No abstract

To examine whether a capacitative Ca2+ entry pathway is present in skeletal muscle, thin muscle fibre bundles were isolated from extensor digitorum longus (EDL) muscle of adult mice, and isometric tension and fura‐2 signals were simultaneously measured. The sarcoplasmic reticulum (SR) in the muscle fibres was successfully depleted of Ca2+ by repetitive treatments with high‐K+ solutions, initially in the absence and then in the presence of a sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor. Depletion of the SR of Ca2+ enabled us for the first time to show convincingly that the vast majority of the voltage‐sensitive Ca2+ store overlaps the caffeine‐sensitive Ca2+ store in intact fibres from mouse EDL muscle. This conclusion was based on the observation that both high‐K+ solution and caffeine failed to cause a contracture in the depleted muscle fibres. The existence of a Ca2+ influx pathway active enough to refill the depleted SR within several minutes was shown in skeletal muscle fibres. Ca2+ entry was sensitive to Ni2+, but resistant to nifedipine and was suppressed by plasma membrane depolarisation. Evidence for store‐operated Ca2+ entry was provided by measurements of Mn2+ entry. Significant acceleration of Mn2+ entry was observed only when the SR was severely depleted of Ca2+. The Mn2+ influx, which was blocked by Ni2+ but not by nifedipine, was inwardly rectifying, as is the case with the Ca2+ entry. These results indicate that the store‐operated Ca2+ entry is similar to the Ca2+ release‐activated Ca2+ channel (CRAC) current described in other preparations.

Recent findings on the ryanodine receptor of vertebrates, a Ca-release channel protein for the caffeine- and ryanodine-sensitive Ca pools, are reviewed in this article. Three distinct genes, i.e., ryr1, ryr2, and ryr3, express different isoforms in specific locations: Ryr1 in skeletal muscle and Purkinje cells of cerebellum; Ryr2 in cardiac muscle and brain, especially cerebellum; Ryr3 in skeletal muscle of nonmammalian vertebrates, the corpus striatum, and limbic cortex of brain, smooth muscles, and the other cells in vertebrates. While only one isoform (Ryr1) is expressed in mammalian skeletal muscles, two isoforms (alpha- and beta-isoforms expressed by ryr1 and ryr3, respectively) are found in nonmammalian vertebrate skeletal muscles. Although the coexistence of two isoforms may merely be related to differentiation and specialization, the biological significance remains to be clarified. Ryanodine receptors in vertebrate skeletal muscles are believed to mediate two different modes of Ca release: Ca(2+)-induced Ca release and action potential-induced Ca release. All results obtained so far with any isoform of ryanodine receptor are related to Ca(2+)-induced Ca release and show very similar characteristics. Ca(2+)-induced Ca release, however, cannot be the underlying mechanism of Ca release on skeletal muscle activation. Susceptibility of the ryanodine receptor's ryanodine-binding activity to modification by physical factors, such as osmolality of the medium, might be related to action potential-induced Ca release. A hypothesis of molecular interaction in view of the plunger model of action potential-induced Ca release is discussed, suggesting that the model could be compatible with Ryr1 and Ryr3, but incompatible with Ryr2. The functional relevance of ryanodine receptor isoforms, especially Ryr3, in brain also remains to be clarified. Among ryr1 gene-related diseases, malignant hyperthermia was the first to be identified; however, there is still the possibility of involvement of the other genes. Central core disease has been added to the list recently. A molecular approach for the diagnosis and treatment of diseases is now in progress.

No abstract

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.

Physiological roles of the members of the synaptophysin family, carrying four transmembrane segments and being basically distributed on intracellular membranes including synaptic vesicles, have not been established yet. Recently, mitsugumin29 (MG29) was identified as a novel member of the synaptophysin family from skeletal muscle. MG29 is expressed in the junctional membrane complex between the cell surface transverse (T) tubule and the sarcoplasmic reticulum (SR), called the triad junction, where the depolarization signal is converted to Ca2+ release from the SR. In this study, we examined biological functions of MG29 by generating knockout mice. The MG29-deficient mice exhibited normal health and reproduction but were slightly reduced in body weight. Ultrastructural abnormalities of the membranes around the triad junction were detected in skeletal muscle from the mutant mice, i.e., swollen T tubules, irregular SR structures, and partial misformation of triad junctions. In the mutant muscle, apparently normal tetanus tension was observed, whereas twitch tension was significantly reduced. Moreover, the mutant muscle showed faster decrease of twitch tension under Ca2+-free conditions. The morphological and functional abnormalities of the mutant muscle seem to be related to each other and indicate that MG29 is essential for both refinement of the membrane structures and effective excitation-contraction coupling in the skeletal muscle triad junction. Our results further imply a role of MG29 as a synaptophysin family member in the accurate formation of junctional complexes between the cell surface and intracellular membranes.

The two ryanodine-binding proteins (RyBPs) have been purified from sarcoplasmic reticulum of bullfrog skeletal muscle by Mono Q column chromatography following solubilization of SR by CHAPS and sucrose density gradient centrifugation. We conclude that the two RyBPs (alpha- and beta-RyBP) are isoforms on the basis (i) that each RyBP is distinguished by a specific polyclonal antibody and (ii) that distinct polypeptides are generated by limited tryptic digestion of the two RyBPs. Monomeric molecular weights for alpha- and beta-RyBP are estimated to be (690 +/- 10) and (570 +/- 10) kDa, respectively, as determined from mobilities on disc SDS-PAGE using the Weber-Osborn buffer system without 6 M urea, which gives an estimate of (590 +/- 10) kDa for RyBP of rabbit skeletal muscle. Similar determination in the presence of 6 M urea gave 630 kDa for alpha-RyBP and unchanged estimates for the other RyBPs. Both RyBPs show [3H]ryanodine-binding activities which are activated by Ca2+, AMPOPCP, and caffeine, and inhibited by ruthenium red, MgCl2, and procaine. beta-RyBP, however, has higher affinity for Ca2+. In the presence of Ca2+ and AMPOPCP, both RyBPs show single homogeneous binding sites for [3H]ryanodine with Kd = 2-5 nM. The values of Bmax for alpha- and beta-RyBP were 320-340 and 320-375 pmol/mg protein, respectively. These results are consistent with the conclusion that a homo-tetramer of each RyBP binds one ryanodine molecule, taking account of the estimated molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)

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