Hiroaki Takekura

The brain ryanodine receptor: a caffeine-sensitive calcium release channel.

The release of stored Ca2+ from intracellular pools triggers a variety of important neuronal processes. Physiological and pharmacological evidence has indicated the presence of caffeine-sensitive intracellular pools that are distinct from the well-characterized inositol 1,4,5,-trisphosphate (IP3)-gated pools. Here we report that the brain ryanodine receptor functions as a caffeine- and ryanodine-sensitive Ca2+ release channel that is distinct from the brain IP3 receptor. The brain ryanodine receptor has been purified 6700-fold with no change in [3H]ryanodine binding affinity and shown to be a homotetramer composed of an approximately 500 kd protein subunit, which is identified by anti-peptide antibodies against the skeletal and cardiac muscle ryanodine receptors. Our results demonstrate that the brain ryanodine receptor functions as a caffeine-sensitive Ca2+ release channel and thus is the likely gating mechanism for intracellular caffeine-sensitive Ca2+ pools in neurons.

Eccentric exercise‐induced morphological changes in the membrane systems involved in excitation—contraction coupling in rat skeletal muscle

Physiological evidence suggests that excitation‐contraction (E—C) coupling failure results from eccentric contraction‐induced muscle injury because of structural and morphological damage to membrane systems directly associated with the E—C coupling processes within skeletal muscle fibres. In this study using rats, we observed the ultrastructural features of the membrane systems of fast‐twitch (FT) and slow‐twitch (ST) muscle fibres involved in E—C coupling following level and downhill running exercise. Our aim was to find out whether mechanically mediated events following eccentric exercise caused disorder in the membrane systems involved in E—C coupling, and how soon after exercise such disorder occurred. We also compared the morphological changes of the membrane systems between ST and FT muscle fibres within the same muscles. Single muscle fibres were dissected from triceps brachii muscles of male Fischer 344 rats after level or downhill (16 deg decline) motor‐driven treadmill running (18 m min−1, 5 min running with 2 min rest interval, 18 bouts). All single muscle fibres were histochemically classified into ST or FT fibres. The membrane systems were visualized using Ca2+–K3Fe(CN)6–OsO4 techniques, and observed by high voltage electron microscopy (120–200 kV). There were four obvious ultrastructural changes in the arrangement of the transverse (t)‐tubules and the disposition of triads after the downhill running exercise: (1) an increase in the number of longitudinal segments of the t‐tubule network, (2) changes in the direction and disposition of triads, (3) the appearance of caveolar clusters, and (4) the appearance of pentads and heptads (close apposition of two or three t‐tubule elements with three or four elements of terminal cisternae of the sarcoplasmic reticulum). The caveolar clusters appeared almost exclusively in the ST fibres immediately after downhill running exercise and again 16 h later. The pentads and heptads appeared almost exclusively in the FT fibres, and their numbers increased dramatically 2–3 days after the downhill running exercise. The eccentric exercise led to the formation of abnormal membrane systems involved in E–C coupling processes. These systems have unique morphological features, which differ between ST and FT fibres, even within the same skeletal muscle, and the damage appears to be concentrated in the FT fibres. These observations also support the idea that eccentric exercise‐ induced E–C coupling failure is due to physical and chemical disruption of the membrane systems involved in the E–C coupling process in skeletal muscle.

RESTORATION OF JUNCTIONAL TETRADS IN DYSGENIC MYOTUBES BY DIHYDROPYRIDINE RECEPTOR CDNA

Excitation-contraction coupling was restored in primary cultures of dysgenic myotubes by transfecting the cells with an expression plasmid encoding the rabbit skeletal muscle dihydropyridine receptor. Dishes containing normal, dysgenic, and transfected myotubes were fixed, freeze-fractured, and replicated for electron microscopy. Numerous small domains in the surface membrane of normal myotubes contain ordered arrays of intramembrane particles in groups of four (tetrads). The disposition of tetrads in the arrays is consistent with alternate positioning of tetrads relative to the underlying feet of the sarcoplasmic reticulum. Dysgenic myotubes have no arrays of tetrads. Some myotubes from successfully transfected cultures have arrays of tetrads with spacings equal to those found in normal myotubes. Thus the dihydropyridine receptor appears to be needed for the formation of tetrads and their association with the sarcoplasmic reticulum feet. This result is consistent with the hypothesis that each tetrad is composed of four dihydropyridine receptors.

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

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

CO-EXPRESSION IN CHO CELLS OF TWO MUSCLE PROTEINS INVOLVED IN EXCITATION-CONTRACTION COUPLING

SummaryRyanodine receptors and dihydropyridine receptors are located opposite each other at the junctions between sarcoplasmic reticulum and either the surface membrane or the transverse tubules in skeletal muscle. Ryanodine receptors are the calcium release channels of the sarcoplasmic reticulum and their cytoplasmic domains form the feet, connecting sarcoplasmic reticulum to transverse tubules. Dihydropyridine receptors are L-type calcium channels that act as the voltage sensors of excitation-contraction coupling: they sense surface membrane and tranverse tubule depolarization and induce opening of the sarcoplasmic reticulum release channels. In skeletal muscle, ryanodine receptors are arranged in extensive arrays and dihydropyridine receptors are grouped into tetrads, which in turn are associated with the four subunits of ryanodine receptors. The disposition allows for a direct interaction between the two sets of molecules.CHO cells were stably transformed with plasmids for skeletal muscle ryanodine receptors and either the skeletal dihydropyridine receptor, or a skeletal-cardiac dihydropyridine receptor chimera (CSk3) which can functionally substitute for the skeletal dihydropyridine receptor, in addition to plasmids for the α2, β and γ subunits. RNA blot hybridization gave positive results for all components. Immunoblots, ryanodine binding, electron microscopy and exposure to caffeine show that the expressed ryanodine receptors forms functional tetrameric channels, which are correctly inserted into the endoplasmic reticulum membrane, and form extensive arrays with the same spacings as in skeletal muscle. Since formation of arrays does not require coexpression of dihydropyridine receptors, we conclude that self-aggregation is an independent property of ryanodine receptors. All dihydropyridine receptor-expressing clones show high affinity binding for dihydropyridine and immunolabelling with antibodies against dihydropyridine receptor. The presence of calcium currents with fast kinetics and immunolabelling for dihydropyridine receptors in the surface membrane of CSk3 clones indicate that CSk3-dihydropyridine receptors are appropriately targeted to the cells plasmalemma. The expressed skeletal-type dihydropyridine receptors, however, remain mostly located within perinuclear membranes. In cells coexpressing functional dihydropyridine receptors and ryanodine receptors, no junctions between feet-bearing endoplasmic reticulum elements and surface membrane are formed, and dihydropyridine receptors do not assemble into tetrads. A separation between dihydropyridine receptors and ryanodine receptors is not unique to CHO cells, but is found also in cardiac muscle, in muscles of invertebrates and, under certain conditions, in skeletal muscle. We suggest that failure to form junctions in co-transfected CHO cell may be due to lack of an essential protein necessary either for the initial docking of the endoplasmic reticulum to the surface membrane or for maintaining the interaction between dihydropyridine receptors and ryanodine receptors. We also conclude that formation of tetrads requires a close interaction between dihydropyridine receptors and ryanodine receptors.

Development of the excitation-contraction coupling apparatus in skeletal muscle: peripheral and internal calcium release units are formed sequentially

SummaryThe development of calcium release units and of transverse tubules has been studied in skeletal muscle fibres from embryonal and newborn chicken. Three constituents of calcium release units: the tetrads, the feet and an internal protein directly associated with junctional surface of the sarcoplasmic reticulum are visualized by various electron microscope techniques. Evidence in the literature indicates that the three components correspond to the voltage sensors, the sarcoplasmic reticulum calcium release channels and the calcium binding protein calsequestrin respectively.We recognize two stages at which important events in membrane morphogenesis occur. The first stage coincides with early myofibrillogenesis (starting at approximately embryonal day E5.5), and it involves the assembly of calcium release units at the periphery of the muscle fibre in which feet and the internal protein are identified. Groups of tetrads also are present at very early stages and their disposition indicates a relation to the feet of peripheral couplings. Thus three major components of the excitation-contraction coupling pathway are in place as soon as myofibrils develop. The density of groups of tetrads in the surface membrane of primary and secondary fibres is similar, despite differences in developmental stages. The second stage involves the formation of a complex transverse tubule network and of internal sarcoplasmic reticulum-transverse tubule junctions, while peripheral couplings disappear. This stage starts abruptly (between E15 and E16) and simultaneously in primary and secondary fibres. It coincides with the myotube-to-myofibre transition. The two stages are separated by a relatively long intervening period (between E9 and E16). During the latter part of this period some primitive transverse tubules appear, and form junctions with the sarcoplasmic reticulum, but they remain strictly located at the periphery of the fibre and are not numerous. Finally, after the second stage there is a prolonged (up to 4 weeks) period of maturation, during which density of free sarcoplasmic reticulum increases, triads acquire a location at the A-I junction and fibre type differences appear. We conclude that a system for calcium uptake, storage and release exists at the periphery of the myotube during early myogenesis. The complexity of the system and its ability to deliver calcium through the entire fibre develop in parallel to the formation of myofibrils.

Morphological changes in the triads and sarcoplasmic reticulum of rat slow and fast muscle fibres following denervation and immobilization

SummaryWe observed the morphological features of the membrane systems (sarcoplasmic reticulum, transverse tubules and triads) involved with the excitation-contraction coupling in rat soleus and extensor digitorum longus muscle following two disuse protocols: denervation and immobilization. The immobilized positions were: maximum dorsal flexor (soleus were stretched and extensor digitorum longus were shortened), maximum plantar flexor (soleus were shortened and extensor digitorum longus were stretched), and midway between the dorsal flexor and plantar flexor. The arrangement of the membrane systems was disordered following both disuse conditions. Increases in transverse tubule network were apparent; there were clearly more triads than in normal fibres, and pentadic and heptadic structures (i.e., a close approximation of two or three transverse tubule elements with three or four elements of terminal cisternae of sarcoplasmic reticulum) were frequently appeared following both denervation and immobilization. The most notable difference between the influence of denervation and immobilization on the membrane systems is the time at which the pentads and heptads appeared. They appeared much earlier (1 week after denervation) in denervated than in immobilized (3 or 4 weeks after immobilization) muscle fibres. On the other hand, the frequency of pentads and heptads is clearly related to the fibre type (significantly higher in extensor digitorum longus) and to extent of atrophy. The different influences of immobilization in each leg position suggest that disuse, but with neurotrophic factor(s), influences on the membrane systems were affected by sarcomere length, and the neurotrophic factor(s) and muscle activity were not always necessary to form mew membrane systems in disuse skeletal muscle fibres.

Plasticity of the transverse tubules following denervation and subsequent reinnervation in rat slow and fast muscle fibres

We have studied the effects of short term denervation followed by reinnervation on the ultrastructure of the membrane systems and on the content of and distribution of key proteins involved in calcium regulation of fast-twitch (FT) extensor digitorum longus (EDL) and slow-twitch (ST) soleus (SOL) muscle fibres. Ischiadic nerve freezing resulted in total lack of neuromuscular transmission for 3 days followed by a slow recovery, but no decline in twitch force elicited by direct stimulation. The latter measurements indicate no significant atrophy within this time frame. The membrane systems of skeletal muscle fibres were visualized using Ca2+-K3Fe(CN)6-OsO4 techniques and observed using a high voltage electron microscope. [3H]nitrendipine binding was used to detect levels of dihydropyridine receptor (DHPR) expression. The Ca2+ pumping free sarcoplasmic reticulum domains were not affected by the denervation, but the Ca2+ release domains were dramatically increased, particularly in the FT-EDL muscle fibres. The increase is evidenced by a doubling up of the areas of contacts between SR and transverse (t-) tubules, so that in place of the normal triadic arrangement, pentadic and heptadic junctions, formed by multiple interacting layers of ST and t-tubules are seen. Frequency of pentads and heptads increases and declines in parallel to the denervation and reinnervation but with a delay. Immunofluorecence and electron microscopy observations show presence of DHPR and ryanodine receptor clusters at pentads and heptads junctions. A significant (P > 0.01) positive correlation between the level of [3H]nitrendipine binding component and the frequency pentads and heptads was observed in both the FT-EDL and ST-SOL muscle fibres indicating that overexpression of DHPRs accompanies the build up extra junctional contacts. The results indicate that denervation reversibly affects the domains of the membrane systems involved in excitation-contraction coupling.

Differentiation of membrane systems during development of slow and fast skeletal muscle fibres in chicken

SummaryThe disposition of transverse (T) tubules, sarcoplasmic reticulum (SR) and T-SR junctions (triads) and the width of Z lines are matched to contractile properties in adult muscle fibres. We have studied the development of the membrane systems in the slow anterior (ALD) and the fast posterior (PLD) latissimus dorsi of the chicken in ovo (E14–E21) and after hatching (D1–D30). T tubules, SR, triads and Z lines were visualized using DiIC16[3] labelling for confocal microscopy and either Ca-osmium-ferrocyanide or standard procedures for electron microscopy. Anterior latissimus dorsi and PLD have similar, slow twitches in early development (E14–E16), but PLD suddenly becomes faster starting at E17–E18. We find that in coincidence with the differentiation of faster contraction properties (starting at E18–E19) density of triads is significantly higher and width of Z lines is narrower in PLD. The SR also begins to acquire fibre-type specific characteristics at this time. Early development of T tubules, on the other hand, is quite similar in the two muscles. Peripherally-located, longitudinally-oriented T tubules, and the first T networks crossing the fibre center appear earlier in ALD (E14–E15 and E16) than in PLD (E14–E16 and E17), but have similar dispositions. The final fibre-type specific distribution of T tubules is achieved after hatching. Some T tubules-rich fibres in the ALD, presumably future fast fibres, develop extensive T tubule networks at early stages. Location of triads at the Z line in pectoralis occurs in three steps: an initial location of longitudinally oriented triads at the A-I junction; a subsequent move to the Z lines and finally a rotation to a transverse orientation.

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

Excitation‐contraction coupling in skeletal muscle involves junctions (triads and dyads) between sarcoplasmic reticulum (SR) and transverse (T) ‐ tubules. Two proteins of the junctional SR, ryanodine receptors (RyRs) and triadin and one protein of T tubules, dihydropyridine receptors (DHPRs) are located at these junctions. We studied the targeting of DHPRs and triadin to T‐tubules and SR in skeletal muscles of dyspedic mouse embryos lacking RyR1. In normal differentiating muscle fibers DHPRs, triadin and RyRs are located in intensely immunolabeled foci that are randomly distributed across the fiber. Correlation with electron microscopy and with previous studies indicates that the foci represent the location of triads and dyads. In dyspedic fibers DHPRs and triadin antibodies stain internal foci of the two proteins; RyR antibodies are completely negative. The appearance and location of the foci in dyspedic fibers is similar to that of normal muscle, but their fluorescent intensity is weaker. The SR Ca‐ATPase has more diffuse distribution than triadin in both normal and dyspedic fibers. These observations indicate that an interaction with RyRs is not necessary for the appropriate targeting of DHPRs or triadin to junctional domains of T tubules and SR respectively. Dev Dyn 1999;214:372–380.