Three-dimensional architecture of the calcium channel/foot structure of sarcoplasmic reticulum

The calcium channel responsible for the release of Ca2+ from the sarcoplasmic reticulum of skeletal muscle during excitation-contraction coupling has recently been identified and purified. The isolated calcium channel has been identified morphologically with the 'foot' structures which are associated with the junctional face membrane of the terminal cisternae of sarcoplasmic reticulum. In situ, the foot structure extends across the gap of the triad junction from the terminal cisternae of the reticulum to the transverse tubule. We describe here the three-dimensional architecture (3.7 nm resolution) of the calcium channel/foot structure from fast-twitch rabbit skeletal muscle, which we determined from electron micrographs of isolated, non-crystalline structures that had been tilted in the electron microscope. The reconstruction reveals two different faces and an internal structure in which stain accumulates at several interconnected locations, which could empty into the junctional gap of the triad junction. The detailed architecture of the channel complex is relevant to understanding both the physical path followed by calcium ions during excitation-contraction coupling and the association of the terminal cisternae and the transverse tubules in the triad junction.     

Metabolic stabilization of endplate acetylcholine receptors regulated by Ca2+ influx associated with muscle activity.

During formation of the neuromuscular junction, acetylcholine receptors in the endplate membrane become metabolically stabilized under neural control, their half-life increasing from about 1 day to about 10 days. The metabolic stability of the receptors is regulated by the electrical activity induced in the muscle by innervation. We report here that metabolic stabilization of endplate receptors but not of extrajunctional receptors can be induced in the absence of muscle activity if muscles are treated with the calcium ionophore A23187. Acetylcholine receptor stabilization was also induced by culturing non-stimulated muscle in elevated K+ with the Ca2+ channel activator (+)-SDZ202-791. Conversely, activity-dependent receptor stabilization is prevented in muscle stimulated in the presence of the Ca2+ channel blockers (+)-PN200-110 or D-600. Treatment of muscles with ryanodine, which induces Ca2+ release from the sarcoplasmic reticulum in the absence of activity, does not cause stabilization of junctional receptors. Evidently, muscle activity induces metabolic acetylcholine receptor stabilization by way of an influx of Ca2+ ions through dihydropyridine-sensitive Ca2+ channels in the endplate membrane, whereas Ca2+ released from the sarcoplasmic reticulum is ineffective in this developmental process.     

Crystal structure of the calcium pump with a bound ATP analogue

P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across cell and organelle membranes. Here, we describe the crystal structure of the Ca2+ pump of skeletal muscle sarcoplasmic reticulum, a representative member of the P-type ATPase superfamily, with an ATP analogue, a Mg2+ and two Ca2+ ions in the respective binding sites. In this state, the ATP analogue reorganizes the three cytoplasmic domains (A, N and P), which are widely separated without nucleotide, by directly bridging the N and P domains. The structure of the P-domain itself is altered by the binding of the ATP analogue and Mg2+. As a result, the A-domain is tilted so that one of the transmembrane helices moves to lock the cytoplasmic gate of the transmembrane Ca2+-binding sites. This appears to be the mechanism for occluding the bound Ca2+ ions, before releasing them into the lumen of the sarcoplasmic reticulum.     

Opening of dihydropyridine calcium channels in skeletal muscle membranes by inositol trisphosphate

In many non-muscle cells, D-inositol 1,4,5-trisphosphate (InsP3) has been shown to release Ca2+ from intracellular stores, presumably from the endoplasmic reticulum. It is thought to be a ubiquitous second messenger that is produced in, and released from, the plasma membrane in response to extracellular receptor stimulation. By analogy, InsP3 in muscle cells has been postulated to open calcium channels in the sarcoplasmic reticulum (SR) membrane, which is the intracellular Ca2+ store that releases Ca2+ during muscle contraction. We report here that InsP3 may have a second site of action. We show that InsP3 opens dihydropyridine-sensitive Ca2+ channels in a vesicular preparation of rabbit skeletal muscle transverse tubules. InsP3-activated channels and channels activated by a dihydropyridine agonist in the same preparation have similar slope conductance and extrapolated reversal potential and are blocked by a dihydropyridine antagonist. This suggests that in skeletal muscle, InsP3 can modulate Ca2+ channels of transverse tubules from plasma membrane, in contrast to the previous suggestion that the functional locus of InsP3 is exclusively in the sarcoplasmic reticulum membrane.     

Structural changes in the calcium pump accompanying the dissociation of calcium

In skeletal muscle, calcium ions are transported (pumped) against a concentration gradient from the cytoplasm into the sarcoplasmic reticulum, an intracellular organelle. This causes muscle cells to relax after cytosolic calcium increases during excitation. The Ca(2+) ATPase that carries out this pumping is a representative P-type ion-transporting ATPase. Here we describe the structure of this ion pump at 3.1 A resolution in a Ca(2+)-free (E2) state, and compare it with that determined previously for the Ca(2+)-bound (E1Ca(2+)) state. The structure of the enzyme stabilized by thapsigargin, a potent inhibitor, shows large conformation differences from that in E1Ca(2+). Three cytoplasmic domains gather to form a single headpiece, and six of the ten transmembrane helices exhibit large-scale rearrangements. These rearrangements ensure the release of calcium ions into the lumen of sarcoplasmic reticulum and, on the cytoplasmic side, create a pathway for entry of new calcium ions.     

The structure of phospholamban and its MD simulations

Phospholamban is an important protein with responsibility for regulating the activity of the sarcoplasmic reticulum Ca2+ pump through reversible phosphorylation.And its three-dimensional structure in living cell has been a focus of attention.In the current case, we summarized the investigations on phospholamban structure, and on this base, employed long time-scale molecular dy-namics simulations to study its structure systematically.The first 22 residues from one chain of phospholamban in bellflower structu...     

Inositol 1,4,5-trisphosphate induces calcium release from sarcoplasmic reticulum of skeletal muscle

The sarcoplasmic reticulum of skeletal muscle is a specialized form of endoplasmic reticulum that controls myoplasmic calcium concentration and, therefore, the contraction-relaxation cycle. Ultrastructural studies have shown that the sarcoplasmic reticulum is a continuous but heterogeneous membranous network composed of longitudinal tubules that surround myofibrils and terminal cisternae. These cisternae are junctionally associated, via bridging structures called 'feet', with sarcolemmal invaginations (the transverse tubules) to form the triadic junction. Following transverse tubule depolarization, a signal, transmitted along the triadic junction, triggers Ca2+ release from terminal cisternae, but the mechanism of this coupling is still unknown. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) has recently been shown to mobilize Ca2+ from intracellular stores, referable to endoplasmic reticulum, in a variety of cell types (see ref. 8 for review), including smooth muscle cells of the porcine coronary artery and canine cardiac muscle cells. Here we show that Ins(1,4,5)P3 releases Ca2+ from isolated, purified sarcoplasmic reticulum fractions of rabbit fast-twitch skeletal muscle, the effect being more pronounced on a fraction of terminal cisternae that contains morphologically intact feet structures; and elicits isometric force development in chemically skinned muscle fibres.     

TRIC channels are essential for Ca2+ handling in intracellular stores

Cell signalling requires efficient Ca2+ mobilization from intracellular stores through Ca2+ release channels, as well as predicted counter-movement of ions across the sarcoplasmic/endoplasmic reticulum membrane to balance the transient negative potential generated by Ca2+ release. Ca2+ release channels were cloned more than 15 years ago, whereas the molecular identity of putative counter-ion channels remains unknown. Here we report two TRIC (trimeric intracellular cation) channel subtypes that are differentially expressed on intracellular stores in animal cell types. TRIC subtypes contain three proposed transmembrane segments, and form homo-trimers with a bullet-like structure. Electrophysiological measurements with purified TRIC preparations identify a monovalent cation-selective channel. In TRIC-knockout mice suffering embryonic cardiac failure, mutant cardiac myocytes show severe dysfunction in intracellular Ca2+ handling. The TRIC-deficient skeletal muscle sarcoplasmic reticulum shows reduced K+ permeability, as well as altered Ca2+ 'spark' signalling and voltage-induced Ca2+ release. Therefore, TRIC channels are likely to act as counter-ion channels that function in synchronization with Ca2+ release from intracellular stores.     

Purification and reconstitution of the calcium release channel from skeletal muscle

The calcium release channel from rabbit muscle sarcoplasmic reticulum (SR) has been purified and reconstituted as a functional unit in lipid bilayers. Electron microscopy reveals the four-leaf clover structure previously described for the 'feet' that span the transverse tubule (T)-SR junction. Ca2+ release from the SR induced by T-system depolarization during excitation-contraction coupling in muscle may thus be effected through a direct association of the T-system with SR Ca2+-release channels.     

Nature and site of phospholamban regulation of the Ca2+ pump of sarcoplasmic reticulum

The rapid removal of Ca2+ ions from the cytosol, necessary for the efficient relaxation of cardiac muscle cells, is performed by the Ca2+-pumping ATPase of the sarcoplasmic reticulum. The calcium pump is activated by cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban, an integral membrane protein of the sarcoplasmic reticulum. Using a heterobifunctional crosslinking agent which can be cleaved and photoactivated, we provide evidence for a direct interaction between the two proteins. Only the non-phosphorylated form of phospholamban interacts with the ATPase, demonstrating that phospholamban is an endogenous inhibitor that is removed from the ATPase by phosphorylation. Non-phosphorylated phospholamban interacts only with the calcium-free conformation of the ATPase and is released when it is converted to the calcium-bound state. We localized the site of interaction to a single peptide isolated after cyanogen bromide cleavage of the ATPase. The peptide derives from a domain just C-terminal to the aspartyl phosphate of the active site. This domain is unique to ATPases of the sarcoplasmic reticulum in that it has no homology with any other phosphorylation-type ion pump. The domain occurs in both slow- and fast-twitch isoforms of the ATPase, even though phospholamban is not expressed in fast-twitch muscles.     

力竭运动对大鼠骨骼肌肌浆网钙转运的影响

采用递增负荷力竭运动方式观察急性运动对肌浆网(SR)钙转运能力的影响,发现运动后股四头肌SR Ca2+-ATPase活性和Ca2+摄取速率均明显下降,表现为SR Ca2+-ATPase活性和Ca2+摄取速率分别下降51.12%(P<0.01)和17.72%(P<0.01);而脂质过氧化物浓度则显著升高,增加48.85%(P<0.01).提示急性运动后SR Ca2+-ATP ase 活性和Ca2+摄取速率下降可能是脂质过氧化增高的结果,而Ca2+摄取功能下降可能与运动性疲劳密切相关.     

Ryanodine receptor gene is a candidate for predisposition to malignant hyperthermia

Malignant hyperthermia (MH) is a potentially lethal condition in which sustained muscle contracture, with attendant hypercatabolic reactions and elevation in body temperature, are triggered by commonly used inhalational anaesthetics and skeletal muscle relaxants. In humans, the trait is usually inherited in an autosomal dominant fashion, but in halothane-sensitive pigs with a similar phenotype, inheritance of the disease is autosomal recessive or co-dominant. A simple and accurate non-invasive test for the gene is not available and predisposition to the disease is currently determined through a halothane- and/or caffeine-induced contracture test on a skeletal muscle biopsy. Because Ca2+ is the chief regulator of muscle contraction and metabolism, the primary defect in MH is believed to lie in Ca2+ regulation. Indeed, several studies indicate a defect in the Ca2+ release channel of the sarcoplasmic reticulum, making it a prime candidate for the altered gene product in predisposed individuals. We have recently cloned complementary DNA and genomic DNA encoding the human ryanodine receptor (the Ca2(+)-release channel of the sarcoplasmic reticulum) and mapped the ryanodine receptor gene (RYR) to region q13.1 of human chromosome 19 (ref. 14), in close proximity to genetic markers that have been shown to map near the MH susceptibility locus in humans and the halothane-sensitive gene in pigs. As a more definitive test of whether the RYR gene is a candidate gene for the human MH phenotype, we have carried out a linkage study with MH families to determine whether the MH phenotype segregates with chromosome 19q markers, including markers in the RYR gene. Co-segregation of MH with RYR markers, resulting in a lod score of 4.20 at a linkage distance of zero centimorgans, indicates that MH is likely to be caused by mutations in the RYR gene.     

Regions of the skeletal muscle dihydropyridine receptor critical for excitation-contraction coupling

It is thought that in skeletal muscle excitation-contraction (EC) coupling, the release of Ca2+ from the sarcoplasmic reticulum is controlled by the dihydropyridine (DHP) receptor in the transverse tubular membrane, where it serves as the voltage sensor. We have shown previously that injection of an expression plasmid carrying the skeletal muscle DHP receptor complementary DNA restores EC coupling and L-type calcium current that are missing in skeletal muscle myotubes from mutant mice with muscular dysgenesis. This restored coupling resembles normal skeletal muscle EC coupling, which does not require entry of extracellular Ca2+. By contrast, injection into dysgenic myotubes of an expression plasmid carrying the cardiac DHP receptor cDNA produces L-type calcium current and cardiac-type EC coupling, which does require entry of extracellular Ca2+. To identify the regions responsible for this important functional difference between the two structurally similar DHP receptors, we have expressed various chimaeric DHP receptor cDNAs in dysgenic myotubes. The results obtained indicate that the putative cytoplasmic region between repeats II and III of the skeletal muscle DHP receptor is an important determinant of skeletal-type EC coupling.     

Inactivation of the sarcoplasmic reticulum calcium channel by protein kinase.

The ryanodine receptor protein of skeletal muscle sarcoplasmic reticulum (SR) membranes is a calcium ion channel which allows movement of calcium from the SR lumen into the cytoplasm during muscle activation. Gating of this channel is modulated by a number of physiologically important substances including calcium. Interestingly, calcium has both activating and inactivating effects which are concentration- and tissue-specific. In skeletal muscle, calcium-dependent inactivation of calcium release occurs at concentrations reached physiologically, suggesting that calcium may modulate the release process by a negative feedback mechanism. To determine the cellular mechanism responsible for calcium-dependent inactivation, we have investigated the ability of protein phosphorylation to affect single channel gating behaviour using the patch clamp technique. Here we demonstrate that the ryanodine receptor protein/calcium release channel of skeletal muscle SR is inactivated under conditions permissive for protein phosphorylation. This inactivation is reversed by the application of phosphatase and prevented by a peptide inhibitor specific for calcium/calmodulin-dependent protein kinase II. The results provide evidence for an endogenous protein kinase which is closely associated with the ryanodine receptor protein and regulates channel gating.     

Inositol 1,4,5-trisphosphate activates a channel from smooth muscle sarcoplasmic reticulum

Inositol 1,4,5-trisphosphate (InsP3) can initiate calcium release into the cytoplasm in a variety of cells. From experiments using permeabilized cells, membrane vesicles, and patch-clamp techniques, it has been suggested that InsP3 acts by directly opening calcium channels. Here, we show that InsP3 induced openings of channels in planar lipid bilayers into which vesicles made from aortic muscle sarcoplasmic reticulum (SR) were incorporated. Activation of channels by InsP3 was not observed when vesicles made from SR of cardiac or skeletal muscle were incorporated into planar lipid bilayers. The present study demonstrates for the first time unique properties of an InsP3-gated calcium channel in sarcoplasmic reticulum vesicles from vascular smooth muscle. This InsP3-activated channel from aortic SR differs strikingly from the calcium-gated calcium channel of striated muscle SR in single-channel conductance and pharmacology.     

The amino-terminal disease hotspot of ryanodine receptors forms a cytoplasmic vestibule

Many physiological events require transient increases in cytosolic Ca(2+) concentrations. Ryanodine receptors (RyRs) are ion channels that govern the release of Ca(2+) from the endoplasmic and sarcoplasmic reticulum. Mutations in RyRs can lead to severe genetic conditions that affect both cardiac and skeletal muscle, but locating the mutated residues in the full-length channel structure has been difficult. Here we show the 2.5?? resolution crystal structure of a region spanning three domains of RyR type 1 (RyR1), encompassing amino acid residues 1-559. The domains interact with each other through a predominantly hydrophilic interface. Docking in RyR1 electron microscopy maps unambiguously places the domains in the cytoplasmic portion of the channel, forming a 240-kDa cytoplasmic vestibule around the four-fold symmetry axis. We pinpoint the exact locations of more than 50 disease-associated mutations in full-length RyR1 and RyR2. The mutations can be classified into three groups: those that destabilize the interfaces between the three amino-terminal domains, disturb the folding of individual domains or affect one of six interfaces with other parts of the receptor. We propose a model whereby the opening of a RyR coincides with allosterically coupled motions within the N-terminal domains. This process can be affected by mutations that target various interfaces within and across subunits. The crystal structure provides a framework to understand the many disease-associated mutations in RyRs that have been studied using functional methods, and will be useful for developing new strategies to modulate RyR function in disease states.     

Ca2+ signalling between single L-type Ca2+ channels and ryanodine receptors in heart cells

Ca2+-induced Ca2+ release is a general mechanism that most cells use to amplify Ca2+ signals. In heart cells, this mechanism is operated between voltage-gated L-type Ca2+ channels (LCCs) in the plasma membrane and Ca2+ release channels, commonly known as ryanodine receptors, in the sarcoplasmic reticulum. The Ca2+ influx through LCCs traverses a cleft of roughly 12 nm formed by the cell surface and the sarcoplasmic reticulum membrane, and activates adjacent ryanodine receptors to release Ca2+ in the form of Ca2+ sparks. Here we determine the kinetics, fidelity and stoichiometry of coupling between LCCs and ryanodine receptors. We show that the local Ca2+ signal produced by a single opening of an LCC, named a 'Ca2+ sparklet', can trigger about 4-6 ryanodine receptors to generate a Ca2+ spark. The coupling between LCCs and ryanodine receptors is stochastic, as judged by the exponential distribution of the coupling latency. The fraction of sparklets that successfully triggers a spark is less than unity and declines in a use-dependent manner. This optical analysis of single-channel communication affords a powerful means for elucidating Ca2+-signalling mechanisms at the molecular level.     

Oestrogen protects FKBP12.6 null mice from cardiac hypertrophy

FK506 binding proteins 12 and 12.6 (FKBP12 and FKBP12.6) are intracellular receptors for the immunosuppressant drug FK506 (ref. 1). The skeletal muscle ryanodine receptor (RyR1) is isolated as a hetero-oligomer with FKBP12 (ref. 2), whereas the cardiac ryanodine receptor (RyR2) more selectively associates with FKBP12.6 (refs 3, 4, 5). FKBP12 modulates Ca2+ release from the sarcoplasmic reticulum in skeletal muscle and developmental cardiac defects have been reported in FKBP12-deficient mice, but the role of FKBP12.6 in cardiac excitation-contraction coupling remains unclear. Here we show that disruption of the FKBP12.6 gene in mice results in cardiac hypertrophy in male mice, but not in females. Female hearts are normal, despite the fact that male and female knockout mice display similar dysregulation of Ca2+ release, seen as increases in the amplitude and duration of Ca2+ sparks and calcium-induced calcium release gain. Female FKBP12.6-null mice treated with tamoxifen, an oestrogen receptor antagonist, develop cardiac hypertrophy similar to that of male mice. We conclude that FKBP12.6 modulates cardiac excitation-contraction coupling and that oestrogen plays a protective role in the hypertrophic response of the heart to Ca2+ dysregulation.     

Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution

Calcium ATPase is a member of the P-type ATPases that transport ions across the membrane against a concentration gradient. Here we have solved the crystal structure of the calcium ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1a) at 2.6 A resolution with two calcium ions bound in the transmembrane domain, which comprises ten alpha-helices. The two calcium ions are located side by side and are surrounded by four transmembrane helices, two of which are unwound for efficient coordination geometry. The cytoplasmic region consists of three well separated domains, with the phosphorylation site in the central catalytic domain and the adenosine-binding site on another domain. The phosphorylation domain has the same fold as haloacid dehalogenase. Comparison with a low-resolution electron density map of the enzyme in the absence of calcium and with biochemical data suggests that large domain movements take place during active transport.     

Involvement of dihydropyridine receptors in excitation-contraction coupling in skeletal muscle

The transduction of action potential to muscle contraction (E-C coupling) is an example of fast communication between plasma membrane events and the release of calcium from an internal store, which in muscle is the sarcoplasmic reticulum (SR). One theory is that the release channels of the SR are controlled by voltage-sensing molecules or complexes, located in the transverse tubular (T)-membrane, which produce, as membrane voltage varies, 'intramembrane charge movements', but nothing is known about the structure of such sensors. Receptors of the Ca-channel-blocking dihydropyridines present in many tissues, are most abundant in T-tubular muscle fractions from which they can be isolated as proteins. Fewer than 5% of muscle dihydropyridines are functional Ca channels; there is no known role for the remainder in skeletal muscle physiology. We report here that low concentrations of a dihydropyridine inhibit charge movements and SR calcium release in parallel. The effect has a dependence on membrane voltage analogous to that of specific binding of dihydropyridines. We propose specifically that the molecule that generates charge movement is the dihydropyridine receptor.