Distribution and Function of Cardiac Ryanodine Receptor Clusters in Live Ventricular Myocytes

The cardiac Ca²⁺ release channel (ryanodine receptor, RyR2) plays an essential role in excitation-contraction coupling in cardiac muscle cells. Effective and stable excitation-contraction coupling critically depends not only on the expression of RyR2, but also on its distribution. Despite its importance, little is known about the distribution and organization of RyR2 in living cells. To study the distribution of RyR2 in living cardiomyocytes, we generated a knock-in mouse model expressing a GFP-tagged RyR2 (GFP-RyR2). Confocal imaging of live ventricular myocytes isolated from the GFP-RyR2 mouse heart revealed clusters of GFP-RyR2 organized in rows with a striated pattern. Similar organization of GFP-RyR2 clusters was observed in fixed ventricular myocytes. Immunofluorescence staining with the anti-α-actinin antibody (a z-line marker) showed that nearly all GFP-RyR2 clusters were localized in the z-line zone. There were small regions with dislocated GFP-RyR2 clusters. Interestingly, these same regions also displayed dislocated z-lines. Staining with di-8-ANEPPS revealed that nearly all GFP-RyR2 clusters were co-localized with transverse but not longitudinal tubules, whereas staining with MitoTracker Red showed that GFP-RyR2 clusters were not co-localized with mitochondria in live ventricular myocytes. We also found GFP-RyR2 clusters interspersed between z-lines only at the periphery of live ventricular myocytes. Simultaneous detection of GFP-RyR2 clusters and Ca²⁺ sparks showed that Ca²⁺ sparks originated exclusively from RyR2 clusters. Ca²⁺ sparks from RyR2 clusters induced no detectable changes in mitochondrial Ca²⁺ level. These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes.     

Ca2+ sparks and embers of mammalian muscle. Properties of the sources

Ca2+ sparks of membrane-permeabilized rat muscle cells were analyzed to derive properties of their sources. Most events identified in longitudinal confocal line scans looked like sparks, but 23% (1,000 out of 4,300) were followed by long-lasting embers. Some were preceded by embers, and 48 were "lone embers." Average spatial width was approximately 2 microm in the rat and 1.5 microm in frog events in analogous solutions. Amplitudes were 33% smaller and rise times 50% greater in the rat. Differences were highly significant. The greater spatial width was not a consequence of greater open time of the rat source, and was greatest at the shortest rise times, suggesting a wider Ca2+ source. In the rat, but not the frog, spark width was greater in scans transversal to the fiber axis. These features suggested that rat spark sources were elongated transversally. Ca2+ release was calculated in averages of sparks with long embers. Release current during the averaged ember started at 3 or 7 pA (depending on assumptions), whereas in lone embers it was 0.7 or 1.3 pA, which suggests that embers that trail sparks start with five open channels. Analysis of a spark with leading ember yielded a current ratio ranging from 37 to 160 in spark and ember, as if 37-160 channels opened in the spark. In simulations, 25-60 pA of Ca2+ current exiting a point source was required to reproduce frog sparks. 130 pA, exiting a cylindric source of 3 microm, qualitatively reproduced rat sparks. In conclusion, sparks of rat muscle require a greater current than frog sparks, exiting a source elongated transversally to the fiber axis, constituted by 35-260 channels. Not infrequently, a few of those remain open and produce the trailing ember.     

Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release

Aberrant Zn²⁺ homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn²⁺ modulates cardiac ryanodine receptor gating and Ca²⁺ dynamics in isolated cardiomyocytes. We reveal that Zn²⁺ is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn²⁺ concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca²⁺. At concentrations of free Zn²⁺ >1 nm, Zn²⁺ is the main activating ligand, and the dependence on Ca²⁺ is removed. Zn²⁺ is therefore a higher affinity activator of RyR2 than Ca²⁺. Millimolar levels of free Zn²⁺ were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn²⁺ modulates both the frequency and amplitude of Ca²⁺ waves in a concentration-dependent manner and that physiological levels of Zn²⁺ elicit Ca²⁺ release in the absence of activating levels of cytosolic Ca²⁺. This highlights a new role for intracellular Zn²⁺ in shaping Ca²⁺ dynamics in cardiomyocytes through modulation of RyR2 gating.     

A calcium conducting channel akin to a calcium pump

Summary Calcium conducting channels were studied in blebs of sarcoplasmic reticulum described by Stein & Palade (1988). The calcium channels had at least three conductance states (70 pS, 50 pS and 37 pS) and were weakly selective for calcium ions, with a permeability ratio Ca²⁺ to K⁺ of about 3.4. The open probability of the channel was strongly voltage dependent, decreasing at positive membrane voltages. 10 m ryanodine and 5 m ruthenium red had no effect on this channel; neither did millimolar concentrations of ATP, Mg²⁺, caffeine, and Ca²⁺, implying that the calcium conducting channels are not ryanodine receptors. Several calcium pump inhibitors—namely, vanadate, AlF ₄ ^– , reactive red 120, and cyclopiazonic acid—had obvious effects on the calcium conducting channels, suggesting that the calcium conducting channel of SR membrane blebs is some form of the SR calcium pump.We thank the National Science Foundation for steadfast support.We thank Drs. F. Cohen, A. Fox, R. Levis and E. Rios for much useful help and criticism and Dr. G. Inesi for sending us his paper while in press.     

Dynamic interreceptor coupling: a novel working mechanism of two-dimensional ryanodine receptor array

Ryanodine receptors (RyRs) usually form two-dimensional regular array in sarcoplasmic reticulum membranes in muscle cells. The inter-RyRs coupling may be essential for the maintenance of quiescent Ca2+ release in resting state, as well as for the coordinated activation and rapid termination of RyR-mediated Ca2+ release during excitation-contraction coupling. In our previous work, we have reported that the inter-RyRs interaction is modulated by RyR channel's functional state, which inspired us to propose a novel working mechanism of RyR array: "dynamic inter-RyR coupling". In this work, we built a simple model based on cellular automata and the Monte-Carlo method to quantitatively investigate the roles of intermolecular coupling and its modulation in regulating the signaling capabilities of RyR array. Our simulation results showed that with a suitable inter-RyR coupling strength, the combination of rest stability and high response efficiency, namely optimal signal/noise ratio, of Ca2+ signaling could be achieved. Moreover, we also found the continued coupling between open RyRs would delay the system termination rate. The coacquisition of robust termination of array opening relied on the proper decrease of coupling strength between activated RyRs. Obviously, such temporally asymmetric coupling would simultaneously endow the system with physiologically relevant resting stability and fast termination.     

Biochemical characterization of the Ca2+ release channel of skeletal and cardiac sarcoplasmic reticulum

Summary Rapid mixing-vesicle ion flux and planar lipid bilayer-single channel measurements have shown that a high-conductance, ligand-gated Ca²⁺ release channel is present in heavy, junctional-derived membrane fractions of skeletal and cardiac muscle sarcoplasmic reticulum. Using the release channel-specific probe, ryanodine, a 30S protein complex composed of polypeptides of M_r 400 000 has been isolated from cardiac and skeletal muscle. Reconstitution of the complex into planar lipid bilayers has revealed a Ca²⁺ conductance with properties characteristic of the native Ca²⁺ release channel.     

Calmodulin binding to the 3614-3643 region of RyR1 is not essential for excitation-contraction coupling in skeletal myotubes

Calmodulin is a ubiquitous Ca(2+) binding protein that modulates the in vitro activity of the skeletal muscle ryanodine receptor (RyR1). Residues 3614-3643 of RyR1 comprise the CaM binding domain and mutations within this region result in a loss of both high-affinity Ca(2+)-bound calmodulin (CaCaM) and Ca(2+)-free CaM (apoCaM) binding (L3624D) or only CaCaM binding (W3620A). To investigate the functional role of CaM binding to this region of RyR1 in intact skeletal muscle, we compared the ability of RyR1, L3624D, and W3620A to restore excitation-contraction (EC) coupling after expression in RyR1-deficient (dyspedic) myotubes. W3620A-expressing cells responded normally to 10 mM caffeine and 500 microM 4-chloro-m-cresol (4-cmc). Interestingly, L3624D-expressing cells displayed a bimodal response to caffeine, with a large proportion of cells ( approximately 44%) showing a greatly attenuated response to caffeine. However, high and low caffeine-responsive L3624D-expressing myotubes exhibited Ca(2+) transients of similar magnitude after activation by 4-cmc (500 microM) and electrical stimulation. Expression of either L3624D or W3620A in dyspedic myotubes restored both L-type Ca(2+) currents (retrograde coupling) and voltage-gated SR Ca(2+) release (orthograde coupling) to a similar degree as that observed for wild-type RyR1, although L-current density was somewhat larger and activated at more hyperpolarized potentials in W3620A-expressing myotubes. The results indicate that CaM binding to the 3614-3643 region of RyR1 is not essential for voltage sensor activation of RyR1.     

Appraisal of the physiological relevance of two hypotheses for the mechanism of calcium release from the mammalian cardiac sarcoplasmic reticulum: calcium-induced release versus charge-coupled release

Recent studies correlating the calcium current with, respectively, the clamp-imposed voltage and the calcium current in intact isolated mammalian cardiac myocytes are reviewed. The major findings are the following: [1] With the exception of one group, all investigators agree that a calcium transient is never observed in the absence of a calcium current. In addition, there is a good correlation between voltage dependence of the calcium current and that of the calcium transient, although this correlation may vary among the cardiac tissues from different animal species. [2] Repolarization clamp pulses from highly positive potentials produce a tail current which is associated with a tail calcium transient. [3] The calcium transient is inhibited when the calcium current is blocked by calcium deprivation or substitution, or by the addition of calcium current antagonists, despite the fact that sarcoplasmic reticulum still contains calcium that can be released by caffeine (with inhibition of this release by ryanodine). These three findings are strongly in favor of a calcium-induced release of calcium and against the hypothesis of charge-movement-coupled release of calcium from the sarcoplasmic reticulum. [4] The only finding that would be more in favor of the latter hypothesis (although till reconciliable with the former) is that repolarization occurring before the rapid rise of calcium transient is complete curtails the calcium transient. Thus, the possibility that charge movement might somehow regulate calcium-induced release of calcium cannot be excluded.     

Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor

Type 1 ryanodine receptors (RyR1s) release Ca²⁺ from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca²⁺ release response in HEK293 cells and bound the RyR-specific ligand [³H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K⁺ conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca²⁺ release in HEK293 cells, low [³H]ryanodine binding levels, and channels that were not regulated by Ca²⁺ and did not conduct Ca²⁺ in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes.     

Ryanodine as a functional probe of the skeletal muscle sarcoplasmic reticulum Ca2+ release channel

Ryanodine is a neutral plant alkaloid which functions as a probe for an intracellular Ca²⁺ release channel (ryanodine receptor) in excitable tissues. Using [³H]ryanodine, a 30 S protein complex comprised of four polypeptides of M_r 565,000 has been isolated and functionally reconstituted into planar lipid bilayers. The effects of salt concentration and divalent cations on skeletal muscle sarcoplasmic reticulum [³H]ryanodine binding and Ca²⁺ release channel activity have been compared. These studies suggest that ryanodine is a good probe for investigating the function of the release channel.     

Interactions of a Reversible Ryanoid (21-Amino-9α-Hydroxy-Ryanodine) with Single Sheep Cardiac Ryanodine Receptor Channels

The binding of ryanodine to a high affinity site on the sarcoplasmic reticulum Ca²⁺-release channel results in a dramatic alteration in both gating and ion handling; the channel enters a high open probability, reduced-conductance state. Once bound, ryanodine does not dissociate from its site within the time frame of a single channel experiment. In this report, we describe the interactions of a synthetic ryanoid, 21-amino-9α-hydroxy-ryanodine, with the high affinity ryanodine binding site on the sheep cardiac sarcoplasmic reticulum Ca²⁺-release channel. The interaction of 21-amino-9α-hydroxy-ryanodine with the channel induces the occurrence of a characteristic high open probability, reduced-conductance state; however, in contrast to ryanodine, the interaction of this ryanoid with the channel is reversible under steady state conditions, with dwell times in the modified state lasting seconds. By monitoring the reversible interaction of this ryanoid with single channels under voltage clamp conditions, we have established a number of novel features of the ryanoid binding reaction. (a) Modification of channel function occurs when a single molecule of ryanoid binds to the channel protein. (b) The ryanoid has access to its binding site only from the cytosolic side of the channel and the site is available only when the channel is open. (c) The interaction of 21-amino-9α-hydroxy-ryanodine with its binding site is influenced strongly by transmembrane voltage. We suggest that this voltage dependence is derived from a voltage-driven conformational alteration of the channel protein that changes the affinity of the binding site, rather than the translocation of the ryanoid into the voltage drop across the channel.     

Block of the sheep cardiac sarcoplasmic reticulum Ca2+-release channel by tetra-alkyl ammonium cations

Summary The purified ryanodine receptor channel of the sheep cardiac muscle sarcoplasmic reticulum (SR) membrane functions as a calcium-activated cation-selective channel under voltage-clamp conditions following reconstitution into planar phospholipid bilayers. We have investigated the effects of the tetra-alkyl ammonium (TAA) cations, (C _n H_2n+1)₄N⁺ and the trimethyl ammonium cations, ethyltrimethyl ammonium and propyltrimethyl ammonium, on potassium conductance through the receptor channel. Small TAA cations (n = 1–3) and the trimethyl ammonium derivatives act as asymmetric, voltage-dependent blockers of potassium current. Quantitative analysis of the voltage dependence of block indicates that the conduction pathway of the sheep cardiac SR ryanodine receptor channel contains two distinct sites for the interaction of these small organic cations. Sites are located at approximately 50% for tetramethyl ammonium (TMA ⁺) and 90% for tetraethyl ammonium (TEA⁺) and tetrapropyl ammonium (TPrA⁺) of the voltage drop across the channel from the cytosolic face of the protein. The chemical substitution of an ethyl or propyl group for one of the methyl groups in TMA⁺ increases the voltage dependence of block to a level similar to that of TEA ⁺ and TPrA⁺. The zero-voltage dissociation constant (K _b(0)) falls with the increasing number of methyl and methylene groups for those blockers acting 90% of the way across the voltage drop. This is interpreted as suggesting a hydrophobic binding site at this point in the conduction pathway. The degree of block increases as the concentration of small TAA cations is raised. The concentration dependence of tetraethyl ammonium block indicates that the cation interacts with a single site within the conduction pathway with a K _m of 9.8±1.7 mm (mean±sd) at 40 mV. Larger TAA cations (n = 4–5) do not induce voltage-dependent block of potassium current of the form seen with the smaller TAA cations. These data support the contention that the sheep cardiac SR ryanodine receptor channel may be occupied by at most one ion at a time and suggest that a large proportion of the voltage drop falls over a relatively wide region of the conduction pathway.This work was supported by funds from the Medical Research Council and the British Heart Foundation. We would like to thank Richard Montgomery for his considerable help with the chemical synthesis. We are grateful to Drs. John Chambers, Nick Price and staff for showing us the intricacies of NMR spectroscopy.     

Endoplasmic Reticulum Dysfunction and Ca<Superscript>2<Emphasis Type="Bold">+</Emphasis></Superscript> Deregulation in Isolated CA1 Neurons during Oxygen and Glucose Deprivation

Intracellular calcium ([Ca²⁺]_i) plays a pivotal role in neuronal ischemia. The aim of the present study was to investigate the routes of Ca²⁺ entry during non-excitotoxic oxygen and glucose deprivation (OGD) in acutely dissociated rat CA1 neurons. During OGD the fluo-3/fura red ratio reflecting [Ca²⁺]_i increased rapidly and irreversibly. [Ca²⁺]_i increased to the same degree in Ca²⁺ depleted medium, and also when both the ryanodine receptors (RyR) and the inositol 1,4,5-trisphosphate (IP₃) receptors were blocked. When the endoplasmic reticulum (ER) Ca²⁺ stores were emptied with thapsigargin no increase in [Ca²⁺]_i was observed independent of extracellular Ca²⁺. The OGD induced Ca²⁺ deregulation in isolated CA1 neurons is not prevented by removing Ca²⁺, or by blocking the IP₃– or RyR receptors. However, when SERCA was blocked, no increase in [Ca²⁺]_i was observed suggesting that SERCA dysfunction represents an important mechanism for ischemic Ca²⁺ overload.     

A Point Mutation in the Ubiquitin Ligase RNF170 That Causes Autosomal Dominant Sensory Ataxia Destabilizes the Protein and Impairs Inositol 1,4,5-Trisphosphate Receptor-mediated Ca2+ Signaling

RNF170 is an endoplasmic reticulum membrane ubiquitin ligase that contributes to the ubiquitination of activated inositol 1,4,5-trisphosphate (IP₃) receptors, and also, when point mutated (arginine to cysteine at position 199), causes autosomal dominant sensory ataxia (ADSA), a disease characterized by neurodegeneration in the posterior columns of the spinal cord. Here we demonstrate that this point mutation inhibits RNF170 expression and signaling via IP₃ receptors. Inhibited expression of mutant RNF170 was seen in cells expressing exogenous RNF170 constructs and in ADSA lymphoblasts, and appears to result from enhanced RNF170 autoubiquitination and proteasomal degradation. The basis for these effects was probed via additional point mutations, revealing that ionic interactions between charged residues in the transmembrane domains of RNF170 are required for protein stability. In ADSA lymphoblasts, platelet-activating factor-induced Ca²⁺ mobilization was significantly impaired, whereas neither Ca²⁺ store content, IP₃ receptor levels, nor IP₃ production were altered, indicative of a functional defect at the IP₃ receptor locus, which may be the cause of neurodegeneration. CRISPR/Cas9-mediated genetic deletion of RNF170 showed that RNF170 mediates the addition of all of the ubiquitin conjugates known to become attached to activated IP₃ receptors (monoubiquitin and Lys⁴⁸- and Lys⁶³-linked ubiquitin chains), and that wild-type and mutant RNF170 have apparently identical ubiquitin ligase activities toward IP₃ receptors. Thus, the Ca²⁺ mobilization defect seen in ADSA lymphoblasts is apparently not due to aberrant IP₃ receptor ubiquitination. Rather, the defect likely reflects abnormal ubiquitination of other substrates, or adaptation to the chronic reduction in RNF170 levels.     

Molecular Determinants of Calpain-dependent Cleavage of Junctophilin-2 Protein in Cardiomyocytes

Junctophilin-2 (JP2), a membrane-binding protein that provides a structural bridge between the plasmalemma and sarcoplasmic reticulum, is essential for precise Ca²⁺-induced Ca²⁺ release during excitation-contraction coupling in cardiomyocytes. In animal and human failing hearts, expression of JP2 is decreased markedly, but the molecular mechanisms underlying JP2 down-regulation remain incompletely defined. In mouse hearts, ischemia/reperfusion injury resulted in acute JP2 down-regulation, which was attenuated by pretreatment with the calpain inhibitor MDL-28170 or by transgenic overexpression of calpastatin, an endogenous calpain inhibitor. Using a combination of computational analysis to predict calpain cleavage sites and in vitro calpain proteolysis reactions, we identified four putative calpain cleavage sites within JP2 with three N-terminal and one C-terminal cleavage sites. Mutagenesis defined the C-terminal region of JP2 as the predominant calpain cleavage site. Exogenous expression of putative JP2 cleavage fragments was not sufficient to rescue Ca²⁺ handling in JP2-deficient cardiomyocytes, indicating that cleaved JP2 is non-functional for normal Ca²⁺-induced Ca²⁺ release. These data provide new molecular insights into the posttranslational regulatory mechanisms of JP2 in cardiac diseases.