Toshiharu Oba

Further Characterization of the Type 3 Ryanodine Receptor (RyR3) Purified from Rabbit Diaphragm

We characterized type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm by immunoaffinity chromatography using a specific antibody. The purified receptor was free from 12-kDa FK506-binding protein, although it retained the ability to bind 12-kDa FK506-binding protein. Negatively stained images of RyR3 show a characteristic rectangular structure that was indistinguishable from RyR1. The location of the D2 segment, which exists uniquely in the RyR1 isoform, was determined as the region around domain 9 close to the corner of the square-shaped assembly, with use of D2-directed antibody as a probe. The RyR3 homotetramer had a single class of high affinity [3H]ryanodine-binding sites with a stoichiometry of 1 mol/mol. In planar lipid bilayers, RyR3 displayed cation channel activity that was modulated by several ligands including Ca2+, Mg2+, caffeine, and ATP, which is consistent with [3H]ryanodine binding activity. RyR3 showed a slightly larger unit conductance and a longer mean open time than RyR1. Whereas RyR1 showed two classes of channel activity with distinct open probabilities (P o), RyR3 displayed a homogeneous and steeply Ca2+-dependent activity withP o ∼1. RyR3 was more steeply affected in the channel activity by sulfhydryl-oxidizing and -reducing reagents than RyR1, suggesting that the channel activity of RyR3 may be transformed more precipitously by the redox state. This is also a likely explanation for the difference in the Ca2+ dependence of RyR3 between [3H]ryanodine binding and channel activity.

Sulfhydryls associated with H2O2-induced channel activation are on luminal side of ryanodine receptors.

The mechanism underlying H2O2-induced activation of frog skeletal muscle ryanodine receptors was studied using skinned fibers and by measuring single Ca2+-release channel current. Exposure of skinned fibers to 3-10 mM H2O2 elicited spontaneous contractures. H2O2 at 1 mM potentiated caffeine contracture. When the Ca2+-release channels were incorporated into lipid bilayers, open probability ( P o) and open time constants were increased on intraluminal addition of H2O2 in the presence of cis catalase, but unitary conductance and reversal potential were not affected. Exposure to cis H2O2 at 1.5 mM failed to activate the channel in the presence of trans catalase. Application of 1.5 mM H2O2 to the transside of a channel that had been oxidized by cis p-chloromercuriphenylsulfonic acid (pCMPS; 50 μM) still led to an increase in P o, comparable to that elicited by trans 1.5 mM H2O2 without pCMPS. Addition of cis pCMPS to channels that had been treated with or without trans H2O2 rapidly resulted in high P o followed by closure of the channel. These results suggest that oxidation of luminal sulfhydryls in the Ca2+-release channel may contribute to H2O2-induced channel activation and muscle contracture.

Modulation of the Ca2+ channel voltage sensor and excitation-contraction coupling by silver.

Ag+ (0.5-10 microM) is known to produce a transient contraction of intact frog skeletal muscle fibers followed by complete inhibition of excitation-contraction (E-C) coupling. We have carried out physiological and biochemical experiments to investigate the basis of this effect. Dihydropyridine (DHP) Ca2+ channel blockers, which inhibit the voltage sensor of the Ca2+ channel, completely inhibit Ag+ contractions. Removal of extracellular Ca2+, or blockade of Ca2+ entry with cadmium, does not inhibit Ag+ contractions. Activation of the Ca2+ channels voltage sensor with the Ca2+ channel agonists Bay K 8644 or with perchlorate, potentiates the Ag(+)-induced contraction. Ag+ binds to the partially purified rabbit skeletal muscle Ca2+ channel and inhibits DHP binding (IC50 = 1.1 microM) and sulfhydryl (SH) reactivity (IC50 = 0.11 microM) over the concentration range where it inhibits E-C coupling. Oxidation of free SH groups by H2O2 or their reaction with DTNB prevents Ag+ contractions, while DTT reduction of oxidized SH groups restores Ag+ contractions. These results suggest that Ag+ binds to critical SH groups on the DHP receptor Ca2+ channel, resulting in modification of the channels voltage sensor and the failure of E-C coupling.

Silver ion-induced tension development and membrane depolarization in frog skeletal muscle fibres

Silver ions elicit dose-dependently a transient contracture in single fibres of bull-frog toe muscle placed in 0-Ca2+, Cl−-free MOPS solution containing 3 mM Mg2+ and NO3−. To elucidate the mechanisms involved, changes in membrane potential and in tension development were continuously measured following exposure to Ag+. The effect of Ag+ on contraction in fibres in which the membrane had been depolarized by elevating the external K+ concentration was also examined. The major findings of this investigation are as follows. (1) The mechanical threshold was shifted towards more negative potentials by 5 mV (−51 to −56 mV), when Ca2+ and Cl− in the Ringers solution were replaced with Mg2+ and NO3−, respectively. (2) On the exposure of the fibres to 5 μM Ag+, the membrane potential decreased by 1.6 mV from −87.8 mV and tension was developed. (3) In fibres soaked in a solution containing 10 mM K+ (corresponding to a membrane potential of −69.5 mV), 5 μM Ag+ produced a large contracture similar to that seen in the control solution. (4) The Ag+-induced contracture was inactivated when more than 20 mM K+ was used. (5) The membrane depolarization evoked by either 20 or 50 μM Hg2+ did not produce contraction. (6) Muscle fibres which had been exposed to 20 μM Hg2+ for 5 min responded to 5 μM Ag+ by a transient tension development. These findings strongly suggest that Ag+-induced tension development is not associated with depolarization of the surface membrane but rather is caused by specific actions of Ag+ on membrane proteins in the T-tubules.

Role of Amino-terminal Half of the S4-S5 Linker in Type 1 Ryanodine Receptor (RyR1) Channel Gating

The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca2+ itself, i.e. Ca2+-induced Ca2+ release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr4825–Ser4829) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca2+ release, single-channel current recordings, and [3H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca2+ sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.

Electromechanical and morphological observations on single muscle fibers in developing dystrophic mouse

Abstract Electrophysiologic characteristics and ultrastructural features of single muscle fibers of the dystrophic mouse (C57BL/6J-dy2J) at various stages of disease were investigated and comparison with those of normal muscle. Resting membrane potentials of the extensor digitorum longus muscle fibers from both normal and dystrophic young mice (to 3 weeks) were between −90 and −40 mV. With advancing ages to 7 to 9 weeks, the membrane potentials of normal fibers increased and tended to be within a narrow range around the mean value (−74 mV), whereas those of dystrophic fibers remained at the same value as at 3 weeks (−63 mV), with large deviations. The twitch tension of single fibers induced by direct stimulation was drastically reduced in dystrophic muscle, approximately from one-half to one-third of what it had been in normal fibers. Three-dimensional structures of the T-system in normal muscle observed under a conventional electron microscope revealed the formation of a dense network of tubules along the A-I boundaries of the myofibrils. In dystrophic muscle, the network was poorly developed and the tubules looked disrupted, although the arrangement of myofilaments was preserved relatively well even in the advanced stages. It seems likely that the drastic reduction of contractile force in dystrophic muscle is due to the defect of the internal membrane system, rather than the degeneration of contractile elements.

Hypocalcemic Induced Increase in Creatine Kinase in Rats

Calcium plays an important role in various myopathies. We report on an animal model with increased plasma creatine kinase (CK) resulting from hypocalcemia that will provide clues for studying human hypocalcemic myopathy. Male Wistar rats were pair-fed either a control or a calcium- and vitamin D3-deficient diet for 1, 2, 3, 4, or 5-6 weeks after weaning (3 weeks old). In the deficient diet-fed rats, plasma creatine kinase was increased and was accompanied by marked hypocalcemia. The omission of calcium and vitamin D3 from the diet for 1 or 2 weeks was enough to cause increased plasma creatine kinase; the creatine kinase ratio of deficient diet-fed rats to controls was 4.84 (1,777 IU L(-1)/367 IU L(-1)), and the calcium ion ratio was 0.41 (1.8 mg dL(-1)/4.4 mg dL(-1)) after 2 weeks. These values returned to control levels on treatment of the rats with the control diet and 1alpha-OH-vitamin D3 for 1 week.

Involvement of thiols in the induction of inward current induced by silver in frog skeletal muscle membrane

Exposure of voltage-clamped frog skeletal muscle fibres to silver caused a maintained inward current which could be carried by Ca2+, Mg2+ or Na+. Inorganic Ca2+ channel blockers and dithiothreitol (SH reducing agent) diminished this current, but a Na+ channel blocker did not. Thus, silver activates the Ca2+ channel by acting on SH groups in a Ca2+ channel protein.

The effect of changing free Ca2+ on light diffraction intensity and correlation with tension development in skinned fibers of frog skeletal muscle

The relationship between the diffraction intensity change of the first order line and tension development was examined in mechanically skinned single fibers from the dorsal head of the semitendinosus of frogs. Passive stretch of the fibers resulted in an increase in intensity over the range of sarcomere lengths from 2.5 to 3.6 μm, indicating that the intensity is a function of sarcomere length. Activation of skinned fibers caused a decrease in the intensity, at all sarcomere lengths, where the thick and thin filaments overlapped. The magnitude of the intensity decrease and that of the tension development depended on the Ca2+ concentration in the medium. The drop of intensity-pCa and the tension-pCa curves showed a similarly steep S-shape within a range of 0.5 pCa unit, although the intensity-pCa curve shifted to the left; the pCa for 50% decrease in light signal was 6.48 and that for 50% tension development was 6.40. Caffeine (25 mM) added to the medium produced a decrease in the intensity of skinned fibers with the simultaneous development of tension, thereby indicating that caffeine induces a release of Ca2+ from the sarcoplasmic reticulum and disorder in the filaments ensues. Changes in diffraction intensity with electrical stimulation to the intact single fiber were similar, although a more striking summation was observed in the optical response, as compared to the tension development. These results suggest that tension development upon stimulation can be monitored by assessing the magnitude of diffraction intensity decrease in the first order line, except for some shift in the short fiber.

Similar inhibitory effects of dantrolene sodium on twitch tension and on silver ion-induced contracture in skeletal muscle

To determine the mechanism by which Ag+ induces a transient contracture in skeletal muscle, the effect of dantrolene sodium on the Ag+ contracture was examined and the findings compared with those for the twitch, tetanus and caffeine contracture. The inhibition of twitch by dantrolene was equivalent to that of the Ag+ contracture at concentrations of 1, 2 or 5 microM of dantrolene. The tetanus tension was slightly inhibited by dantrolene, but not the caffeine contracture. These observations suggest that the Ag+ contracture may be governed by the same mechanism as that involved in the development of twitch tension.