SPR methods, the region 459-482 in SOAR of STIM2, which corresponds with residues 368-391 in SOAR of STIM1, was found to interact with Ca 2+ -CaM 55 . ...
Store-operated calcium entry (SOCE) is a major pathway for calcium ions influx into cells and has a critical role in various cell functions. Here we demonstrate that calcium-bound cal-modulin (Ca 2+-CaM) binds to the core region of activated STIM1. This interaction facilitates slow Ca 2+-dependent inactivation after Orai1 channel activation by wild-type STIM1 or a constitutively active STIM1 mutant. We define the CaM-binding site in STIM1, which is adjacent to the STIM1-Orai1 coupling region. The binding of Ca 2+-CaM to activated STIM1 disrupts the STIM1-Orai1 complex and also disassembles STIM1 oligomer. Based on these results we propose a model for the calcium-bound CaM-regulated deactivation of SOCE.
... Adjacent to the SOAR domain there is a proline-and histidine-rich region whose function is still unclear [78,88]. Close to the end of the C-terminal region there is a calmodulin-binding region and a polybasic lysine-rich region [88,89]. ...
Store-operated Ca²⁺ entry (SOCE) is a ubiquitous mechanism for Ca²⁺ influx in mammalian cells with important physiological implications. Since the discovery of SOCE more than three decades ago, the mechanism that communicates the information about the amount of Ca²⁺ accumulated in the intracellular Ca²⁺ stores to the plasma membrane channels and the nature of these channels have been matters of intense investigation and debate. The stromal interaction molecule-1 (STIM1) has been identified as the Ca²⁺ sensor of the intracellular Ca²⁺ compartments that activates the store-operated channels. STIM1 regulates two types of store-dependent channels: the Ca²⁺ release-activated Ca²⁺ (CRAC) channels, formed by Orai1 subunits, that conduct the highly Ca²⁺ selective current ICRAC and the cation permeable store-operated Ca²⁺ (SOC) channels, which consist of Orai1 and TRPC1 proteins and conduct the non-selective current ISOC. While the crystal structure of Drosophila CRAC channel has already been solved, the architecture of the SOC channels still remains unclear. The dynamic interaction of STIM1 with the store-operated channels is modulated by a number of proteins that either support the formation of the functional STIM1-channel complex or protect the cell against Ca²⁺ overload.
... Wie im vorherigen Abschnitt gezeigt, wiesen die PS1 Deletionszellen eine 7,7-fach erhöhte Das intrazelluläre APP Fragment AICD entsteht nach Prozessierung von APP durch die γ-Sekretase und bildet mit dem nukleären Adapterprotein Fe65 und der Histonacetyl-transferase Tip60 einen Komplex, welcher vermutlich transkriptionell aktiv ist [Cao and Südhof, 2001] [Brandman et al., 2007;Stathopulos et al., 2006;Zheng et al., 2011] Rana et al., 2015]. . In der C-terminalen Region wurden zuvor bereits weitere STIM2 Regionen mit einer im Vergleich zur CAD-Domäne niedrigeren Affinität für CaM charakterisiert (C-Terminus: 1mM [Bauer et al., 2008;Bhardwaj et al., 2013] ...
... Adjacent to the CAD region, a proline-and histidine-rich (P/H) region is located at a similar position to that of the serineand proline-rich (S/P) region observed in STIM1, whose function is still unclear (Williams et al. 2001;Ercan et al. 2012). At the end of the C-terminal region there is a CaM-binding region (Bauer et al. 2008) and a polybasic lysine (Lys)-rich domain involved in the interaction with PIP 2 , phosphatidylinositol 1,4,5-trisphosphate (PIP 3 ) and CaM (Ercan et al. 2009;Bhardwaj et al. 2013). In contrast to STIM1, STIM2 also contains a consensus sequence for a di-lysine ER-retention signal (K(X)KXX) in this region, restricting its expression to the ER (Ercan et al. 2012). ...
An endoplasmic reticulum (ER)-resident protein that regulates cytosolic and ER free-Ca(2+) concentration by induction of store-operated calcium entry. That is the original definition of STIM2 and its function. While its activity strongly depends on the amount of calcium stored in the ER, its function goes further to intracellular signalling and gene expression. Initially undercovered by the prominent function of STIM1, STIM2 became to be vital in mice, gradually emerging as an important player in the nervous system, and cooperating with STIM1 in the immune system. STIM2 has also been proposed as a relevant player in pathological conditions related to ageing, Alzheimer and Huntington's diseases, autoimmune disorders and cancer. The discovery of additional functions, together with new splicing forms with opposite roles, clarified existing controversies about STIM2 function in SOCE. Being essential for life, but apparently not for development, new available data demonstrated a complex and still intriguing behaviour that this review summarizes, updating the current knowledge about STIM2 function. This article is protected by copyright. All rights reserved.
... CaM interacts with the lysine-rich domain of STIM1. It is believed that this interaction could be involved in the control of STIM1 translocation to ER-PM junctions or in STIM-Orai1 disassembling (Bauer et al. 2008). It has been reported that CaM binds to a region in the C-terminal of TRPC1 (758-793) thus regulating Ca 2+ -dependent inactivation of this channel (Singh et al. 2002). ...
Store Operated Ca2+ Entry (SOCE), the main Ca2+ influx mechanism in non-excitable cells, is implicated in the immune response and has been reported to be affected in several pathologies including cancer. The basic molecular constituents of SOCE are Orai, the pore forming unit, and STIM, a multidomain protein with at least two principal functions: one is to sense the Ca2+ content inside the lumen of the endoplasmic reticulum(ER) and the second is to activate Orai channels upon depletion of the ER. The link between Ca2+ depletion inside the ER and Ca2+ influx from extracellular media is through a direct association of STIM and Orai, but for this to occur, both molecules have to interact and form clusters where ER and plasma membrane (PM) are intimately apposed. In recent years a great number of components have been identified as participants in SOCE regulation, including regions of plasma membrane enriched in cholesterol and sphingolipids, the so called lipid rafts, which recruit a complex platform of specialized microdomains, which cells use to regulate spatiotemporal Ca2+ signals.
... CaM interacts with the lysine-rich domain of STIM1. It is believed that this interaction could be involved in the control of STIM1 translocation to ER-PM junctions or in STIM-Orai1 disassembling (Bauer et al. 2008). It has been reported that CaM binds to a region in the C-terminal of TRPC1 (758-793) thus regulating Ca 2+ -dependent inactivation of this channel (Singh et al. 2002). ...
Store-operated calcium entry (SOCE) occurs at specialized regions where the endoplasmic reticulum and plasma membranes are closely apposed. Several molecules converge in these junctions to form a complex that spatiotemporally circumscribes SOCE signaling. We have named recently this complex as SOCIC (Store Operated Calcium Influx Complex). There is a growing list of SOCIC members, including the Ca2+ sensor and channel activator STIM1, the Orai and TRPC1 channels, SOCE regulators as CaM and CRACR2A, and SOCE-regulated proteins as SERCA and adenylyl cyclases. Considering that under physiological conditions Ca2+ entry is transient, SOCIC should be a dynamic structure that goes through assembly and disassembly cycles depending on cell requirements, and on the depleted state of intracellular Ca2+ stores. Moreover SOCIC seems to assembly at specialized regions of plasma membrane known as lipid rafts. In this chapter we discuss the evidence supporting the idea that SOCE occurs at microdomains and introduce the SOCIC components known so far. Then we illustrate some ideas on how this complex is assembled and disassembled. Finally we address the evidence of physiological and pathological implications of the microdomain organization of SOCE.
Cell polarization enables restriction of signalling into microdomains. Polarization of lymphocytes following formation of a mature immunological synapse (IS) is essential for calcium-dependent T-cell activation. Here, we analyse calcium microdomains at the IS with total internal reflection fluorescence microscopy. We find that the subplasmalemmal calcium signal following IS formation is sufficiently low to prevent calcium-dependent inactivation of ORAI channels. This is achieved by localizing mitochondria close to ORAI channels. Furthermore, we find that plasma membrane calcium ATPases (PMCAs) are re-distributed into areas beneath mitochondria, which prevented PMCA up-modulation and decreased calcium export locally. This nano-scale distribution—only induced following IS formation—maximizes the efficiency of calcium influx through ORAI channels while it decreases calcium clearance by PMCA, resulting in a more sustained NFAT activity and subsequent activation of T cells.
The calcium ion (Ca(2+)) is a ubiquitous second messenger that is crucial for the regulation of a wide variety of cellular processes. The diverse transient signals transduced by Ca(2+) are mediated by intracellular -Ca(2+)-binding proteins. Calcium ions shuttle into and out of the cytosol, transported across membranes by channels, exchangers, and pumps that regulate flux across the ER, mitochondrial and plasma membranes. Calcium regulates both rapid events, such as cytoskeleton remodelling or release of vesicle contents, and slower ones, such as transcriptional changes. Moreover, sustained cytosolic calcium elevations can lead to unwanted cellular activation or apoptosis. Calmodulin represents the most significant of the Ca(2+)-binding proteins and is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile novel protein-protein interactions that calmodulin participates in, we probed a high-content recombinant human protein array with fluorophore-labelled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human proteins expressed from a human brain cDNA library. We describe the identification of a high affinity interaction between calmodulin and the single-pass transmembrane proteins STIM1 and STIM2 that localise to the ER. Translocation of STIM1 and STIM2 from the endoplasmic reticulum to the plasma membrane is a key step in store operated calcium entry in the cell.
Secretagogin is a calcium-binding protein whose expression is characterised in neuroendocrine, pancreatic, and retinal cells. We have used an array-based proteomic approach with the prokaryotically expressed human protein array (hEx1) and the eukaryotically expressed human protein array (Protoarray) to identify novel calcium-regulated interaction networks of secretagogin. Screening of these arrays with fluorophore-labelled secretagogin in the presence of Ca(2+) ions led to the identification of 12 (hEx1) and 6 (Protoarray) putative targets. A number of targets were identified in both array screens. The putative targets from the hEx1 array were expressed, purified, and subjected to binding analysis using surface plasmon resonance. This identified binding affinities for nine novel secretagogin targets with equilibrium dissociation constants in the 100 pM to 10 nM range. Six of the novel target proteins have important roles in vesicle trafficking; SNAP-23, ARFGAP2, and DOC2alpha are involved in regulating fusion of vesicles to membranes, kinesin 5B and tubulin are essential for transport of vesicles in the cell, and rootletin builds up the rootlet, which is believed to function as scaffold for vesicles. Among the targets are two enzymes, DDAH-2 and ATP-synthase, and one oncoprotein, myeloid leukaemia factor 2. This screening method identifies a role for secretagogin in secretion and vesicle trafficking interacting with several proteins integral to these processes.
Store-operated Ca2+ entry (SOCE) is an ubiquitous and major mechanism for Ca2+ influx in mammalian cells with important physiological relevance. Since the discovery of SOCE in 1986 both, the mechanism that communicates the amount of Ca2+ accumulated in the intracellular Ca2+ stores to the plasma membrane channels and the nature of the capacitative channels, have been a matter of intense investigation. During the last decade, two of the major elements of SOCE, STIM1, the Ca2+ sensor of the intracellular Ca2+ compartments, and Orai1, the protein forming the channel that conducts the capacitative Ca2+ release-activated current I
CRAC, were identified. Together with these proteins, different homologues, including STIM2, Orai2 and Orai3, were identified, although their relevance in SOCE has not been fully characterized yet. Before the identification of STIM1 and Orai1, TRPC proteins were found to be involved in SOCE in different cell types, more likely conducting the non-selective capacitative current described as I
SOC. Current evidence indicates that STIM1, Orai1 and TRPC proteins dynamically interact forming a ternary complex that mediates SOCE in a number of cellular models. The dynamic interaction of STIM1 with Orai1, TRPCs or both might provide an explanation to the distinct capacitative currents described in different cell types.
Ca2+ entry in non-excitable cells is mainly carried by store-operated channels among which the CRAC channel is best characterized. Its two limiting molecular components are represented by the Ca2+ sensor protein STIM1 located in the endoplasmic reticulum and Orai1 in the plasma membrane. STIM1 senses a decrease of the Ca2+ content in internal stores and triggers its accumulation into puncta like structures resulting in coupling to as well as activation of Orai1 channels. The STIM1–Orai coupling process is determined by an interaction via their C-termini. This review highlights recent developments on domains particularly within the cytosolic part of STIM1 that govern this interaction.
The discovery of molecular players in capacitative calcium (Ca(2+)) entry, also referred to as store-operated Ca(2+) entry (SOCE), supposed a great advance in the knowledge of cellular mechanisms of Ca(2+) entry, which are essential for a broad range of cellular functions. The identification of STIM1 and STIM2 proteins as the sensors of Ca(2+) stored in the endoplasmic reticulum unraveled the mechanism by which depletion of intracellular Ca(2+) stores is communicated to store-operated Ca(2+) channels located in the plasma membrane, triggering the activation of SOCE and intracellular Ca(2+)-dependent signaling cascades. Initial studies suggested a dominant function of STIM1 in SOCE and SOCE-dependent cellular functions compared to STIM2, especially those that participate in immune responses. Consequently, most of the subsequent studies focused on STIM1. However, during the last years, STIM2 has been demonstrated to play a more relevant and complex function than initially reported, being even important to sustain normal life in mice. These studies have led to reconsider the role of STIM2 in SOCE and its relevance in cellular physiology. This review is intended to summarize and provide an overview of the current data available about this exciting isoform, STIM2, and its actual position together with STIM1 in the mechanism of SOCE.
Black phosphorus nanosheets (BPNSs) have substantially promoted biomedical nanotechnology due to their unique photothermal and chemotherapeutic properties. However, there is still a limited molecular understanding of the effects of bio-nano interfaces on BPNSs and the subsequent impacts on physiological systems. Here, it is showed that black phosphorus-corona complexes (BPCCs) could function as immune modulators to promote the polarization of macrophages. Mechanistically, BPCCs could interact with calmodulin to activate stromal interaction molecule 2 and facilitate Ca2+ influx in macrophages, which induced the activation of p38 and NF-κB and polarized M0 macrophages to the M1 phenotype. As a result, BPCC-activated macrophages show greater migration towards cancer cells, 1.3-1.9 times higher cellular cytotoxicity and effective phagocytosis of cancer cells. These findings offer insights into the development of potential and unique applications of corona on BPNSs in nanomedicine.
The Ca(2+) depletion of the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca(2+) entry (SOCE) pathway that sustains long-term Ca(2+) signals critical for cellular functions. ER Ca(2+) depletion initiates the oligomerization of stromal interaction molecules (STIM) that control SOCE activation, but whether ER Ca(2+) refilling controls STIM de-oligomerization and SOCE termination is not known. Here, we correlate the changes in free luminal ER Ca(2+) concentrations ([Ca(2+)](ER)) and in STIM1 oligomerization, using fluorescence resonance energy transfer (FRET) between CFP-STIM1 and YFP-STIM1. We observed that STIM1 de-oligomerized at much lower [Ca(2+)](ER) levels during store refilling than it oligomerized during store depletion. We then refilled ER stores without adding exogenous Ca(2+) using a membrane-permeable Ca(2+) chelator to provide a large reservoir of buffered Ca(2+). This procedure rapidly restored pre-stimulatory [Ca(2+)](ER) levels but did not trigger STIM1 de-oligomerization, the FRET signals remaining elevated as long as the external [Ca(2+)] remained low. STIM1 dissociation evoked by Ca(2+) readmission was prevented by SOC channel inhibition and was associated with cytosolic Ca(2+) elevations restricted to STIM1 puncta, indicating that Ca(2+) acts on a cytosolic target close to STIM1 clusters. These data indicate that the refilling of ER Ca(2+) stores is not sufficient to induce STIM1 de-oligomerization and that localized Ca(2+) elevations in the vicinity of assembled SOCE complexes are required for the termination of SOCE.
Receptor-evoked Ca2+ influx is a central component of the Ca2+ signal. A ubiquitous form of Ca2+ influx is activated by depletion of endoplasmic Ca2+ stores, the SOC channels. The two established Ca2+ influx channels that are activated by depletion of the endoplasmic reticulum Ca2+ store are the TRPC channels and the newly discovered Orai channels. The two channels can function independently and are gated by different STIM1 domains. Yet, the two channels also affect the function of each other by competition for STIM1 and by yet unresolved mechanism in which their function is required for their mutual activity. This chapter will discuss the evidence for the regulation of the Orai and TRPC channels by STIM1 and the interrelations between the two activities. The function and properties of the Orai channels and their regulation by STIM1 is extensively covered in other chapters and will only be briefly discussed here as they relate to the function of TRPC channels.
Diverse physiological processes are regulated differentially by Ca(2+) oscillations through the common regulatory hub calmodulin. The capacity of calmodulin to combine specificity with promiscuity remains to be resolved. Here we propose a mechanism based on the molecular properties of calmodulin, its two domains with separate Ca(2+) binding affinities, and target exchange rates that depend on both target identity and Ca(2+) occupancy. The binding dynamics among Ca(2+), Mg(2+), calmodulin, and its targets were modeled with mass-action differential equations based on experimentally determined protein concentrations and rate constants. The model predicts that the activation of calcineurin and nitric oxide synthase depends nonmonotonically on Ca(2+)-oscillation frequency. Preferential activation reaches a maximum at a target-specific frequency. Differential activation arises from the accumulation of inactive calmodulin-target intermediate complexes between Ca(2+) transients. Their accumulation provides the system with hysteresis and favors activation of some targets at the expense of others. The generality of this result was tested by simulating 60 000 networks with two, four, or eight targets with concentrations and rate constants from experimentally determined ranges. Most networks exhibit differential activation that increases in magnitude with the number of targets. Moreover, differential activation increases with decreasing calmodulin concentration due to competition among targets. The results rationalize calmodulin signaling in terms of the network topology and the molecular properties of calmodulin.
All eukaryotic cells have adapted the use of the calcium ion (Ca2+) as a universal signaling element through the evolution of a toolkit of Ca2+sensor, buffer and effector proteins. Among these toolkit components, integral and peripheral proteins decorate biomembranes and coordinate the movement of Ca2+between compartments, sense these concentration changes and elicit physiological signals. These changes in compartmentalized Ca2+levels are not mutually exclusive as signals propagate between compartments. For example, agonist induced surface receptor stimulation can lead to transient increases in cytosolic Ca2+sourced from endoplasmic reticulum (ER) stores; the decrease in ER luminal Ca2+can subsequently signal the opening surface channels which permit the movement of Ca2+from the extracellular space to the cytosol. Remarkably, the minuscule compartments of mitochondria can function as significant cytosolic Ca2+sinks by taking up Ca2+in a coordinated manner. In non-excitable cells, inositol 1,4,5 trisphosphate receptors (IP3Rs) on the ER respond to surface receptor stimulation; stromal interaction molecules (STIMs) sense the ER luminal Ca2+depletion and activate surface Orai1 channels; surface Orai1 channels selectively permit the movement of Ca2+from the extracellular space to the cytosol; uptake of Ca2+into the matrix through the mitochondrial Ca2+uniporter (MCU) further shapes the cytosolic Ca2+levels. Recent structural elucidations of these key Ca2+toolkit components have improved our understanding of how they function to orchestrate precise cytosolic Ca2+levels for specific physiological responses. This chapter reviews the atomic-resolution structures of IP3R, STIM1, Orai1 and MCU elucidated by X-ray crystallography, electron microscopy and NMR and discusses the mechanisms underlying their biological functions in their respective compartments within the cell.
Signaling molecules produced in the pancreatic β-cell following mitochondrial oxidation of glycolytic intermediate metabolites and oxidative phosphorylation trigger Ca2+-dependent signaling pathways that regulate insulin exocytosis. Much is known about ATP-sensitive K+ and voltage-gated Ca2+ currents that contribute to Ca2+-dependent signal transduction in β-cells and insulin secretion, but relatively little is known about other Ca2+ channels that regulate β-cell Ca2+ signaling dynamics and insulin secretion. In a wide range of eukaryotic cells, store-operated Ca2+ entry (SOCE) plays a critical role regulating spatial and temporal changes in cytoplasmic Ca2+ concentration, endoplasmic reticulum (ER) Ca2+ homeostasis, gene expression, protein biosynthesis, and cell viability. Although SOCE has been proposed to play important roles in β-cell Ca2+ signaling and insulin secretion, the underlying molecular mechanisms remain undefined. In this chapter, we provide both an overview of our current understanding of ionic currents regulated by ER Ca2+ stores in insulin-secreting cells and a review of studies in other cell systems that have identified the molecular basis and regulation of SOCE.
Plasma-membrane-localized Orai1 ion channel subunits interacting with ER-localized STIM1 molecules comprise the major subunit composition responsible for calcium release-activated calcium channels. STIM1 "translates" the Ca(2+) store content into Orai1 activity, making it a store-operated channel. Surprisingly, in addition to being the physical activator, STIM1 also modulates Orai1 properties, including its inactivation and permeation (see Chapter 1). STIM1 is thus more than a pure Orai1 activator. Within the past 7 years following the discovery of STIM and Orai proteins, the molecular mechanisms of STIM1/Orai1 activity and their functional importance have been studied in great detail. Much less is currently known about the other isoforms STIM2, Orai2, and Orai3. In this chapter, we summarize the current knowledge about STIM2, Orai2, and Orai3 properties and function. Are these homologues mainly modulators of predominantly STIM1/Orai1-mediated complexes or do store-dependent or -independent functions such as regulation of basal Ca(2+) concentration and activation of Orai3-containing complexes by arachidonic acid or by estrogen receptors point toward their "true" physiological function? Is Orai2 the Orai1 of neurons? A major focus of the review is on the functional relevance of STIM2, Orai2, and Orai3, some of which still remains speculative.
Stromal interaction molecule 1 (STIM1) along with Orai1 mediates extracellular Ca2+ entry into the cytosol through a store-operated Ca2+ entry (SOCE) mechanism in various tissues including skeletal muscle. However, the role(s) of STIM2, a homolog of STIM1, in skeletal muscle has not been well addressed. The present study, first, was focused on searching for STIM2-binding proteins from among proteins mediating skeletal muscle functions. This study used a binding assay, quadrupole time-of-flight mass spectrometry, and co-immunoprecipitation assay with bona-fide STIM2- and SERCA1a-expressing rabbit skeletal muscle. The region for amino acids from 453 to 729 of STIM2 binds to sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a). Next, oxalate-supported 45Ca2+-uptake experiments and various single-myotube Ca2+ imaging experiments using STIM2-knockdown mouse primary skeletal myotubes have suggested that STIM2 attenuates SERCA1a activity during skeletal muscle contraction, which contributes to the intracellular Ca2+ distribution between the cytosol and the SR at rest. In addition, STIM2 regulates Ca2+ movement through RyR1 during skeletal muscle contraction as well as SOCE. Therefore, via regulation of SERCA1a activity, STIM2 regulates both intracellular Ca2+ distribution and Ca2+ movement in skeletal muscle, which makes it both similar to, yet different from, STIM1.
Considerable progress has been made elucidating the molecular mechanisms of calcium (Ca²⁺) sensing by stromal interaction molecules (STIMs) and the basis for Orai channel activity. This chapter focuses on the available high-resolution structural details of STIM and Orai proteins with respect to the regulation of store-operated Ca²⁺ entry (SOCE). Solution structures of the Ca²⁺-sensing domains of STIM1 and STIM2 are reviewed in detail, crystal structures of cytosolic coiled-coil STIM fragments are discussed, and an overview of the closed Drosophila melanogaster Orai hexameric structure is provided. Additionally, we highlight structures of human Orai1 N-terminal and C-terminal domains in complex with calmodulin and human STIM1, respectively. Ultimately, the accessible structural data are discussed in terms of potential mechanisms of action and cohesiveness with functional observations.
Cysteines are among the least abundant amino acids found in proteins. Due to their unique nucleophilic thiol group, they are able to undergo a broad range of chemical modifications besides their known role in disulfide formation, such as S-sulfenylation (-SOH), S-sulfinylation (-SO(2)H), S-sufonylation (-SO(3)H), S-glutathionylation (-SSG), and S-sulfhydration (-SSH), among others. These posttranslational modifications can be irreversible and act as transitional modifiers or as reversible on-off switches for the function of proteins. Disturbances of the redox homeostasis, for example, in situations of increased oxidative stress, can contribute to a range of diseases. Because Ca²⁺ signaling mediated by store-operated calcium entry (SOCE) is involved in a plethora of cellular responses, the cross-talk between reactive oxygen species (ROS) and Ca²⁺ is critical for homeostatic control. Identification of calcium regulatory protein targets of thiol redox modifications is needed to understand their role in biology and disease.
The basic paradigm of a mechanism for calcium influx triggered after a reduction on calcium store content implies a sensor of calcium concentration on the endoplasmic reticulum (the stores) and a calcium channel immersed on the plasma membrane. These two basic components are STIM and Orai, the most fundamental and minimal molecular constituents of the store-operated calcium entry mechanism. However, even when minimal components can be reduced to these two proteins, the intricate process involved in approximating two cellular membranes (endoplasmic reticulum, ER and plasma membrane, PM) require the participation of several other components, many of which remain unidentified to this date. Here we review several of the proteins identified as constituents of the so-called store-operated calcium influx complex (SOCIC) and discuss their role in modulating this complex phenomenon.
Ca2+ signaling depends on a tight regulation of the intracellular Ca2+ concentration. Alterations in basal Ca2+ can lead to various diseases and likely contribute to development of abnormal growth. Different regulators such as calmodulin and Ca2+ pumps limit cytosolic [Ca2+] and their down-regulation by siRNA lead to an increased basal [Ca2+]. Another important regulator is the stromal interaction molecule 2 (STIM2) that shows a reduction in basal [Ca2+] following knock down. The two known isoforms of STIM, STIM1 and STIM2, are ER resident membrane proteins which sense the Ca2+ content of the ER via their luminal EF-hands.
We studied the effect of KAR-2 on cytosolic Ca(2+) level in human neutrophils by using a fluorescent dye (Fura-2) trapped in the cells. KAR-2 is a semisynthetic bis-indole derivative that shares vinblastine anti-microtubular properties, but does not share the vinblastine antagonistic effect on calmodulin. Therefore KAR-2 offers a convenient mean of studying the effect of microtubule destabilization, without concomitant calmodulin alterations. We found that KAR-2 induces Ca(2+) release from intracellular stores, whereby the stores are depleted. In addition KAR-2 reduces store-operated entry of extracellular Ca(2+) induced by agonists such as thapsigargin or ATP. On the other hand, in Ca(2+) refilled cells, KAR-2 promotes limited entry of extracellular Ca(2+) in the absence of agonist, but still interferes prominently with Ca(2+) entry triggered by ATP and with Ca(2+) uptake by intracellular stores. We suggest that Ca(2+) traffic through the plasma membrane is operated by two diverse pathways: the prominent pathway is interfered with by microtubule destabilization, while an alternate and minor pathway is actually favored (or uncovered) following microtubule destabilization.
The three-dimensional solution structure of the complex between calcium-bound calmodulin (Ca(2+)-CaM) and a 26-residue synthetic peptide comprising the CaM binding domain (residues 577 to 602) of skeletal muscle myosin light chain kinase, has been determined using multidimensional heteronuclear filtered and separated nuclear magnetic resonance spectroscopy. The two domains of CaM (residues 6 to 73 and 83 to 146) remain essentially unchanged upon complexation. The long central helix (residues 65 to 93), however, which connects the two domains in the crystal structure of Ca(2+)-CaM, is disrupted into two helices connected by a long flexible loop (residues 74 to 82), thereby enabling the two domains to clamp residues 3 to 21 of the bound peptide, which adopt a helical conformation. The overall structure of the complex is globular, approximating an ellipsoid of dimensions 47 by 32 by 30 angstroms. The helical peptide is located in a hydrophobic channel that passes through the center of the ellipsoid at an angle of approximately 45 degrees with its long axis. The complex is mainly stabilized by hydrophobic interactions which, from the CaM side, involve an unusually large number of methionines. Key residues of the peptide are Trp4 and Phe17, which serve to anchor the amino- and carboxyl-terminal halves of the peptide to the carboxyl- and amino-terminal domains of CaM, respectively. Sequence comparisons indicate that a number of peptides that bind CaM with high affinity share this common feature containing either aromatic residues or long-chain hydrophobic ones separated by a stretch of 12 residues, suggesting that they interact with CaM in a similar manner.
STIM1 (where STIM is stromal interaction molecule) is a candidate tumour suppressor gene that maps to human chromosome 11p15.5, a region implicated in a variety of cancers, particularly embryonal rhabdomyosarcoma. STIM1 codes for a transmembrane phosphoprotein whose structure is unrelated to that of any other known proteins. The precise pathway by which STIM1 regulates cell growth is not known. In the present study we screened gene databases for STIM1-related sequences, and have identified and characterized cDNA sequences representing a single gene in humans and other vertebrates, which we have called STIM2. We identified a single STIM homologue in Drosophila melanogaster (D-Stim) and Caenorhabditis elegans, but no homologues in yeast. STIM1, STIM2 and D-Stim have a conserved genomic organization, indicating that the vertebrate family of two STIM genes most probably arose from a single ancestral gene. The three STIM proteins each contain a single SAM (sterile alpha-motif) domain and an unpaired EF hand within the highly conserved extracellular region, and have coiled-coil domains that are conserved in structure and position within the cytoplasmic region. However, the STIM proteins diverge significantly within the C-terminal half of the cytoplasmic domain. Differential levels of phosphorylation appear to account for two molecular mass isoforms (105 and 115 kDa) of STIM2. We demonstrate by mutation analysis and protein sequencing that human STIM2 initiates translation exclusively from a non-AUG start site in vivo. STIM2 is expressed ubiquitously in cell lines, and co-precipitates with STIM1 from cell lysates. This association into oligomers in vivo indicates a possible functional interaction between STIM1 and STIM2. The structural similarities between STIM1, STIM2 and D-STIM suggest conserved biological functions.
The intracellular calcium sensor protein calmodulin (CaM) interacts with a large number of proteins to regulate their biological functions in response to calcium stimulus. This molecular recognition process is diverse in its mechanism, but can be grouped into several classes based on structural and sequence information. We have developed a web-based database (http://calcium.uhnres.utoronto.ca/ctdb) for this family of proteins containing CaM binding sites or, as we propose to call it herein, CaM recruitment signaling (CRS) motifs. At present the CRS motif found in approximately 180 protein sequences in the databases can be divided into four subclasses, each subclass representing a distinct structural mode of molecular recognition involving CaM. The database can predict a putative CRS location within a given protein sequence, identify the subclass to which it may belong, and structural and biophysical parameters such as hydrophobicity, hydrophobic moment, and propensity for alpha-helix formation.
The calcium ion (Ca(2+)) is a ubiquitous second messenger that is crucial for the regulation of a wide variety of cellular processes. The diverse transient signals transduced by Ca(2+) are mediated by intracellular Ca(2+)-binding proteins, also known as Ca(2+) sensors. A key obstacle to studying many Ca(2+)-sensing proteins is the difficulty in identifying the numerous downstream target interactions that respond to Ca(2+)-induced conformational changes. Among a number of Ca(2+) sensors in the eukaryotic cell, calmodulin (CaM) is the most widespread and the best studied. Employing the mRNA display technique, we have scanned the human proteome for CaM-binding proteins and have identified and characterized a large number of both known and previously uncharacterized proteins that interact with CaM in a Ca(2+)-dependent manner. The interactions of several identified proteins with Ca(2+)/CaM were confirmed by using pull-down assays and coimmunoprecipitation. Many of the CaM-binding proteins identified belong to protein families such as the DEAD/H box proteins, ribosomal proteins, proteasome 26S subunits, and deubiquitinating enzymes, suggesting the possible involvement of Ca(2+)/CaM in different signaling pathways. The selection method described herein could be used to identify the binding partners of other calcium sensors on the proteome-wide scale.
Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.
Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.
As the sole Ca2+ entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx is important in Ca2+ signalling and many other cellular processes. A calcium-release-activated calcium (CRAC) channel in T lymphocytes is the best-characterized SOC influx channel and is essential to the immune response, sustained activity of CRAC channels being required for gene expression and proliferation. The molecular identity and the gating mechanism of SOC and CRAC channels have remained elusive. Previously we identified Stim and the mammalian homologue STIM1 as essential components of CRAC channel activation in Drosophila S2 cells and human T lymphocytes. Here we show that the expression of EF-hand mutants of Stim or STIM1 activates CRAC channels constitutively without changing Ca2+ store content. By immunofluorescence, EM localization and surface biotinylation we show that STIM1 migrates from endoplasmic-reticulum-like sites to the plasma membrane upon depletion of the Ca2+ store. We propose that STIM1 functions as the missing link between Ca2+ store depletion and SOC influx, serving as a Ca2+ sensor that translocates upon store depletion to the plasma membrane to activate CRAC channels.
Calcium signaling pathways control a variety of cellular events such as gene transcription, protein phosphorylation, nucleotide metabolism, and ion transport. These pathways often involve a large number of calcium-binding proteins collectively known as the calmodulin or EF-hand protein superfamily. Many EF-hand proteins undergo a large conformational change upon binding to Ca(2+) and target proteins. All members of the superfamily share marked sequence homology and similar structural features required to sense Ca(2+). Despite such structural similarities, the functional diversity of EF-hand calcium-binding proteins is extraordinary. Calmodulin itself can bind >300 different proteins, and the many members of the neuronal calcium sensor and S100 protein families collectively recognize a largely different set of target proteins. Recent biochemical and structural studies of many different EF-hand proteins highlight remarkable similarities and variations in conformational responses to the common ligand Ca(2+) and their respective cellular targets. In this review, we examine the essence of molecular recognition activities and the mechanisms by which calmodulin superfamily proteins control a wide variety of Ca(2+) signaling processes.
Receptor-induced Ca²⁺ signals are key to the function of all cells and involve release of Ca²⁺ from endoplasmic reticulum (ER) stores, triggering Ca²⁺ entry through plasma membrane (PM) “store-operated channels” (SOCs). The identity of SOCs and their coupling to store depletion remain molecular and mechanistic mysteries. The single transmembrane-spanning Ca²⁺-binding protein, STIM1, is necessary in this coupling process and is proposed to function as an ER Ca²⁺ sensor to provide the trigger for SOC activation. Here we reveal that, in addition to being an ER Ca²⁺ sensor, STIM1 functions within the PM to control operation of the Ca²⁺ entry channel itself. Increased expression levels of STIM1 correlate with a gain in function of Ca²⁺ release-activated Ca²⁺ (CRAC) channel activity. Point mutation of the N-terminal EF hand transforms the CRAC channel current (I CRAC) into a constitutively active, Ca²⁺ store-independent mode. Mutants in the EF hand and cytoplasmic C terminus of STIM1 alter operational parameters of CRAC channels, including pharmacological profile and inactivation properties. Last, Ab externally applied to the STIM1 N-terminal EF hand blocks both I CRAC in hematopoietic cells and SOC-mediated Ca²⁺ entry in HEK293 cells, revealing that STIM1 has an important functional presence within the PM. The results reveal that, in addition to being an ER Ca²⁺ sensor, STIM1 functions within the PM to exert control over the operation of SOCs. As a cell surface signaling protein, STIM1 represents a key pharmacological target to control fundamental Ca²⁺-regulated processes including secretion, contraction, metabolism, cell division, and apoptosis.
• calcium signaling
• calcium channel
• patch-clamp
• mast cells
• T lymphocytes
Stromal interaction molecule 1 (STIM1) has recently been identified by our group and others as an endoplasmic reticulum (ER) Ca²⁺ sensor that responds to ER Ca²⁺ store depletion and activates Ca²⁺ channels in the plasma membrane (PM). The molecular mechanism by which STIM1 transduces signals from the ER lumen to the PM is not yet understood. Here we developed a live-cell FRET approach and show that STIM1 forms oligomers within 5 s after Ca²⁺ store depletion. These oligomers rapidly dissociated when ER Ca²⁺ stores were refilled. We further show that STIM1 formed oligomers before its translocation within the ER network to ER–PM junctions. A mutant STIM1 lacking the C-terminal polybasic PM-targeting motif oligomerized after Ca²⁺ store depletion but failed to form puncta at ER–PM junctions. Using fluorescence recovery after photobleaching measurements to monitor STIM1 mobility, we show that STIM1 oligomers translocate on average only 2 μm to reach ER–PM junctions, arguing that STIM1 ER-to-PM signaling is a local process that is suitable for generating cytosolic Ca²⁺ gradients. Together, our live-cell measurements dissect the STIM1 ER-to-PM signaling relay into four sequential steps: (i) dissociation of Ca²⁺, (ii) rapid oligomerization, (iii) spatially restricted translocation to nearby ER–PM junctions, and (iv) activation of PM Ca²⁺ channels.
• Ca2+ release-activated Ca2+
• fluorescence recovery after photobleaching
• FRET
• store-operated Ca2+ influx
STIM1 and Orai1 are essential components of Ca2+ release-activated Ca2+ channels (CRACs). After endoplasmic reticulum Ca2+ store depletion, STIM1 in the endoplasmic reticulum aggregates and migrates toward the cell periphery to co-localize with
Orai1 on the opposing plasma membrane. Little is known about the roles of different domains of STIM1 and Orai1 in protein
clustering, migration, interaction, and, ultimately, opening CRAC channels. Here we demonstrate that the coiled-coil domain
in the C terminus of STIM1 is crucial for its aggregation. Amino acids 425–671 of STIM1, which contain a serine-proline-rich
region, are important for the correct targeting of the STIM1 cluster to the cell periphery after calcium store depletion.
The polycationic region in the C-terminal tail of STIM1 also helps STIM1 targeting but is not essential for CRAC channel activation.
The cytoplasmic C terminus but not the N terminus of Orai1 is required for its interaction with STIM1. We further identify
a highly conserved region in the N terminus of Orai1 (amino acids 74–90) that is necessary for CRAC channel opening. Finally,
we show that the transmembrane domain of Orai1 participates in Orai1-Orai1 interactions.
STIM1 and CRACM1 (or Orai1) are essential molecular components mediating store-operated Ca2+ entry (SOCE) and Ca2+ release-activated Ca2+ (CRAC) currents. Although STIM1 acts as a luminal Ca2+ sensor in the endoplasmic reticulum (ER), the function of STIM2 remains unclear. Here we reveal that STIM2 has two distinct modes of activating CRAC channels: a store-operated mode that is activated through depletion of ER Ca2+ stores by inositol 1,4,5-trisphosphate (InsP3) and store-independent activation that is mediated by cell dialysis during whole-cell perfusion. Both modes are regulated by calmodulin (CaM). The store-operated mode is transient in intact cells, possibly reflecting recruitment of CaM, whereas loss of CaM in perfused cells accounts for the persistence of the store-independent mode. The inhibition by CaM can be reversed by 2-aminoethoxydiphenyl borate (2-APB), resulting in rapid, store-independent activation of CRAC channels. The aminoglycoside antibiotic G418 is a highly specific and potent inhibitor of STIM2-dependent CRAC channel activation. The results reveal a novel bimodal control of CRAC channels by STIM2, the store dependence and CaM regulation, which indicates that the STIM2/CRACM1 complex may be under the control of both luminal and cytoplasmic Ca2+ levels.
The crystal structure of calcium-bound calmodulin (Ca(2+)-CaM) bound to a peptide analog of the CaM-binding region of chicken
smooth muscle myosin light chain kinase has been determined and refined to a resolution of 2.4 angstroms (A). The structure
is compact and has the shape of an ellipsoid (axial ratio approximately 2:1). The bound CaM forms a tunnel diagonal to its
long axis that engulfs the helical peptide, with the hydrophobic regions of CaM melded into a single area that closely covers
the hydrophobic side of the peptide. There is a remarkably high pseudo-twofold symmetry between the closely associated domains.
The central helix of the native CaM is unwound and expanded into a bend between residues 73 and 77. About 185 contacts (less
than 4 A) are formed between CaM and the peptide, with van der Waals contacts comprising approximately 80% of this total.
113Cd-NMR studies of solutions of cadmium-loaded calmodulin (Cd4CaM) and the tetradecapeptide mastoparan in different ratios show that mastoparan binds to Cd4CaM with high affinity. The off-rate of protein- bound mastoparan is found to be 40 s-1 or less. The binding of one molecule of mastoparan to Cd4CaM is observed to affect all four metal-binding sites, indicating that both the N-terminal and C-terminal globular domains of the protein undergo conformational changes.
Calmodulin (CaM) acts as an intracellular calcium sensor that translates the Ca2+ signal into a variety of cellular processes. Ca(2+)-CaM recognition of a short polypeptide segment in target proteins induces conformational changes in both CaM and the target, enabling the target protein to become functionally active. The solution and crystal structures of Ca(2+)-CaM bound to peptides derived from three CaM-dependent enzymes reveal structural features that are common in target recognition by Ca(2+)-CaM. Phosphorylation of the target proteins at sites in or near the CaM-binding region modulates binding of CaM, thereby providing an additional mechanism of functional regulation. The structural aspects of target recognition by Ca(2+)-CaM are discussed using mainly the three-dimensional structural information obtained with nuclear magnetic resonance spectroscopy and X-ray diffraction methods.
Recent structural studies on calmodulin complexes with anthrax adenylyl cyclase and rat Ca2+-activated K+ channel have uncovered unexpected ways by which calmodulin interacts with target proteins.
The complex between calmodulin and the calmodulin-binding portion of smMLCKp has been studied. Electrostatic interactions have been anticipated to be important in this system where a strongly negative protein binds a peptide with high positive charge. Electrostatic interactions were probed by varying the pH in the range from 4 to 11 and by charge deletions in CaM and smMLCKp. The change in net charge of CaM from approximately -5 at pH 4.5 to -15 at pH 7.5 leaves the binding constant virtually unchanged. The affinity was also unaffected by mutations in CaM and charge substitutions in the peptide. The insensitivity of the binding constant to pH may seem surprising, but it is a consequence of the high charge on both protein and peptide. At low pH it is further attenuated by a charge regulation mechanism. That is, the protein releases a number of protons when binding the positively charged peptide. We speculate that the role of electrostatic interactions is to discriminate against unbound proteins rather than to increase the affinity for any particular target protein.
Calmodulin (CaM) operates as a Ca(2+) sensor and is known to interact with and regulate hundreds of proteins involved in a great many aspects of cellular function. It is of considerable interest to understand the balance of forces in complex formation of CaM with its target proteins. Here we have studied the importance of electrostatic interactions in the complex between CaM and a peptide derived from smooth-muscle myosin light-chain kinase by experimental methods and Monte Carlo simulations of electrostatic interactions. We show by Monte Carlo simulations that, in agreement with experimental data, the binding affinity between CaM and highly charged peptides is surprisingly insensitive to changes in the net charge of both the protein and peptide. We observe an increase in the binding affinity between oppositely charged partners with increasing salt concentration from zero to 100 mM, showing that formation of globular CaM-kinase type complexes is facilitated at physiological ionic strength. We conclude that ionic interactions in complex formation are optimized at pH and saline similar to the cell environment, which probably overrules the electrostatic repulsion between the negatively charged Ca(2+)-binding domains of CaM. We propose a conceivable rationalization of CaM electrostatics associated with interdomain repulsion.
Calmodulin is an essential Ca2+-binding protein that binds to a variety of targets that carry out critical signaling functions. We describe the proteomic characterization of mouse brain Ca2+-calmodulin-binding proteins that were purified using calmodulin affinity chromatography. Proteins in the eluates from four different affinity chromatography experiments were identified by 1-DE and in-gel digestion followed by LC-MS/MS. Parallel experiments were performed using two related control-proteins belonging to the EF-hand family. After comparing the results from the different experiments, we were able to exclude a significant number of proteins suspected to bind in a nonspecific manner. A total of 140 putative Ca2+-calmodulin-binding proteins were identified of which 87 proteins contained calmodulin-binding motifs. Among the 87 proteins that contained calmodulin-binding motifs, 48 proteins have not previously been shown to interact with calmodulin and 39 proteins were known calmodulin-binding proteins. Many proteins with ill-defined functions were identified as well as a number of proteins that at the time of the analysis were described only as ORFs. This study provides a functional framework for studies on these previously uncharacterized proteins.
Reconstitution studies of a protein from domain fragments can furnish important insights into the distinctive role of particular domain interactions and how they affect biophysical properties important for function. Using isothermal titration calorimetry (ITC) and a number of spectroscopic and chromatographic tools, including CD, fluorescence and NMR spectroscopy, size-exclusion chromatography and non-denaturing agarose gel electrophoresis, we have investigated the reconstitution of the ubiquitous Ca2+-sensor protein calmodulin (CaM) and its globular domains from fragments comprising one or two EF-hands. The studies were carried out with and without the target peptide from smooth muscle myosin light chain kinase (smMLCKp). The CaM-target complex can be reconstituted from the three components consisting of the target peptide and the globular domains TR1C and TR2C. In the absence of peptide, there is no evidence for association of the globular domains. The globular domains can further be reconstituted from their corresponding native subdomains. The dissociation constant, K(D), in 2 mM Tris-HCl (pH 7.5), for the subdomain complexes, EF1:EF2 and EF3:EF4, was determined with ITC to 9.3 x 10(-7) M and 5.9 x 10(-8) M, respectively. Thus, the affinity between the two C-terminal subdomains, located within TR2C, is stronger by a factor of 16 than that between the corresponding subdomains within TR1C. These observations are corroborated by the spectroscopic and chromatographic investigations.
The coupling mechanism between endoplasmic reticulum (ER) Ca(2+) stores and plasma membrane (PM) store-operated channels (SOCs) remains elusive [1-3]. STIM1 was shown to play a crucial role in this coupling process [4-7]; however, the role of the closely related STIM2 protein remains undetermined. We reveal that STIM2 is a powerful SOC inhibitor when expressed in HEK293, PC12, A7r5, and Jurkat T cells. This contrasts with gain of SOC function in STIM1-expressing cells. While STIM1 is expressed in both the ER and plasma membrane, STIM2 is expressed only intracellularly. Store depletion induces redistribution of STIM1 into distinct "puncta." STIM2 translocates into puncta upon store depletion only when coexpressed with STIM1. Double labeling shows coincidence of STIM1 and STIM2 within puncta, and immunoprecipitation reveals direct interactions between STIM1 and STIM2. Independent of store depletion, STIM2 colocalizes with and blocks the function of a STIM1 EF-hand mutant that preexists in puncta and is constitutively coupled to activate SOCs. Thus, whereas STIM1 is a required mediator of SOC activation, STIM2 is a powerful inhibitor of this process, interfering with STIM1-mediated SOC activation at a point downstream of puncta formation. The opposing functions of STIM1 and STIM2 suggest they may play a coordinated role in controlling SOC-mediated Ca(2+) entry signals.
Receptor-evoked Ca2+ signalling involves Ca2+ release from the endoplasmic reticulum, followed by Ca2+ influx across the plasma membrane. Ca2+ influx is essential for many cellular functions, from secretion to transcription, and is mediated by Ca2+-release activated Ca2+ (I(crac)) channels and store-operated calcium entry (SOC) channels. Although the molecular identity and regulation of I(crac) and SOC channels have not been precisely determined, notable recent findings are the identification of STIM1, which has been indicated to regulate SOC and I(crac) channels by functioning as an endoplasmic reticulum Ca2+ sensor, and ORAI1 (ref. 7) or CRACM1 (ref. 8)--both of which may function as I(crac) channels or as an I(crac) subunit. How STIM1 activates the Ca2+ influx channels and whether STIM1 contributes to the channel pore remains unknown. Here, we identify the structural features that are essential for STIM1-dependent activation of SOC and I(crac) channels, and demonstrate that they are identical to those involved in the binding and activation of TRPC1. Notably, the cytosolic carboxyl terminus of STIM1 is sufficient to activate SOC, I(crac) and TRPC1 channels even when native STIM1 is depleted by small interfering RNA. Activity of STIM1 requires an ERM domain, which mediates the selective binding of STIM1 to TRPC1, 2 and 4, but not to TRPC3, 6 or 7, and a cationic lysine-rich region, which is essential for gating of TRPC1. Deletion of either region in the constitutively active STIM1(D76A) yields dominant-negative mutants that block native SOC channels, expressed TRPC1 in HEK293 cells and I(crac) in Jurkat cells. These observations implicate STIM1 as a key regulator of activity rather than a channel component, and reveal similar regulation of SOC, I(crac) and TRPC channel activation by STIM1.
Deviations in basal Ca2+ levels interfere with receptor-mediated Ca2+ signaling as well as endoplasmic reticulum (ER) and mitochondrial function. While defective basal Ca2+ regulation has been linked to various diseases, the regulatory mechanism that controls basal Ca2+ is poorly understood. Here we performed an siRNA screen of the human signaling proteome to identify regulators of basal Ca2+ concentration and found STIM2 as the strongest positive regulator. In contrast to STIM1, a recently discovered signal transducer that triggers Ca2+ influx in response to receptor-mediated depletion of ER Ca2+ stores, STIM2 activated Ca2+ influx upon smaller decreases in ER Ca2+. STIM2, like STIM1, caused Ca2+ influx via activation of the plasma membrane Ca2+ channel Orai1. Our study places STIM2 at the center of a feedback module that keeps basal cytosolic and ER Ca2+ concentrations within tight limits.