Calsequestrin is an inhibitor of skeletal muscle ryanodine receptor calcium release channels

Nicole A Beard, Magdalena M Sakowska, Angela F. Dulhunty, Derek R Laver

    Research output: Contribution to journalArticle

    112 Citations (Scopus)

    Abstract

    We provide novel evidence that the sarcoplasmic reticulum calcium binding protein, calsequestrin, inhibits native ryanodine receptor calcium release channel activity. Calsequestrin dissociation from junctional face membrane was achieved by increasing luminal (trans) ionic strength from 250 to 500 mM with CsCl or by exposing the luminal side of ryanodine receptors to high [Ca(2+)] (13 mM) and dissociation was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Calsequestrin dissociation caused a 10-fold increase in the duration of ryanodine receptor channel opening in lipid bilayers. Adding calsequestrin back to the luminal side of the channel after dissociation reversed this increased activity. In addition, an anticalsequestrin antibody added to the luminal solution reduced ryanodine receptor activity before, but not after, calsequestrin dissociation. A population of ryanodine receptors (approximately 35%) may have initially lacked calsequestrin, because their activity was high and was unaffected by increasing ionic strength or by anticalsequestrin antibody: their activity fell when purified calsequestrin was added and they then responded to antibody. In contrast to native ryanodine receptors, purified channels, depleted of triadin and calsequestrin, were not inhibited by calsequestrin. We suggest that calsequestrin reduces ryanodine receptor activity by binding to a coprotein, possibly to the luminal domain of triadin.

    Original languageEnglish
    Pages (from-to)310-320
    Number of pages11
    JournalBiophysical Journal
    Volume82
    Issue number1 Pt 1
    DOIs
    Publication statusPublished - Jan 2002

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    Calsequestrin
    Ryanodine Receptor Calcium Release Channel
    Skeletal Muscle
    Osmolar Concentration
    Antibodies
    Calcium-Binding Proteins
    Lipid Bilayers
    Sarcoplasmic Reticulum
    Sodium Dodecyl Sulfate
    Polyacrylamide Gel Electrophoresis
    Western Blotting

    Cite this

    Beard, Nicole A ; Sakowska, Magdalena M ; Dulhunty, Angela F. ; Laver, Derek R. / Calsequestrin is an inhibitor of skeletal muscle ryanodine receptor calcium release channels. In: Biophysical Journal. 2002 ; Vol. 82, No. 1 Pt 1. pp. 310-320.
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    abstract = "We provide novel evidence that the sarcoplasmic reticulum calcium binding protein, calsequestrin, inhibits native ryanodine receptor calcium release channel activity. Calsequestrin dissociation from junctional face membrane was achieved by increasing luminal (trans) ionic strength from 250 to 500 mM with CsCl or by exposing the luminal side of ryanodine receptors to high [Ca(2+)] (13 mM) and dissociation was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Calsequestrin dissociation caused a 10-fold increase in the duration of ryanodine receptor channel opening in lipid bilayers. Adding calsequestrin back to the luminal side of the channel after dissociation reversed this increased activity. In addition, an anticalsequestrin antibody added to the luminal solution reduced ryanodine receptor activity before, but not after, calsequestrin dissociation. A population of ryanodine receptors (approximately 35{\%}) may have initially lacked calsequestrin, because their activity was high and was unaffected by increasing ionic strength or by anticalsequestrin antibody: their activity fell when purified calsequestrin was added and they then responded to antibody. In contrast to native ryanodine receptors, purified channels, depleted of triadin and calsequestrin, were not inhibited by calsequestrin. We suggest that calsequestrin reduces ryanodine receptor activity by binding to a coprotein, possibly to the luminal domain of triadin.",
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    Calsequestrin is an inhibitor of skeletal muscle ryanodine receptor calcium release channels. / Beard, Nicole A; Sakowska, Magdalena M; Dulhunty, Angela F.; Laver, Derek R.

    In: Biophysical Journal, Vol. 82, No. 1 Pt 1, 01.2002, p. 310-320.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Calsequestrin is an inhibitor of skeletal muscle ryanodine receptor calcium release channels

    AU - Beard, Nicole A

    AU - Sakowska, Magdalena M

    AU - Dulhunty, Angela F.

    AU - Laver, Derek R

    PY - 2002/1

    Y1 - 2002/1

    N2 - We provide novel evidence that the sarcoplasmic reticulum calcium binding protein, calsequestrin, inhibits native ryanodine receptor calcium release channel activity. Calsequestrin dissociation from junctional face membrane was achieved by increasing luminal (trans) ionic strength from 250 to 500 mM with CsCl or by exposing the luminal side of ryanodine receptors to high [Ca(2+)] (13 mM) and dissociation was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Calsequestrin dissociation caused a 10-fold increase in the duration of ryanodine receptor channel opening in lipid bilayers. Adding calsequestrin back to the luminal side of the channel after dissociation reversed this increased activity. In addition, an anticalsequestrin antibody added to the luminal solution reduced ryanodine receptor activity before, but not after, calsequestrin dissociation. A population of ryanodine receptors (approximately 35%) may have initially lacked calsequestrin, because their activity was high and was unaffected by increasing ionic strength or by anticalsequestrin antibody: their activity fell when purified calsequestrin was added and they then responded to antibody. In contrast to native ryanodine receptors, purified channels, depleted of triadin and calsequestrin, were not inhibited by calsequestrin. We suggest that calsequestrin reduces ryanodine receptor activity by binding to a coprotein, possibly to the luminal domain of triadin.

    AB - We provide novel evidence that the sarcoplasmic reticulum calcium binding protein, calsequestrin, inhibits native ryanodine receptor calcium release channel activity. Calsequestrin dissociation from junctional face membrane was achieved by increasing luminal (trans) ionic strength from 250 to 500 mM with CsCl or by exposing the luminal side of ryanodine receptors to high [Ca(2+)] (13 mM) and dissociation was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Calsequestrin dissociation caused a 10-fold increase in the duration of ryanodine receptor channel opening in lipid bilayers. Adding calsequestrin back to the luminal side of the channel after dissociation reversed this increased activity. In addition, an anticalsequestrin antibody added to the luminal solution reduced ryanodine receptor activity before, but not after, calsequestrin dissociation. A population of ryanodine receptors (approximately 35%) may have initially lacked calsequestrin, because their activity was high and was unaffected by increasing ionic strength or by anticalsequestrin antibody: their activity fell when purified calsequestrin was added and they then responded to antibody. In contrast to native ryanodine receptors, purified channels, depleted of triadin and calsequestrin, were not inhibited by calsequestrin. We suggest that calsequestrin reduces ryanodine receptor activity by binding to a coprotein, possibly to the luminal domain of triadin.

    KW - Antibodies

    KW - Calcium Chloride

    KW - Calcium-Transporting ATPases

    KW - Calsequestrin

    KW - Egtazic Acid

    KW - Lipid Bilayers

    KW - Muscle, Skeletal

    KW - Phosphatidylethanolamines

    KW - Rabbits

    KW - Ryanodine Receptor Calcium Release Channel

    KW - Sarcoplasmic Reticulum

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    DO - 10.1016/S0006-3495(02)75396-4

    M3 - Article

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    SP - 310

    EP - 320

    JO - Biophysical Journal

    JF - Biophysical Journal

    SN - 0006-3495

    IS - 1 Pt 1

    ER -