Mg<sup>2+</sup> activates the ryanodine receptor type 2 (RyR2) at intermediate Ca<sup>2+</sup> concentrations

Chugun, Akihito; Satō, Osamu; Takeshima, Hiroshi; Ogawa, Yasuo

doi:10.1152/ajpcell.00275.2006

Cited by 20 publications

(29 citation statements)

References 44 publications

(88 reference statements)

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“…Loss of Mg 2+ inhibition would be possible through competition at H sites and a conformational state at the Ca 2+ biphasic plateau, which reduces the affinity of Mg 2+ at L sites relative to conformations at low (3 and 10μM) and high (100μM) Ca 2+ . Alternatively, Mg 2+ binding could impart an activating effect on H sites, as suggested for rat RyR2 [31]. This further supports allosteric coupling between H and L sites and suggests Ca 2+ may contribute along with DHPR [18] and oxidation [19] to relieving the inherent physiological block provided by Mg 2+ .…”

Section: Mg 2+ Inhibitionsupporting

confidence: 59%

“…It is also noteworthy that previously published whole cell Ca 2+ imaging experiments indicate these chimeras expressed in 1B5 cells are functional, with all three constructs exhibiting calcium-induced Ca 2+ release (CICR) and Ch-4 and −21 additionally engaging skeletal-type EC-coupling [20]. Interestingly, a recent [ 3 H]Ry-binding study with membranes isolated from rat ventricular muscle showed a similar biphasic Ca 2+ activation curve for RyR2, which was reverted to a monophasic binding profile in the presence of Mg 2+ [31]. Collectively these results support a mechanism by which coordinated Ca 2+ activation sites regulate RyR conformations that bind ryanodine with high affinity.…”

Section: Resultsmentioning

confidence: 94%

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Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras

Voss

Allen

Pessah

et al. 2008

Biochemical and Biophysical Research Communications

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We studied cation regulation of wild type ryanodine receptor type 1 ( WT RyR1), type 3 ( WT RyR3) and RyR3/RyR1 chimeras (Ch) expressed in 1B5 dyspedic myotubes. Using [ 3 H]ryanodine binding to sarcoplasmic reticulum (SR) membranes, Ca 2+ titrations with WT RyR3 and three chimeras show biphasic activation that is allosterically coupled to attenuated inhibition relative to WT RyR1. Chimeras show biphasic Mg 2+ inhibition profiles at 3 and 10μM Ca 2+ , no observable inhibition at 20μM Ca 2+ and monophasic inhibition at 100μM Ca 2+ . Ca 2+ imaging of intact myotubes expressing Ch-4 exhibit caffeine-induced Ca 2+ transients with inhibition kinetics that are significantly slower than those expressing WT RyR1 or WT RyR3. Four new aspects of RyR regulation are evident: 1) high affinity (H) activation and low affinity (L) inhibition sites are allosterically coupled, 2) Ca 2+ facilitates removal of the inherent Mg 2+ block, 3) WT RyR3 exhibits reduced cooperativity between H activation sites when compared to WT RyR1, and 4) uncoupling of these sites in Ch-4 results in decreased rates of inactivation of caffeine-induced Ca transients.Keywords ryanodine receptors; calcium-induced calcium release; channel regulation Three isoforms of wild type ryanodine receptors ( WT RyR1, 2 and 3) are expressed in specialized regions of endoplasmic/sarcoplasmic reticulum (ER/SR) in most mammalian cells where they function as Ca 2+ Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Materials and Methods Chimeric RyR1/RyR3 constructsSpecific primers were designed for PCR amplification of the selected fragments using WT RyR1 as a template. Amplified fragments from WT RyR1 encoding aa 1681-2217 (Ch-17), 1924-2446 (Ch-21) and 1681-3770 (Ch-4) were inserted, in frame, into the endogenous restriction site(s) of HSV-RyR3 plasmid as described previously [20]. All chimeric constructs were cloned into the HSV-1 amplicon vector and packaged using a helper virus-free packaging system [22]. Cell culture, infection and membrane preparation1B5 myoblasts were cultured and differentiated into myotubes as described previously [20] and [23]. Plates with differentiated myotubes were infected with virion containing wild type and RyR1/RyR3 chimeric cDNA for 2 h and membrane extracts were prepared 36 h after infection. Myotubes were homogenized and membrane fractions obtained by differential centrifugation as described previously [24].[ cold buffer, placed into 5 ml scintillation cocktail (ScintiVerse; Fisher Scientific) and radioactivity counted. Equations for binding analysisCurve fitting was ...

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Section: Mg 2+ Inhibitionsupporting

confidence: 59%

Section: Resultsmentioning

confidence: 94%

Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras

Voss

Allen

Pessah

et al. 2008

Biochemical and Biophysical Research Communications

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“…Very recently, Mg 2+ has been suggested to also exhibit a stimulating effect on RYR2 in an intermediate concentration range of Ca 2+ (10 -100 μ M) ( Chugun et al, 2007 ). These authors showed that Mg 2+ renders the RYR2 more sensitive to modulators of CICR, such as caffeine, ␤ , ␥ -methylene ATP, procaine, and calmodulin.…”

Section: Control Of Intracellular Camentioning

confidence: 99%

Modulation of the Local SR Ca2+ Release by Intracellular Mg2+ in Cardiac Myocytes

Gusev

Niggli

2008

The Journal of General Physiology

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In cardiac muscle, Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) defines the amplitude and time course of the Ca2+ transient. The global elevation of the intracellular Ca2+ concentration arises from the spatial and temporal summation of elementary Ca2+ release events, Ca2+ sparks. Ca2+ sparks represent the concerted opening of a group of ryanodine receptors (RYRs), which are under the control of several modulatory proteins and diffusible cytoplasmic factors (e.g., Ca2+, Mg2+, and ATP). Here, we examined by which mechanism the free intracellular Mg2+ ([Mg2+]free) affects various Ca2+ spark parameters in permeabilized mouse ventricular myocytes, such as spark frequency, duration, rise time, and full width, at half magnitude and half maximal duration. Varying the levels of free ATP and Mg2+ in specifically designed solutions allowed us to separate the inhibition of RYRs by Mg2+ from the possible activation by ATP and Mg2+-ATP via the adenine binding site of the channel. Changes in [Mg2+]free generally led to biphasic alterations of the Ca2+ spark frequency. For example, lowering [Mg2+]free resulted in an abrupt increase of spark frequency, which slowly recovered toward the initial level, presumably as a result of SR Ca2+ depletion. Fitting the Ca2+ spark inhibition by [Mg2+]free with a Hill equation revealed a Ki of 0.1 mM. In conclusion, our results support the notion that local Ca2+ release and Ca2+ sparks are modulated by Mg2+ in the intracellular environment. This seems to occur predominantly by hindering Ca2+-dependent activation of the RYRs through competitive Mg2+ occupancy of the high-affinity activation site of the channels. These findings help to characterize CICR in cardiac muscle under normal and pathological conditions, where the levels of Mg2+ and ATP can change.

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“…The magnitude of the reported inhibition of RyR2 by Ca 2+ CaM is often exceedingly small (Yamaguchi et al, 2004;Xu and Meissner, 2004) and, interestingly, there are also rare reports suggesting that CaM may activate RyR2 (Fruen et al, 2000;Chugun et al, 2007 Xu and Meissner, 2004) and Ca 2+ -spark generation in cardiac cells (Lukyanenko and Gyorke, 1999;Ai et al, 2005;Guo et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

“…It has been well documented that Ca 2+ CaM can cause partial inhibition of both cardiac and skeletal (RyR1) isoforms of RyR (Fruen et al, 2000;Balshaw et al, 2001). Additionally, apoCaM is known to activate RyR1, but has little effect on RyR2 Tripathy et al, 1995;Fruen et al, 2000).The magnitude of the reported inhibition of RyR2 by Ca 2+ CaM is often exceedingly small (Yamaguchi et al, 2004;Xu and Meissner, 2004) and, interestingly, there are also rare reports suggesting that CaM may activate RyR2 (Fruen et al, 2000;Chugun et al, 2007 Xu and Meissner, 2004) and Ca 2+ -spark generation in cardiac cells (Lukyanenko and Gyorke, 1999;Ai et al, 2005;Guo et al, 2006).As CaM can be immunoprecipitated with RyR2 (Ai et al, 2005), and as the t1/2 for [ 35 S]CaM dissociation from cardiac SR is approximately 9 min for Ca 2+ CaM and 40 s for apoCaM (Balshaw et al, 2001), it is likely that some CaM is always bound tightly to RyR2 in situ, and may provide a certain level of 'constitutive' modulation of channel function. To experimentally investigate this modulation in cardiac cells, it is necessary first to remove the CaM attached to RyR2.…”

mentioning

confidence: 99%

Ca²⁺‐calmodulin can activate and inactivate cardiac ryanodine receptors

Sigalas

Bent

Kitmitto

et al. 2009

British J Pharmacology

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) on the gating of native sheep RyR2, reconstituted into bilayers. Suramin displaces CaM from RyR2 and we have used a gel-shift assay to provide evidence of the mechanism underlying this effect. Finally, using suramin to displace endogenous CaM from RyR2 in permeabilized cardiac cells, we have investigated the effects of 50 nmol·L -1 CaM on sarcoplasmic reticulum (SR) Ca 2+-release. Key results: Ca 2+CaM activated or inhibited single RyR2, but activation was much more likely at low (50-100 nmol·L -1 ) concentrations. Also, suramin displaced CaM from a peptide of the CaM binding domain of RyR2, indicating that, like the skeletal isoform (RyR1), suramin directly competes with CaM for its binding site on the channel. Pre-treatment of rat permeabilized ventricular myocytes with suramin to displace CaM, followed by addition of 50 nmol·L -1 CaM to the mock cytoplasmic solution caused an increase in the frequency of spontaneous Ca 2+ -release events. Application of caffeine demonstrated that 50 nmol·L -1 CaM reduced SR Ca 2+ content. Conclusions and implications: We describe for the first time how Ca 2+CaM is capable, not only of inactivating, but also of activating RyR2 channels in bilayers in a CaM kinase II-independent manner. Similarly, in cardiac cells, CaM stimulates SR Ca 2+-release and the use of caffeine suggests that this is a RyR2-mediated effect. Abbreviations: AIP, autocamtide-2-related inhibitory peptide; CaM, calmodulin; CaMKII, CaM kinase II; EC-coupling, excitation-contraction coupling; RyR2, cardiac ryanodine receptor; RyRF, RyR2 peptide (RSKKAVWHKLL-SKQRKRAVVACFRMAPLYNLP); SR, sarcoplasmic reticulum British Journal of Pharmacology IntroductionThe cardiac ryanodine receptor (RyR2; nomenclature follows Alexander et al., 2008) is the pathway for the release of intracellular Ca 2+ during excitation-contraction (EC) coupling. It also acts as a scaffolding protein localizing numerous other proteins to the dyadic cleft regions. Calmodulin (CaM) binds very tightly to RyR2, but the physiological role of this direct association is unclear (Balshaw et al., 2001;Ai et al., 2005). CaM is a Ca 2+ -sensing protein, which contains four Ca 2+ -binding sites, two on the amino and two on the carboxyl lobe. CaM interacts with numerous proteins, usually in the Ca 2+ -bound form (Ca 2+ CaM), but can also modulate certain proteins in the non-Ca 2+ -bound form (apoCaM). It has been well documented that Ca 2+ CaM can cause partial inhibition of both cardiac and skeletal (RyR1) isoforms of RyR (Fruen et al., 2000;Balshaw et al., 2001). Additionally, apoCaM is known to activate RyR1, but has little effect on RyR2 Tripathy et al., 1995;Fruen et al., 2000).The magnitude of the reported inhibition of RyR2 by Ca 2+ CaM is often exceedingly small (Yamaguchi et al., 2004;Xu and Meissner, 2004) and, interestingly, there are also rare reports suggesting that CaM may activate RyR2 (Fruen et al., 2000;Chugun et al., 2007 Xu and Meissner, 2004) and Ca 2+ -spark generation in cardiac cells (Lukyanenko and Gyorke, 1999;Ai et al., 2005;Guo ...

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Mg²⁺ activates the ryanodine receptor type 2 (RyR2) at intermediate Ca²⁺ concentrations

Cited by 20 publications

References 44 publications

Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras

Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras

Modulation of the Local SR Ca2+ Release by Intracellular Mg2+ in Cardiac Myocytes

Ca²⁺‐calmodulin can activate and inactivate cardiac ryanodine receptors

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Mg2+ activates the ryanodine receptor type 2 (RyR2) at intermediate Ca2+ concentrations

Cited by 20 publications

References 44 publications

Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras

Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras

Modulation of the Local SR Ca2+ Release by Intracellular Mg2+ in Cardiac Myocytes

Ca2+‐calmodulin can activate and inactivate cardiac ryanodine receptors

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Mg²⁺ activates the ryanodine receptor type 2 (RyR2) at intermediate Ca²⁺ concentrations

Ca²⁺‐calmodulin can activate and inactivate cardiac ryanodine receptors