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 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.

Physiological role of Ca(2+)-activated and voltage-dependent K+ currents in rabbit coronary myocytes

1994 ◽  
Vol 266 (6) ◽  
pp. C1523-C1537 ◽  
Author(s):  
N. Leblanc ◽  
X. Wan ◽  
P. M. Leung
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Physiological Role ◽  
Cell Voltage ◽  
Resting Membrane Potential ◽  
Physiological Level ◽  
Steady State Current ◽  
Voltage Dependent ◽  
A Minor ◽  
And Function

The properties and function of Ca(2+)-activated K+ (KCa) and voltage-dependent K+ (IK) currents of rabbit coronary myocytes were studied under whole cell voltage-clamp conditions (22 degrees C). Inhibition of KCa by tetraethylammonium chloride (1-10 mM) or charybdotoxin (50-100 nM) suppressed noisy outward rectifying current elicited by 5-s voltage steps or ramp at potentials > 0 mV, reduced the hump of the biphasic ramp current-voltage relation, and shifted by less than +5 mV the potential at which no net steady-state current is recorded (Enet; index of resting membrane potential). Inhibition of steady-state inward Ca2+ currents [ICa(L)] by nifedipine (1 microM) displaced Enet by -11 mV. Analysis of steady-state voltage dependence of IK supported the existence of a "window" current between -50 and 0 mV. 4-Aminopyridine (2 mM) blocked a noninactivating component of IK evoked between -30 and -40 mV, abolished the hump current during ramps, and shifted Enet by more than +15 mV; hump current persisted during 2-min ramp depolarizations and peaked near the maximum overlap of the steady-state activation and inactivation curves of IK (about -22 mV). A threefold rise in extracellular Ca2+ concentration (1.8-5.4 mM) enhanced time-dependent outward K+ current (6.7-fold at +40 mV) and shifted Enet by -30 mV. It is concluded that, under steady-state conditions, IK and ICa(L) play a major role in regulating resting membrane potential at a physiological level of intracellular Ca2+ concentration, with a minor contribution from KCa. However, elevation of intracellular Ca2+ concentration enhances KCa and hyperpolarizes the myocyte to limit Ca2+ entry through ICa(L).

Angiotensin II and potassium activate different calcium entry mechanisms in rat adrenal glomerulosa cells

1989 ◽  
Vol 122 (1) ◽  
pp. 361-370 ◽  
Author(s):  
A. Spät ◽  
I. Balla ◽  
T. Balla ◽  
E. J. Cragoe ◽  
Gy. Hajnóczky ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Angiotensin Ii ◽  
Calcium Channels ◽  
Small Reduction ◽  
Nickel Ions ◽  
Ca Uptake ◽  
Voltage Dependent ◽  
Aldosterone Production ◽  
Glomerulosa Cells ◽  
Isolated Rat

ABSTRACT Initial 45Ca uptake was measured in isolated rat glomerulosa cells. A small reduction in membrane potential produced by increasing the K+ concentration from 2 to 3·6 mmol/l stimulated 45Ca uptake by about 35%, while a bigger depolarization induced by 18·5 mmol K+/l increased the uptake by about 100%. Since Ca2+ influx was already activated at a calculated membrane potential below −70 mV, and was found to be sensitive to the dihydropyridine antagonist nifedipine (1 μmol/l), but insensitive to nickel ions (100 μmol/l), it does not meet the criteria established for T- or L-type voltage-dependent Ca2+ channels. Exposure of glomerulosa cells to angiotensin II (AII) for 10 min also enhanced the rate of 45Ca influx. The effect of AII was not sensitive to 1 μmol nifedipine/l, but was strongly inhibited by 5-(N-4-chlorobenzyl)-N-(2′,4′-dimethyl)benzamil (CBDMB, 30 μmol/l), an inhibitor of the Na+/Ca2+ antiporter. These observations suggest that during the sustained phase of stimulation with AII, a CBDMB-sensitive mechanism, rather than dihydropyridine-sensitive calcium channels, is involved in Ca2+ uptake in rat glomerulosa cells. The bulk Ca2+ influx did not correlate with aldosterone production; however, the maintained activity of different Ca2+ entry mechanisms seems to be essential for AII-induced aldosterone production. Journal of Endocrinology (1989) 122, 361–370

scholarly journals Transcription inhibition by the depsipeptide antibiotic salinamide A

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
David Degen ◽  
Yu Feng ◽  
Yu Zhang ◽  
Katherine Y Ebright ◽  
Yon W Ebright ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Rna Synthesis ◽  
Antibacterial Drug ◽  
Transcription Inhibition ◽  
Bacterial Rna ◽  
Cellular Target ◽  
Nucleotide Addition ◽  
And Function

We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipeptide antibiotic salinamide A (Sal), and we report that Sal inhibits RNAP through a novel binding site and mechanism. We show that Sal inhibits RNA synthesis in cells and that mutations that confer Sal-resistance map to RNAP genes. We show that Sal interacts with the RNAP active-center ‘bridge-helix cap’ comprising the ‘bridge-helix N-terminal hinge’, ‘F-loop’, and ‘link region’. We show that Sal inhibits nucleotide addition in transcription initiation and elongation. We present a crystal structure that defines interactions between Sal and RNAP and effects of Sal on RNAP conformation. We propose that Sal functions by binding to the RNAP bridge-helix cap and preventing conformational changes of the bridge-helix N-terminal hinge necessary for nucleotide addition. The results provide a target for antibacterial drug discovery and a reagent to probe conformation and function of the bridge-helix N-terminal hinge.

Effects of ropivacaine on membrane potential and voltage-dependent calcium channel current in single guinea-pig ventricular myocytes

2002 ◽  
Vol 16 (4) ◽  
pp. 273-278 ◽  
Author(s):  
Noboru Hatakeyama ◽  
Masana Yamada ◽  
Nobuko Shibuya ◽  
Mitsuaki Yamazaki ◽  
Shigeo Yamamura ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Guinea Pig ◽  
Calcium Channel ◽  
Ventricular Myocytes ◽  
Channel Current ◽  
Voltage Dependent Calcium Channel ◽  
Voltage Dependent

scholarly journals The Voltage Sensor in Voltage-Dependent Ion Channels

2000 ◽  
Vol 80 (2) ◽  
pp. 555-592 ◽  
Author(s):  
Francisco Bezanilla
Keyword(s):  
Membrane Potential ◽  
Conformational Changes ◽  
Structural Information ◽  
Noise Analysis ◽  
Voltage Sensor ◽  
Fluorescence Labeling ◽  
Gating Current ◽  
Gating Currents ◽  
Gating Charge ◽  
Voltage Dependent

In voltage-dependent Na, K, or Ca channels, the probability of opening is modified by the membrane potential. This is achieved through a voltage sensor that detects the voltage and transfers its energy to the pore to control its gate. We present here the theoretical basis of the energy coupling between the electric field and the voltage, which allows the interpretation of the gating charge that moves in one channel. Movement of the gating charge constitutes the gating current. The properties are described, along with macroscopic data and gating current noise analysis, in relation to the operation of the voltage sensor and the opening of the channel. Structural details of the voltage sensor operation were resolved initially by locating the residues that make up the voltage sensor using mutagenesis experiments and determining the number of charges per channel. The changes in conformation are then analyzed based on the differential exposure of cysteine or histidine-substituted residues. Site-directed fluorescence labeling is then analyzed as another powerful indicator of conformational changes that allows time and voltage correlation of local changes seen by the fluorophores with the global change seen by the electrophysiology of gating currents and ionic currents. Finally, we describe the novel results on lanthanide-based resonance energy transfer that show small distance changes between residues in the channel molecule. All of the electrophysiological and the structural information are finally summarized in a physical model of a voltage-dependent channel in which a change in membrane potential causes rotation of the S4 segment that changes the exposure of the basic residues from an internally connected aqueous crevice at hyperpolarized potentials to an externally connected aqueous crevice at depolarized potentials.

scholarly journals Voltage-dependent structural models of the human Hv1 proton channel from long-timescale molecular dynamics simulations

2020 ◽  
Vol 117 (24) ◽  
pp. 13490-13498 ◽  
Author(s):  
Andrew D. Geragotelis ◽  
Mona L. Wood ◽  
Hendrik Göddeke ◽  
Liang Hong ◽  
Parker D. Webster ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Membrane Potential ◽  
Conformational Changes ◽  
Structural Model ◽  
Ph Regulation ◽  
Conformational Dynamics ◽  
Structural Models ◽  
Dynamics Simulation ◽  
Proton Channel ◽  
Voltage Dependent

The voltage-gated Hv1 proton channel is a ubiquitous membrane protein that has roles in a variety of cellular processes, including proton extrusion, pH regulation, production of reactive oxygen species, proliferation of cancer cells, and increased brain damage during ischemic stroke. A crystal structure of an Hv1 construct in a putative closed state has been reported, and structural models for the channel open state have been proposed, but a complete characterization of the Hv1 conformational dynamics under an applied membrane potential has been elusive. We report structural models of the Hv1 voltage-sensing domain (VSD), both in a hyperpolarized state and a depolarized state resulting from voltage-dependent conformational changes during a 10-μs-timescale atomistic molecular dynamics simulation in an explicit membrane environment. In response to a depolarizing membrane potential, the S4 helix undergoes an outward displacement, leading to changes in the VSD internal salt-bridge network, resulting in a reshaping of the permeation pathway and a significant increase in hydrogen bond connectivity throughout the channel. The total gating charge displacement associated with this transition is consistent with experimental estimates. Molecular docking calculations confirm the proposed mechanism for the inhibitory action of 2-guanidinobenzimidazole (2GBI) derived from electrophysiological measurements and mutagenesis. The depolarized structural model is also consistent with the formation of a metal bridge between residues located in the core of the VSD. Taken together, our results suggest that these structural models are representative of the closed and open states of the Hv1 channel.

Voltage and beat dependence of Ca2+ transient in feline ventricular myocytes

1989 ◽  
Vol 257 (3) ◽  
pp. H746-H759 ◽  
Author(s):  
W. H. duBell ◽  
S. R. Houser
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Sarcoplasmic Reticulum ◽  
Voltage Clamp ◽  
Ventricular Myocytes ◽  
Rest Period ◽  
Voltage Dependent ◽  
Early Peak ◽  
Comparable Magnitude ◽  
Voltage Relationship

The positive contractile staircase after a period of rest is attributable to a positive staircase in the magnitude of the Ca2+ transient. The present study used voltage-clamp techniques and the fluorescent Ca2+ indicator, indo-1, to examine the effects of membrane potential, the duration of depolarization, and the slow inward Ca2+ current (Isi) in the regulation of the magnitude of the steady-state Ca2+ transient and the development of the steady state during the positive staircase. In the steady state, the Ca2+ transient was greatest at +10 mV, the potential at which Isi was also the greatest. However, the Isi-voltage relationship was much more bell-shaped than the Ca2+ transient-voltage relationship. The magnitude and duration of the steady-state Ca2+ transient was not affected by pulse durations as short as 25 ms. However, prolonged voltage pulses were essential to maintain the steady state. The development of the positive staircase was very voltage dependent. After a rest period, a positive staircase was seen when voltage-clamp drives were done to +30 mV but not when done to -10 mV, potentials that elicit Isi of comparable magnitude. These results support the idea that the early peak of Isi can act as a trigger for release of Ca2+ from the sarcoplasmic reticulum. However, sarcoplasmic reticulum Ca2+ loading is dependent on prolonged depolarization and may be mediated through Na+-Ca2+ exchange.

Voltage-dependent effects of isoproterenol on cytosolic Ca concentration in rat heart

1987 ◽  
Vol 252 (4) ◽  
pp. H697-H703 ◽  
Author(s):  
S. S. Sheu ◽  
V. K. Sharma ◽  
M. Korth
Keyword(s):  
Membrane Potential ◽  
Calcium Concentration ◽  
Ventricular Myocytes ◽  
Cytosolic Calcium ◽  
Resting Membrane Potential ◽  
Beta Adrenoceptor ◽  
Adrenoceptor Agonist ◽  
Voltage Dependent ◽  
K Concentration ◽  
Ca2 Channels

The effect of the beta-adrenoceptor agonist, isoproterenol, on cytosolic calcium concentration ([Ca2+]i) was studied with the Ca2+-sensitive fluorescent indicator quin 2 in enzymatically dissociated rat ventricular myocytes. Under conditions in which cells have normal polarized resting membrane potential, isoproterenol (1 microM) produced a decrease in [Ca2+]i. In contrast, in the depolarized cells (by raising extracellular K+ concentration to 50 mM), isoproterenol (1 microM) caused an increase in [Ca2+]i. This isoproterenol-induced increase in [Ca2+]i in depolarized cells could be reversed by prior exposure of the cells to the Ca2+ channel blocker, verapamil (5 microM). The results indicate that isoproterenol can either decrease or increase [Ca2+]i depending on membrane potential. The actual effect of isoproterenol on [Ca2+]i at any given membrane potential probably reflects the relative contributions of isoproterenol-induced stimulation of Ca2+ buffering or effluxing activities (which favor a decrease in [Ca2+]i) and enhancement of Ca2+ influx through voltage-sensitive Ca2+ channels (which favors an increase in [Ca2+]i).

Role of the Cilia Membrane in Control of Microtubule Sliding

1980 ◽  
Vol 38 ◽  
pp. 612-615
Author(s):  
Edna S. Kaneshiro
Keyword(s):  
Control Mechanism ◽  
Beat Frequency ◽  
Surface Membrane ◽  
Ciliary Membrane ◽  
Ciliary Beating ◽  
Ciliary Reversal ◽  
Voltage Dependent ◽  
Reversal Mechanism ◽  
And Function

It is currently believed that ciliary beating results from microtubule sliding which is restricted in regions to cause bending. Cilia beat can be modified to bring about changes in beat frequency, cessation of beat and reversal in beat direction. In ciliated protozoans these modifications which determine swimming behavior have been shown to be related to intracellular (intraciliary) Ca2+ concentrations. The Ca2+ levels are in turn governed by the surface ciliary membrane which exhibits increased Ca2+ conductance (permeability) in response to depolarization. Mutants with altered behaviors have been isolated. Pawn mutants fail to exhibit reversal of the effective stroke of ciliary beat and therefore cannot swim backward. They lack the increased inward Ca2+ current in response to depolarizing stimuli. Both normal and pawn Paramecium made leaky to Ca2+ by Triton extrac¬tion of the surface membrane exhibit backward swimming only in reactivating solutions containing greater than IO-6 M Ca2+ Thus in pawns the ciliary reversal mechanism itself is left operational and only the control mechanism at the membrane is affected. The topographic location of voltage-dependent Ca2+ channels has been identified as a component of the ciliary mem¬brane since the inward Ca2+ conductance response is eliminated by deciliation and the return of the response occurs during cilia regeneration. Since the ciliary membrane has been impli¬cated in the control of Ca2+ levels in the cilium and therefore is the site of at least one kind of control of microtubule sliding, we have focused our attention on understanding the structure and function of the membrane.

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