Orai1 pore residues control CRAC channel inactivation independently of calmodulin

Mullins, Franklin M.; Yen, Michelle; Lewis, Richard S.

doi:10.1085/jgp.201511437

Cited by 46 publications

(78 citation statements)

References 55 publications

(71 reference statements)

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“…A mechanism in which the linker sequence binds via hydrophobic interactions with a ring of W76 side chains would be consistent with our results showing that significant levels of inactivation require bulky hydrophobic residues at position 76 (W76F/Y; Fig. 5 in Mullins et al, 2016). Measuring the effects of combined mutations in the linker, W76, and ID STIM may provide a means of testing for functional interactions of the linker with the ID STIM -W76 module.…”

Section: Discussionsupporting

confidence: 88%

“…For shorthand, we refer to the inactivation supported by STIM1 1-469 as "residual inactivation." We attribute this increased level of inactivation for STIM1 , like that observed for Orai1 W76A in Mullins et al (2016), to a EGTA in the intracellular solution (P < 0.01 at 120 mV; Fig. 1, A and B), as previously reported for CDI of endogenous CRAC channels (Hoth and Penner, 1993;Zweifach and Lewis, 1995).…”

Section: Transfectionsupporting

confidence: 84%

“…Estimates of unitary Ca 2+ current (i Ca ) were made by adjusting the unitary Na + current (i Na ) by the relative magnitude of macroscopic I Ca and I Na as measured using rapid solution exchanges and correcting for the residual level of CDI in 2 mM Ca 2+ Ringer's ( Fig. S3; Mullins et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

“…In principle, W76 could form the binding site for a proteinaceous blocking particle, form a hydrophobic gate, or enable conformational changes leading to closure at another site. The polyanionic character of ID STIM initially suggested the possibility that it might interact with the three rings of positively charged residues in the pore (R83, K87, and R91) to act as a blocking particle, thus explaining why introduction of negative charges at these pore sites inhibit CDI (Mullins et al, 2016). However, if a blocking particle is involved, it cannot be ID STIM , as inactivation clearly occurs in its absence in Y80A Orai1 channels (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Cells with a high mCh-STIM1/GFP-Orai1 ratio were selected for recording by the relative fluorescence of mCherry and GFP as described in Mullins et al (2016).…”

mentioning

confidence: 99%

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The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca²⁺-dependent inactivation

Mullins¹,

Lewis²

2016

J Cell Biol

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Ca 2+ release-activated Ca 2+ (CRAC) channels are prototypic store operated channels that are encoded by the Orai family of plasma membrane proteins and activated by depletion of Ca 2+ from the ER (Prakriya and Lewis, 2015). The ER protein STIM1 is best recognized for its function in sensing ER Ca 2+ depletion and, in response, activating CRAC channels in the plasma membrane. However, an early clue that STIM1 is more than an activating ligand for Orai1 was the observation that channels activated by an isolated fragment of STIM1 (the CRAC activation domain or CAD; STIM1 aa 342-448), failed to show characteristic fast Ca 2+ -dependent inactivation (CDI) in response to Ca 2+ entry . A series of C-terminal truncations identified a region of STIM1 (aa 470-491) that is required for CDI, termed the inactivation domain of STIM, or ID STIM . Additional evidence for a role of STIM1 in CDI came from observations that in heterologous systems the STIM1/Orai1 expression ratio influences the extent of CDI, with a ratio of 2-4:1 being Correspondence to Richard S. Lewis: r s l e w i s @ s t a n f o r d . e d u ; or Franklin M. Mullins: f m u l l i n s @ s t a n f o r d . e d u Abbreviations used in this paper: CDI, Ca 2+ -dependent inactivation; CRAC, Ca 2+ release-activated Ca 2+ ; DVF, divalent-free; ID STIM , inactivation domain of STIM.required for CDI to approach the level seen for native CRAC channels (Scrimgeour et al., 2009;Hoover and Lewis, 2011).A highly negatively charged region within ID STIM ( 475 DDVDDMDEE 483 ) attracted attention from several groups as a potential Ca 2+ binding site for CDI, based on analogy to the highly acidic Ca 2+ bowl binding site of the BK channel (Cox, 2011). Consistent with such a role, alanine or glycine substitutions for various combinations of acidic residues within this region reduced CDI, and full neutralization of all acidic residues eliminated CDI (Derler et al., 2009b;Lee et al., 2009;Mullins et al., 2009 The inactivation domain of STIM1 (ID STIM : amino acids 470-491) has been described as necessary for Ca 2+ -dependent inactivation (CDI) of Ca 2+ release-activated Ca 2+ (CRAC) channels, but its mechanism of action is unknown. Here we identify acidic residues within ID STIM that control the extent of CDI and examine functional interactions of ID STIM with Orai1 pore residues W76 and Y80. Alanine scanning revealed three ID STIM residues (D476/D478/ D479) that are critical for generating full CDI. Disabling ID STIM by a triple alanine substitution for these three residues ("STIM1 3A") or by truncation of the entire domain (STIM1 1-469 ) reduced CDI to the same residual level observed for the Orai1 pore mutant W76A (approximately one third of the extent seen with full-length STIM1). Results of noise analysis showed that STIM1 1-469 and Orai1 W76A mutants do not reduce channel open probability or unitary Ca 2+ conductance, factors that determine local Ca 2+ accumulation, suggesting that they diminish CDI instead by inhibiting the CDI gating mechanism. We tested for functional coupli...

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Section: Discussionsupporting

confidence: 88%

Section: Transfectionsupporting

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…Cells with a high mCh-STIM1/GFP-Orai1 ratio were selected for recording by the relative fluorescence of mCherry and GFP as described in Mullins et al (2016).…”

mentioning

confidence: 99%

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The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca²⁺-dependent inactivation

Mullins¹,

Lewis²

2016

J Cell Biol

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Multiple Evidenz für einen ungewöhnlichen Wechselwirkungsmodus zwischen Calmodulin und Orai‐Proteinen

et al. 2017

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Calmodulin (CaM) bindet an die meisten Zielproteine durch Umwickeln einer amphiphatischen a-Helix. Das N-terminale Ende von Orai-Proteinen enthält ein solches konserviertes CaM-Bindungssegment, der molekulare Bindungsmechanismus ist jedochs trittig. Hier wurden die Bindungsgleichgewichte, die entsprechenden Kinetiken sowie die Bindungskräfte zwischen einzelnen Calmodulinmolekülen und den konservierten Helices von Orai1 oder Orai3 untersucht.Die Resultate zeigten übereinstimmend eine schrittweise Bindung von zwei einzelnen Peptiden an die beiden Lappen von CaM. Eine Affinitätsteigerung wurde beobachtet, wenn zwei Orai-Peptide an ihren C-Termini entweder dimerisiert oder mit hoher lateraler Dichte immobilisiert wurden, wodurch die enge Nachbarschaft dieser N-terminalen Helices in den nativen Orai-Oligomeren nachgebildet wurde.A naloge Kontrollexperimente mit Myosin-Leichtketten-Kinase von glatten Muskelzellen zeigten nur die konventionelle 1:1-Bindung und bestätigten die Beweiskraft unserer Methoden.

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Detailed Evidence for an Unparalleled Interaction Mode between Calmodulin and Orai Proteins

et al. 2017

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Calmodulin (CaM) binds most of its targets by wrapping around an amphipathic a-helix. The N-terminus of Orai proteins contains ac onserved CaM-binding segment but the binding mechanismh as been only partially characterized. Here,m icroscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy( AFM) were employed to study the binding equilibria, the kinetics,a nd the single-molecule interaction forces involved in the binding of CaM to the conserved helical segments of Orai1 and Orai3. The results consistently indicated stepwise binding of two separate target peptides to the two lobes of CaM. An unparalleled high affinity was found when two Orai peptides were dimerized or immobilized at high lateral density,thereby mimicking the close proximity of the N-termini in native Orai oligomers.T he analogous experiments with smooth muscle myosin light chain kinase (smMLCK) showed only the expected 1:1b inding,c onfirming the validity of our methods. Orai1andOrai3areCa2+ channels in the plasma membrane of non-excitable cells which are activated by Ca 2+ depletion of the endoplasmic reticulum (ER);areduction of [Ca 2+ ]inthe ER causes the stromal interaction molecule (STIM) in the ER membrane to oligomerize,w hereupon it binds to Orai and activates its channel function. [1][2][3][4] Much less is known about the Ca 2+ -dependent inactivation (CDI) of Orai which is important in the regulation of the intracellular Ca 2+ level. In anumber of studies,calmodulin (CaM) was found to bind to ahighly conserved N-terminal segment of Orai1/3, and to act as an egative regulator of channel function, [5][6][7] although this was lately questioned.[8] Thec rystal structure of the complex between CaM and the isolated CaM-binding segment of Orai1 (aa69-88) revealed an unusual extended conformation of CaM with only its C-terminal lobe binding one Orai1 peptide.[9] Parallel pulldown and isothermal titration calorimetry (ITC) experiments suggested stepwise binding of two Orai1 69-88 peptides with different affinities,o ne on the Cterminal lobe of CaM (K d = 1.1 mm)a nd one on the Nterminal lobe (K d = 4.6 mm). Motivated by these findings,weaimed at acomprehensive characterization of this interaction mechanism, for the following reasons:( i) the measured K d values (1.1 and 4.6 mm)a re much higher than those usually reported for Ca 2+ -induced CaM binding (typically 10 À7 to 10 À11 m) [10] and were probably influenced by the high calmodulin concentration used in ITC,( ii)the transitions between monovalent and bivalent bond formation have not yet been addressed, and, most prominently,( iii)quantitative information about the kinetics of stepwise association and dissociation is still missing. Here,t hese questions were addressed by complementary in vitro methods.E quilibrium measurements by MST yielded the distinct affinities of both binding steps at equilibrium, SPR provided information about the kinetic rate constants,a nd AFM revealed the interaction forces of monovalent and bivalent binding,aswell as the...

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Orai1 pore residues control CRAC channel inactivation independently of calmodulin

Abstract: Researchers have reevaluated the role of calmodulin and previously identified calmodulin binding sites in the mechanism by which Ca2+-release activated Ca2+ channels can be inactivated as Ca2+ ions enter cells.

Cited by 46 publications

References 55 publications

The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca²⁺-dependent inactivation

The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca²⁺-dependent inactivation

Multiple Evidenz für einen ungewöhnlichen Wechselwirkungsmodus zwischen Calmodulin und Orai‐Proteinen

Detailed Evidence for an Unparalleled Interaction Mode between Calmodulin and Orai Proteins

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Orai1 pore residues control CRAC channel inactivation independently of calmodulin

Abstract: Researchers have reevaluated the role of calmodulin and previously identified calmodulin binding sites in the mechanism by which Ca2+-release activated Ca2+ channels can be inactivated as Ca2+ ions enter cells.

Cited by 46 publications

References 55 publications

The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca2+-dependent inactivation

The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca2+-dependent inactivation

Multiple Evidenz für einen ungewöhnlichen Wechselwirkungsmodus zwischen Calmodulin und Orai‐Proteinen

Detailed Evidence for an Unparalleled Interaction Mode between Calmodulin and Orai Proteins

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The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca²⁺-dependent inactivation

The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca²⁺-dependent inactivation