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Ca2+ release induced by myotoxin a, a radio-labellable probe having novel Ca2+ release properties in sarcoplasmic reticulum
British Journal of Pharmacology
Abstract
Myotoxin a (MYTX), a polypeptide toxin purified from the venom of prairie rattlesnakes ( Crotalus viridis viridis ) induced Ca ²⁺ release from the heavy fraction (HSR) but not the light fraction of skeletal sarcoplasmic reticulum at concentrations higher than 1 μ m , followed by spontaneous Ca ²⁺ reuptake by measuring extravesicular Ca ²⁺ concentrations using the Ca ²⁺ electrode.
The rate of ⁴⁵ Ca ²⁺ release from HSR vesicles was markedly accelerated by MYTX in a concentration‐dependent manner in the range of concentrations between 30 nM and 10 μ m , indicating the most potent Ca ²⁺ releaser in HSR.
The Ca ²⁺ dependency of MYTX‐induced ⁴⁵ Ca ²⁺ release has a bell‐shaped profile but it was quite different from that of caffeine, an inducer of Ca ²⁺ ‐induced Ca ²⁺ release.
⁴⁵ Ca ²⁺ release induced by MYTX was remarkable in the range of pCa between 8 and 3, whereas that by caffeine was prominent in the range of pCa, i.e., between 7 and 5.5.
MYTX‐induced ⁴⁵ Ca ²⁺ release consists of both early and late components. The early component caused by MYTX at low concentrations (30–300 nM) completed within 20 s, while the late component induced by it at higher concentrations (>0.3μ m ) was maintained for at least 1 min.
Both the components were almost completely inhibited by inhibitors of Ca ²⁺ release such as Mg ²⁺ , ruthenium red and spermine.
⁴⁵ Ca ²⁺ release induced by caffeine or β,γ‐methyleneadenosine 5′‐triphosphate (AMP‐PCP) was completely inhibited by high concentrations of procaine. Procaine abolished the early component but not the late one, suggesting that at least the early component is mediated through Ca ²⁺ ‐induced Ca ²⁺ release channels.
On the basis of these results, the character of Ca ²⁺ release induced by MYTX was quite different from that caused by caffeine or AMP‐PCP, suggesting that MYTX induces Ca ²⁺ release having novel properties in HSR. MYTX is the first polypeptide Ca ²⁺ inducer and has become a useful pharmacological tool for clarifying the mechanism of Ca ²⁺ release from skeletal muscle SR.
... Intramuscular injection of MYTX caused immediate and sustained local skeletal muscle contracture, followed by vacuolation of the SR after 12^24 h and degeneration of the myo¢brils without damage of the membranes of sarcolemma and SR after 48^72 h [18,20,21]. Recently, we found that MYTX strongly induced Ca 2 release from the heavy fraction of SR (HSR) containing RyR [22]. Although MYTX-induced Ca 2 release from HSR was completely inhibited by Mg 2 and ruthenium red, Ca 2 channel blockers [22], [ 125 I]MYTX binding to HSR was not a¡ected by these blockers [23]. ...
... Recently, we found that MYTX strongly induced Ca 2 release from the heavy fraction of SR (HSR) containing RyR [22]. Although MYTX-induced Ca 2 release from HSR was completely inhibited by Mg 2 and ruthenium red, Ca 2 channel blockers [22], [ 125 I]MYTX binding to HSR was not a¡ected by these blockers [23]. Since [ 125 I]MYTX did not bind to the puri¢ed RyR [24] but the MYTXinduced Ca 2 release was abolished by pretreatment with ryanodine [25], MYTX is assumed to bind to important regulatory proteins in Ca 2 release, which are distinct from the RyR. ...
... The 45 Ca 2 release from HSR passively preloaded with 45 Ca 2 was measured at 0³C as described previously [22]. After 12 h preincubation of HSR (20 mg/ml) at 0³C in a solution containing 5 mM 45 CaCl 2 , 90 mM KCl and 5 mM Tris-maleate (pH 7.0), HSR (5 Wl) was diluted with 500 Wl of an icecold reaction medium containing 500 WM CaCl 2 with various concentrations of EGTA, 90 mM KCl and 50 mM MOPS-Tris (pH 7.0) in the presence or absence of the test substance. ...
The molecular mechanism of Ca(2+) release by myotoxin a (MTYX), a polypeptide toxin isolated from the venom of prairie rattlesnakes (Crotalus viridis viridis), was investigated in the heavy fraction of sarcoplasmic reticulum (HSR) of rabbit skeletal muscles. [(125)I]MYTX bound to four HSR proteins (106, 74, 53 and 30 kDa) on polyvinylidene difluoride (PVDF) membrane. DIDS, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, bound predominantly to 30 kDa protein on the PVDF membrane, the molecular weight of which was similar to one of the MYTX binding proteins. The maximum (45)Ca(2+) release induced by caffeine (30 mM) was further increased in the presence of MYTX (10 microM) or DIDS (30 microM), whereas that induced by DIDS (30 microM) was not affected by MYTX (10 microM). MYTX inhibited [(3)H]DIDS binding to HSR in a concentration-dependent manner. Furthermore, [(125)I]MYTX binding to 30 kDa protein was inhibited by DIDS in a concentration-dependent manner. These results suggest that MYTX and DIDS release Ca(2+) from HSR in a common mechanism. The 30 kDa protein may be a target protein for the Ca(2+) releasing action of MYTX and DIDS.
... Da es sich hierbei um einen TTX-sensitiven Effekt zu handeln scheint, wird vermutet, dass diese Wirkungsweise von Crotamin über entsprechende Natriumkanäle der Plasmamembran vermittelt wird (Filho et al. 1978;Tsai et al. 1981;Hong et al. 1985;Brazil et al. 1993). (Furukawa et al. 1994;Ohkura et al. 1994;Ohkura et al. 1995). ...
... Da es sich hierbei um einen TTX-sensitiven Effekt zu handeln scheint, wird vermutet, dass diese Wirkungsweise von Crotamin über entsprechende Natriumkanäle der Plasmamembran vermittelt wird (Filho et al. 1978;Tsai et al. 1981;Hong et al. 1985;Brazil et al. 1993). (Furukawa et al. 1994;Ohkura et al. 1994;Ohkura et al. 1995). ...
Für das Verständnis der pharmakologischen Aktivität von Proteinen und Peptiden ist es wichtig, den Zusammenhang zwischen Molekülstruktur und Wirkungsweise zu untersuchen. Schlangengifte setzen sich aus einer Vielzahl von Komponenten zusammen, die in unterschiedlichster Weise den Organismus der Beutetiere beeinflussen und schädigen. Sie stellen daher eine reiche Quelle für Substanzen mit biologischer Wirksamkeit dar. Die Isolierung der einzelnen Komponenten bietet somit eine effektive Möglichkeit, pharmakologisch aktive Proteine und Peptide zu gewinnen. Der Fokus der vorliegenden Arbeit lag auf der Isolierung und Charakterisierung von Einzelkomponenten aus dem Gift der südamerikanischen Klapperschlange Crotalus durissus terrificus. Es konnten zwei der Hauptbestandteile, Crotamin, sowie eine Mischung an Crotoxin B Isoformen, mittels RP-HPLC isoliert und durch massenspektrometrische Untersuchungen identifiziert werden. Im Weiteren lag der Schwerpunkt auf der Charakterisierung der Interaktion von Crotamin mit künstlichen Membranen. Crotamin ist ein amphiphatisches, hoch basisches Polypeptid mit einer Größe von 4,9 kDa und stark positiver Oberflächenladung. Zunächst als Neurotoxin beschrieben, sind für Crotamin mittlerweile eine Vielzahl weiterer Wirkungen berichtet worden. Zu diesen gehören myotoxische, analgetische, antimikrobielle und antitumorale Effekte. Die Wirkungsweise beruht teilweise auf einer direkten Interaktion mit den Lipiden der Membran. Crotamin werden dabei sowohl membranbeeinflussende Eigenschaften, als auch die Fähigkeit zur Translokation in die Zelle zugeschrieben. Die vorliegende Arbeit zeigt deutlich den Einfluss der Lipidkomposition von Membranen auf die membranbeeinflussenden Eigenschaften von Crotamin. In Monolayern aus Asolektin führte die Anwesenheit von Cholesterin zu einem langsameren Einbau von Crotamin. Zudem verursachte sowohl die Anwesenheit bivalenter Kationen, als auch die Erhöhung des lateralen Drucks im Monolayer, einen beschleunigten Einbau von Crotamin. Des Weiteren konnte gezeigt werden, dass Crotamin die Fluidität von Vesikelmembranen verändert. Bei reinen Asolektinvesikeln führte Crotamin zu festeren, bei DOPC-Vesikeln zu fluideren Membranen. Bei Vesikeln, deren Membran zusätzlich Cholesterin enthielt, führte Crotamin unabhängig von der gewählten Grundsubstanz (Asolektin oder DOPC) zu fluideren Membranen. Das Ausmaß der Fluidisierung war jedoch abhängig von der jeweiligen Cholesterinkonzentration. Je mehr Cholesterin die Vesikelmembranen enthielten, desto geringer die Wirkung von Crotamin. Generell war hierbei der Einfluss der Cholesterinkonzentration bei asolektinhaltigen Vesikeln größer als bei DOPC-haltigen. Demnach wirkte Cholesterin den membranbeeinflussenden Eigenschaften von Crotamin entgegen. Vermutlich beruhte dies unter anderem auf der geringeren Fluidität von cholesterinhaltigen Membranen. Zudem könnte für die polaren Kopfgruppen der Lipide die Wahrscheinlichkeit zur Interaktion mit kationischen Molekülen wie Crotamin durch die Anwesenheit von Cholesterin verringert sein. Der stärkere Einfluss von Crotamin auf die Fluidität von asolektinhaltigen Membranen könnte auf den, in Asolektin enthaltenen, Lipiden mit negativer Ladung beruhen. Diese könnten, auf Grund einer negativeren Oberflächenladung der Membran, zu einer stärkeren Anziehung von Crotamin führen. Außerdem könnte Crotamin, durch die Interaktion mit negativ geladenen Lipiden, die Bildung von Domänen verursachen. Die Untersuchungen dieser Arbeit lassen somit den Schluss zu, dass sowohl Cholesterin als auch Lipide mit negativer Ladung die Wirkungsweise von Crotamin auf Membranen verändern. Zudem wird sie durch die Anwesenheit bivalenter Kationen in der umgebenden Lösung und dem lateralen Membrandruck beeinflusst. Darüber hinaus konnte gezeigt werden, dass Crotamin die Kalziumhomöostase von neuronalen Zellen beeinflusst. Hierbei verursachte Crotamin einen konzentrationsabhängigen Anstieg der intrazellulären freien Kalziumkonzentration.
... AVPIAQK is derived from the N-terminus of the second mitochondria-derived activator of caspase (Smac) protein. Induction of caspase-3 activity was observed after the treatment of HeLa cells with SmacN7-K (FITC)-CyLoP-1 in comparison with K(FITC)-CyLoP-1 or even with SmacN7-K(FITC)-Tat (49)(50)(51)(52)(53)(54)(55)(56)(57), which is derived from Tat, one of the best characterized and explored CPPs. CyLoP-1 or Smac-N7, as well as the Tat derivative peptide, failed to increase the caspase-3 activity alone. ...
... MYTX is closely related to crotamine and also demonstrates CPP activity [50]. Several studies carried out in the 1980s and 1990s using radiolabeled MYTX showed that it induces Ca 2? release from the sarcoplasmic reticulum (SR) of skeletal muscle cells [51][52][53], probably by binding to calsequestrin [54], ATP/ ADP translocase [55] and inhibiting Ca 2? -ATPase [56]. The attachment of MYTX to Ca 2? -ATPase is believed to cause uncoupling of the calcium pump [56]. ...
Toxins have been shown to have many biological functions and to constitute a rich source of drugs and biotechnological tools. We focus on toxins that not only have a specific activity, but also contain residues responsible for transmembrane penetration, which can be considered bioportides—a class of cell-penetrating peptides that are also intrinsically bioactive. Bioportides are potential tools in pharmacology and biotechnology as they help deliver substances and nanoparticles to intracellular targets. Bioportides characterized so far are peptides derived from human proteins, such as cytochrome c (CYCS), calcitonin receptor (camptide), and endothelial nitric oxide synthase (nosangiotide). However, toxins are usually disregarded as potential bioportides. In this review, we discuss the inclusion of some toxins and molecules derived thereof as a new class of bioportides based on structure activity relationship, minimization, and biological activity studies. The comparative analysis of the amino acid residue composition of toxin-derived bioportides and their short molecular variants is an innovative analytical strategy which allows us to understand natural toxin multifunctionality in vivo and plan novel pharmacological and biotechnological products. Furthermore, we discuss how many bioportide toxins have a rigid structure with amphiphilic properties important for both cell penetration and bioactivity.
... Several studies have been carried out evaluating the effect of crotamine or myotoxin-alpha on heavy sarcoplasmic reticulum preparations. These assays showed that these toxins promote calcium release from the sarcoplasmic reticulum via the opening of the ryanodine receptor (RyR), as assessed using several pharmacological agents [39][40][41]. It has been speculated that crotamine binds to proteins associated with the RyR, such as calsequestrin [42], because no direct binding to the RyR was observed [43]. ...
Crotamine is a rattlesnake-derived toxin that causes fast-twitch muscle paralysis. As a cell-penetrating polypeptide, crotamine has been investigated as an experimental anti-cancer and immunotherapeutic agent. We hypothesized that molecules targeting crotamine could be designed to study its function and intervene in its adverse activities. Here, we characterize synthetic crotamine and show that, like the venom-purified toxin, it induces hindlimb muscle paralysis by affecting muscle contraction and inhibits KCNA3 (Kv1.3) channels. Synthetic crotamine, labeled with a fluorophore, displayed cell penetration, subcellular myofiber distribution, ability to induce myonecrosis, and bind to DNA and heparin. Here, we used this functionally validated synthetic polypeptide to screen a combinatorial phage display library for crotamine-binding cyclic peptides. Selection for tryptophan-rich peptides was observed, binding of which to crotamine was confirmed by ELISA and gel shift assays. One of the peptides (CVWSFWGMYC), synthesized chemically, was shown to bind both synthetic and natural crotamine and to block crotamine-DNA binding. In summary, our study establishes a functional synthetic substitute to the venom-derived toxin and identifies peptides that could further be developed as probes to target crotamine.
Key messages
Synthetic crotamine was characterized as a functional substitute for venom-derived crotamine based on myotoxic effects.
A combinatorial peptide library was screened for crotamine-binding peptides.
Tryptophan-rich peptides were shown to bind to crotamine and interfere with its DNA binding.
Crotamine myofiber distribution and affinity for tryptophan-rich peptides provide insights on its mechanism of action.
... Although classi®ed as a myotoxin, crotamine does not act by disrupting the integrity of muscle cell plasma membrane. Instead, it induces a sodium in¯ux and contracture (Chang and Tseng, 1978), together with vacuolation and calcium release from sarcoplasmic reticulum (Cameron and Tu, 1978; Furukawa et al., 1994). Such pharmacological action is not expected to cause rapid plasma membrane rupture nor LDH release. ...
A rapid in vitro cytolytic effect of some myotoxic phospholipases A2 (PLA2s) isolated from the venoms of Viperidae snakes has been previously described. This study was undertaken to investigate if cytolytic activity is a common property of the myotoxic proteins from this group. Murine endothelial cells (tEnd) and skeletal muscle myotubes (C2C12) were utilized as targets. The release of lactic dehydrogenase was quantified as a measure of cell damage, 3 h after exposure of cells to the different PLA2s, including representatives from the genera Bothrops, Agkistrodon, Trimeresurus, Crotalus (family Viperidae), and Notechis (family Elapidae). All of the group II myotoxic PLA2s tested displayed rapid cytolytic activity when tested in the micromolar range of concentrations (8–32 μM). In contrast, the group I myotoxic PLA2 notexin was devoid of this activity. Aspartate-49 and lysine-49 PLA2 group II variants showed a comparable cytolytic effect. Skeletal muscle myotubes, obtained after fusion and differentiation of C2C12 myoblasts, were significantly more susceptible to the cytolytic action of myotoxins than endothelial cells, previously reported to be more susceptible than undifferentiated myoblasts under the same assay conditions. Cytolytic activity appears to be a common characteristic of group II myotoxic PLA2s of the Viperidae. Bee venom PLA2, a group III enzyme of known myotoxicity, also displayed cytotoxic activity on C2C12 myotubes, being devoid of activity on endothelial cells. These results suggest that in vitro differentiated skeletal muscle myotubes may represent a suitable model target for the study of myotoxic PLA2s of the structural group II found in snake venoms.
... Although specific for its binding site, the effect of ryanodine is largely dependent on the free Ca2+ concentration and the activity of muscle (Jenden & Fairhurst, 1969) making it difficult to study the effect of ryanodine in resting muscle. Xanthone and norathyriol represent novel chemicals with structures different from known Ca2+ release inducers (Palade, 1987b;Abramson et al., 1988;Valdivia et al., 1992;Beeler & Gable, 1993;Furukawa et al., 1994). The data from this study suggest that norathyriol may be a useful tool for studying the internal Ca21 pool of skeletal muscle in either isolated membrane vesicles or intact muscle. ...
. Effects of xanthone and its derivative, 1,3,6,7‐tetrahydroxyxanthone (norathyriol), on Ca ²⁺ release and ryanodine binding were studied in isolated sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle.
. Both xanthone and norathyriol dose‐dependently induced Ca ²⁺ release from the actively loaded SR vesicles which was blocked by ruthenium red, a specific Ca ²⁺ release inhibitor, and Mg ²⁺ .
. Xanthone and norathyriol also dose‐dependently increased apparent [ ³ H]‐ryanodine binding. Norathyriol, but not xanthone, produced a synergistic effect on binding activation when added concurrently with caffeine.
. In the presence of Mg ²⁺ , which inhibits ryanodine binding, both caffeine and norathyriol, but not xanthone, could restore the binding to the level observed in the absence of Mg ²⁺ .
. Xanthone activated the Ca ²⁺ ‐ATPase activity of isolated SR vesicles dose‐dependently reaching 70% activation at 300 μ m .
. When tested in mouse diaphragm, norathyriol potentiated the muscle contraction followed by twitch depression and contracture in either a Ca ²⁺ ‐free bathing solution or one containing 2.5 mM Ca ²⁺ . These norathyriol‐induced effects on muscle were inhibited by pretreatment with ruthenium red or ryanodine.
. These data suggest that xanthone and norathyriol can induce Ca ²⁺ release from the SR of skeletal muscle through a direct interaction with the Ca ²⁺ release channel, also known as the ryanodine receptor.
... Traditionally, venom from many poisonous snakes, lizards, and scorpions has been a rich source of peptide toxins that target various voltage-and ligand-gated channels including the CRCs (Furukawa et al., 1994;Morrissette et al., 1995Morrissette et al., , 1996. Recently, from the venom of the African scorpion Pandinus imperator, two factors, imperatoxin activator (IpTx a ) and imperatoxin inhibitor (IpTx i ), were isolated that selectively activated and inhibited, respectively, the CRCs (Valdivia et al., 1992;El-Hayek et al., 1995). ...
Single-channel and [3H]ryanodine binding experiments were carried out to examine the effects of imperatoxin activator (IpTxa), a 33 amino acid peptide isolated from the venom of the African scorpion Pandinus imperator, on rabbit skeletal and canine cardiac muscle Ca2+ release channels (CRCs). Single channel currents from purified CRCs incorporated into planar lipid bilayers were recorded in 250 mM KCl media. Addition of IpTxa in nanomolar concentration to the cytosolic (cis) side, but not to the lumenal (trans) side, induced substates in both ryanodine receptor isoforms. The substates displayed a slightly rectifying current-voltage relationship. The chord conductance at -40 mV was approximately 43% of the full conductance, whereas it was approximately 28% at a holding potential of +40 mV. The substate formation by IpTxa was voltage and concentration dependent. Analysis of voltage and concentration dependence and kinetics of substate formation suggested that IpTxa reversibly binds to the CRC at a single site in the voltage drop across the channel. The rate constant for IpTxa binding to the skeletal muscle CRC increased e-fold per +53 mV and the rate constant of dissociation decreased e-fold per +25 mV applied holding potential. The effective valence of the reaction leading to the substate was approximately 1.5. The IpTxa binding site was calculated to be located at approximately 23% of the voltage drop from the cytosolic side. IpTxa induced substates in the ryanodine-modified skeletal CRC and increased or reduced [3H]ryanodine binding to sarcoplasmic reticulum vesicles depending on the level of channel activation. These results suggest that IpTxa induces subconductance states in skeletal and cardiac muscle Ca2+ release channels by binding to a single, cytosolically accessible site different from the ryanodine binding site.
... Although classi®ed as a myotoxin, crotamine does not act by disrupting the integrity of muscle cell plasma membrane. Instead, it induces a sodium in¯ux and contracture (Chang and Tseng, 1978), together with vacuolation and calcium release from sarcoplasmic reticulum (Cameron and Tu, 1978; Furukawa et al., 1994). Such pharmacological action is not expected to cause rapid plasma membrane rupture nor LDH release. ...
A rapid in vitro cytolytic effect of some myotoxic phospholipases A2 (PLA2s) isolated from the venoms of Viperidae snakes has been previously described. This study was undertaken to investigate if cytolytic activity is a common property of the myotoxic proteins from this group. Murine endothelial cells (tEnd) and skeletal muscle myotubes (C2C12) were utilized as targets. The release of lactic dehydrogenase was quantified as a measure of cell damage, 3 h after exposure of cells to the different PLA2s, including representatives from the genera Bothrops, Agkistrodon, Trimeresurus, Crotalus (family Viperidae), and Notechis (family Elapidae). All of the group II myotoxic PLA2s tested displayed rapid cytolytic activity when tested in the micromolar range of concentrations (8-32 microM). In contrast, the group I myotoxic PLA2 notexin was devoid of this activity. Aspartate-49 and lysine-49 PLA2 group II variants showed a comparable cytolytic effect. Skeletal muscle myotubes, obtained after fusion and differentiation of C2C12 myoblasts, were significantly more susceptible to the cytolytic action of myotoxins than endothelial cells, previously reported to be more susceptible than undifferentiated myoblasts under the same assay conditions. Cytolytic activity appears to be a common characteristic of group II myotoxic PLA2s of the Viperidae. Bee venom PLA2, a group III enzyme of known myotoxicity, also displayed cytotoxic activity on C2C12 myotubes, being devoid of activity on endothelial cells. These results suggest that in vitro differentiated skeletal muscle myotubes may represent a suitable model target for the study of myotoxic PLA2s of the structural group II found in snake venoms.
... It is known to cause reduction in resting membrane potential and increase in membrane conductance by a tetrodotoxin-sensitive, i.e., Na + -channel mediated mechanism (Hong and Chang, 1985; Brazil and Fontana, 1993). It also induces Ca 2+ release from the heavy fraction of the sarcoplasmic reticulum, by interacting with calsequestrin (Furukawa et al., 1994; Ohkura et al., 1994 Ohkura et al., , 1995), resulting in swelling of the sarcoplasmic reticulum and necrosis of skeletal muscle. Cloning and nucleotide sequence of crotamine genes of C. durissus terri®cus has been reported by Smith and Schmidt (1990). ...
A cDNA phage library was constructed from venom glands of a single adult specimen of crotamine-plus Crotalus durissus terrificus (South American rattlesnake) captured in a known region. Fifteen crotamine positive clones were isolated using a PCR-based screening protocol and sequenced. These complete cDNAs clones were grouped for maximal alignment into six distinct nucleotide sequences. The crotamine cDNAs, with 340-360 bases, encompass open reading frame of 198 nucleotides with 5' and 3' untranslated regions of variable size, signal peptide sequence, one crotamine isoform message, and putative poly(A+) signal. Of these six different crotamine cDNA precursors, two predict the identical amino acid sequence previously described by Laure (1975), and the other four a crotamine isoform precursor where the Leucine residue at position 19 is replaced by isoleucine by a single base change. On the other hand, nucleotide variation was observed in the 5' and 3' untranslated regions, with one interesting variant containing an 18 base pair deletion at the 5' untranslated region which results in the usual ATG initiator being replaced by the rarely used GUG start codon. Comparison by Northern blot analysis of poly(A+) RNA from venom glands of a crotamine-plus specimen to total and poly(A+) RNA from a crotamine-minus snake indicated that crotamine transcripts were not expressed in the crotamine-minus specimen.
... After ultrafiltration, the sample was incubated in the SDS sample buffer overnight at 20 mC. SDS\PAGE was conducted as described by Laemmli [34]. After electrophoretic separation on 7.5 % resolving gel with a 4 % stacking gel, analysis of ["#&I- Tyr$]mastoparan binding to HSR proteins was performed by using an image analyser (Molecular Imager GS-363 ; Bio-Rad Laboratories). ...
The ryanodine receptor, a Ca(2+)-releasing channel in sarcoplasmic reticulum (SR), plays an important role in the excitation-contraction coupling of skeletal muscle. In a previous study [Hirata, Nakahata and Ohizumi (2000) Mol. Pharmacol. 57, 1235-1242], we reported that mastoparan caused Ca(2+) release through ryanodine receptor from the heavy fraction of SR (HSR) isolated from rabbit skeletal muscle, and that it specifically bound to a 97 kDa protein which was distinct from Ca(2+)-pump or triadin. The present study was undertaken to identify and characterize the 97 kDa mastoparan-binding protein. The 97 kDa protein was purified from solubilized HSR by DEAE-Sepharose column chromatography and preparative SDS/PAGE. The partial amino acid sequence of the purified 97 kDa protein was matched with that of glycogen phosphorylase (GP). The proteolytic cleavage pattern of the 97 kDa protein was identical with that of GP. Furthermore, [(125)I-Tyr(3)]mastoparan specifically bound to GP. Interestingly, mastoparan-induced Ca(2+) release was inhibited by exogenous addition of GP-a, and mastoparan dissociated GP from HSR. These results indicate that the 97 kDa mastoparan-binding protein is GP, which negatively regulates Ca(2+) release from HSR. There may be a functional cross-talk between Ca(2+) release from HSR and glycogenolysis for energy supply mediated through GP in skeletal muscles.
Numerous neurotoxins that alter Na⁺-channel function have been shown to be useful tools for characterizing Na⁺ channels. Polypeptide blockers of voltage-dependent K⁺ channels (dendrotoxins, etc.) and Ca²⁺-activated K⁺ channels (apamine, etc.) have been studied extensively by numerous investigators. Peptide toxins, calciseptine and ω-conotoxins have been attracting much attention as inhibitors of L-type and N-type Ca²⁺ channels, respectively, while ω-conotoxins-MVIIC and ω-agatoxin IVA have been used as new types of Ca²⁺-channel blockers. Ryanodine and bromoeudistomin D analogues have been extensively used to elucidate Ca²⁺-release-channel functions and to purify its target protein. Polypeptide toxins (myotoxin a, etc.) and macrolides (FK 506, etc.) are useful Ca²⁺ releasers with a novel mechanism, while natural products such as thapsigargin and gingerol have been used as modulators of Ca²⁺-pumping ATPase. Some modulators of the function of myosin (purealin, etc.) and actin (goniodomin A, etc.) have been demonstrated to be important chemical probes for understanding the physiological roles of the contractile proteins in structural changes and their interaction in muscle contraction. A large number of protein kinase inhibitors (staurosporine, etc.) and phosphatase inhibitors (okadaic acid, etc.) are widely used as first-choice reagents for studying protein phosphorylation. These natural products have become essential tools for studying the regulatory mechanism of cellular ion movements, muscle contraction and protein phosphorylation.
In both the heavy and light fractions of fragmented sarcoplasmic reticulum (SR) vesicles from the fast skeletal muscle, about 27 min after beginning the active Ca2+ uptake, the extravesicular Ca2+ concentration suddenly increased to reach a steady level (delayed Ca2+ release). Phosphatidylinositol 4,5-bisphosphate (PIP2) not only shortened the rime to delayed Ca2+ release but also induced prompt Ca2+ release from the heavy fraction of SR. Delayed Ca2+ release and prompt Ca2+ release stimulated by 100 mu M PIP2 were not modified by ruthenium red. PIP2 (>0.1 mu M) markedly accelerated the rate of Ca-45(2+) efflux from SR vesicles in a concentration-dependent manner. The PIP2-induced Ca-45(2+) efflux was potentiated by ruthenium red but profoundly inhibited by La3+. The concentration-response curve for Ca2+ or Mg2+ in PIP2-induced Ca-45(2+) release was clearly different from that in the Ca2+-induced Ca2+ release. PIP2 caused a concentration-dependent increase in Ca2+ release from SR of chemically skinned fibers from skeletal muscle. Furthermore, [H-3]ryanodine or [H-3]methyl-7-bromoeudistomin D (MBED) binding to SR was increased by PIP2 in a concentration-dependent manner. These observations present the first evidence that PIP2 most likely activates two types of SR Ca2+ release channels whose properties are entirely different from those of Ca2+-induced Ca2+ release channels (the ryanodine receptor 1).
The lectin himehabu lectin (HHL) has recently been isolated from crude venom of the snake Trimeresurus okinavensis.
Ca2+-electrode and fluorescent Ca2+-indicator experiments showed that HHL induced release of Ca2+ from the heavy fraction of skeletal muscle sarcoplasmic reticulum (HSR). The release of Ca2+ induced by caffeine from HSR was abolished by ryanodine, Mg2+ and ruthenium red, typical inhibitors of Ca2+-release channels, whereas that induced by HHL was only partially reduced by these inhibitors. HHL, unlike caffeine, had no effect on [3H]ryanodine binding to HSR.
These results suggest that HHL induces release of Ca2+ which is at least partially mediated through Ca2+-release channels with novel pharmacological properties.
Abstract Myotoxin a and a group of closely related, small, basic toxins cause myonecrotic destruction of muscle tissue upon envenomation. The sarcoplasmic reticulum swells and eventually breaks down to small vesicles. Degeneration of myofibrils and myofilaments ensues and loss of the classic striation pattern is apparent. These toxins exhibit high sequence similarity as well as sequence microheterogeneity. A conformational heterogeneity was recently discovered in myotoxin a. The existence of myotoxin a in two forms in equilibrium in solution hinders the generation of a well defined three-dimensional structure. The difference, if any, in the biological activity of the two forms has not been established yet. Recent biochemical studies indicate that myotoxin a is a potent Ca2+ releasing agent that binds to calsequestrin in the lumen of the sarcoplasmic reticulum.
Triadin, a calsequestrin-anchoring transmembrane protein of the sarcoplasmic reticulum (SR), was successfully purified from the heavy fraction of SR (HSR) of rabbit skeletal muscle with an anti-triadin immunoaffinity column. Since depletion of triadin from solubilized HSR with the column increased the [3H]ryanodine binding activity, we tested a possibility of triadin for a negative regulator of the ryanodine receptor/Ca2+ release channel (RyR). Purified triadin not only inhibited [3H]ryanodine binding to the solubilized HSR but also reduced openings of purified RyR incorporated into the planar lipid bilayers. On the other hand, calsequestrin, an endogenous activator of RyR [Kawasaki and Kasai (1994) Biochem. Biophys. Res. Commun. 199, 1120−1127; Ohkura et al. (1995) Can. J. Physiol. Pharmacol. 73, 1181−1185] potentiated [3H]ryanodine binding to the solubilized HSR. Ca2+ dependency of [3H]ryanodine binding to the solubilized HSR was reduced by triadin, whereas that was enhanced by calsequestrin. Interestingly, [3H]ryanodine binding to the solubilized HSR potentiated by calsequestrin was reduced by triadin. Immunostaining with anti-triadin antibody proved that calsequestrin inhibited the formation of oligomeric structure of triadin. These results suggest that triadin inhibits the RyR activity and that RyR is regulated by both triadin and calsequestrin, probably through an interaction between them. In this paper, triadin has been first demonstrated to have an inhibitory role in the regulatory mechanism of the RyR.
[3H]9-Methyl-7-bromoeudistomin D ([3H]MBED), the most powerful Ca2+ releaser from sarcoplasmic reticulum, specifically bound to the brain microsomes. Caffeine competitively inhibited [3H]MBED binding. [3H]MBED binding was markedly blocked by procaine, whereas that was enhanced by adenosine-5'-(beta,gamma-methylene)triphosphate. The Bmax value was 170 times more than that of [3H]ryanodine binding. The profile of sucrose-density gradient centrifugation of solubilized microsomes indicated that [3H]MBED binding protein was different from [3H]ryanodine binding protein. These results suggest that there are MBED/caffeine-binding sites in brain that are distinct from the ryanodine receptor and that MBED becomes an essential molecular probe for characterizing caffeine-binding protein in the central nervous system.
Myotoxin alpha (MYTX), a polypeptide toxin purified from the venom of prairie rattlesnakes (Crotalus viridis viridis), induced Ca2+ release from the heavy fraction of skeletal sarcoplasmic reticulum (HSR), using a Ca2+ electrode. The effect of MYTX was nearly abolished by pretreatment with ryanodine, an alkaloid-based Ca2+ channel blocker. In the stopped-flow experiments, MYTX increased the choline+ permeability of HSR in the presence of calsequestrin (CS). Single channel recording experiments showed that in the presence of CS, the channel currents were markedly enhanced by MYTX applied to the cis side, but not to the trans side. However, in the absence of CS, MYTX failed to cause the excitatory effect in both the experiments. These results suggest that CS is essential for MYTX-induced Ca2+ release through the Ca2+ release channels in skeletal HSR.
Quinolidomicin A1, a 60-membered macrolide purified from an actinomycete Micromonospora sp. markedly induced 45Ca2+ release from the heavy fraction of skeletal muscle sarcoplasmic reticulum (HSR), but induced only slightly from the light fraction of sarcoplasmic reticulum (LSR), showing a lack of the ionophoretic activity even at a high concentration (300 microM). This was also confirmed by measuring the 45Ca2+ transport activity of quinolidomicin A1 across an organic solvent barrier. Quinolidomicin A1 (3-300 microM) increased 45Ca2+ release from HSR with an EC50 value of approx. 20 microM. The potency of quinolidomicin A1 was approx. 100-fold higher than that of caffeine. The bell-shaped profile of Ca2+ dependence for quinolidomicin A1 was different from that for caffeine. Blockers of Ca2+ release channels such as Mg2+ (10 mM), procaine (10 mM) and ruthenium red (10 microM) partially blocked quinolidomicin A1 (30 microM)-induced 45Ca2+ release from HSR. At 0 degrees C, quinolidomicin A1-induced 45Ca2+ release was ascertained not to be due to the inhibition of Ca2+ ATPase by the ATPase assay. Quinolidomicin A1 potentiated [3H]ryanodine binding to HSR with a decrease in KD but without a change in Bmax. These results suggest that quinolidomicin A1-induced Ca2+ release from HSR is consisted of two components, which are both sensitive and insensitive to blockers of Ca2+ release channels, and that the former component is associated with the ryanodine receptor.
Myotoxin a, from the venom of the prairie rattlesnake, Crotalus viridis viridis, exists as a temperature-dependent equilibrium of two interconverting forms. Reverse-phase high-performance liquid chromatography (RP-HPLC) shows that the two forms interconvert slowly enough at 25 degrees C to be seen as two separate peaks with a molar ratio of c. 1:4. Each peak can be isolated and individually injected to give the same two peaks in the same ratio of areas. The two peaks merge during chromatography at elevated temperatures, indicating an increase in the rate of interconversion. At low temperature, c. 5 degrees C, the individual peaks can be isolated and maintained for several days without reaching equilibrium. Mass analysis by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry shows that myotoxin a is present in both RP-HPLC peaks, suggesting that the two resolved forms are conformational isomers. Capillary zone electrophoresis (CZE) also shows two resolved, but interconvertible peaks over a range of pH values. Furthermore, RP-HPLC chromatograms of myotoxin a at concentrations from 0.013 mM to 0.41 mM maintain a consistent ratio of peak areas, without evidence of dimerization. Two-dimensional 1H-NMR nuclear Overhauser enhancement spectroscopy indicates the presence of a cis-proline peptide bond, consistent with an equilibrium mixture of cis-trans isomers; however, addition of peptidyl-prolyl cis-trans isomerase (PPI) does not enhance the rate of equilibration of the RP-HPLC peaks isolated at c. 5 degrees C.
The goal of this review has been to describe the current state of the pharmacology of ryanodine and related compounds relative to the vertebrate RyRs. Resolution of questions concerning the molecular properties of RyR channel function and the contributions made by the RyR isoforms to cellular signaling in a variety of tissues will require the production of new pharmacological agents directed against these proteins. Novel naturally occurring ryanodine congeners have been identified, and significant advances have been made in developing chemical approaches that permit the structure of ryanodine to be derivatized in selective ways. Moreover, several of these changes have yielded compounds that differ in their binding affinities and in their abilities to modify the properties of the RyR channels. These advances give substance to the possibility of designing the required pharmacological agents based on rational design changes of the structure ryanodine.
Numerous neurotoxins that alter Na(+)-channel function have been shown to be useful tools for characterizing Na+ channels. Polypeptide blockers of voltage-dependent K+ channels (dendrotoxins, etc.) and Ca(2+)-activated K+ channels (apamine, etc.) have been studied extensively by numerous investigators. Peptide toxins, calciseptine and omega-conotoxins have been attracting much attention as inhibitors of L-type and N-type Ca2+ channels, respectively, while omega-conotoxins-MVIIC and omega-agatoxin IVA have been used as new types of Ca(2+)-channel blockers. Ryanodine and bromoeudistomin D analogues have been extensively used to elucidate Ca(2+)-release-channel functions and to purify its target protein. Polypeptide toxins (myotoxin alpha, etc.) and macrolides (FK 506, etc.) are useful Ca2+ releasers with a novel mechanism, while natural products such as thapsigargin and gingerol have been used as modulators of Ca(2+)-pumping ATPase. Some modulators of the function of myosin (purealin, etc.) and actin (goniodomin A, etc.) have been demonstrated to be important chemical probes for understanding the physiological roles of the contractile proteins in structural changes and their interaction in muscle contraction. A large number of protein kinase inhibitors (staurosporine, etc.) and phosphatase inhibitors (okadaic acid, etc.) are widely used as first-choice reagents for studying protein phosphorylation. These natural products have become essential tools for studying the regulatory mechanism of cellular ion movements, muscle contraction and protein phosphorylation.
Visceral smooth muscle cells (VSMC) play an essential role, through changes in their contraction-relaxation cycle, in the maintenance of homeostasis in biological systems. The features of these cells differ markedly by tissue and by species; moreover, there are often regional differences within a given tissue. The biophysical features used to investigate ion channels in VSMC have progressed from the original extracellular recording methods (large electrode, single or double sucrose gap methods), to the intracellular (microelectrode) recording method, and then to methods for recording from membrane fractions (patch-clamp, including cell-attached patch-clamp, methods). Remarkable advances are now being made thanks to the application of these more modern biophysical procedures and to the development of techniques in molecular biology. Even so, we still have much to learn about the physiological features of these channels and about their contribution to the activity of both cell and tissue. In this review, we take a detailed look at ion channels in VSMC and at receptor-operated ion channels in particular; we look at their interaction with the contraction-relaxation cycle in individual VSMC and especially at the way in which their activity is related to Ca2+ movements and Ca2+ homeostasis in the cell. In sections II and III, we discuss research findings mainly derived from the use of the microelectrode, although we also introduce work done using the patch-clamp procedure. These sections cover work on the electrical activity of VSMC membranes (sect. II) and on neuromuscular transmission (sect. III). In sections IV and V, we discuss work done, using the patch-clamp procedure, on individual ion channels (Na+, Ca2+, K+, and Cl-; sect. IV) and on various types of receptor-operated ion channels (with or without coupled GTP-binding proteins and voltage dependent and independent; sect. V). In sect. VI, we look at work done on the role of Ca2+ in VSMC using the patch-clamp procedure, biochemical procedures, measurements of Ca2+ transients, and Ca2+ sensitivity of contractile proteins of VSMC. We discuss the way in which Ca2+ mobilization occurs after membrane activation (Ca2+ influx and efflux through the surface membrane, Ca2+ release from and uptake into the sarcoplasmic reticulum, and dynamic changes in Ca2+ within the cytosol). In this article, we make only limited reference to vascular smooth muscle research, since we reviewed the features of ion channels in vascular tissues only recently.
Myotoxin a, a small basic polypeptide from prairie rattlesnakes (Crotalus viridis viridis), induces myonecrosis and binds to a single class of binding sites in skeletal muscle sarcoplasmic reticulum. In the present study, [125I]myotoxin a with a high specific activity was prepared and it was shown to bind mainly to microsomes in rat whole brain. [125I]Myotoxin a was further shown to bind to microsomes prepared from all regions tested in brain. Its specific binding to whole brain microsomes was of approximately 1.9 times lower affinity (KD = 0.76 microM; Bmax = 13.1 nmol/mg) than that to skeletal muscle sarcoplasmic reticulum. [125I]Myotoxin a binding to brain microsomes was displaced by unlabeled myotoxin a with an IC50 value of 4.5 microM. [125I]Myotoxin a binding was markedly reduced by treatment of microsomes with trypsin, suggesting that the binding site of [125I]myotoxin a is partially proteins. The binding was significantly inhibited by Mg2+ at concentrations above 1 mM. Having looked at several drugs, we noted that [125I]myotoxin a binding was noncompetitively inhibited by spermine, whereas it was enhanced by heparin. On the other hand, the i.c.v. injection of myotoxin a in mice induced potent convulsive effects at 0.05 nmol/mouse or more. This paper is the first to show that the specific binding site of myotoxin a is present in mouse brain and that myotoxin a is a novel peptidic convulsant in mice.
A peptide toxin isolated from the Chinese scorpion Buthus martensi Karsch (BmK-PL) stimulated Ca2+-release channel activity in both triad membranes and reconstituted ryanodine receptors partially purified from rabbit skeletal muscle. In [3H]ryanodine binding experiments, the toxin increased the affinity of ryanodine for the receptor, from a Kd of 24.3 nM to 2.9 nM, which is an enhancement similar to that seen with known receptor activators, such as ATP and high concentrations of KCl. In contrast, toxin enhancement was not observed with purified receptors, although intrinsic binding activity and stimulation by the conventional receptor activators were retained. In single channel recordings of Ca2+-release activity, the toxin increased the open channel probability (Po) from 0.019 to 0.043 (226% of control) in triad preparations. Further toxin enhancement of Po from 0.07 to 0.37 (529% of control) was observed using partially-purified receptors in the presence of ATP. When purified receptors were assayed in the presence of ATP, however, they showed a high value of Po (0.33) and no further increase was observed following application of the toxin. Results derived from two different experimental methods consistently suggest that a molecule(s) required for toxin-induced enhancement is absent from the purified receptor preparation. Western blot analysis of receptors prepared using three different protocols showed that triadin was missing from the purified receptor preparation. The scorpion toxin minimally enhanced Ca2+-release channel activity of cardiac preparations. From these results, we conclude that the toxin preferentially increases the activity of skeletal-muscle ryanodine receptors by an indirect mechanism, possibly binding to associated protein molecule(s). Triadin is a strong candidate for such a molecule.
Amentoflavone, like caffeine, caused a concentration-dependent increase in Ca2+ release from the heavy fraction of fragmented sarcoplasmic reticulum of rabbit skeletal muscle. The Ca2+ -releasing activity of amentoflavone was approximately 20 times more potent than that of caffeine. The bell-shaped profile of Ca2+ dependence for amentoflavone was almost the same as that for caffeine. Typical blockers of Ca2+ -induced Ca2+ release channels, such as Mg2+, procaine and ruthenium red, inhibited markedly amentoflavone- and caffeine-induced 45Ca2+ release. The maximum 45Ca2+ release in response to amentoflavone was not changed by caffeine, but was further increased by adenosine-5'-(beta,gamma-methylene) triphosphate. This compound enhanced [3H]ryanodine binding to the heavy fraction of fragmented sarcoplasmic reticulum with a decrease in K(D) but without a change in Bmax. These results suggest that amentoflavone, which does not contain a nitrogen atom, probably binds to the caffeine-binding site in Ca2+ channels and thus potentiates Ca2+ -induced Ca2+ release from the heavy fraction of fragmented sarcoplasmic reticulum.
In both the heavy and light fractions of fragmented sarcoplasmic reticulum (SR) vesicles from the fast skeletal muscle, about 27 min after beginning the active Ca2+ uptake, the extravesicular Ca2+ concentration suddenly increased to reach a steady level (delayed Ca2+ release). Phosphatidylinositol 4,5-bisphosphate (PIP2) not only shortened the time to delayed Ca2+ release but also induced prompt Ca2+ release from the heavy fraction of SR. Delayed Ca2+ release and prompt Ca2+ release stimulated by 100 microM PIP2 were not modified by ruthenium red. PIP2 (>0.1 microM) markedly accelerated the rate of 45Ca2+ efflux from SR vesicles in a concentration-dependent manner. The PIP(2)-induced 45Ca2+ efflux was potentiated by ruthenium red but profoundly inhibited by La3+. The concentration-response curve for Ca2+ or Mg2+ in PIP2-induced 45Ca2+ release was clearly different from that in the Ca(2+)-induced Ca2+ release. PIP2 caused a concentration-dependent increase in Ca2+ release from SR of chemically skinned fibers from skeletal muscle. Furthermore, [3H]ryanodine or [3H]methyl-7-bromoeudistomin D (MBED) binding to SR was increased by PIP2 in a concentration-dependent manner. These observations present the first evidence that PIP2 most likely activates two types of SR Ca2+ release channels whose properties are entirely different from those of Ca(2+)-induced Ca2+ release channels (the ryanodine receptor 1).
Xestoquinone (XQN) (3 x 10(-7) to 3 x 10(-3) M), isolated from the sea sponge Xestospongia sapra, induced a concentration-dependent Ca(2+) release from the heavy fraction of fragmented sarcoplasmic reticulum (HSR) of rabbit skeletal muscle with an EC(50) value of approximately 30 microM. On the basis of the EC(50), XQN is 10 times more potent than caffeine. Dithiothreitol completely blocked XQN-induced Ca(2+) release from HSR without affecting that induced by caffeine. Caffeine-induced Ca(2+) release was reduced markedly by Mg(2+), procaine, and ruthenium red, agents that are known to block release of Ca(2+) from sarcoplasmic reticulum, whereas that induced by XQN was not inhibited. The bell-shaped profile of Ca(2+) dependence for XQN was significantly shifted upward in a wider range of pCa (between 7 and 3), whereas that for caffeine was shifted to the left in a narrower range of pCa (between 8 and 7). The maximum response to caffeine in (45)Ca(2+) release was not affected by 9-methyl-7-bromoeudistomin D, whereas the response was further increased by XQN. XQN caused a concentration-dependent decrease in [(3)H]ryanodine binding to HSR. This effect of XQN also was abolished in the presence of dithiothreitol. Scatchard analysis revealed that the mode of inhibition by XQN was noncompetitive in [(3)H]ryanodine binding to HSR. These results indicate that sulfhydryl groups are involved in both the XQN effect on ryanodine binding and on Ca(2+) release.
Bisprasin, a unique bromotyrosine derivative containing a disulfide linkage, was isolated from a marine sponge of Dysidea spp. This compound caused a concentration-dependent (from 10 to 30 microM) increase in the (45)Ca(2+) release from the heavy fraction of skeletal muscle sarcoplasmic reticulum (HSR) of rabbit skeletal muscle in the same way as does caffeine. The 50% effective concentrations of bisprasin and caffeine were approximately 18 microM and 1.2 mM, respectively, indicating that the (45)Ca(2+)-releasing activity of bisprasin was approximately 70 times more potent than that of caffeine in HSR. The bell-shaped profile of Ca(2+) dependence for bisprasin was almost the same as that for caffeine. Typical blockers of Ca(2+)-induced Ca(2+) release channels, such as Mg(2+), procaine, and ruthenium red, inhibited markedly bisprasin- and caffeine-induced (45)Ca(2+) release from HSR. This compound, like caffeine, significantly enhanced [(3)H]ryanodine binding to HSR. Scatchard analysis of [(3)H]ryanodine binding to HSR revealed that bisprasin and caffeine decreased the K(D) value without affecting the B(max) value, suggesting that both the drugs facilitate the opening of ryanodine receptor channels. The bisprasin- and caffeine-induced increases in [(3)H]ryanodine binding were further enhanced by adenosine-5'-(beta, gamma-methylene)triphosphate. These results suggest that the pharmacological properties of bisprasin are almost similar to those of caffeine, except for its 70-fold higher potency. Here, we present the first report on the pharmacological properties of bisprasin, which, like caffeine, induces Ca(2+) release from skeletal muscle SR mediated through the ryanodine receptor.
Mastoparan (MP) and radiolabeled [Tyr(3)]MP caused a transient Ca(2+) release from the heavy fraction of sarcoplasmic reticulum, which was inhibited by ryanodine. MP enhanced [(3)H]ryanodine binding in a concentration-dependent manner with an EC(50) value of approximately 0.3 microM. The (45)Ca(2+) release was accelerated by MP, [Tyr(3)]MP, or caffeine in a concentration-dependent manner. The EC(50) values for MP, [Tyr(3)]MP, and caffeine were approximately 2. 0 microM, 7.7 microM, and 1.8 mM, respectively. MP, like caffeine, shifted the stimulatory limb of a bell-shaped curve of Ca(2+) dependence to the left. (45)Ca(2+) release induced by caffeine was completely inhibited by typical blockers of Ca(2+)-induced Ca(2+) release, such as Mg(2+), ruthenium red, or procaine. However, (45)Ca(2+) release induced by MP was completely inhibited by Mg(2+), but it was only partially inhibited by ruthenium red or procaine. The rate of (45)Ca(2+) release induced by MP was further increased in the presence of caffeine, showing that the MP binding site is different from that of caffeine on Ca(2+) release channels. We succeeded in the synthesis of (125)I-[Tyr(3)]MP with a high specific activity. (125)I-[Tyr(3)]MP bound specifically to heavy fraction of sarcoplasmic reticulum with a K(d) value of 4.0 microM and a B(max) value of 3.0 nmol/mg. Furthermore, (125)I-[Tyr(3)]MP specifically cross-linked to the 97-kDa protein without direct binding to ryanodine receptor. The protein was not triadin or Ca(2+)-pump, because antitriadin antibody and anti-Ca(2+)-pump antibody did not immunoprecipitate the protein. These results suggest that the 97-kDa MP-binding protein may have an important role in the excitation-contraction coupling of skeletal muscle.
Buthotus judaicus toxin 1 (BjTx-1) and toxin 2 (BjTx-2), two novel peptide activators of ryanodine receptors (RyR), were purified from the
venom of the scorpion B. judaicus. Their amino acid sequences differ only in 1 residue out of 28 (residue 16 corresponds to Lys in BjTx-1 and Ile in BjTx-2).
Despite a slight difference in EC50, both toxins increased binding of [3H]ryanodine to skeletal sarcoplasmic reticulum at micromolar concentrations but had no effect on cardiac or liver microsomes.
Their activating effect was Ca2+-dependent and was synergized by caffeine. B. judaicus toxins also increased binding of [3H]ryanodine to the purified RyR1, suggesting that a direct protein-protein interaction mediates the effect of the peptides.
BjTx-1 and BjTx-2 induced Ca2+ release from Ca2+-loaded sarcoplasmic reticulum vesicles in a dose-dependent manner and induced the appearance of long lived subconductance
states in skeletal RyRs reconstituted into lipid bilayers. Three-dimensional structural modeling reveals that a cluster of
positively charged residues (Lys11 to Lys16) is a prominent structural motif of both toxins. A similar structural motif is believed to be important for activation of
RyRs by imperatoxin A (IpTxa), another RyR-activating peptide (Gurrola, G. B., Arevalo, C., Sreekumar, R., Lokuta, A. J., Walker, J. W., and Valdivia,
H. H. (1999) J. Biol. Chem. 274, 7879-7886). Thus, it is likely that B. judaicus toxins and imperatoxin A bind to RyRs by means of electrostatic interactions that lead to massive conformational changes
in the channel protein. The different affinity and structural diversity of this family of scorpion peptides makes them excellent
peptide probes to identify RyR domains that trigger the channel to open.
The mechanism of Ca2+ release from sarcoplasmic reticulum, which triggers contraction in skeletal muscle, remains the key unresolved problem in excitation-contraction coupling. Recently, we have described the isolation of purified fractions referable to terminal and longitudinal cisternae of sarcoplasmic reticulum. Junctional terminal cisternae are distinct in that they have a low net energized Ca2+ loading, which can be enhanced 5-fold or more by addition of ruthenium red. The loading rate, normalized for calcium pump protein content, then approaches that of longitudinal cisternae of sarcoplasmic reticulum. We now find that the ruthenium red-enhanced Ca2+ loading rate can be blocked by the previous addition of ryanodine. The inhibition constant is in the nanomolar range (20-180 nM). Ryanodine and ruthenium red have no effect on the Ca2+ loading rate of longitudinal cisternae. Direct binding studies with [3H]ryanodine localized the receptors to the terminal cisternae and not to longitudinal cisternae. Scatchard analysis of the binding data gives a dissociation constant for ryanodine in the range of the drug action on the terminal cisternae (approximately 100 nM range) with approximately 4 to 20 pmol bound per mg of protein. Ryanodine is known to be toxic in animals, leading to irreversible muscle contractures. These studies provide evidence on the mode of action of ryanodine and its localization to the terminal cisternae. The low concentration at which the drug is effective appears to account for its toxicity. Ryanodine locks the Ca2+ release channels in the "open state," so that Ca2+ is not reaccumulated and the muscle fiber cannot relax.
Bromo-eudistomin D induced a contraction of the chemically skinned fibers from skeletal muscle at concentrations of 10 microM or more. This contractile response to bromo-eudistomin D was completely blocked by 10 mM procaine. The extravascular Ca2+ concentrations of the heavy fractions of the fragmented sarcoplasmic reticulum (HSR) were measured directly by a Ca2+ electrode to examine the effect of bromo-eudistomin D on the sarcoplasmic reticulum. After the HSR was loaded with Ca2+ by the ATP-dependent Ca2+ pump, the addition of 10 microM bromo-eudistomin D caused Ca2+ release that was followed by spontaneous Ca2+ reuptake. In the presence of 2 microM ruthenium red or 4 mM MgCl2, no Ca2+ release was induced by 20 microM bromo-eudistomin D. The rate of 45Ca2+ efflux from HSR, which had been passively preloaded with 45Ca2+, was accelerated 7 times by 10 microM bromo-eudistomin D. The concentration of bromo-eudistomin D for half-maximum effect on the apparent efflux rate was 1.5 microM, while that of caffeine was 0.6 mM. The bromo-eudistomin D-evoked efflux of 45Ca2+ was abolished by 2 microM ruthenium red or 0.5 mM MgCl2. Bromo-eudistomin D was found to be 400 times more potent than caffeine in its Ca2+-releasing action but was similar in its action in other respects. These results indicate that bromo-eudistomin D may induce Ca2+ release from the sarcoplasmic reticulum through physiologically relevant Ca2+ channels.
Calcium ions that have been preloaded into isolated SR subfractions in the presence of ATP and pyrophosphate may be released upon addition of a large number of diverse pharmacologic substances in a manner that is effectively blocked by ruthenium red and other organic polyamines. Effective blocking substances include certain antibiotics (neomycin, gentamicin, streptomycin, clindamycin, kanamycin, and tobramycin), naturally occurring polyamines (spermine and spermidine), and a number of basic polypeptides and proteins (polylysine, polyarginine, certain histones, and protamine). These agents have only one feature in common: the presence of several amino groups. Ruthenium red, neomycin, spermine, and protamine all appear to act by blocking SR Ca2+ channels since unidirectional 45Ca2+ efflux from the vesicles is strongly inhibited by these agents. Functions ascribable to the SR Ca2+ pump are largely unaffected by these agents. Since inositol 1,4,5-trisphosphate is ineffective at inducing Ca2+ release under these conditions, we conclude that these polyamines may directly block SR Ca2+ channels at very low concentrations by a mechanism unrelated to effects on inositol 1,4,5-trisphosphate production.
The ryanodine receptor of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle has been purified and found by electron microscopy to be equivalent to the feet structures that are involved in situ in the junctional association of transverse tubules with terminal cisternae of SR. We now find that when the purified receptor is incorporated into vesicle-derived planar bilayers, it forms Ca2+-specific channels, which are dependent on submicromolar Ca2+ for activity. In the presence of 1 mM ATP, the channel shows essentially no activity at 10 nM Ca2+ but becomes highly activated at 50 nM Ca2+. At suboptimal Ca2+ levels (100 nM), the channel is strongly activated by 1 mM ATP and can be blocked by ruthenium red, both effects being prevented by higher Ca2+ levels (1 microM). Mg2+, added from the cis side at millimolar concentrations, blocks Ca2+ flux through the channel from trans to cis (equivalent to flux from luminal to myoplasmic compartment). Ryanodine stabilizes the open state of the channel and blocks the action of ruthenium red to close the channel. Thus, the purified ryanodine receptor incorporated into a bilayer has the Ca2+-channel characteristics consistent with the calcium release observed in isolated terminal cisternae vesicles. Furthermore, ryanodine induced the appearance of a sublevel gating mode characterized by long open conductance states, which were integral multiples of the smallest observed conductance, 3.8 pS in 50 mM Ca2+. The purified receptor consists essentially of a single-sized high molecular weight polypeptide (Mr. approximately equal to 360,000), which on reconstitution forms the square rectangles diagnostic of the feet structures. We conclude that the identity of the Ca2+-release channel of SR is the foot structure, which consists of an oligomer of the high molecular weight polypeptide.
This study is concerned with the characterization of the morphology of the calcium release channel of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle, which is involved in excitation-contraction coupling. We have previously purified the ryanodine receptor and found it to be equivalent to the feet structures, which are involved, in situ, in the junctional association of transverse tubules with terminal cisternae of SR. The receptor is an oligomer of a single high molecular weight polypeptide and when incorporated into phospholipid bilayers, has channel conductance which is characteristic of calcium release in terminal cisternae of SR. The purified channel can be observed by electron microscopy using different methods of sample preparation, with complementary views being observed by negative staining, double staining, thin section and rotary shadowing electron microscopy. Three views can be observed and interpreted: (a) a square face which, in situ, is junctionally associated with the transverse tubule or junctional face membrane; (b) a rectangle equivalent to the side view; and (c) a diamond shape equivalent to the side view, of which the base portion appears to be equivalent to the transmembrane segment. Negative staining reveals detailed substructure of the channel. A computer averaged view of the receptor displays fourfold symmetry and ultrastructural detail. The dense central mass is divided into four domains with a 2-nm hole in the center, and is enclosed within an outer frame which has a pinwheel appearance. Double staining shows substructure of the square face in the form of parallel linear arrays (six/face). The features of the isolated receptor can be correlated with the structure observed in terminal cisternae vesicles. Sections tangential to the junctional face membrane reveal that the feet structures (23-nm squares) overlap so as to enclose smaller square spaces of approximately 14 nm/side. We suggest that this is equivalent to the transverse tubule face and that the terminal cisternae face is smaller (approximately 17 nm/face) and has larger alternating spaces as a consequence of the tapered sides of the foot structures. Image reconstruction analysis appears to be feasible and should provide the three-dimensional structure of the channel.
The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified as a single 450,000-dalton polypeptide from CHAPS-solubilized triads using immunoaffinity chromatography. The purified receptor had a [3H]ryanodine-binding capacity (Bmax) of 490 pmol/mg and a binding affinity (Kd) of 7.0 nM. Using planar bilayer recording techniques, we show that the purified receptor forms cationic channels selective for divalent ions. Ryanodine receptor channels were identical to the Ca-release channels described in native sarcoplasmic reticulum using the same techniques. In the present work, four criteria were used to establish this identity: (a) activation of channels by micromolar Ca and millimolar ATP and inhibition by micromolar ruthenium red, (b) a main channel conductance of 110 +/- 10 pS in 54 mM trans Ca, (c) a long-term open state of lower unitary conductance induced by ryanodine concentrations as low as 20 nM, and (d) a permeability ratio PCa/PTris approximately equal to 14. In addition, we show that the purified ryanodine receptor channel displays a saturable conductance in both monovalent and divalent cation solutions (gamma max for K and Ca = 1 nS and 172 pS, respectively). In the absence of Ca, channels had a broad selectivity for monovalent cations, but in the presence of Ca, they were selectively permeable to Ca against K by a permeability ratio PCa/PK approximately equal to 6. Receptor channels displayed several equivalent conductance levels, which suggest an oligomeric pore structure. We conclude that the 450,000-dalton polypeptide ryanodine receptor is the Ca-release channel of the sarcoplasmic reticulum and is the target site of ruthenium red and ryanodine.
The architecture of the junctional sarcoplasmic reticulum (SR) and transverse tubule (T tubule) membranes and the morphology of the two major proteins isolated from these membranes, the ryanodine receptor (or foot protein) and the dihydropyridine receptor, have been examined in detail. Evidence for a direct interaction between the foot protein and a protein component of the junctional T tubule membrane is presented. Comparisons between freeze-fracture images of the junctional SR and rotary-shadowed images of isolated triads and of the isolated foot protein, show that the foot protein has two domains. One is the large hydrophilic foot which spans the junctional gap and is composed of four subunits. The other is a hydrophobic domain which presumably forms the SR Ca2+-release channel and which also has a fourfold symmetry. Freeze-fracture images of the junctional T tubule membranes demonstrate the presence of diamond-shaped clusters of particles that correspond exactly in position to the subunits of the feet protein. These results suggest the presence of a large junctional complex spanning the two junctional membranes and intervening gap. This junctional complex is an ideal candidate for a mechanical coupling hypothesis of excitation-contraction coupling at the triadic junction.
The ryanodine receptor has been purified from junctional terminal cisternae of fast skeletal muscle sarcoplasmic reticulum (SR). The ryanodine receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and stabilized by addition of phospholipids. The solubilized receptor showed the same [3H]ryanodine binding properties as the original SR vesicles in terms of affinity, Ca2+ dependence, and salt dependence. Purification of the ryanodine receptor was performed by sequential column chromatography on heparin-agarose and hydroxylapatite in the presence of CHAPS. The purified receptor bound 393 +/- 65 pmol of ryanodine/mg of protein (mean +/- S.E., n = 5). The purified receptor showed three bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr of 360,000, 330,000, and 175,000. Densitometry indicates that these are present in the ratio of 2/1/1, suggesting a monomer Mr of 1.225 X 10(6) and supported by gel exclusion chromatography in CHAPS. Electron microscopy of the purified preparation showed the square shape of 210 A characteristic of and comparable in size and shape to the feet structures of junctional terminal cisternae of SR, indicating that ryanodine binds directly to the feet structures. From the ryanodine binding data, the stoichiometry between ryanodine binding sites to the number of feet structures is estimated to be about 2. Since the ryanodine receptor is coupled to Ca2+ gating, the present finding suggests that the ryanodine receptor and Ca2+ release channel represent a functional unit, the structural unit being the foot structure which, in situ, is junctionally associated with the transverse tubules. It is across this triad junction that the signal for Ca2+ release is expressed. Thus, the foot structure appears to directly respond to the signal from transverse tubules, causing the release of Ca2+ from the junctional face membrane of the terminal cisternae of SR.
Release of Ca2+ from a heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum was triggered by several different methods: (a) increasing extravesicular [Ca2+] [( CaO2+] from about 0.1 microM to 10 microM), (b), adding caffeine, (c) adding quercetin, and (d) substituting a solution containing equimolar choline+ for K+-containing solution (depolarization-induced Ca2+ release). The maximal rate of Ca2+ release triggered by caffeine or quercetin in the presence of 12.5 microM [CaO2+] (21-25 nmol of Ca2+/mg/s) is similar to that of the depolarization-induced Ca2+ release (19 nmol of Ca2+/mg/s), as determined by stopped flow spectrometry of changes in [CaO2+] with arsenazo III. The release is transient and all of the released Ca2+ is reaccumulated. The rates of Ca2+ release triggered by caffeine, quercetin, or membrane depolarization sharply decrease at high [CaO2+], suggesting a negative feedback effect of the released Ca2+. Inhibition of the release pathway allows the sarcoplasmic reticulum to reaccumulate Ca2+. The rate of Ca2+ release triggered by caffeine or quercetin, but not that triggered by membrane depolarization, is also reduced upon decreasing [CaO2+] to the submicromolar range. Passive efflux of intravesicular Ca2+ in solutions containing lower [CaO2+] in the absence of Mg.ATP is attenuated at about the same time (congruent to 1 min) regardless of the amounts of Ca2+ released, indicating that the opened Ca2+ channels close spontaneously. These results suggest that kinetically identical channels are responsible for Ca2+ release independent of the methods of triggering and this in vitro release is consistent with the physiological mechanism both in terms of the rapidity and the reversibility of Ca2+ release.
3H-Labeled 9-methyl-7-bromoeudistomin D ([3H] MBED), the most powerful inducer of Ca2+ release from sarcoplasmic reticulum (SR), was successfully prepared with a high specific activity of 10.2 Ci/mmol. [3H]MBED bound to terminal cisternae (TC) of skeletal muscle SR in a replacable and saturable manner, indicating the existence of its specific binding site. Caffeine inhibited the [3H]MBED binding to the TC-SR membranes from skeletal muscle with an IC50 value of 0.8 mM, in close agreement with a concentration that causes Ca2+ release from SR. Scatchard analysis gave values of KD = 40 nM and Bmax = 10 pmol/mg protein. The KD value was increased by caffeine, while that of Bmax was not changed, indicating a competitive mode of inhibition. Adenosine 5'-(beta, gamma-methylene)triphosphate enhanced [3H]MBED binding, but ryanodine and Ca2+ did not affect it. [3H]MBED binding to TC-SR membranes was inhibited by procaine, a representative blocker of Ca(2+)-induced Ca2+ release channels, whereas that was not changed by Mg2+, suggesting that procaine but not Mg2+ may exert its inhibitory effect on Ca(2+)-induced Ca2+ release by affecting the caffeine-binding sites. These results suggest that MBED shares the same binding site as that of caffeine in TC-SR. The [3H]MBED is the first radiolabeled ligand for caffeine-binding sites in Ca2+ release channels and thus may provide an essential biochemical tool for elucidating this site.
It is now well known that the contractile system of muscle fibres becomes activated when the intracellular calcium ion concentration increases to about 1 to 10 µM and that it is turned off when it is lowered to below about 0.1 µM. Here, we shall describe the structures and mechanisms that raise and lower the intracellular free calcium ion concentration quickly during the contraction-relaxation cycle of various types of muscle. In very thin muscle fibres, such as those of the frog heart (Fabiato 1983), vertebrate smooth muscle (Deth and van Breemen 1974), and in the myotome of Branchiostoma lanceolatum (Hagiwara et al. 1971; but cf. Melzer 1982a, b), the activating calcium ions seem to originate, at least partly, from the extracellular fluid and enter the cell through calcium channels of the cell membrane during activation. Bianchi and Shanes (1959) detected a small calcium influx even in stimulated skeletal muscle, but Sandow (1965) calculated that the amount of calcium entering the stimulated muscle cell is too small and its diffusion into the interior of the fibre too slow (cf. also Hill 1948) to account for the rapid onset of contraction. In these fast and wide-fibred muscles the activator calcium is released from and recuperated by the sarcoplasmic reticulum (Hasselbach 1964) during contraction and relaxation (Sects. 2.2 and 2.3).
Ca release from the sarcoplasmic reticulum (SR) is one of the most important steps in excitation–contraction coupling of skeletal muscle. This chapter describes the physiological release of Ca from the SR, various modes of Ca release from the SR, and the physiological significance of various Ca release mechanisms. Ca ion is the mediator of information of action potentials to the contractile machinery; however, the physiological source of the mediator Ca is not yet unequivocally established. The inhibitors of Ca-induced Ca release––procaine and adenine––were shown not to inhibit contraction of living skeletal muscle fibers induced by the depolarization of the surface membrane. Studies of the ionic composition of the lumen of the SR by electron-probe analysis show that there are no significant differences between the ionic compositions in the lumen of the SR and that in the cytoplasm except for Ca ion. The essential part of the physiological Ca release mechanism is almost entirely unknown; therefore, further studies, especially by using preparations, such as improved cut fibers, that retain the physiological tubule (T)–SR coupling mechanism, but have easy access to sarcoplasm so that its composition can be altered at will, are necessary.
The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified as a single 450,000-dalton polypeptide from CHAPS-solubilized triads using immunoaffinity chromatography. The purified receptor had a [3H]ryanodine-binding capacity (Bmax) of 490 pmol/mg and a binding affinity (Kd) of 7.0 nM. Using planar bilayer recording techniques, we show that the purified receptor forms cationic channels selective for divalent ions. Ryanodine receptor channels were identical to the Ca-release channels described in native sarcoplasmic reticulum using the same techniques. In the present work, four criteria were used to establish this identity: (a) activation of channels by micromolar Ca and millimolar ATP and inhibition by micromolar ruthenium red, (b) a main channel conductance of 110 +/- 10 pS in 54 mM trans Ca, (c) a long-term open state of lower unitary conductance induced by ryanodine concentrations as low as 20 nM, and (d) a permeability ratio PCa/PTris approximately equal to 14. In addition, we show that the purified ryanodine receptor channel displays a saturable conductance in both monovalent and divalent cation solutions (gamma max for K and Ca = 1 nS and 172 pS, respectively). In the absence of Ca, channels had a broad selectivity for monovalent cations, but in the presence of Ca, they were selectively permeable to Ca against K by a permeability ratio PCa/PK approximately equal to 6. Receptor channels displayed several equivalent conductance levels, which suggest an oligomeric pore structure. We conclude that the 450,000-dalton polypeptide ryanodine receptor is the Ca-release channel of the sarcoplasmic reticulum and is the target site of ruthenium red and ryanodine.
This study is concerned with the characterization of the morphology of the calcium release channel of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle, which is involved in excitation-contraction coupling. We have previously purified the ryanodine receptor and found it to be equivalent to the feet structures, which are involved, in situ, in the junctional association of transverse tubules with terminal cisternae of SR. The receptor is an oligomer of a single high molecular weight polypeptide and when incorporated into phospholipid bilayers, has channel conductance which is characteristic of calcium release in terminal cisternae of SR. The purified channel can be observed by electron microscopy using different methods of sample preparation, with complementary views being observed by negative staining, double staining, thin section and rotary shadowing electron microscopy. Three views can be observed and interpreted: (a) a square face which, in situ, is junctionally associated with the transverse tubule or junctional face membrane; (b) a rectangle equivalent to the side view; and (c) a diamond shape equivalent to the side view, of which the base portion appears to be equivalent to the transmembrane segment. Negative staining reveals detailed substructure of the channel. A computer averaged view of the receptor displays fourfold symmetry and ultrastructural detail. The dense central mass is divided into four domains with a 2-nm hole in the center, and is enclosed within an outer frame which has a pinwheel appearance. Double staining shows substructure of the square face in the form of parallel linear arrays (six/face). The features of the isolated receptor can be correlated with the structure observed in terminal cisternae vesicles. Sections tangential to the junctional face membrane reveal that the feet structures (23-nm squares) overlap so as to enclose smaller square spaces of approximately 14 nm/side. We suggest that this is equivalent to the transverse tubule face and that the terminal cisternae face is smaller (approximately 17 nm/face) and has larger alternating spaces as a consequence of the tapered sides of the foot structures. Image reconstruction analysis appears to be feasible and should provide the three-dimensional structure of the channel.
The repeated contractions which can be observed in skinned fibres in appropriate concentrations of caffeine, Ca2+ and chelating agent suggest that calcium release is a regenerative process in which calcium itself causes the release of calcium from the reticulum.
This review summarizes present knowledge about calcium release and discusses their physiological significance. Descriptions and discussions are mainly confined to skeletal muscle, especially that of amphibians, since there are fewer available data on other kinds of muscle.
The primary structure of myotoxin a, a myotoxin protein from the venom of the North American rattlesnake Crotalus viridis viridis, was determined and the position of the disulfide bonds assigned. The toxin was isolated, carboxymethylated, and cleaved by cyanogen bromide, and the resultant peptides were isolated. The cyanogen bromide peptides were subjected to amino acid sequence analysis. In order to assign the positions of the three disulfide bonds, the native toxin was cleaved sequentially with cyanogen bromide and trypsin. A two peptide unit connected by one disulfide bond was isolated and characterized, and a three-peptide unit connected by two disulfide bonds was isolated. One peptide in the three-peptide unit was identified as Cys-Cys-Lys. In order to establish the linkages between the peptides and Cys-Cys-Lys, one cycle of Edman degradation was carried out such that the Cys-Cys bond was cleaved. Upon isolation and analysis of the cleavage products, the disulfide bonds connecting the three peptides were determined. The positions of the disulfide bridges of myotoxin a were determined to be totally different from those of neurotoxins isolated from snake venoms. The sequence of myotoxin a was compared with the sequences of other snake venom toxins using the computer program RELATE to determine whether myotoxin a is similar to any other types of toxins. From the computer analysis, myotoxin a did not show any close relationship to other toxins except crotamine from the South American rattlesnake Crotalus durissus terrificus.
A previously unknown polypeptide myotoxin, designated myotoxin a, was isolated for the first time from prairie rattlesnake (Crotalus viridis viridis) venom. Electrophoretic homogeneity of myotoxin a was shown in beta-alanine disc gel polyacrylamide gel electrophoresis and in isoelectric focusing gel electrophoresis. Molecular weight and isoelectric point estimates of 4100 and 9.6 were obtained by gel filtration and isoelectric focusing gel electrophoresis, respectively. Amino acid composition showed a total of 39 amino acid residues, with 10 lysine residues and two disulfide bridges. When the two disulfide brides were reduced and alkylated, the myotoxic activity was abolished, indicating that the disulfide bridges of myotoxin a are essential for its biological activity. The loss of the biological activity is probably due to a marked change in secondary structure. The circular dichroic spectrum indicates that the chemically modified, inactive myotoxin exhibits typical random-coil conformation.
We report the purification of two peptides, called "imperatoxin inhibitor" and "imperatoxin activator," from the venom of the scorpion Pandinus imperator targeted against ryanodine receptor Ca(2+)-release channels. Imperatoxin inhibitor has a M(r) of approximately 10,500, inhibits [3H]ryanodine binding to skeletal and cardiac sarcoplasmic reticulum with an ED50 of approximately 10 nM, and blocks openings of skeletal and cardiac Ca(2+)-release channels incorporated into planar bilayers. In whole-cell recordings of cardiac myocytes, imperatoxin inhibitor decreased twitch amplitude and intracellular Ca2+ transients, suggesting a selective blockade of Ca2+ release from the sarcoplasmic reticulum. Imperatoxin activator has a M(r) of approximately 8700, stimulates [3H]ryanodine binding in skeletal but not cardiac sarcoplasmic reticulum with an ED50 of approximately 6 nM, and activates skeletal but not cardiac Ca(2+)-release channels. These ligands may serve to selectively "turn on" or "turn off" ryanodine receptors in fragmented systems and whole cells.
Release of calcium from intracellular stores occurs by two pathways, an inositol 1,4,5-trisphosphate (InsP3)-gated channel and a calcium-gated channel (ryanodine receptor). Using specific antibodies, both receptors were found in Purkinje cells of cerebellum. We have now compared the functional properties of the channels corresponding to the two receptors by incorporating endoplasmic reticulum vesicles from canine cerebellum into planar bilayers. InsP3-gated channels were observed most frequently. Another channel type was activated by adenine nucleotides or caffeine, inhibited by ruthenium red, and modified by ryanodine, characteristics of the ryanodine receptor/channel6. The open probability of both channel types displayed a bell-shaped curve for dependence on calcium. For the InsP3-gated channel, the maximum probability of opening occurred at 0.2 microM free calcium, with sharp decreases on either side of the maximum. Maximum activity for the ryanodine receptor/channel was maintained between 1 and 100 microM calcium. Thus, within the physiological range of cytoplasmic calcium, the InsP3-gated channel itself allows positive feedback and then negative feedback for calcium release, whereas the ryanodine receptor/channel behaves solely as a calcium-activated channel. The existence in the same cell of two channels with different responses to calcium and different ligand sensitivities provides a basis for complex patterns of intracellular calcium regulation.
9-Methyl-7-bromoeudistomin D (MBED), a derivative of eudistomin D isolated from a marine tunicate, induced Ca++ release from the heavy fraction of fragmented sarcoplasmic reticulum (HSR) in the same way as that of caffeine, followed by spontaneous Ca++ reuptake in the Ca++ electrode experiment. The rate of 45Ca++ efflux from HSR vesicles was accelerated markedly by MBED or caffeine in a concentration-dependent manner. The 50% effective concentrations of MBED and caffeine were approximately 1 microM and 1 mM, respectively, indicating that MBED is 1000 times more potent than caffeine in HSR. Procaine, ruthenium red or Mg++ caused concentration-dependent inhibition of MBED-triggered Ca++ release from HSR. The bell-shaped profile of Ca++ dependence for MBED is very similar to that of caffeine. The caffeine-produced maximum response of 45Ca++ efflux was increased further by adenosine-5'-(beta, gamma-methyl-ene)triphosphate, whereas that was not changed by MBED. MBED also caused Ca++ release from sarcoplasmic reticulum (SR) of chemically skinned fibers. These stimulatory effects of MBED on the Ca++ release from skeletal muscle SR were almost indistinguishable from those of caffeine except the difference in potencies. The [3H]ryanodine binding to junctional terminal cisternae membranes was not inhibited by MBED or caffeine. MBED did not cause Ca++ release from the light fraction of fragmented SR and turbidity change of mitochondrial suspension. These observations suggest a most likely idea that MBED binds to the caffeine-binding site in the Ca channel protein and thus produces the potentiation of Ca(++)-induced Ca++ release from SR.(ABSTRACT TRUNCATED AT 250 WORDS)
The Ca2+-ryanodine receptor complex is a functional unit at the terminal cisternae (TC) of the sarcoplasmic reticulum (SR) whose proteins comprise the Ca2+ release channels which may be involved in excitation-contraction coupling. Ca2+, Mg2+, caffeine, and adenine nucleotides, but not inositol 1,4,5-trisphosphate, may exert their inotropic effects on skeletal muscle SR by direct allosteric modulation of the [3H]ryanodine-binding site. Micromolar Ca2+ is primarily responsible for activating [3H]ryanodine binding by regulating receptor site density, affinity, and cooperativity. Mg2+ reduces the sensitivity to Ca2+ activation by directly competing with Ca2+ for the activator site. However, inhibition by Mg2+ is overcome in the presence of beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP; 1 mM) or caffeine (20 mM). Caffeine dramatically increases the affinity of the Ca2+ activator site for Ca2+, whereas AMP-PCP or cAMP enhances the gating efficiency or the lifetime of the open state of the TC SR channel. A kinetic model is proposed for four functional domains of the Ca2+-ryanodine receptor complex: the Ca2+-regulatory domain which binds Ca2+ with microM affinity is primarily responsible for gating the Ca2+ channel of the TC SR in a cooperative manner, and is inhibited by mM Mg2+ by direct competition for the activator site which appears to contain critical sulfhydryl groups; a Ca2+-activate alkaloid binding domain in close proximity to the channel which binds ryanodine with nM affinity and rapidly occludes upon complex formation; a domain which binds caffeine with low (greater than mM) affinity and directly influences the sensitivity of the Ca2+-regulatory site; and a domain which binds adenine nucleotides with intermediate affinity (less than mM), does not require phosphorylation, and intensifies the Ca2+ signal which triggers opening of the Ca2+-release channel.
Gingerol, isolated as a potent cardiotonic agent from the rhizome of ginger, stimulated the Ca2+-pumping activity of fragmented sarcoplasmic reticulum (SR) prepared from rabbit skeletal and dog cardiac muscles. The extravesicular Ca2+ concentrations of the heavy fraction of the fragmented SR (HSR) were measured directly with a Ca2+ electrode to examine the effect of gingerol on the SR. Gingerol (3-30 microM) accelerated the Ca2+-pumping rate of skeletal and cardiac SR in a concentration-dependent manner. The rate of 45Ca2+ uptake of HSR was also increased markedly by 30 microM gingerol without affecting the 45Ca2+ efflux from HSR. Furthermore, gingerol activated Ca2+-ATPase activities of skeletal and cardiac SR (EC50, 4 microM). The activation of SR Ca2+-ATPase activity by gingerol (30 microM) was completely reversed by 100-fold dilution with the fresh saline solution. Kinetic analysis of activating effects of gingerol suggests that the activation of SR Ca2+-ATPase is uncompetitive and competitive with respect to Mg . ATP at concentrations of 0.2-0.5 mM and above 1 mM, respectively. Kinetic analysis also suggests that the activation by gingerol is mixed-type with respect to free Ca2+ and this enzyme is activated probably due to the acceleration of enzyme-substrate complex breakdown. Gingerol had no significant effect on sarcolemmal Ca2+-ATPase, myosin Ca2+-ATPase, actin-activated myosin ATPase and cAMP-phosphodiesterase activities, indicating that the effect of gingerol is rather specific to SR Ca2+-ATPase activity. Gingerol may provide a valuable chemical tool for studies aimed at clarifying the regulatory mechanisms of SR Ca2+-pumping systems and the causal relationship between the Ca2+-pumping activity of SR and muscle contractility.
The calcium release channel from rabbit muscle sarcoplasmic reticulum (SR) has been purified and reconstituted as a functional unit in lipid bilayers. Electron microscopy reveals the four-leaf clover structure previously described for the 'feet' that span the transverse tubule (T)-SR junction. Ca2+ release from the SR induced by T-system depolarization during excitation-contraction coupling in muscle may thus be effected through a direct association of the T-system with SR Ca2+-release channels.
The calcium channel responsible for the release of Ca2+ from the sarcoplasmic reticulum of skeletal muscle during excitation-contraction coupling has recently been identified and purified. The isolated calcium channel has been identified morphologically with the 'foot' structures which are associated with the junctional face membrane of the terminal cisternae of sarcoplasmic reticulum. In situ, the foot structure extends across the gap of the triad junction from the terminal cisternae of the reticulum to the transverse tubule. We describe here the three-dimensional architecture (3.7 nm resolution) of the calcium channel/foot structure from fast-twitch rabbit skeletal muscle, which we determined from electron micrographs of isolated, non-crystalline structures that had been tilted in the electron microscope. The reconstruction reveals two different faces and an internal structure in which stain accumulates at several interconnected locations, which could empty into the junctional gap of the triad junction. The detailed architecture of the channel complex is relevant to understanding both the physical path followed by calcium ions during excitation-contraction coupling and the association of the terminal cisternae and the transverse tubules in the triad junction.
The preparations of novel phosphodiesterase inhibitors, 8-acetoxy-5-iodo-6-methoxypyrido[3,4-b]indole, 5,7-dibromo-6-hydroxypyrido[3,4-b]indole, 5,7-dichloro-6-hydroxypyrido[3,4-b]-indole and 8-acetoxy-5-bromo-6-methoxypyrido[3,4-b]indole, are described together with concentrations giving 50% inhibition against cyclic AMP phosphodiesterase, i.e. 3 X 10(-6), 3 X 10(-6), 7 X 10(-6) and 1 X 10(-5) M, respectively. The relative potency of these eudistomin derivatives is discussed in terms of the chemical structures compared with those of other inactive eudistomins and derivatives.
Myotoxin a is a muscle-damaging toxin isolated from the venom of Crotalus viridis viridis. Its interaction with the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles purified from rabbit skeletal muscle was investigated. Myotoxin a inhibited Ca2+ loading and stimulated Ca2+-dependent ATPase without affecting unidirectional Ca2+ efflux. Its action was dose, time, and temperature dependent. Myotoxin a partially blocked the binding of specific anti-(rabbit SR Ca2+-ATPase) antibodies. It is concluded that myotoxin a attaches to the SR Ca2+-ATPase and uncouples Ca2+ uptake from Ca2+-dependent ATP hydrolysis. Myotoxin a also prevented the formation of decavanadate-induced two-dimensional crystalline arrays of the SR Ca2+-ATPase.
A simple and rapid colorimetric assay for measuring the high affinity Ca2+-ATPase activity in subcellular fractions is presented. With this method a one-step addition of a malachite green/molybdate/polyvinyl alcohol reagent to the assay mixture at the end of the incubation period is all that is required for the spectrophotometric quantification of the phosphomolybdate-malachite green complex. The presence of polyvinyl alcohol allows the quantification of released phosphate without having to separate it from protein. We have validated this assay by characterizing the high affinity Ca2+-ATPase activity in isolated rat liver microsomes. Comparable Ca2+-ATPase activities in rat liver microsomes and adipocyte plasma membranes were found when measured with this colorimetric assay and an isotopic assay. This method is applicable to the measurement of other types of ATPase activities.
It is suggested that a link in excitation-contraction coupling involves
the movement of a fixed amount of charge free to move between different
locations across the membrane.
Free calcium appears to trigger the release of stored calcium from the sarcoplasmic reticulum of skinned skeletal muscle fibers immersed in solutions with a low concentration of magnesium ion.
Since the discovery of the ATP-dependent Ca2+ transport by SR a little over two decades ago, remarkable progress has been made in understanding the kinetic mechanism of Ca2+ transport and ATP hydrolysis and the role of phosphorylated enzyme intermediates in the energetics of active ion transport. Significant information has accumulated on the structure and composition of the SR membrane, on the primary amino acid sequence of the Ca2+-pump protein, and on the adaptive changes in the Ca2+-transport function during embryonic development and muscle activity. The discovery of the charge movement as a step in EC coupling and the use of novel optical probes for analyzing potential and calcium transients in living muscle changed the enigma of EC coupling into a well-defined problem that is clearly open to rational solutions. Studies on the structure, composition, and function of the isolated components of the T-SR system have just begun. The effectiveness of this approach will depend on successful maintenance of the functionally intact structure of the T-SR junction during the disruption of the muscle, which is required for the isolation of pure membrane elements. Reconstitution of a functionally competent junctional complex from isolated components is the ultimate aim of these studies, but the path toward that goal is so difficult that much of the mechanism of EC coupling may be solved by electrophysiologists, before reconstitution is achieved. The avalanche of information on Ca2+ releases induced by various agents under diverse and sometimes ill-defined conditions led to formulation of a series of hypothetical mechanisms. Of these, Ca2+-induced Ca2+ release promises to be an important element of the physiological Ca2+-release process, but few of the other proposed mechanisms can be eliminated from consideration at this stage. The impressive progress of the last few years has left several fundamental problems largely unsolved. Among these are the physical mode of translocation of Ca2+ across the membrane and the molecular mechanism of the coupling of Ca2+ transport to ATP hydrolysis; the regulation of the concentration of the Ca2+-pump protein and calcium in the SR of fast and slow skeletal, cardiac, and smooth muscles; the gating mechanisms that regulate the graded release of calcium from the SR and the composition and biochemical characterization of the triad; the role of SR membrane potential in the regulation of Ca2+ fluxes in vivo; the permeability of SR membranes in living muscle; the functional significance of protein-protein interactions in the SR with respect to Ca2+ transport and permeability control.(ABSTRACT TRUNCATED AT 400 WORDS)
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
Drug-induced Ca2l plasmic reticulum: III. Block of Ca2l-induced Ca2l organic polyamines channels in planer release from sarcop release from isolated sarco-release by PESSAH, I Ca2+-activated ryanodine binding: mechanisms of sensitivity modulation by Mg2+, caffeine, and adenine nucleotides
- P -J Palade
- J Gilmore
- M Mascal
PALADE, P. (1987). Drug-induced Ca2l plasmic reticulum: III. Block of Ca2l-induced Ca2l organic polyamines. J. Biol. Chem., 262, 6149-6154. channels in planer release from sarcop-J., GILMORE, J., MASCAL, M., release from isolated sarco-release by PESSAH, I.N., STAMBUK, R.A. & CASIDA, J.E. (1987). Ca2+-activated ryanodine binding: mechanisms of sensitivity modulation by Mg2+, caffeine, and adenine nucleotides. Mol
Excitation-contraction coupling and the mechan-ism of muscle contraction Bell-reticulum of cerebellum. snake (Crotalis viridis viridis)
- S Ebashi
EBASHI, S. (1991). Excitation-contraction coupling and the mechan-ism of muscle contraction. Annu. Rev. Physiol., 53, 1-16. I., WATRAS, J. & EHRLICH, B.E. (1991). Bell-reticulum of cerebellum. snake (Crotalis viridis viridis)
Regenerative calcium release within muscle cells & release channels with ryanodine in rMYOTOXIN a, A NOVEL Ca2 RELEASER239 FOX Amino acid sequence and Disulfide bond assignment of myotoxin a isolated from the venom of prairie rattlesnake (Crotalus viridis viridis) Biochemis-try
- L E Ford
- R J Y Podolsky
- .-I Adachi
- M Kobayashi
- M Kobayashi
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