Transmembrane orientation of the N-terminal and C-terminal ends of the ryanodine receptor in the sarcoplasmic reticulum of rabbit skeletal muscle.
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ABSTRACT:
SUBMITTER: Marty I
PROVIDER: S-EPMC1137923 | biostudies-other | 1994 Mar
REPOSITORIES: biostudies-other
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Project description:DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate), an anion channel blocker, triggers Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). The present study characterized the effects of DIDS on rabbit skeletal single Ca2+-release channel/RyR1 (ryanodine receptor type 1) incorporated into a planar lipid bilayer. When junctional SR vesicles were used for channel incorporation (native RyR1), DIDS increased the mean P(o) (open probability) of RyR1 without affecting unitary conductance when Cs+ was used as the charge carrier. Lifetime analysis of single RyR1 activities showed that 10 microM DIDS induced reversible long-lived open events (P(o)=0.451+/-0.038) in the presence of 10 microM Ca2+, due mainly to a new third component for both open and closed time constants. However, when purified RyR1 was examined in the same condition, 10 microM DIDS became considerably less potent (P(o)=0.206+/-0.025), although the caffeine response was similar between native and purified RyR1. Hence we postulated that a DIDS-binding protein, essential for the DIDS sensitivity of RyR1, was lost during RyR1 purification. DIDS-affinity column chromatography of solubilized junctional SR, and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis of the affinity-column-associated proteins, identified four major DIDS-binding proteins in the SR fraction. Among them, aldolase was the only protein that greatly potentiated DIDS sensitivity. The association between RyR1 and aldolase was further confirmed by co-immunoprecipitation and aldolase-affinity batch-column chromatography. Taken together, we conclude that aldolase is physically associated with RyR1 and could confer a considerable potentiation of the DIDS effect on RyR1.
| S-EPMC1609923 | biostudies-literature
Project description:Antibodies were raised against synthetic peptides corresponding to the N-terminal (residues 2-17) and C-terminal (residues 691-706) ends of rabbit skeletal muscle triadin, a 95 kDa protein located in the sarcoplasmic reticulum membrane at the triad junction. The specificity of the antibodies generated was tested by ELISA and Western blot analysis. These tests demonstrated the ability of the antibodies to react specifically with the proteins. The anti-N-terminus antibodies bound to sarcoplasmic reticulum vesicles, indicating that the N-terminal end of the membrane-embedded triadin is exposed on the cytoplasmic side of the vesicles. In contrast, the anti-C-terminus antibodies were able to react with sarcoplasmic reticulum vesicles only after permeabilization of the vesicles with a detergent, indicating that the C-terminal end is exposed on the luminal side of the vesicles. These immunological data were complemented by proteolysis experiments using carboxypeptidases and endoproteinase Arg C. A mixture of carboxypeptidases A, B and Y was used to induce degradation of the C-terminal end of triadin in sarcoplasmic reticulum vesicles. This degradation, and a concomitant loss of reactivity of the anti-C-terminus antibodies in Western blots, was observed only when the vesicles were permeabilized, providing further evidence for the luminal localization of the C-terminal end of triadin. Treatment of sarcoplasmic reticulum vesicles with endoproteinase Arg C resulted in the removal of the N-terminal end of triadin, probably due to cleavage after Arg-34. This is a further indication of the cytoplasmic localization of the N-terminal end of triadin (and of its first 34 amino acids). When the proteolysis with endoproteinase Arg C was carried out with permeabilized vesicles, the cleavage occurred after Arg-141 or Arg-157, indicating that at least one of these residues is luminal.
| S-EPMC1136716 | biostudies-other
Project description:Antibody was raised in chickens against purified sarcoplasmic-reticulum Ca2+-activated ATPase (Ca2+-ATPase). The immunological relationship between the Ca2+-ATPase of fast-muscle and slow-muscle sarcoplasmic reticulum was investigated by a one-step and a two-step competitive enzyme-linked immunosorbent assay (ELISA). The results show marked antigenic differences between the membrane-bound Ca2+-ATPase of the sarcoplasmic-reticulum vesicles from fast muscle and slow muscle, beside differences in the membrane content of ATPase protein.
| S-EPMC1163077 | biostudies-other
Project description:With the use of a [(3)H]ryanodine binding assay, the modulation of skeletal muscle ryanodine receptor (RyR1) by Zn(2+) was investigated. In the presence of 100 microM free Ca(2+) concentration ([Ca(2+)](f)) as activator, the equilibrium [(3)H]ryanodine binding to heavy sarcoplasmic reticulum vesicles was biphasically modulated by Zn(2+). The binding was increased by a free Zn(2+) concentration ([Zn(2+)](f)) of less than 1 microM; a peak binding, approx. 140% of the control (without added Zn(2+)) was obtained at 0.3 microM [Zn(2+)](f). An inhibitory effect of Zn(2+) became obvious with a [Zn(2+)](f) of more than 1 microM; the [Zn(2+)](f) for producing half inhibition was 2.7+/-0.5 microM (mean+/-S.D.). Scatchard analysis indicated that the increase in the binding induced by low [Zn(2+)](f) was due to a decrease in K(d), whereas both an increase in K(d) and a possible decrease in B(max) were responsible for the decrease in binding induced by high [Zn(2+)](f). The binding in the presence of micromolar [Zn(2+)](f) showed a biphasic time course. In the presence of 3 microM [Zn(2+)](f), after reaching a peak with an increased rate of initial binding, the binding gradually declined. The decline phase could be prevented by decreasing [Zn(2+)](f) to 0.5 microM or by adding 2 mM dithiothreitol, a thiol-reducing agent. The [Ca(2+)](f) dependence of binding was changed significantly by Zn(2+), whereas Ca(2+) had no clear effect on the [Zn(2+)](f) dependence of binding. Moreover, some interactions were found in the effects between Zn(2+) and other RyR1 modulators. It is indicated that Zn(2+) can modulate the activation sites and inactivation sites for Ca(2+) on RyR1. The physiological significance of the effects of Zn(2+) on ryanodine binding is discussed.
| S-EPMC1220757 | biostudies-other
Project description:TRPV1 represents a non-selective cation channel activated by capsaicin, acidosis and high temperature. In the central nervous system where TRPV1 is highly expressed, its physiological role in nociception is clearly identified. In skeletal muscle, TRPV1 appears implicated in energy metabolism and exercise endurance. However, how as a Ca(2+) channel, it contributes to intracellular calcium concentration ([Ca(2+)]i) maintenance and muscle contraction remains unknown. Here, as in rats, we report that TRPV1 is functionally expressed in mouse skeletal muscle. In contrast to earlier reports, our analysis show TRPV1 presence only at the sarcoplasmic reticulum (SR) membrane (preferably at the longitudinal part) in the proximity of SERCA1 pumps. Using intracellular Ca(2+) imaging, we directly accessed to the channel functionality in intact FDB mouse fibers. Capsaicin and resiniferatoxin, both agonists as well as high temperature (45°C) elicited an increase in [Ca(2+)]i. TRPV1-inhibition by capsazepine resulted in a strong inhibition of TRPV1-mediated functional responses and abolished channel activation. Blocking the SR release (with ryanodine or dantrolene) led to a reduced capsaicin-induced Ca(2+) elevation suggesting that TRPV1 may participate to a secondary SR Ca(2+) liberation of greater amplitude. In conclusion, our experiments point out that TRPV1 is a functional SR Ca(2+) leak channel and may crosstalk with RyR1 in adult mouse muscle fibers.
| S-EPMC3594164 | biostudies-literature
Project description:Skeletal muscle stores Ca²(+) in the sarcoplasmic reticulum (SR) and releases it to initiate contraction, but the concentration of luminal Ca²(+) in the SR ([Ca²(+)](SR)) and the amount that is released by physiological or pharmacological stimulation has been difficult to measure. Here we present a novel, yet simple and direct, method that provides the first quantitative estimates of static content and dynamic changes in [Ca²(+)](SR) in mammalian skeletal muscle, to our knowledge. The method uses fluo-5N loaded into the SR of single, mammalian skeletal muscle cells (murine flexor digitorum brevis myofibers) and confocal imaging to detect and calibrate the signals. Using this method, we have determined that [Ca²(+)](SR, free) is 390 ?M. 4-Chloro-m-cresol, an activator of the skeletal muscle ryanodine receptor, reduces [Ca²(+)](SR, free) to ?8 ?M, when values are corrected for background fluorescence from cytoplasmic pools of dye. Prolonged electrical stimulation (10 s) at 50 Hz releases 88% of the SR Ca²(+) content, whereas stimulation at 1 Hz (10 s) releases only 20%. Our results lay the foundation for molecular modeling of the dynamics of luminal SR Ca²(+) and for future studies of the role of SR Ca²(+) in healthy and diseased mammalian muscle.
| S-EPMC2955505 | biostudies-literature
Project description:Small ankyrin 1 (sAnk1; Ank1.5) is a ~20 kDa protein of striated muscle that concentrates in the network compartment of the sarcoplasmic reticulum (nSR). We used siRNA targeted to sAnk1 to assess its role in organizing the sarcoplasmic reticulum (SR) of skeletal myofibers in vitro. siRNA reduced sAnk1 mRNA and protein levels and disrupted the organization of the remaining sAnk1. Sarcomeric proteins were unchanged, but two other proteins of the nSR, SERCA and sarcolipin, decreased significantly in amount and segregated into distinct structures containing sarcolipin and sAnk1, and SERCA, respectively. Exogenous sAnk1 restored SERCA to its normal distribution. Ryanodine receptors and calsequestrin in the junctional SR, and L-type Ca(2+) channels in the transverse tubules were not reduced, although their striated organization was mildly altered. Consistent with the loss of SERCA, uptake and release of Ca(2+) were significantly inhibited. Our results show that sAnk1 stabilizes the nSR and that its absence causes the nSR to fragment into distinct membrane compartments.
| S-EPMC3215573 | biostudies-literature
Project description:Myosin VI (MVI) is a unique unconventional motor moving backwards on actin filaments. In non-muscle cells, it is involved in cell migration, endocytosis and intracellular trafficking, actin cytoskeleton dynamics, and possibly in gene transcription. An important role for MVI in striated muscle functioning was suggested in a report showing that a point mutation (H236R) within the MVI gene was associated with cardiomyopathy (Mohiddin et al., J Med Genet 41:309-314, 2004). Here, we have addressed MVI function in striated muscle by examining its expression and distribution in rat hindlimb skeletal muscle. We found that MVI was present predominantly at the muscle fiber periphery, and it was also localized within muscle nuclei. Analysis of both the hindlimb and cardiac muscle longitudinal sections revealed ~3 μm striation pattern, corresponding to the sarcoplasmic reticulum. Moreover, MVI was detected in the sarcoplasmic reticulum fractions isolated from skeletal and cardiac muscle. The protein also localized to the postsynaptic region of the neuromuscular junction. In denervated muscle, the defined MVI distribution pattern was abolished and accompanied by significant increase in its amount in the muscle fibers. In addition, we have identified several novel potential MVI-binding partners, which seem to aid our observations that in striated muscle MVI could be involved in postsynaptic trafficking as well as in maintenance of and/or transport within the sarcoplasmic reticulum and non-sarcomeric cytoskeleton.
| S-EPMC3656228 | biostudies-literature
Project description:Depolarization of skeletal muscle fibers induces sarcoplasmic reticulum (SR) Ca(2+) release and contraction that progressively decline while depolarization is maintained. Voltage-dependent inactivation of SR Ca(2+) release channels and SR Ca(2+) depletion are the two processes proposed to explain the decline of SR Ca(2+) release during long-lasting depolarizations. However, the relative contribution of these processes, especially under physiological conditions of activation, is not clearly established. Using Fura-2 and Fluo-5N to monitor cytosolic and SR Ca(2+) changes, respectively, in voltage-controlled mouse muscle fibers, we show that 2-min conditioning depolarizations reduce voltage-activated cytosolic Ca(2+) signals with a V1/2 of -53 mV but also induce SR Ca(2+) depletion that decreased the releasable pool of Ca(2+) with the same voltage sensitivity. In contrast, measurement of SR Ca(2+) changes indicated that SR Ca(2+) release channels were inactivated after SR had been depleted and in response to much higher depolarizations with a V1/2 of -13 mV. In response to trains of action potentials, cytosolic Ca(2+) signals decayed with time, whereas SR Ca(2+) changes remained stable over 1-min stimulation, demonstrating that SR Ca(2+) depletion is exclusively responsible for the decline of SR Ca(2+) release under physiological conditions of excitation. These results suggest that previous studies using steady-state inactivation protocols to investigate the voltage dependence of Ca(2+) release inactivation in fact probed the voltage dependence of SR Ca(2+) depletion, and that SR Ca(2+) depletion is the only process that leads to Ca(2+) release decline during continuous stimulation of skeletal muscle.
| S-EPMC3639577 | biostudies-literature
Project description:We have previously shown that inhibition of the spontaneous contractile activity of cultured embryonic-chick skeletal-muscle fibres with tetrodotoxin (TTX) leads to decreased sarcoplasmic-reticulum Ca(2+)-transport rates and steady-state concentrations of the high-energy Ca(2+)-ATPase phosphoenzyme intermediate [Charuk & Holland (1983) Exp. Cell Res. 144, 143-157]. In the present study we used a monoclonal antibody to the Ca(2+)-ATPase to show that there is a decreased amount of enzyme accumulated by contraction-inhibited myotubes. Indirect immunofluorescence microscopy using the monoclonal antibody to the Ca(2+)-ATPase also revealed a disordered subcellular organization of the sarcotubular system in contraction-inhibited myotubes. The biogenesis of sarcoplasmic-reticulum proteins in TTX-paralysed myofibres was studied by labelling cells with [35S]methionine before isolation of the active Ca(2+)-pump membrane fraction. Protein turnover was selectively increased in that fraction from TTX-treated muscle cultures. Electrophoretic analysis and quantitative fluorography confirmed that decreased accumulation of the Ca(2+)-ATPase enzyme in contraction-inhibited myotubes was associated with increased turnover of this protein. The present results demonstrate that biogenesis of the sarcoplasmic-reticulum Ca(2+)-ATPase is regulated by the contractile activity of skeletal-muscle fibres.
| S-EPMC1130792 | biostudies-other