Ryanodine Receptors

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Mark Nelson - One of the best experts on this subject based on the ideXlab platform.

  • Ryanodine Receptors calcium signaling and regulation of vascular tone in the cerebral parenchymal microcirculation
    Microcirculation, 2013
    Co-Authors: Fabrice Dabertrand, Mark Nelson, Joseph E Brayden
    Abstract:

    The cerebral blood supply is delivered by a surface network of pial arteries and arterioles from which arise (parenchymal) arterioles that penetrate into the cortex and terminate in a rich capillary bed. The critical regulation of CBF, locally and globally, requires precise vasomotor regulation of the intracerebral microvasculature. This vascular region is anatomically unique as illustrated by the presence of astrocytic processes that envelope almost the entire basolateral surface of PAs. There are, moreover, notable functional differences between pial arteries and PAs. For example, in pial VSMCs, local calcium release events (“calcium sparks”) through Ryanodine receptor (RyR) channels in SR membrane activate large conductance, calcium-sensitive potassium channels to modulate vascular diameter. In contrast, VSMCs in PAs express functional RyR and BK channels, but under physiological conditions, these channels do not oppose pressure-induced vasoconstriction. Here, we summarize the roles of Ryanodine Receptors in the parenchymal microvasculature under physiologic and pathologic conditions, and discuss their importance in the control of CBF.

  • trpv4 forms a novel ca2 signaling complex with Ryanodine Receptors and bkca channels
    Circulation Research, 2005
    Co-Authors: Scott Earley, Thomas J Heppner, Mark Nelson, Joseph E Brayden
    Abstract:

    Vasodilatory factors produced by the endothelium are critical for the maintenance of normal blood pressure and flow. We hypothesized that endothelial signals are transduced to underlying vascular smooth muscle by vanilloid transient receptor potential (TRPV) channels. TRPV4 message was detected in RNA from cerebral artery smooth muscle cells. In patch-clamp experiments using freshly isolated cerebral myocytes, outwardly rectifying whole-cell currents with properties consistent with those of expressed TRPV4 channels were evoked by the TRPV4 agonist 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) (5 micromol/L) and the endothelium-derived arachidonic acid metabolite 11,12 epoxyeicosatrienoic acid (11,12 EET) (300 nmol/L). Using high-speed laser-scanning confocal microscopy, we found that 11,12 EET increased the frequency of unitary Ca2+ release events (Ca2+ sparks) via Ryanodine Receptors located on the sarcoplasmic reticulum of cerebral artery smooth muscle cells. EET-induced Ca2+ sparks activated nearby sarcolemmal large-conductance Ca2+-activated K+ (BKCa) channels, measured as an increase in the frequency of transient K+ currents (referred to as "spontaneous transient outward currents" [STOCs]). 11,12 EET-induced increases in Ca2+ spark and STOC frequency were inhibited by lowering external Ca2+ from 2 mmol/L to 10 micromol/L but not by voltage-dependent Ca2+ channel inhibitors, suggesting that these responses require extracellular Ca2+ influx via channels other than voltage-dependent Ca2+ channels. Antisense-mediated suppression of TRPV4 expression in intact cerebral arteries prevented 11,12 EET-induced smooth muscle hyperpolarization and vasodilation. Thus, we conclude that TRPV4 forms a novel Ca2+ signaling complex with Ryanodine Receptors and BKCa channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca2+-induced Ca2+ release in response to an endothelial-derived factor.

  • differential regulation of sk and bk channels by ca2 signals from ca2 channels and Ryanodine Receptors in guinea pig urinary bladder myocytes
    The Journal of Physiology, 2002
    Co-Authors: Gerald M Herrera, Mark Nelson
    Abstract:

    Small-conductance (SK) and large-conductance (BK) Ca2+-activated K+ channels are key regulators of excitability in urinary bladder smooth muscle (UBSM) of guinea-pig. The overall goal of this study was to define how SK and BK channels respond to Ca2+ signals from voltage-dependent Ca2+ channels (VDCCs) in the surface membrane and from Ryanodine-sensitive Ca2+ release channels or Ryanodine Receptors (RyRs) in the sarcoplasmic reticulum (SR) membrane. To characterize the role of SK channels in UBSM, the effects of the SK channel blocker apamin on phasic contractions were examined. Apamin caused a dose-dependent increase in the amplitude of phasic contractions over a broad concentration range (10−10 to 10−6m). To determine the effects of Ca2+ signals from VDCCs and RyRs to SK and BK channels, whole cell membrane current was measured in isolated myocytes bathed in physiological solutions. Depolarization (-70 to +10 mV for 100 ms) of isolated myocytes caused an inward Ca2+ current (ICa), followed by an outward current. The outward current was reduced in a dose-dependent manner by apamin (10−10 to 10−6m), and designated ISK. ISK had a mean amplitude of 53.8 ± 6.1 pA or ∼1.4 pA pF−1 at +10 mV. The amplitude of ISK correlated with the peak ICa. Blocking ICa abolished ISK. In contrast, ISK was insensitive to the RyR blocker Ryanodine (10 μM). These data indicate that Ca2+ signals from VDCCs, but not from RyRs, activate SK channels. BK channel currents (IBK) were isolated from other currents by using the BK channel blockers tetraethylammonium ions (TEA+; 1 mm) or iberiotoxin (200 nm). Voltage steps evoked transient and steady-state IBK components. Transient BK currents have previously been shown to result from BK channel activation by local Ca2+ release through RyRs (‘Ca2+ sparks’). Transient BK currents were inhibited by Ryanodine (10 μM), as expected, and had a mean amplitude of 152.6 pA at +10 mV. The mean number of transient BK currents during a voltage step (range 0 to 3) correlated with ICa. There was a long delay (52.4 ± 2.7 ms) between activation of ICa and the first transient BK current. In contrast, Ryanodine did not affect the steady-state BK current (mean amplitude 135.4 pA) during the voltage step. The steady-state BK current was reduced 95 % by inhibition of VDCCs, suggesting that this process depends largely on Ca2+ entry through VDCCs and not Ca2+ release through RyRs. These results indicate that Ca2+ entry through VDCCs activates both BK and SK channels, but Ca2+ release (Ca2+ sparks) through RyRs activates only BK channels.

  • regulation of urinary bladder smooth muscle contractions by Ryanodine Receptors and bk and sk channels
    American Journal of Physiology-regulatory Integrative and Comparative Physiology, 2000
    Co-Authors: Gerald M Herrera, Thomas J Heppner, Mark Nelson
    Abstract:

    This study examines the roles of voltage-dependent Ca2+ channels (VDCC), Ryanodine Receptors (RyRs), large-conductance Ca2+-activated K+ (BK) channels, and small-conductance Ca2+-activated K+ (SK) ...

  • functional coupling of Ryanodine Receptors to kca channels in smooth muscle cells from rat cerebral arteries
    The Journal of General Physiology, 1999
    Co-Authors: Guillermo J Perez, Adrian D Bonev, Joseph B Patlak, Mark Nelson
    Abstract:

    The relationship between Ca2+ release (“Ca2+ sparks”) through Ryanodine-sensitive Ca2+ release channels in the sarcoplasmic reticulum and KCa channels was examined in smooth muscle cells from rat cerebral arteries. Whole cell potassium currents at physiological membrane potentials (−40 mV) and intracellular Ca2+ were measured simultaneously, using the perforated patch clamp technique and a laser two-dimensional (x–y) scanning confocal microscope and the fluorescent Ca2+ indicator, fluo-3. Virtually all (96%) detectable Ca2+ sparks were associated with the activation of a spontaneous transient outward current (STOC) through KCa channels. A small number of sparks (5 of 128) were associated with currents smaller than 6 pA (mean amplitude, 4.7 pA, at −40 mV). Approximately 41% of STOCs occurred without a detectable Ca2+ spark. The amplitudes of the Ca2+ sparks correlated with the amplitudes of the STOCs (regression coefficient 0.8; P 104-fold) during a Ca2+ spark is consistent with local Ca2+ during a spark being in the order of 1–100 μM. Therefore, the increase in fractional fluorescence (F/Fo) measured during a Ca2+ spark (mean 2.04 F/Fo or ∼310 nM Ca2+) appears to significantly underestimate the local Ca2+ that activates KCa channels. These results indicate that the majority of Ryanodine Receptors that cause Ca2+ sparks are functionally coupled to KCa channels in the surface membrane, providing direct support for the idea that Ca2+ sparks cause STOCs.

Heping Cheng - One of the best experts on this subject based on the ideXlab platform.

  • orphaned Ryanodine Receptors in the failing heart
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Longsheng Song, W J Lederer, Eric A Sobie, Stacey L Mcculle, William C Balke, Heping Cheng
    Abstract:

    Heart muscle is characterized by a regular array of proteins and structures that form a repeating functional unit identified as the sarcomere. This regular structure enables tight coupling between electrical activity and Ca2+ signaling. In heart failure, multiple cellular defects develop, including reduced contractility, altered Ca2+ signaling, and arrhythmias; however, the underlying causes of these defects are not well understood. Here, in ventricular myocytes from spontaneously hypertensive rats that develop heart failure, we identify fundamental changes in Ca2+ signaling that are related to restructuring of the spatial organization of the cells. Myocytes display both a reduced ability to trigger sarcoplasmic reticulum Ca2+ release and increased spatial dispersion of the transverse tubules (TTs). Remodeled TTs in cells from failing hearts no longer exist in the regularly organized structures found in normal heart cells, instead moving within the sarcomere away from the Z-line structures and leaving behind the sarcoplasmic reticulum Ca2+ release channels, the Ryanodine Receptors (RyRs). These orphaned RyRs appear to be responsible for the dyssynchronous Ca2+ sparks that have been linked to blunted contractility and, probably, Ca2+-dependent arrhythmias in diverse models of heart failure. We conclude that the increased spatial dispersion of the TTs and orphaned RyRs lead to the loss of local control and Ca2+ instability in heart failure.

  • ca2 signalling between single l type ca2 channels and Ryanodine Receptors in heart cells
    Nature, 2001
    Co-Authors: Shiqiang Wang, Longsheng Song, Edward G Lakatta, Heping Cheng
    Abstract:

    Ca2+-induced Ca2+ release is a general mechanism that most cells use to amplify Ca2+ signals1,2,3,4,5. In heart cells, this mechanism is operated between voltage-gated L-type Ca2+ channels (LCCs) in the plasma membrane and Ca2+ release channels, commonly known as Ryanodine Receptors, in the sarcoplasmic reticulum3,4,5. The Ca2+ influx through LCCs traverses a cleft of roughly 12 nm formed by the cell surface and the sarcoplasmic reticulum membrane, and activates adjacent Ryanodine Receptors to release Ca2+ in the form of Ca2+ sparks6. Here we determine the kinetics, fidelity and stoichiometry of coupling between LCCs and Ryanodine Receptors. We show that the local Ca2+ signal produced by a single opening of an LCC, named a ‘Ca2+ sparklet’, can trigger about 4–6 Ryanodine Receptors to generate a Ca2+ spark. The coupling between LCCs and Ryanodine Receptors is stochastic, as judged by the exponential distribution of the coupling latency. The fraction of sparklets that successfully triggers a spark is less than unity and declines in a use-dependent manner. This optical analysis of single-channel communication affords a powerful means for elucidating Ca2+-signalling mechanisms at the molecular level.

  • local control models of cardiac excitation contraction coupling a possible role for allosteric interactions between Ryanodine Receptors
    The Journal of General Physiology, 1999
    Co-Authors: Michael D Stern, James S K Sham, Longsheng Song, Heping Cheng, Huangtian Yang, Kenneth R Boheler, Eduardo Rios
    Abstract:

    In cardiac muscle, release of activator calcium from the sarcoplasmic reticulum occurs by calcium- induced calcium release through Ryanodine Receptors (RyRs), which are clustered in a dense, regular, two-dimensional lattice array at the diad junction. We simulated numerically the stochastic dynamics of RyRs and L-type sarcolemmal calcium channels interacting via calcium nano-domains in the junctional cleft. Four putative RyR gating schemes based on single-channel measurements in lipid bilayers all failed to give stable excitation–contraction coupling, due either to insufficiently strong inactivation to terminate locally regenerative calcium-induced calcium release or insufficient cooperativity to discriminate against RyR activation by background calcium. If the Ryanodine receptor was represented, instead, by a phenomenological four-state gating scheme, with channel opening resulting from simultaneous binding of two Ca2+ ions, and either calcium-dependent or activation-linked inactivation, the simulations gave a good semiquantitative accounting for the macroscopic features of excitation–contraction coupling. It was possible to restore stability to a model based on a bilayer-derived gating scheme, by introducing allosteric interactions between nearest-neighbor RyRs so as to stabilize the inactivated state and produce cooperativity among calcium binding sites on different RyRs. Such allosteric coupling between RyRs may be a function of the foot process and lattice array, explaining their conservation during evolution.

  • termination of ca2 release by a local inactivation of Ryanodine Receptors in cardiac myocytes
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: James S K Sham, Longsheng Song, Edward G Lakatta, Ye Chen, Lihua Deng, Michael D Stern, Heping Cheng
    Abstract:

    In heart, a robust regulatory mechanism is required to counteract the regenerative Ca2+-induced Ca2+ release from the sarcoplasmic reticulum. Several mechanisms, including inactivation, adaptation, and stochastic closing of Ryanodine Receptors (RyRs) have been proposed, but no conclusive evidence has yet been provided. We probed the termination process of Ca2+ release by using a technique of imaging local Ca2+ release, or “Ca2+ spikes”, at subcellular sites; and we tracked the kinetics of Ca2+ release triggered by L-type Ca2+ channels. At 0 mV, Ca2+ release occurred and terminated within 40 ms after the onset of clamp pulses (0 mV). Increasing the open-duration and promoting the reopenings of Ca2+ channels with the Ca2+ channel agonist, FPL64176, did not prolong or trigger secondary Ca2+ spikes, even though two-thirds of the sarcoplasmic reticulum Ca2+ remained available for release. Latency of Ca2+ spikes coincided with the first openings but not with the reopenings of L-type Ca2+ channels. After an initial maximal release, even a multi-fold increase in unitary Ca2+ current induced by a hyperpolarization to −120 mV failed to trigger additional release, indicating absolute refractoriness of RyRs. When the release was submaximal (e.g., at +30 mV), tail currents did activate additional Ca2+ spikes; confocal images revealed that they originated from RyRs unfired during depolarization. These results indicate that Ca2+ release is terminated primarily by a highly localized, use-dependent inactivation of RyRs but not by the stochastic closing or adaptation of RyRs in intact ventricular myocytes.

C F Louis - One of the best experts on this subject based on the ideXlab platform.

Andrew R Marks - One of the best experts on this subject based on the ideXlab platform.

  • dysfunctional Ryanodine Receptors in the heart new insights into complex cardiovascular diseases
    Journal of Molecular and Cellular Cardiology, 2013
    Co-Authors: Steven O Marx, Andrew R Marks
    Abstract:

    Calcium dependent signaling is highly regulated in cardiomyocytes and determines the force of cardiac muscle contraction. The cardiac Ryanodine Receptors (RyR2) play important roles in health and disease. Modulation of RyR2 by phosphorylation is required for sympathetic regulation of cardiac function. Abnormal regulation of RyR2 contributes to heart failure, and atrial and ventricular arrhythmias. RyR2 channels are oxidized, nitrosylated, and hyperphosphorylated by protein kinase A (PKA) in heart failure, resulting in “leaky” channels. These leaky RyR2 channels contribute to depletion of calcium from the sarcoplasmic reticulum, resulting in defective cardiac excitation–contraction coupling. In this review, we discuss both the importance of PKA and calcium/calmodulin-dependent kinase II (CaMKII) regulation of RyR2 in health, and how altered phosphorylation, nitrosylation and oxidation of RyR2 channels lead to cardiac disease. Correcting these defects using either genetic manipulation (knock-in) in mice, or specific and novel small molecules ameliorates the RyR2 dysfunction, reducing the progression to heart failure and the incidence of arrhythmias. This article is part of a Special Issue entitled “Calcium Signaling in Heart”.

  • enhancing calstabin binding to Ryanodine Receptors improves cardiac and skeletal muscle function in heart failure
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Xander H T Wehrens, Steven Reiken, Stephan E Lehnart, Roel Van Der Nagel, Raymond Morales, Zhenzhuang Cheng, Shixiang Deng, Leon J De Windt, Donald W Landry, Andrew R Marks
    Abstract:

    Abstract Abnormalities in intracellular calcium release and reuptake are responsible for decreased contractility in heart failure (HF). We have previously shown that cardiac Ryanodine Receptors (RyRs) are protein kinase A-hyperphosphorylated and depleted of the regulatory subunit calstabin-2 in HF. Moreover, similar alterations in skeletal muscle RyR have been linked to increased fatigability in HF. To determine whether restoration of calstabin binding to RyR may ameliorate cardiac and skeletal muscle dysfunction in HF, we treated WT and calstabin-2-/- mice subjected to myocardial infarction (MI) with JTV519. JTV519, a 1,4-benzothiazepine, is a member of a class of drugs known as calcium channel stabilizers, previously shown to increase calstabin binding to RyR. Echocardiography at 21 days after MI demonstrated a significant increase in ejection fraction in WT mice treated with JTV519 (45.8 ± 5.1%) compared with placebo (31.1 ± 3.1%; P < 0.05). Coimmunoprecipitation experiments revealed increased amounts of calstabin-2 bound to the RyR2 channel in JTV519-treated WT mice. However, JTV519 did not show any of these beneficial effects in calstabin-2-/- mice with MI. Additionally, JTV519 improved skeletal muscle fatigue in WT and calstabin-2-/- mice with HF by increasing the binding of calstabin-1 to RyR1. The observation that treatment with JTV519 improved cardiac function in WT but not calstabin-2-/- mice indicates that calstabin-2 binding to RyR2 is required for the beneficial effects in failing hearts. We conclude that JTV519 may provide a specific way to treat the cardiac and skeletal muscle myopathy in HF by increasing calstabin binding to RyR. calcium FKBP12.6 myocardial infarction contractility

  • coupled gating between cardiac calcium release channels Ryanodine Receptors
    Circulation Research, 2001
    Co-Authors: Steven O Marx, Jana Gaburjakova, Marta Gaburjakova, Charles Henrikson, Karol Ondrias, Andrew R Marks
    Abstract:

    Abstract —Excitation-contraction coupling in heart muscle requires the activation of Ca2+-release channels/type 2 Ryanodine Receptors (RyR2s) by Ca2+ influx. RyR2s are arranged on the sarcoplasmic reticular membrane in closely packed arrays such that their large cytoplasmic domains contact one another. We now show that multiple RyR2s can be isolated under conditions such that they remain physically coupled to one another. When these coupled channels are examined in planar lipid bilayers, multiple channels exhibit simultaneous gating, termed “coupled gating.” Removal of the regulatory subunit, the FK506 binding protein (FKBP12.6), functionally but not physically uncouples multiple RyR2 channels. Coupled gating between RyR2 channels may be an important regulatory mechanism in excitation-contraction coupling as well as in other signaling pathways involving intracellular Ca2+ release.

  • phosphorylation dependent regulation of Ryanodine Receptors a novel role for leucine isoleucine zippers
    Journal of Cell Biology, 2001
    Co-Authors: Steven O Marx, Jana Gaburjakova, Marta Gaburjakova, Steven Reiken, Yuji Hisamatsu, Yiming Yang, Nora Rosemblit, Andrew R Marks
    Abstract:

    Ryanodine Receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function.

  • Ryanodine Receptors calcium release channels in heart failure and sudden cardiac death
    Journal of Molecular and Cellular Cardiology, 2001
    Co-Authors: Andrew R Marks
    Abstract:

    Abstract Calcium (Ca 2+ ) ions are second messengers in signaling pathways in all types of cells. They regulate muscle contraction, electrical signals which determine the cardiac rhythm and cell growth pathways in the heart. In the past decade cDNA cloning has provided clues as to the molecular structure of the intracellular Ca 2+ release channels (Ryanodine Receptors, RyR, and inositol 1,4,5-trisphosphate Receptors, IP3R) on the sarcoplasmic and endoplasmic reticulum (SR/ER) and an understanding of how these molecules regulate Ca 2+ homeostasis in the heart is beginning to emerge. The intracellular Ca 2+ release channels form a distinct class of ion channels distinguished by their structure, size, and function. Both RyRs and IP3Rs have gigantic cytoplasmic domains that serve as scaffolds for modulatory proteins that regulate the channel pore located in the carboxy terminal 10% of the channel sequence. The channels are tetramers comprised of four RyR or IP3R subunits. RyR2 is required for excitation-contraction (EC) coupling in the heart. Using co-sedimentation and co-immunoprecipitation we have defined a macromolecular complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases PP1 and PP2A, and an anchoring protein mAKAP. We have shown that protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and regulates the channel open probability (P o ). In failing human hearts RyR2 is PKA hyperphosphorylated resulting in defective channel function due to increased sensitivity to Ca 2+ -induced activation.

Longsheng Song - One of the best experts on this subject based on the ideXlab platform.

  • orphaned Ryanodine Receptors in the failing heart
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Longsheng Song, W J Lederer, Eric A Sobie, Stacey L Mcculle, William C Balke, Heping Cheng
    Abstract:

    Heart muscle is characterized by a regular array of proteins and structures that form a repeating functional unit identified as the sarcomere. This regular structure enables tight coupling between electrical activity and Ca2+ signaling. In heart failure, multiple cellular defects develop, including reduced contractility, altered Ca2+ signaling, and arrhythmias; however, the underlying causes of these defects are not well understood. Here, in ventricular myocytes from spontaneously hypertensive rats that develop heart failure, we identify fundamental changes in Ca2+ signaling that are related to restructuring of the spatial organization of the cells. Myocytes display both a reduced ability to trigger sarcoplasmic reticulum Ca2+ release and increased spatial dispersion of the transverse tubules (TTs). Remodeled TTs in cells from failing hearts no longer exist in the regularly organized structures found in normal heart cells, instead moving within the sarcomere away from the Z-line structures and leaving behind the sarcoplasmic reticulum Ca2+ release channels, the Ryanodine Receptors (RyRs). These orphaned RyRs appear to be responsible for the dyssynchronous Ca2+ sparks that have been linked to blunted contractility and, probably, Ca2+-dependent arrhythmias in diverse models of heart failure. We conclude that the increased spatial dispersion of the TTs and orphaned RyRs lead to the loss of local control and Ca2+ instability in heart failure.

  • the ca2 leak paradox and rogue Ryanodine Receptors sr ca2 efflux theory and practice
    Progress in Biophysics & Molecular Biology, 2006
    Co-Authors: Eric A Sobie, Longsheng Song, W J Lederer, Silvia Guatimosim, Leticia Gomezviquez, Hali Hartmann, Saleet M Jafri
    Abstract:

    Abstract Ca 2+ efflux from the sarcoplasmic reticulum (SR) is routed primarily through SR Ca 2+ release channels (Ryanodine Receptors, RyRs). When clusters of RyRs are activated by trigger Ca 2+ influx through L-type Ca 2+ channels (dihydropyridine Receptors, DHPR), Ca 2+ sparks are observed. Close spatial coupling between DHPRs and RyR clusters and the relative insensitivity of RyRs to be triggered by Ca 2+ together ensure the stability of this positive-feedback system of Ca 2+ amplification. Despite evidence from single channel RyR gating experiments that phosphorylation of RyRs by protein kinase A (PKA) or calcium-calmodulin dependent protein kinase II (CAMK II) causes an increase in the sensitivity of the RyR to be triggered by [Ca 2+ ] i there is little clear evidence to date showing an increase in Ca 2+ spark rate. Indeed, there is some evidence that the SR Ca 2+ content may be decreased in hyperadrenergic disease states. The question is whether or not these observations are compatible with each other and with the development of arrhythmogenic extrasystoles that can occur under these conditions. Furthermore, the appearance of an increase in the SR Ca 2+ “leak” under these conditions is perplexing. These and related complexities are analyzed and discussed in this report. Using simple mathematical modeling discussed in the context of recent experimental findings, a possible resolution to this paradox is proposed. The resolution depends upon two features of SR function that have not been confirmed directly but are broadly consistent with several lines of indirect evidence: (1) the existence of unclustered or “rogue” RyRs that may respond differently to local [Ca 2+ ] i in diastole and during the [Ca 2+ ] i transient; and (2) a decrease in cooperative or coupled gating between clustered RyRs in response to physiologic phosphorylation or hyper-phosphorylation of RyRs in disease states such as heart failure. Taken together, these two features may provide a framework that allows for an improved understanding of cardiac Ca 2+ signaling.

  • ca2 signalling between single l type ca2 channels and Ryanodine Receptors in heart cells
    Nature, 2001
    Co-Authors: Shiqiang Wang, Longsheng Song, Edward G Lakatta, Heping Cheng
    Abstract:

    Ca2+-induced Ca2+ release is a general mechanism that most cells use to amplify Ca2+ signals1,2,3,4,5. In heart cells, this mechanism is operated between voltage-gated L-type Ca2+ channels (LCCs) in the plasma membrane and Ca2+ release channels, commonly known as Ryanodine Receptors, in the sarcoplasmic reticulum3,4,5. The Ca2+ influx through LCCs traverses a cleft of roughly 12 nm formed by the cell surface and the sarcoplasmic reticulum membrane, and activates adjacent Ryanodine Receptors to release Ca2+ in the form of Ca2+ sparks6. Here we determine the kinetics, fidelity and stoichiometry of coupling between LCCs and Ryanodine Receptors. We show that the local Ca2+ signal produced by a single opening of an LCC, named a ‘Ca2+ sparklet’, can trigger about 4–6 Ryanodine Receptors to generate a Ca2+ spark. The coupling between LCCs and Ryanodine Receptors is stochastic, as judged by the exponential distribution of the coupling latency. The fraction of sparklets that successfully triggers a spark is less than unity and declines in a use-dependent manner. This optical analysis of single-channel communication affords a powerful means for elucidating Ca2+-signalling mechanisms at the molecular level.

  • local control models of cardiac excitation contraction coupling a possible role for allosteric interactions between Ryanodine Receptors
    The Journal of General Physiology, 1999
    Co-Authors: Michael D Stern, James S K Sham, Longsheng Song, Heping Cheng, Huangtian Yang, Kenneth R Boheler, Eduardo Rios
    Abstract:

    In cardiac muscle, release of activator calcium from the sarcoplasmic reticulum occurs by calcium- induced calcium release through Ryanodine Receptors (RyRs), which are clustered in a dense, regular, two-dimensional lattice array at the diad junction. We simulated numerically the stochastic dynamics of RyRs and L-type sarcolemmal calcium channels interacting via calcium nano-domains in the junctional cleft. Four putative RyR gating schemes based on single-channel measurements in lipid bilayers all failed to give stable excitation–contraction coupling, due either to insufficiently strong inactivation to terminate locally regenerative calcium-induced calcium release or insufficient cooperativity to discriminate against RyR activation by background calcium. If the Ryanodine receptor was represented, instead, by a phenomenological four-state gating scheme, with channel opening resulting from simultaneous binding of two Ca2+ ions, and either calcium-dependent or activation-linked inactivation, the simulations gave a good semiquantitative accounting for the macroscopic features of excitation–contraction coupling. It was possible to restore stability to a model based on a bilayer-derived gating scheme, by introducing allosteric interactions between nearest-neighbor RyRs so as to stabilize the inactivated state and produce cooperativity among calcium binding sites on different RyRs. Such allosteric coupling between RyRs may be a function of the foot process and lattice array, explaining their conservation during evolution.

  • termination of ca2 release by a local inactivation of Ryanodine Receptors in cardiac myocytes
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: James S K Sham, Longsheng Song, Edward G Lakatta, Ye Chen, Lihua Deng, Michael D Stern, Heping Cheng
    Abstract:

    In heart, a robust regulatory mechanism is required to counteract the regenerative Ca2+-induced Ca2+ release from the sarcoplasmic reticulum. Several mechanisms, including inactivation, adaptation, and stochastic closing of Ryanodine Receptors (RyRs) have been proposed, but no conclusive evidence has yet been provided. We probed the termination process of Ca2+ release by using a technique of imaging local Ca2+ release, or “Ca2+ spikes”, at subcellular sites; and we tracked the kinetics of Ca2+ release triggered by L-type Ca2+ channels. At 0 mV, Ca2+ release occurred and terminated within 40 ms after the onset of clamp pulses (0 mV). Increasing the open-duration and promoting the reopenings of Ca2+ channels with the Ca2+ channel agonist, FPL64176, did not prolong or trigger secondary Ca2+ spikes, even though two-thirds of the sarcoplasmic reticulum Ca2+ remained available for release. Latency of Ca2+ spikes coincided with the first openings but not with the reopenings of L-type Ca2+ channels. After an initial maximal release, even a multi-fold increase in unitary Ca2+ current induced by a hyperpolarization to −120 mV failed to trigger additional release, indicating absolute refractoriness of RyRs. When the release was submaximal (e.g., at +30 mV), tail currents did activate additional Ca2+ spikes; confocal images revealed that they originated from RyRs unfired during depolarization. These results indicate that Ca2+ release is terminated primarily by a highly localized, use-dependent inactivation of RyRs but not by the stochastic closing or adaptation of RyRs in intact ventricular myocytes.