Nagomi Kurebayashi

Depletion of Ca2+ in the sarcoplasmic reticulum stimulates Ca2+ entry into mouse skeletal muscle fibres

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.

Ryanodine receptor isoforms in excitation-contraction coupling.

Three genomically distinct isoforms of RyR are now known. RyR1 homologue is the primary isoform in skeletal muscles, whereas in cardiac muscles it is RyR2 homologue. RyR3 homologue occurs ubiquitously in many cells, but the biological function is little known, partly because of its minuscule amount in mammalian cells. The difference among RyR isoforms may not be so great in CICR activity, in other words, in the interaction of RyR isoforms with Ca2+, adenine nucleotides and caffeine. Species specificity among RyR1 homologues may be more important in the apparent difference between RyR1 and RyR3 homologues. CICR is likely to be the dominant underlying mechanism for E-C coupling in the cardiac muscle and probably in cells other than the skeletal muscle where the significance of CICR is controversial in physiological contraction. In E-C coupling of skeletal muscle (DICR), the reciprocal tight interactions between DHPR and RyR1 are critically required. The alpha 1 subunit of DHPR was only the main target of our current interests in the interaction with RyR1; the involvement of auxiliary subunits of alpha 2/delta and beta subunits and their mutual interactions, however, are also important. DICR and CICR in RyR1 share common properties of stimulation by concentrated solutes and modulation by luminal calcium or Ca2+, suggesting that the main difference between the two Ca2+ release mechanisms may be in the gating mechanism of the channel. Further investigations are required to understand molecular interactions during E-C coupling.

Discrimination of Ca2+-ATPase activity of the sarcoplasmic reticulum from actomyosin-type ATPase activity of myofibrils in skinned mammalian skeletal muscle fibres: distinct effects of cyclopiazonic acid on the two ATPase activities

SummaryWe have developed a procedure to discriminate actomyosin-type ATPase activity from Ca2+-ATPase activity of sarcoplasmic reticulum (SR) in mechanically skinned fibres, determining simultaneously their Ca2+-induced tension and accompanying ATPase activity. When they were treated with an alkaline CyDTA-containing solution of low ionic strength which was reported to remove troponin C, the fibres showed a considerable amount of Ca2+-dependent ATPase activity, in spite of having little or no Ca2+-induced isometric tension. The residual ATPase activity is ascribed to the Ca2+-ATPase activity of SR, because it is completely abolished by 1% CHAPS treatment for 10 min. This conclusion is also supported by the finding that the Ca2+-dependence of the ATPase activity is very similar to that of Ca2+-ATPase of SR isolated from rabbit skeletal muscle, and that the estimated activity is consistent with the reported values of direct determinations. On the other hand, treatment with a detergent such as CHAPS or Triton X-100 removes SR activities (ATPase and Ca-uptake), leaving Ca2+-induced tension and actomyosin-type ATPase activity unchanged. This procedure indicated that the contribution of Ca2+-ATPase activity of SR may be minimal in total steady-state ATPase activity of mechanically skinned mammalian skeletal muscle fibres. Successive CyDTA and CHAPS treatments eliminated both Ca2+-induced tension and ATPase activity, which were recovered by the addition of troponin C. Using these procedures, we also examined the effect of cyclopiazonic acid (CPA) which was reported to be a specific inhibitor of Ca2+-ATPase of SR. Ca2+-ATPase activity of SR in skinned fibres was inhibited completely by 10 μm CPA and held to one-half by about 0.2 μm. This effect was only partially reversible. CPA at 10μm or higher concentrations showed Ca2+-sensitizing action on myofibrils, which was readily reversible. CPA at 3μm inhibited almost completely the Ca2+-ATPase activity of SR, while it had no effect on either actomyosin-type ATPase or isometric tension of myofibrils.

Role of Mg2+ in Ca2+-Induced Ca2+ Release through Ryanodine Receptors of Frog Skeletal Muscle: Modulations by Adenine Nucleotides and Caffeine

Mg(2+) serves as a competitive antagonist against Ca(2+) in the high-affinity Ca(2+) activation site (A-site) and as an agonist of Ca(2+) in the low-affinity Ca(2+) inactivation site (I-site) of the ryanodine receptor (RyR), which mediates Ca(2+)-induced Ca(2+) release (CICR). This paper presents the quantitative determination of the affinities for Ca(2+) and Mg(2+) of A- and I-sites of RyR in frog skeletal muscles by measuring [(3)H]ryanodine binding to purified alpha- and beta-RyRs and CICR activity in skinned fibers. There was only a minor difference in affinity at most between alpha- and beta-RyRs. The A-site favored Ca(2+) 20- to 30-fold over Mg(2+), whereas the I-site was nonselective between the two cations. The RyR in situ showed fivefold higher affinities for Ca(2+) and Mg(2+) of both sites than the purified alpha- and beta-RyRs with unchanged cation selectivity. Adenine nucleotides, whose stimulating effect was found to be indistinguishable between free and complexed forms, did not alter the affinities for cations in either site, except for the increased maximum activity of RyR. Caffeine increased not only the affinity of the A-site for Ca(2+) alone, but also the maximum activity of RyR with otherwise minor changes. The results presented here suggest that the rate of CICR in frog skeletal muscles appears to be too low to explain the physiological Ca(2+) release, even though Mg(2+) inhibition disappears.

Putative Roles of Type 3 Ryanodine Receptor Isoforms (RyR3)

Ca(2+)-release from the sarcoplasmic or endoplasmic reticulum, the intracellular Ca(2+) store, is mediated by the ryanodine receptor (RyR) and/or the inositol trisphosphate receptor (IP3R). While IP3R is a ligand(IP3)-operated channel, RyR can be gated by a ligand (Ca(2+)) and/or mechanical coupling with the voltage sensor. There are three genetically distinct isoforms among RyR in mammals: RyR1-3. RyR1, the primary isoform in the skeletal muscle, can be gated by direct or indirect coupling with the conformation change of the alpha 1S subunit of dihydropyridine receptor (DHPR) on the T-tubules (transversely invaginated sarcolemma) upon depolarization of skeletal muscles or by the increased cytoplasmic Ca(2+) (Ca(2+)-induced Ca(2+) release, CICR). RyR2, the primary isoform in the cardiac ventricular muscle (and, in a lesser amount, the brain), can be gated by Ca(2+) which flows in through DHPR, especially the alpha1C subunit on depolarization. RyR3 is distributed ubiquitously in various tissues and may be coexpressed with RyR1 and RyR2. RyR3 is considered to be similar to RyR2 in the respect that it can be activated by Ca(2+), in view of the lack of available evidence to show the activation by the alpha1S subunit. Therefore, it is anticipated that RyR3 might take part through CICR in Ca(2+) signaling in smooth muscle and other non-muscle cells. To address the possible involvement of the CICR mechanism in the Ca(2+) signal transduction, it is critical to assess the effect of Mg(2+) on the CICR activity and the cytoplasmic concentration of Mg(2+). In this brief review, our discussion focuses on the effects of Ca(2+) and Mg(2+) on the activity of RyR3.

Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research

Background Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming. Methodology/Principal Findings Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8×His), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8×His and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry. Conclusions and Significance The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.

Increase by trifluoperazine in calcium sensitivity of myofibrils in a skinned fibre from frog skeletal muscle.

1. Since it has been demonstrated that trifluoperazine (TFP) increases the affinity for Ca2+ of troponin C as well as calmodulin, the effect of TFP was examined on the Ca2+‐induced tension in mechanically skinned fibres isolated from frog skeletal muscle and on Ca2+‐dependent ATPase activity of myofibrils from similar frog skeletal muscle. 2. Lower concentrations of TFP increased the Ca2+ sensitivity of myofibrils without a change in the maximum tension, giving rise to a less steep tension‐pCa relationship. This effect was reversible although thorough washes were necessary. The drug also enhanced myofibrillar ATPase activity, not only at low Ca2+ concentrations but also at saturating high Ca2+ concentrations. The increased affinity of troponin C for Ca2+ is difficult to accept as the sole explanation for the stimulatory effect of TFP. 3. Half of the maximum stimulating effect was obtained between 10 and 30 microM‐TFP, which is similar to the reported apparent inhibition constant (Ki) for calmodulin‐dependent enzyme reactions. However, the stimulating effect of TFP cannot be attributed to its inhibition of calmodulin because of the finding that this effect was independent of Ca2+. Earlier published results (e.g. Klee & Vanaman, 1982) also support this conclusion. 4. Studies on myofibrillar ATPase activity suggest that the stimulating effect of TFP is not identical in its underlying action with those of caffeine and quercetin, which are also known as Ca2+‐sensitizing drugs, having a similar eventual effect on tension development. 5. Higher concentrations of TFP decreased the maximum tension induced by high concentrations of Ca2+, while enhancing the tension in the presence of low concentrations of Ca2+. Analogous findings for ATPase activity were also made. TFP concentration for half the maximum depression was about 10 times higher than that for half the maximum stimulation. This suggests that different site(s) are involved in the stimulatory and inhibitory effects of TFP, although there may be some sites in common. 6. Discussion favours the stimulating effects of TFP as being caused considerably by the affected molecular interactions among myosin, actin, tropomyosin and troponin.

Two ryanodine receptor isoforms in nonmammalian vertebrate skeletal muscle: Possible roles in excitation–contraction coupling and other processes

The ryanodine receptor (RyR) is a Ca(2+) release channel in the sarcoplasmic reticulum in vertebrate skeletal muscle and plays an important role in excitation-contraction (E-C) coupling. Whereas mammalian skeletal muscle predominantly expresses a single RyR isoform, RyR1, skeletal muscle of many nonmammalian vertebrates expresses equal amounts of two distinct isoforms, α-RyR and β-RyR, which are homologues of mammalian RyR1 and RyR3, respectively. In this review we describe our current understanding of the functions of these two RyR isoforms in nonmammalian vertebrate skeletal muscle. The Ca(2+) release via the RyR channel can be gated by two distinct modes: depolarization-induced Ca(2+) release (DICR) and Ca(2+)-induced Ca(2+) release (CICR). In frog muscle, α-RyR acts as the DICR channel, whereas β-RyR as the CICR channel. However, several lines of evidence suggest that CICR by β-RyR may make only a minor contribution to Ca(2+) release during E-C coupling. Comparison of frog and mammalian RyR isoforms highlights the marked differences in the patterns of Ca(2+) release mediated by RyR1 and RyR3 homologues. Interestingly, common features in the Ca(2+) release patterns are noticed between β-RyR and RyR1. We will discuss possible roles and significance of the two RyR isoforms in E-C coupling and other processes in nonmammalian vertebrate skeletal muscle.

Effect of luminal calcium on Ca2+ release channel activity of sarcoplasmic reticulum in situ.

Ca2+ influx into empty SR in the absence of Ca2+ pump activity was determined in skinned frog skeletal muscle fibers and compared with Ca2+ efflux from loaded SR (i.e., Ca2+ release) to deepen our understanding of the properties of the Ca2+ release channel (CRC). Calcium content in SR increased approximately in a first-order kinetics and finally reached the equilibrium level determined by cytoplasmic Ca2+ ([Ca2+]c). Because AMP caused an increase in the rate of Ca2+ influx, and procaine, Mg2+, and high concentrations of Ca2+ caused a characteristic decrease, the major Ca2+ influx pathway was concluded to be the CRC, as is true of Ca2+ release. The apparent rate constant (k(app)) of Ca2+ efflux did not significantly change when the loading level was decreased to one-third. At a given [Ca2+]c, the same equilibrium level of calcium in SR was attained with a similar k(app) by both Ca2+ influx and Ca2+ efflux. The relationship between [Ca2+]c and calcium in SR indicated the Ca2+ binding sites in SR. These results, together with the anticipated effects of these Ca2+ buffer sites on kinetics, are consistent with the idea that luminal Ca2+ inhibits the CRC.

TRANSIENT KINETICS FOR Ca UPTAKE BY FRAGMENTED SARCOPLASMIC RETICULUM FROM BULLFROG SKELETAL MUSCLE WITH REFERENCE TO THE RATE OF RELAXATION OF LIVING MUSCLE

The transient kinetics of Ca uptake for fragmented sarcoplasmic reticulum from bullfrog skeletal muscle was determined spectrophotometrically in the presence of tetramethylmurexide as a Ca indicator by a stopped-flow method in view of understanding the time course of muscle relaxation in terms of the transient Ca uptake rate. The initial uptake rate depended on the concentration of MgATP, the pH of the medium, and the concentration of Mg 2+ . Under the optimum conditions for the initial uptake rate, i.e. pH 6.80, 1 mM Mg 2+ and 0.5 mM MgATP, the transient time course of Ca uptake was composed of two components of first-order kinetics: the rapid component of an apparent rate constant of 40–60 s −1 and the slow component of an apparent rate constant of 2–3 s −1 at 15°C in the presence of 0.35 mg protein/ml. High concentrations of MgATP, 0.5 mM or more, are required for the observation of the rapid component. Both apparent rate constants appear to be proportional to the amount of FSR in the medium. The ratio of the amplitude for the rapid component to that for the slow one was estimated to be 1:5–10. The temperature coefficient, Q 10 , for the initial uptake rate was 3.0, which corresponded to that for the relaxation rate of living muscle. The time course of muscle relaxation was discussed in reference with these observations, taking account of sarcoplasmic reticulum content (9mg protein/g muscle wet weight), and myoplasmic Ca 2+ concentration.