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

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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

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