Luminal Ca2+ Regulation of RyR1 Ca2+ Channel Leak Activation and Inactivation in Sarcoplasmic Reticulum Membrane Vesicles

2020 ◽  
Author(s):  
Chris Palahniuk ◽  
Mark Mutawe ◽  
James Gilchrist
Keyword(s):  
Membrane Vesicles ◽  
Ruthenium Red ◽  
Sarcoplasmic Reticulum Membrane ◽  
Channel Opening ◽  
Complex Organization ◽  
Complete Disruption ◽  
In Trans ◽  
Functional States ◽  
Opening And Closing ◽  
Ca2 Channels

In this study, we tested the hypothesis that RyR1 Ca2+ channel closure is sensitive to outward trans-SR membrane Ca2+ gradients established by SERCA1 pumping. To perform these studies we employed stopped-flow rapid-kinetic fluorescence methods to measure and assess how variation in trans-SR membrane Ca2+ distribution affects evolution of RyR1 Ca2+ leaks in RyR1/CASQ1/SERCA1-rich HSR vesicles. Our studies showed that rapid filling of a Mag-Fura-2-sensitive free Ca2+ pool during SERCA1-mediated Ca2+ sequestration appears to be a crucial condition allowing RyR1 Ca2+ channels to close once reloading of luminal Ca2+ stores is complete. Disruption in the filling of this pool caused activation of ruthenium red-inhibitable RyR1 Ca2+ leaks suggesting that SERCA1 pump formation of outward Ca2+ gradients is an important aspect of Ca2+ flux control channel opening and closing. In addition, our observed ryanodine-induced shift in luminal Ca2+ from free to a CTC.Ca+-sensitive, CASQ1-associated bound compartment, underscores the complex organization and regulation of luminal Ca2+. Our study provides, strong evidence that RyR1 functional states directly and indirectly influence the compartmentation of luminal Ca2+. This, in turn, is influenced by the activity of SERCA1 pumps to both fill luminal pools while synchronously reducing Ca2+ levels on the cytosolic face of RyR1 channels.

scholarly journals Distinct Mg2+-dependent Steps Rate Limit Opening and Closing of a Single CFTR Cl− Channel

2002 ◽  
Vol 119 (6) ◽  
pp. 545-559 ◽  
Author(s):  
Athanasios G. Dousmanis ◽  
Angus C. Nairn ◽  
David C. Gadsby
Keyword(s):  
Open Channel ◽  
Tight Binding ◽  
Single Channels ◽  
Channel Opening ◽  
Subsequent Exposure ◽  
Cl Channels ◽  
Excised Patches ◽  
Atp Binding And Hydrolysis ◽  
Opening And Closing ◽  
Rate Limit

The roles played by ATP binding and hydrolysis in the complex mechanisms that open and close cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels remain controversial. In this work, the contributions made by ATP and Mg2+ ions to the gating of phosphorylated cardiac CFTR channels were evaluated separately by measuring the rates of opening and closing of single channels in excised patches exposed to solutions in which [ATP] and [Mg2+] were varied independently. Channel opening was found to be rate-limited not by the binding of ATP alone, but by a Mg2+-dependent step that followed binding of both ATP and Mg2+. Once a channel had opened, sudden withdrawal of all Mg2+ and ATP could prevent it from closing for tens of seconds. But subsequent exposure of such an open channel to Mg2+ ions alone could close it, and the closing rate increased with [Mg2+] over the micromolar range (half maximal at ∼50 μM [Mg2+]). A simple interpretation is that channel closing is stoichiometrically coupled to hydrolysis of an ATP molecule that remains tightly associated with the open CFTR channel despite continuous washing. If correct, that ATP molecule appears able to reside for over a minute in the catalytic site that controls channel closing, implying that the site must entrap, or have an intrinsically high apparent affinity for, ATP, even without a Mg2+ ion. Such stabilization of the open-channel conformation of CFTR by tight binding, or occlusion, of an ATP molecule echoes the stabilization of the active conformation of a G protein by GTP.

Bastadins relate ryanodine-sensitive and -insensitive Ca2+ efflux pathways in skeletal SR and BC3H1 cells

1997 ◽  
Vol 272 (2) ◽  
pp. C601-C614 ◽  
Author(s):  
I. N. Pessah ◽  
T. F. Molinski ◽  
T. D. Meloy ◽  
P. Wong ◽  
E. D. Buck ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Steady State ◽  
Ruthenium Red ◽  
Loading Capacity ◽  
Fk 506 ◽  
High Affinity ◽  
Plasmic Reticulum ◽  
Efflux Pathway ◽  
The Relationship ◽  
Ca2 Channels

Bastadins potently interact with the FK-506-binding protein of 12 kDa (FKBP12)-ryanodine receptor (Ry1R) complex in skeletal muscle to enhance a high-affinity ryanodine binding conformation (M. M. Mack, T. F. Molinski, E. D. Buck, and I. N. Pessah. J. Biol. Chem. 269: 23236-23249, 1994). Bastadins are used to examine the relationship between ryanodine-sensitive and ryanodine-insensitive Ca2+ efflux pathways that coexist in junctional sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle and differentiated BC3H1 cells. Complete block of caffeine-sensitive Ca2+ channels with micromolar ryanodine or ruthenium red does not alter the steady-state loading capacity of SR. Inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps with thapsigargin unmasks a ryanodine- and ruthenium red-insensitive Ca2+ efflux pathway. Bastadin 5 alone does not inhibit Ca2+ efflux unmasked by inhibition of SERCA pumps, but, in combination with blocking concentrations of ryanodine or ruthenium red, it eliminates the ryanodine-insensitive Ca2+ "leak" and enhances steady-state loading capacity of SR vesicles approximately 2.5-fold. These actions of bastadins occur in the same concentration range that enhances the number of high-affinity binding sites for [3H]ryanodine (50% effective concentration of approximately 2 microM). Similar effects on SR Ca2+ transport are found with FK-506 and ryanodine in combination. Block of Ry1R in intact BC3H1 cells with ryanodine does not eliminate the prominent Ca2+ leak unmasked by thapsigargin. A membrane-permeant mixture of bastadins in combination with ryanodine nearly eliminates the Ca2+ leak unmasked by thapsigargin, even though the Ca2+ stores are replete. The requirement of both a known Ry1R blocker and bastadins in combination provides a pharmacological link between ryanodine-sensitive Ca2+ channels and ryanodine-insensitive leak pathways in isolated junctional SR and BC3H1 cells. Together, these results strongly suggest that bastadins, through their modulatory actions on the FKBP12-Ry1R complex, convert ryanodine-insensitive leak states into ryanodine-sensitive channels that recognize [3H]ryanodine with high affinity.

Water movement during channel opening and closing

1987 ◽  
Vol 19 (4) ◽  
pp. 351-358 ◽  
Author(s):  
Joshua Zimmerberg ◽  
V. Adrian Parsegian
Keyword(s):  
Water Movement ◽  
Channel Opening ◽  
Opening And Closing

scholarly journals Calcium-gated calcium channels in sarcoplasmic reticulum of rabbit skinned skeletal muscle fibers.

1986 ◽  
Vol 87 (2) ◽  
pp. 289-303 ◽  
Author(s):  
P Volpe ◽  
G Salviati ◽  
A Chu
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Isometric Force ◽  
Muscle Fibers ◽  
Ruthenium Red ◽  
Scattering Method ◽  
Tension Development ◽  
Maximal Force ◽  
Skeletal Muscle Fibers ◽  
Ca2 Channels

The action of ruthenium red (RR) on Ca2+ loading by and Ca2+ release from the sarcoplasmic reticulum (SR) of chemically skinned skeletal muscle fibers of the rabbit was investigated. Ca2+ loading, in the presence of the precipitating anion pyrophosphate, was monitored by a light-scattering method. Ca2+ release was indirectly measured by following tension development evoked by caffeine. Stimulation of the Ca2+ loading rate by 5 microM RR was dependent on free Ca2+, being maximal at pCa 5.56. Isometric force development induced by 5 mM caffeine was reversibly antagonized by RR. IC50 for the rate of tension rise was 0.5 microM; that for the extent of tension was 4 microM. RR slightly shifted the steady state isometric force/pCa curve toward lower pCa values. At 5 microM RR, the pCa required for half-maximal force was 0.2 log units lower than that of the control, and maximal force was depressed by approximately 16%. These results suggest that RR inhibited Ca2+ release from the SR and stimulated Ca2+ loading into the SR by closing Ca2+-gated Ca2+ channels. Previous studies on isolated SR have indicated the selective presence of such channels in junctional terminal cisternae.

scholarly journals The transverse distribution of phospholipids in the membranes of Golgi subfractions of rat hepatocytes

1984 ◽  
Vol 219 (1) ◽  
pp. 261-272 ◽  
Author(s):  
J A Higgins
Keyword(s):  
Phospholipase C ◽  
Morphological Changes ◽  
Residual Activity ◽  
Membrane Vesicles ◽  
Rat Hepatocytes ◽  
Membrane Bilayer ◽  
Transverse Distribution ◽  
Golgi Vesicles ◽  
Golgi Membranes ◽  
In Trans

The transverse distribution of phospholipids in the membranes of subfractions of the Golgi complex was investigated by using phospholipase C and 2,4,6-trinitrobenzenesulphonic acid as probes. In trans-enriched Golgi membranes, 26% of the phosphatidylethanolamine is available for reaction with trinitrobenzenesulphonate or for hydrolysis by phospholipase C, and 72% of the phosphatidylcholine is hydrolysed by phospholipase C. In cis-enriched Golgi membranes, 45% of the phosphatidylethanolamine is available for reaction with trinitrobenzenesulphonate and for hydrolysis by phospholipase C, and 95% of the phosphatidylcholine is hydrolysed by phospholipase C. Under the conditions used with either probe the contents of the Golgi vesicles labelled with either [3H]palmitic acid or [14C]leucine were retained. Galactosyltransferase activity of the membrane vesicles was partially inhibited by the experimental procedures used to investigate the transverse distribution of phospholipids. However, the residual activity was latent, suggesting that the vesicles remained closed. Trinitrobenzenesulphonic acid caused no detectable morphological change in either Golgi fraction. Phospholipase C treatment caused morphological changes, including fusion of vesicles and the appearance of ‘signet-ring’ profiles in some vesicles; however, the vesicles remained closed and the bilayer was retained. It appears, therefore, that neither probe causes major disruption of the Golgi vesicles nor gains access to the inner surface of the membrane bilayer. These observations suggest that phospholipids have a transverse asymmetry in Golgi membranes, that this distribution differs in trans and cis membranes, and that the phospholipid structure of Golgi membranes is inconsistent with a simple flow of membrane bilayer from endoplasmic reticulum to Golgi membranes to plasma membrane.

scholarly journals Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels

2015 ◽  
Vol 145 (4) ◽  
pp. 261-283 ◽  
Author(s):  
Luiz A. Poletto Chaves ◽  
David C. Gadsby
Keyword(s):  
Atp Hydrolysis ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Dimer Interface ◽  
Binding Domains ◽  
Channel Opening ◽  
Order Of Magnitude ◽  
Signature Sequence ◽  
Dependent Modification ◽  
Opening And Closing

Cystic fibrosis transmembrane conductance regulator (CFTR) channel opening and closing are driven by cycles of adenosine triphosphate (ATP) binding–induced formation and hydrolysis-triggered disruption of a heterodimer of its cytoplasmic nucleotide-binding domains (NBDs). Although both composite sites enclosed within the heterodimer interface contain ATP in an open CFTR channel, ATP hydrolysis in the sole catalytically competent site causes channel closure. Opening of the NBD interface at that site then allows ADP–ATP exchange. But how frequently, and how far, the NBD surfaces separate at the other, inactive composite site remains unclear. We assessed separation at each composite site by monitoring access of nucleotide-sized hydrophilic, thiol-specific methanothiosulfonate (MTS) reagents to interfacial target cysteines introduced into either LSGGQ-like ATP-binding cassette signature sequence (replacing equivalent conserved serines: S549 and S1347). Covalent MTS-dependent modification of either cysteine while channels were kept closed by the absence of ATP impaired subsequent opening upon ATP readdition. Modification while channels were opening and closing in the presence of ATP caused macroscopic CFTR current to decline at the same speed as when the unmodified channels shut upon sudden ATP withdrawal. These results suggest that the target cysteines can be modified only in closed channels; that after modification the attached MTS adduct interferes with ATP-mediated opening; and that modification in the presence of ATP occurs rapidly once channels close, before they can reopen. This interpretation was corroborated by the finding that, for either cysteine target, the addition of the hydrolysis-impairing mutation K1250R (catalytic site Walker A Lys) similarly slowed, by an order of magnitude, channel closing on ATP removal and the speed of modification by MTS reagent in ATP. We conclude that, in every CFTR channel gating cycle, the NBD dimer interface separates simultaneously at both composite sites sufficiently to allow MTS reagents to access both signature-sequence serines. Relatively rapid modification of S1347C channels by larger reagents—MTS-glucose, MTS-biotin, and MTS-rhodamine—demonstrates that, at the noncatalytic composite site, this separation must exceed 8 Å.

scholarly journals Immunocytochemical Localization of a Calmodulinlike Protein in Bacillus subtilis Cells

1999 ◽  
Vol 181 (15) ◽  
pp. 4605-4610 ◽  
Author(s):  
Delfina C. Dominguez ◽  
Hank Adams ◽  
James H. Hageman
Keyword(s):  
Bacillus Subtilis ◽  
Cellular Localization ◽  
Time Course ◽  
Cell Envelope ◽  
Membrane Vesicles ◽  
Cross Reactivity ◽  
Bovine Brain ◽  
Ruthenium Red ◽  
Thin Sections ◽  
Calcium Ion Transport

ABSTRACT To determine possible functions of the calmodulinlike protein ofBacillus subtilis, the time course of its expression during sporulation and its cellular localization were studied. The protein was expressed in a constitutive manner from the end of logarithmic growth through 8 h of sporulation as determined by antibody cross-reactivity immunoblots and enzyme-linked immunosorbent assays (ELISAs). In partially purified extracts, the immunopositive protein comigrated upon electrophoresis with a protein which selectively bound [45Ca]CaCl2, ruthenium red, and Stains-all. Previous studies showed increased extractability of the calmodulinlike protein from B. subtilis cells when urea and 2-mercaptoethanol were used in breakage buffers, implying that the protein might be partially associated with the membrane fraction. This was confirmed by demonstrating that isolated membrane vesicles ofB. subtilis also gave positive immunological tests with Western blotting and ELISAs. To more precisely locate the protein in cells, thin sections of late-log-phase cells, sporulating cells, and free spores were reacted first with bovine brain anticalmodulin specific antibodies and then with gold-conjugated secondary antibodies; the thin sections were examined by transmission electron microscopy. The calmodulinlike protein was found almost exclusively associated with the cell envelope of these fixed, sectioned cells. A possible function of the calmodulinlike protein in sensing calcium ions or regulating calcium ion transport is suggested.

scholarly journals L-Type Ca2+ Channel Regulation by Calmodulin and CaBP1

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1811
Author(s):  
James B. Ames
Keyword(s):  
Open Probability ◽  
Apparent Dissociation Constant ◽  
Iq Motif ◽  
Channel Regulation ◽  
Channel Inactivation ◽  
Channel Opening ◽  
Binding Data ◽  
Functional Implications ◽  
Sensor Proteins ◽  
Ca2 Channels

L-type voltage-gated Ca2+ channels (CaV1.2 and CaV1.3, called CaV) interact with the Ca2+ sensor proteins, calmodulin (CaM) and Ca2+ binding Protein 1 (CaBP1), that oppositely control Ca2+-dependent channel activity. CaM and CaBP1 can each bind to the IQ-motif within the C-terminal cytosolic domain of CaV, which promotes increased channel open probability under basal conditions. At elevated cytosolic Ca2+ levels (caused by CaV channel opening), Ca2+-bound CaM binding to CaV is essential for promoting rapid Ca2+-dependent channel inactivation (CDI). By contrast, CaV binding to CaBP1 prevents CDI and promotes Ca2+-induced channel opening (called CDF). In this review, I provide an overview of the known structures of CaM and CaBP1 and their structural interactions with the IQ-motif to help understand how CaM promotes CDI, whereas CaBP1 prevents CDI and instead promotes CDF. Previous electrophysiology studies suggest that Ca2+-free forms of CaM and CaBP1 may pre-associate with CaV under basal conditions. However, previous Ca2+ binding data suggest that CaM and CaBP1 are both calculated to bind to Ca2+ with an apparent dissociation constant of ~100 nM when CaM or CaBP1 is bound to the IQ-motif. Since the neuronal basal cytosolic Ca2+ concentration is ~100 nM, nearly half of the neuronal CaV channels are suggested to be bound to Ca2+-bound forms of either CaM or CaBP1 under basal conditions. The pre-association of CaV with calcified forms of CaM or CaBP1 are predicted here to have functional implications. The Ca2+-bound form of CaBP1 is proposed to bind to CaV under basal conditions to block CaV binding to CaM, which could explain how CaBP1 might prevent CDI.

scholarly journals Broken symmetry in the human BK channel

2018 ◽  
Author(s):  
Lige Tonggu ◽  
Liguo Wang
Keyword(s):  
Ion Channels ◽  
Binding Sites ◽  
Bk Channel ◽  
Excitable Cells ◽  
Local Conformation ◽  
Channel Opening ◽  
Significant Movement ◽  
Em Method ◽  
The Common ◽  
Functional States

ABSTRACTVoltage-gated and ligand-modulated ion channels play critical roles in excitable cells. To understand the interplay among voltage-sensing, ligand-binding and channel opening, the structures of ion channels in various functional states need to be determined. Here, the “random spherically constrained” (RSC) single-particle cryo-EM method was employed to study the human large conductance voltage- and calcium-activated potassium (hBK or hSlo1) channels reconstituted into liposomes. The hBK structure has been determined at 3.5 Å resolution in the absence of Ca2+. Instead of the common four-fold symmetry observed in ligand-modulated ion channels, a two-fold symmetry was observed in hBK. Two opposing subunits in the Ca2+ sensing gating ring rotate around the center of each subunit, which results in the movement of the assembly and flexible interfaces and Ca2+ binding sites. Despite the significant movement, the local conformation of the assembly interfaces and Ca2+ binding sites remains the same among the four subunits.

Ca2+ release from Ca2+ stores, particularly from ryanodine-sensitive Ca2+ stores, is required for the induction of LTD in cultured cerebellar Purkinje cells

1995 ◽  
Vol 74 (5) ◽  
pp. 2184-2188 ◽  
Author(s):  
K. Kohda ◽  
T. Inoue ◽  
K. Mikoshiba
Keyword(s):  
Purkinje Cells ◽  
Specific Inhibitor ◽  
Cell Voltage ◽  
Ruthenium Red ◽  
Mouse Embryos ◽  
Voltage Gated ◽  
Inward Currents ◽  
Cerebellar Purkinje Cells ◽  
Ca2 Channels

1. Primary-cultured cerebellar Purkinje cells (PCs) from mouse embryos were whole cell voltage clamped, and L-glutamate (Glu) was applied iontophoretically to the dendrite. Long-term depression (LTD) of Glu-evoked currents was induced through the conjunction of repeated depolarizations and Glu applications. 2. Thapsigargin, a specific inhibitor of Ca(2+)-ATPase on the endoplasmic reticulum, and ryanodine and ruthenium red, inhibitors of the ryanodine receptor, blocked the induction of LTD. 3. Thapsigargin and ryanodine alone did not affect influx of Ca2+ through voltage-gated Ca2+ channels and inward currents evoked by Glu applications. 4. Our results suggest that Ca2+ release from internal stores, particularly from ryanodine-sensitive stores, is necessary for the induction of LTD in cultured PCs.

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