Search

Many proteins are phosphorylated at more than one phosphorylation site to achieve precise tuning of protein function and/or integrate a multitude of signals into the activity of one protein. Increasing the number of phosphorylation sites significantly broadens the complexity of molecular mechanisms involved in processing multiple phosphorylation sites by one or more distinct kinases. The cardiac ryanodine receptor (RYR2) is a well-established multiple phospho-target of kinases activated in response to β-adrenergic stimulation because this Ca2+ channel is a critical component of Ca2+ handling machinery which is responsible for β-adrenergic enhancement of cardiac contractility. Our review presents a selective overview of the extensive, often conflicting, literature which focuses on identifying reliable lines of evidence to establish if multiple RYR2 phosphorylation is achieved randomly or in a specific sequence, and whether phosphorylation at individual sites is functionally specific and additive or similar and can therefore be substituted.

Background: Clinical treatment of heart failure is still suffering from limited efficacy and unfavorable side effects. The recently developed group of agents, the myosin motor activators, act directly on cardiac myosin resulting in an increased force generation and prolongation of contraction. The lead molecule, omecamtiv mecarbil is now in human 3 stage. In addition to the promising clinical data published so far, there are new in vitro results indicating that the effect of omecamtiv mecarbil on contractility is rate-dependent. Furthermore, omecamtiv mecarbil was shown to activate cardiac ryanodine receptors, an effect that may carry proarrhythmic risk. Methods: These new results, together with the controversial effects of the drug on cardiac oxygen consumption, are critically discussed in this review in light of the current literature on omecamtiv mecarbil. Results: In therapeutically relevant concentrations the beneficial inotropic effect of the agent is not likely affected by these new results - in accordance with the good clinical data. At supratherapeutic concentrations, however, activation of cardiac ryanodine receptors may increase arrhythmia propensity, and the stronger effect on diastolic than systolic cell shortening, observed at higher pacing frequencies, may decrease or offset the inotropic effect of omecamtiv mecarbil. Conclusion: Further studies with definitely supratherapeutical concentrations of omecamtiv mecarbil should be designed to map the actual risk of these potentially harmful side-effects.

Notwithstanding major advances in psychotherapeutics, their efficacy and specificity remain limited. The slow onset of beneficial outcomes and numerous adverse effects of widely used medications remain of chief concern, warranting in-depth studies. The majority of frontline therapies are thought to enhance the endogenous monoaminergic drive, to initiate a cascade of molecular events leading to lasting functional and structural plasticity. They also involve alterations in trophic factor signalling, including brain-derived neurotrophic factor (BDNF), VGF (non-acronymic), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), glial cell-derived neurotrophic factor (GDNF), and others. In several major mental disorders, emerging data suggest protective and restorative effects of trophic factors in preclinical models, when applied on their own. Antidepressant outcomes of VGF and FGF2, for instance, were shown in experimental animals, while BDNF and GDNF prove useful in the treatment of addiction, schizophrenia, and autism spectrum disorders. The main challenge with the effective translation of these and other findings in the clinic is the knowledge gap in action mechanisms with potential risks, as well as the lack of effective platforms for validation under clinical settings. Herein, we review the state-of-the-art and advances in the therapeutic use of trophic factors in several major neuropsychiatric disorders.

Iron, an essential element for most living organism, participates in a wide variety of physiological processes. Disturbance in iron homeostasis has been associated with numerous pathologies, particularly in the heart and brain, which are the most susceptible organs. Under iron-overload conditions, the generation of reactive oxygen species leads to impairment in Ca2+ signaling, fundamentally implicated in cardiac and neuronal physiology. Since iron excess is accompanied by increased expression of iron-storage protein, ferritin, we examined whether ferritin has an effect on the ryanodine receptor - isoform 2 (RYR2), which is one of the major components of Ca2+ signaling. Using the method of planar lipid membranes, we show that ferritin induced an abrupt, permanent blockage of the RYR2 channel. The ferritin effect was strongly voltage dependent and competitively antagonized by cytosolic TEA+, an impermeant RYR2 blocker. Our results collectively indicate that monomeric ferritin highly likely blocks the RYR2 channel by a direct electrostatic interaction within the wider region of the channel permeation pathway.

Dysregulation of the cardiac ryanodine receptor (RYR2) by luminal Ca

Ca2+ plays a critical role in several processes involved in skeletal and cardiac muscle contraction. One key step in cardiac excitation-contraction (E-C) coupling is the activation of the cardiac ryanodine receptor (RYR2) by cytosolic Ca2+ elevations. Although this process is not critical for skeletal E-C coupling, the activation and inhibition of the skeletal ryanodine receptor (RYR1) seem to be important for overall muscle function. The RYR1 and RYR2 channels fall within the large category of Ca2+ -binding proteins that harbour highly selective Ca2+ -binding sites to receive and translate the various Ca2+ signals into specific functional responses. However, little is known about the precise localization of these sites within the cytosolic assembly of both RYR isoforms, although several experimental lines of evidence have highlighted their EF-hand nature. EF-hand proteins share a common helix-loop-helix structural motif with highly conserved residues involved in Ca2+ coordination. The first step in predicting EF-hand positive regions is to compare the primary protein structure with the EF-hand motif by employing available bioinformatics tools. Although this simple method narrows down search regions, it does not provide solid evidence regarding which regions bind Ca2+ in both RYR isoforms. In this review, we seek to highlight the key findings and experimental approaches that should strengthen our future efforts to identify the cytosolic Ca2+ -binding sites responsible for activation and inhibition in the RYR1 channel, as much less work has been conducted on the RYR2 channel.

A growing body of evidence suggests that the regulation of cardiac ryanodine receptor (RYR2) by luminal Ca(2+) is mediated by luminal binding sites located on the RYR2 channel itself and/or its auxiliary protein, calsequestrin. The localization and structure of RYR2-resident binding sites are not known because of the lack of a high-resolution structure of RYR2 luminal regions. To obtain the first structural insight, we probed the RYR2 luminal face stripped of calsequestrin by alkaline earth metal divalents (M(2+): Mg(2+), Ca(2+), Sr(2+) or Ba(2+)). We show that the RYR2 response to caffeine at the single-channel level is significantly modified by the nature of luminal M(2+). Moreover, we performed competition experiments by varying the concentration of luminal M(2+) (Mg(2+), Sr(2+) or Ba(2+)) from 8 mM to 53 mM and investigated its ability to compete with 1mM luminal Ca(2+). We demonstrate that all tested M(2+) bind to exactly the same RYR2 luminal binding sites. Their affinities decrease in the order: Ca(2+)>Sr(2+)>Mg(2+)~Ba(2+), showing a strong correlation with the M(2+) affinity of the EF-hand motif. This indicates that the RYR2 luminal binding regions and the EF-hand motif likely share some structural similarities because the structure ties directly to the function.

Electrophysiological methods are the “gold standard” for ion channel research, providing valuable information about function and regulation even at the single-channel level. This is of critical importance in attempts to understand the molecular role of these transmembrane proteins in various diseases that remain ineffectively treated, leading to the development of novel ion channel therapeutics. To investigate their molecular function, ion channels are usually incorporated into model membrane systems such as planar lipid membranes (BLMs); however, this process is still challenging. This approach requires BLMs with the sufficient mechanical and electrical stability and the ability to integrate spherical membrane systems enriched in ion channel proteins. We review relevant discoveries in membrane biophysics that could provide a useful platform for electrophysiologists to achieve a notable improvement in the experimental throughput of the BLM method.

Due to the limited results achieved in the clinical treatment of heart failure, a new inotropic strategy of myosin motor activation has been developed. The lead molecule of myosin activator agents is omecamtiv mecarbil, which binds directly to the heavy chain of the cardiac β-myosin and enhances cardiac contractility by lengthening the lifetime of the acto-myosin complex and increasing the number of the active force-generating cross-bridges. In the absence of relevant data, the effect of omecamtiv mecarbil on canine cardiac ryanodine receptors (RyR 2) has been investigated in the present study by measuring the electrical activity of single RyR 2 channels incorporated into planar lipid bilayer. When applying 100nM Ca2+ concentration on the cis side ([Ca2+]cis) omecamtiv mecarbil (1-10µM) significantly increased the open probability and opening frequency of RyR 2, while the mean closed time was reduced. Mean open time was increased moderately by 10µM omecamtiv mecarbil. When [Ca2+]cis was elevated to 322 and 735nM, the effect of omecamtiv mecarbil on open probability was evident only at higher (3-10µM) concentrations. All effects of omecamtiv mecarbil were fully reversible upon washout. Omecamtiv mecarbil (up to 10µM) had no effect on the open probability of RyR 1, isolated from either canine or rabbit skeletal muscles. It is concluded that the direct stimulatory action of omecamtiv mecarbil on RyR 2 has to be taken into account when discussing the mechanism of action or the potential side effects of the compound.

The synergic effect of luminal Ca2+, cytosolic Ca2+, and cytosolic adenosine triphosphate (ATP) on activation of cardiac ryanodine receptor (RYR2) channels was examined in planar lipid bilayers. The dose–response of RYR2 gating activity to ATP was characterized at a diastolic cytosolic Ca2+ concentration of 100 nM over a range of luminal Ca2+ concentrations and, vice versa, at a diastolic luminal Ca2+ concentration of 1 mM over a range of cytosolic Ca2+ concentrations. Low level of luminal Ca2+ (1 mM) significantly increased the affinity of the RYR2 channel for ATP but without substantial activation of the channel. Higher levels of luminal Ca2+ (8–53 mM) markedly amplified the effects of ATP on the RYR2 activity by selectively increasing the maximal RYR2 activation by ATP, without affecting the affinity of the channel to ATP. Near-diastolic cytosolic Ca2+ levels (

The objective of this work was to identify and further characterize potential changes in the functional profile of the cardiac ryanodine receptor (RyR2) channel caused by the coupled gating phenomenon. By reconstituting an ion channel into a planar lipid membrane, we showed that coupled RyR2 channels were activated by cytosolic Ca(2+) with similar efficacy and potency as reported for the single RyR2 channel. In contrast, all examined parameters of gating kinetics were affected by the functional interaction between channels. Ignoring brief closings during main open events, the average open and closed times were considerably prolonged and the frequency of opening was reduced. Interestingly, when luminal Ca(2+) was used as a charge carrier, Ca(2+)-activated coupled RyR2 channels did not exhibit a sudden switch from slow to fast gating kinetics at an open probability of 0.5 as reported for the single RyR2 channel. Regarding flicker gating, the average closed time was significantly shorter and the frequency of closing was greatly enhanced. Furthermore, in contrast to the single RyR2 channel, both parameters for coupled channels were independent of cytosolic Ca(2+). Selected permeation properties of coupled RyR2 channels were comparable to those found for the single RyR2 channel. The Ca(2+) current amplitude-luminal Ca(2+) relationship displayed a simple saturation and the channel selectivity for Ba(2+) and Ca(2+) ions was similar. Our results suggest that the major targets influenced by coupled gating are likely the gates of individual RyR2 channels recruited into a functional complex, thus ensuring the correlation of Ca(2+) fluxes.

Aim: Two or more RYR2 channels reconstituted into a bilayer lipid membrane (BLM) can open and close either independently (single gating) or simultaneously (coupled gating). The coupled gating phenomenon has been suggested as an attractive candidate for a termination mechanism of Ca 2+ release from the sarcoplasmic reticulum, required for periodic contraction and relaxation of cardiac muscle. Methods: Using the method of reconstitution of a channel into the BLM, we investigated the potential effect of luminal Ca 2+ on the stability of the interaction between coupled RYR2 channels isolated from the rat heart. We introduced a new parameter - the coupling stability - for each detected simultaneous opening and closing and further averaged values for experiments performed under identical conditions. Results: We found that the coupling stability during simultaneous opening of RYR2 channels was significantly lower in comparison with the simultaneous closing under the same experimental conditions. Furthermore, high concentration of luminal Ca 2+ (53 mmol L -1 ) as well as the absence of luminal Ca 2+ noticeably destabilized functional coupling between coupled RYR2 channels during opening, in contrast to lower tested concentrations (8-20 mmol L -1 ). Conclusions: We provide experimental evidence that the strength of interaction between coupled RYR2 channels depends on the functional state of the channels. Furthermore, we show, for the first time, the regulation role of luminal Ca 2+ in the inter-RYR2 functional coupling in the rat heart.

Planar lipid bilayers represent a versatile platform for studying the functions of various membrane proteins as well as the development of biosensors. Despite the continuing technological progress in the fabrication of low-noise bilayer setups with mechanically and electrically stable planar bilayers, there is still a lack of software utilities for assistance during bilayer formation. We present here a multipurpose software tool, the bilayer lipid membrane (BLM) Analyzer, which performs high-resolution measurements of bilayer capacitance and resistance using saw-tooth voltage stimulation. Based on the measured values of capacitance and resistance, the BLM Analyzer detects formation, stabilization, and breakage of lipid bilayer, automatically selects appropriate stimulus protocol, compensates for voltage offsets, and issues sound and voice alerts informing about the state of the measurement cycle. The principle of the BLM Analyzer is based on the integration of current responses within four equivalent time segments. It provides capacitance estimates with standard deviation of several femtofarads at temporal resolution of several tens of milliseconds. The functions of the BLM Analyzer were tested experimentally by monitoring formation and thinning of planar lipid bilayer.

The type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium (Ca2+) release channel required for skeletal muscle excitation–contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are “leaky.” RyR1 PKA hyperphosphorylation correlated with impaired SR Ca2+ release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF.

The cardiac ryanodine receptor/calcium release channel (RyR2) on the sarcoplasmic reticulum (SR) comprises a macromolecular complex that includes a kinase and two phosphatases that are bound to the channel via targeting proteins. We previously found that the RyR2 is protein kinase A (PKA)-hyperphosphorylated in end-stage human heart failure. Because heart failure is a progressive disease that often evolves from hypertrophy, we analyzed the RyR2 macromolecular complex in several animal models of cardiomyopathy that lead to heart failure, including hypertrophy, and at different stages of disease progression. We now show that RyR2 is PKA-hyperphosphorylated in diverse models of heart failure and that the degree of RyR2 PKA phosphorylation correlates with the degree of cardiac dysfunction. Interestingly, we show that RyR2 PKA hyperphosphorylation can be lost during perfusion of isolated hearts due to the activity of the endogenous phosphatases in the RyR2 macromolecular complex. Moreover, infusion of isoproterenol resulted in PKA phosphorylation of RyR2 in rat, indicating that systemic catecholamines can activate phosphorylation of RyR2 in vivo. These studies extend our previous analyses of the RyR2 macromolecular complex, show that both the kinase and phosphatase activities in the macromolecular complex are regulated physiologically in vivo, and suggest that RyR2 PKA hyperphosphorylation is likely a general feature of heart failure.

β-Adrenergic receptor (βAR) signaling, which elevates intracellular cAMP and enhances cardiac contractility, is severely impaired in the failing heart. Protein kinase A (PKA) is activated by cAMP, but the long-term physiological effect of PKA activation on cardiac function is unclear. To investigate the consequences of chronic cardiac PKA activation in the absence of upstream events associated with βAR signaling, we generated transgenic mice that expressed the catalytic subunit of PKA in the heart. These mice developed dilated cardiomyopathy with reduced cardiac contractility, arrhythmias, and susceptibility to sudden death. As seen in human heart failure, these abnormalities correlated with PKA-mediated hyperphosphorylation of the cardiac ryanodine receptor/Ca 2+ -release channel, which enhances Ca 2+ release from the sarcoplasmic reticulum, and phospholamban, which regulates the sarcoplasmic reticulum Ca 2+ -ATPase. These findings demonstrate a specific role for PKA in the pathogenesis of heart failure, independent of more proximal events in βAR signaling, and support the notion that PKA activity is involved in the adverse effects of chronic βAR signaling.

Abstract —Excitation-contraction coupling in heart muscle requires the activation of Ca 2+ -release channels/type 2 ryanodine receptors (RyR2s) by Ca 2+ 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 Ca 2+ release.

Local calcium release via RYRs, observed in the form of calcium sparks ([Cheng et al., 1993][1]), is generally accepted to be a principal mediator of calcium homeostasis and excitation–contraction coupling in cardiac myocytes. However, investigation of local calcium release signals is extremely

Excitation-contraction (EC) coupling in muscle requires the activation of intracellular calcium release channels (CRC). Four type 1 ryanodine receptor (RyR1) molecules form each tetrameric CRC. Each RyR1 contains a binding site for the FK506 binding protein (FKBP12), a cis-trans peptidyl-prolyl isomerase that is required for coordinated gating of the four RyR1 subunits comprising the channel. 1,2 When FKBP12 is bound to RyR1, it stabilizes the four subunits that form each CRC. We propose that binding of one FKBP12 to each RyR1 lowers the energy of twisted-amide peptidyl-prolyl bonds and stabilizes RyR1 in a conformation that permits coordinated gating of the four RyR1 subunits.

The lipid bilayer is widely accepted as the basic structure of all biological membranes. Known as BLM (bilayer lipid membrane), it can be prepared artificially. Suitably modified, the BLM serves as a very appropriate model for biological membranes. Recent investigations have verified the high analytical potential of artificial lipid membranes. With a structure and composition almost identical to the lipid moiety of biomembranes, the BLM may serve as an ideal host for receptor molecules of biological origin, thus becoming a transducer which could “see” the environment the way the living cell does. For the construction of lipid bilayer based biosensors; however, stable, easy to prepare and long-lasting lipid membranes are required. With this aim in mind, we have prepared lipid bilayer membranes which use an agar gel as support. This as-BLM (agar-supported BLM) has been shown to possess the same electrical, mechanical and dynamic properties the conventional BLM is famous for, along with the benefits of long-term stability and considerably elevated breakdown voltages. Its preparation on the tip of an agar-filled Teflon tube of 0.5 mm diameter is easy and can be performed even by less-skilled personnel. In an attempt of further miniaturization the concept of the as-BLM was applied to thin-film micro-systems manufactured by standard micro-electronic techniques. The result is a lipid bilayer system, which, while preserving all the essential properties of the bilayer lipid membrane, can serve as a basic building block for cheap, disposable biosensoric systems.

Cardiac ryanodine receptors (RYR2s) 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 + (8 mM, the virtual absence of Ca 2 + ) and luminal Ca 2 + (8 mM), 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.

Evidence obtained in the last two decades indicates that calsequestrin (CSQ2), as the major Ca(2+)-binding protein in the sarcoplasmic reticulum of cardiac myocytes, communicates changes in the luminal Ca(2+) concentration to the cardiac ryanodine receptor (RYR2) channel. This review summarizes the major aspects in the interaction between CSQ2 and the RYR2 channel. The single channel properties of RYR2 channels, discussed here in the context of structural changes in CSQ2 after Ca(2+) binding, are particularly important. We focus on five important questions concerning: (1) the method for reliable detection of CSQ2 on the reconstituted RYR2 channel complex; (2) the power of the procedure to strip CSQ2 from the RYR2 channel complex; (3) structural changes in CSQ2 upon binding of Ca(2+) which cause CSQ2 dissociation; (4) the potential role of CSQ2-independent regulation of the RYR2 activity by luminal Ca(2+); and (5) the vizualization of CSQ2 dissociation from the RYR2 channel complex on the single channel level. We discuss the potential sources of the conflicting experimental results which may aid detailed understanding of the CSQ2 regulatory role. Although we mainly focus on the cardiac isoform of the proteins, some aspects of more extensive work carried out on the skeletal isoform are also discussed.

Anomalies in the permeation properties of the cardiac RyR channel reconstituted into bilayer lipid membranes were investigated systematically. We tested the presence of the anomalous mole fraction effect (AMFE) for the ion conductance and the reversal potential with varying mole fractions of two permeant ions, while the total ion concentration was lower, as in previous studies, to avoid the masking effect of the channel pore saturation with ions. Mixtures of Ba 2+ with other divalents (Ca 2+ , Sr 2+ ), of Ca 2+ with monovalents (Li + , Cs + ), and of Na + with other monovalents (Cs + , Li + ) were used. We revealed a clear anomaly only for the ion conductance measured in the Na + –Cs + and Ca 2+ –Li + mixtures as computed by a Poisson–Nernst–Planck/density functional theory (PNP/DFT) model. Furthermore, we found a significant minimum in the concentration dependence of the reversal potential determined under Li + /Ca 2+ bi-ionic conditions. Our study led to new observations that may have important implications for understanding the mechanisms involved in ion handling in the RyR channel pore; furthermore our results could be useful for further validation of ion permeation models developed for the RyR channel.

The objective of this work was to characterize in more detail the inhibition effect of diisothiocyanatostilbene-2',2-disulfonic acid (DIDS) on anion channels isolated from the rat heart mitochondria. The channels reconstituted into a planar lipid membrane displayed limited powers of discrimination between anions and cations and the ion conductance measured under asymmetric (250/50 mM KCl, cis/trans) and symmetric (150 mM KCl) conditions was approximately 100 pS. DIDS caused a dramatic decrease in the channel activity (IC(50) = 11.7 +/- 3.1 microM) only when it was added to the cis side of a planar lipid membrane. The inhibition was accompanied by the significant prolongation of closings and the shortening of openings within the burst as well as gaps between bursts were prolonged and durations of bursts were reduced. The blockade was complete and irreversible when concentration of DIDS was increased up to 200 microM. Our data indicate that DIDS is an allosteric blocker of mitochondrial anion channels and this specific effect could be used as a tool for reliable identification of anion channels on the functional level.

Ca(2+) released from the sarcoplasmic reticulum (SR) via ryanodine receptor type 2 (RYR2) is the key determinant of cardiac contractility. Although activity of RYR2 channels is primary controlled by Ca(2+) entry through the plasma membrane, there is growing evidence that Ca(2+) in the lumen of the SR can also be effectively involved in the regulation of RYR2 channel function. In the present study, we investigated the effect of luminal Ca(2+) on the response of RYR2 channels reconstituted into a planar lipid membrane to caffeine and Ca(2+) added to the cytosolic side of the channel. We performed two sets of experiments when the channel was exposed to either luminal Ba(2+) or Ca(2+). The given ion served also as a charge carrier. Luminal Ca(2+) effectively shifted the EC(50) for caffeine sensitivity to a lower concentration but did not modify the response of RYR2 channels to cytosolic Ca(2+). Importantly, luminal Ca(2+) exerted an effect on channel gating kinetics. Both the open and closed dwell times were considerably prolonged over the whole range (response to caffeine) or the partial range (response to cytosolic Ca(2+)) of open probability. Our results provide strong evidence that an alteration of the gating kinetics is the result of the interaction of luminal Ca(2+) with the luminally located Ca(2+) regulatory sites on the RYR2 channel complex.

The cardiac ryanodine receptor/calcium release channel (RyR2) on the sarcoplasmic reticulum (SR) comprises a macromolecular complex that includes a kinase and two phosphatases that are bound to the channel via targeting proteins. We previously found that the RyR2 is protein kinase A (PKA)-hyperphosphorylated in end-stage human heart failure. Because heart failure is a progressive disease that often evolves from hypertrophy, we analyzed the RyR2 macromolecular complex in several animal models of cardiomyopathy that lead to heart failure, including hypertrophy, and at different stages of disease progression. We now show that RyR2 is PKA-hyperphosphorylated in diverse models of heart failure and that the degree of RyR2 PKA phosphorylation correlates with the degree of cardiac dysfunction. Interestingly, we show that RyR2 PKA hyperphosphorylation can be lost during perfusion of isolated hearts due to the activity of the endogenous phosphatases in the RyR2 macromolecular complex. Moreover, infusion of isoproterenol resulted in PKA phosphorylation of RyR2 in rat, indicating that systemic catecholamines can activate phosphorylation of RyR2 in vivo. These studies extend our previous analyses of the RyR2 macromolecular complex, show that both the kinase and phosphatase activities in the macromolecular complex are regulated physiologically in vivo, and suggest that RyR2 PKA hyperphosphorylation is likely a general feature of heart failure.

Background β-Adrenergic receptor blockade is one of the most effective treatments for heart failure, a leading cause of mortality worldwide. The use of β-adrenergic receptor blockers in patients with heart failure is counterintuitive, however, because they are known to decrease contractility in normal hearts. The ryanodine receptor (RyR2) on cardiac sarcoplasmic reticulum is the key calcium release channel required for excitation-contraction coupling. In failing hearts, the stoichiometry and function of the RyR2 macromolecular complex is altered. Decreased levels of phosphatases (PP1 and PP2A) and hyperphosphorylation by protein kinase A result in dissociation of the regulatory protein FKBP12.6 and channels with increased open probability. Methods and Results Here, we show that systemic oral administration of a β-adrenergic receptor blocker reverses protein kinase A hyperphosphorylation of RyR2, restores the stoichiometry of the RyR2 macromolecular complex, and normalizes single-channel function in a canine model of heart failure. Conclusions These results may, in part, explain the improved cardiac function observed in heart failure patients treated with β-adrenergic receptor blockers.

The ryanodine receptor (RyR1)/calcium release channel on the sarcoplasmic reticulum of skeletal muscle is comprised of four 565,000-dalton RyR1s, each of which binds one FK506 binding protein (FKBP12). RyR1 is required for excitation-contraction coupling in skeletal muscle. FKBP12, a cis-transpeptidyl-prolyl isomerase, is required for the normal gating of the RyR1 channel. In the absence of FKBP12, RyR1 channels exhibit increased gating frequency, suggesting that FKBP12 “stabilizes” the channel in the open and closed states. We now show that substitution of a Gly, Glu, or Ile for Val2461 in RyR1 prevents FKBP12 binding to RyR1, resulting in channels with increased gating frequency. In the case of the V2461I mutant RyR1, normal channel function can be restored by adding FKBP12.6, an isoform of FKBP12. These data identify Val2461 as a critical residue required for FKBP12 binding to RyR1 and demonstrate the functional role for FKBP12 in the RyR1 channel complex.

The mechanism of activation of the cardiac ryanodine receptor (RyR2) by luminal Ca2+ in the presence of ATP was examined in planar lipid bilayers. The dose response of the RyR2 channel to ATP was characterized at a range of cytosolic (100-400 nM) and luminal (0.01 - 53 mM) Ca2+ concentrations. Luminal Ca2+ markedly increased the maximal open probability in the presence of ATP (POmax) and markedly decreased EC50 for ATP (EC50ATP). Cytosolic Ca2+, without substantially activating the RyR2 channel in the absence of ATP, greatly amplified the effects of 1 mM [Ca2+]L on POmax and EC50ATP. An allosteric model of RyR2 interaction with ATP showed that the increase in RyR2 open probability by luminal Ca2+ in the presence of ATP is induced by a decrease of the ATP dissociation constant (KATP) at low [Ca2+]L, by an increase in the positive allosteric effect of ATP on channel opening (decrease of fATP) at intermediate [Ca2+]L, and by an increase in the stability of the ATP-free RyR2 open state (decrease of KO0) at the highest [Ca2+]L. Increasing [Ca2+]C did not affect KATP but led to a parallel decrease of KO0 and fATP. These results suggest that the allosteric effect of ATP might be mediated by the energy of the ATP-free open state, i.e., indirectly by the effect of the occupancy of the channel by Ca2+ at the cytosolic and luminal sites on the stability of the open state. The increase of RyR2 open probability at diastolic levels of cytosolic Ca2+ by elevated luminal Ca2+ may play a role in the calcium overload induced Ca2+ release.Supported by APVV-0721-10, APVV-0628-10, VEGA2/0118/09 and VEGA 2/0190/10

Local character of calcium release in cardiac myocytes implies independent recruitment of calcium release units by triggering stimuli. Calcium release from individual CRUs also displays quantal character, interpreted either as recruitment of small cohorts of independent RYRs (1,2), or as recruitment of small cohorts of RYR clusters with coupled RYRs gating (3). We tested both interpretations on published experimental data using a model of virtual calcium release units (vCRUs) consisting of 1-10 clusters that were constructed in accordance with the experimentally observed cluster size distribution (4). If RYR gating was independent, vCRUs consisting of 3 or more clusters provided a good agreement between the model and experimental data (1). If RYR gating was coupled, the calcium release flux of vCRUs did not display quantal structure that would correspond to in situ observations. The model of RYR gating (2) combined with the model of independent RYRs in vCRUs (2) matched the experimentally observed calcium dependence of calcium spark frequency (5) under all conditions. However, the Mg2+-binding parameters of RYRs were in accordance with the single-channel observations (6) only for vCRUs composed of 3 or more clusters. In conclusion, these results favor independent over fully coupled RYR gating in situ, and predict the presence of at least 40 RYRs per cardiac calcium release unit.Supported by grants APVV-0441-09, APVV-0721-10, VEGA 2/0190/10, 2/0203/11, and 2/0197/11.References1. Wang SQ et al., PNAS 101: 3979-3984, 20042. Zahradnikova A et al., J. Gen. Physiol. 136: 101-116, 20103. Xie W et al., J. Gen. Physiol. 136: 129-133, 20104. Baddeley D et al., PNAS 106: 22275-22280, 20095. Lukyanenko V, Gyorke S, J Physiol. 521: 575-585, 19996. Zahradnikova A et al., Am J Physiol. 285, C1059-1070, 2003

After the incorporation of the tracheal microsomal membrane into bilayer lipid membrane (BLM), a new single channel permeable for calcium was observed. Using the BLM conditions, 53 mM Ca2+ in trans solution versus 200 nM Ca2+ in cis solution, the single calcium channel current at 0 mV was 1.4-2.1 pA and conductance was 62-75 pS. The channel Ca2+/K+ permeability ratio was 4.8. The open probability (P-open) was in the range of 0.7-0.97. The P-open, measured at -10 mV to +30 mV (trans-cis), was not voltage dependent. The channel was neither inhibited by 10-20 microM ruthenium red, a specific blocker of ryanodine calcium release channel, nor by 10-50 microM heparin, a specific blocker of IP3 receptor calcium release channel, and its activity was not influenced by addition of 0.1 mM MgATP. We suggest that the observed new channel is permeable for calcium, and it is neither identical with the known type 1 or 2 ryanodine calcium release channel, nor type 1 or 2 IP3 receptor calcium release channel.

Several biophysical methods have been used to study the interaction of the amphiphilic hormone derivative ACTHt-24, the wasp venom peptide mastoparan and the synthetic neuropeptide galparan with planar bilayer lipid membranes (BLM) and liposomes. Addition of ACTH1–24, galparan and mastoparan at different concentrations (0.1 μM to 0.1 mM) to BLM from soybean phosphatidylcholine leads to an increase of membrane electrical conductance to an extent that correlates with the activity of the different peptides on mast cells. Addition of ACTH1–24 to a suspension of DMPC + DMPG liposomes leads to an increase of dielectric relaxation time, however the less charged peptides did not influence the dielectric relaxation properties of liposomes. The velocity number [u], measured by ultrasonic velocimetry in suspensions of liposomes from DMPC, decreased after addition of the peptides. This reveals an increase of the adiabatic volume compressibility of the liposomes. The presence of ACTH1–24, galparan and mastoparan in liposomes of DMPC (mole fraction peptide/DMPC=7.7 × 10−4) resulted in different changes of absorption number Δ[αλ] of ultrasound at temperatures below and above the phase transition of DMPC. The results obtained are consistent with the idea that the lipid bilayer may function as a mediator for the receptor-independent stimulation of G-proteins.