Junctional Sarcoplasmic Reticulum Membrane Research Articles

Desmin interacts with STIM1 and coordinates Ca2+ signaling in skeletal muscle.

Stromal interaction molecule 1 (STIM1), the sarcoplasmic reticulum (SR) transmembrane protein, activates store-operated Ca2+ entry (SOCE) in skeletal muscle and, thereby, coordinates Ca2+ homeostasis, Ca2+-dependent gene expression, and contractility. STIM1 occupies space in the junctional SR membrane of the triads and the longitudinal SR at the Z-line. How STIM1 is organized and is retained in these specific subdomains of the SR is unclear. Here, we identified desmin, the major type III intermediate filament protein in muscle, as a binding partner for STIM1 based on a yeast 2-hybrid screen. Validation of the desmin-STIM1 interaction by immunoprecipitation and immunolocalization confirmed that the CC1-SOAR domains of STIM1 interact with desmin to enhance STIM1 oligomerization yet limit SOCE. Based on our studies of desmin-KO mice, we developed a model wherein desmin connected STIM1 at the Z-line in order to regulate the efficiency of Ca2+ refilling of the SR. Taken together, these studies showed that desmin-STIM1 assembles a cytoskeletal-SR connection that is important for Ca2+ signaling in skeletal muscle.

Detection of Ca2+ transients near ryanodine receptors by targeting fluorescent Ca2+ sensors to the triad

In intact muscle fibers, functional properties of ryanodine receptor (RYR)-mediated sarcoplasmic reticulum (SR) Ca2+ release triggered by activation of the voltage sensor CaV1.1 have so far essentially been addressed with diffusible Ca2+-sensitive dyes. Here, we used a domain (T306) of the protein triadin to target the Ca2+-sensitive probe GCaMP6f to the junctional SR membrane, in the immediate vicinity of RYR channels, within the triad region. Fluorescence of untargeted GCaMP6f was distributed throughout the muscle fibers and experienced large Ca2+-dependent changes, with obvious kinetic delays, upon application of voltage-clamp depolarizing pulses. Conversely, T306-GCaMP6f localized to the triad and generated Ca2+-dependent fluorescence transients of lower amplitude and faster kinetics for low and intermediate levels of Ca2+ release than those of untargeted GCaMP6f. By contrast, model simulation of the spatial gradients of Ca2+ following Ca2+ release predicted limited kinetic differences under the assumptions that the two probes were present at the same concentration and suffered from identical kinetic limitations. At the spatial level, T306-GCaMP6f transients within distinct regions of a same fiber yielded a uniform time course, even at low levels of Ca2+ release activation. Similar observations were made using GCaMP6f fused to the γ1 auxiliary subunit of CaV1.1. Despite the probe's limitations, our results point out the remarkable synchronicity of voltage-dependent Ca2+ release activation and termination among individual triads and highlight the potential of the approach to visualize activation or closure of single groups of RYR channels. We anticipate targeting of improved Ca2+ sensors to the triad will provide illuminating insights into physiological normal RYR function and its dysfunction under stress or pathological conditions.

Calsequestrin. Structure, function, and evolution

Calsequestrin is the major Ca2+ binding protein in the sarcoplasmic reticulum (SR), serves as the main Ca2+ storage and buffering protein and is an important regulator of Ca2+ release channels in both skeletal and cardiac muscle. It is anchored at the junctional SR membrane through interactions with membrane proteins and undergoes reversible polymerization with increasing Ca2+ concentration. Calsequestrin provides high local Ca2+ at the junctional SR and communicates changes in luminal Ca2+ concentration to Ca2+ release channels, thus it is an essential component of excitation-contraction coupling. Recent studies reveal new insights on calsequestrin trafficking, Ca2+ binding, protein evolution, protein-protein interactions, stress responses and the molecular basis of related human muscle disease, including catecholaminergic polymorphic ventricular tachycardia (CPVT). Here we provide a comprehensive overview of calsequestrin, with recent advances in structure, diverse functions, phylogenetic analysis, and its role in muscle physiology, stress responses and human pathology.

Measurements of Triadic Calcium in Differentiated Muscle Fibers using a Gcamp Probe Targeted to the Junctional Sr Membrane

With the aim of gaining further insights into sarcoplasmic reticulum (SR) Ca2+ release function in skeletal muscle, we used a domain consisting of the 306 residues (T306) of the N-terminal end of triadin to target the Ca2+-sensitive probe GCaMP6f to the junctional SR membrane of muscle fibers. Muscles from mouse were electroporated with plasmid constructs encoding GCaMP6f or T306-GCaMP6f. Muscle fibers were isolated 1-2 weeks later and voltage-activated fluorescence changes were studied with line-scan confocal microscopy under voltage-clamp control. GCaMP6f fluorescence was homogenous throughout the transfected fibers and experienced large changes upon membrane depolarization in the absence of exogenous Ca2+ buffer in the intracellular solution (e.g. mean relative change of ∼20 in response to a 0.5 s-long pulse from −80 to −15 mV). When compared with simultaneously measured rhod-2 Ca2+ transients, GCaMP6f transients showed obvious signs of kinetic delays with, for instance, the time constant of decay after the end of short depolarizing pulses being 4-5 times that of the rhod-2 transients. In contrast, T306-GCaMP6f expression was localized to the vicinity of one or of a few nuclei. Within these spots, fluorescence yielded a transverse banded pattern consistent with triadic localization. Upon membrane depolarization, T306-GCaMP6f generated fluorescence transients of faster kinetics and much lower relative amplitude than T306-free GCaMP6f (e.g. mean relative change of 3.2 in response to a 0.5 s-long pulse to −15 mV), suggesting reduced affinity and faster kinetics of the SR membrane-bound probe as compared to the free probe. In several fibers, voltage-induced fluorescence changes at different positions of the scanned line showed time-course discrepancies which may reflect distinct behaviors of ryanodine receptor activation. Supported by CNRS, INSERM, Université Lyon 1 and AFM-Téléthon MyoNeurAlp 2.3.1.3.

Simulations of Subcellular Heterogeneity in Calcium Blink Kinetics

In cardiac myocytes, calcium (Ca2+) release from the sarcoplasmic reticulum (SR) depends on local calcium concentrations in both the cytosol and the SR. The local dynamics of SR Ca2+ depletion have been visualized experimentally through the recording of Ca2+ “blinks.” Specifically, it has been shown that blink nadir is relatively reproducible between local Ca2+ release sites whereas blink recovery kinetics are highly variable. We applied a spatially-detailed, stochastic mathematical model to gain insight into the mechanisms underlying this heterogeneity.Novel features of our model include: (1) calculation of Ca2+ fluxes between locations within the junctional SR (JSR) and dyadic spaces; (2) variable numbers and locations of connections between the JSR and the SR network; (3) variable and irregular clusters of ryanodine receptor (RyR) channels in the JSR membrane; (4) spatially-defined localization of SR Ca2+ pumps (SERCA) in JSR and network SR membranes. Using this model, we investigated how different parameters influenced Ca2+ blink amplitude and kinetics. From these simulations we determined that the number of connections between the JSR and the SR network was the most important parameter controlling Ca2+ blink recovery kinetics. For instance, 80% recovery of pre-release JSR [Ca2+] decreased from 400 ms with 1 connection to 125 ms with 5 connections.SERCA density also influenced Ca2+ blink recovery, but the quantitative strength of this effect was relatively smaller (126 ms at nominal SERCA versus 209 ms with SERCA reduced by 80%). The remaining model parameters had only minor effects on blink kinetics. These results indicate the importance of SR Ca2+ diffusion in release kinetics and suggest mechanisms underlying Ca2+ blink heterogeneity.

On the Adjacency Matrix of RyR2 Cluster Structures.

In the heart, electrical stimulation of cardiac myocytes increases the open probability of sarcolemmal voltage-sensitive Ca2+ channels and flux of Ca2+ into the cells. This increases Ca2+ binding to ligand-gated channels known as ryanodine receptors (RyR2). Their openings cause cell-wide release of Ca2+, which in turn causes muscle contraction and the generation of the mechanical force required to pump blood. In resting myocytes, RyR2s can also open spontaneously giving rise to spatially-confined Ca2+ release events known as “sparks.” RyR2s are organized in a lattice to form clusters in the junctional sarcoplasmic reticulum membrane. Our recent work has shown that the spatial arrangement of RyR2s within clusters strongly influences the frequency of Ca2+ sparks. We showed that the probability of a Ca2+ spark occurring when a single RyR2 in the cluster opens spontaneously can be predicted from the precise spatial arrangements of the RyR2s. Thus, “function” follows from “structure.” This probability is related to the maximum eigenvalue (λ 1) of the adjacency matrix of the RyR2 cluster lattice. In this work, we develop a theoretical framework for understanding this relationship. We present a stochastic contact network model of the Ca2+ spark initiation process. We show that λ 1 determines a stability threshold for the formation of Ca2+ sparks in terms of the RyR2 gating transition rates. We recapitulate these results by applying the model to realistic RyR2 cluster structures informed by super-resolution stimulated emission depletion (STED) microscopy. Eigendecomposition of the linearized mean-field contact network model reveals functional subdomains within RyR2 clusters with distinct sensitivities to Ca2+. This work provides novel perspectives on the cardiac Ca2+ release process and a general method for inferring the functional properties of transmembrane receptor clusters from their structure.

Junctophilin-2 protein in cardiac myocytes: progress in physiological and pathophysiological functions

The normal cardiac function relies on highly specialized subcellular architectures.The subcellular domains junctional membrane complexes(JMCs)are essential for excitation-contraction coupling of the myocardium.Junctophilin-2(JPH2)has been widely recognized as the crucial structural protein involved in JMCs.Initial studies limited the role of JPH2 to anchoring junctional sarcoplasmic reticulum(SR)and transverse-tubule(T-tubule) membrane invaginations.Recently,researchers have found an expanded role of JPH2 in the development of postnatal T-tubule in mammals,progression of disease in failing hearts,hypertrophic cardiomyopathy and arrhythmias.In this review we summarized the role of JPH2 in the above physiological or pathophysiological processes and discussed the perspective in future investigation.

Emerging roles of junctophilin-2 in the heart and implications for cardiac diseases.

Cardiomyocytes rely on a highly specialized subcellular architecture to maintain normal cardiac function. In a little over a decade, junctophilin-2 (JPH2) has become recognized as a cardiac structural protein critical in forming junctional membrane complexes (JMCs), which are subcellular domains essential for excitation-contraction coupling within the heart. While initial studies described the structure of JPH2 and its role in anchoring junctional sarcoplasmic reticulum and transverse-tubule (T-tubule) membrane invaginations, recent research has an expanded role of JPH2 in JMC structure and function. For example, JPH2 is necessary for the development of postnatal T-tubule in mammals. It is also critical for the maintenance of the complex JMC architecture and stabilization of local ion channels in mature cardiomyocytes. Loss of this function by mutations or down-regulation of protein expression has been linked to hypertrophic cardiomyopathy, arrhythmias, and progression of disease in failing hearts. In this review, we summarize current views on the roles of JPH2 within the heart and how JPH2 dysregulation may contribute to a variety of cardiac diseases.

Dynamics of Calcium Sparks and SR Calcium Leak During Excitation-Contraction Coupling in Mouse Heart Cells

Stable calcium-induced calcium release (CICR) is critical for maintaining normal cell contraction during cardiac excitation-contraction (EC) coupling in heart cells. The fundamental element of CICR in heart is the calcium (Ca2+) spark which arises from the regenerative release of Ca2+ from a cluster of ryanodine receptors (RyR2) in the junctional sarcoplasmic reticulum membrane. We have shown how stochastic gating of RyR2s could produce an SR Ca2+ leak capable of balancing SR Ca2+-ATPase (SERCA2a) activity under quiescent conditions (Williams et al. BJ 2011). This investigation suggested the surprising finding that a single, or even multiple, RyR2 openings could fail probabilistically to trigger a Ca2+ spark. To further investigate this effect, we expanded upon that formulation to create a detailed, local control model of EC coupling in mouse heart. A number of features were added or modified, including the addition of a novel seven-state Markov chain model of the sarcolemmal L-type Ca2+ channel (LCC). This model features dynamic action-potential (AP) generation, robust Ca2+ spark initiation and termination, realistic [Ca2+]i transients, and true SR Ca2+ pump/leak balance. The model suggests that numerous LCC openings are often required to trigger a single Ca2+ spark. This is consistent with our prior work where multiple RyR2 openings were needed to trigger a spontaneous Ca2+ spark during quiescent conditions. We also investigated how RyR2 mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) influence the dynamics of Ca2+ sparks, “invisible” non-spark Ca2+ leak, [Ca2+]i transients, and SR Ca2+ content. We observe that CPVT mutations can lead to unstable Ca2+ spark dynamics, altering SR Ca2+ content and promoting [Ca2+]i signaling instability. Our new model provides significant insights into the dynamics of local control of CICR under physiological and pathological conditions.

Human junctophilin-2 undergoes a structural rearrangement upon binding PtdIns(3,4,5)P3 and the S101R mutation identified in hypertrophic cardiomyopathy obviates this response

JP2 (junctophilin-2) is believed to hold the transverse tubular and jSR (junctional sarcoplasmic reticulum) membranes in a precise geometry that facilitates excitation–contraction coupling in cardiomyocytes. We have expressed and purified human JP2 and shown using electron microscopy that the protein forms elongated structures ~15 nm long and 2 nm wide. Employing lipid-binding assays and quartz crystal microbalance with dissipation we have determined that JP2 is selective for PS (phosphatidylserine), with a Kd value of ~0.5 μM, with the N-terminal domain mediating this interaction. JP2 also binds PtdIns(3,4,5)P3 at a different site than PS, resulting in the protein adopting a more flexible conformation; this interaction is modulated by both Ca2+ and Mg2+ ions. We show that the S101R mutation identified in patients with hypertrophic cardiomyopathy leads to modification of the protein secondary structure, forming a more flexible molecule with an increased affinity for PS, but does not undergo a structural transition in response to binding PtdIns(3,4,5)P3. In conclusion, the present study provides new insights into the structural and lipid-binding properties of JP2 and how the S101R mutation may have an effect upon the stability of the dyad organization with the potential to alter JP2–protein interactions regulating Ca2+ cycling.

Extinguishing the Sparks

The mechanical contraction of cardiac muscle is triggered by membrane depolarization through a process known as calcium-induced calcium-release (CICR). The structural basis of CICR is exquisite in its detail. At thousands of distinct locations throughout the myocyte, the junctional sarcoplasmic reticulum (JSR) membrane approaches to within ∼12 nm of the t-tubule membrane to form structures known as dyads. Dyads consist of a handful of L-type calcium (Ca2+) channels (LCCs) in the t-tubule membrane, a larger number of ryanodine-sensitive Ca2+-gated Ca2+-release channels (RyR2s) in the closely apposed JSR membrane, regulatory proteins that modify LCC and RyR2 function, and the fluid volume that separates them. Membrane depolarization leads to openings of LCCs, flux of Ca2+ into the dyadic space, Ca2+ binding to and opening of RyR2s, and release of Ca2+ from the JSR into the dyadic space. This Ca2+ then diffuses out of the dyad and binds to the mechanical machinery of the cell. The sum of these Ca2+ release events drives contraction of heart muscle.

Type 1 Inositol (1,4,5)-Trisphosphate Receptor Activates Ryanodine Receptor 1 to Mediate Calcium Spark Signaling in Adult Mammalian Skeletal Muscle

Functional coupling between inositol (1,4,5)-trisphosphate receptor (IP(3)R) and ryanodine receptor (RyR) represents a critical component of intracellular Ca(2+) signaling in many excitable cells; however, the role of this mechanism in skeletal muscle remains elusive. In skeletal muscle, RyR-mediated Ca(2+) sparks are suppressed in resting conditions, whereas application of transient osmotic stress can trigger activation of Ca(2+) sparks that are restricted to the periphery of the fiber. Here we show that onset of these spatially confined Ca(2+) sparks involves interaction between activation of IP(3)R and RyR near the sarcolemmal membrane. Pharmacological prevention of IP(3) production or inhibition of IP(3)R channel activity abolishes stress-induced Ca(2+) sparks in skeletal muscle. Although genetic ablation of the type 2 IP(3)R does not appear to affect Ca(2+) sparks in skeletal muscle, specific silencing of the type 1 IP(3)R leads to ablation of stress-induced Ca(2+) sparks. Our data indicate that membrane-delimited signaling involving cross-talk between IP(3)R1 and RyR1 contributes to Ca(2+) spark activation in skeletal muscle.

Imaging Arrhythmogenic Calcium Signaling in Intact Hearts

Protein complex of the cardiac junctional sarcoplasmic reticulum (SR) membrane formed by type 2 ryanodine receptor, junction, triadin, and calsequestrin is responsible for controlling SR calcium (Ca) release. Increased intracellular calcium (Ca(i)) activates the electrogenic sodium-Ca exchanger current, which is known to be important in afterdepolarization and triggered activities (TAs). Using optical-mapping techniques, it is possible to simultaneously map membrane potential (V (m)) and Ca(i) transient in Langendorff-perfused rabbit ventricles to better define the mechanisms by which V (m) and Ca(i) interactions cause early afterdepolarizations (EADs). Phase 3 EAD is dependent on heterogeneously prolonged action potential duration (APD). Electrotonic currents that flow between a persistently depolarized region and its recovered neighbors underlies the mechanisms of phase 3 EADs and TAs. In contrast, "late phase-3 EAD" is induced by APD shortening, not APD prolongation. In failing ventricles, upregulation of apamin-sensitive Ca-activated potassium (K) channels (I(KAS)) causes APD shortening after fibrillation-defibrillation episodes. Shortened APD in the presence of large Ca(i) transients generates late-phase 3 EADs and recurrent spontaneous ventricular fibrillation. The latter findings suggest that I (KAS) may be a novel antiarrhythmic targets in patients with heart failure and electrical storms.

Calsequestrin-1 is Required for Association but not for Targeting of Triadin to the Junctional Sarcoplasmic Reticulum Membranes

In striated muscles two membrane systems, the sarcolemma and the sarcoplasmic reticulum (SR) are typically arranged in contiguous junctional membrane complexes (JMCs), called triads. Triads are key components in the excitation-contraction coupling mechanism, as they supply the structural elements necessary for efficient crosstalk between voltage-dependent calcium channels (DHPR) on the T-tubules and the ryanodine receptors calcium channels (RyRs), on the SR. Other proteins including triadin, junctin, calsequestrin and junctophillins are selectively localized to JMCs and assemble into regularly arranged complexes. Mechanisms explaining membrane compartmentalization include selective targeting and “trapping” machineries, which may prevent the spread of molecules throughout the membrane surface. However, no specific mechanisms have been yet associated with either assembly or maintenance of JMCs in striated muscles. In previous studies we showed that the mobile fraction of triadin progressively reduced during differentiation, suggesting that a trapping mechanism may be efficiently active in striated muscle and indicate triadin as a good candidate to study protein localization and association to the JMCs.Expression of GFP-tagged triadin proteins and deletion mutants in rat myotubes showed that three regions (named 1, 2 and 3) were required for protein targeting to the JMCs. On the contrary, analysis of protein mobility revealed that association of triadin to the JMC was mediated by region 3 only. GST-pull down and FRET experiments showed that region 3 was able to interact with calsequestrin-1. Interestingly, experiments performed on myotubes from calsequestrin-1 knockout mice revealed that in the absence of calsequestrin, triadin was properly localized to the JMC, although its mobile fraction was dramatically increased. These results suggest that calsequestrin-1 is directly involved in trapping triadin to the JMCs, whereas selective targeting is likely to be mediated by interactions with other proteins.

Spontaneous and voltage-activated Ca2+ release in adult mouse skeletal muscle fibres expressing the type 3 ryanodine receptor.

The physiological properties and role of the type 3 ryanodine receptor (RyR3), a calcium release channel expressed in a wide variety of cell types, remain mysterious. We forced, in vivo, the expression of RyR3 in adult mouse skeletal muscle fibres using a GFP-RyR3 DNA construct. GFP fluorescence was found within spatially restricted regions of muscle fibres where it exhibited a sarcomere-related banded pattern consistent with a localization within or near the junctional sarcoplasmic reticulum membrane. Immunostaining confirmed the presence of RyR3 together with RyR1 within the GFP-positive areas. In approximately 90% of RyR3-positive fibres microinjected with the calcium indicator fluo-3, we detected repetitive spontaneous transient elevations of intracellular Ca2+ that persisted when fibres were voltage-clamped at -80 mV. These Ca2+ transients remained essentially confined to the RyR3 expression region. They ranged from wide local events to propagating Ca2+ waves and were in some cases associated with local contractile activity. When voltage-clamp depolarizations were applied while fluo-3 or rhod-2 fluorescence was measured within the RyR3-expressing region, no voltage-evoked 'spark-like' elementary Ca2+ release event could be detected. Still global voltage-activated Ca2+ release exhibited a prominent early peak within the RyR3-expressing regions. Measurements were also taken from muscles fibres expressing a GFP-RyR1 construct; positive fibres also yielded a local banded pattern of GFP fluorescence but exhibited no spontaneous Ca2+ release. Results demonstrate that RyR3 is a very potent source of voltage-independent Ca2+ release activity. Conversely we find no evidence that it could contribute to the production of discrete voltage-activated Ca2+ release events in differentiated mammalian skeletal muscle.

Protein–protein interactions between triadin and calsequestrin are involved in modulation of sarcoplasmic reticulum calcium release in cardiac myocytes

In cardiac muscle, intracellular Ca2+ release is controlled by a number of proteins including the ryanodine receptor (RyR2), calsequestrin (CASQ2), triadin-1 (Trd) and junctin (Jn) which form a complex in the junctional sarcoplasmic reticulum (SR) membrane. Within this complex, Trd appears to link CASQ2 to RyR2 although the functional significance of this interaction is unclear. In this study, we explored the functional importance of Trd-CASQ2 interactions for intracellular Ca2+ handling in rat ventricular myocytes. A peptide encompassing the homologous CASQ2 binding domain of Trd (residues 206-230 in the rat; TrdPt) was expressed in the lumen of the SR to disrupt Trd-CASQ2 interactions. Myocytes expressing TrdPt exhibited increased responsiveness of SR Ca2+ release to activation by ICa as manifested by flattened and broadened voltage dependency of the amplitude of cytosolic Ca2+ transients. Rhythmically paced, TrdPt-expressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca2+ and membrane potential that was not seen in control cells. In addition, the frequency of spontaneous Ca2+ sparks and Ca2+ waves was significantly increased in TrdPt-expressing, permeabilized myocytes. These alterations in SR Ca2+ release were accompanied by a significant decrease in basal free intra-SR[Ca2+] and total SR Ca2+ content in TrdPt-expressing cells. At the same time a synthetic peptide corresponding to the CASQ2 binding domain of Trd produced no direct effects on the activity of single RyR2 channels incorporated into lipid bilayers while interfering with the ability of CASQ2 to inhibit the RyR2 channel. These results suggest that CASQ2 stabilizes SR Ca2+ release by inhibiting the RyR2 channel through interaction with Trd. They also show that intracellular Ca2+ cycling in the heart relies on coordinated interactions between proteins of the RyR2 channel complex and that disruption of these interactions may represent a molecular mechanism for cardiac disease.

Fast kinetics of calcium dissociation from calsequestrin

We measured the kinetics of calcium dissociation from calsequestrin in solution or forming part of isolated junctional sarcoplasmic reticulum membranes by mixing calsequestrin equilibrated with calcium with calcium-free solutions in a stopped-flow system. In parallel, we measured the kinetics of the intrinsic fluorescence changes that take place following calcium dissociation from calsequestrin. We found that at 25 degrees C calcium dissociation was 10-fold faster for calsequestrin attached to junctional membranes (k = 109 s(-1)) than in solution. These results imply that calcium dissociation from calsequestrin in vivo is not rate limiting during excitation-contraction coupling. In addition, we found that the intrinsic fluorescence decrease for calsequestrin in solution or forming part of junctional membranes was significantly slower than the rates of calcium dissociation. The kinetics of intrinsic fluorescence changes had two components for calsequestrin associated to junctional membranes and only one for calsequestrin in solution; the faster component was 8-fold faster (k = 54.1 s(-1)) than the slower component (k = 6.9 s(-1)), which had the same k value as for calsequestrin in solution. These combined results suggest that the presence of calsequestrin at high concentrations in a restricted space, such as when bound to the junctional membrane, accelerates calcium dissociation and the resulting structural changes, presumably as a result of cooperative molecular interactions.

Sarcoplasmic Reticulum Calcium Release Channels in Ventricles of Older Adult Hamsters

Whether the density of sarcoplasmic reticulum (SR) calcium release channels/ryanodine receptors in the heart declines with age is not clear. We investigated age-related changes in the density of [3H]-ryanodine receptors in crude ventricular homogenates, which contained all ligand binding sites in heart and in isolated junctional SR membranes. Experiments utilized young (120 days) and older adult (300 days) hamsters. [3H]-ryanodine binding site density did not change with age in crude homogenate preparations, although total heart protein concentration increased significantly with age. In contrast, the density of [3H]-ryanodine binding sites decreased markedly in heavy SR membranes purified from older hearts. These results show that demonstration of age-related changes in cardiac ryanodine receptor density depends upon the preparation used. Furthermore, the increase in total ventricular protein with age suggests that normalization of data by membrane protein should be used with caution in studies of aging heart.

Triadin

See related article, pages 651–658 The Ca2+ movements that control many cellular functions, including contraction of striated and smooth muscle cells and vesicular secretion from endocrine cells and nerve terminals, are increasingly recognized to involve macromolecular Ca2+-signaling complexes that, in addition to the key Ca2+-transporting proteins, include large numbers of associated proteins that provide a variety of regulatory, structural and Ca2+-sensing/buffering functions. The importance of working out the subtle interactions within these macromolecular complexes is underscored by the genetic diseases that have been associated with mutations of their constituent proteins. In this issue, Terentyev et al1 use a spectrum of molecular and electrophysiological techniques to demonstrate that triadin (TRD) plays an unexpectedly important role in regulating the ryanodine receptors (RyRs), found primarily in dyadic junctions of cardiac sarcoplasmic reticulum (SR; Figure). It has been previously suggested that TRD and junctin are integral membrane proteins of the junctional SR, and serve as linker proteins from the SR Ca release channel (RyR) to calsequestrin (CSQ) complexes, the major Ca2+-buffer in the lumen of the SR (Figure).2 The large (≈4500 aa) cytoplasmic domain of RyR appears to have multiple binding sites for an ever-growing list of proteins that includes: calmodulin, PKA, FKBP 12.6.3 The major findings of the present communication are that overexpression of TRD leads to 3-fold increase in open probability of RyRs in bilayers, a 60% increase in spontaneous spark frequency with only minor decreases in spark amplitude (≈10%) and SR Ca2+ content (≈30%), as well as a marked alteration in the voltage dependence of Ca2+ release. The authors propose the activity of RyRs to be directly modulated by the level of expression of TRD, most likely mediated by amino acid residues 200 to 224 …

Complementary DNA cloning, genomic characterization and expression analysis of a mammalian gene encoding histidine-rich calcium binding protein

A protein complex present at the junctional sarcoplasmic reticulum (SR) membrane is implicated in the Ca 2+ release process during muscle contraction. The histidine-rich Ca 2+-binding protein (HRC) is an emerging component associated into the SR protein complex. We cloned cDNAs for rat and monkey HRCs, showing a conserved sequence organization in common with other mammalian HRCs. Genomic analysis revealed that each mammalian HRC gene is present as a single copy in the genome, consisting of 6 exons and 5 introns. Developmental expression analysis using mouse embryos and postnatal hearts demonstrated that Hrc transcription begins at 12.5 days postcoitum and its level increases gradually, reaching an adult level in the range 5–20 days after birth. Comparing the Hrc gene and other SR genes, we found that the timing and pattern of gene expression vary among the SR genes and the full-level expression of these genes is achieved in the heart after postnatal day 20. Collectively, our study provides comprehensive information about the structure and expression of the mammalian HRC gene, together with the comparative expression data of the related SR genes.