A simple, fast, one-step method for the purification of the skeletal-muscle ryanodine receptor

In this paper we describe a simple, fast, one-step method for the purification of the skeletal-muscle ryanodine receptor. The ryanodine receptor from CHAPS-solubilized junctional sarcoplasmic-reticulum membranes was adsorbed to a spermine-agarose column and eluted by 2 mM-spermine. The purified receptor, consisting predominantly of a 450 kDa polypeptide on SDS/PAGE, binds [3H]ryanodine with a specific activity of approximately 300 pmol/mg of protein and with a high affinity (KD = 4.7 +/- 2 nM). The purified receptor appears to retain the pharmacological properties of the receptor in the original membranes. The purification resulted in over 80% recovery of the initial ryanodine-binding sites and about 30-96-fold purification. This simple and fast method is highly reproducible and suitable for purification of small as well as large quantities of ryanodine receptor.

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Structural and functional interactions between the Ca2+, ATP, and caffeine binding sites of skeletal muscle ryanodine receptor (RyR1)

Journal of Biological Chemistry ◽

10.1016/j.jbc.2021.101040 ◽

2021 ◽

pp. 101040

Author(s):

Venkat R. Chirasani ◽

Daniel A. Pasek ◽

Gerhard Meissner

Keyword(s):

Skeletal Muscle ◽

Ryanodine Receptor ◽

Binding Sites ◽

Functional Interactions

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Relationship of low affinity [3]ryanodine binding sites to high affinity sites on the skeletal muscle Ca2+ release channel.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)36881-4 ◽

1993 ◽

Vol 268 (28) ◽

pp. 20974-20982

Author(s):

J.P. Wang ◽

D.H. Needleman ◽

S.L. Hamilton

Keyword(s):

Skeletal Muscle ◽

Binding Sites ◽

Release Channel ◽

High Affinity ◽

Relationship Of

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Caffeine sensitivity of native RyR channels from normal and malignant hyperthermic pigs: effects of a DHPR II–III loop peptide

AJP Cell Physiology ◽

10.1152/ajpcell.00311.2003 ◽

2004 ◽

Vol 286 (4) ◽

pp. C821-C830 ◽

Cited By ~ 9

Author(s):

Esther M. Gallant ◽

James Hart ◽

Kevin Eager ◽

Suzanne Curtis ◽

Angela F. Dulhunty

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Malignant Hyperthermia ◽

Lipid Bilayers ◽

Ryanodine Receptor ◽

High Affinity ◽

Enhanced Sensitivity ◽

Skeletal Muscle Contraction ◽

Activation Mechanisms ◽

Standard Tests

Enhanced sensitivity to caffeine is part of the standard tests for susceptibility to malignant hyperthermia (MH) in humans and pigs. The caffeine sensitivity of skeletal muscle contraction and Ca2+ release from the sarcoplasmic reticulum is enhanced, but surprisingly, the caffeine sensitivity of purified porcine ryanodine receptor Ca2+-release channels (RyRs) is not affected by the MH mutation (Arg615Cys). In contrast, we show here that native malignant hyperthermic pig RyRs (incorporated into lipid bilayers with RyR-associated lipids and proteins) were activated by caffeine at 100- to 1,000-fold lower concentrations than native normal pig RyRs. In addition, the results show that the mutant ryanodine receptor channels were less sensitive to high-affinity activation by a peptide (CS) that corresponds to a part of the II–III loop of the skeletal dihydropyridine receptor (DHPR). Furthermore, subactivating concentrations of peptide CS enhanced the response of normal pig and rabbit RyRs to caffeine. In contrast, the caffeine sensitivity of MH RyRs was not enhanced by the peptide. These novel results showed that in MH-susceptible pig muscles 1) the caffeine sensitivity of native RyRs was enhanced, 2) the sensitivity of RyRs to a skeletal II–III loop peptide was depressed, and 3) an interaction between the caffeine and peptide CS activation mechanisms seen in normal RyRs was lost.

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Suramin and the suramin analogue NF307 discriminate among calmodulin-binding sites

Biochemical Journal ◽

10.1042/bj3550827 ◽

2001 ◽

Vol 355 (3) ◽

pp. 827-833 ◽

Cited By ~ 15

Author(s):

Markus KLINGER ◽

Elisa BOFILL-CARDONA ◽

Bernd MAYER ◽

Christian NANOFF ◽

Michael FREISSMUTH ◽

...

Keyword(s):

Skeletal Muscle ◽

Ryanodine Receptor ◽

Binding Sites ◽

Metabotropic Glutamate Receptor ◽

Target Proteins ◽

Porcine Brain ◽

Metabotropic Glutamate ◽

Calmodulin Binding ◽

Brain Membranes ◽

Bacterial Lysates

Calmodulin-binding sites on target proteins show considerable variation in primary sequence; hence compounds that block the access of calmodulin to these binding sites may be more selective than compounds that inactivate calmodulin. Suramin and its analogue NF307 inhibit the interaction of calmodulin with the ryanodine receptor. We have investigated whether inhibition of calmodulin binding to target proteins is a general property of these compounds. Suramin inhibited binding of [125I]calmodulin to porcine brain membranes and to sarcoplasmic reticulum from skeletal muscle (IC50 = 4.9±1.2µM and 19.9±1.8µM, respectively) and blocked the cross-linking of [125I]calmodulin to some, but not all, target proteins in brain membranes by [125I]calmodulin. Four calmodulin-binding proteins were purified [ryanodine receptor-1 (RyR1) from rabbit skeletal muscle, neuronal NO synthase (nNOS) from Sf9 cells, G-protein βγ dimers (Gβγ) from porcine brain and a glutathione S-transferase-fusion protein comprising the C-terminal calmodulin-binding domain of the metabotropic glutamate receptor 7A (GST-CmGluR7A) from bacterial lysates]. Three of the proteins employed (Gβγ, GST-CmGluR7A and RyR1) display a comparable affinity for calmodulin (in the range of 50–70nM). Nevertheless, suramin and NF307 only blocked the binding of Gβγ and RyR1 to calmodulin–Sepharose. In contrast, the association of GST-CmGluR7A and nNOS was not impaired, whereas excess calmodulin uniformly displaced all proteins from the matrix. Thus suramin and NF307 are prototypes of a new class of calmodulin antagonists that do not interact directly with calmodulin but with calmodulin-recognition sites. In addition, these compounds discriminate among calmodulin-binding motifs.

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Structural Determinants of High-Affinity Binding of Ryanoids to the Vertebrate Skeletal Muscle Ryanodine Receptor: A Comparative Molecular Field Analysis

Biochemistry ◽

10.1021/bi00186a006 ◽

1994 ◽

Vol 33 (20) ◽

pp. 6074-6085 ◽

Cited By ~ 34

Author(s):

William Welch ◽

Syed Ahmad ◽

Judith A. Airey ◽

Koert Gerzon ◽

Rod A. Humerickhouse ◽

...

Keyword(s):

Skeletal Muscle ◽

Ryanodine Receptor ◽

Field Analysis ◽

Molecular Field ◽

Affinity Binding ◽

Comparative Molecular Field Analysis ◽

Structural Determinants ◽

High Affinity Binding ◽

High Affinity ◽

Vertebrate Skeletal Muscle

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Fragment D Immunoreacivity: The Influence of Iodination and Calcium Environment

10.1055/s-0039-1684551 ◽

1979 ◽

Author(s):

B. Kudryk ◽

M. Blombäck

Keyword(s):

Conformational Changes ◽

Binding Sites ◽

Specific Activity ◽

High Specific Activity ◽

Antigenic Determinants ◽

Affinity Binding ◽

Compact Structure ◽

High Affinity Binding ◽

High Affinity ◽

Chloramine T

Human fragment D (Fg-Ds) has heen iodinated using both the Chloramine-T and lactoperoildaae methods. The specific activity was similar regardless of the method used. However, binding to a specific antibody was different for each preparation. The antigen labeled by the Chloramine-T method bound to a maximum of 40% the other labeled product bound up to 85%. A correlation between the decree of immunoreactivity and avidity for a fihrinmcnomer conjugate vas found also. Fibrinmonomer bound about twice the ajnount of lactoperoxidase iodinated Fg-Da ae it did the Chloramine-T product. The use of these conjugates in the purification of immunoreactive Fg-Ds of high specific activity will be discussed. High affinity binding sites for calcium have recently been demonstrated in fibrinogen. Tha presence of bound calcium is also believed to protect Fg-Ds f m further digestion by plasmin. This is probably due to the formation of a more compact structure. However, conformational changes for calcium bound fibrinogen or Fg-Ds have not been observed. We tested the immunoreactivity of the lactoperoxidase iodinated Fg-Ds in presence and absence of calcium. Differences were found and this data suggests that soma modification of antigenic determinants takes place as a consequence of calcium in the environment.

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A kinetic model for Ca2+ efflux mediated by the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum

Biochemical Journal ◽

10.1042/bj2450713 ◽

1987 ◽

Vol 245 (3) ◽

pp. 713-721 ◽

Cited By ~ 12

Author(s):

J M McWhirter ◽

G W Gould ◽

J M East ◽

A G Lee

Keyword(s):

Experimental Data ◽

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Kinetic Model ◽

Atpase Activity ◽

Binding Site ◽

Binding Sites ◽

Adenine Nucleotides ◽

Contraction Coupling ◽

High Affinity

We present a model for Ca2+ efflux from vesicles of sarcoplasmic reticulum (SR). It is proposed that efflux is mediated by the Ca2+ + Mg2+-activated ATPase that is responsible for Ca2+ uptake in this system. In the normal ATPase cycle of the ATPase, phosphorylation of the ATPase is followed by a conformational change in which the Ca2+-binding sites change from being outward-facing and of high affinity to being inward-facing and of low affinity. To mediate Ca2+ efflux, it is proposed that the ATPase can adopt a conformation in which the Ca2+-binding sites are of low affinity but still outward-facing. It is shown that experimental data on the rates of Ca2+ efflux can be simulated in terms of this model, with Ca2+-binding-site affinities previously proposed to explain ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227]. Effects of Mg2+ and adenine nucleotides on efflux rates are explained. It is suggested that Ca2+ efflux from SR mediated by the ATPase could be important in excitation-contraction coupling in skeletal muscle.

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Functional and Structural Interactions between Ca2+, ATP and Caffeine Binding Sites of Skeletal Muscle Ryanodine Receptor (RyR1)

Biophysical Journal ◽

10.1016/j.bpj.2020.11.1783 ◽

2021 ◽

Vol 120 (3) ◽

pp. 280a

Author(s):

Venkat R. Chirasani ◽

Daniel A. Pasek ◽

Hannah G. Addis ◽

Naohiro Yamaguchi ◽

Gerhard Meissner

Keyword(s):

Skeletal Muscle ◽

Ryanodine Receptor ◽

Binding Sites ◽

Structural Interactions

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Skeletal muscle ouabain binding sites are reduced in rats with chronic heart failure

Journal of Applied Physiology ◽

10.1152/japplphysiol.00686.2001 ◽

2002 ◽

Vol 92 (6) ◽

pp. 2326-2334 ◽

Cited By ~ 15

Author(s):

Timothy I. Musch ◽

Swen Wolfram ◽

K. Sue Hageman ◽

Joel G. Pickar

Keyword(s):

Heart Failure ◽

Skeletal Muscle ◽

Chronic Heart Failure ◽

Oxygen Uptake ◽

Binding Sites ◽

Ouabain Binding ◽

High Affinity ◽

Lv Dysfunction ◽

Run Time ◽

Fast Twitch

Intrinsic skeletal muscle abnormalities decrease muscular endurance in chronic heart failure (CHF). In CHF patients, the number of skeletal muscle Na+-K+ pumps that have a high affinity for ouabain (i.e., the concentration of [3H]ouabain binding sites) is reduced, and this reduction is correlated with peak oxygen uptake. The present investigation determined whether the concentration of skeletal muscle [3H]ouabain binding sites found during CHF is related to 1) severity of the disease state, 2) muscle fiber type composition, and/or 3) endurance capacity. Four muscles were chosen that represented slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), fast-twitch glycolytic (FG), and mixed fiber types. Measurements were obtained 8–10 wk postsurgery in 23 myocardial infarcted (MI) and 18 sham-operated control (sham) rats. Eighteen rats had moderate left ventricular (LV) dysfunction [LV end-diastolic pressure (LVEDP) < 20 mmHg], and five had severe LV dysfunction (LVEDP > 20 mmHg). Rats with severe LV dysfunction had significant pulmonary congestion and were likely in a chronic state of compensated congestive failure as indicated by an approximately twofold increase in both lung and right ventricle weight. Run time to fatigue and maximal oxygen uptake (V˙o 2 max) were significantly reduced (↓39 and ↓28%, respectively) in the rats with severe LV dysfunction and correlated with the magnitude of LV dysfunction as indicated by LVEDP (run time: r = 0.60, n = 21, P < 0.01 and V˙o 2 max: r = 0.93, n = 13, P < 0.01). In addition, run time to fatigue was significantly correlated withV˙o 2 max ( r = 0.87, n = 15, P < 0.01). The concentration of [3H]ouabain binding sites (Bmax) was significantly reduced (21–28%) in the three muscles comprised primarily of oxidative fibers [soleus: 259 ± 14 vs. 188 ± 17; plantaris: 295 ± 17 vs. 229 ± 18; red portion of gastrocnemius: 326 ± 17 vs. 260 ± 14 pmol/g wet tissue wt]. In addition, Bmax was significantly correlated withV˙o 2 max (soleus: r = 0.54, n = 15, P < 0.05; plantaris: r = 0.59, n = 15, P < 0.05; red portion of gastrocnemius: r = 0.65, n = 15, P < 0.01). These results suggest that downregulation of Na+-K+ pumps that possess a high affinity for ouabain in oxidative skeletal muscle may play an important role in the exercise intolerance that attends severe LV dysfunction in CHF.

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