Marta Gaburjakova

Comparison of the Effects Exerted by Luminal Ca2+ on the Sensitivity of the Cardiac Ryanodine Receptor to Caffeine and Cytosolic Ca2+

Ca2+ 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 Ca2+ entry through the plasma membrane, there is growing evidence that Ca2+ 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 Ca2+ on the response of RYR2 channels reconstituted into a planar lipid membrane to caffeine and Ca2+ added to the cytosolic side of the channel. We performed two sets of experiments when the channel was exposed to either luminal Ba2+ or Ca2+. The given ion served also as a charge carrier. Luminal Ca2+ effectively shifted the EC50 for caffeine sensitivity to a lower concentration but did not modify the response of RYR2 channels to cytosolic Ca2+. Importantly, luminal Ca2+ 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 Ca2+) of open probability. Our results provide strong evidence that an alteration of the gating kinetics is the result of the interaction of luminal Ca2+ with the luminally located Ca2+ regulatory sites on the RYR2 channel complex.

Functional interaction between calsequestrin and ryanodine receptor in the heart

Evidence obtained in the last two decades indicates that calsequestrin (CSQ2), as the major Ca2+-binding protein in the sarcoplasmic reticulum of cardiac myocytes, communicates changes in the luminal Ca2+ 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 Ca2+ 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 Ca2+ which cause CSQ2 dissociation; (4) the potential role of CSQ2-independent regulation of the RYR2 activity by luminal Ca2+; 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.

Coupled gating modifies the regulation of cardiac ryanodine receptors by luminal Ca2

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+) (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+) (90nM) 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.

The cardiac ryanodine receptor: Looking for anomalies in permeation properties

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.

Cardiac ryanodine receptor: Selectivity for alkaline earth metal cations points to the EF-hand nature of luminal binding sites.

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.

Challenging quantal calcium signaling in cardiac myocytes

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

Omecamtiv mecarbil activates ryanodine receptors from canine cardiac but not skeletal muscle

&NA; 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 &bgr;‐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 100 nM Ca2+ concentration on the cis side ([Ca2+]cis) omecamtiv mecarbil (1–10 &mgr;M) 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 10 &mgr;M omecamtiv mecarbil. When [Ca2+]cis was elevated to 322 and 735 nM, the effect of omecamtiv mecarbil on open probability was evident only at higher (3–10 &mgr;M) concentrations. All effects of omecamtiv mecarbil were fully reversible upon washout. Omecamtiv mecarbil (up to 10 &mgr;M) 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.

BLM Analyzer: a software tool for experiments on planar lipid bilayers.

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.

Properties of a new calcium-permeable single channel from tracheal microsomes

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.

Omecamtiv Mecarbil: A Myosin Motor Activator Agent with Promising Clinical Performance and New in vitro Results

BACKGROUNDnClinical 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.nnnMETHODSnThese 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.nnnRESULTSnIn 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.nnnCONCLUSIONnFurther studies with definitely supratherapeutical concentrations of omecamtiv mecarbil should be designed to map the actual risk of these potentially harmful side-effects.