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1.
Store-operated Ca2+ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca2+ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent ISOC, activated in response to Ca2+ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated ICRAC; the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(684EE685). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca2+ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd3+, removal of extracellular Ca2+, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca2+-containing, but not Ca2+-free, medium. Consistent with this, ICRAC is activated in cells pretreated with thapsigargin in Ca2+-free medium while ISOC is activated in cells pretreated in Ca2+-containing medium. Significantly, TRPC1 function is required for sustained KCa activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca2+ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca2+ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE. 相似文献
2.
Calcium release-activated calcium (CRAC) channels are highly calcium ion (Ca2+)-selective channels in the plasma membrane. The transient drop of endoplasmic reticulum Ca2+ level activates its calcium sensor stromal interaction molecule (STIM) and then triggers the gating of the CRAC channel pore unit Orai. This process involves a variety of activities of the immune system. Therefore, understanding how the activation and regulation of the CRAC channel can be accomplished is essential. Here we briefly summarize the recent progress on Orai gating and its regulation by 2-aminoethoxydiphenylborate (2-APB) obtained from structural biology studies, biochemical and electrophysiological measurements, as well as molecular modeling. Indeed, integration between experiments and computations has further deepened our understanding of the channel gating and regulation. 相似文献
3.
Prevailing models postulate that high Ca2+ selectivity of Ca2+ release-activated Ca2+ (CRAC) channels arises from tight Ca2+ binding to a high affinity site within the pore, thereby blocking monovalent ion flux. Here, we examined the contribution of high affinity Ca2+ binding for Ca2+ selectivity in recombinant Orai3 channels, which function as highly Ca2+-selective channels when gated by the endoplasmic reticulum Ca2+ sensor STIM1 or as poorly Ca2+-selective channels when activated by the small molecule 2-aminoethoxydiphenyl borate (2-APB). Extracellular Ca2+ blocked Na+ currents in both gating modes with a similar inhibition constant (Ki; ∼25 µM). Thus, equilibrium binding as set by the Ki of Ca2+ blockade cannot explain the differing Ca2+ selectivity of the two gating modes. Unlike STIM1-gated channels, Ca2+ blockade in 2-APB–gated channels depended on the extracellular Na+ concentration and exhibited an anomalously steep voltage dependence, consistent with enhanced Na+ pore occupancy. Moreover, the second-order rate constants of Ca2+ blockade were eightfold faster in 2-APB–gated channels than in STIM1-gated channels. A four-barrier, three–binding site Eyring model indicated that lowering the entry and exit energy barriers for Ca2+ and Na+ to simulate the faster rate constants of 2-APB–gated channels qualitatively reproduces their low Ca2+ selectivity, suggesting that ion entry and exit rates strongly affect Ca2+ selectivity. Noise analysis indicated that the unitary Na+ conductance of 2-APB–gated channels is fourfold larger than that of STIM1-gated channels, but both modes of gating show a high open probability (Po; ∼0.7). The increase in current noise during channel activation was consistent with stepwise recruitment of closed channels to a high Po state in both cases, suggesting that the underlying gating mechanisms are operationally similar in the two gating modes. These results suggest that both high affinity Ca2+ binding and kinetic factors contribute to high Ca2+ selectivity in CRAC channels. 相似文献
4.
5.
Depletion of intracellular Ca2 + stores in mammalian cells results in Ca2 + entry across the plasma membrane mediated primarily by Ca2 + release-activated Ca2 + (CRAC) channels. Ca2 + influx through these channels is required for the maintenance of homeostasis and Ca2 + signaling in most cell types. One of the main features of native CRAC channels is fast Ca2 +-dependent inactivation (FCDI), where Ca2 + entering through the channel binds to a site near its intracellular mouth and causes a conformational change, closing the channel and limiting further Ca2 + entry. Early studies suggested that FCDI of CRAC channels was mediated by calmodulin. However, since the discovery of STIM1 and Orai1 proteins as the basic molecular components of the CRAC channel, it has become apparent that FCDI is a more complex phenomenon. Data obtained using heterologous overexpression of STIM1 and Orai1 suggest that, in addition to calmodulin, several cytoplasmic domains of STIM1 and Orai1 and the selectivity filter within the channel pore are required for FCDI. The stoichiometry of STIM1 binding to Orai1 also has emerged as an important determinant of FCDI. Consequently, STIM1 protein expression levels have the potential to be an endogenous regulator of CRAC channel Ca2 + influx. This review discusses the current understanding of the molecular mechanisms governing the FCDI of CRAC channels, including an evaluation of further experiments that may delineate whether STIM1 and/or Orai1 protein expression is endogenously regulated to modulate CRAC channel function, or may be dysregulated in some pathophysiological states. 相似文献
6.
Although highly selective Ca2+ entry pathways play a critical role in agonist-activated Ca2+ signals in non-excitable cells, only with the recent discovery of the Orai proteins have the first insights into the molecular nature of these pathways been possible. To date, just two such highly Ca2+-selective “Orai channels” have been identified in native cells—the storeoperated CRAC channels and the store-independent, arachidonic acid-activated ARC channels. Studies have shown that the functional CRAC channel pore is formed by a tetrameric arrangement of Orai1 subunits, whilst a heteropentamer of three Orai1 subunits and two Orai3 subunits forms the functional ARC channel pore. Importantly, this inclusion of Orai3 subunits in the ARC channel structure has been shown to play a specific role in determining the selectivity of these channels for activation by arachidonic acid. Using an approach based on the expression of various concatenated constructs, we examined the basis for this Orai3-dependent effect on selectivity for arachidonic acid. We show that, whilst heteropentamers containing only one Orai3 subunit are sensitive to arachidonic acid, specific selectivity for activation by this fatty acid is only achieved on inclusion of the second Orai3 subunit in the pentamer. Further studies identified the cytosolic N-terminal domain of Orai3 as the region specifically responsible for this switch in selectivity. Substitution of just this domain into an otherwise complete single Orai1 subunit within a concatenated 31111 pentamer is sufficient to change the resulting channel from one that is predominantly store-operated, to one that is exclusively activated by arachidonic acid.Key words: STIM1, Orai1, Orai3, calcium channel, calcium entry, arachidonic acid 相似文献
7.
Anna Amcheslavsky Mona?L. Wood Andriy?V. Yeromin Ian Parker J.?Alfredo Freites Douglas?J. Tobias Michael?D. Cahalan 《Biophysical journal》2015,108(2):237-246
Upon endoplasmic reticulum Ca2+ store depletion, Orai channels in the plasma membrane are activated directly by endoplasmic reticulum-resident STIM proteins to generate the Ca2+-selective, Ca2+ release-activated Ca2+ (CRAC) current. After the molecular identification of Orai, a plethora of functional and biochemical studies sought to compare Orai homologs, determine their stoichiometry, identify structural domains responsible for the biophysical fingerprint of the CRAC current, identify the physiological functions, and investigate Orai homologs as potential therapeutic targets. Subsequently, the solved crystal structure of Drosophila Orai (dOrai) substantiated many findings from structure-function studies, but also revealed an unexpected hexameric structure. In this review, we explore Orai channels as elucidated by functional and biochemical studies, analyze the dOrai crystal structure and its implications for Orai channel function, and present newly available information from molecular dynamics simulations that shed light on Orai channel gating and permeation. 相似文献
8.
9.
Gwack Y Srikanth S Oh-Hora M Hogan PG Lamperti ED Yamashita M Gelinas C Neems DS Sasaki Y Feske S Prakriya M Rajewsky K Rao A 《Molecular and cellular biology》2008,28(17):5209-5222
ORAI1 is a pore subunit of the store-operated Ca2+ release-activated Ca2+ (CRAC) channel. To examine the physiological consequences of ORAI1 deficiency, we generated mice with targeted disruption of the Orai1 gene. The results of immunohistochemical analysis showed that ORAI1 is expressed in lymphocytes, skin, and muscle of wild-type mice and is not expressed in Orai1−/− mice. Orai1−/− mice with the inbred C57BL/6 background showed perinatal lethality, which was overcome by crossing them to outbred ICR mice. Orai1−/− mice were small in size, with eyelid irritation and sporadic hair loss resembling the cyclical alopecia observed in mice with keratinocyte-specific deletion of the Cnb1 gene. T and B cells developed normally in Orai1−/− mice, but B cells showed a substantial decrease in Ca2+ influx and cell proliferation in response to B-cell receptor stimulation. Naïve and differentiated Orai1−/− T cells showed substantial reductions in store-operated Ca2+ entry, CRAC currents, and cytokine production. These features are consistent with the severe combined immunodeficiency and mild extraimmunological symptoms observed in a patient with a missense mutation in human ORAI1 and distinguish the ORAI1-null mice described here from a previously reported Orai1 gene-trap mutant mouse which may be a hypomorph rather than a true null. 相似文献
10.
A Structural Model of the Pore-Forming Region of the Skeletal Muscle Ryanodine Receptor (RyR1)
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Srinivas Ramachandran Adrian W. R. Serohijos Le Xu Gerhard Meissner Nikolay V. Dokholyan 《PLoS computational biology》2009,5(4)
Ryanodine receptors (RyRs) are ion channels that regulate muscle contraction by releasing calcium ions from intracellular stores into the cytoplasm. Mutations in skeletal muscle RyR (RyR1) give rise to congenital diseases such as central core disease. The absence of high-resolution structures of RyR1 has limited our understanding of channel function and disease mechanisms at the molecular level. Here, we report a structural model of the pore-forming region of RyR1. Molecular dynamics simulations show high ion binding to putative pore residues D4899, E4900, D4938, and D4945, which are experimentally known to be critical for channel conductance and selectivity. We also observe preferential localization of Ca2+ over K+ in the selectivity filter of RyR1. Simulations of RyR1-D4899Q mutant show a loss of preference to Ca2+ in the selectivity filter as seen experimentally. Electrophysiological experiments on a central core disease mutant, RyR1-G4898R, show constitutively open channels that conduct K+ but not Ca2+. Our simulations with G4898R likewise show a decrease in the preference of Ca2+ over K+ in the selectivity filter. Together, the computational and experimental results shed light on ion conductance and selectivity of RyR1 at an atomistic level. 相似文献
11.
Thermodynamic measurements of ion binding to the Streptomyces lividans K+ channel were carried out using isothermal titration calorimetry, whereas atomic structures of ion-bound and ion-free conformations of the channel were characterized by x-ray crystallography. Here we use these assays to show that the ion radius dependence of selectivity stems from the channel's recognition of ion size (i.e., volume) rather than charge density. Ion size recognition is a function of the channel's ability to adopt a very specific conductive structure with larger ions (K+, Rb+, Cs+, and Ba2+) bound and not with smaller ions (Na+, Mg2+, and Ca2+). The formation of the conductive structure involves selectivity filter atoms that are in direct contact with bound ions as well as protein atoms surrounding the selectivity filter up to a distance of 15 Å from the ions. We conclude that ion selectivity in a K+ channel is a property of size-matched ion binding sites created by the protein structure. 相似文献
12.
Zhengzheng Li Lin Liu Yongqiang Deng Wei Ji Wen Du Pingyong Xu Liangyi Chen Tao Xu 《Cell research》2011,21(2):305-315
The Ca2+ release-activated Ca2+ (CRAC) channel pore is formed by Orai1 and gated by STIM1 after intracellular Ca2+ store depletion. To resolve how many STIM1 molecules are required to open a CRAC channel, we fused different numbers of Orai1 subunits with functional two-tandem cytoplasmic domains of STIM1 (residues 336-485, designated as S domain). Whole-cell patch clamp recordings of these chimeric molecules revealed that CRAC current reached maximum at a stoichiometry of four Orai1 and eight S domains. Further experiments indicate that two-tandem S domains specifically interact with the C-terminus of one Orai1 subunit, and CRAC current can be gradually increased as more Orai1 subunits can interact with S domains or STIM1 proteins. Our data suggest that maximal opening of one CRAC channel requires eight STIM1 molecules, and support a model that the CRAC channel activation is not in an “all-or-none” fashion but undergoes a graded process via binding of different numbers of STIM1. 相似文献
13.
Store-operated Ca2+ entry (SOCE) is a widespread mechanism to elevate the intracellular Ca2+ concentrations and stimulate downstream signaling pathways affecting proliferation, secretion, differentiation and death in different cell types. In immune cells, immune receptor stimulation induces intracellular Ca2+ store depletion that subsequently activates Ca2+-release-activated-Ca2+ (CRAC) channels, a prototype of store-operated Ca2+ (SOC) channels. CRAC channel opening leads to activation of diverse downstream signaling pathways affecting proliferation, differentiation, cytokine production and cell death. Recent identification of STIM1 as the endoplasmic reticulum Ca2+ sensor and Orai1 as the pore subunit of CRAC channels has provided the much-needed molecular tools to dissect the mechanism of activation and regulation of CRAC channels. In this review, we discuss the recent advances in understanding the associating partners and posttranslational modifications of Orai1 and STIM1 proteins that regulate diverse aspects of CRAC channel function. 相似文献
14.
HCN channels are thought to be structurally similar to Kv channels, but show much lower selectivity for K+. The ∼3.3 Å selectivity filter of K+ channels is formed by the pore-lining sequence XT(V/I)GYG, with X usually T, and is held stable by key residues in the P-loop. Differences in the P-loop sequence of HCN channels (eg. the pore-lining sequence L478C479IGYG) suggest these residues could account for differences in selectivity between these channel families. Despite being expressed, L478T/C479T HCN4 channels did not produce current. Since threonine in the second position is highly conserved in K+ channels, we also studied C479T channels. Based on permeability ratios (PX/PK), C479T HCN4 channels (K+(1)>Rb+(0.85)>Cs+(0.59)>Li+(0.50)≥Na+(0.49)) were less selective than WT rabbit HCN4 (K+(1)>Rb+(0.48)>Cs+(0.31)≥Na+(0.29)>Li+(0.03)), indicating that the TIGYG sequence is insufficient to confer K+ selectivity to HCN channels. C479T HCN4 channels had an increased permeability to large organic cations than WT HCN4 channels, as well as increased unitary K+ conductance, and altered channel gating. Collectively, these results suggest that HCN4 channels have larger pores than K+ channels and replacement of the cysteine at position 479 with threonine further increases pore size. Furthermore, selected mutations in other regions linked previously to pore stability in K+ channels (ie. S475D, S475E and F471W/K472W) were also unable to confer K+ selectivity to C479T HCN4 channels. Our findings establish the presence of the TIGYG pore-lining sequence does not confer K+ selectivity to rabbit HCN4 channels, and suggests that differences in selectivity of HCN4 versus K+ channels originate from differences outside the P-loop region. 相似文献
15.
Sonal Srikanth Hea-Jin Jung Bernard Ribalet Yousang Gwack 《The Journal of biological chemistry》2010,285(7):5066-5075
Store-operated Ca2+ entry (SOCE) due to activation of Ca2+ release-activated Ca2+ (CRAC) channels leads to sustained elevation of cytoplasmic Ca2+ and activation of lymphocytes. CRAC channels consisting of four pore-forming Orai1 subunits are activated by STIM1, an endoplasmic reticulum Ca2+ sensor that senses intracellular store depletion and migrates to plasma membrane proximal regions to mediate SOCE. One of the fundamental properties of CRAC channels is their Ca2+-dependent fast inactivation. To identify the domains of Orai1 involved in fast inactivation, we have mutated residues in the Orai1 intracellular loop linking transmembrane segment II to III. Mutation of four residues, V151SNV154, at the center of the loop (MutA) abrogated fast inactivation, leading to increased SOCE as well as higher CRAC currents. Point mutation analysis identified five key amino acids, N153VHNL157, that increased SOCE in Orai1 null murine embryonic fibroblasts. Expression or direct application of a peptide comprising the entire intracellular loop or the sequence N153VHNL157 blocked CRAC currents from both wild type (WT) and MutA Orai1. A peptide incorporating the MutA mutations had no blocking effect. Concatenated Orai1 constructs with four MutA monomers exhibited high CRAC currents lacking fast inactivation. Reintroduction of a single WT monomer (MutA-MutA-MutA-WT) was sufficient to fully restore fast inactivation, suggesting that only a single intracellular loop can block the channel. These data suggest that the intracellular loop of Orai1 acts as an inactivation particle, which is stabilized in the ion permeation pathway by the N153VHNL157 residues. These results along with recent reports support a model in which the N terminus and the selectivity filter of Orai1 as well as STIM1 act in concert to regulate the movement of the intracellular loop and evoke fast inactivation. 相似文献
16.
The inactivation domain of STIM1 (IDSTIM: amino acids 470–491) has been described as necessary for Ca2+-dependent inactivation (CDI) of Ca2+ release–activated Ca2+ (CRAC) channels, but its mechanism of action is unknown. Here we identify acidic residues within IDSTIM that control the extent of CDI and examine functional interactions of IDSTIM with Orai1 pore residues W76 and Y80. Alanine scanning revealed three IDSTIM residues (D476/D478/D479) that are critical for generating full CDI. Disabling IDSTIM by a triple alanine substitution for these three residues (“STIM1 3A”) or by truncation of the entire domain (STIM11–469) 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 STIM11–469 and Orai1 W76A mutants do not reduce channel open probability or unitary Ca2+ conductance, factors that determine local Ca2+ accumulation, suggesting that they diminish CDI instead by inhibiting the CDI gating mechanism. We tested for functional coupling between IDSTIM and the Orai1 pore by double-mutant cycle analysis. The effects on CDI of mutations disabling IDSTIM or W76 were not additive, demonstrating that IDSTIM and W76 are strongly coupled and act in concert to generate full-strength CDI. Interestingly, disabling IDSTIM and W76 separately gave opposite results in Orai1 Y80A channels: channels with W76 but lacking IDSTIM generated approximately two thirds of the WT extent of CDI but those with IDSTIM but lacking W76 completely failed to inactivate. Together, our results suggest that Y80 alone is sufficient to generate residual CDI, but acts as a barrier to full CDI. Although IDSTIM is not required as a Ca2+ sensor for CDI, it acts in concert with W76 to progress beyond the residual inactivated state and enable CRAC channels to reach the full extent of inactivation. 相似文献
17.
Takumi Kawasaki Takehiko Ueyama Ingo Lange Stefan Feske Naoaki Saito 《The Journal of biological chemistry》2010,285(33):25720-25730
Ca2+ signals through store-operated Ca2+ (SOC) channels, activated by the depletion of Ca2+ from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca2+ sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca2+ entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKCβ both resulted in increased Ca2+ influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30. 相似文献
18.
Qinghuan Xiao Andrew Prussia Kuai Yu Yuan-yuan Cui H. Criss Hartzell 《The Journal of general physiology》2008,132(6):681-692
Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of Cl− ion channels that are activated by intracellular Ca2+. At present, the structures and mechanisms responsible for Ca2+ sensing remain unknown. Here, we have used a combination of molecular modeling, density functional–binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in Ca2+ sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential for Ca2+ sensing by hBest1. The cluster of five amino acids (293–308) is crucial for normal channel gating by Ca2+ because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by Ca2+. Using homology models built on the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca2+ with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca2+-binding loop (312–323) reduced the apparent Ca2+ affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca2+-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350–390) that is required for Ca2+ activation and plays a role in current rundown. These experiments identify a region of hBest1 (312–323) that is involved in the gating of hBest1 by Ca2+ and suggest a model in which Ca2+ binding to EF1 activates the channel in a process that requires the acidic domain (293–308) and another regulatory domain (350–390). Many of the ~100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca2+ sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca2+. 相似文献
19.
Anna Amcheslavsky Olga Safrina Michael D. Cahalan 《The Journal of general physiology》2014,143(5):621-631
After endoplasmic reticulum (ER) Ca2+ store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident stromal interacting molecule (STIM) proteins to form the Ca2+-selective Ca2+ release-activated Ca2+ (CRAC) channel. Of the three human Orai channel homologues, only Orai3 can be activated by high concentrations (>50 µM) of 2-aminoethyl diphenylborinate (2-APB). 2-APB activation of Orai3 occurs without STIM1–Orai3 interaction or store depletion, and results in a cationic, nonselective current characterized by biphasic inward and outward rectification. Here we use cysteine scanning mutagenesis, thiol-reactive reagents, and patch-clamp analysis to define the residues that assist in formation of the 2-APB–activated Orai3 pore. Mutating transmembrane (TM) 1 residues Q83, V77, and L70 to cysteine results in potentiated block by cadmium ions (Cd2+). TM1 mutants E81C, G73A, G73C, and R66C form channels that are not sensitive to 2-APB activation. We also find that Orai3 mutant V77C is sensitive to block by 2-aminoethyl methanethiosulfonate (MTSEA), but not 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). Block induced by reaction with MTSEA is state dependent, as it occurs only when Orai3-V77C channels are opened by either 2-APB or by cotransfection with STIM1 and concurrent passive store depletion. We also analyzed TM3 residue E165. Mutation E165A in Orai3 results in diminished 2-APB–activated currents. However, it has little effect on store-operated current density. Furthermore, mutation E165C results in Cd2+-induced block that is state dependent: Cd2+ only blocks 2-APB–activated, not store-operated, mutant channels. Our data suggest that the dilated pore of 2-APB–activated Orai3 is lined by TM1 residues, but also allows for TM3 E165 to approach the central axis of the channel that forms the conducting pathway, or pore. 相似文献
20.
Calcium flux through store-operated calcium entry is a central regulator of intracellular calcium signaling. The two key components of the store-operated calcium release-activated calcium channel are the Ca2+-sensing protein stromal interaction molecule 1 (STIM1) and the channel pore-forming protein Orai1. During store-operated calcium entry activation, calcium depletion from the endoplasmic reticulum triggers a series of conformational changes in STIM1 that unmask a minimal Orai1-activating domain (CRAC activation region (CAD)). To gate Orai1 channels, the exposed STIM1-activating domain binds to two sites in Orai1, one in the N terminus and one in the C terminus. Whether the two sites operate as distinct binding domains or cooperate in CAD binding is unknown. In this study, we show that the N and C-terminal domains of Orai1 synergistically contribute to the interaction with STIM1 and couple STIM1 binding with channel gating and modulation of ion selectivity. 相似文献