scholarly journals Orai1 pore residues control CRAC channel inactivation independently of calmodulin

2016 ◽  
Vol 147 (2) ◽  
pp. 137-152 ◽  
Author(s):  
Franklin M. Mullins ◽  
Michelle Yen ◽  
Richard S. Lewis
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Negative Charge ◽  
Noise Analysis ◽  
Dominant Negative ◽  
Side Chain ◽  
Open Probability ◽  
Crac Channels ◽  
Channel Inactivation ◽  
Crac Channel

Ca2+ entry through CRAC channels causes fast Ca2+-dependent inactivation (CDI). Previous mutagenesis studies have implicated Orai1 residues W76 and Y80 in CDI through their role in binding calmodulin (CaM), in agreement with the crystal structure of Ca2+–CaM bound to an Orai1 N-terminal peptide. However, a subsequent Drosophila melanogaster Orai crystal structure raises concerns about this model, as the side chains of W76 and Y80 are predicted to face the pore lumen and create a steric clash between bound CaM and other Orai1 pore helices. We further tested the functional role of CaM using several dominant-negative CaM mutants, none of which affected CDI. Given this evidence against a role for pretethered CaM, we altered side-chain volume and charge at the Y80 and W76 positions to better understand their roles in CDI. Small side chain volume had different effects at the two positions: it accelerated CDI at position Y80 but reduced the extent of CDI at position W76. Positive charges at Y80 and W76 permitted partial CDI with accelerated kinetics, whereas introducing negative charge at any of five consecutive pore-lining residues (W76, Y80, R83, K87, or R91) completely eliminated CDI. Noise analysis of Orai1 Y80E and Y80K currents indicated that reductions in CDI for these mutations could not be accounted for by changes in unitary current or open probability. The sensitivity of CDI to negative charge introduced into the pore suggested a possible role for anion binding in the pore. However, although Cl− modulated the kinetics and extent of CDI, we found no evidence that CDI requires any single diffusible cytosolic anion. Together, our results argue against a CDI mechanism involving CaM binding to W76 and Y80, and instead support a model in which Orai1 residues Y80 and W76 enable conformational changes within the pore, leading to CRAC channel inactivation.

scholarly journals Physiological CRAC channel activation and pore properties require STIM1 binding to all six Orai1 subunits

2018 ◽  
Vol 150 (10) ◽  
pp. 1373-1385 ◽  
Author(s):  
Michelle Yen ◽  
Richard S. Lewis
Keyword(s):  
Single Channel ◽  
Divalent Cations ◽  
Ion Selectivity ◽  
Functional Coupling ◽  
Open Probability ◽  
Channel Activation ◽  
Channel Current ◽  
Crac Channels ◽  
Crac Channel ◽  
Pore Properties

The binding of STIM1 to Orai1 controls the opening of store-operated CRAC channels as well as their extremely high Ca2+ selectivity. Although STIM1 dimers are known to bind directly to the cytosolic C termini of the six Orai1 subunits (SUs) that form the channel hexamer, the dependence of channel activation and selectivity on the number of occupied binding sites is not well understood. Here we address these questions using dimeric and hexameric Orai1 concatemers in which L273D mutations were introduced to inhibit STIM1 binding to specific Orai1 SUs. By measuring FRET between fluorescently labeled STIM1 and Orai1, we find that homomeric L273D mutant channels fail to bind STIM1 appreciably; however, the L273D SU does bind STIM1 and contribute to channel activation when located adjacent to a WT SU. These results suggest that STIM1 dimers can interact with pairs of neighboring Orai1 SUs. Surprisingly, a single L273D mutation within the Orai1 hexamer reduces channel open probability by ∼90%, triples the size of the single-channel current, weakens the Ca2+ binding affinity of the selectivity filter, and lowers the selectivity for Na+ over Cs+ in the absence of divalent cations. These findings reveal a surprisingly strong functional coupling between STIM1 binding and CRAC channel gating and pore properties. We conclude that under physiological conditions, all six Orai1 SUs of the native CRAC channel bind STIM1 to effectively open the pore and generate the signature properties of extremely low conductance and high ion selectivity.

scholarly journals Divergence of Ca2+ selectivity and equilibrium Ca2+ blockade in a Ca2+ release-activated Ca2+ channel

2014 ◽  
Vol 143 (3) ◽  
pp. 325-343 ◽  
Author(s):  
Megumi Yamashita ◽  
Murali Prakriya
Keyword(s):  
Rate Constants ◽  
Noise Analysis ◽  
Entry And Exit ◽  
Open Probability ◽  
High Affinity ◽  
Crac Channels ◽  
Equilibrium Binding ◽  
Na Currents ◽  
Eyring Model ◽  
Monovalent Ion

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.

scholarly journals Susceptibility of β1 Na+-K+ Pump Subunit to Glutathionylation and Oxidative Inhibition Depends on Conformational State of Pump

2012 ◽  
Vol 287 (15) ◽  
pp. 12353-12364 ◽  
Author(s):  
Chia-Chi Liu ◽  
Alvaro Garcia ◽  
Yasser A. Mahmmoud ◽  
Elisha J. Hamilton ◽  
Keyvan Karimi Galougahi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Membrane Potential ◽  
Angiotensin Ii ◽  
Conformational Changes ◽  
Ventricular Myocytes ◽  
Conformational State ◽  
Side Chain ◽  
Voltage Dependent ◽  
Cytosolic Glutathione ◽  
And Function

Glutathionylation of cysteine 46 of the β1 subunit of the Na+-K+ pump causes pump inhibition. However, the crystal structure, known in a state analogous to an E2·2K+·Pi configuration, indicates that the side chain of cysteine 46 is exposed to the lipid bulk phase of the membrane and not expected to be accessible to the cytosolic glutathione. We have examined whether glutathionylation depends on the conformational changes in the Na+-K+ pump cycle as described by the Albers-Post scheme. We measured β1 subunit glutathionylation and function of Na+-K+-ATPase in membrane fragments and in ventricular myocytes. Signals for glutathionylation in Na+-K+-ATPase-enriched membrane fragments suspended in solutions that preferentially induce E1ATP and E1Na3 conformations were much larger than signals in solutions that induce the E2 conformation. Ouabain further reduced glutathionylation in E2 and eliminated an increase seen with exposure to the oxidant peroxynitrite (ONOO−). Inhibition of Na+-K+-ATPase activity after exposure to ONOO− was greater when the enzyme had been in the E1Na3 than the E2 conformation. We exposed myocytes to different extracellular K+ concentrations to vary the membrane potential and hence voltage-dependent conformational poise. K+ concentrations expected to shift the poise toward E2 species reduced glutathionylation, and ouabain eliminated a ONOO−-induced increase. Angiotensin II-induced NADPH oxidase-dependent Na+-K+ pump inhibition was eliminated by conditions expected to shift the poise toward the E2 species. We conclude that susceptibility of the β1 subunit to glutathionylation depends on the conformational poise of the Na+-K+ pump.

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

2016 ◽  
Vol 147 (2) ◽  
pp. 153-164 ◽  
Author(s):  
Franklin M. Mullins ◽  
Richard S. Lewis
Keyword(s):  
Noise Analysis ◽  
Functional Coupling ◽  
Open Probability ◽  
Strongly Coupled ◽  
Functional Interactions ◽  
Crac Channels ◽  
Gating Mechanism ◽  
Dependent Inactivation ◽  
Residual Level ◽  
Double Mutant Cycle

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.

scholarly journals Structural modeling of the hERG potassium channel and associated drug interactions

2021 ◽  
Author(s):  
Jan Maly ◽  
Aiyana Emigh ◽  
Kevin DeMarco ◽  
Kazuharu Furutani ◽  
Jon T. Sack ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Conformational Changes ◽  
Drug Binding ◽  
Selectivity Filter ◽  
Herg Channel ◽  
Side Chain ◽  
Ion Conduction ◽  
Drug Induced ◽  
Channel Inactivation ◽  
Conduction Pathway

The voltage-gated potassium channel, KV11.1, encoded by the human Ether-a-go-go-Related Gene (hERG) is expressed in cardiac myocytes, where it is crucial for the membrane repolarization of the action potential. Gating of hERG channel is characterized by rapid, voltage-dependent, C-type inactivation, which blocks ion conduction and is suggested to involve constriction of the selectivity filter. Mutations S620T and S641A/T within the selectivity filter region of hERG have been shown to alter the voltage-dependence of channel inactivation. Because hERG channel blockade is implicated in a number of drug-induced arrhythmias associated with both the open and inactivated states, we simulated the effects of these mutations to elucidate conformational changes associated with hERG channel inactivation and differences in drug binding between the two states. Rosetta modeling of the S641A fast-inactivating mutation revealed a lateral shift of F627 side chain in the selectivity filter into the central channel axis along the ion conduction pathway and formation of a fenestration region below the selectivity filter. Rosetta modeling of the non-inactivating mutations S620T and S641T suggested a potential molecular mechanism preventing F627 side chain from shifting into the ion conduction pathway during the proposed inactivation process. Furthermore, we used Rosetta docking to explore the binding mechanism of highly selective and potent hERG blockers - dofetilide, terfenadine, and E4031. Our results correlate well with existing experimental evidence involving interactions of these drugs with key hERG residues Y652 and F656 inside the pore and reveal potential ligand binding interactions within fenestration region in an inactivated state.

scholarly journals Separation and Characterization of Currents through Store-operated CRAC Channels and Mg2+-inhibited Cation (MIC) Channels

2002 ◽  
Vol 119 (5) ◽  
pp. 487-508 ◽  
Author(s):  
Murali Prakriya ◽  
Richard S. Lewis
Keyword(s):  
Calcium Release ◽  
Divalent Cations ◽  
Noise Analysis ◽  
Crac Channels ◽  
Unitary Conductance ◽  
New Information ◽  
Crac Channel ◽  
High Doses ◽  
Whole Cell Recordings

Although store-operated calcium release–activated Ca2+(CRAC) channels are highly Ca2+-selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na+and Cs+with a unitary conductance of ∼40 pS, and that intracellular Mg2+modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg2+-inhibited cation (MIC) channels that open as Mg2+is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8–10 mM intracellular Mg2+to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca2+to divalent-free extracellular solution evokes Na+current through open CRAC channels (Na+-ICRAC) that is initially eightfold larger than the preceding Ca2+current and declines by ∼80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs+(PCs/PNa= 0.13 vs. 1.2 for MIC). Neither the decline in Na+-ICRACnor its low Cs+permeability are affected by intracellular Mg2+(90 μM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca2+channels.

scholarly journals Regulation of Store-Operated Ca2+ Entry by SARAF

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1887
Author(s):  
Inbal Dagan ◽  
Raz Palty
Keyword(s):  
Molecular Mechanisms ◽  
Feedback Mechanism ◽  
Cellular Biology ◽  
Excitable Cells ◽  
Major Pathway ◽  
Negative Feedback Mechanism ◽  
Crac Channels ◽  
Dependent Inactivation ◽  
Crac Channel ◽  
The One

Calcium (Ca2+) signaling plays a dichotomous role in cellular biology, controlling cell survival and proliferation on the one hand and cellular toxicity and cell death on the other. Store-operated Ca2+ entry (SOCE) by CRAC channels represents a major pathway for Ca2+ entry in non-excitable cells. The CRAC channel has two key components, the endoplasmic reticulum Ca2+ sensor stromal interaction molecule (STIM) and the plasma-membrane Ca2+ channel Orai. Physical coupling between STIM and Orai opens the CRAC channel and the resulting Ca2+ flux is regulated by a negative feedback mechanism of slow Ca2+ dependent inactivation (SCDI). The identification of the SOCE-associated regulatory factor (SARAF) and investigations of its role in SCDI have led to new functional and molecular insights into how SOCE is controlled. In this review, we provide an overview of the functional and molecular mechanisms underlying SCDI and discuss how the interaction between SARAF, STIM1, and Orai1 shapes Ca2+ signaling in cells.

Replacement of the interchain disulfide bridge-forming amino acids A7 and B7 by glutamate impairs the structure and activity of insulin

2004 ◽  
Vol 385 (12) ◽  
pp. 1171-1175 ◽  
Author(s):  
Zhan-Yun Guo ◽  
Xiao-Yuan Jia ◽  
You-Min Feng
Keyword(s):  
Biological Activity ◽  
Disulfide Bonds ◽  
Negative Charge ◽  
Disulfide Bridge ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Insulin Analogs ◽  
High Biological Activity ◽  
Chemical Groups ◽  
Structure And Activity

Abstract Insulin contains three disulfide bonds, one intrachain bond, A6–A11, and two interchain bonds, A7–B7 and A20–B19. Site-directed mutagenesis results (the two cysteine residues of disulfide A7–B7 were replaced by serine) showed that disulfide A7–B7 is crucial to both the structure and activity of insulin. However, chemical modification results showed that the insulin analogs still retained relatively high biological activity when A7Cys and B7Cys were modified by chemical groups with a negative charge. Did the negative charge of the modification groups restore the loss of activity and/or the disturbance of structure of these insulin analogs caused by deletion of disulfide A7–B7? To answer this question, an insulin analog with both A7Cys and B7Cys replaced by Glu, which has a long side-chain and a negative charge, was prepared by protein engineering, and its structure and activity were analyzed. Both the structure and activity of the present analog are very similar to that of the mutant with disulfide A7–B7 replaced by Ser, but significantly different from that of wild-type insulin. The present results suggest that removal of disulfide A7–B7 will result in serious loss of biological activity and the native conformation of insulin, even if the disulfide is replaced by residues with a negative charge.

scholarly journals Crystal structures of Ca2+–calmodulin bound to NaV C-terminal regions suggest role for EF-hand domain in binding and inactivation

2019 ◽  
Vol 116 (22) ◽  
pp. 10763-10772 ◽  
Author(s):  
Bernd R. Gardill ◽  
Ricardo E. Rivera-Acevedo ◽  
Ching-Chieh Tung ◽  
Filip Van Petegem
Keyword(s):  
Crystal Structure ◽  
Binding Sites ◽  
Binding Mode ◽  
Conformational Switch ◽  
Channel Inactivation ◽  
Dependent Inactivation ◽  
Ef Hand ◽  
Inherited Arrhythmia ◽  
A Site

Voltage-gated sodium (NaV) and calcium channels (CaV) form targets for calmodulin (CaM), which affects channel inactivation properties. A major interaction site for CaM resides in the C-terminal (CT) region, consisting of an IQ domain downstream of an EF-hand domain. We present a crystal structure of fully Ca2+-occupied CaM, bound to the CT of NaV1.5. The structure shows that the C-terminal lobe binds to a site ∼90° rotated relative to a previous site reported for an apoCaM complex with the NaV1.5 CT and for ternary complexes containing fibroblast growth factor homologous factors (FHF). We show that the binding of FHFs forces the EF-hand domain in a conformation that does not allow binding of the Ca2+-occupied C-lobe of CaM. These observations highlight the central role of the EF-hand domain in modulating the binding mode of CaM. The binding sites for Ca2+-free and Ca2+-occupied CaM contain targets for mutations linked to long-QT syndrome, a type of inherited arrhythmia. The related NaV1.4 channel has been shown to undergo Ca2+-dependent inactivation (CDI) akin to CaVs. We present a crystal structure of Ca2+/CaM bound to the NaV1.4 IQ domain, which shows a binding mode that would clash with the EF-hand domain. We postulate the relative reorientation of the EF-hand domain and the IQ domain as a possible conformational switch that underlies CDI.

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