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Junctional feet and particles in triads of a fast-twitch muscle
Abstract
Structural details of junctional feet in triads of fish muscle are described. These feet have a less dense central core and contact both sarcoplasmic reticulum and T-tubule membranes at tetragonally disposed sites. The distribution of intramembraneous particles differs at the junctional T-membrane, and the junction is asymmetric.
... Ca V 1.1 are detected in the electron microscope by the technique of freeze fracture, which reveals the position of the α1 subunit as a prominent, tall particle. In normal calcium release units (CRUs), Ca V 1.1 particles are grouped in small clusters of four, termed junctional tetrads (Franzini-Armstrong and Nunzi, 1983; an example is in Fig. 1 B). The grouping of Ca V 1.1 is directly dependent on isoform-specific interactions of the two skeletal muscle isoforms, Ca V 1.1 and RYR1, as demonstrated by the fact that Ca V 1.1 do not cluster into tetrads in CRUs of dyspedic (RYR1-null) myotubes or in cardiac myocytes where the two cardiac isoforms (Ca V 1.2 and RYR2) are present. ...
... Fig. 6 exemplifies images from multiple sources and illustrates some of the problems found in their analysis. Fig. 6 A is from the toadfish swim bladder, in the first study that revealed tetrads (Franzini-Armstrong and Nunzi, 1983); in this case, only one of the two rows of feet is visible, which makes this type of image not suitable for quantitative analysis. Fig. 6 B is from a later study of the toadfish swim bladder (Block et al., 1988); Fig. 6 C shows the result of the alignment/idealization approach to the image in Fig. 6 B. Fig. 6 D is a junction in a stac3 −/− zebrafish rescued by expression of WT stac3 (Linsley et al., 2017). ...
... Quantitative analysis of triadic junctions. (A) T tubule of a toadfish swim bladder(Franzini-Armstrong and Nunzi, 1983). The quantitative analysis is not possible, as the couplon or couplons are only partially imaged. ...
Calcium for contraction of skeletal muscles is released via tetrameric ryanodine receptor (RYR1) channels of the sarcoplasmic reticulum (SR), which are assembled in ordered arrays called couplons at junctions where the SR abuts T tubules or plasmalemma. Voltage-gated Ca ²⁺ (Ca V 1.1) channels, found in tubules or plasmalemma, form symmetric complexes called Ca V tetrads that associate with and activate underlying RYR tetramers during membrane depolarization by conveying a conformational change. Intriguingly, Ca V tetrads regularly skip every other RYR tetramer within the array; therefore, the RYRs underlying tetrads (named V), but not the voltage sensor–lacking (C) RYRs, should be activated by depolarization. Here we hypothesize that the checkerboard association is maintained solely by reversible binary interactions between Ca V s and RYRs and test this hypothesis using a quantitative model of the energies that govern Ca V 1.1–RYR1 binding, which are assumed to depend on number and location of bound Ca V s. A Monte Carlo simulation generates large statistical samples and distributions of state variables that can be compared with quantitative features in freeze-fracture images of couplons from various sources. This analysis reveals two necessary model features: (1) the energy of a tetramer must have wells at low and high occupation by Ca V s, so that Ca V s positively cooperate in binding RYR (an allosteric effect), and (2) a large energy penalty results when two Ca V s bind simultaneously to adjacent RYR protomers in adjacent tetramers (a steric clash). Under the hypothesis, V and C channels will eventually reverse roles. Role reversal justifies the presence of sensor-lacking C channels, as a structural and functional reserve for control of muscle contraction.
... a sleevelike manner, and is composed of two distinct portions: (a) the terminal cisternae which are junctionally associated with the transverse tubule, and (b) the longitudinal cisternae or longitudinal SR, which connect medially with the two terminal cisternae. The terminal cisternae are connected to the transverse tubule via junctional structures referred to as "feet" (6,13,14, 20,31,36). The major component of the SR membrane is the Ca 2÷ pump protein, which has a molecular weight of ~ 115,000. ...
... In support of these findings, indirect immunoferritin labeling of frozen sections indicated that the Ca 2+ pump protein was localized in the longitudinal SR and in the nonjunctional regions of the terminal cisternae (18). The model of the terminal cisternae and the junctional face presented here (Fig. 8, f a n d h) builds upon and extends that of the SR/transverse tubule junction presented by Franzini-Armstrong and Franzini-Armstrong and Nunzini (11,13,14) and others (e.g., 6,19,20). ...
... The membranes within muscle have been extensively studied in situ (7,10,11,13,14,19,20,31). The availability of isolated terminal cisternae makes possible a detailed study of morphology which cannot be readily observed in situ. ...
We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of sarcoplasmic reticulum (SR), which retain morphologically intact junctional "feet" structures similar to those observed in situ. The fraction is largely devoid of transverse tubule, plasma membrane, mitochondria, triads (transverse tubules junctionally associated with terminal cisternae), and longitudinal cisternae, as shown by thin-section electron microscopy of representative samples. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae (light SR) obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes, i.e., the junctional face membrane and the Ca2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca2+ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend approximately 12 nm from the membrane surface and can be clearly visualized in thin section through using tannic acid enhancement, by negative staining and by freeze-fracture electron microscopy. Sections of the terminal cisternae, cut tangential to and intersecting the plane of the junctional face, reveal a checkerboardlike lattice of alternating, square-shaped feet structures and spaces each 20 nm square. Structures characteristic of the Ca2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca2+ pump protein is observed in the nonjunctional membrane on the remainder of the same vesicle. Likewise, freeze-fracture replicas reveal regions of the P face containing ropelike strands instead of the high density of the 7-8-nm particles referable to the Ca2+ pump protein. The intravesicular content of the terminal cisternae, mostly Ca2+-binding protein (calsequestrin), is organized in the form of strands, sometimes appearing paracrystalline, and attached to the inner face of the membrane in the vicinity of the junctional feet. The terminal cisternae preparation is distinct from previously described heavy SR fractions in that it contains the highest percentage of junctional face membrane with morphologically well-preserved junctional feet structures.
... These channels, still sometimes referred as 1,4-dihydropyridine receptors (DHPRs) because of their responsivity to pharmacological agonists and antagonists from this class of drugs, are abundantly expressed in skeletal muscle, where they are physically tethered to juxtapositioned type 1 ryanodine receptors (RyR1s) in the triad junctions. Freeze-fracture electron microscopy studies revealed that Ca V 1.1s are arranged into tetrads (4 channel groups), with each individual Ca V 1.1 aligning with a single RyR1 homotetramer (87 . Structure and dimensions of voltage-gated Ca 21 (Ca V ) channels. ...
... These data strongly suggested that normal Ca 21 sparks were likely produced by a cluster of RyRs opening in synchrony. The results described above were consistent with classic electron microscopy studies (87,360,361) and, more recently, fluorescence superresolution microscopy (28,49,133,362) showing that RyRs organize in clusters/arrays in the endo/sarcoplasmic reticulum of muscle and neurons (FIGURE 8A). In cardiac and smooth muscle, it has been estimated that these clusters contain $20-40 ryanodine receptors, respectively (133,362,363). ...
Ion channels play a central role in the regulation of nearly every cellular process. Dating back to the classic 1952 Hodgkin–Huxley model of the generation of the action potential, ion channels have always been thought of as independent agents. A myriad of recent experimental findings exploiting advances in electrophysiology, structural biology, and imaging techniques, however, have posed a serious challenge to this long-held axiom, as
several classes of ion channels appear to open and close in a coordinated, cooperative manner. Ion channel cooperativity ranges from variable-sized oligomeric cooperative gating in voltage-gated, dihydropyridine-sensitive CaV1.2 and CaV1.3 channels to obligatory dimeric assembly and gating of voltage-gated NaV1.5 channels.
Potassium channels, transient receptor potential channels, hyperpolarization cyclic nucleotide-activated channels, ryanodine receptors (RyRs), and inositol trisphosphate receptors (IP3Rs) have also been shown to gate cooperatively. The implications of cooperative gating of these ion channels range from fine-tuning excitation contraction
coupling in muscle cells to regulating cardiac function and vascular tone, to modulation of action potential and conduction velocity in neurons and cardiac cells, and to control of pacemaking activity in the heart. In this review, we discuss the mechanisms leading to cooperative gating of ion channels, their physiological consequences, and how alterations in cooperative gating of ion channels may induce a range of clinically significant pathologies.
Key Words: calcium signaling; channel clustering; cooperative gating; excitability; stochastic self-assembly
... These channels, still sometimes referred as 1,4-dihydropyridine receptors (DHPRs) because of their responsivity to pharmacological agonists and antagonists from this class of drugs, are abundantly expressed in skeletal muscle, where they are physically tethered to juxtapositioned type 1 ryanodine receptors (RyR1s) in the triad junctions. Freeze-fracture electron microscopy studies revealed that Ca V 1.1s are arranged into tetrads (4 channel groups), with each individual Ca V 1.1 aligning with a single RyR1 homotetramer (87 . Structure and dimensions of voltage-gated Ca 21 (Ca V ) channels. ...
... The results described above were consistent with classic electron microscopy studies (87,360,361) and, more recently, fluorescence superresolution microscopy (28,49,133,362) showing that RyRs organize in clusters/arrays in the endo/sarcoplasmic reticulum of muscle and neurons (FIGURE 8A). In cardiac and smooth muscle, it has been estimated that these clusters contain $20-40 ryanodine receptors, respectively (133,362,363). ...
Abstract
Ion channels play a central role in the regulation of nearly every cellular process. Dating back to the classic 1952 Hodgkin–Huxley model of the generation of the action potential, ion channels have always been thought of as independent agents. A myriad of recent experimental findings exploiting advances in electrophysiology, structural biology, and imaging techniques, however, have posed a serious challenge to this long-held axiom, as
several classes of ion channels appear to open and close in a coordinated, cooperative manner. Ion channel cooperativity ranges from variable-sized oligomeric cooperative gating in voltage-gated, dihydropyridine-sensitive CaV1.2 and CaV1.3 channels to obligatory dimeric assembly and gating of voltage-gated NaV1.5 channels.
Potassium channels, transient receptor potential channels, hyperpolarization cyclic nucleotide-activated channels, ryanodine receptors (RyRs), and inositol trisphosphate receptors (IP3Rs) have also been shown to gate cooperatively. The implications of cooperative gating of these ion channels range from fine-tuning excitation contraction
coupling in muscle cells to regulating cardiac function and vascular tone, to modulation of action potential and conduction velocity in neurons and cardiac cells, and to control of pacemaking activity in the heart. In this review, we discuss the mechanisms leading to cooperative gating of ion channels, their physiological consequences, and how alterations in cooperative gating of ion channels may induce a range of clinically significant pathologies.
calcium signaling; channel clustering; cooperative gating; excitability; stochastic self-assembly
... These channels, still sometimes referred as 1,4-dihydropyridine receptors (DHPRs) because of their responsivity to pharmacological agonists and antagonists from this class of drugs, are abundantly expressed in skeletal muscle, where they are physically tethered to juxtapositioned type 1 ryanodine receptors (RyR1s) in the triad junctions. Freeze-fracture electron microscopy studies revealed that Ca V 1.1s are arranged into tetrads (4 channel groups), with each individual Ca V 1.1 aligning with a single RyR1 homotetramer (87 . Structure and dimensions of voltage-gated Ca 21 (Ca V ) channels. ...
... The results described above were consistent with classic electron microscopy studies (87,360,361) and, more recently, fluorescence superresolution microscopy (28,49,133,362) showing that RyRs organize in clusters/arrays in the endo/sarcoplasmic reticulum of muscle and neurons (FIGURE 8A). In cardiac and smooth muscle, it has been estimated that these clusters contain $20-40 ryanodine receptors, respectively (133,362,363). ...
Ion channels play a central role in the regulation of nearly every cellular process. Dating back to the classic 1952 Hodgkin-Huxley model of the generation of the action potential, ion channels have always been thought of as independent agents. A myriad of recent experimental findings exploiting advances in electrophysiology, structural biology, and imaging techniques, however, have posed a serious challenge to this long-held axiom as several classes of ion channels appear to open and close in a coordinated, cooperative manner. Ion channel cooperativity ranges from variable-sized oligomeric cooperative gating in voltage-gated, dihydropyridine-sensitive Cav1.2 and Cav1.3 channels to obligatory dimeric assembly and gating of voltage-gated Nav1.5 channels. Potassium channels, transient receptor potential channels, hyperpolarization cyclic nucleotide-activated channels, ryanodine receptors (RyRs), and inositol trisphosphate receptors (IP3Rs) have also been shown to gate cooperatively. The implications of cooperative gating of these ion channels range from fine tuning excitation-contraction coupling in muscle cells to regulating cardiac function and vascular tone, to modulation of action potential and conduction velocity in neurons and cardiac cells, and to control of pace-making activity in the heart. In this review, we discuss the mechanisms leading to cooperative gating of ion channels, their physiological consequences and how alterations in cooperative gating of ion channels may induce a range of clinically significant pathologies.
... At specific regions, these come geometrically close (~ 12 nm) to, whilst remaining electrically isolated from, terminal cisternal membranes of the SR Ca 2+ store. The resulting T-SR triad and dyad junctions are strategic to excitation-contraction coupling [11][12][13] . In cardiac muscle, tubular ...
... The structural parameters describing sarcomere, surface, T-tubular and SR membrane structure, and distributions, densities and electron microscope ultrastructure of their T-SR junctional regions required for such modelling were available for amphibian skeletal muscle [11][12][13][39][40][41][42][43][44] . [Ca 2+ ] gradients through the resulting formalized geometrical model of a typical T-SR junction in both resting and stimulated muscle fibres were then resolved by finite element method (FEM) solutions of basic Fick diffusion equations. ...
A finite element analysis modelled diffusional generation of steady-state Ca2+ microdomains within skeletal muscle transverse (T)-tubular-sarcoplasmic reticular (SR) junctions, sites of ryanodine receptor (RyR)-mediated SR Ca2+ release. It used established quantifications of sarcomere and T-SR anatomy (radial diameter d=220nm; axial distance w=12nm). Its boundary SR Ca2+ influx densities,Jinflux, reflected step impositions of influxes, Φinflux=Jinfluxπd24, deduced from previously measured Ca2+ signals following muscle fibre depolarization. Predicted steady-state T-SR junctional edge [Ca2+], [Ca2+]edge, matched reported corresponding experimental cytosolic [Ca2+] elevations given diffusional boundary effluxΦefflux=D[Ca2+]edgeλ(πdw), established cytosolic Ca2+ diffusion coefficients (D=4×107nm2/s) and exit length λ=9.2nm. Dependences of predicted [Ca2+]edge upon Jinflux then matched those of experimental [Ca2+] upon Ca2+ release through their entire test voltage range. The resulting model consistently predicted elevated steady-state T-SR junctional ~ µM-[Ca2+] elevations radially declining from maxima at the T-SR junction centre along the entire axial T-SR distance. These [Ca2+] heterogeneities persisted through 104- and fivefold, variations in D and w around, and fivefold reductions in d below, control values, and through reported resting muscle cytosolic [Ca2+] values, whilst preserving the flux conservation (Φinflux=Φefflux) condition, Ca2+edge=λdJinflux4Dw. Skeletal muscle thus potentially forms physiologically significant ~ µM-[Ca2+] T-SR microdomains that could regulate cytosolic and membrane signalling molecules including calmodulin and RyR, These findings directly fulfil recent experimental predictions invoking such Ca2+ microdomains in observed regulatory effects upon Na+ channel function, in a mechanism potentially occurring in similar restricted intracellular spaces in other cell types.
... In summary, considering that: i) each couplon has an approximately round shape and that ii) EM sections cut a random chord of the round shape circle, the average area of individual couplons was estimated using the following equations: 1) Mean diameter of each couplon: the average measured chord (y) is related to the diameter of the average circle (D) by y = πD/4.2) Mean area of each couplon: (D/2) 2 x π d. The number of RYR2-feet in each couplon was estimated assuming that: a) the couplon is filled with RYR2-feet, which form ordered arrays touching each other as in31,53,54 ; and b) each RYR2-foot occupies an area of approximately 29x29nm53,54 . ...
... In summary, considering that: i) each couplon has an approximately round shape and that ii) EM sections cut a random chord of the round shape circle, the average area of individual couplons was estimated using the following equations: 1) Mean diameter of each couplon: the average measured chord (y) is related to the diameter of the average circle (D) by y = πD/4.2) Mean area of each couplon: (D/2) 2 x π d. The number of RYR2-feet in each couplon was estimated assuming that: a) the couplon is filled with RYR2-feet, which form ordered arrays touching each other as in31,53,54 ; and b) each RYR2-foot occupies an area of approximately 29x29nm53,54 . ...
Rationale: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare disease, manifested by syncope or sudden death in children or young adults under stress conditions. Mutations in the Ca ²⁺ release channel/ryanodine receptor (RyR2) gene account for about 60% of the identified mutations. Recently, we found and described a mutation in RyR2 N-terminal domain, RyR2 R420Q .
Objective: To determine the arrhythmogenic mechanisms of this mutation.
Methods and Results: Ventricular tachycardias under stress conditions were observed in both CPVT patients and KI mice. During action potential recording (by patch-clamp in KI mouse cardiomyocytes and by microelectrodes in mutant hiPSC-CM) we observed an increased occurrence of delayed after-depolarizations (DADs) under isoproterenol stimulation, associated with increased Ca ²⁺ waves during confocal Ca ²⁺ recording in both mouse and human RyR2 R420Q cardiomyocytes. In addition, Ca ²⁺ -induced Ca ²⁺ -release, as well as a rough indicator of fractional Ca ²⁺ release, were higher and Ca ²⁺ sparks longer in the RyR2 R420Q expressing cells. At the ultrastructural nanodomain level, we observed smaller RyR2 clusters and widened junctional sarcoplasmic reticulum (jSR) measured by g-STED super-resolution and electronic microscopy, respectively. The increase in jSR width might be due to the impairment of RyR2 R420Q binding to junctophilin-2, as there were less junctophilin-2 co-immunoprecipitated with RyR2 R420Q . At the single current level, the RyR2R420Q channel dwells longer in the open state at low [Ca ²⁺ ] i , but there is predominance of a subconductance state. The latter might be correlated with an enhanced interaction between the N-terminus and the core solenoid, a RyR2 inter-domain association that has not been previously implicated in the pathogenesis of arrhythmias and sudden cardiac death.
Conclusions: The RyR2 R420Q CPVT mutation modifies the interdomain interaction of the channel and weaken its association with junctophillin-2. These defects may underlie both nanoscale disarrangement of the dyad and channel dysfunction.
... Franzini-Armstrong and Nunzi (1983) described three morphological types of junctional feet, as follows: (a) Type 1 or parallel lines: feet which are elongated in a plane parallel to the adjacent membranes of the SR and t-tubule; (b) Type 2: feet forming single dense columns between the apposed membranes they touch and (c) Type 3 or pillars: feet with distinctly less dense centres which give them of two lines perpendicular on the facing membranes they unite (Franzini-Armstrong and Nunzi, 1983). ...
... Type 1 of junctional feet is variable in regards of the extent of the attachment of feet to the facing membranes (Franzini-Armstrong and Nunzi, 1983). We found symmetrical (sTs) and asymmetrical (aTs) triads, built-up on longitudinal cuts by two thick junctional feet or by a thin and a thick one. ...
Specific ultrastructural anatomy of masticatory muscles is commonly referred to a general pattern assigned to striated muscles. Junctional feet consisting of calcium channels of the sarcoplasmic reticulum (i.e. the ryanodine receptors, RyRs)physically connected to the calcium channels of the t-tubules build triads within striated muscles. Functional RyRs were demonstrated in the nuclear envelopes of pancreas and of a skeletal muscle derived cell line, but not in muscle in situ. It was hypothesized that ryanodine receptors (RyRs)could also exist in the nuclear envelope in the masseter muscle, thus aiming at studying this by transmission electron microscopy. There were identified paired and consistent subsarcolemmal clusters of mitochondria, appearing as outpockets of the muscle fibers, usually flanking an endomysial microvessel. It was observed on grazing longitudinal cuts that the I-band-limited mitochondria were not strictly located in a single intermyofibrillar space but continued transversally over the I-band to the next intermyofibrillar space. It appeared that the I-band-limited transverse mitochondria participate with the column-forming mitochondria in building a rather incomplete mitochondrial reticulum of the masseter muscle. Subsarcolemmal nuclei presented nuclear envelope-associated RyRs. Moreover, t-tubules were contacting the nuclear envelope and they were seemingly filled from the perinuclear space. This could suggest that nucleoplasmic calcium could contribute to balance the cytosolic concentration via pre-built anatomical routes: (i)indirectly, via the RyRs of the nuclear envelope and (ii)directly via the communication of t-tubules and sarcoplasmic reticulum through the perinuclear space.
... One is a voltage dependent L-type calcium channel, Cav1.1, identified as the dihydropyridine receptor, or DHPR. Molecules of DHPR, arranged in groups of four or tetrads, are located at the surface and transverse tubule membranes of muscle fibres (Franzini-Armstrong and Nunzi 1983;Schwartz et al. 1985;Campbell et al. 1987;Rios and Brum 1987;Paolini et al. 2004). The other channel is a large homo-tetramer, present in the sarcoplasmic reticulum (SR) membrane and recognized as the isoform 1 of the calcium release channel or ryanodine receptor, RyR1, (Sutko et al. 1985;Block et al. 1988;Smith et al. 1988). ...
... The other channel is a large homo-tetramer, present in the sarcoplasmic reticulum (SR) membrane and recognized as the isoform 1 of the calcium release channel or ryanodine receptor, RyR1, (Sutko et al. 1985;Block et al. 1988;Smith et al. 1988). In the triadic junctional region, the two types of membranes come into close apposition, with the two types of channel facing each other, and with each DHPR molecule of a tetrad in register, vis a vis, with one subunit of the RyR tetramer, forming Ca 2? Release Units, CRU (Block et al. 1988;Franzini-Armstrong and Nunzi 1983;Franzini-Armstrong and Protasi 1997;Protasi 2002;Paolini et al. 2004). Upon fibre depolarization, DHPRs undergo conformational changes that lead to activation of junctional RyR1 and to Ca 2? release. ...
Muscle fibres, isolated from frog tibialis anterior and mouse flexor digitorum brevis (FDB) were loaded with the fast dye MagFluo-4 to study the effects of potentiators caffeine, nitrate, Zn(2+) and perchlorate on Ca(2+) transients elicited by single action potentials. Overall, the potentiators doubled the transients amplitude and prolonged by about 1.5-fold their decay time. In contrast, as shown here for the first time, nitrate and Zn(2+), but not caffeine, activated a late, secondary component of the transient rising phase of frog but not mouse, fibres. The rise time was increased from 1.9 ms in normal solution (NR) to 3.3 ms (nitrate) and 4.4 ms (Zn(2+)). In NR, a single exponential, fitted the rising phase of calcium transients of frog (τ1 = 0.47 ms) and mouse (τ1 = 0.28 ms). In nitrate and Zn(2+) only frog transients showed a secondary exponential component, τ2 = 0.72 ms (nitrate) and 0.94 ms, (Zn(2+)). We suggest that nitrate and Zn(2+) activate a late slower component of the ΔF/F signals of frog but not of mouse fibres, possibly promoting Ca(2+) induced Ca(2+) release at level of the RyR3, that in frog muscle fibres are localized in the para-junctional region of the triads and are absent in mouse FDB muscle fibres.
... Note: In addition to Cav1.1 and RyR1, many other proteins form part of the T-tubule-junctional SR complex (e.g., FKPB12, triadin, junctin, Casq1) and are not shown here. Panels (B,C) are based on references: (Franzini-Armstrong and Porter, 1964;Franzini-Armstrong and Nunzi, 1983;Block et al., 1988;Franzini-Armstrong and Jorgensen, 1994;Franzini-Armstrong and Kish, 1995;Franzini-Armstrong and Protasi, 1997). channels, the skeletal muscle voltage-gated sodium channel, localized at the sarcolemma and through the transverse (T)tubule system of the myofiber ( Figure 1B). ...
... RyR1s are organized in regular checkered arrays within the terminal cisternae of the junctional SR (Franzini-Armstrong and Nunzi, 1983;Franzini-Armstrong et al., 1998). Ca v 1.1 channels are clustered in groups of four (or tetrads) in the Ttubule membrane portion that is in the vicinity of junctional SR (Block et al., 1988). ...
The skeletal muscle Ca²⁺ release channel, also known as ryanodine receptor type 1 (RyR1), is the largest ion channel protein known and is crucial for effective skeletal muscle contractile activation. RyR1 function is controlled by Cav1.1, a voltage gated Ca²⁺ channel that works mainly as a voltage sensor for RyR1 activity during skeletal muscle contraction and is also fine-tuned by Ca²⁺, several intracellular compounds (e.g., ATP), and modulatory proteins (e.g., calmodulin). Dominant and recessive mutations in RyR1, as well as acquired channel alterations, are the underlying cause of various skeletal muscle diseases. The aim of this mini review is to summarize several current aspects of RyR1 function, structure, regulation, and to describe the most common diseases caused by hereditary or acquired RyR1 malfunction.
... There appear to be tradeoffs in sonic fiber morphology. The SR constitutes about a third of the fiber volume (Franzini-Armstrong and Nunzi, 1983), and its intimate association with myofibrils ensures that calcium ions released from the SR are therefore close to binding sites on myofibrils. However, the cost is the exclusion of mitochondria and glycogen granules from proximity to sites that require energy. ...
My research has been devoted to neuromuscular control of sound production in toadfish, catfish, and other species along with an occasional foray into hearing. Toadfish utilize a heart shaped swim bladder and superfast muscles with small fibers and an unusual ultrastructure. Both sonic motor neurons and muscle fibers increase in size and number for multiple years, and large muscle fibers fragment and likely divide, maintaining energetic efficiency. Toadfish sonic muscles drive the swim bladder directly (a forced response), and the sound waveform parallels bladder movement. The forced response differs from traditional interpretations of swim bladders as underwater resonant bubbles. High water content in the swim bladder wall inhibits resonance by viscous damping at shallow but likely less effectively at deeper depths, suggesting both notions might apply. Catfish produce sounds with their pectoral spines by rubbing a ridged surface on the dorsal process against a rough surface on the cleithrum: a series of quick jerks produce sounds via a slip-stick mechanism. Recent discoveries on other species reveal novel adaptations for sound production and suggestions are made for future work.
... Structural descriptions of skeletal or cardiac muscle, sarcomeres and their surface, T-tubular and SR membranes localize the processes coupling T-tubular membrane potential changes and SR Ca 2+ release to T-SR triadic or dyadic junctions [3][4][5]. ...
Skeletal and cardiac muscle excitation–contraction coupling commences with Nav1.4/Nav1.5-mediated, surface and transverse (T-) tubular, action potential generation. This initiates feedforward, allosteric or Ca²⁺-mediated, T-sarcoplasmic reticular (SR) junctional, voltage sensor-Cav1.1/Cav1.2 and ryanodine receptor-RyR1/RyR2 interaction. We review recent structural, physiological and translational studies on possible feedback actions of the resulting SR Ca²⁺ release on Nav1.4/Nav1.5 function in native muscle. Finite-element modelling predicted potentially regulatory T-SR junctional [Ca²⁺]TSR domains. Nav1.4/Nav1.5, III-IV linker and C-terminal domain structures included Ca²⁺ and/or calmodulin-binding sites whose mutations corresponded to specific clinical conditions. Loose-patch-clamped native murine skeletal muscle fibres and cardiomyocytes showed reduced Na⁺ currents (INa) following SR Ca²⁺ release induced by the Epac and direct RyR1/RyR2 activators, 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate and caffeine, abrogated by the RyR inhibitor dantrolene. Conversely, dantrolene and the Ca²⁺-ATPase inhibitor cyclopiazonic acid increased INa. Experimental, catecholaminergic polymorphic ventricular tachycardic RyR2-P2328S and metabolically deficient Pgc1β−/− cardiomyocytes also showed reduced INa accompanying [Ca²⁺]i abnormalities rescued by dantrolene- and flecainide-mediated RyR block. Finally, hydroxychloroquine challenge implicated action potential (AP) prolongation in slowing AP conduction through modifying Ca²⁺ transients. The corresponding tissue/organ preparations each showed pro-arrhythmic, slowed AP upstrokes and conduction velocities. We finally extend discussion of possible Ca²⁺-mediated effects to further, Ca²⁺, K⁺ and Cl⁻, channel types.
This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’.
... Ca V 1.1 channels are clustered in groups of four within the plasma membrane of triad junctions. Each group of four Ca V 1.1 channels is known as a "tetrad" and each tetrad is juxtaposed to one leaf every other quatrefoil RyR1 in the SR membrane forming a checkerboard-like pattern [2][3][4] (Figure 1c). This unique ultrastructure is a prerequisite for the intermolecular communication between Ca V 1.1 and RyR1 that supports excitation-contraction (EC) coupling in skeletal muscle [2]. ...
The CaV1.1 voltage-gated Ca²⁺ channel carries L-type Ca²⁺ current and is the voltage-sensor for excitation-contraction (EC) coupling in skeletal muscle. Significant breakthroughs in the EC coupling field have often been close on the heels of technological advancement. In particular, CaV1.1 was the first voltage-gated Ca²⁺ channel to be cloned, the first ion channel to have its gating current measured and the first ion channel to have an effectively null animal model. Though these innovations have provided invaluable information regarding how CaV1.1 detects changes in membrane potential and transmits intra- and inter-molecular signals which cause opening of the channel pore and support Ca²⁺ release from the sarcoplasmic reticulum remain elusive. Here, we review current perspectives on this topic including the recent application of functional site-directed fluorometry.
... Furthermore, as we describe here, these structures are significantly upregulated by humming activity, and previous studies did not use actively humming fish. We were also surprised to note that stacks (or CEUs) have never been reported in the sonic muscles of Opsanus tau (oyster toadfish), despite the fact that the muscle has been the subject of numerous investigations by electron microscopy, included several by Prof. Clara Franzini-Armstrong's laboratory (Franzini-Armstrong and Nunzi, 1983;Ferguson et al., 1984;Block et al., 1988;Appelt et al., 1991). A careful re-examination of a large number of samples, based on our current knowledge of CEUs identifying features, confirms this absence (data not shown). ...
Over the past two decades, mounting evidence has demonstrated that a mechanism known as store-operated Ca2+ entry (SOCE) plays a crucial role in sustaining skeletal muscle contractility by facilitating Ca2+ influx from the extracellular space during sarcoplasmic reticulum (SR) Ca2+ depletion. We recently demonstrated that, in exercised fast-twitch muscle from mice, the incidence of Ca2+ entry units (CEUs), newly described intracellular junctions between dead-end longitudinal transverse tubular (T-tubule) extensions and stacks of sarcoplasmic reticulum (SR) flat cisternae, strictly correlate with both the capability of fibers to maintain contractions during fatigue and enhanced Ca2+ influx via SOCE. Here, we tested the broader relevance of this result across vertebrates by searching for the presence of CEUs in the vocal muscles of a teleost fish adapted for extended, high-frequency activity. Specifically, we examined active vs. inactive superfast sonic muscles of plainfin midshipman (Porichthys notatus). Interestingly, muscles from actively humming territorial males had a much higher incidence of CEU SR stacks relative to territorial males that were not actively vocalizing, strengthening the concept that assembly of these structures is dynamic and use-dependent, as recently described in exercised muscles from mice. Our results support the hypothesis that CEUs represent a conserved mechanism, across vertebrates, for enabling high levels of repetitive muscle activity, and also provide new insights into the adaptive mechanisms underlying the unique properties of superfast midshipman sonic muscles.
... In cardiac muscle the DHPRs are arranged variably. 65,66,240,241 The regions of the skeletal muscle DHPR and of RyR1 that mediate this process have been investigated thoroughly but there is still little evidence to suggest that the two proteins are in direct physical contact. In the DHPR, a chimeric and biochemical approach has been used to identify cytosolic loops that may come into contact with RyR. ...
Binding of Ryanodine receptor peptides with calmodulin and crystal structure and docking of the ryanodine receptor SPRY2 domain
... BC3H1 is a cell line that expresses major proteins of CRUs, DHPR and RyR, and has frequent large PCs Armstrong and Nunzi 1983;Franzini-Armstrong 1983,1984, 1999Leung et al., 1988;Block et al., 1988;McPherson et al., 1991;Takekura et al., 1994b;Protasi et al., 1997;Gach et al., 2008. ...
This project was initiated with the aim of preserving for the future a portion of the extensive muscle ultrastructural image archive accumulated over a 50 year period by the author and her laboratory. Between the late 1950s and current times electron microscopy (EM) evolved. The early EM revealed the ultrastructural identification and definition of cell organelles and laid the foundation for cell biology. This continues with he discovery of new structural organizations and organelles. Currently EM images provide the essential basis for identification of structural changes associated with mutational experiments and newly discovered pathology. The most recent development of techniques allowing near atomic level of resolution brings microscopy to modern times. This evolution has been greatly enriched by the rebirth of light microscopy, fueled by dynamic views of events based on video techniques, by the availability of specific targeting of cell components and by confocal microscopy that allows 3-D structural reconstructs. Correlation between light and electron microscopy, when appropriately done, is highly revealing. The images in this publication present descriptive and structure-function correlations covering the whole animal kingdom with some areas more extensively illustrated than others. The first part classifies the images based in their site of origin, the second part identifies functional correlations, the third part introduces results of mutational experiments, the fourth part covers aging and pathology examples, the fifth part is a guide to the techniques. The images are presented with a numerical identification correlated with a very succinct description. References are limited to the directly relevant published material from the author’s laboratory that are helpful in extending the limited legends and in assigning contributions. With very few exceptions, references to the literature are not given. The author asks for the forgiveness of the many friends, trainees, collaborators and competitors whose contributions and priorities are not recognized. The work has been stimulated by interactions with teachers and advisors. Keith R. Porter, father of the endoplasmic reticulum and sarcoplasmic reticulum instilled admiration for Nature’s work and was always been a guiding light in the author’s work. Richard J. Podolsky and Andrew F. Huxley taught the importance of structural correlations. Past and present trainees and collaborators, whose names appear in the reference list, have added depth of understanding. In the case of skeletal muscle much necessary information is gained by using the muscle bible: “Myology”, A.G. Engel and C. Franzini-Armstrong, Eds. 3rd Edition, McGraw Hill, N.Y. 2004.
... BC3H1 is a cell line that expresses major proteins of CRUs, DHPR and RyR, and has frequent large PCs Armstrong and Nunzi 1983;Franzini-Armstrong 1983,1984, 1999Leung et al., 1988;Block et al., 1988;McPherson et al., 1991;Takekura et al., 1994b;Protasi et al., 1997;Gach et al., 2008. ...
This project was initiated with the aim of preserving for the future a portion of the extensive muscle ultrastructural image archive accumulated over a 50 year period by the author and her laboratory. Between the late 1950s and current times electron microscopy (EM) evolved. The early EM revealed the ultrastructural identification and definition of cell organelles and laid the foundation for cell biology. This continues with he discovery of new structural organizations and organelles. Currently EM images provide the essential basis for identification of structural changes associated with mutational experiments and newly discovered pathology. The most recent development of techniques allowing near atomic level of resolution brings microscopy to modern times. This evolution has been greatly enriched by the rebirth of light microscopy, fueled by dynamic views of events based on video techniques, by the availability of specific targeting of cell components and by confocal microscopy that allows 3-D structural reconstructs. Correlation between light and electron microscopy, when appropriately done, is highly revealing. The images in this publication present descriptive and structure-function correlations covering the whole animal kingdom with some areas more extensively illustrated than others. The first part classifies the images based in their site of origin, the second part identifies functional correlations, the third part introduces results of mutational experiments, the fourth part covers aging and pathology examples, the fifth part is a guide to the techniques. The images are presented with a numerical identification correlated with a very succinct description. References are limited to the directly relevant published material from the author’s laboratory that are helpful in extending the limited legends and in assigning contributions. With very few exceptions, references to the literature are not given. The author asks for the forgiveness of the many friends, trainees, collaborators and competitors whose contributions and priorities are not recognized. The work has been stimulated by interactions with teachers and advisors. Keith R. Porter, father of the endoplasmic reticulum and sarcoplasmic reticulum instilled admiration for Nature’s work and was always been a guiding light in the author’s work. Richard J. Podolsky and Andrew F. Huxley taught the importance of structural correlations. Past and present trainees and collaborators, whose names appear in the reference list, have added depth of understanding. In the case of skeletal muscle much necessary information is gained by using the muscle bible: “Myology”, A.G. Engel and C. Franzini-Armstrong, Eds. 3rd Edition, McGraw Hill, N.Y. 2004.
... clearly showing he same disposition of tetrads as the T tubules of toadfish. Franzini- Armstrong and Nunzi 1983;Franzini-Armstrong 1983,1984, 1999Leung et al., 1988;Block et al., 1988;McPherson et al., 1991;Takekura et al., 1994b;Protasi et al., 1997;Gach et al., 2008. ...
This project was initiated with the aim of preserving for the future a portion of the extensive muscle ultrastructural image archive accumulated over a 50 year period by the author and her laboratory. Between the late 1950s and current times electron microscopy (EM) evolved. The early EM revealed the ultrastructural identification and definition of cell organelles and laid the foundation for cell biology. This continues with he discovery of new structural organizations and organelles. Currently EM images provide the essential basis for identification of structural changes associated with mutational experiments and newly discovered pathology. The most recent development of techniques allowing near atomic level of resolution brings microscopy to modern times. This evolution has been greatly enriched by the rebirth of light microscopy, fueled by dynamic views of events based on video techniques, by the availability of specific targeting of cell components and by confocal microscopy that allows 3-D structural reconstructs. Correlation between light and electron microscopy, when appropriately done, is highly revealing. The images in this publication present descriptive and structure-function correlations covering the whole animal kingdom with some areas more extensively illustrated than others. The first part classifies the images based in their site of origin, the second part identifies functional correlations, the third part introduces results of mutational experiments, the fourth part covers aging and pathology examples, the fifth part is a guide to the techniques. The images are presented with a numerical identification correlated with a very succinct description. References are limited to the directly relevant published material from the author’s laboratory that are helpful in extending the limited legends and in assigning contributions. With very few exceptions, references to the literature are not given. The author asks for the forgiveness of the many friends, trainees, collaborators and competitors whose contributions and priorities are not recognized. The work has been stimulated by interactions with teachers and advisors. Keith R. Porter, father of the endoplasmic reticulum and sarcoplasmic reticulum instilled admiration for Nature’s work and was always been a guiding light in the author’s work. Richard J. Podolsky and Andrew F. Huxley taught the importance of structural correlations. Past and present trainees and collaborators, whose names appear in the reference list, have added depth of understanding. In the case of skeletal muscle much necessary information is gained by using the muscle bible: “Myology”, A.G. Engel and C. Franzini-Armstrong, Eds. 3rd Edition, McGraw Hill, N.Y. 2004.
... Besides the surface sarcolemma, Nav1.4 occurs in the transverse (T-) tubules 17,18 themselves accounting for ~ 80% of total muscle membrane 19 , thereby contributing substantial I Na . T-tubular membranes come into close proximity to SR terminal cisternal membranes containing the RyR1-Ca 2+ release channels, leaving electron-microscopically demonstrable narrow, ~ 15 nm, T-SR gaps permitting the Ca v 1.1-RyR1 interactions strategic to excitation-contraction coupling [24][25][26] . Although ultimately continuous with remaining cytosol, T-SR gaps form restricted intracellular diffusion spaces close to the RyR1-Ca 2+ release sites potentially generating microdomains with local Ca 2+ concentrations, [Ca 2+ ] TSR , distinct from the remaining bulk [Ca 2+ ] i , that might modulate T-tubular, even if not surface membrane Na v 1.4 function (Fig. 7). ...
Skeletal muscle Na ⁺ channels possess Ca ²⁺ - and calmodulin-binding sites implicated in Nav1.4 current ( I Na ) downregulation following ryanodine receptor (RyR1) activation produced by exchange protein directly activated by cyclic AMP or caffeine challenge, effects abrogated by the RyR1-antagonist dantrolene which itself increased I Na . These findings were attributed to actions of consequently altered cytosolic Ca ²⁺ , [Ca ²⁺ ] i , on Na v 1.4. We extend the latter hypothesis employing cyclopiazonic acid (CPA) challenge, which similarly increases [Ca ²⁺ ] i , but through contrastingly inhibiting sarcoplasmic reticular (SR) Ca ²⁺ -ATPase. Loose patch clamping determined Na ⁺ current ( I Na ) families in intact native murine gastrocnemius skeletal myocytes, minimising artefactual [Ca ²⁺ ] i perturbations. A bespoke flow system permitted continuous I Na comparisons through graded depolarizing steps in identical stable membrane patches before and following solution change. In contrast to the previous studies modifying RyR1 activity, and imposing control solution changes, CPA (0.1 and 1 µM) produced persistent increases in I Na within 1–4 min of introduction. CPA pre-treatment additionally abrogated previously reported reductions in I Na produced by 0.5 mM caffeine. Plots of peak current against voltage excursion demonstrated that 1 µM CPA increased maximum I Na by ~ 30%. It only slightly decreased half-maximal activating voltages ( V 0.5 ) and steepness factors ( k ), by 2 mV and 0.7, in contrast to the V 0.5 and k shifts reported with direct RyR1 modification. These paradoxical findings complement previously reported downregulatory effects on Nav1.4 of RyR1-agonist mediated increases in bulk cytosolic [Ca ²⁺ ]. They implicate possible local tubule-sarcoplasmic triadic domains containing reduced [Ca ²⁺ ] TSR in the observed upregulation of Nav1.4 function following CPA-induced SR Ca ²⁺ depletion.
... b Side (top) and upper (bottom) views of cryo-EM reconstruction of the RyR1 (PDB, 5TAL) [175] and four superimposed Cav1.1 α1 subunits (PDB 5GJW) [100], forming a tetrad. This model was created in Chimera [174] using the cryo-EM maps with relative location of the Cav1.1 subunits as suggested by Samsó [118] RyR1s are arranged in a regular array within the terminal cisternae of the junctional SR [89,110,111]. Similarly, Cav1.1 channels are clustered in groups of four (or tetrads) in the TT membrane that is adjacent to the junctional SR [89,112], with a Cav1.1 tetrad facing every other RyR1 in the junctional SR RyR1 array (Fig. 11b). ...
The process by which muscle fiber electrical depolarization is linked to activation of muscle contraction is known as excitation-contraction coupling (ECC). Our understanding of ECC has increased enormously since the early scientific descriptions of the phenomenon of electrical activation of muscle contraction by Galvani that date back to the end of the eighteenth century. Major advances in electrical and optical measurements, including muscle fiber voltage clamp to reveal membrane electrical properties, in conjunction with the development of electron microscopy to unveil structural details provided an elegant view of ECC in skeletal muscle during the last century. This surge of knowledge on structural and biophysical aspects of the skeletal muscle was followed by breakthroughs in biochemistry and molecular biology, which allowed for the isolation, purification, and DNA sequencing of the muscle fiber membrane calcium channel/transverse tubule (TT) membrane voltage sensor (Cav1.1) for ECC and of the muscle ryanodine receptor/sarcoplasmic reticulum Ca2+ release channel (RyR1), two essential players of ECC in skeletal muscle. In regard to the process of voltage sensing for controlling calcium release, numerous studies support the concept that the TT Cav1.1 channel is the voltage sensor for ECC, as well as also being a Ca2+ channel in the TT membrane. In this review, we present early and recent findings that support and define the role of Cav1.1 as a voltage sensor for ECC.
... The disposition of DHPR tetrads in T tubules is linked to that of feet. (A-D) The first images of DHPR tetrads were seen in T tubules of a small fish (A; Franzini-Armstrong andNunzi, 1983) and in the plasmalemma of frog slow fibers(B-D;Franzini-Armstrong, 1984), where it was determined that the distance between tetrads is twice the distance between feet. (E-I) DHPR tetrads in T tubules of 4-d-old zebrafish larvae. ...
The concept of excitation–contraction coupling is almost as old as Journal of General Physiology . It was understood as early as the 1940s that a series of stereotyped events is responsible for the rapid contraction response of muscle fibers to an initial electrical event at the surface. These early developments, now lost in what seems to be the far past for most young investigators, have provided an endless source of experimental approaches. In this Milestone in Physiology, I describe in detail the experiments and concepts that introduced and established the field of excitation–contraction coupling in skeletal muscle. More recent advances are presented in an abbreviated form, as readers are likely to be familiar with recent work in the field.
... 9,10 The interaction between RyR1 and DHPR takes place at the triad junctions, specialized regions of the muscle fiber of close apposition between endoplasmic/sarcoplasmic reticulum (ER/SR) and transverse (T) tubules (tubular invaginations of the plasma membrane). [11][12][13][14] In heart, RyR2 is activated by Cav1.2 without direct interaction., 15 via a calcium-induced calcium release mechanism. 16,17 RyR mutations lead to deregulation of calcium homeostasis and can have severe consequences. ...
Signal transduction by the ryanodine receptor (RyR) is essential in many excitable cells including all striated contractile cells and some types of neurons. While its transmembrane domain is a classic tetrameric, six-transmembrane cation channel, the cytoplasmic domain is uniquely large and complex, hosting a multiplicity of specialized domains. The overall outline and substructure readily recognizable by electron microscopy make RyR a geometrically well-behaved specimen. Hence, for the last two decades, the 3D structural study of the RyR has tracked closely the technological advances in electron microscopy, cryo-electron microscopy (cryoEM), and computerized 3D reconstruction. This review summarizes the progress in the structural determination of RyR by cryoEM and, bearing in mind the leap in resolution provided by the recent implementation of direct electron detection, analyzes the first near-atomic structures of RyR. These reveal a complex orchestration of domains controlling the channel's function, and help to understand how this could break down as a consequence of disease-causing mutations.
... Figure 2 shows some of our initial micrographs from FIB-milled fast-twitch toadfish swim bladder, which has previously been extensively characterized by TEM of conventional thin-section and freeze-fracture specimens by Dr. C. Franzini-Armstrong, who assisted us with the preparation of the swimbladder muscle. 24 For convenience, for our initial work to demonstrate feasibility of FIB-milling for cryo-ET, the muscle was fixed with 2.5% glutaraldehyde, but the specimens remained fully hydrated and no heavy metal stains were used. In longitudinal sections, as expected, triad junctions are prevalent, and the major structural features agree remarkably well with those described previously, albeit at reduced contrast because we are imaging native muscle. ...
Cryo-electron tomography (cryo-ET) has emerged as perhaps the only practical technique for revealing nanometer-level three-dimensional structural details of subcellular macromolecular complexes in their native context, inside the cell. As currently practiced, the specimen should be 0.1- 0.2 microns in thickness to achieve optimal resolution. Thus, application of cryo-ET to intact frozen (vitreous) tissues, such as skeletal muscle, requires that they be sectioned. Cryo-ultramicrotomy is notoriously difficult and artifact-prone when applied to frozen cells and tissue, but a new technique, focused ion beam milling (cryo-FIB), shows great promise for “thinning” frozen biological specimens. Here we describe our initial results in applying cryo-FIB and cryo-ET to triad junctions of skeletal muscle.
... Indeed, there is the handedness problem in which cytoplasmic or transmembrane sides of a RyR may not be distinguishable in 2D images. The regular arrangement of RyRs and the size of the junctional gap are remarkably constant throughout the animal kingdom and in different fiber types, 7 indicating that each RyR should have constant distance to membrane of a T-tubule. In nature, T-tubules are invaginations of the cell membrane and the invaginations form a series of tubes. ...
Rabbit muscle vesicles derived from sarcoplasmic reticulum were used as a material in studying networks of ryanodine receptors by cryo electron tomography. Three-dimensional analysis reveals the dynamical features of these networks. It was found that the connection angles were rotated along the transmembrane axis of ryanodine receptors. Majority of the connections was observed at domains 6/6 of ryanodine receptors while a small group of connections were showed at domains 9/10. The flexible rotation and connection shift seem to facilitate the extension of an annular network on the wall of the sarcoplasmic reticulum in a triad.
... Closely associated with feet are the regions of exterior membranes containing the DHPR (Flucher, Morton et al. 1990;Jorgensen, Shen et al. 1993). In normal muscle RyR1 feet are placed tetragonally, and groups of four DHPRs, called tetrads ( Figure 5), are associated with alternate RyRs forming a related array (Franzini-Armstrong and Nunzi 1983;Protasi, Franzini-Armstrong et al. 1997). Tetrads are visible in freeze-fracture replicas, that form orderly clusters either on the surface membrane, presumably at sites of peripheral couplings or in the junctional region of T tubules. ...
BIN1 is a membrane tubulating protein and it consists of the BAR domain which binds membranes and has tubulating property; the PI motif which binds phosphoinositides and is expressed only in skeletal muscle; the CLAP domain binds clathrin and AP2 and is present exclusively in brain isoforms of BIN1; the MBD is involved in c-Myc binding and the SH3 domain is involved in interactions with prolin-rich proteins. BIN1 is an ubiquitously expressed protein with the highest expression in skeletal muscle. Mutations in amphiphysin 2 / BIN1 were found to cause autosomal recessive centronuclear myopathy (ARCNM, OMIM 255200). Mutations in patients were found in all the protein domains and include two mutations leading to a premature stop codon in the last exon 20. The PI motive, encoded by exon 11, is upregulated during myogenesis. The aim of this research was to better understand the role of BIN1 in healthy muscle and in the pathology of CNM. For this purpose, by using targeted homologous recombination in ES cells, we generated two knockout mouse models: BIN1 exon 11 and BIN1 exon 20, with exon 11 and 20 deleted, respectively. The deletion of exon 20 disrupts the SH3 domain, involved in interactions with different proteins, amongst which is dynamin 2 and induced a considerable loss of the total BIN1 protein expression. The total and muscle specific deletions of exon 20 were perinatally lethal. A disrupted T-tubules organization was observed in knockout mice, showing an importance of BIN1 during the T-tubule biogenesis. Interestingly, deletion induced in adult mice did not affect muscle function and organization. In order to understand the role of the muscle specific PI motif, we characterized the BIN1 exon 11 KO mice. Even at 12 months of age the muscle function in mice was not compromised by this deletion. However, further examination showed impairment of skeletal muscle regeneration. This work revealed that in vivo, BIN1 is necessary during the T-tubules biogenesis and dispensable for muscle maintenance, whereas the skeletal muscle specific PI motif of BIN1 is involved in muscle regeneration. Its function in muscle is tightly regulated by isoform switch and intramolecular binding. Understanding these features will help us step forward towards successful therapy in ARCNM and MD patients.
... Dans le muscle squelettique adulte, les ULC forment principalement des triades (Figure 22). Les pieds font face à des structures situées dans la membrane du tubule T, les tétrades, composées de 4 DHPR (Block et coll., 1988 ;Franzini-Armstrong et Nunzi, 1983). La preuve directe de la formation de ces tétrades par le DHPR provient d'études menées sur le modèle de la souris dysgénique (mutant qui n'exprime pas α 1S ). ...
Excitation-contraction (EC) coupling of skeletal muscle depends upon interactions between the membrane voltage-sensor (dihydropyridine receptor, DHPR), and the sarcoplasmic reticulum calcium release channel (ryanodine receptor, RyR). The DHPR stands within two membrane structure enriched in cholesterol, the transverse tubule system and the caveolae. Caveolae also contain the nitric oxide (NO) synthase. The present work provides new insights into the functional modulation of EC coupling by cholesterol and NO, using electrophysiological techniques and fluorescence measurements on isolated muscle cells. The membrane cholesterol content regulates the voltage-sensing and calcium channel function of the DHPR. NO specifically affects the RyR function. At physiological levels, NO modulates the channel activation by membrane depolarization; excess NO maintains some RyR within an activated state
... These considerations invite comparison between the cortical ER and the SR of muscle. Close and well-characterized junctional appositions between terminal cisternae of the SR and tubular invaginations of the sarcolemma (T-system) participate in signal transduction between the cell surface and the interior (Franzini-Armstrong and Nunzi, 1983;Block et al. 1988). During excitation/contraction coupling, membrane depolarizations traveling along the sarcolemma are propagated down the T-system. ...
Structural observations provide persuasive evidence for the existence of a cortical network of endoplasmic reticulum (ER) in a large number of plant and animal cells. The network in plants generally possesses a polygonal pattern in which smooth, tubular elements are joined by intervening lamellar segments. The individual elements of ER are often positioned extremely close to the plasma membrane (PM), and may form appositional contacts, but fusion does not occur. The network arises at cytokinesis and establishes continuity between the cortical ER of daughter cells in the form of tightly furled membrane tubules that traverse the plasmodesmata. The specific function of the cortical ER complex is unknown but different possibilities seem attractive. It may serve key roles in anchoring the cytoskeleton and in facilitating secretion. The cortical ER might also participate in the communication of signals between the exterior of the cell and cytoplasm. As a consequence of its ability to release and/or sequester Ca, the ER could control the cytoplasmic activity of this ion and thus a host of physiologically and developmentally important reactions.
Excitation-contraction coupling (ECC) is a fundamental mechanism in control of skeletal muscle contraction and occurs at triad junctions, where dihydropyridine receptors (DHPRs) on transverse tubules sense excitation signals and then cause calcium release from the sarcoplasmic reticulum via coupling to type 1 ryanodine receptors (RyR1s), inducing the subsequent contraction of muscle filaments. However, the molecular mechanism remains unclear due to the lack of structural details. Here, we explored the architecture of triad junction by cryo–electron tomography, solved the in situ structure of RyR1 in complex with FKBP12 and calmodulin with the resolution of 16.7 Angstrom, and found the intact RyR1-DHPR supercomplex. RyR1s arrange into two rows on the terminal cisternae membrane by forming right-hand corner-to-corner contacts, and tetrads of DHPRs bind to RyR1s in an alternating manner, forming another two rows on the transverse tubule membrane. This unique arrangement is important for synergistic calcium release and provides direct evidence of physical coupling in ECC.
Mutations in RyR alter the cell's Ca2+ homeostasis and can cause serious health problems for which few effective therapies are available. Until recently, there was little structural context for the hundreds of mutations linked to muscular disorders reported for this large channel. Growing knowledge of the three-dimensional structure of RyR starts to illustrate the fine control of Ca2+ release. Current efforts directed towards understanding how disease mutations impinge in such processes will be crucial for future design of novel therapies. In this review article we discuss the up-to-date information about mutations according to their role in the 3D structure, and classified them to provide context from a structural perspective.
Dans le muscle squelettique, des invaginations du sarcolemme appelées cavéoles et leur composant principal Cav-3 seraient impliqués dans la formation des tubules transverses, des structures musculaires permettant de propager le potentiel d'action dans la fibre musculaire. Pourtant, ce mécanisme demeure à ce jour inconnu. L’importance des cavéoles et de Cav-3 est accentuée par l’existence de défauts dans l’organisation et la fonction des cavéoles dans le cas de cavéolinopathies, des maladies neuromusculaires autosomiques dominantes dues à des mutations dans le gène CAV-3 et dont les mécanismes physiopathologiques sont à ce jour incompris. L’objectif de mon projet était de comprendre le rôle des cavéoles dans la formation précoce des tubules-T. Une technique de microscopie corrélative combinant de la fluorescence à super résolution et de la microscopie électronique sur répliques de métal a permis d’examiner en détails les composants moléculaires des cavéoles et des tubules-T dans des myotubes extensivement différenciés. J’ai ainsi montré l'organisation des cavéoles sur des plateformes de Bin1 formant ainsi une nouvelle structure en anneaux semblant optimiser la tubulation de la membrane afin d’initier la formation des tubules-T. Ces anneaux ainsi que la tubulation des membranes par Bin1 sont altérés dans le cas de défauts d’expression de Cav-3 et dans les myotubes de patients cavéolinopathes. Mes travaux suggèrent que les anneaux de cavéoles constituent le site d’initiation des tubules-T et apportent les bases d’une caractérisation de la biogénèse des tubules-T dans le muscle squelettique et dans la physiopathologie des cavéolinopathies.
Historically, ryanodine receptors (RyRs) have presented unique challenges for high-resolution structural determination despite long-standing interest in their role in excitation–contraction coupling. Owing to their large size (nearly 2.2 MDa), high-resolution structures remained elusive until the advent of cryogenic electron microscopy (cryo-EM) techniques. In recent years, structures for both RyR1 and RyR2 have been solved at near-atomic resolution. Furthermore, recent reports have delved into their more complex structural associations with key modulators – proteins such as the dihydropyridine receptor (DHPR), FKBP12/12.6, and calmodulin (CaM), as well as ions and small molecules including Ca²⁺, ATP, caffeine, and PCB95. This review addresses the modulation of RyR1 and RyR2, in addition to the impact of such discoveries on intracellular Ca²⁺ dynamics and biophysical properties.
Ryanodine receptors (RyRs) are large intracellular calcium release channels that play a crucial role in coupling excitation to contraction in both cardiac and skeletal muscle cells. In addition, they are expressed in other cell types where their function is less well understood. Hundreds of mutations in the different isoforms of RyR have been associated with inherited myopathies and cardiac arrhythmia disorders. The structure of these important drug targets remained elusive for a long time, despite decades of intensive research. In the recent years, a technical revolution in the field of single particle cryogenic electron microscopy (SP cryo-EM) allowed solving high-resolution structures of the skeletal and cardiac RyR isoforms. Together with the structures of individual domains solved by X-ray crystallography, this resulted in an unprecedented understanding of the structure, gating and regulation of these largest known ion channels. In this chapter we describe the recently solved high-resolution structures of RyRs, discuss molecular details of the channel gating, regulation and the disease mutations. Additionally, we highlight important questions that require further progress in structural studies of RyRs.
During the complex series of events leading to muscle contraction, the initial electric signal coming from motor neurons is transformed into an increase in calcium concentration that triggers sliding of myofibrils. This process, referred to as excitation–contraction coupling, is reliant upon the calcium-release complex, which is restricted spatially to a sub-compartment of muscle cells (“the triad”) and regulated precisely. Any dysfunction in the calcium-release complex leads to muscle impairment and myopathy. Various causes can lead to alterations in excitation–contraction coupling and to muscle diseases. The latter are reviewed and classified into four categories: (i) mutation in a protein of the calcium-release complex; (ii) alteration in triad structure; (iii) modification of regulation of channels; (iv) modification in calcium stores within the muscle. Current knowledge of the pathophysiologic mechanisms in each category is described and discussed.
The myocardial sarcolemma is not structurally uniform. Characteristic specializations occur in areas of the membrane. These specialized areas, namely, the transverse tubules (TT), caveolae and intercalated discs of the heart muscle cell have been described with conventional electron microscopy (both thin-section and freeze-fracture). However, some important aspects of the structure of these specializations and in some cases their functions are still unclear. Our understanding of the mechanisms by which they are formed and maintained in the plasma membrane is still fragmentary. The ultra-structure of the most important junctions formed by the sarcolemma (i.e., junction of TT with the sarcoplasmic reticulum (SR) and the gap junction formed by sarcolemma of adjacent myocardial cells) is still not satisfactorily understood in terms of their function. In this review of the ultrastructure of the sarcolemma and its specializations, the intent is not for an extensive review; there are excellent reviews for this already published (1,2). Rather, the focus here is on two areas: (1) to view the ultrastructure of sarcolemma with state of the art freeze-fracture techniques (e.g., ultra-rapid freezing and deep-etch rotary shadowing) and (2) to point out some of the complexities of the ultrastructure for which we still require more information.
A historical approach is a simple way for reviewing the background necessary for discussions of the current concerns. 1947 is a convenient starting point; in this field, as well as in others, a number of contributions appeared two to three years after the war, that defined the field for years to come. Heilbrunn and Wiercinski [1] essentially demonstrated Ca2+ to be the second messenger that controls contraction, and proposed that Ca2+ entered the myoplasm from outside; A.V. Hill [2] demonstrated, with a simple diffusion calculation, the need for a propagation mechanism of activation, other than diffusion from the outside. This system was identified in the fifties: in 1958 Huxley and Taylor [3] suceeded in depolarizing localized regions of the membrane of a fiber with an extracellular electrode placed very close to the fiber, a predecessor of patch clamp. The depolarization only induced contractions (localized to the underlying zone) when the pipette was placed over the I band, but not over the A band. It is interesting that C. Franzini — Armstrong, when reviewing the field, always starts at this time. This result obviously motivated her, as it did other morphologists, to search for a structure underlying these localized effects. Her celebrated studies have a predecessor, closer to home, E. Veratti [4] who had described the T tubular system in 1902, using the Golgi staining technique. Apparently his contemporaries, when presented with the pictures, only said se non è vero è ben trovato, and ignored the result, wich was only exploited in the sixties and seventies by Franzini — Armstrong [4 bis], B. Eisenberg [5], L. Peachey [6] and others. Figure 1 shows a view of a thick transversal cut of a fiber bundle, with Golgi stain (Peachey and Eisenberg [7]), demonstrating how the T system effectively reduces the (diffusion) distances in the transversal direction to under a micrometer. As the morphological description of the T system progressed, so did that of the sarcoplasmic reticulum (Sr).
The sonic muscle of the oyster toadfish Opsanus tau produces unfused contractions at over 200 Hz for mating call production, requiring extreme muscle fiber synchronization. This multiply innervated muscle is sexually dimorphic and grows for life by fiber proliferation and hypertrophy. Previous descriptions of its multiple innervation did not consider fish size or sex. We examined neuromuscular junction (NMJ) development in adult fish of both sexes between 123 and 343 mm in total length (24.7-790 g in mass). The NMJ was a tubelike trough that varied in length from 8 to 178 mu m. Troughs were usually straight, although some consisted of consecutive ovals and some were branched. Median length of NMJs increased linearly with fish length (r(2) = .40; p = .002) from 58 to 75 mu m. Modal lengths were mostly between 50 and 60 mu m and did not increase ontogenetically, indicating that the median increase was caused by a greater number of large junctions in older fish. Median interval between NMJs (measured from the beginning of one junction to the next) ranged from 92 to 116 pm and did not vary with fish size (r(2) = .06; p = .285). Considering muscle fiber elongation, the data indicate an increase from 60 to 140 NMJs per fiber during fish growth. There were no sexual differences in NMJ length or spacing. In view of the slow conduction velocity of sonic muscle fibers, the addition of new NMJs and the relatively constant distance between them supports rapid and synchronized contraction necessary for sound production in both sexes. (C) 1998 John Wiley & Sons, Inc.
Fish sounds are often associated with agonistic and courtship behavior, and in many cases sounds are produced by males with sexually dimorphic sound-producing mechanisms. This review focuses on the sounds and sexually dimorphic sonic neuromuscular system of the oyster toadfish Opsanus tau, compares it with findings on a related toadfish, the midshipman Porichthys notatus and finally reviews data on steroid levels and their effect on seasonal changes in mass of the sonic muscles and sound characteristics in the weakfish Cynoscion regalis. Highlights of the work on the oyster toadfish include the following. (1) Quantitative seasonal changes in the male boatwhistle mating call in a manner suggesting that steroids modulate the output of central nervous system pattern generators for fundamental frequency and duration. (2) Larger sonic muscles in males comprised of a greater number of smaller and therefore more energy efficient fibers than in females. Male muscles have greater activities of anaerobic and aerobic enzymes and enzyme activities and muscle size are increased by androgens. (3) Neurons in the spinal sonic motor nucleus (SMN) are small in females and either small (S) or large (L) in males. The same three morphs are present in Porichthys, but there are major differences in somatic growth patterns, dendrite development and sonic abilities among morphs in the two species. Development of sexual dimorphism in the SMN suggests major differences between amniotic vertebrates (birds and mammals) and fishes, particularly with regard to perinatal critical periods and cell death. (4) As in other vertebrates, toadfish sonic centers in the central nervous system (including the supracommissural nucleus of the ventral telencephalon, nucleus preopticus parvocellularis anterior, the torus semicircularis and the inferior reticular nucleus) possess gonadal steroid-concentrating neurons. Although endocrine investigation of sound production has been attempted in only three species, its relevance to important life history events of commercially-important species and the availability of large numbers of basic biological questions suggest the field is ripe for further investigation.
It is generally accepted that the transverse tubules (T-tubules), a membrane system produced by invaginations of the plasma membrane in skeletal muscle, play a central role in the process of coupling the depolarization of the plasma membrane and muscle contraction (excitation-contraction coupling). However, the detailed molecular mechanism by which the T-tubules communicate with the intracellular membrane system of the sarcoplasmic reticulum (SR) where calcium is stored is largely unknown. Ultrastructural studies of skeletal muscle show that the T-tubules are connected to the SR at both sides through specific structures (the “feet”), forming a structural and functional unit known as a triad(1)
Contraction of all types of muscle fibers is activated by an increase in the cytoplasmic concentration of calcium ions. In the skeletal muscle fibers of vertebrates the calcium required for activation is released rapidly from an internal membrane system, the sarcoplasmic reticulum (SR). In other muscles release from the SR and influx through the surface membrane may contribute in variable proportion to the increase of intracellular calcium needed for myofibrillar activation. Most striated muscle fibers have extensive tubular invaginations of the surface membrane, forming networks called the transverse (T) tubular systems. Individual components are called transverse (T) tubules, even though their orientation is not always transverse to the long axis of the fiber.
The dramatic event by which skeletal muscle, when stimulated, converts chemical energy into mechanical work has fascinated and puzzled physiologists for a long time(1) The interest with which the process of muscular activation has been studied was probably also aroused by the possibility of measuring accurately both the electrical activity associated with the outer membranous system and the mechanical output. The whole sequence of events which bridges these two processes has been intuitively called excitation-contraction coupling(2) Most of the current knowledge concerning this coupling is based on experiments performed in recent years, particularly on single amphibian and crustacean muscle fibers, a point to be borne in mind in any discussion on this subject.
For more than 100 years plasmodesmata have been known as fine channels, of a cytoplasmic nature, that connect neighbouring plant cells through the prominent and rigid carbohydrate walls that separate the cells (Tangl 1879). However, our understanding of the structure and function of plasmodesmata is surprisingly poor compared with their anticipated major roles in intercellular communication between plant cells, i.e. the symplastic transport of water and solutes as well as the channelling of biophysical and biochemical signals from cell to cell (Gunning and Robards 1976a).
Elucidation of the molecular structure of the calcium release channels (CRC) has provided substantial advances towards understanding of the molecular basis for excitation-contraction (EC) coupling. On the basis of cloning studies it is now appreciated that the calcium release channels of the sarcoplasmic and endoplasmic reticula comprise a distinct gene family. This distinct gene family currently includes two major channel types, ryanodine receptors (RyR) and inositol 1,4,5-trisphosphate receptors (IP3R). Each of these major channel types in turn is represented by three forms (Figure 1). Additional structural complexity is achieved by the presence of alternative splicing in the IP3R generating specific neuronal and non-neuronal forms.
There is a diversity of signaling proteins that bind Ca2+ at regulatory sites and that are activated by increases in its level. These are the Ca2+ effectors. Common Ca2+-binding sites on proteins include the EF-hand motif and the C2 domain. However, some EF-hands have lost or never even acquired the ability to bind Ca2+, for example, the EF-hands of phospholipase C. The Ca2+ dissociation constants of C2 domains also vary widely and again, some do not bind Ca2+ at all. Many Ca2+ effectors are without either C2 domains or EF-hands, but acquire their Ca2+ dependence through the Ca2+ sensor protein calmodulin, which in some cases forms an integral subunit. When Ca2+ signals are transient, an effector must be able detect the change and initiate a response before the concentration subsides. Ca2+ signals are often repetitive, taking the form of trains of spikes or pulses with periods of the order of minutes and it is likely that there are specific effectors that can respond to this form of temporal encoding. Calmodulin and its isoform troponin C are the most prominent Ca2+-sensing proteins in animal cells. Such Ca2+-sensitive proteins usually have one or more high affinity binding sites and these may be in the form of EF-hand motifs, C2 domains, or other structures. Some of the paradigms of calcium signaling like triggering neurotransmitter secretion, initiation of contraction in skeletal muscle, and smooth muscle contraction have also been discussed.
The skeletal muscle fiber is a giant multinucleate cell, extending centimeters in length, and up to 100μ in diameter. The bulk of the mass of the fast twitch skeletal muscle fiber is occupied by myofibrils that run the length of fiber. There can be hundreds of myofibrils in a single muscle fiber. The myofibril consists of a linear array of sarcomeres (approximately 2.5 μ long and 1 μ in diameter), the structural units that carry out muscle contraction and relaxation. The number of sarcomeres in a fibril depends on the length of the fiber. Muscle contraction and relaxation are controlled by the intra-fiber free Ca++ concentration, [Ca++]i, in the myoplasm that bathes the muscle filaments of the sarcomeres. In turn, the [Ca++]i is controlled by a network of membranes that regulate and coordinate the process in time and space. This chapter illustrates the structure of a mammalian striated-muscle fiber. It also illustrates a longitudinal section of a rabbit's skeletal muscle fiber. The chapter presents molecular biology approach for the study of sarcoplasmic reticulum (SR). It discusses the reconstitution approach to correlate membrane composition with structure and function and to characterize the nature of lipid–protein interactions in the SR membrane.
Calcium ion (Ca2+) is an important second messenger in signal transduction. This chapter discusses how Ca2+ elevation within the cytosol can activate downstream pathways. The first step must be the binding of Ca2+ to specific proteins which then carry the message forwards. There are important signaling proteins that bind Ca2+ at regulatory sites and that can be activated by local or global increases in Ca2+ level. Common Ca2+-binding sites on proteins include the EF-hand motif and the C2 domain. Ca2+ is also bound at loops in a structure term the endonexin fold. Elevated Ca2+ can activate a wide range of Ca2+-sensitive regulatory enzymes. Calmodulin and its isoform troponin C of skeletal muscle, represent the major Ca2+-sensing proteins in animal cells. Ca2+, through its interaction with calmodulin, can cause the activation of more than 100 different enzymes. The chapter describes several Ca2+-calmodulin dependent enzymes, including Ca2+/calmodulin-dependent kinases (CaM-kinases), calcineurin, phosphorylase kinase, multifunctional Ca2+-activated protein kinases, and nitric oxide synthase (NOS). There are also many enzymes that can respond to changes in Ca2+ directly and are not regulated by calmodulin, including calpain, synaptotagmin, diacylglycerol (DAG), recoverin, and cytoskeletal proteins. The exchange of information between nerve cells, and between nerve and muscle, involves the release of neurotransmitter substances at synapses, in which Ca2+ plays a critical role. The contraction of all types of muscle depends on an increase in intracellular Ca2+. In vertebrate striated muscle, rapid mechanisms have evolved that are mediated by transient increases in cytosol Ca2+. Ca2+ is also involved in andergenic control of cardiac muscle contraction.
Malignant hyperthermia (MH) is a pharmacogenetic disorder with autosomal dominant inheritance. In susceptible individuals, a MH crisis may be triggered by commonly used halogenated anaesthetics (halothane, isoflurane) or muscle relaxants (succhinylcholine). The main symptoms are hypermetabolism and muscle rigidity. Without treatment, death will occur in more than 80% of cases. Although a genetic-chip based diagnostic approach is under development, the invasive in vitro contracture test (IVCT) remains the “gold standard” to diagnose the disorder. Central core disease (CCD) is a slowly progressive myopathy characterised by muscle weakness and hypotonia; affected individuals show delayed motor development and remain physically compromised. Multi-minicore (MmD) disease is a more severe, rare, autosomal recessive myopathy characterised histologically by the presence of multi-minicores in only a small number of sarcomeres. So far, no effective therapy has been developed to treat muscle weakness in CCD and MmD patients and their diagnosis is difficulton the basis of clinical findings alone and a histological examinationof muscle tissue is essential. MmD, CCD and MH are thought to result from a defect in the components involved in excitation-contraction mechanisms and all three diseases are linked to point mutations in the gene encoding the sarcoplasmic reticulum ryanodine receptor calcium release channel (RYR1). The aim of the thesis is to increase our knowledge of the underlying mechanisms which lead to the three different pathologies from mutations in the same gene, namely the ryanodine receptor type 1. Cultured skeletal muscle cells as well as immortalized B-lymphocyte cell lines were used to assay the underlying functional effects of RYR1 mutations, both cell types having the advantage of naturally expressing the ryanodine receptor type 1. The first element of my thesis reports our investigations of the functional characteristics of the ryanodine receptor in cells carrying the following RYR1 mutations: (i) V2168M mutation linked with Malignant hyperthermia; (ii) 2 substitutions, I4898T and R4893W and 1 deletion R4214-F4216 associated with central core disease and (iii) 3 substitutions P3527S, V4849I and R999H associated with CCD/MmD mixed phenotypes. The second aim of my thesis deals with the downstream effects of Ca2+ dysregulation, in particular, the possible role of the ryanodine receptor in the immune system. For this purpose, we have established whether RYR1 mutations influence the release of two cytokines:
viinterleukin-1β and interleukin-6 and if so whether the latter effect may influence the clinical symptoms of MH, CCD or MmD. In the long run, this work may help to develop a non-invasive approach for the diagnosis of MH susceptibility as well as new concepts for the treatment of these muscular pathologies.
This chapter discusses the different types and tools to characterize calcium channels. It focuses on the voltage-dependent calcium channels in plasma membranes from mammalian electrically excitable cells. The calcium channel drugs are treated as structural and functional tools. The chapter also presents some electrophysiological studies of calcium channels. Calcium channels are among the Methuselahs of ion channels. The diversity and the complexity of the physiological control and pharmacological modulation of the calcium channel family have become apparent. The plasma membrane of excitable cells contains several types of calcium channels. They are distinguished by voltage dependence, unitary conductance, selectivity, and by pharmacology. In many cells, multiple channel types are observed, but there are also some membranes where only one type appears to exist. The different types (termed L, T, and N) are well characterized, especially in heart and neuronal tissue. The L type is predominant in heart and smooth muscle cells; the L, T and N types are found in nerve cells. The calcium channels can be characterized biochemically only by specificity of drug binding.
In skeletal muscle a rise in the cytosolic calcium concentration is the first trigger able to initiate the contraction of the sarcomere. Intracellular calcium levels are tightly controlled by channels and pumps, and it is not surprising that many inherited skeletal muscle disorders arise from mutations altering the players regulating calcium ions concentration (Betzenhauser et al., 2010). In this chapter, we will focus on the pathologies linked to the sarcoplasmic reticulum calcium channel-RyR1 mutations.
This study is concerned with the characterization of the morphology of the calcium release channel of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle, which is involved in excitation-contraction coupling. We have previously purified the ryanodine receptor and found it to be equivalent to the feet structures, which are involved, in situ, in the junctional association of transverse tubules with terminal cisternae of SR. The receptor is an oligomer of a single high molecular weight polypeptide and when incorporated into phospholipid bilayers, has channel conductance which is characteristic of calcium release in terminal cisternae of SR. The purified channel can be observed by electron microscopy using different methods of sample preparation, with complementary views being observed by negative staining, double staining, thin section and rotary shadowing electron microscopy. Three views can be observed and interpreted: (a) a square face which, in situ, is junctionally associated with the transverse tubule or junctional face membrane; (b) a rectangle equivalent to the side view; and (c) a diamond shape equivalent to the side view, of which the base portion appears to be equivalent to the transmembrane segment. Negative staining reveals detailed substructure of the channel. A computer averaged view of the receptor displays fourfold symmetry and ultrastructural detail. The dense central mass is divided into four domains with a 2-nm hole in the center, and is enclosed within an outer frame which has a pinwheel appearance. Double staining shows substructure of the square face in the form of parallel linear arrays (six/face). The features of the isolated receptor can be correlated with the structure observed in terminal cisternae vesicles. Sections tangential to the junctional face membrane reveal that the feet structures (23-nm squares) overlap so as to enclose smaller square spaces of approximately 14 nm/side. We suggest that this is equivalent to the transverse tubule face and that the terminal cisternae face is smaller (approximately 17 nm/face) and has larger alternating spaces as a consequence of the tapered sides of the foot structures. Image reconstruction analysis appears to be feasible and should provide the three-dimensional structure of the channel.
The consequences of ionic current flow from the T system to the sarcoplasmic reticulum (SR) of skeletal muscle are examined. The Appendix analyzes a simple model in which the conductance gx, linking T system and SR, is in series with a parallel resistor and capacitor having fixed values. The conductance gx is supposed to increase rapidly with depolarization and to decrease slowly with repolarization. Nonlinear transient currents computed from this model have some of the properties of gating currents produced by intramembrane charge movement. In particular, the integral of the transient current upon depolarization approximates that upon repolarization. Thus, equality of nonlinear charge movement can occur without intramembrane charge movement. A more complicated model is used in the text to fit the structure of skeletal muscle and other properties of its charge movement. Rectification is introduced into gx and the membrane conductance of the terminal cisternae to give asymmetry in the time-course of the transient currents and saturation in the curve relating charge movement to depolarization, respectively. The more complex model fits experimental data quite well if the longitudinal tubules of the sarcoplasmic reticulum are isolated from the terminal cisternae by a substantial resistance and if calcium release from the terminal cisternae is, for the most part, electrically silent. Specific experimental tests of the model are proposed, and the implications for excitation-contraction coupling are discussed.
The structure of the membranes of sarcoplasmic reticulum (SR), tubular (T) system, and sarcolemma has been studied by freeze fracture in leg muscles of the Tarantula spider. Two regions of the sarcoplasmic reticulum can be differentiated by the distribution of particles on the fracture faces: a junctional SR, at the dyads, and a longitudinal SR, elsewhere. The dyads are asymmetric junctions, the disposition of particles in the apposed membranes of SR and T tubules being different from one another and from the regular arrangement of feet in the junctional gap. It is concluded that no channels can be visualized to directly connect SR- and T-system lumina.
The neuromuscular junctions and nonjunctional sarcolemmas of mammalian skeletal muscle fibers were studied by conventional thin-section electron microscopy and freeze-fracture techniques. A modified acetylcholinesterase staining procedure that is compatible with light microscopy, conventional thin-section electron microscopy, and freeze-fracture techniques is described. Freeze-fracture replicas were utilized to visualize the internal macromolecular architecture of the nerve terminal membrane, the chemically excitable neuromuscular junction postsynaptic folds, and the electrically excitable nonjunctional sarcolemma. The nerve terminal membrane is characterized by two parallel rows of 100–110-Å particles which may be associated with synpatic vesicle fusion and release. On the postsynpatic folds, irregular rows of densely packed 110–140-Å particles were observed and evidence is assembled which indicates that these large transmembrane macromolecules may represent the morphological correlate for functional acetylcholine receptor activity in mammalian motor endplates. Differences in the size and distribution of particles in mammalian as compared with amphibian and fish postsynaptic junctional membranes are correlated with current biochemical and electron micrograph autoradiographic data. Orthogonal arrays of 60-Å particles were observed in the split postsynaptic sarcolemmas of many diaphragm myofibers. On the basis of differences in the number and distribution of these "square" arrays within the sarcolemmas, two classes of fibers were identified in the diaphragm. Subsequent confirmation of the fiber types as fast- and slow-twitch fibers (Ellisman et al. 1974. J. Cell Biol. 63[2, Pt. 2]:93 a. [Abstr.]) may indicate a possible role for the square arrays in the electrogenic mechanism. Experiments in progress involving specific labeling techniques are expected to permit positive identification of many of these intriguing transmembrane macromolecules.
The structure of the junction between sarcoplasmic reticulum (SR) and transverse tubular (T) system at the triad has been studied in twitch fibers of the frog. The junction is formed by flattened surfaces of the SR lateral sacs and the T-system tubule, which face each other at a distance of 120–140 A. At periodic intervals of about 300 A, the SR membrane forms small projections, whose tips are joined to the T system membrane by some amorphous material. The SR projections and the amorphous material are here called SR feet. The feet are disposed in two parallel rows, two such rows being present on either side of the T-system tubule. The junctional area between the feet is apparently empty. The feet cover no more than 30% of the T system surface area and 3% of the total SR area. The functional significance of this interpretation of the junctional structure is discussed.
The bat cricothyroid muscle is believed to participate in the production of the short bursts of frequency modulated ultrasound
which these animals use as an echolocation device. The evidence seems to indicate that this muscle must be extremely fast
acting. It possesses a very well developed sarcoplasmic reticulum, consisting of intercommunicating longitudinal and transverse
tubular elements. The transverse elements, situated at the level of the junction between the A and the I bands, are tripartite
complexes of tubules called triads, and these are sometimes replaced by more complex structures, the pentads. The intermediate
element of the triad appears as a slender continuous tubule, which can be shown to come into close contact with the sarcolemma
and also to share with it certain common staining properties. The longitudinal components of the reticulum consist of very
numerous tubules which link successive triads to each other and anastomose to form multiple layers of close-meshed reticula
in the interfibrillar sarcoplasm. Both the longitudinal and the transverse elements of the sarcoplasmic reticulum form a continuous
network across the muscle fiber. It is suggested that the extraordinary development of the sarcoplasmic reticulum in the bat
cricothyroid is related to the unusual physiological properties of this muscle.
When various cations, including Ca2+, are in the fixative, both sarcoplasmic reticulum (SR) of whole skeletal muscle and isolated SR vesicles collapse to form pentalaminate "compound membranes" that result from the apparent fusion of the lumenal lamellae of the membranous envelope of the SR. The process may be reversed by subsequently soaking the tissue in 1 M NaCl. An identical morphological phenomenon is observed in unfixed quickly frozen isolated frog skeletal muscle fibers, the cation in that case coming from endogenous sources. The hypothesis is advanced that the collapse is an in vivo process mediated by the sequestration of Ca2+ after contraction. The resulting obliteration of the SR lumen would have the effect of displacing the SR contents into the junctional SR, as well as electrically isolating the free SR from the junctional SR during relaxation. As a consequence, resistive coupling between the plasmalemma and the junctional SR becomes a plausible mechanism for the translation of the action potential into Ca2+ release, since the bulk of the SR membrane capacitance would now remain separated from the plasmalemma during relaxation.
A method is presented for recording extrinsic optical signals from segments of single skeletal muscle fibers under current or voltage clamp conditions. Such segments, which are cut from intact fibers, are maintained in a relaxed state, while exhbiting otherwise normal physiological properties, including healthy delayed rectifier currents. Extrinsic fluorescence changes are demonstrated, using the permeant potentiometric probe, Nile Blue A. These changes vary nonlinearly with the controlled surface membrane potential, in a manner which suggests that they arise from potential changes in the sarcoplasmic reticulum. According to this interpretation, a simple model based on the gating charge movement implicated in excitation-contraction coupling, provides a self-consistent description of the voltage dependence of the signal that requires no additional parameters.
The membrane systems of skeletal muscle were examined after tannic acid fixation. A new structure consisting of bridges spanning the junctional gap is described, and a model is proposed in which the cytoplasmic but not the luminal membrane leaflets of the transverse tubule and of the junctional sarcoplasmic reticulum (SR) are continuous. The globular particles (presumably the Ca-binding proteins) within the terminal cisternae were arranged in longitudinal rows and appeared adherent to the junctional membrane. The junctional gap was present in negatively stained, frozen thin sections of fixed muscles. Negatively staining material occured within the junctional gap. The cytoplasmic leaflets of the longitudinal, intermediate, and terminal cisterna regions of the SR exhibited a thick coat of densely staining material compatible with the presence of the Ca-ATPase. Similar bridges were also observed at the surface membrane-SR close coupling sites of vascular smooth muscle.
The receptor-rich postsynaptic membrane of the elasmobranch electric organ was fixed by quick-freezing and then viewed by freeze-fracture, deep-etching and rotary-replication. Traditional freeze-fracture revealed a distinct, geometrical pattern of shallow 8.5-nm bumps on the E fracture-face, similar to the lattice which has been seen before in chemically fixed material, but seen less clearly than after quick-freezing. Fracture plus deep-etching brought into view on the true outside of this membrane a similar geometrical pattern of 8.5-nm projections rising out of the membrane surface. The individual projections looked like structures that have been seen in negatively stained or deep-etched membrane fragments and have been identified as individual acetylcholine receptor molecules. The surface protrusions were twice as abundant as the large intramembrane particles that characterize the fracture faces of this membrane, which have also been considered to be receptor molecules. Particle counts have always been too low to match the estimates of postsynaptic receptor density derived from physiological and biochemical studies; counts of surface projections, however, more closely matched these estimates. Rotary-replication of quick-frozen, etched postsynaptic membranes enhanced the visibility of these surface protuberances and illustrated that they often occur in dimers, tetramers, and ordered rows. The variations in these surface patterns suggested that in vivo, receptors in the postsynaptic membrane may tend to pack into "liquid crystals" which constantly appear, flow, and disappear in the fluid environment of the membrane. Additionally, deep-etching revealed a distinct web of cytoplasmic filaments beneath the postsynaptic membrane, and revealed the basal lamina above it; and delineated possible points of contact between these structures and the membrane proper.
Direct stimulation of single muscle fibers from Xenopus laevis at a frequency of 1 Hz results in a decline of the peak isometric twitch tension after about 200 twitches. Fibers were chemically fixed in glutaraldehyde after a varying number of twitches and at several fatigue levels, and the ultrastructural appearance was compared with that of resting fibers treated by identical fixation methods. No gross structural abnormalities were observed but subtle changes occurred. The mitochondria of stimulated fibers contain granules of normal size and number. The inner crista width is constant but the matrix width is increased on stimulation. These changes would not compromise ATP production. The myofibrils are normal except for a slight swelling in the myosin lattice. The transverse system (T system) and sarcoplasmic reticulum are intact. The minor diameter of the transverse tubule (T tubule) is increased slightly in stimulated fibers. The gap between the T-TC membranes stays constant at about 110 A, but tiny connecting pillars are seen to cross this gap more frequently in stimulated fibers (21 +/- 5% triads) than in resting fibers (8 +/- 6%). In stimulated fibers there is a marked increase in the electron dense content of the terminal cisternae (TC). Inasmuch as the observed structural changes correlate with the number of twitches but not with the fatigue level, it is concluded that TC density and T-TC pillar formation are related to the normal mechanisms of excitation-contraction coupling.
Transverse tubules were labeled with [3H]ouabain and isolated from skeletal muscle microsomes. When the labeled transverse tubules were incubated with a fresh unlabeled microsomal preparation in isotonic sucrose, they combined with the terminal cisternae fraction of microsomes and could be observed in the terminal cisternae/triad region of a sucrose density gradient. No attachment between transverse tubules and longitudinal reticulum or mitochondria was detectable. Complete recombination between transverse tubules and terminal cisternae was attained in a medium containing 0.42 M potassium cacodylate even when only a portion of the transverse tubules reattached to terminal cisternae in isotonic sucrose. When the incubation medium contained 0.42 M KCl, NaCl, potassium methylsulfate, or choline chloride, no recombination occurred. Recombination could be observed between purified terminal cisternae and transverse tubules in 0.6 M potassium cacodylate but was not observed in isotonic sucrose under these conditions. When isolated transverse tubules and terminal cisternae were recombined in the presence of 0.6 M potassium cacodylate and the medium was then changed to 0.6 M KCl, the KCl did not break the reformed junction. The 0.6 M KCl treatment did not disrupt the junction between transverse tubules and terminal cisternae of triad junctions which were isolated initially. Observations by electron microscopy of the transverse tubules and terminal cisternae after they had been recombined in the presence of potassium cacodylate revealed the presence of tubular vesicles in close juxtaposition to spherical vesicles which contained electron dense matter. The appearance of the reformed junction was mainly that of diadic entities consisting of a transverse tubule and terminal cisternae vesicle in apposition. More rarely triads were observed, but these might represent triads which had not been detached by the French Press treatment. The electron dense matter of the terminal cisternae was not necessarily at the junction. The data are consistent with the view that the transverse tubule/terminal cisternae junction may be reformed in vitro. This process is under ionic control which may represent a physiological control mechanism of junction formation.
The structure of the triadic junction in frog slow fibers has been studied and compared with that of twitch fibers. The junctional gap is wider (by approximately 13%) in slow fibers. The junctional feet have the same size and disposition as in twitch fibers, although the size and shape of the junctional areas are different. It is concluded that the role of triads in slow fibers is the same as in twitch fibers
The neuromuscular junctions and nonjunctional sarcolemmas of mammalian skeletal muscle fibers were studied by conventional thin-section electron microscopy and freeze-fracture techniques. A modified acetylcholinesterase staining procedure that is compatible with light microscopy, conventional thin-section electron microscopy, and freeze-fracture techniques is described. Freeze-fracture replicas were utilized to visualize the internal macromolecular architecture of the nerve terminal membrane, the chemically excitable neuromuscular junction postsynaptic folds, and the electrically excitable nonjunctional sarcolemma. The nerve terminal membrane is characterized by two parallel rows of 100-110-A particles which may be associated with synpatic vesicle fusion and release. On the postsynpatic folds, irregular rows of densely packed 110-140-A particles were observed and evidence is assembled which indicates that these large transmembrane macromolecules may represent the morphological correlate for functional acetylcholine receptor activity in mammalian motor endplates. Differences in the size and distribution of particles in mammalian as compared with amphibian and fish postsynaptic junctional membranes are correlated with current biochemical and electron micrograph autoradiographic data. Orthogonal arrays of 60-A particles were observed in the split postsynaptic sarcolemmas of many diaphragm myofibers. On the basis of differences in the number and distribution of these "square" arrays within the sarcolemmas, two classes of fibers were identified in the diaphragm. Subsequent confirmation of the fiber types as fast- and slow-twitch fibers (Ellisman et al. 1974. J. Cell Biol.63[2, Pt. 2]:93 a. [Abstr.]) may indicate a possible role for the square arrays in the electrogenic mechanism. Experiments in progress involving specific labeling techniques are expected to permit positive identification of many of these intriguing transmembrane macromolecules.
The contractile response of turtle oviduct smooth muscle to acetylcholine after 30 min of incubation of muscles in Ca-free, 4 mM ethylene (bis) oxyethylenenitrilotetraacetic acid (EGTA) solutions at room temperature was greater than the contractile response after 30 min of incubation in the Ca-free medium at 37 degrees C. Incubation in Ca-free solution at 37 degrees C before stimulation with acetylcholine in Ca-free solutions at room temperature also reduced the contractile response, suggesting that activator calcium was lost from the fibers at a faster rate at higher temperatures. Electron micrographs of turtle oviduct smooth muscle revealed a sarcoplasmic reticulum (SR) occupying approximately 4% of the nucleus- and mitochondria-free cell volume. Incubation of oviduct smooth muscle with ferritin confirmed that the predominantly longitudinally oriented structures described as the SR did not communicate with the extracellular space. The SR formed fenestrations about the surface vesicles, and formed close contacts (couplings) with the surface membrane and surface vesicles in oviduct and vena caval smooth muscle; it is suggested that these are sites of electromechanical coupling. Calculation of the calcium requirements for smooth muscle contraction suggest that the amount of SR observed in the oviduct smooth muscle could supply the activator calcium for the contractions observed in Ca-free solutions. Incubation of oviduct smooth muscle in hypertonic solutions increased the electron opacity of the fibers. A new feature of some of the surface vesicles observed in oviduct, vena caval, and aortic smooth muscle was the presence of approximately 10 nm striations running approximately parallel to the openings of the vesicles to the extracellular space. Thick, thin, and intermediate filaments were observed in turtle oviduct smooth muscle, although the number of thick filaments seen in the present study appeared less than that previously found in mammalian smooth muscles.
The membrane potential of frog single muscle fibers in solutions containing tetrodotoxin was controlled with a two-electrode voltage clamp. Local contractions elicited by 100-ms square steps of depolarization were observed microscopically and recorded on cinefilm. The absence of myofibrillar folding with shortening to striation spacings below 1.95 microm served as a criterion for activation of the entire fiber cross section. With depolarizing steps of increasing magnitude, shortening occurred first in the most superficial myofibrils and spread inward to involve axial myofibrils as the depolarization was increased. In contractions in which the entire fiber cross section shortened actively, both the extent of shortening and the velocity of shortening at a given striation spacing could be graded by varying the magnitude of the depolarization step. The results provide evidence that the degree of activation of individual myofibrils can be graded with membrane depolarization.
The structure of the membranes of sarcoplasmic reticulum (SR), tubular (T) system, and sarcolemma has been studied by freeze fracture in leg muscles of the Tarantula spider. Two regions of the sarcoplasmic reticulum can be differentiated by the distribution of particles on the fracture faces: a junctional SR, at the dyads, and a longitudinal SR, elsewhere. The dyads are asymmetric junctions, the disposition of particles in the apposed membranes of SR and T tubules being different from one another and from the regular arrangement of feet in the junctional gap. It is concluded that no channels can be visualized to directly connect SR- and T-system lumina.
The organization of the indirect flight muscle of an aphid (Hemiptera-Homoptera) is described. The fibers of this muscle contain an extensive though irregularly disposed complement of T system tubules, derived as open invaginations from the cell surface and from the plasma membrane sheaths accompanying the tracheoles within the fiber. The sarcoplasmic reticulum is reduced to small vesicles applied to the T system surfaces, the intermembrane gap being traversed by blocks of electron-opaque material resembling that of septate desmosomes. The form and distribution of the T system and sarcoplasmic reticulum membranes in flight muscles of representatives of the major insect orders is described, and the extreme reduction of the reticulum cisternae in all asynchronous fibers (to which group the aphid flight muscle probably belongs), and the high degree of their development in synchronous fibers is documented and discussed in terms of the contraction physiology of these muscle cells.
With the use of two intracellular microelectrodes and a circuit designed to compensate for the effects of stray capacitances around the electrodes, transfer impedance measurements were made at frequencies from 0.5 to 1000 c/s on frog sartorius muscle fibers bathed in 7.5 mM K Ringer solution. Complete AC cable analyses performed at 46, 100, 215, 464, and 1000 c/s showed that the fibers behaved as ideal one-dimensional cables having purely resistive internal impedances (R(i) = 102 +/- 11 Omega cm). Two circuits were considered for fiber inside-outside impedance, a four lumped parameter circuit and a parallel resistance and capacitance shunted by the input impedance of a lattice model for the T-system. Least squares fits to fiber input impedance phase angles were better with the latter circuit than with the former. With the use of the lattice model the specific capacitance of both the surface and transverse tubule membranes was found to be 1 microF/cm(2) and the internal resistivity of the tubules to be about 300 Omega cm.
Cardiac muscle fibers of the hummingbird and finch have no transverse tubules and are smaller in diameter than those of mammalian hearts. The fibers are connected by intercalated discs which are composed of desmosomes and f. adherentes; small nexuses are often interspersed. As in cardiac muscle of several other animals, the junctional SR of the couplings is highly structured in these two birds but, in addition, and after having lost sarcolemmal contact, the junctional SR continues beyond the coupling to extend deep into the interior of the cells and to form belts around the Z-I regions of the sarcomeres. This portion of the sarcoplasmic reticulum, which we have named "extended junctional SR," and which is so prominent and invariant a feature of cardiac cells of hummingbirds and finches, has not been observed in chicken cardiac cells. The morphological differences between these species of birds may be related to respective differences in heart rates characteristic for these birds.
Approximately 60-70% of the total fiber calcium was localized in the terminal cisternae (TC) in resting frog muscle as determined by electron-probe analysis of ultrathin cryosections. During a 1.2 s tetanus, 59% (69 mmol/kg dry TC) of the calcium content of the TC was released, enough to raise total cytoplasmic calcium concentration by approximately 1 mM. This is equivalent to the concentration of binding sites on the calcium-binding proteins (troponin and parvalbumin) in frog muscle. Calcium release was associated with a significant uptake of magnesium and potassium into the TC, but the amount of calcium released exceeded the total measured cation accumulation by 62 mEq/kg dry weight. It is suggested that most of the charge deficit is apparent, and charge compensation is achieved by movement of protons into the sarcoplasmic reticulum (SR) and/or by the movement of organic co- or counterions not measured by energy dispersive electron-probe analysis. There was no significant change in the sodium or chlorine content of the TC during tetanus. The unchanged distribution of a permeant anion, chloride, argues against the existence of a large and sustained transSR potential during tetanus, if the chloride permeability of the in situ SR is as high as suggested by measurements on fractionated SR. The calcium content of the longitudinal SR (LSR) during tetanus did not show the LSR to be a major site of calcium storage and delayed return to the TC. The potassium concentration in the LSR was not significantly different from the adjacent cytoplasmic concentration. Analysis of small areas of I-band and large areas, including several sarcomeres, suggested that chloride is anisotropically distributed, with some of it probably bound to myosin. In contrast, the distribution of potassium in the fiber cytoplasm followed the water distribution. The mitochondrial concentration of calcium was low and did not change significantly during a tetanus. The TC of both tetanized and resting freeze-substituted muscles contained electron-lucent circular areas. The appearance of the TC showed no evidence of major volume changes during tetanus, in agreement with the estimates of unchanged (approximately 72%) water content of the TC obtained with electron-probe analysis.
The junction between the T system and sarcoplasmic reticulum (SR) of frog skeletal muscle was examined in resting and contracting muscles. Pillars, defined as pairs of electron-opaque lines bounding an electron-lucent interior, were seen spanning the gap between T membrane and SR. Feet, defined previously in images of heavily stained preparations, appear with electron-opaque interiors and as such are distinct from the pillars studied here. Amorphous material was often present in the gap between T membrane and SR. Sometimes the amorphous material appeared as a thin line parallel to the membranes; sometimes it seemed loosely organized at the sites where feet have been reported. Resting single fibers contained 39 +/- 14.3 (mean +/- SD; n = 9 fibers) pillars/micrometer2 of tubule membrane. Single fibers, activated by a potassium-rich solution at 4 degrees C, contained 66 +/- 12.9 pillars/micrometer2 (n = 8) but fibers contracting in response to 2 mM caffeine contained 33 +/- 8.6/micrometer2 (n = 5). Pillar formation occurs when fibers are activated electrically, but not when calcium is released directly from the SR; and so we postulate that pillar formation is a step in excitation-contraction coupling.
The consequences of ionic current flow from the T system to the sarcoplasmic reticulum (SR) of skeletal muscle are examined. The Appendix analyzes a simple model in which the conductance gx, linking T system and SR, is in series with a parallel resistor and capacitor having fixed values. The conductance gx is supposed to increase rapidly with depolarization and to decrease slowly with repolarization. Nonlinear transient currents computed from this model have some of the properties of gating currents produced by intramembrane charge movement. In particular, the integral of the transient current upon depolarization approximates that upon repolarization. Thus, equality of nonlinear charge movement can occur without intramembrane charge movement. A more complicated model is used in the text to fit the structure of skeletal muscle and other properties of its charge movement. Rectification is introduced into gx and the membrane conductance of the terminal cisternae to give asymmetry in the time-course of the transient currents and saturation in the curve relating charge movement to depolarization, respectively. The more complex model fits experimental data quite well if the longitudinal tubules of the sarcoplasmic reticulum are isolated from the terminal cisternae by a substantial resistance and if calcium release from the terminal cisternae is, for the most part, electrically silent. Specific experimental tests of the model are proposed, and the implications for excitation-contraction coupling are discussed.
A method is presented for recording extrinsic optical signals from segments of single skeletal muscle fibers under current or voltage clamp conditions. Such segments, which are cut from intact fibers, are maintained in a relaxed state, while exhbiting otherwise normal physiological properties, including healthy delayed rectifier currents. Extrinsic fluorescence changes are demonstrated, using the permeant potentiometric probe, Nile Blue A. These changes vary nonlinearly with the controlled surface membrane potential, in a manner which suggests that they arise from potential changes in the sarcoplasmic reticulum. According to this interpretation, a simple model based on the gating charge movement implicated in excitation-contraction coupling, provides a self-consistent description of the voltage dependence of the signal that requires no additional parameters.
Approximately 60-70% of the total fiber calcium was localized in the terminal cisternae (TC) in resting frog muscle as determined by electron-probe analysis of ultrathin cryosections. During a 1.2 s tetanus, 59% (69 mmol/kg dry TC) of the calcium content of the TC was released, enough to raise total cytoplasmic calcium concentration by approximately 1 mM. This is equivalent to the concentration of binding sites on the calcium-binding proteins (troponin and parvalbumin) in frog muscle. Calcium release was associated with a significant uptake of magnesium and potassium into the TC, but the amount of calcium released exceeded the total measured cation accumulation by 62 mEq/kg dry weight. It is suggested that most of the charge deficit is apparent, and charge compensation is achieved by movement of protons into the sarcoplasmic reticulum (SR) and/or by the movement of organic co- or counterions not measured by energy dispersive electron-probe analysis. There was no significant change in the sodium or chlorine content of the TC during tetanus. The unchanged distribution of a permeant anion, chloride, argues against the existence of a large and sustained transSR potential during tetanus, if the chloride permeability of the in situ SR is as high as suggested by measurements on fractionated SR. The calcium content of the longitudinal SR (LSR) during tetanus did not show the LSR to be a major site of calcium storage and delayed return to the TC. The potassium concentration in the LSR was not significantly different from the adjacent cytoplasmic concentration. Analysis of small areas of I-band and large areas, including several sarcomeres, suggested that chloride is anisotropically distributed, with some of it probably bound to myosin. In contrast, the distribution of potassium in the fiber cytoplasm followed the water distribution. The mitochondrial concentration of calcium was low and did not change significantly during a tetanus. The TC of both tetanized and resting freeze-substituted muscles contained electron-lucent circular areas. The appearance of the TC showed no evidence of major volume changes during tetanus, in agreement with the estimates of unchanged (approximately 72%) water content of the TC obtained with electron-probe analysis.
The organization of the indirect flight muscle of an aphid (Hemiptera-Homoptera) is described. The fibers of this muscle contain an extensive though irregularly disposed complement of T system tubules, derived as open invaginations from the cell surface and from the plasma membrane sheaths accompanying the tracheoles within the fiber. The sarcoplasmic reticulum is reduced to small vesicles applied to the T system surfaces, the intermembrane gap being traversed by blocks of electron-opaque material resembling that of septate desmosomes. The form and distribution of the T system and sarcoplasmic reticulum membranes in flight muscles of representatives of the major insect orders is described, and the extreme reduction of the reticulum cisternae in all asynchronous fibers (to which group the aphid flight muscle probably belongs), and the high degree of their development in synchronous fibers is documented and discussed in terms of the contraction physiology of these muscle cells.
The ultrastructure of chicken and frog cardiac muscle are compared and then contrasted with the ultrastructure of mammalian cardiac muscle. Both chicken and frog cardiac muscle have no transverse tubules, remarkably few nexuses and no prominent M-lines. M-fibers of both animals are small (2–5 μ) in diameter and contain dense granules. Chicken cardiac muscle like mammalian cardiac muscle has very well developed sarcoplasmic reticulum and couplings. The latter do not occur in frog cardiac muscle and the former is poorly developed in that muscle. Morphologic evidence is presented in the frog and chicken heart that would tend to attribute to the sarcoplasmic reticulum a transport function for electron-dense material (presumably proteinaceous) the possible significance of which is discussed. Purkinje fibers were identified in the form of a network on the endocardial surface of both atria and ventricles of chicken hearts. The topography of these fibers corresponds to that of a population of fibers in small mammalian hearts that, and unlike ventricular fibers in those animals, does not have transverse tubules.
Light and heavy sarcoplasmic reticulum vesicles were isolated from rabbit leg muscle using a combination of differential centrifugation and isophycnic zonal ultracentrifugation. Light sarcoplasmic reticulum vesicles obtained from the 30–32.5% and heavy sarcoplasmic reticulum vesicles obtained from the 38.5–42% sucrose regions of the linear sucrose gradient were determined to be free of surface and mitochondrial membrane contamination by marker enzyme analysis and electron microscopy. Thin sections of the light vesicles revealed empty vesicles of various sizes and shapes. Freeze-fracture replicas of the light vesicles showed an asymmetric distribution of intramembranous particles with the same orientation and distribution as the longitudinal sarcoplasmic reticulum in vivo. Heavy vesicles appeared as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The cytoplasmic surface of the membrane was decorated by membrane projections, closely resembling the ‘feet’ which join the sarcoplasmic reticulum to the transverse tubules in the intact muscle fiber. Freeze-fracture replicas of the heavy vesicles revealed an asymmetric distribution of particles which in some areas of the vesicle's surface are larger and less densely aggregated than those of the light vesicles. In the best quality replicas, some regions of the luminal leaflet were not smooth but showed evidence of pits. These structural details are characteristic of the area of sarcoplasmic reticulum membrane which is covered by the ‘feet’ in the intact muscle.
The SR network in immature skeletal muscle fibers forms a lacelike network of diverging and converging tubules oriented both longitudinally and transversely around the fibril. The network is irregularly oriented and does not appear to be differentiated at various fibril striation levels as in adult fibers. Triads in immature fibers are fewer in number and are oriented predominantly longitudinally while in the adult they are numerous and encircle the fibril transversely at the A-I junction.Densely staining material within SR apposed at the T or sarcolemma has been shown to be structural in nature. Periodic bridges traverse a space approximately 100 Å wide between apposed membranes. A density coextensive with the apposed SR limiting membrane is located near the inside surface of the membrane. Densities appear to form connections between the coextensive density and the apposed SR limiting membrane. Additional dense structures are located in the expanded SR segment between the coextensive density and the unapposed SR membrane.Observations on fetal and very young rats suggest that the earliest differentiation of structures within apposed SR is dependent upon close apposition of undifferentiated SR network tubule segments at the T or sarcolemma. The coextensive density is seen only in apposed SR showing periodic bridges traversing the space between apposed membranes. The formation of bridges between apposed membranes would therefore appear to be prerequisite to differentiation of the coextensive density and its connections to the apposed SR limiting membrane. It is suggested that expansion of the apposed SR tubule in a plane perpendicular to apposition accommodates additional structures between the coextensive density and the unapposed SR limiting membrane. Structures showing expansion characteristics of apposed SR are never found unless the SR is indeed apposed at the T or the sarcolemma.Observations on fibers from fetal and newborn to 35-day rats have shown that the predominantly longitudinally oriented triads of immature fibers change orientation. The change from predominantly longitudinally to transversely oriented triads is complete in the larger fibers from animals 10 to 15 days postnatal. It is of interest to note that this change in orientation corresponds to the observed marked decrease in E-C latency.
THE nature of intramembranous particles has recently been discussed in relation to the mode of freeze fracturing1,2. It has been argued that particles that do not show complementarity (complementary pits on the opposite fracture face) are a reflection of protein penetrating the membrane. The protein giving rise to an intramembraneous particle is plastically deformed during the fracturing procedure and is probably pulled out to one of the two fracture faces. Moreover we have argued that particles showing complementarity (pits) are possibly of lipidic origin. This latter hypothesis was mainly based on the determination of the nature of the intramembranous particles of the outer membrane of Escherichia coli. These complementary particles were shown to be determined by lipo-polysaccharide3,4. We show here that lipid can by itself form intramembranous particles showing complementarity and that these particles may be inverted micelles of phospholipid sandwiched between lipid monolayers.
Vascular smooth muscle cells in mouse heart contain prominent membrane systems (“sarcotubules”). One of these systems consists of vesicular structures whose unit membranes are continuous with the sarcolemma and which occur either as single caveolae, more complex tubules, or branched chains of fused caveolae. Such caveolar systems are both analogous to, and homologous with, the T or T-axial tubular systems of striated muscle cells. A second system of membranes, the sarcoplasmic reticulum (SR), comprises tubules and saccules that often come into close association with the sarcolemma but apparently are not open to the extracellular space. In addition to forming “couplings” with the sarcolemma, the SR often comes into close contact with mitochondria and caveolae. The ultrastructural complexity of these membrane systems in coronary vascular smooth muscle equals or surpasses that of smooth muscle cells in the large blood vessels that have been extensively studied by other investigators.
Freeze-fracture analysis of triadic junctions utilizing an abbreviated glycerination schedule reveals regularly arranged intramembranous subunits within junctional portions of sarcoplasmic reticulum terminal cisterns. The subunits appear as round to square 20-nm elevations of the junctional terminal cistern E-face; the complementary P-face is characterized by a concentration of small particles, the detailed arrangement of which is less obvious. The E-face subunits may contain a pore, and their spatial arrangement corresponds to the tetragonal array of dimples or feet previously described within the junctional gap. No corresponding subunits have been detected within T-tubular membranes. Stereo techniques and complementary replica analysis have been utilized for detailed study of the subunits as well as associated features of the fracture faces of both T-tubular and terminal cistern junctional and nonjunctional membranes. The new findings are discussed in relation to structural models which might explain the mechanisms of the triadic junction involved in excitation-contraction coupling.
We studied the morphology of rabbit psoas muscle fixed at increasing intervals of time in a chemical skinning solution (Wood et al., 1975), or after skinning and storage for times up to 1 week. The storage solution, in which the chemically skinned muscled fibers were kept at -20 degrees C, had the same ionic composition as the skinning solution but was made with 50% (v/v) glycerol. Progressive structural changes occurred in fibers exposed to skinning solution. The structural changes were essentially complete after 24-48 hr in skinning solution and no further changes were detected in fibers stored for periods up to 1 week. Structural changes were: (i) holes or gaps in the plasma membrane; (ii) swelling of mitochondria and disorganization of their internal structure; (iii) slight swelling of the sarcoplasmic reticulum; (iv) disappearance of sarcoplasmic reticulum (SR) feet from triadic gaps. Other changes included loss of glycogen between fibrils and extraction of myoplasm, or the change of its staining properties. All architectural elements of the SR, except "feet", remained during skinning and storage, and the SR remained able to accumulate calcium. The morphology of the myofilaments during chemical skinning and during storage did not differ from control fibers. We conclude that chemical skinning alters the gross structure of the plasma membrane and mitochondria, but produces minimal changes in the sarcoplasmic reticulum and contractile proteins.
Flight muscle fibers of Anax imperator nymphs, in different developmental stages are analyzed for several morphological features, such as the arragnement and numerical ratio of actin and myosin filaments, the pattern of the T system and sarcoplasmic reticulum, the number of microtubules and the fractional volume of mitochondria in each fiber. The T system is initially represented by longitudinal grooves on the cell surface, joined with vesicles of the sarcoplasmic reticulum; this pattern rapidly changes and the grooves start to break up into longitudinal segments. The thin to thick filament ratio is at first quite high (about 4-4.5:1) but rapidly falls to the final (3:1) when the myofibrils are well developed at the fiber periphery. Statistical analyses show that the measured values are significantly different in the various stages of development, also indicating a progressive reduction of the ratio variability. The reduction of thin to thick filament ratio and the variance decrease fit quite well with the hypothesis that the synthesis of actin and myosin depends on independently regulated messenger RNA molecules.
In mouse myocardial cells, the sarcoplasmic reticulum (SR) forms specialized complexes (“couplings”) with both the sarcolemma and its extension, the system of transverse (T) and axial (Ax) tubules. As well as contributing to conventional “diads” and “triads,” the junctional region of the SR (J-SR) often forms complexes having special configurations, the latter including: (1) couplings in which J-SR is wrapped partially or completely around T-Ax tubules; (2) “reversed triads” in which a single J-SR saccule is flanked by two T-Ax tubules; (3) “pentad” and “sextad” couplings with multiple J-SR and T-Ax elements; and (4) “bidirectional peripheral couplings” in which J-SR is sandwiched between closely apposed folds of the sarcolemma, a condition frequently observed in the region of the intercalated disc. In the last three configurations, electron-opaque junctional processes are formed on both sides of the flattened sac of J-SR, rather than on a single side as in conventional couplings.
EXCITATION-contraction (E-C) coupling in striated muscle1,2 involves depolarisation of the surface membrane that propagates through invaginations of the surface membrane, the transverse tubular (T) system, to the interior of the fibre. Stored Ca is then released from the adjacent sarcoplasmic reticulum (SR), and activates the contractile proteins. The mechanism of release of Ca from the SR is not definitely known, although it is thought to be triggered by the T-tubule action potential either through electrical coupling, possibly involving voltage dependent charge movement3-5 across the T-SR foot processes6-7 and depolarisation of the SR2,8,9 and/or a chemical coupling involving a Ca induced Ca release9-12. Depolarisation of the sarcoplasmic reticulum in skinned muscle fibres by Cl induces Ca release2. The evaluation of the various models of E-C coupling requires some knowledge of whether the ionic distribution across the SR membrane can give rise to a potential and, specifically, whether the content of the SR resembles that of the extracellular or of the intracellular fluid. So, to measure the ionic composition of individual elements of the sarcoplasmic reticulum in situ, we used electron probe X-ray microanalysis of frozen-dried thin section of a fast striated muscle. The data we report here show that neither Cl nor Na are compartmentalised in the SR, and that their concentrations in the SR (on a dry weight basis) do not differ from those in the surrounding cytoplasm. Ca was found to be sequestered in the sarcoplasmic reticulum confirming other studies (refs 13, 14 and see refs 1, 2 for reviews). These findings (albeit on a dry weight basis) do not support the existence of a Cl or Na potential across the SR membrane in resting muscle.
The structure of the membranes limiting the sarcoplasmic reticulum (SR) and transverse (T) tubules in frog and fish muscle fibers has been studied by freeze fracture. Emphasis is placed on the structure of the membranes at the triad, where thin sections have previously shown that rows of regularly disposed "feet" join SR and T exposed fracture faces allows the following conclusions. 1) The SR membrane is continuous and identical in appearance along the whole sarcomere. Thus the SR is a single uninterrupted compartment and it is likely that the major function of the reticulum, calcium accumulation is performed by the membrane limiting the lateral sacs of the triad, as well as the longitudinal tubules. 2) At the level of the junction with the T tublue, the SR presents a strikingly different number, size and arrangement of particles and pits. This distinct portion of the SR membrane extends father than the area covered by the junctional "feet" and no correlation can be found between the disposition of particles within the membrane and that of the feet on the membrane surface. 3) The T system membrane presents few prominent particles on its junctional face, but these are far less numerous than the feet. 4) Thus, no visible preformed channels exist between SR and T system lumina and it is suggested that direct electrical coupling between the two membranes during excitation is unlikely.
It is suggested that a link in excitation-contraction coupling involves
the movement of a fixed amount of charge free to move between different
locations across the membrane.
Triad junctions in skeletal muscle of young newts and adult frogs have been examined after a wide variety of fixation procedures. When collidine-osmium is employed, the T-tubules of younger larval newt muscle appear as dilated channels which retain normal junctional relations with adjacent terminal cisterns. In older muscle, or after other fixation techniques, the T-tubule is narrow. This latter condition is less useful for analysis of junctional morphology, particularly when sections parallel to and including the junction are utilized. The images (including stereo electron micrographs) suggest that a given triad junction contains a gap distance of about 150 Å bridged by an array of cementing materials. “Dimples” in the terminal cistern membrane bring it into tight or close junctional proximity to the T-tubule membrane. The overall appearance resembles an intermediate junction within which small foci of tight or close junctional contact exist. Evidence of direct pore-like membrane continuities between T-tubules and terminal cisterns was not observed.
Rattlesnake body and shaker muscles were studied using light microscopy and histochemistry. Five myofiber types are distinguishable in the body musculature. The majority are large diameter fast twitch fibers with high alkaline-stable ATPase activity and few mitochondria. In the shaker muscle the major fiber differs from all body fibers in that myofibrils do not entirely fill the fibers. The myofibrils branch repeatedly with one another, which leaves large areas of sarcoplasm devoid of filaments and gives the fibers a characteristic mottled appearance. Mitochondria and glycogen deposits are very numerous. Shaker fibers have high alkaline stable ATPase activity and, in addition, stain intensely for NADH-TR and alpha GPD. Myofibers of the shaker muscle are unusual in that they are extremely fast contracting yet are highly fatigue resistant.
Membranes of two crayfish muscles with different contraction speeds were studied with freeze-fracture replicas and thin sections. A fast-contracting, short sarcomere phasic muscle, the tail flexor, and a slowly contracting long sarcomere tonic muscle, the carpopodite flexor, were chosen for this study. Membranes examined included the plasmalemma, clefts, T-system, Z-tubules and sarcoplasmic reticulum (SR). We found distinct differences in the distribution of these membranes: T-system and clefts are more elaborate in the tail flexor, while SR is more extensive in the leg flexor. The tail flexor apparently lacks Z-tubules. These differences were more obvious in freeze-fracture replicas than in thin sections. In freeze-fracture replicas, both junctional and non-junctional T-tubule membranes can be distinguished from Z-tubules by content of intramembranous particles. The junctional regions of T-system and surface membranes contain large (10-11 nm) intramembranous particles that are absent from non-junctional parts of these membranes. There is also a class of particles on the junctional SR fracture faces that differs from intramembranous particles on non-junctional SR. These junctional specialization are similar in long and short sarcomere fibres.
The shaker muscle of the rattlesnake is able to sustain a rate of contraction around 50/sec for at least 3 hr. Most of the muscle is composed of a single, major fiber type which contains large numbers of small myofibrils, abundant sarcomplasmic reticulum, numerous mitochondria, and large deposits of glycogen. The neuromuscsular junction of the major fiber type is extensive with several nerve terminal expansions and junctional folds that are both numerous and deep. The structure of the major fiber type suggests that it must be responsible both for the speed of contraction as well as for the indefatigability of the shaker muscle. The ultrastructure of the much less numerous minor fibers in the shaker muscle suggest that their role may be a tonic one.
We examine here the proposition that membrane lipids1-4, rather than intrinsic membrane proteins5-7, are the principal structural elements of the strands comprising tight junctions. Our evidence, which is based on direct rapid freezing of newly formed tight junctions between rat prostate epithelial cells, indicates that individual tight junction strands are pairs of inverted cylindrical micelles sandwiched between linear fusions of the external membrane leaflets of adjacent cells. Although individual tight junction strands appear as continuous cylinders when fractured near the frozen surface, where ice crystals have not damaged the plasma membrane, they appear as rows of particles when fractured deeper in the frozen tissue. We now interpret these tight junction particles as remnants of intramembrane cylinders disrupted during freezing The morphology and dimensions of the intact cylinders correspond to those of lipids in the cylindrical hexagonal II phase8,9 and this suggests that tight junction formation requires a phase transition of the planar lipid bilayer similar to that invoked in models of membrane fusion10,11. Our morphological interpretation explains the known functional properties of tight junctions.
Freeze fracture analysis of intramembranous particle density in skeletal muscle plasma dystrophy from 7 patients with Duchenne muscular dystrophy (DMD), 5 patients with facioscapulohumeral muscular dystrophy (FSH) and 5 patients with myotonic dystrophy (MyD) were carried out. Marked depletion of intramembranous particles including orthogonal arrays was noted in DMD while only orthogonal arrays were significantly decreased in FSH. No abnormalities were noted in MyD.
Bilayers incorporating phospholipids that are capable, in isolation, of
forming non-bilayer structures show `pits and particles' or `cusps' by
freeze-fracture electron microscopy1-6. These points of very
high curvature in the bilayer have been interpreted either as inverted
micelles within a single bilayer2,3 or intersecting
bilayers4,5, or as intermembrane sites of
attachment1,6. We have used extremely rapid freezing and
cleavage at very low temperature to look at the
cardiolipin-phosphatidylcholine system and report here a high incidence
of cross-fractured liposomes, which shows that even at lower
CaCl2 concentrations they are filled with membranous
structures. More interestingly, the arrays of pits and particles are
closely aligned and associated with two further kinds of asymmetric
bilayer deformation. Flat areas in the bilayer seem to represent sites
of particularly adherent interbilayer contact and deep imaginations
appear to represent sites where fusion with internal bilayers has
occurred.
A classification of the sarcoplasmic reticulum (SR) membrane into areas devoted to different functions is described. The junctional (j) SR is covered by feet, it faces towards the transverse tubules, and it is probably devoted to receiving a signal from them. The free (f) SR contains the calcium pump. Isolated SR forms two fractions: light and heavy SR. The heavy fraction is composed of the lateral sacs of the triad, containing calsequestrin, and its membrane comprises both f and j SR. The light fraction is entirely composed of fSR membrane. It is proposed that the feet join jSR membrane to particles contained within the transverse tubules and that they play a direct role in excitation-contraction coupling.
Although freeze-fracture electron microscopy is normally analysed with the presumption that particles represent proteins embedded within the membrane, particles which appeared to represent inverted lipid micelles within membranes have been reported. I have now confirmed the occurrence of particles in protein-free liposomes, but have found that they behave like intermembrane attachment sites rather than structures within a membrane.