Flux regulation of cardiac ryanodine receptor channels

Department of Molecular Physiology and Biophysics, Rush University Medical Center, Chicago, IL 60612, USA.
The Journal of General Physiology (Impact Factor: 4.79). 12/2009; 135(1):15-27. DOI: 10.1085/jgp.200910273
Source: PubMed


The cardiac type 2 ryanodine receptor (RYR2) is activated by Ca2+-induced Ca2+ release (CICR). The inherent positive feedback of CICR is well controlled in cells, but the nature of this control is debated. Here, we explore how the Ca2+ flux (lumen-to-cytosol) carried by an open RYR2 channel influences its own cytosolic Ca2+ regulatory sites as well as those on a neighboring channel. Both flux-dependent activation and inhibition of single channels were detected when there were super-physiological Ca2+ fluxes (>3 pA). Single-channel results indicate a pore inhibition site distance of 1.2 +/- 0.16 nm and that the activation site on an open channel is shielded/protected from its own flux. Our results indicate that the Ca2+ flux mediated by an open RYR2 channel in cells (approximately 0.5 pA) is too small to substantially regulate (activate or inhibit) the channel carrying it, even though it is sufficient to activate a neighboring RYR2 channel.

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Available from: Maura Porta, Apr 09, 2014
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    • "feed-through regulation (Liu et al., 2010). The immunity may arise because Ca 2+ is already occupying the cytosolic activation site(s) of the open RyR2 and thus the fluxed Ca 2+ can have little effect (Liu et al., 2010). Note that cytosolic Ca 2+ activation sites on nearby RyR2s may not be occupied. "
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    ABSTRACT: Release of Ca2+ from the sarcoplasmic reticulum (SR) drives contractile function of cardiac myocytes. Luminal Ca2+ regulation of SR Ca2+ release is fundamental not only in physiology but also in physiopathology because abnormal luminal Ca2+ regulation is known to lead to arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and/or sudden cardiac arrest, as inferred from animal model studies. Luminal Ca2+ regulates ryanodine receptor (RyR)2-mediated SR Ca2+ release through mechanisms localized inside the SR; one of these involves luminal Ca2+ interacting with calsequestrin (CASQ), triadin, and/or junctin to regulate RyR2 function. CASQ2-RyR2 regulation was examined at the single RyR2 channel level. Single RyR2s were incorporated into planar lipid bilayers by the fusion of native SR vesicles isolated from either wild-type (WT), CASQ2 knockout (KO), or R33Q-CASQ2 knock-in (KI) mice. KO and KI mice have CPVT-like phenotypes. We show that CASQ2(WT) action on RyR2 function (either activation or inhibition) was strongly influenced by the presence of cytosolic MgATP. Function of the reconstituted CASQ2(WT)–RyR2 complex was unaffected by changes in luminal free [Ca2+] (from 0.1 to 1 mM). The inhibition exerted by CASQ2(WT) association with the RyR2 determined a reduction in cytosolic Ca2+ activation sensitivity. RyR2s from KO mice were significantly more sensitive to cytosolic Ca2+ activation and had significantly longer mean open times than RyR2s from WT mice. Sensitivity of RyR2s from KI mice was in between that of RyR2 channels from KO and WT mice. Enhanced cytosolic RyR2 Ca2+ sensitivity and longer RyR2 open times likely explain the CPVT-like phenotype of both KO and KI mice.
    The Journal of General Physiology 07/2013; 142(2). DOI:10.1085/jgp.201311022 · 4.79 Impact Factor
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    • "Voltage dependence, observed with high flux of luminal divalent ions has been attributed to the interaction of the M2+ moving through the pore with activating and inhibitory sites in the cytosolic side of the channel [15], [17], [18], [19]. "
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    ABSTRACT: Cardiac ryanodine receptor (RyR2) function is modulated by Ca(2+) and Mg(2+). To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+) binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+) or Sr(2+). This activation was interfered by Mg(2+) and Ba(2+) acting at low affinity M(2+)-unspecific binding sites. When testing the effects of luminal M(2+) as current carriers, all M(2+) increased maximal RyR2 open probability (compared to Cs(+)), suggesting the existence of low affinity activating M(2+)-unspecific sites at the luminal surface. Responses to M(2+) vary from channel to channel (heterogeneity). However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)). Kinetics of RyR2 with mixtures of luminal Ba(2+)/Ca(2+) and additive action of luminal plus cytosolic Ba(2+) or Mg(2+) suggest luminal M(2+) differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+)/Sr(2+)-specific sites, which stabilize high P(o) mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca(2+) activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage.
    PLoS ONE 10/2011; 6(10):e26693. DOI:10.1371/journal.pone.0026693 · 3.23 Impact Factor
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    • "The brief opening of either an L-type Ca 2+ channel or an RYR2 under diastolic conditions leads to a local elevation of Ca 2+ in the subspace ([Ca 2+ ] subspace ) from the normal [Ca 2+ ] i of 100 nM to 10 µM (Cannell and Soeller, 1997; Soeller and Cannell, 1997; Sobie et al., 2002). This elevation of [Ca 2+ ] subspace , albeit brief (<1 ms), is sufficient to activate the RYR2 cluster to produce a Ca 2+ spark, a process termed Ca 2+ -induced Ca 2+ release, or CICR (Fabiato, 1983; Cheng et al., 1993; Cannell et al., 1994a,b; Sobie et al., 2002; Cheng and Lederer, 2008; Györke and Terentyev, 2008; Liu et al., 2010). The spatial organization of the jSR enables reliable activation of Ca 2+ sparks by action potentials, but the relative insensitivity of RYR2s to calcium protects the cell from instability and enables Ca 2+ sparks during diastole to remain isolated from neighboring jSR spark sites. "
    The Journal of General Physiology 08/2010; 136(2):135-42. DOI:10.1085/jgp.201010406 · 4.79 Impact Factor
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