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.     

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.     

Distribution of two distinct Ca2+-ATPase-like proteins and their relationships to the agonist-sensitive calcium store in adrenal chromaffin cells

Many cellular functions are regulated by activation of cell-surface receptors that mobilize calcium from internal stores sensitive to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The nature of these internal calcium stores and their localization in cells is not clear and has been a subject of debate. It was originally suggested that the Ins(1,4,5)P3-sensitive store is the endoplasmic reticulum, but a new organelle, the calciosome, identified by its possession of the calcium-binding protein, calsequestrin, and a Ca2+-ATPase-like protein of relative molecular mass 100,000 (100K), has been described as a potential Ins(1,4,5)P3-sensitive calcium store. Direct evidence on whether the calciosome is the Ins(1,4,5)P3-sensitive store is lacking. Using monoclonal antibodies raised against the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, we show that bovine adrenal chromaffin cells contain two Ca2+-ATPase-like proteins with distinct subcellular distributions. A 100K Ca2+-ATPase-like protein is diffusely distributed, whereas a 140K Ca2+-ATPase-like protein is restricted to a region in close proximity to the nucleus. In addition, Ins(1,4,5)P3-generating agonists result in a highly localized rise in cytosolic calcium concentration ([Ca2+]i) initiated in a region close to the nucleus, whereas caffeine results in a rise in [Ca2+]i throughout the cytoplasm. Our results indicate that chromaffin cells possess two calcium stores with distinct Ca2+-ATPases and that the organelle with the 100K Ca2+-ATPase is not the Ins(1,4,5)P3-sensitive store.     

A saturable receptor for 32P-inositol-1,4,5-triphosphate in hepatocytes and neutrophils

Several receptors for neurotransmitters, hormones and growth factors cause accelerated phosphodiesteratic breakdown of polyphosphoinositides when activated. One of the soluble products of this reaction, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) is thought to act as a second messenger signalling the release of Ca2+ from intracellular stores. In support of this hypothesis, several studies have shown that Ins(1,4,5)P3 releases sequestered Ca2+ from permeable cells and microsomes. On the basis of certain structural requirements for Ca2+-releasing activity by inositol phosphates, it has been postulated that Ins(1,4,5)P3 acts by binding to a specific intracellular receptor, probably on a component of the endoplasmic reticulum. Here we report that 32P-Ins(1,4,5)P3 binds to a specific saturable site in permeabilized guinea pig hepatocytes and rabbit neutrophils, and that the properties of this binding site suggest that it is the physiological receptor for Ins(1,4,5)P3.     

Putative receptor for inositol 1,4,5-trisphosphate similar to ryanodine receptor

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) serves as an intracellular second messenger for several neurotransmitters, hormones and growth factors by initiating calcium release from intracellular stores. A cerebellar Ins(1,4,5)P3 receptor has been characterized biochemically and shown by immunocytochemistry to be present in intracellular membranes in Purkinje cells. We show that a previously described Purkinje-cell messenger RNA encodes a protein of relative molecular mass 260,000 (260 K) with the same properties as the cerebellar Ins(1,4,5)P3 receptor. Its sequence is partially homologous to the skeletal muscle ryanodine receptor. By immunocytochemistry and electron microscopy the protein is shown to be present in all parts of the endoplasmic reticulum, including those that extend into axon terminals and dendritic spines. Our results indicate that gated calcium release from intracellular stores in muscle and Purkinje cells uses similar calcium-channel proteins localized in analogous intracellular compartments. This implies that the intracellular calcium stores in the endoplasmic reticulum of neurons extend into presynaptic terminals and dendritic spines where they may play a direct role in regulating the efficacy of neurotransmission.     

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.     

Irreversible inhibition of Ca2+ release in saponin-treated macrophages by the photoaffinity derivative of inositol-1, 4, 5-trisphosphate

D-myo-inositol-1,4,5-trisphosphate (InsP3) is a putative intracellular second messenger for the mobilization of Ca2+ from intracellular stores, in particular, the endoplasmic reticulum. Specific binding sites on the endoplasmic reticulum may participate in the InsP3-induced release of Ca2+ from the Ca2+ pool. To examine the specific binding sites on the endoplasmic reticulum, we synthesized an arylazide derivative of InsP3 for photoaffinity labelling; InsP3 coupled to p-azidobenzoic acid (InsP3-pAB) using N,N'-carbonyldiimidazole (CDI) was obtained at a 9-11% yield. Here, we report that InsP3-pAB, but not an arylazide derivative of inositol-1,4-bisphophate (Ins(1,4)P2), causes the irreversible inhibition of InsP3-induced release of Ca2+ in saponin-permeabilized photo-irradiated macrophages. The irreversible inhibition by InsP3-pAB after photo-irradiation was prevented by a 10-fold excess of unmodified InsP3.     

Rapid mobilization of Ca2+ from rat insulinoma microsomes by inositol-1,4,5-trisphosphate

Several hormones and neurotransmitters raise the cytosolic free Ca2+ concentration by stimulating the influx of Ca2+ and/or by mobilizing stored Ca2+. However, the link between the agonist receptor on the cell surface and the organelle(s) from which Ca2+ is mobilized is unknown. One feature of the agonists that increase cytosolic Ca2+ is their rapid induction of phosphatidylinositol turnover and polyphosphoinositide hydrolysis; in some tissues this leads, within seconds, to a marked accumulation of the water-soluble products, inositol 1,4-bisphosphate ( Ins1 , 4P2 ) and inositol-1,4,5- trisphosphate ( Ins1 ,4, 5P3 ), suggesting that these might mediate Ca2+ mobilization from internal pools. Such an action of Ins1 ,4, 5P3 has recently been inferred from studies with permeabilized pancreatic acinar cells and hepatocytes. Here we show directly that Ins1 ,4, 5P3 rapidly releases Ca2+ from a microsomal fraction of rat insulinoma but not from mitochondria or secretory granules. Moreover, this response is transient and desensitizes the microsomes to subsequent Ins1 ,4, 5P3 additions. These results suggest that Ins1 ,4, 5P3 functions as a cellular messenger inducing early mobilization of Ca2+ from the endoplasmic reticulum.     

Restoration of dysgenic muscle contraction and calcium channel function by co-culture with normal spinal cord neurons

Muscular dysgenesis (mdg) is a spontaneous recessive lethal mutation in the mouse. The disease is characterized by a total lack of excitation-contraction coupling in embryonic skeletal muscle. This developmental abnormality is associated with a drastic deficiency in the expression of voltage-sensitive Ca2+ channels in skeletal muscle without alteration of the properties of voltage-sensitive Na+ channels or of voltage-sensitive Ca2+ channels in cardiac and neuronal cells. Membrane couplings between sarcoplasmic reticulum and the transverse tubules, known as triads, were also found to be drastically altered in embryonic muscle of the homozygous mutant (mdg/mdg). Triads in the mdg/mdg muscle were less numerous, disorganized and lacked spaced densities. This paper shows that co-culture of mdg/mdg myotubes with normal spinal cord neurons re-establishes Ca2+ channel activity, contraction and normal triad organization. The decrease thus cannot be due to a mutation of the Ca2+ channel as previously suggested. Normal nerve cells may supply an essential factor to mutant muscle cells.     

The structural basis of calcium transport by the calcium pump

The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding, Ca2+ translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca2+-ATPase. Phosphorylation of the enzyme triggers the onset of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca2+ release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical gradients required for key functions in eukaryotic cells.     

Ion channels activated by inositol 1,4,5-trisphosphate in plasma membrane of human T-lymphocytes

Hydrolysis of membrane-associated phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)-P2) to water soluble inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is a common response by many different kinds of cells to a wide variety of external stimuli (see refs 1 and 2 for review). Ins (1,4,5)P3 is a putative second messenger which increases intracellular Ca2+ by mobilizing internal Ca2+ stores, a hypothesis which has been substantiated by studies with chemically permeabilized cells and with isolated microsomal membrane fractions. But the possibility that Ins(1,4,5)P3 could induce in intact cells an influx of external Ca2+ through transmembrane channels, originally hypothesized by Michell in 1975, has never been directly tested. We report here single-channel recordings of an Ins(1,4,5)P3-activated conductance in excised patches of T-lymphocyte plasma membrane. The Ins(1,4,5)P3-activated transmembrane channel appears to be identical to the recently described mitogen-regulated, voltage-insensitive Ca2+ permeable channel involved in T-cell activation. We suggest that Ins(1,4,5)P3 acts as the second messenger mediating transmembrane Ca2+ influx through specific Ca2+-permeable channels in mitogen-stimulated T-cell activation.     

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.     

Inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons

Inositol 1,4,5-trisphosphate (InsP3) mediates the effects of several neurotransmitters, hormones and growth factors by mobilizing Ca2+ from a vesicular, non-mitochondrial intracellular store. Many studies have indirectly suggested the endoplasmic reticulum (ER) to be the site of InsP3 action, though some have implicated the plasma membrane or a newly described smooth surfaced structure, termed the calciosome. Using antibodies directed against a purified InsP3-receptor glycoprotein, of relative molecular mass 260,000, in electron microscope immunocytochemical studies of rat cerebellar Purkinje cells, we have now localized the InsP3 receptor to ER, including portions of the rough endoplasmic reticulum, a population of smooth-membrane-bound organelles (smooth ER), a portion of subplasmalemmal cisternae and the nuclear membrane, but not to mitochondria or the cell membrane. These results suggest that in cerebellar Purkinje cells, InsP3-induced intracellular calcium release is not the property of a single organelle, but is effected by specialized portions of both rough and smooth ER, and possibly by other smooth surfaced structures. The present findings are the first immunocytochemical demonstration of an InsP3 receptor within a cell.     

Purified inositol 1,4,5-trisphosphate receptor mediates calcium flux in reconstituted lipid vesicles

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), a second messenger molecule involved in actions of neurotransmitters, hormones and growth factors, releases calcium from vesicular non-mitochondrial intracellular stores. An Ins(1,4,5)P3 binding protein, purified from brain membranes, has been shown to be phosphorylated by cyclic-AMP-dependent protein kinase and localized by immunohistochemical techniques to intracellular particles associated with the endoplasmic reticulum. Although the specificity of the Ins(1,4,5)P3 binding protein for inositol phosphates and the high affinity of the protein for Ins(1,4,5)P3 indicate that it is a physiological Ins(1,4,5)P3 receptor mediating calcium release, direct evidence for this has been difficult to obtain. Also, it is unclear whether a single protein mediates both the recognition of Ins(1,4,5)P3 and calcium transport or whether these two functions involve two or more distinct proteins. In the present study we report reconstitution of the purified Ins(1,4,5)P3 binding protein into lipid vesicles. We show that Ins(1,4,5)P3 and other inositol phosphates stimulate calcium flux in the reconstituted vesicles with potencies and specificities that match the calcium releasing actions of Ins(1,4,5)P3. These results indicate that the purified Ins(1,4,5)P3 binding protein is a physiological receptor responsible for calcium release.     

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.     

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.     

Kinetics of smooth and skeletal muscle activation by laser pulse photolysis of caged inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) can stimulate skinned smooth and skeletal muscle to contract by initiating Ca2+ release from the sarcoplasmic reticulum. Whether this process is an integral component of the in vivo muscle activation mechanism was tested by releasing InsP3 rapidly within skinned muscle fibers of rabbit main pulmonary artery and frog semitendinosus. InsP3 was liberated on laser pulse photolysis of a photolabile but biologically inactive precursor of InsP3 termed caged InsP3. Caged InsP3 is a mixture of compounds in which InsP3 is esterified with 1(2-nitrophenyl)diazoethane (probably at the P4- or P5-position). Photochemical release of InsP3 induced a full contraction in both muscles at physiological free Mg2+ concentrations, but only in the smooth muscle were the InsP3 concentration (0.5 microM) and the activation rate compatible with the in vivo physiological response. Endogenous InsP3-specific phosphatase activity was present in smooth muscle and had about 35-fold greater activity than that in the skeletal-muscle preparation. Caged InsP3 was not susceptible to phosphatases in either preparation.     

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.     

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.