scholarly journals Effects of pH on phosphorylation of the Ca2+-ATPase of sarcoplasmic reticulum by inorganic phosphate

1997 ◽  
Vol 321 (3) ◽  
pp. 671-676 ◽  
Author(s):  
Yamin M. KHAN ◽  
J. Malcolm EAST ◽  
Anthony G. LEE
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Equilibrium Constant ◽  
Fluorescence Intensity ◽  
Inorganic Phosphate ◽  
Ph Dependence ◽  
Phosphorylated Form ◽  
Phosphorylation Reaction ◽  
Effects Of Ph ◽  
High Concentrations

The fluorescence intensity of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SR) labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin has been shown to decrease on phosphorylation of the ATPase with Pi, this providing a convenient measure of the level of phosphorylation. Comparison of the fluorescence decrease observed with ATP and with high concentrations of Pi fix the value of the equilibrium constant for the phosphorylation reaction E2PMg ⇌ E2PiMg at pH 6.0 at about 2. Studies of the pH-dependence of phosphorylation show that H2PO4- and HPO42- bind to the ATPase with equal affinity, but that only binding of H2PO4- leads to phosphorylation, described by an equilibrium constant of 2.3. Luminal Ca2+ can bind to a pair of sites on the ATPase, with affinities of 1.3ȕ103 and 1.7ȕ103 M-1 for the unphosphorylated and phosphorylated forms of the ATPase respectively, with stronger binding of Ca2+ to the phosphorylated form resulting in an increase in the effective equilibrium constant for phosphorylation.

scholarly journals Effects of polycations on Ca2+ binding to the Ca2+-ATPase

1995 ◽  
Vol 308 (2) ◽  
pp. 493-499 ◽  
Author(s):  
G Hughes ◽  
Y M Khan ◽  
J M East ◽  
A G Lee
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Equilibrium Constant ◽  
Fluorescence Intensity ◽  
Binding Sites ◽  
Ruthenium Red ◽  
Basic Peptide ◽  
Basic Peptides

Spermine and polyarginine have been shown to increase the rate of dissociation of Ca2+ from the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum. They also decrease the affinity of the ATPase for Mg2+ as detected by changes in the fluorescence intensity of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin (DMC). Polyarginine itself also decreases the fluorescence intensity of DMC-labelled ATPase. These results are consistent with binding of spermine and polyarginine to a gating site controlling the rate of access of Ca2+ to its binding sites on the ATPase. A basic peptide PLN-(1-25) corresponding to residues 1-25 of phospholamban had no effect on the rate of dissociation of Ca2+ or on the fluorescence of DMC-labelled ATPase. Spermine, polyarginine and PLN-(1-25) all increased the equilibrium constant E1/E2, and spermine and polyarginine increased the rate of Ca2+ binding to the ATPase, consistent with an increase in the rate of the E2-->E1 transition. Spermine displaced Tb3+ and Ruthenium Red from the ATPase, consistent with binding in the stalk region of the ATPase. Polyarginine and PLN-(1-25), however, had no effect on Tb3+ or Ruthenium Red binding, suggesting a greater specificity in binding basic peptides to the ATPase than spermine.

Lactate inhibits Ca2+-activated Ca2+-channel activity from skeletal muscle sarcoplasmic reticulum

1997 ◽  
Vol 82 (2) ◽  
pp. 447-452 ◽  
Author(s):  
Terence G. Favero ◽  
, Anthony C. Zable ◽  
, David Colter ◽  
Jonathan J. Abramson
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Single Channel ◽  
Channel Activity ◽  
Release Channel ◽  
Muscle Lactate ◽  
Tension Development ◽  
Contraction Coupling ◽  
Single Channel Activity ◽  
High Concentrations

Favero, Terence G., Anthony C. Zable, David Colter, and Jonathan J. Abramson. Lactate inhibits Ca2+-activated Ca2+-channel activity from skeletal muscle sarcoplasmic reticulum. J. Appl. Physiol. 82(2): 447–452, 1997.—Sarcoplasmic reticulum (SR) Ca2+-release channel function is modified by ligands that are generated during about of exercise. We have examined the effects of lactate on Ca2+- and caffeine-stimulated Ca2+ release, [3H]ryanodine binding, and single Ca2+-release channel activity of SR isolated from rabbit white skeletal muscle. Lactate, at concentrations from 10 to 30 mM, inhibited Ca2+- and caffeine-stimulated [3H]ryanodine binding to and inhibited Ca2+- and caffeine-stimulated Ca2+ release from SR vesicles. Lactate also inhibited caffeine activation of single-channel activity in bilayer reconstitution experiments. These findings suggest that intense muscle activity, which generates high concentrations of lactate, will disrupt excitation-contraction coupling. This may lead to decreases in Ca2+ transients promoting a decline in tension development and contribute to muscle fatigue.

scholarly journals A model for the uptake and release of Ca2+ by sarcoplasmic reticulum

1987 ◽  
Vol 245 (3) ◽  
pp. 739-749 ◽  
Author(s):  
G W Gould ◽  
J M McWhirter ◽  
J M East ◽  
A G Lee
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
External Medium ◽  
Spontaneous Release ◽  
Calculated Concentration ◽  
Effects Of Ph ◽  
Hydrolysis Of ◽  
Uptake And Release

On addition of ATP to vesicles derived from the sarcoplasmic reticulum (SR) of skeletal muscle, Ca2+ is accumulated from the external medium. Following uptake, spontaneous release of Ca2+ occurs in the presence or in the absence of ATP. These processes of Ca2+ uptake and release were simulated by using the models derived for ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227; Stefanova, Napier, East & Lee (1987) Biochem. J. 245, 723-730] and for Ca2+ release from passively loaded vesicles [McWhirter, Gould, East & Lee (1987) Biochem. J. 245, 713-722]. The simulations are consistent with measurements of the effects of pH, K+, Ca2+ and Mg2+ on uptake and release of Ca2+. The increase in maximal Ca2+ accumulation observed in the presence of maleate is explained in terms of complexing of Ca2+ and maleate within the SR. The calculated concentration of ADP generated by hydrolysis of ATP has a large effect on the simulations. The effects of an ATP-regenerating system on the measured Ca2+ uptake is explained in terms of both removal of ADP and precipitation of Ca3(PO4)2 within the vesicles. It is concluded that both the process of Ca2+ uptake and the process of Ca2+ release seen with SR vesicles can be interpreted quantitatively in terms solely of the properties of the Ca2+ + Mg2+-activated ATPase.

A Mechanism for Both Capacitative Ca2+ Entry and Excitation-Contraction Coupled Ca2+ Release by the Sarcoplasmic Reticulum of Skeletal Muscle Cells

2002 ◽  
Vol 227 (6) ◽  
pp. 425-431 ◽  
Author(s):  
Mohammad Naimul Islam ◽  
Bisni Narayanan ◽  
Raymond S. Ochs
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Sarcoplasmic Reticulum ◽  
Muscle Cells ◽  
Cell Types ◽  
Release Channel ◽  
Close Proximity ◽  
High Concentrations ◽  
Combined Channel ◽  
Uncompetitive Inhibitor

We have previously established that L6 skeletal muscle cell cultures display capacitative calcium entry (CCE), a phenomenon established with other cells in which Ca2+ uptake from outside cells increases when the endoplasmic reticulum (sarcoplasmic reticulum in muscle, or SR) store is decreased. Evidence for CCE rested on the use of thapsigargin (Tg), an inhibitor of the SR CaATPase and consequently transport of Ca2+ from cytosol to SR, and measurements of cytosolic Ca2+. When Ca2+ is added to Ca2+-free cells in the presence of Tg, the measured cytosolic Ca2+ rises. This has been universally interpreted to mean that as SR Ca2+ is depleted, exogenous Ca2+ crosses the plasma membrane, but accumulates in the cytosol due to CaATPase inhibition. Our goal in the present study was to examine CCE in more detail by measuring Ca2+ in both the SR lumen and the cytosol using established fluorescent dye techniques for both. Surprisingly, direct measurement of SR Ca2+ in the presence of Tg showed an increase in luminal Ca2+ concentration in response to added exogenous Ca2+. While we were able to reproduce the conventional demonstration of CCE—an increase of Ca2+ in the cytosol in the presence of thapsigargin—we found that this process was inhibited by the prior addition of ryanodine (Ry), which inhibits the SR Ca2+ release channel, the ryanodine receptor (RyR). This was also unexpected if Ca2+ enters the cytosol first. When Ca2+ was added prior to Ry, the later was unable to exert any inhibition. This implies a competitive interaction between Ca2+ and Ry at the RyR. In addition, we found a further paradox: we had previously found Ry to be an uncompetitive inhibitor of Ca2+ transport through the RyR during excitation-contraction coupling. We also found here that high concentrations of Ca2+ inhibited its own uptake, a known feature of the RyR. We confirmed that Ca2+ enters the cells through the dihydropyridine receptor (DHPR, also known as the L-channel) by demonstrating inhibition by diltiazem. A previous suggestion to the contrary had used Mn2+ in place of direct Ca2+ measurements; we showed that Mn2+ was not inhibited by diltiazem and was not capacitative, and thus not an appropriate probe of Ca2+ flow in muscle cells. Our findings are entirely explained by a new model whereby Ca2+ enters the SR from the extracellular space directly through a combined channel formed from the DHPR and the RyR. These are known to be in close proximity in skeletal muscle. Ca2+ subsequently appears in the cytosol by egress through a separate, unoccupied RyR, explaining Ry inhibition. We suggest that upon excitation, the DHPR, in response to the electrical field of the plasma membrane, shifts to an erstwhile-unoccupied receptor, and Ca2+ is released from the now open RyR to trigger contraction. We discuss how this model also resolves existing paradoxes in the literature, and its implications for other cell types.

Effects of polymyxin B on the sarcoplasmic reticulum membrane

1989 ◽  
Vol 9 (5) ◽  
pp. 573-578 ◽  
Author(s):  
T. B. Ktenas ◽  
T. G. Sotiroudis ◽  
A. E. Evangelopoulos
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Fluorescence Intensity ◽  
Cyclic Peptide ◽  
Fluorescein Isothiocyanate ◽  
Polymyxin B ◽  
The Other ◽  
Peptide Antibiotic ◽  
Phosphorylase Kinase ◽  
Sarcoplasmic Reticulum Membrane

Polymyxin B, a cyclic peptide antibiotic, inhibits Ca2+-ATPase, p-nitrophenyl phosphatase and phosphorylase kinase activities associated with rabbit skeletal muscle sarcoplasmic reticulum membranes; 50% inhibition is induced by 100 μM, 130μM and 550 μM of polymyxin respectively. The fluorescence intensity of fluorescein isothiocyanate-labeled Ca2+-ATPase, decreases in the presence of polymyxin (50% of the total decrease at 70 μM polymyxin). On the other hand, the polypeptide inhibits calmodulin-dependent endogenous phosphorylation of 60 kDa, 20 kDa and 14 kDa membrane proteins, while an increase of calmodulin-dependent phosphorylation is observed in 132 kDa and 86 kDa proteins.

Impaired calcium release during fatigue

2008 ◽  
Vol 104 (1) ◽  
pp. 296-305 ◽  
Author(s):  
D. G. Allen ◽  
G. D. Lamb ◽  
H. Westerblad
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Action Potential ◽  
Inorganic Phosphate ◽  
Calcium Release ◽  
Voltage Sensor ◽  
Functional Importance ◽  
Intracellular Atp ◽  
Channel Opening ◽  
Sensor Activation

Impaired calcium release from the sarcoplasmic reticulum (SR) has been identified as a contributor to fatigue in isolated skeletal muscle fibers. The functional importance of this phenomenon can be quantified by the use of agents, such as caffeine, which can increase SR Ca2+ release during fatigue. A number of possible mechanisms for impaired calcium release have been proposed. These include reduction in the amplitude of the action potential, potentially caused by extracellular K+ accumulation, which may reduce voltage sensor activation but is counteracted by a number of mechanisms in intact animals. Reduced effectiveness of SR Ca2+ channel opening is caused by the fall in intracellular ATP and the rise in Mg2+ concentrations that occur during fatigue. Reduced Ca2+ available for release within the SR can occur if inorganic phosphate enters the SR and precipitates with Ca2+. Further progress requires the development of methods that can identify impaired SR Ca2+ release in intact, blood-perfused muscles and that can distinguish between the various mechanisms proposed.

scholarly journals A kinetic model for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum

1986 ◽  
Vol 237 (1) ◽  
pp. 217-227 ◽  
Author(s):  
G W Gould ◽  
J M East ◽  
R J Froud ◽  
J M McWhirter ◽  
H I Stefanova ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Kinetic Model ◽  
Binding Sites ◽  
Ph Dependence ◽  
High Affinity ◽  
Phosphorylated Form ◽  
Low Concentrations ◽  
Kinetics Of ◽  
Single Binding Site ◽  
Micromolar Range

The Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum exhibits complex kinetics of activation with respect to ATP. ATPase activity is pH-dependent, with similar pH-activity profiles at high and low concentrations of ATP. Low concentrations of Ca2+ in the micromolar range activate the ATPase, whereas activity is inhibited by Ca2+ at millimolar concentrations. The pH-dependence of this Ca2+ inhibition and the effect of the detergent C12E8 (dodecyl octaethylene glycol monoether) on Ca2+ inhibition are similar to those observed on activation by low concentrations of Ca2+. On the basis of these and other studies we present a kinetic model for the ATPase. The ATPase is postulated to exist in one of two conformations: a conformation (E1) of high affinity for Ca2+ and MgATP and a conformation (E2) of low affinity for Ca2+ and MgATP. Ca2+ binding to E2 and to the phosphorylated form E2P are equal. Proton binding at the Ca2+-binding sites in the E1 and E2 conformations explains the pH-dependence of Ca2+ effects. Binding of MgATP to the phosphorylated intermediate E1′PCa2 and to E2 modulate the rates of the transport step E1′PCa-E2′PCa2 and the return of the empty Ca2+ sites to the outside surface of the sarcoplasmic reticulum, as well as the rate of dephosphorylation of E2P. Only a single binding site for MgATP is postulated.

scholarly journals Comparison of the effects of inorganic phosphate on caffeine-induced Ca2+ release in fast- and slow-twitch mammalian skeletal muscle

2008 ◽  
Vol 294 (1) ◽  
pp. C97-C105 ◽  
Author(s):  
Giuseppe S. Posterino ◽  
Stacey L. Dunn
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Inorganic Phosphate ◽  
Fiber Type ◽  
Muscle Fibers ◽  
Creatine Phosphate ◽  
Mammalian Skeletal Muscle ◽  
Time Integral ◽  
Slow Twitch ◽  
Fast Twitch

We compared the effects of 50 mM Pi on caffeine-induced Ca2+ release in mechanically skinned fast-twitch (FT) and slow-twitch (ST) skeletal muscle fibers of the rat. The time integral (area) of the caffeine response was reduced by ∼57% (FT) and ∼27% (ST) after 30 s of exposure to 50 mM Pi in either the presence or absence of creatine phosphate (to buffer ADP). Differences in the sarcoplasmic reticulum (SR) Ca2+ content between FT and ST fibers [∼40% vs. 100% SR Ca2+ content (pCa 6.7), respectively] did not contribute to the different effects of Pi observed; underloading the SR of ST fibers so that the SR Ca2+ content approximated that of FT fibers resulted in an even smaller (∼21%), but not significant, reduction in caffeine-induced Ca2+ release by Pi. These observed differences between FT and ST fibers could arise from fiber-type differences in the ability of the SR to accumulate Ca2+-Pi precipitate. To test this, fibers were Ca2+ loaded in the presence of 50 mM Pi. In FT fibers, the maximum SR Ca2+ content (pCa 6.7) was subsequently increased by up to 13 times of that achieved when loading for 2 min in the absence of Pi. In ST fibers, the SR Ca2+ content was only doubled. These data show that Ca2+ release in ST fibers was less affected by Pi than FT fibers, and this may be due to a reduced capacity of ST SR to accumulate Ca2+-Pi precipitate. This may account, in part, for the fatigue-resistant nature of ST fibers.

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