Julio A. Copello

Removal of FKBP12.6 Does Not Alter the Conductance and Activation of the Cardiac Ryanodine Receptor or the Susceptibility to Stress-induced Ventricular Arrhythmias

The 12.6-kDa FK506-binding protein (FKBP12.6) is considered to be a key regulator of the cardiac ryanodine receptor (RyR2), but its precise role in RyR2 function is complex and controversial. In the present study we investigated the impact of FKBP12.6 removal on the properties of the RyR2 channel and the propensity for spontaneous Ca2+ release and the occurrence of ventricular arrhythmias. Single channel recordings in lipid bilayers showed that FK506 treatment of recombinant RyR2 co-expressed with or without FKBP12.6 or native canine RyR2 did not induce long-lived subconductance states. [3H]Ryanodine binding studies revealed that coexpression with or without FKBP12.6 or treatment with or without FK506 did not alter the sensitivity of RyR2 to activation by Ca2+ or caffeine. Furthermore, single cell Ca2+ imaging analyses demonstrated that HEK293 cells co-expressing RyR2 and FKBP12.6 or expressing RyR2 alone displayed the same propensity for spontaneous Ca2+ release or store overload-induced Ca2+ release (SOICR). FK506 increased the amplitude and decreased the frequency of SOICR in HEK293 cells expressing RyR2 with or without FKBP12.6, indicating that the action of FK506 on SOICR is independent of FKBP12.6. As with recombinant RyR2, the conductance and ligand-gating properties of single RyR2 channels from FKBP12.6-null mice were indistinguishable from those of single wild type channels. Moreover, FKBP12.6-null mice did not exhibit enhanced susceptibility to stress-induced ventricular arrhythmias, in contrast to previous reports. Collectively, our results demonstrate that the loss of FKBP12.6 has no significant effect on the conduction and activation of RyR2 or the propensity for spontaneous Ca2+ release and stress-induced ventricular arrhythmias.

Single ryanodine receptor channel basis of caffeine's action on Ca2+ sparks.

Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca(2+) sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca(2+) level on both sides of the channel. Cytosolic Ca(2+) enhanced RyR2 caffeine affinity, whereas luminal Ca(2+) essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC(50) of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca(2+) spark frequency ∼75% and single RyR2 opening frequency ∼150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca(2+) sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms.

Halothane modulation of skeletal muscle ryanodine receptors: dependence on Ca2+, Mg2+, and ATP

Malignant hyperthermia (MH) susceptibility is a genetic disorder of skeletal muscle associated with mutations in the ryanodine receptor isoform 1 (RyR1) of sarcoplasmic reticulum (SR). In MH-susceptible skeletal fibers, RyR1-mediated Ca(2+) release is highly sensitive to activation by the volatile anesthetic halothane. Indeed, studies with isolated RyR1 channels (using simple Cs(+) solutions) found that halothane selectively affects mutated but not wild-type RyR1 function. However, studies in skeletal fibers indicate that halothane can also activate wild-type RyR1-mediated Ca(2+) release. We hypothesized that endogenous RyR1 agonists (ATP, lumenal Ca(2+)) may increase RyR1 sensitivity to halothane. Consequently, we studied how these agonists affect halothane action on rabbit skeletal RyR1 reconstituted into planar lipid bilayers. We found that cytosolic ATP is required for halothane-induced activation of the skeletal RyR1. Unlike RyR1, cardiac RyR2 (much less sensitive to ATP) responded to halothane even in the absence of this agonist. ATP-dependent halothane activation of RyR1 was enhanced by cytosolic Ca(2+) (channel agonist) and counteracted by Mg(2+) (channel inhibitor). Dantrolene, a muscle relaxant used to treat MH episodes, did not affect RyR1 or RyR2 basal activity and did not interfere with halothane-induced activation. Studies with skeletal SR microsomes confirmed that halothane-induced RyR1-mediated SR Ca(2+) release is enhanced by high ATP-low Mg(2+) in the cytosol and by increased SR Ca(2+) load. Thus, physiological or pathological processes that induce changes in cellular levels of these modulators could affect RyR1 sensitivity to halothane in skeletal fibers, including the outcome of halothane-induced contracture tests used to diagnose MH susceptibility.

CGP-37157 Inhibits the Sarcoplasmic Reticulum Ca2+ ATPase and Activates Ryanodine Receptor Channels in Striated Muscle

7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one [CGP-37157 (CGP)], a benzothiazepine derivative of clonazepam, is commonly used as a blocker of the mitochondrial Na+/Ca2+ exchanger. However, evidence suggests that CGP could also affect other targets, such as L-type Ca2+ channels and plasmalemma Na+/Ca2+ exchanger. Here, we tested the possibility of a direct modulation of ryanodine receptor channels (RyRs) and/or sarco/endoplasmic reticulum Ca2+-stimulated ATPase (SERCA) by CGP. In the presence of ruthenium red (inhibitor of RyRs), CGP decreased SERCA-mediated Ca2+ uptake of cardiac and skeletal sarcoplasmic reticulum (SR) microsomes (IC50 values of 6.6 and 9.9 μM, respectively). The CGP effects on SERCA activity correlated with a decreased Vmax of ATPase activity of SERCA-enriched skeletal SR fractions. CGP (≥5 μM) also increased RyR-mediated Ca2+ leak from skeletal SR microsomes. Planar bilayer studies confirmed that both cardiac and skeletal RyRs are directly activated by CGP (EC50 values of 9.4 and 12.0 μM, respectively). In summary, we found that CGP inhibits SERCA and activates RyR channels. Hence, the action of CGP on cellular Ca2+ homeostasis reported in the literature of cardiac, skeletal muscle, and other nonmuscle systems requires further analysis to take into account the contribution of all CGP-sensitive Ca2+ transporters.

Modulation of cardiac ryanodine receptor channels by alkaline earth cations.

Cardiac ryanodine receptor (RyR2) function is modulated by Ca2+ and Mg2+. To better characterize Ca2+ and Mg2+ binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M2+: Mg2+, Ca2+, Sr2+, Ba2+) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M2+ binding to high affinity activating sites at the cytosolic channel surface, specific for Ca2+ or Sr2+. This activation was interfered by Mg2+ and Ba2+ acting at low affinity M2+-unspecific binding sites. When testing the effects of luminal M2+ as current carriers, all M2+ increased maximal RyR2 open probability (compared to Cs+), suggesting the existence of low affinity activating M2+-unspecific sites at the luminal surface. Responses to M2+ vary from channel to channel (heterogeneity). However, with luminal Ba2+or Mg2+, RyR2 were less sensitive to cytosolic Ca2+ and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca2+or Sr2+). Kinetics of RyR2 with mixtures of luminal Ba2+/Ca2+ and additive action of luminal plus cytosolic Ba2+ or Mg2+ suggest luminal M2+ differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca2+/Sr2+-specific sites, which stabilize high Po mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca2+ activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M2+ binding sites (specific for Ca2+ and unspecific for Ca2+/Mg2+) that dynamically modulate channel activity and gating status, depending on SR voltage.

Cross-reactivity of Ryanodine Receptors with Plasma Membrane Ion Channel Modulators

Various pharmacological agents designed to modulate plasma membrane ion channels seem to significantly affect intracellular Ca2+ signaling when acting on their target receptor. Some agents could also cross-react (modulate channels or receptors beyond their putative target) with intracellular Ca2+ transporters. This study investigated the potential of thirty putative modulators of either plasma membrane K+, Na+, or transient receptor potential (TRP) channels to cross-react with intracellular Ca2+ release channels [i.e., ryanodine receptors (RyRs)] from skeletal muscle sarcoplasmic reticulum (SR). Screening for cross-reactivity of these various agents was performed by measuring the rate of spontaneous Ca2+ leak or caffeine-induced Ca2+ release from SR microsomes. Four of the agents displayed a strong cross-reactivity and were further evaluated with skeletal RyR (RyR1) reconstituted into planar bilayers. 6,12,19,20,25,26-Hexahydro-5,27:13,18:21,24-trietheno-11,7-metheno-7H-dibenzo [b,n][1,5,12,16]tetraazacyclotricosine-5, 13-diium dibromide (UCL 1684; K+ channel antagonist) and lamotrigine (Na+ channel antagonist) were found to significantly inhibit the RyR1-mediated caffeine-induced Ca2+ release. TRP channel agonists anandamide and (−)menthol were found to inhibit and activate RyR1, respectively. High concentrations of nine other agents produced partial inhibition of RyR1-mediated Ca2+ release from SR microsomes. Various pharmacological agents, especially TRP modulators, also inhibited a minor RyR1-independent component of the SR Ca2+ leak. Overall, ∼43% of the agents selected cross-reacted with RyR1-mediated and/or RyR1-independent Ca2+ leak from intracellular stores. Thus, cross-reactivity should be considered when using these classes of pharmacological agents to determine the role of plasmalemmal channels in Ca2+ homeostasis.

Voltage-Dependent Modulation of Cardiac Ryanodine Receptors (RyR2) by Protamine

It has been reported that protamine (>10 µg/ml) blocks single skeletal RyR1 channels and inhibits RyR1-mediated Ca2+ release from sarcoplasmic reticulum microsomes. We extended these studies to cardiac RyR2 reconstituted into planar lipid bilayers. We found that protamine (0.02–20 µg/ml) added to the cytosolic surface of fully activated RyR2 affected channel activity in a voltage-dependent manner. At membrane voltage (Vm; SR lumen - cytosol) = 0 mV, protamine induced conductance transitions to several intermediate states (substates) as well as full block of RyR2. At Vm>10 mV, the substate with the highest level of conductance was predominant. Increasing Vm from 0 to +80 mV, decreased the number of transitions and residence of the channel in this substate. The drop in current amplitude (full opening to substate) had the same magnitude at 0 and +80 mV despite the ∼3-fold increase in amplitude of the full opening. This is more similar to rectification of channel conductance induced by other polycations than to the action of selective conductance modifiers (ryanoids, imperatoxin). A distinctive effect of protamine (which might be shared with polylysines and histones but not with non-peptidic polycations) is the activation of RyR2 in the presence of nanomolar cytosolic Ca2+ and millimolar Mg2+ levels. Our results suggest that RyRs would be subject to dual modulation (activation and block) by polycationic domains of neighboring proteins via electrostatic interactions. Understanding these interactions could be important as such anomalies may be associated with the increased RyR2-mediated Ca2+ leak observed in cardiac diseases.

K201 (JTV519) is a Ca2+-Dependent Blocker of SERCA and a Partial Agonist of Ryanodine Receptors in Striated Muscle.

K201 (JTV-519) may prevent abnormal Ca2+ leak from the sarcoplasmic reticulum (SR) in the ischemic heart and skeletal muscle (SkM) by stabilizing the ryanodine receptors (RyRs; RyR1 and RyR2, respectively). We tested direct modulation of the SR Ca2+-stimulated ATPase (SERCA) and RyRs by K201. In isolated cardiac and SkM SR microsomes, K201 slowed the rate of SR Ca2+ loading, suggesting potential SERCA block and/or RyR agonism. K201 displayed Ca2+-dependent inhibition of SERCA-dependent ATPase activity, which was measured in microsomes incubated with 200, 2, and 0.25 µM Ca2+ and with the half-maximal K201 inhibitory doses (IC50) estimated at 130, 19, and 9 µM (cardiac muscle) and 104, 13, and 5 µM (SkM SR). K201 (≥5 µM) increased RyR1-mediated Ca2+ release from SkM microsomes. Maximal K201 doses at 80 µM produced ∼37% of the increase in SkM SR Ca2+ release observed with the RyR agonist caffeine. K201 (≥5 µM) increased the open probability (Po) of very active (“high-activity”) RyR1 of SkM reconstituted into bilayers, but it had no effect on “low-activity” channels. Likewise, K201 activated cardiac RyR2 under systolic Ca2+ conditions (∼5 µM; channels at Po ∼0.3) but not under diastolic Ca2+ conditions (∼100 nM; Po < 0.01). Thus, K201-induced the inhibition of SR Ca2+ leak found in cell-system studies may relate to potentially potent SERCA block under resting Ca2+ conditions. SERCA block likely produces mild SR depletion in normal conditions but could prevent SR Ca2+ overload under pathologic conditions, thus precluding abnormal RyR-mediated Ca2+ release.

Eudistomin D and Penaresin Derivatives as Modulators of Ryanodine Receptor Channels and Sarcoplasmic Reticulum Ca2+ ATPase in Striated Muscle

Eudistomin D (EuD) and penaresin (Pen) derivatives are bioactive alkaloids from marine sponges found to induce Ca2+ release from striated muscle sarcoplasmic reticulum (SR). Although these alkaloids are believed to affect ryanodine receptor (RyR) gating in a “caffeine-like” manner, no single-channel study confirmed this assumption. Here, EuD and MBED (9-methyl-7-bromoeudistomin D) were contrasted against caffeine on their ability to modulate the SR Ca2+ loading/leak from cardiac and skeletal muscle SR microsomes as well as the function of RyRs in planar bilayers. The effects of these alkaloids on [3H]ryanodine binding and SR Ca2+ ATPase (SERCA) activity were also tested. MBED (1–5 μM) fully mimicked maximal activating effects of caffeine (20 mM) on SR Ca2+ leak. At the single-channel level, MBED mimicked the agonistic action of caffeine on cardiac RyR gating (i.e., stabilized long openings characteristic of “high-open-probability” mode). EuD was a partial agonist at the maximal doses tested. The tested Pen derivatives displayed mild to no agonism on RyRs, SR Ca2+ leak, or [3H]ryanodine binding studies. Unlike caffeine, EuD and some Pen derivatives significantly inhibited SERCA at concentrations required to modulate RyRs. Instead, MBEDs affinity for RyRs (EC50 ∼0.5 μM) was much larger than for SERCA (IC50 > 285 μM). In conclusion, MBED is a potent RyR agonist and, potentially, a better choice than caffeine for microsomal and cell studies due to its reported lack of effects on adenosine receptors and phosphodiesterases. As a high-affinity caffeine-like probe, MBED could also help identify the caffeine-binding site in RyRs.

Effect of Phosphorylation, Cytoskeleton and FK-506 Binding Protein on the Gating of Coupled Skeletal Ryanodine Receptors

In skeletal muscle fibers, local Ca2+ sparks and global Ca2+ transients arise from the synchronous activation of arrays of skeletal ryanodine receptors (RyR1) in the sarcoplasmic reticulum. Marx et al. (1998) first described that synchronous Ca2+ signaling in cells could be explained by the coordinated gating of neighboring RyR1; i.e. coupled gating. We have previously reported that coupled gating of multiple RyR1 requires luminal Ca2+ as current carrier and ATP/Mg2+ in the cytosolic solution. As ATP is the most effective nucleotide for coupled gating in the presence of cytosolic Mg2+, the role of ATP to modulate RyR1 as a substrate of protein kinases to phosphorylate the channel or to stabilize RyR1-RyR1 interactions via the cytoskeleton remains possible. Consequently, we investigate the role of kinases/phosphatases and cytoskeleton modulators (colchicine and cytochalasin D) on RyR-mediated Ca2+ leak from SR microsomes as well as on coupled RyR1. We also investigated role of FKBP12 in coupled RyR1 gating by adding FKBP12 to partially coupled RyR1 or rapamycin to coupled RyR1 in planar lipid bilayers. Our results suggest the RyR1-RyR1 interactions that are essential for coupled gating were not significantly affected by addition of kinases/phosphatases, cytoskeleton modulators or the addition/removal of FKBP12 by rapamycin. Yet, some of the agents affected the overall activity of the RyR1. (Supported by NIH R01 GM078665).