The Experts below are selected from a list of 168 Experts worldwide ranked by ideXlab platform
Sidney Fleischer - One of the best experts on this subject based on the ideXlab platform.
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phosphorylation with protein kinases modulates calcium loading of Terminal Cisternae of sarcoplasmic reticulum from skeletal muscle
Cell Calcium, 1995Co-Authors: M Mayrleitner, Hansgeorg Schindler, R Chandler, Sidney FleischerAbstract:Abstract We previously found in single channel studies that ryanodine receptor (RyR) channel activity can be made insensitive to block by Mg 2+ when Terminal Cisternae of sarcoplasmic reticulum, incorporated into planar bilayers, are treated with protein kinase A (PKA) or Ca 2+ /calmodulin dependent protein kinase type II (CamPK II), and then again made sensitive by treatment with protein phosphatases [Hain J. Nath S. Mayrleitner M. Fleischer S. Schindler H. (1994) Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. Biophys. J., 67, 1823–1833]. In this study, modulation by protein kinases and phosphatases on net Ca 2+ uptake by TC is presented. Phosphorylation of TC vesicles with PKA, CamPK II, or protein kinase C (PKC) reduced the calcium loading rate of TC vesicles 3-fold, 2.1-fold and 1.7-fold, respectively, measured in the presence of 1 MM MgCl 2 . There is no effect when AMP-PNP is substituted for ATP. Phosphorylation of the RyR was also measured by incororation of [γ- 32 P]-phosphate from ATP. A phosphorylation stoichiometry of 1.94 ± 0.1 ( 32 P/RyR) for PKA, 0.89 ± 0.08 for CamPK II and 0.95 ± 0.16 for PKC was obtained under these conditions. A study of the time dependence of phosphorylation with PKA and CamPK shows a direct correlation of reduction in calcium loading rate with increased phosphorylation of the ryanodine receptor. Treatment with protein phosphatase 1 enhanced the calcium loading rate again, after it was reduced by PKA phosphorylation. Investigation of the magnesium dependency shows that even at higher [Mg 2+ ] (6 mM), PKA phosphorylated TC vesicles have a 2.3-fold reduced calcium loading rate indicating insensitivity to block by Mg 2+ . Protein kinases have no effect on Ca 2+ -ATPase activity in TC or on Ca 2+ loading in longitudinal tubules which are devoid of RyR. We interpret the decreased net Ca 2+ uptake as being due to enhanced Ca 2+ leakage via the RyR. These studies show, at a macroscopic level, that the calcium release channel is modulated by kinases/phosphatases and support our previous findings with single channel studies that the ryanodine receptor must be phosphorylated to be active under physiological Mg 2+ concentrations.
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characterization of an exchange reaction between soluble fkbp 12 and the fkbp ryanodine receptor complex modulation by fkbp mutants deficient in peptidyl prolyl isomerase activity
Journal of Biological Chemistry, 1995Co-Authors: A P Timerman, Gregory Wiederrecht, Alice I. Marcy, Sidney FleischerAbstract:Abstract FKBP-12 (FKBP), the soluble receptor for the immunosuppresant drug FK-506, is tightly bound to the calcium release channel (CRC)/ryanodine receptor (RyR) of skeletal muscle Terminal Cisternae (TC) of sarcoplasmic reticulum with a stoichiometry of 4 mol of FKBP per tetrameric RyR complex. FKBP displays cis/trans-peptidyl-prolyl isomerase (PPIase) activity which is inhibited by FK-590 or rapamycin. In skeletal muscle TC, FK-590 or rapamycin binds to and dissociates FKBP from the RyR in a time- and temperature-dependent manner which increases the open probability of the channel. Therefore, the net energized Ca2+ uptake rate of TC vesicles devoid of FKBP is reduced due to the increased leak of Ca2+ from the TC specifically via the RyR, which is reversed upon rebinding of FKBP. Thus, the RyR is modulated by FKBP (Timerman, A. P., Ogunbumni, E., Freund, E. A., Wiederrecht, G., Marks, A. R., and Fleischer, S.(1993) J. Biol. Chem. 268, 22922-22999; Mayrleitner, M., Timerman, A. P., Wiederrecht, G., and Fleischer S.(1994) Cell Calcium 15, 99-108). We now find that FKBP can be displaced from the FKBP•RyR complex by exchange with FKBP in solution. The EC50 for exchange is 0.30 μM for wild type FKBP versus 0.6 to 2.4 μM for three different site-directed mutants that are practically devoid of any measurable PPIase activity. Substitution of wild-type FKBP on the RyR complex with these PPIase-deficient mutants did not alter the Ca2+ flux of TC vesicles, whereas dissociation of FKBP from TC with FK-590 increased the Ca2+ leak rate. Our studies show that, in vivo, the FKBP•RyR complex is in equilibrium with the cytosolic pool of FKBP (∼3 μM) and suggest that modulation of the CRC by FKBP is independent of PPIase activity.
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phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle
Biophysical Journal, 1994Co-Authors: J Hain, Sidney Fleischer, M Mayrleitner, Sati Nath, Hansgeorg SchindlerAbstract:The modulation of the calcium release channel (CRC) by protein kinases and phosphatases was studied. For this purpose, we have developed a microsyringe applicator to achieve sequential and multiple treatments with highly purified kinases and phosphatases applied directly at the bilayer surface. Terminal Cisternae vesicles of sarcoplasmic reticulum from rabbit fast twitch skeletal muscle were fused to planar lipid bilayers, and single-channel currents were measured at zero holding potential, at 0.15 microM free Ca2+, +/- 0.5 mM ATP and +/- 2.6 mM free Mg2+. Sequential dephosphorylation and rephosphorylation rendered the CRC sensitive and insensitive to block by Mg2+, respectively. Channel recovery from Mg2+ block was obtained by exogenous protein kinase A (PKA) or by Ca2+/calmodulin-dependent protein kinase II (CalPK II). Somewhat different characteristics were observed with the two kinases, suggesting two different states of phosphorylation. Channel block by Mg2+ was restored by dephosphorylation using protein phosphatase 1 (PPT1). Before application of protein kinases or phosphatases, channels were found to be "dephosphorylated" (inactive) in 60% and "phosphorylated" (active) in 40% of 51 single-channel experiments based on the criterion of sensitivity to block by Mg2+. Thus, these two states were interconvertable by treatment with exogenously added protein kinases and phosphatases. Endogenous Ca2+/calmodulin-dependent protein kinase (end CalPK) had an opposite action to exogenous CalPK II. Previously, dephosphorylated channels using PPT (Mg2+ absent) were blocked in the closed state by action of endogenous CalPK. This block was removed to normal activity by the action of either PPT or by exogenous CalPK II. Our findings are consistent with a physiological role for phosphorylation/dephosphorylation in the modulation of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. A corollary of our studies is that only the phosphorylated channel is active under physiological conditions (mM Mg2+). Our studies suggest that phosphorylation can be at more than one site and, depending on the site, can have different functional consequences on the CRC.
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the calcium release channel of sarcoplasmic reticulum is modulated by fk 506 binding protein effect of fkbp 12 on single channel activity of the skeletal muscle ryanodine receptor
Cell Calcium, 1994Co-Authors: M Mayrleitner, A P Timerman, Gregory Wiederrecht, Sidney FleischerAbstract:The calcium release channel/ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum is tightly associated with the immunophilin FK-506 binding protein (FKBP-12). The immunosuppressant drug FK-506 effectively dissociates FKBP-12 from the calcium release channel of Terminal Cisternae (TC) vesicles. Furthermore, calcium flux measurements of TC indicate that FKBP-12 stabilizes the closed conformation of the calcium release channel of TC [Timerman AP, Ogunbunmi E, Freund EA, Wiederrecht G, Marks AM, Fleischer S. (1993) J. Biol. Chem., 268, 22992-22999]. In this report, the effect of FKBP on single channel recordings of the calcium release channel/ryanodine receptor of TC is measured directly. Single channel recordings of the ryanodine receptor were obtained by fusion of TC vesicles into planar bilayers. The channel devoid of FKBP, retains key diagnostic features. That is, activation by Ca2+ and ryanodine, inhibition by Mg2+ (mM) and ruthenium red (microM), and its unitary conductance remain the same. Recordings of the calcium release channel obtained from the FKBP-deficient TC vesicles, as compared with control TC, have greater open probability and longer mean open times in a free calcium concentration range of 70 nM to 1.2 microM. The sensitivity of the channel to caffeine is also enhanced by the removal of FKBP. The enhanced channel activation of FKBP-deficient TC is reversed by rebinding recombinant FKBP-12 in a cyclical fashion. We conclude that FKBP modifies the channel behavior of the calcium release channel of skeletal muscle sarcoplasmic reticulum.
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the calcium release channel of sarcoplasmic reticulum is modulated by fk 506 binding protein dissociation and reconstitution of fkbp 12 to the calcium release channel of skeletal muscle sarcoplasmic reticulum
Journal of Biological Chemistry, 1993Co-Authors: A P Timerman, Gregory Wiederrecht, E Ogunbumni, E Freund, A R Marks, Sidney FleischerAbstract:The ryanodine receptor/calcium release channel (CRC) of rabbit skeletal muscle Terminal Cisternae (TC) of sarcoplasmic reticulum (SR) has been found to be tightly associated with FK-506 binding protein (FKBP-12), the cytosolic receptor (immunophilin) for the immunosuppressant drug FK-506 (Jayaraman, T., Brillantes, A. M., Timerman, A. P., Fleischer, S., Erdjument-Bromage, H., Tempst, P., and Marks, A. (1992) J. Biol. Chem. 267, 9474-9477). In this study, a procedure is described to dissociate FKBP from TC and reconstitute human recombinant FKBP-12 back to the ryanodine receptor so that the role of the immunophilin on CRC activity can be assessed. Titration of TC vesicles with FK-506 dissociates FKBP from the ryanodine receptor. Sedimentation of FK-506-treated vesicles effectively separates the TC from the soluble FKBP-FK506 complex which remains in the supernatant. The FKBP-deficient TC vesicles have altered functional characteristics: 1) the ATP-stimulated calcium uptake rate of TC vesicles is reduced 2-fold; and 2) the threshold concentration of caffeine required to induce calcium release from TC vesicles is decreased. These changes appear to reflect modification of the calcium release channel since: 1) severalfold higher concentrations of FK-506 do not alter the calcium uptake rate of either longitudinal tubules of SR, or TC vesicles in the presence of ruthenium red; 2) human recombinant FKBP reassociates with FKBP-deficient TC but not with control TC or longitudinal tubules of SR; and 3) the reduced Ca2+ uptake rate in FKBP-deficient TC is restored to control values in the FKBP-reconstituted TC. These studies demonstrate that FKBP-12 modulates the CRC of rabbit skeletal muscle sarcoplasmic reticulum.
A P Timerman - One of the best experts on this subject based on the ideXlab platform.
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characterization of an exchange reaction between soluble fkbp 12 and the fkbp ryanodine receptor complex modulation by fkbp mutants deficient in peptidyl prolyl isomerase activity
Journal of Biological Chemistry, 1995Co-Authors: A P Timerman, Gregory Wiederrecht, Alice I. Marcy, Sidney FleischerAbstract:Abstract FKBP-12 (FKBP), the soluble receptor for the immunosuppresant drug FK-506, is tightly bound to the calcium release channel (CRC)/ryanodine receptor (RyR) of skeletal muscle Terminal Cisternae (TC) of sarcoplasmic reticulum with a stoichiometry of 4 mol of FKBP per tetrameric RyR complex. FKBP displays cis/trans-peptidyl-prolyl isomerase (PPIase) activity which is inhibited by FK-590 or rapamycin. In skeletal muscle TC, FK-590 or rapamycin binds to and dissociates FKBP from the RyR in a time- and temperature-dependent manner which increases the open probability of the channel. Therefore, the net energized Ca2+ uptake rate of TC vesicles devoid of FKBP is reduced due to the increased leak of Ca2+ from the TC specifically via the RyR, which is reversed upon rebinding of FKBP. Thus, the RyR is modulated by FKBP (Timerman, A. P., Ogunbumni, E., Freund, E. A., Wiederrecht, G., Marks, A. R., and Fleischer, S.(1993) J. Biol. Chem. 268, 22922-22999; Mayrleitner, M., Timerman, A. P., Wiederrecht, G., and Fleischer S.(1994) Cell Calcium 15, 99-108). We now find that FKBP can be displaced from the FKBP•RyR complex by exchange with FKBP in solution. The EC50 for exchange is 0.30 μM for wild type FKBP versus 0.6 to 2.4 μM for three different site-directed mutants that are practically devoid of any measurable PPIase activity. Substitution of wild-type FKBP on the RyR complex with these PPIase-deficient mutants did not alter the Ca2+ flux of TC vesicles, whereas dissociation of FKBP from TC with FK-590 increased the Ca2+ leak rate. Our studies show that, in vivo, the FKBP•RyR complex is in equilibrium with the cytosolic pool of FKBP (∼3 μM) and suggest that modulation of the CRC by FKBP is independent of PPIase activity.
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the calcium release channel of sarcoplasmic reticulum is modulated by fk 506 binding protein effect of fkbp 12 on single channel activity of the skeletal muscle ryanodine receptor
Cell Calcium, 1994Co-Authors: M Mayrleitner, A P Timerman, Gregory Wiederrecht, Sidney FleischerAbstract:The calcium release channel/ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum is tightly associated with the immunophilin FK-506 binding protein (FKBP-12). The immunosuppressant drug FK-506 effectively dissociates FKBP-12 from the calcium release channel of Terminal Cisternae (TC) vesicles. Furthermore, calcium flux measurements of TC indicate that FKBP-12 stabilizes the closed conformation of the calcium release channel of TC [Timerman AP, Ogunbunmi E, Freund EA, Wiederrecht G, Marks AM, Fleischer S. (1993) J. Biol. Chem., 268, 22992-22999]. In this report, the effect of FKBP on single channel recordings of the calcium release channel/ryanodine receptor of TC is measured directly. Single channel recordings of the ryanodine receptor were obtained by fusion of TC vesicles into planar bilayers. The channel devoid of FKBP, retains key diagnostic features. That is, activation by Ca2+ and ryanodine, inhibition by Mg2+ (mM) and ruthenium red (microM), and its unitary conductance remain the same. Recordings of the calcium release channel obtained from the FKBP-deficient TC vesicles, as compared with control TC, have greater open probability and longer mean open times in a free calcium concentration range of 70 nM to 1.2 microM. The sensitivity of the channel to caffeine is also enhanced by the removal of FKBP. The enhanced channel activation of FKBP-deficient TC is reversed by rebinding recombinant FKBP-12 in a cyclical fashion. We conclude that FKBP modifies the channel behavior of the calcium release channel of skeletal muscle sarcoplasmic reticulum.
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the calcium release channel of sarcoplasmic reticulum is modulated by fk 506 binding protein dissociation and reconstitution of fkbp 12 to the calcium release channel of skeletal muscle sarcoplasmic reticulum
Journal of Biological Chemistry, 1993Co-Authors: A P Timerman, Gregory Wiederrecht, E Ogunbumni, E Freund, A R Marks, Sidney FleischerAbstract:The ryanodine receptor/calcium release channel (CRC) of rabbit skeletal muscle Terminal Cisternae (TC) of sarcoplasmic reticulum (SR) has been found to be tightly associated with FK-506 binding protein (FKBP-12), the cytosolic receptor (immunophilin) for the immunosuppressant drug FK-506 (Jayaraman, T., Brillantes, A. M., Timerman, A. P., Fleischer, S., Erdjument-Bromage, H., Tempst, P., and Marks, A. (1992) J. Biol. Chem. 267, 9474-9477). In this study, a procedure is described to dissociate FKBP from TC and reconstitute human recombinant FKBP-12 back to the ryanodine receptor so that the role of the immunophilin on CRC activity can be assessed. Titration of TC vesicles with FK-506 dissociates FKBP from the ryanodine receptor. Sedimentation of FK-506-treated vesicles effectively separates the TC from the soluble FKBP-FK506 complex which remains in the supernatant. The FKBP-deficient TC vesicles have altered functional characteristics: 1) the ATP-stimulated calcium uptake rate of TC vesicles is reduced 2-fold; and 2) the threshold concentration of caffeine required to induce calcium release from TC vesicles is decreased. These changes appear to reflect modification of the calcium release channel since: 1) severalfold higher concentrations of FK-506 do not alter the calcium uptake rate of either longitudinal tubules of SR, or TC vesicles in the presence of ruthenium red; 2) human recombinant FKBP reassociates with FKBP-deficient TC but not with control TC or longitudinal tubules of SR; and 3) the reduced Ca2+ uptake rate in FKBP-deficient TC is restored to control values in the FKBP-reconstituted TC. These studies demonstrate that FKBP-12 modulates the CRC of rabbit skeletal muscle sarcoplasmic reticulum.
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fk506 binding protein associated with the calcium release channel ryanodine receptor
Journal of Biological Chemistry, 1992Co-Authors: Thottala Jayaraman, Anne Marie B Brillantes, A P Timerman, Hediye Erdjumentbromage, Paul Tempst, Sidney Fleischer, Andrew R. MarksAbstract:Abstract The calcium release channel (CRC)/ryanodine receptor (RyRec) has been identified as the foot structure of the sarcoplasmic reticulum (SR) and provides the pathway for calcium efflux required for excitation-contraction coupling in skeletal muscle. The CRC has previously been reported to consist of four identical 565-kDa protomers. We now report the identification of a 12-kDa protein which is tightly associated with highly purified RyRec from rabbit skeletal muscle SR. N-Terminal amino acid sequencing and cDNA cloning demonstrates that the 12-kDa protein from fast twitch skeletal muscle is the binding protein for the immunosuppressant drug FK506. In humans, FK506 binds to the 12-kDa FK506-binding protein (FKBP12) and blocks calcium-dependent T cell activation. We find that FKBP12 and the RyRec are tightly associated in skeletal muscle SR on the basis of: 1) co-purification through sequential heparin-agarose, hydroxylapatite, and size exclusion chromatography columns; 2) coimmunoprecipitation of the RyRec and FKBP12 with anti-FKBP12 antibodies; and 3) subcellular localization of both proteins to the Terminal Cisternae of the SR, and not in the longitudinal tubules of SR, in fast twitch skeletal muscle. The molar ratio of FKBP12 to RyRec in highly purified RyRec preparations is approximately 1:4, indicating that one FKBP12 molecule is associated with each calcium release channel/foot structure.
Angela F. Dulhunty - One of the best experts on this subject based on the ideXlab platform.
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single channel activity of the ryanodine receptor calcium release channel is modulated by fk 506
FEBS Letters, 1994Co-Authors: Gerard P Ahern, Pauline R. Junankar, Angela F. DulhuntyAbstract:The immunosuppressant drug FK-506 (3-20 microM) increased the open probability of ryanodine receptor calcium release channels, formed by incorporation of Terminal Cisternae vesicles from rabbit skeletal muscle into lipid bilayers, with cis (cytoplasmic) calcium concentrations between 10(-7) M and 10(-3) M. FK-506 increased mean current and channel open time and induced long sojourns at subconductance levels that were between 28% and 38% of the maximum conductance and were distinct from the ryanodine-induced subconductance level at about 45% of the maximum conductance. FK-506 relieved the Ca2+ inactivation of the ryanodine receptor seen at 10(-3) M Ca2+. The results are consistent with FK-506 removal of FK-506 binding protein from the ryanodine receptor.
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Extra-Junctional Ryanodine Receptors in the Terminal Cisternae of Mammalian Skeletal Muscle Fibres
Proceedings of The Royal Society B: Biological Sciences, 1992Co-Authors: Angela F. Dulhunty, Pauline R. Junankar, Carolyn StanhopeAbstract:The distribution of ryanodine receptor calcium-release channels over the Terminal Cisternae (TC) membrane of skeletal muscle fibres was examined by using immunogold electron microscopy. Two monoclonal antibodies (5C3 and 8E2) that bound to monomers of the ryanodine receptor protein on Western blots of SDS-polyacrylamide gels were used to locate calcium-release channels in longitudinal sections of rat sternomastoid and diaphragm fibres. Up to 21% of 5C3 binding on TC membranes was extra-junctional, compared with 46% for 8E2. Binding of 8E2 to the fibres was less than half that of 5C3, possibly because of steric shielding of the 8E2 antigenic site at the junction. The distances between neighbouring particles in clusters was 20-40 nm, i.e. the distance between subunits of the ryanodine receptor or between neighbouring foot structures. We suggest that, during activation, extra-junctional ryanodine receptors may release Ca$^{2+}$ directly into the myoplasm, rather than into the restricted space of the triad junction.
Alfredo Margreth - One of the best experts on this subject based on the ideXlab platform.
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functional behaviour of the ryanodine receptor ca2 release channel in vesiculated derivatives of the junctional membrane of Terminal Cisternae of rabbit fast muscle sarcoplasmic reticulum
Cell Calcium, 1997Co-Authors: Ernesto Damiani, Gianantonio Tobaldin, Elena Bortoloso, Alfredo MargrethAbstract:Abstract We have devised a novel procedure, employing Chaps rather than Triton [Costello B., Chadwick C., Saito A., Chu A., Maurer A., Fleischer S. J Cell Biol 1986; 103 : 741–753], for obtaining vesiculated derivatives of the junctional face membrane (JFM) domain of isolated Terminal Cisternae (TC) from fast skeletal muscle of the rabbit. Enriched JFM is minimally contaminated with junctional transverse tubules. The characteristic ultrastructural features and the most essential features of TC function relating to this membrane domain — i.e. both the Ca 2+ -release system and the Ca 2+ and calmodulin (CaM)-dependent protein kinase (CaM I PK) system — appear to be retained in enriched JFM. We show that our isolation procedure, yielding up to a 2.5-fold enrichment in ryanodine receptor (RyR) protein and in the maximum number of high affinity [ 3 H]-ryanodine binding sites, does not alter the assembly for integral proteins associated with the receptor in its native membrane environment, i.e. FKBP-12, triadin and the structurally related protein junctin [Jones L.R., Zhang L., Sanborn K., Jorgensen A., Kelley J. J Biol Chem 1995; 270 : 30787–30796] having, in common, the property to bind calsequestrin (CS) in overlays in the presence of EGTA. The substrate specificity of endogenous CaM I PK is also the same as that of parent TC vesicles. Phosphorylation of mainly triadin and of a high M r polypeptide, and not of the RyR, is the most remarkable common property. Retention of peripheral proteins, like CS and histidine-rich Ca 2+ -binding protein, although not that of endogenous CaM, and of a unique set of CaM-binding proteins, unlike that of junctional SR-specific integral proteins, is shown to be influenced by the concentration of Ca 2+ during incubation of TC vesicles with Chaps. Characterization of RyR functional behaviour with [ 3 H]-ryanodine has indicated extensive similarities between the enriched JFM and parent TC vesicles, as far as the characteristic bell shaped Ca 2+ -dependence of [ 3 H]-ryanodine binding and the dose-dependent sensitization to Ca 2+ by caffeine, reflecting the inherent properties of SR Ca 2+ -release channel, as well as concerning the stimulation of [ 3 H]-ryanodine binding by increasing concentrations of KCI. Stabilizing the RyR in a maximally active state by optimizing concentrations of KCI (1 M), at also optimal concentrations of Ca 2+ (pCa 4), rendered the receptor less sensitive to inhibition by 1 μM CaM, to a greater extent in the case of enriched JFM. That was not accounted for by any significant difference in the IC 50 concentrations of CaM, varying between 40 nM to approximately 80 nM, at low-intermediate and at high KCI concentrations, respectively. Additional results with enriched JFM using doxorubicin, a pharmacological Ca 2+ channel allosteric modifier, strengthen the hypothesis that the conformational state at which RyR is stabilized, according to the experimental assay conditions for [ 3 H]-ryanodine binding, directly influences CaM-sensitivity.
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identification of triadin and of histidine rich ca2 binding protein as substrates of 60 kda calmodulin dependent protein kinase in junctional Terminal Cisternae of sarcoplasmic reticulum of rabbit fast muscle
Biochemical and Biophysical Research Communications, 1995Co-Authors: Ernesto Damiani, Enzo Picello, Leopoldo Saggin, Alfredo MargrethAbstract:Abstract The endogenous calmodulin-protein kinase system of sarcoplasmic reticulum Terminal Cisternae of rabbit fast-twitch muscle was studied. Investigation of a single Ca 2+ -channel in Terminal Cisternae fused to planar lipid bilayers demonstrated that the endogenous kinase inhibits the channel, although it remained unclear whether the phosphorylation sites are on the channel protein or on other junctional sarcoplasmic reticulum specific proteins [Hain et al., (1994) Biophys. J. 67 , 1823-1833]. Our results, which show that two junctional sarcoplasmic reticulum specific proteins,i.e., triadin and histidine-rich, Ca 2+ -binding protein, but not the ryanodine receptor/Ca 2+ -channel protein, are phosphorylated by membrane-bound 60 kDa protein kinase, seem to be able to resolve this ambiguity. Furthermore,such aprobably specific protein isoform of calmodulin-protein kinase, by its substrate specificity and exposure to the cytoplasmic side of Terminal Cisternae at the junctional membrane domain and based on protease sensitivity, also seems to possess some of the potential requirements for a regulatory role in the functional state of the Ca 2+ -channel.
M Mayrleitner - One of the best experts on this subject based on the ideXlab platform.
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phosphorylation with protein kinases modulates calcium loading of Terminal Cisternae of sarcoplasmic reticulum from skeletal muscle
Cell Calcium, 1995Co-Authors: M Mayrleitner, Hansgeorg Schindler, R Chandler, Sidney FleischerAbstract:Abstract We previously found in single channel studies that ryanodine receptor (RyR) channel activity can be made insensitive to block by Mg 2+ when Terminal Cisternae of sarcoplasmic reticulum, incorporated into planar bilayers, are treated with protein kinase A (PKA) or Ca 2+ /calmodulin dependent protein kinase type II (CamPK II), and then again made sensitive by treatment with protein phosphatases [Hain J. Nath S. Mayrleitner M. Fleischer S. Schindler H. (1994) Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. Biophys. J., 67, 1823–1833]. In this study, modulation by protein kinases and phosphatases on net Ca 2+ uptake by TC is presented. Phosphorylation of TC vesicles with PKA, CamPK II, or protein kinase C (PKC) reduced the calcium loading rate of TC vesicles 3-fold, 2.1-fold and 1.7-fold, respectively, measured in the presence of 1 MM MgCl 2 . There is no effect when AMP-PNP is substituted for ATP. Phosphorylation of the RyR was also measured by incororation of [γ- 32 P]-phosphate from ATP. A phosphorylation stoichiometry of 1.94 ± 0.1 ( 32 P/RyR) for PKA, 0.89 ± 0.08 for CamPK II and 0.95 ± 0.16 for PKC was obtained under these conditions. A study of the time dependence of phosphorylation with PKA and CamPK shows a direct correlation of reduction in calcium loading rate with increased phosphorylation of the ryanodine receptor. Treatment with protein phosphatase 1 enhanced the calcium loading rate again, after it was reduced by PKA phosphorylation. Investigation of the magnesium dependency shows that even at higher [Mg 2+ ] (6 mM), PKA phosphorylated TC vesicles have a 2.3-fold reduced calcium loading rate indicating insensitivity to block by Mg 2+ . Protein kinases have no effect on Ca 2+ -ATPase activity in TC or on Ca 2+ loading in longitudinal tubules which are devoid of RyR. We interpret the decreased net Ca 2+ uptake as being due to enhanced Ca 2+ leakage via the RyR. These studies show, at a macroscopic level, that the calcium release channel is modulated by kinases/phosphatases and support our previous findings with single channel studies that the ryanodine receptor must be phosphorylated to be active under physiological Mg 2+ concentrations.
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phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle
Biophysical Journal, 1994Co-Authors: J Hain, Sidney Fleischer, M Mayrleitner, Sati Nath, Hansgeorg SchindlerAbstract:The modulation of the calcium release channel (CRC) by protein kinases and phosphatases was studied. For this purpose, we have developed a microsyringe applicator to achieve sequential and multiple treatments with highly purified kinases and phosphatases applied directly at the bilayer surface. Terminal Cisternae vesicles of sarcoplasmic reticulum from rabbit fast twitch skeletal muscle were fused to planar lipid bilayers, and single-channel currents were measured at zero holding potential, at 0.15 microM free Ca2+, +/- 0.5 mM ATP and +/- 2.6 mM free Mg2+. Sequential dephosphorylation and rephosphorylation rendered the CRC sensitive and insensitive to block by Mg2+, respectively. Channel recovery from Mg2+ block was obtained by exogenous protein kinase A (PKA) or by Ca2+/calmodulin-dependent protein kinase II (CalPK II). Somewhat different characteristics were observed with the two kinases, suggesting two different states of phosphorylation. Channel block by Mg2+ was restored by dephosphorylation using protein phosphatase 1 (PPT1). Before application of protein kinases or phosphatases, channels were found to be "dephosphorylated" (inactive) in 60% and "phosphorylated" (active) in 40% of 51 single-channel experiments based on the criterion of sensitivity to block by Mg2+. Thus, these two states were interconvertable by treatment with exogenously added protein kinases and phosphatases. Endogenous Ca2+/calmodulin-dependent protein kinase (end CalPK) had an opposite action to exogenous CalPK II. Previously, dephosphorylated channels using PPT (Mg2+ absent) were blocked in the closed state by action of endogenous CalPK. This block was removed to normal activity by the action of either PPT or by exogenous CalPK II. Our findings are consistent with a physiological role for phosphorylation/dephosphorylation in the modulation of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. A corollary of our studies is that only the phosphorylated channel is active under physiological conditions (mM Mg2+). Our studies suggest that phosphorylation can be at more than one site and, depending on the site, can have different functional consequences on the CRC.
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the calcium release channel of sarcoplasmic reticulum is modulated by fk 506 binding protein effect of fkbp 12 on single channel activity of the skeletal muscle ryanodine receptor
Cell Calcium, 1994Co-Authors: M Mayrleitner, A P Timerman, Gregory Wiederrecht, Sidney FleischerAbstract:The calcium release channel/ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum is tightly associated with the immunophilin FK-506 binding protein (FKBP-12). The immunosuppressant drug FK-506 effectively dissociates FKBP-12 from the calcium release channel of Terminal Cisternae (TC) vesicles. Furthermore, calcium flux measurements of TC indicate that FKBP-12 stabilizes the closed conformation of the calcium release channel of TC [Timerman AP, Ogunbunmi E, Freund EA, Wiederrecht G, Marks AM, Fleischer S. (1993) J. Biol. Chem., 268, 22992-22999]. In this report, the effect of FKBP on single channel recordings of the calcium release channel/ryanodine receptor of TC is measured directly. Single channel recordings of the ryanodine receptor were obtained by fusion of TC vesicles into planar bilayers. The channel devoid of FKBP, retains key diagnostic features. That is, activation by Ca2+ and ryanodine, inhibition by Mg2+ (mM) and ruthenium red (microM), and its unitary conductance remain the same. Recordings of the calcium release channel obtained from the FKBP-deficient TC vesicles, as compared with control TC, have greater open probability and longer mean open times in a free calcium concentration range of 70 nM to 1.2 microM. The sensitivity of the channel to caffeine is also enhanced by the removal of FKBP. The enhanced channel activation of FKBP-deficient TC is reversed by rebinding recombinant FKBP-12 in a cyclical fashion. We conclude that FKBP modifies the channel behavior of the calcium release channel of skeletal muscle sarcoplasmic reticulum.
