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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    • "In both cases, they observed that multiple RYR channels opened and closed in a concerted fashion and referred to this behavior as " coupled gating " . Subsequent studies of our group and others brought the further evidence about coupled RYR channels [7] [8] [9] [10] [11] [12]; and thus, proving a strong platform for studying this phenomenon primarily from the biophysical point of view. "
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    ABSTRACT: Cardiac ryanodine receptors (RYR2) infrequently exhibit coupled gating that is manifested by synchronous opening and closing. To better characterize this phenomenon, we investigated the regulation of coupled RYR2 channels by luminal Ca(2+)focusing on effects that are likely mediated by the true luminal activation mechanism. By reconstituting an ion channel into a planar lipid bilayer and using substantially lower concentration of luminal Ba(2+) (8mM, the virtual absence of Ca(2+)) and luminal Ca(2+) (8mM), we show that response of coupled RYR2 channels to caffeine at a diastolic cytosolic Ca(2+) (90 nM) was affected by luminal Ca(2+) in a similar manner as for the single RYR2 channel except the gating behavior. Whereas, the single RYR2 channel responded to luminal Ca(2+) by prolongation in open and closed times, coupled RYR2 channels seemed to be resistant in this respect. In summary, we conclude that the class of Ca(2+) sites located on the luminal face of coupled RYR2 channels that is responsible for the channel potentiation by luminal Ca(2+) is functional and not structurally hindered by the channel coupling. Thus, the idea about non-functional luminal Ca(2+) sites as a source of the apparent gating resistance of coupled RYR2 channels to luminal Ca(2+) appears to be ruled out.
    Biochimica et Biophysica Acta 11/2013; 1838(3). DOI:10.1016/j.bbamem.2013.11.005 · 4.66 Impact Factor
    • "Another intracellular Ca 2+ -release channel that plays a central role in the generation and regulation of intracellular Ca 2+ signals is the ryanodine receptor (RyR) channel (Smith et al. 1988; Fill and Copello 2002). Until recently, electrophysiological studies of the conductance and permeability properties (Smith et al. 1988; Lindsay et al. 1991; Tinker et al. 1992; Tinker and Williams 1992, 1993, 1995; Tu et al. 1994; Fill and Copello 2002; Kettlun et al. 2003) and ligand regulation (Ashley and Williams 1990; Meissner 1994; Schiefer et al. 1995; Fill and Copello 2002; Fill and Ramos 2004; Laver 2010; Liu et al. 2010) of single wild-type and mutant (Zhao et al. 1999; Gao et al. 2000; Du et al. 2001; Chen et al. 2002; Wang et al. 2005; Xu et al. 2006) RyR channels have been performed exclusively by reconstituting the channels into artificial planar lipid bilayers. However, like the InsP 3 R, the RyR is localized to the nuclear envelope as well as the ER or sarcoplasmic reticulum (Gerasimenko and Gerasimenko 2004; Marius et al. 2006). "
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    ABSTRACT: The modulation of cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) is a universal intracellular signaling pathway that regulates numerous cellular physiological processes. Ubiquitous intracellular Ca(2+)-release channels localized to the endoplasmic/sarcoplasmic reticulum-inositol 1,4,5-trisphosphate receptor (InsP3R) and ryanodine receptor (RyR) channels-play a central role in [Ca(2+)]i signaling in all animal cells. Despite their intracellular localization, electrophysiological studies of the single-channel permeation and gating properties of these Ca(2+)-release channels using the powerful patch-clamp approach have been possible by application of this technique to isolated nuclei because the channels are present in membranes of the nuclear envelope. Here we provide a concise description of how nuclear patch-clamp experiments have been used to study single-channel properties of different InsP3R channels in the outer nuclear membrane. We compare this with other methods for studying intracellular Ca(2+) release. We also briefly describe application of the technique to InsP3R channels in the inner nuclear membrane and to channels in the outer nuclear membrane of HEK293 cells expressing recombinant RyR.
    Cold Spring Harbor Protocols 09/2013; 2013(9). DOI:10.1101/pdb.top066217 · 4.63 Impact Factor
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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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