B-adrenergic stimulation increases RyR activity via intracellular Ca2+ and Mg2+ regulation

Jiao Li, Mohammad Imtiaz, Nicole BEARD, Angela F. Dulhunty, Rick Thorne, Dirk vanHelden

Research output: Contribution to journalArticle

20 Citations (Scopus)
2 Downloads (Pure)

Abstract

Here we investigate how beta-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca2+ and Mg2+ and the role of these changes in SR Ca2+ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 mu M isoproterenol or without (control) and snap frozen in liquid N-2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal beta-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of beta-adrenergic stimulation consistent with other reports that beta-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca2+],1 mM, beta-adrenergic stimulation increased luminal Ca2+ activation of single RyR channels, decreased luminal Mg2+ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg2+. At cytoplasmic [Ca2+] >1 mu M, beta-adrenergic stimulation only decreased cytoplasmic Mg2+ and Ca2+ inhibition of RyRs. The K-a and maximum levels of cytoplasmic Ca2+ activation site were not affected by beta-adrenergic stimulation. Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, beta-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca2+ binding to the luminal Ca2+ site and decreasing its affinity for luminal Mg2+ and 2) decreasing affinity of the low-affinity Ca2+/Mg2+ cytoplasmic inhibition site. However in systole, beta-adrenergic stimulation is mediated mainly by the latter
Original languageEnglish
Pages (from-to)1-14
Number of pages14
JournalPLoS One
Volume8
Issue number3
DOIs
Publication statusPublished - 2013

Fingerprint

Ryanodine Receptor Calcium Release Channel
Adrenergic Agents
ryanodine receptors
calcium
Phosphorylation
phosphorylation
heart
Isoproterenol
Chemical activation
Diastole
Systole
Rats
Perfusion
Western Blotting
Western blotting
Liquids
liquids
rats

Cite this

Li, Jiao ; Imtiaz, Mohammad ; BEARD, Nicole ; Dulhunty, Angela F. ; Thorne, Rick ; vanHelden, Dirk. / B-adrenergic stimulation increases RyR activity via intracellular Ca2+ and Mg2+ regulation. In: PLoS One. 2013 ; Vol. 8, No. 3. pp. 1-14.
@article{5b05899403714a4fbe298523a465fae7,
title = "B-adrenergic stimulation increases RyR activity via intracellular Ca2+ and Mg2+ regulation",
abstract = "Here we investigate how beta-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca2+ and Mg2+ and the role of these changes in SR Ca2+ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 mu M isoproterenol or without (control) and snap frozen in liquid N-2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal beta-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69{\%} and 15{\%}, respectively. These levels were increased to 83{\%} and 60{\%}, respectively, after 60 s of beta-adrenergic stimulation consistent with other reports that beta-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca2+],1 mM, beta-adrenergic stimulation increased luminal Ca2+ activation of single RyR channels, decreased luminal Mg2+ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg2+. At cytoplasmic [Ca2+] >1 mu M, beta-adrenergic stimulation only decreased cytoplasmic Mg2+ and Ca2+ inhibition of RyRs. The K-a and maximum levels of cytoplasmic Ca2+ activation site were not affected by beta-adrenergic stimulation. Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, beta-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca2+ binding to the luminal Ca2+ site and decreasing its affinity for luminal Mg2+ and 2) decreasing affinity of the low-affinity Ca2+/Mg2+ cytoplasmic inhibition site. However in systole, beta-adrenergic stimulation is mediated mainly by the latter",
keywords = "cardiac muscle, calcium release, ryanodine receptors",
author = "Jiao Li and Mohammad Imtiaz and Nicole BEARD and Dulhunty, {Angela F.} and Rick Thorne and Dirk vanHelden",
year = "2013",
doi = "10.1371/journal.pone.0058334",
language = "English",
volume = "8",
pages = "1--14",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "3",

}

B-adrenergic stimulation increases RyR activity via intracellular Ca2+ and Mg2+ regulation. / Li, Jiao; Imtiaz, Mohammad; BEARD, Nicole; Dulhunty, Angela F.; Thorne, Rick; vanHelden, Dirk.

In: PLoS One, Vol. 8, No. 3, 2013, p. 1-14.

Research output: Contribution to journalArticle

TY - JOUR

T1 - B-adrenergic stimulation increases RyR activity via intracellular Ca2+ and Mg2+ regulation

AU - Li, Jiao

AU - Imtiaz, Mohammad

AU - BEARD, Nicole

AU - Dulhunty, Angela F.

AU - Thorne, Rick

AU - vanHelden, Dirk

PY - 2013

Y1 - 2013

N2 - Here we investigate how beta-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca2+ and Mg2+ and the role of these changes in SR Ca2+ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 mu M isoproterenol or without (control) and snap frozen in liquid N-2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal beta-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of beta-adrenergic stimulation consistent with other reports that beta-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca2+],1 mM, beta-adrenergic stimulation increased luminal Ca2+ activation of single RyR channels, decreased luminal Mg2+ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg2+. At cytoplasmic [Ca2+] >1 mu M, beta-adrenergic stimulation only decreased cytoplasmic Mg2+ and Ca2+ inhibition of RyRs. The K-a and maximum levels of cytoplasmic Ca2+ activation site were not affected by beta-adrenergic stimulation. Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, beta-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca2+ binding to the luminal Ca2+ site and decreasing its affinity for luminal Mg2+ and 2) decreasing affinity of the low-affinity Ca2+/Mg2+ cytoplasmic inhibition site. However in systole, beta-adrenergic stimulation is mediated mainly by the latter

AB - Here we investigate how beta-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca2+ and Mg2+ and the role of these changes in SR Ca2+ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 mu M isoproterenol or without (control) and snap frozen in liquid N-2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal beta-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of beta-adrenergic stimulation consistent with other reports that beta-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca2+],1 mM, beta-adrenergic stimulation increased luminal Ca2+ activation of single RyR channels, decreased luminal Mg2+ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg2+. At cytoplasmic [Ca2+] >1 mu M, beta-adrenergic stimulation only decreased cytoplasmic Mg2+ and Ca2+ inhibition of RyRs. The K-a and maximum levels of cytoplasmic Ca2+ activation site were not affected by beta-adrenergic stimulation. Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, beta-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca2+ binding to the luminal Ca2+ site and decreasing its affinity for luminal Mg2+ and 2) decreasing affinity of the low-affinity Ca2+/Mg2+ cytoplasmic inhibition site. However in systole, beta-adrenergic stimulation is mediated mainly by the latter

KW - cardiac muscle

KW - calcium release

KW - ryanodine receptors

UR - http://purl.org/au-research/grants/nhmrc/1003985

U2 - 10.1371/journal.pone.0058334

DO - 10.1371/journal.pone.0058334

M3 - Article

VL - 8

SP - 1

EP - 14

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 3

ER -