Evidence for conformational coupling between two calcium channels.

Ryanodine receptor 1 (RyR1, the sarcoplasmic reticulum Ca(2+) release channel) and alpha(1S)dihydropyridine receptor (DHPR, the surface membrane voltage sensor) of skeletal muscle belong to separate membrane systems but are functionally and structurally linked. Four alpha(1S)DHPRs associated with the four identical subunits of a RyR form a tetrad. We treated skeletal muscle cell lines with ryanodine, at concentrations that block RyRs, and determined whether this treatment affects the distance between DHPRs in the tetrad. We find a substantial ( approximately 2-nm) shift in the alpha(1S)DHPR positions, indicating that ryanodine induces large conformational changes in the RyR1 cytoplasmic domain and that the alpha(1S)DHPR-RyR complex acts as a unit.

Disruption of Caenorhabditis elegans muscle structure and function caused by mutation of troponin I.

Caenorhabditis elegans strains mutant for the unc-27 gene show abnormal locomotion and muscle structure. Experiments revealed that unc-27 is one of four C. elegans troponin I genes and that three mutant alleles truncate the protein: recessive and presumed null allele e155 terminates after nine codons; semidominant su142sd eliminates the inhibitory and C-terminal regions; and semidominant su195sd abbreviates the extreme C-terminus. Assays of in vivo muscular performance at high and low loads indicated that su142sd is most deleterious, with e155 least and su195sd intermediate. Microscopy revealed in mutant muscle a prevalent disorder of dense body positioning and a less well defined sarcomeric structure, with small islands of thin filaments interspersed within the overlap region of A bands and even within the H zone. The mutants' rigid paralysis and sarcomeric disarray are consistent with unregulated contraction of the sarcomeres, in which small portions of each myofibril shorten irregularly and independently of one another, thereby distorting the disposition of filaments. The exacerbated deficits of su142sd worms are compatible with involvement in vivo of the N-terminal portion of troponin I in enhancing force production, and the severe impairment associated with su195sd highlights importance of the extreme C-terminus in the protein's inhibitory function.

MUP-4 is a novel transmembrane protein with functions in epithelial cell adhesion in Caenorhabditis elegans.

Tissue functions and mechanical coupling of cells must be integrated throughout development. A striking example of this coupling is the interactions of body wall muscle and hypodermal cells in Caenorhabditis elegans. These tissues are intimately associated in development and their interactions generate structures that provide a continuous mechanical link to transmit muscle forces across the hypodermis to the cuticle. Previously, we established that mup-4 is essential in embryonic epithelial (hypodermal) morphogenesis and maintenance of muscle position. Here, we report that mup-4 encodes a novel transmembrane protein that is required for attachments between the apical epithelial surface and the cuticular matrix. Its extracellular domain includes epidermal growth factor-like repeats, a von Willebrand factor A domain, and two sea urchin enterokinase modules. Its intracellular domain is homologous to filaggrin, an intermediate filament (IF)-associated protein that regulates IF compaction and that has not previously been reported as part of a junctional complex. MUP-4 colocalizes with epithelial hemidesmosomes overlying body wall muscles, beginning at the time of embryonic cuticle maturation, as well as with other sites of mechanical coupling. These findings support that MUP-4 is a junctional protein that functions in IF tethering, cell-matrix adherence, and mechanical coupling of tissues.

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine.

The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca(2+) release from endoplasmic reticulum/sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high- and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200-500 microM ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, P(o) < 0.005) and brief (<250 micros) gating events and insensitivity to Ca(2+). Addition of ryanodine restores Ca(2+)-dependent channel activity exhibiting full, 3/4, 1/2, and 1/4 substates. This evidence suggests that, whereas ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.

Structural alterations in cardiac calcium release units resulting from overexpression of junctin.

Junctin is a 26 kDa membrane protein that binds to calsequestrin, triadin, and ryanodine receptors (RyRs) within the junctional sarcoplasmic reticulum of calcium release units. The sequence of junctin includes a short N-terminal cytoplasmic domain a single transmembrane domain, and a highly charged C-terminal domain located in the sarcoplasmic reticulum lumen. Dog and mouse junctins are highly conserved at the transmembrane domains, but the luminal domains are more divergent. To probe the contribution of junctin to the architecture of calcium release units in heart, we engineered transgenic mice overexpressing canine junctin and examined the left ventricular myocardium by electron microscopy. Overall architecture of calcium release units is similar in control myocardium and in myocardium overexpressing junctin by 5-10-fold. In both myocardia, junctional SR cisternae are closely associated with exterior membranes (plasmalemma and transverse tubules). The cisternae are flat; they contain a string of calsequestrin beads and are lined by a row of feet, or RyRs, on the side facing the exterior membranes. T tubule surface density, measured as the perimeter of T tubule profiles v area of section, is the same in transgenic and control myocardia (305 v 289 nm/nm(2)). Three changes affecting the junctional SR architecture are apparent in the myocardium overexpressing junctin. One is a more tightly zippered appearance of the junctional SR cisternae. The width of the junctional SR is narrower and less variable in overexpressing than in control myocardium and the calsequestrin content is more compact. A second change is the extension of zippered junctional SR domains to non-junctional regions, which we term "frustrated" junctional SR. A third change is an increase in the extent of association between SR and T tubules. In junctin overexpressing myocardium junctional SR cisternae cover approximately 45% of the surface of all T tubule profiles, while in control myocardium the coverage approximately 30%. Junctional associations between SR and T tubules are increased in size. We conclude that the increase in junctin expression affects the packing of calsequestrin in the junctional SR and facilitates the association of SR and T tubules.

Sequential docking, molecular differentiation, and positioning of T-Tubule/SR junctions in developing mouse skeletal muscle.

Skeletal muscle Ca(2+) release units (CRUs) are junctions of the surface membrane/T-tubule system and the sarcoplasmic reticulum (SR) that function in excitation-contraction coupling. They contain high concentrations of dihydropyridine receptors (DHPRs) in the T-tubules and of ryanodine receptors (RyR) in the SR and they are positioned at specific locations in the sarcomere. In order to characterize the sequence of developmental steps leading to the specific molecular and structural organization of CRUs, we applied a range of imaging techniques that allowed us to follow the differentiation of the membrane compartments and the expression of junctional proteins in developing mouse diaphragm muscle. We find that docking of the two membrane systems precedes the incorporation of the RyRs into the junctions, and that T-tubule/SR junctions are formed and positioned at the I-A interface at a stage when the orientation of T-tubule is predominantly longitudinal. Thus, the sequence of developmental events is first the docking of T-tubules and SR, secondly the incorporation of RyR in the junctions, thirdly the positioning of the junctions in the sarcomere, and only much later the transverse orientation of the T-tubules. These sequential stages suggests an order of inductive processes for the molecular differentiation and structural organization of the CRUs in skeletal muscle development.

RYR1 and RYR3 have different roles in the assembly of calcium release units of skeletal muscle.

Calcium release units (CRUs) are junctions between the sarcoplasmic reticulum (SR) and exterior membranes that mediates excitation contraction (e-c) coupling in muscle cells. In skeletal muscle CRUs contain two isoforms of the sarcoplasmic reticulum Ca(2+)release channel: ryanodine receptors type 1 and type 3 (RyR1 and RyR3). 1B5s are a mouse skeletal muscle cell line that carries a null mutation for RyR1 and does not express either RyR1 or RyR3. These cells develop dyspedic SR/exterior membrane junctions (i.e., dyspedic calcium release units, dCRUs) that contain dihydropyridine receptors (DHPRs) and triadin, two essential components of CRUs, but no RyRs (or feet). Lack of RyRs in turn affects the disposition of DHPRs, which is normally dictated by a linkage to RyR subunits. In the dCRUs of 1B5 cells, DHPRs are neither grouped into tetrads nor aligned in two orthogonal directions. We have explored the structural role of RyR3 in the assembly of CRUs in 1B5 cells independently expressing either RyR1 or RyR3. Either isoform colocalizes with DHPRs and triadin at the cell periphery. Electron microscopy shows that expression of either isoform results in CRUs containing arrays of feet, indicating the ability of both isoforms to be targeted to dCRUs and to assemble in ordered arrays in the absence of the other. However, a significant difference between RyR1- and RyR3-rescued junctions is revealed by freeze fracture. While cells transfected with RyR1 show restoration of DHPR tetrads and DHPR orthogonal alignment indicative of a link to RyRs, those transfected with RyR3 do not. This indicates that RyR3 fails to link to DHPRs in a specific manner. This morphological evidence supports the hypothesis that activation of RyR3 in skeletal muscle cells must be indirect and provides the basis for failure of e-c coupling in muscle cells containing RyR3 but lacking RyR1 (see the accompanying report, ).

Transport of Ca2+ from sarcoplasmic reticulum to mitochondria in rat ventricular myocytes.

Studies with electron microscopy have shown that sarcoplasmic reticulum (SR) and mitochondria locate close to each other in cardiac muscle cells. We investigated the hypothesis that this proximity results in a transient exposure of mitochondrial Ca2+ uniporter (CaUP) to high concentrations of Ca2+ following Ca2+ release from the SR and thus an influx of Ca2+ into mitochondria. Single ventricular myocytes of rat were skinned by exposing them to a physiological solution containing saponin (0.2 mg/ml). Cytosolic Ca2+ concentration ([Ca2+]c) and mitochondrial Ca2+ concentration ([Ca2+]m) were measured with fura-2 and rhod2, respectively. Application of caffeine (10 mM) induced a concomitant increase in [Ca2+]c and [Ca2+]m. Ruthenium red, at concentrations that block CaUP but not SR release, diminished the caffeine-induced increase in [Ca2+]m but not [Ca2+]c. In the presence of 1 mM BAPTA, a Ca2+ chelator, the caffeine-induced increase in [Ca2+]m was reduced substantially less than [Ca2+]c. Moreover, inhibition of SR Ca2+ pump with two different concentrations of thapsigargin caused an increase in [Ca2+]m, which was related to the rate of [Ca2+]c increase. Finally, electron microscopy showed that sites of junctions between SR and T tubules from which Ca2+ is released, or Ca2+ release units, CRUs, are preferentially located in close proximity to mitochondria. The distance between individual SR Ca2+ release channels (feet or ryanodine receptors) is very short, ranging between approximately 37 and 270 nm. These results are consistent with the idea that there is a preferential coupling of Ca2+ transport from SR to mitochondria in cardiac muscle cells, because of their structural proximity.

Tomographic 3D reconstruction of quick-frozen, Ca2+-activated contracting insect flight muscle.

Motor actions of myosin were directly visualized by electron tomography of insect flight muscle quick-frozen during contraction. In 3D images, active cross-bridges are usually single myosin heads, bound preferentially to actin target zones sited midway between troponins. Active attached bridges (approximately 30% of all heads) depart markedly in axial and azimuthal angles from Rayment's rigor acto-S1 model, one-third requiring motor domain (MD) tilting on actin, and two-thirds keeping rigor contact with actin while the light chain domain (LCD) tilts axially from approximately 105 degrees to approximately 70 degrees. The results suggest the MD tilts and slews on actin from weak to strong binding, followed by swinging of the LCD through an approximately 35 degrees axial angle, giving an approximately 13 nm interaction distance and an approximately 4-6 nm working stroke.

Shape, size, and distribution of Ca(2+) release units and couplons in skeletal and cardiac muscles.

Excitation contraction (e-c) coupling in skeletal and cardiac muscles involves an interaction between specialized junctional domains of the sarcoplasmic reticulum (SR) and of exterior membranes (either surface membrane or transverse (T) tubules). This interaction occurs at special structures named calcium release units (CRUs). CRUs contain two proteins essential to e-c coupling: dihydropyridine receptors (DHPRs), L-type Ca(2+) channels of exterior membranes; and ryanodine receptors (RyRs), the Ca(2+) release channels of the SR. Special CRUs in cardiac muscle are constituted by SR domains bearing RyRs that are not associated with exterior membranes (the corbular and extended junctional SR or EjSR). Functional groupings of RyRs and DHPRs within calcium release units have been named couplons, and the term is also loosely applied to the EjSR of cardiac muscle. Knowledge of the structure, geometry, and disposition of couplons is essential to understand the mechanism of Ca(2+) release during muscle activation. This paper presents a compilation of quantitative data on couplons in a variety of skeletal and cardiac muscles, which is useful in modeling calcium release events, both macroscopic and microscopic ("sparks").

Correct targeting of dihydropyridine receptors and triadin in dyspedic mouse skeletal muscle in vivo.

Excitation-contraction coupling in skeletal muscle involves junctions (triads and dyads) between sarcoplasmic reticulum (SR) and transverse (T) -tubules. Two proteins of the junctional SR, ryanodine receptors (RyRs) and triadin and one protein of T tubules, dihydropyridine receptors (DHPRs) are located at these junctions. We studied the targeting of DHPRs and triadin to T-tubules and SR in skeletal muscles of dyspedic mouse embryos lacking RyR1. In normal differentiating muscle fibers DHPRs, triadin and RyRs are located in intensely immunolabeled foci that are randomly distributed across the fiber. Correlation with electron microscopy and with previous studies indicates that the foci represent the location of triads and dyads. In dyspedic fibers DHPRs and triadin antibodies stain internal foci of the two proteins; RyR antibodies are completely negative. The appearance and location of the foci in dyspedic fibers is similar to that of normal muscle, but their fluorescent intensity is weaker. The SR Ca-ATPase has more diffuse distribution than triadin in both normal and dyspedic fibers. These observations indicate that an interaction with RyRs is not necessary for the appropriate targeting of DHPRs or triadin to junctional domains of T tubules and SR respectively.

The sarcoplasmic reticulum and the control of muscle contraction.

Activation of muscle contraction is a rapid event that is initiated by electrical activity in the surface membrane and transverse (T) tubules. This is followed by release of calcium from the inner membrane system, the sarcoplasmic reticulum (SR). Using electron microscopy (EM), K. R. Porter and his laboratory defined the SR, the unique junctions between SR and T tubules, and the continuity between T tubules and surface membrane. Current research in this area centers on the interaction between T tubules and SR. This is mediated by 2 well-identified calcium channels: the dihydropyridine receptors (DHPRs) that act as voltage sensors in the T tubules, and the ryanodine receptors (RyRs) or calcium release channels of the SR. The relative positions of these 2 molecules differ significantly in skeletal and cardiac muscle, and this correlates well with known functional differences in the control of contraction. Molecular biology experiments combined with EM indicate that DHPRs are linked to RyRs in skeletal but probably not in cardiac muscle.

Regulation of Ca2+ signaling in transgenic mouse cardiac myocytes overexpressing calsequestrin.

To probe the physiological role of calsequestrin in excitation-contraction coupling, transgenic mice overexpressing cardiac calsequestrin were developed. Transgenic mice exhibited 10-fold higher levels of calsequestrin in myocardium and survived into adulthood, but had severe cardiac hypertrophy, with a twofold increase in heart mass and cell size. In whole cell-clamped transgenic myocytes, Ca2+ channel- gated Ca2+ release from the sarcoplasmic reticulum was strongly suppressed, the frequency of occurrence of spontaneous or Ca2+ current-triggered "Ca2+ sparks" was reduced, and the spark perimeter was less defined. In sharp contrast, caffeine-induced Ca2+ transients and the resultant Na+-Ca2+ exchanger currents were increased 10-fold in transgenic myocytes, directly implicating calsequestrin as the source of the contractile-dependent pool of Ca2+. Interestingly, the proteins involved in the Ca2+-release cascade (ryanodine receptor, junctin, and triadin) were downregulated, whereas Ca2+-uptake proteins (Ca2+-ATPase and phospholamban) were unchanged or slightly increased. The parallel increase in the pool of releasable Ca2+ with overexpression of calsequestrin and subsequent impairment of physiological Ca2+ release mechanism show for the first time that calsequestrin is both a storage and a regulatory protein in the cardiac muscle Ca2+-signaling cascade. Cardiac hypertrophy in these mice may provide a novel model to investigate the molecular determinants of heart failure.

Role of ryanodine receptors in the assembly of calcium release units in skeletal muscle.

In muscle cells, excitation-contraction (e-c) coupling is mediated by "calcium release units," junctions between the sarcoplasmic reticulum (SR) and exterior membranes. Two proteins, which face each other, are known to functionally interact in those structures: the ryanodine receptors (RyRs), or SR calcium release channels, and the dihydropyridine receptors (DHPRs), or L-type calcium channels of exterior membranes. In skeletal muscle, DHPRs form tetrads, groups of four receptors, and tetrads are organized in arrays that face arrays of feet (or RyRs). Triadin is a protein of the SR located at the SR-exterior membrane junctions, whose role is not known. We have structurally characterized calcium release units in a skeletal muscle cell line (1B5) lacking Ry1R. Using immunohistochemistry and freeze-fracture electron microscopy, we find that DHPR and triadin are clustered in foci in differentiating 1B5 cells. Thin section electron microscopy reveals numerous SR-exterior membrane junctions lacking foot structures (dyspedic). These results suggest that components other than Ry1Rs are responsible for targeting DHPRs and triadin to junctional regions. However, DHPRs in 1B5 cells are not grouped into tetrads as in normal skeletal muscle cells suggesting that anchoring to Ry1Rs is necessary for positioning DHPRs into ordered arrays of tetrads. This hypothesis is confirmed by finding a "restoration of tetrads" in junctional domains of surface membranes after transfection of 1B5 cells with cDNA encoding for Ry1R.

Contractile impairment and structural alterations of skeletal muscles from knockout mice lacking type 1 and type 3 ryanodine receptors.

Skeletal muscle contraction is triggered by the release of Ca2+ from the sarcoplasmic reticulum through the type 1 ryanodine receptor (RyR1). Recently it has been shown that also the type 3 isoform of ryanodine receptor (RyR3), which is expressed in some mammalian skeletal muscles, may participate in the regulation of skeletal muscle contraction. Here we report the generation and the characterization of double mutant mice carrying a targeted disruption of both the RyR1 and the RyR3 genes (RyR1-/-;RyR3-/-). Skeletal muscles from mice homozygous for both mutations are unable to contract in response to caffeine and to ryanodine. In addition, they show a very poor capability to develop tension when directly activated with micromolar [Ca2+]i after membrane permeabilization which indicates either poor development or degeneration of the myofibrils. This was confirmed by biochemical analysis of contractile proteins. Electron microscopy confirms small size of myofibrils and shows complete absence of feet (RyRs) in the junctional SR.

Comparative ultrastructure of Ca2+ release units in skeletal and cardiac muscle.

The sarcoplasmic reticulum (SR) of striated muscle fibers interacts with exterior membranes (surface membrane and transverse tubules) to form junctions that are involved in the internal release of calcium during excitation-contraction coupling. Release of calcium through the ryanodine receptors (RyRs) or calcium release channels of the SR is under the control of the L type calcium channels or dihydropyridine receptors (DHPRs) of exterior membranes. Interacting clusters of the two proteins constitute calcium release units. The cytoplasmic domains of RyRs are visible as large electron-dense structures (the feet) with four identical subunits in the junctional gap separating SR from exterior membranes. In freeze-fracture replicas of skeletal muscle, large intramembrane particles are grouped into clusters of tetrads in the exterior membranes, and the tetrads are located in correspondence of the four subunits of the feet. Lack of tetrads in dysgenic muscle fibers with a null mutation for DHPRs and appearance of the tetrads after transfection with cDNA for DHPR indicate identity of tetrads with four DHPRs. In cardiac muscle, DHPRs are located at the sites of SR-surface junctions, but they are not grouped into tetrads. This is consistent with a possible direct DHPR-RyR interaction in skeletal but not in cardiac muscle. The size and distribution of SR-surface junctions in skeletal and cardiac muscles provide further clues to their function.

Ryanodine receptors of striated muscles: a complex channel capable of multiple interactions.

The ryanodine receptor (RyR) is a high-conductance Ca2+ channel of the sarcoplasmic reticulum in muscle and of the endoplasmic reticulum in other cells. In striated muscle fibers, RyRs are responsible for the rapid release of Ca2+ that activates contraction. Ryanodine receptors are complex molecules, with unusually large cytoplasmic domains containing numerous binding sites for agents that control the state of activity of the channel-forming domain of the molecule. Structural considerations indicate that long-range interactions between cytoplasmic and intramembrane domains control channel function. Ryanodine receptors are located in specialized regions of the SR, where they are structurally and functionally associated with other intrinsic proteins and, indirectly, also with the luminal Ca2(+)-binding protein calsequestrin. Activation of RyRs during the early part of the excitation-contraction coupling cascade is initiated by the activity of surface-membrane Ca2+ channels, the dihydropyridine receptors (DHPRs). Skeletal and cardiac muscles contain different RyR and DHPR isoforms and both contribute to the diversity in cardiac and skeletal excitation-contraction coupling mechanisms. The architecture of the sarcoplasmic reticulum-surface junctions determines the types of RyR-DHPR interactions in the two muscle types.

Coordinated incorporation of skeletal muscle dihydropyridine receptors and ryanodine receptors in peripheral couplings of BC3H1 cells.

Rapid release of calcium from the sarcoplasmic reticulum (SR) of skeletal muscle fibers during excitation-contraction (e-c) coupling is initiated by the interaction of surface membrane calcium channels (dihydropyridine receptors; DHPRs) with the calcium release channels of the SR (ryanodine receptors; RyRs, or feet). We studied the early differentiation of calcium release units, which mediate this interaction, in BC3H1 cells. Immunofluorescence labelings of differentiating myocytes with antibodies against alpha1 and alpha2 subunits of DHPRs, RyRs, and triadin show that the skeletal isoforms of all four proteins are abundantly expressed upon differentiation, they appear concomitantly, and they are colocalized. The transverse tubular system is poorly organized, and thus clusters of e-c coupling proteins are predominantly located at the cell periphery. Freeze fracture analysis of the surface membrane reveals tetrads of large intramembrane particles, arranged in orderly arrays. These appear concomitantly with arrays of feet (RyRs) and with the appearance of DHPR/RyS clusters, confirming that the four components of the tetrads correspond to skeletal muscle DHPRs. The arrangement of tetrads and feet in developing junctions indicates that incorporation of DHPRs in junctional domains of the surface membrane proceeds gradually and is highly coordinated with the formation of RyR arrays. Within the arrays, tetrads are positioned at a spacing of twice the distance between the feet. The incorporation of individual DHPRs into tetrads occurs exclusively at positions corresponding to alternate feet, suggesting that the assembly of RyR arrays not only guides the assembly of tetrads but also determines their characteristic spacing in the junction.