Rabbit Skeletal Muscle Sarcoplasmic Reticulum Research Articles

Deciphering Functions of Intracellular Formaldehyde: Linking Cancer and Aldehyde Metabolism.

Carnosine (beta-alanyl-L-histidine), which is present in millimolar concentrations in skeletal muscles, induces Ca2+ release from the heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum by activation ruthenium red-sensitive Ca-release channels. The effect of carnosine is dose-dependent, which indicates the presence of saturable carnosine-binding sites in the Ca-release channel molecule. The half-maximal Ca2+ release is observed in the presence of 8.7 mM carnosine. At the same time, carnosine addition to the medium increases the affinity of sarcoplasmic reticulum Ca-channels for the Ca-release activators, caffeine and adenine nucleotides. It is concluded that carnosine is an endogenous regulator of skeletal muscle sarcoplasmic reticulum Ca-channels which modulates the affinity of these channels for different ligands.

Discovery of Tricyclic Clerodane Diterpenes as Sarco/Endoplasmic Reticulum Ca(2+)-ATPase Inhibitors and Structure-Activity Relationships.

Tricyclic clerodane diterpenes (TCDs) are natural compounds that often show potent cytotoxicity for cancer cells, but their mode of action remains elusive. A computationally based similarity search (CDRUG), combined with principal component analysis (ChemGPS-NP) and docking calculations (GOLD 5.2), suggested TCDs to be inhibitors of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump, which is also the target of the sesquiterpene lactone thapsigargin. Biochemical studies were performed with 11 TCDs on purified rabbit skeletal muscle sarcoplasmic reticulum membranes, which are highly enriched with the SERCA1a isoform. Casearborin D (2) exhibited the highest affinity, with a KD value of 2 μM and giving rise to complete inhibition of SERCA1a activity. Structure-activity relationships revealed that functionalization of two acyl side chains (R1 and R4) and the hydrophobicity imparted by the aliphatic chain at C-9, as well as a C-3,C-4 double bond, play crucial roles for inhibitory activity. Docking studies also suggested that hydrophobic interactions in the binding site, especially with Phe256 and Phe834, may be important for a strong inhibitory activity of the TCDs. In conclusion, a novel class of SERCA inhibitory compounds is presented.

SRP-35, a newly identified protein of the skeletal muscle sarcoplasmic reticulum, is a retinol dehydrogenase

In the present study we provide evidence that SRP-35, a protein we identified in rabbit skeletal muscle sarcoplasmic reticulum, is an all-trans-retinol dehydrogenase. Analysis of the primary structure and tryptic digestion revealed that its N-terminus encompasses a short hydrophobic sequence bound to the sarcoplasmic reticulum membrane, whereas its C-terminal catalytic domain faces the myoplasm. SRP-35 is also expressed in liver and adipocytes, where it appears in the post-microsomal supernatant; however, in skeletal muscle, SRP-35 is enriched in the longitudinal sarcoplasmic reticulum. Sequence comparison predicts that SRP-35 is a short-chain dehydrogenase/reductase belonging to the DHRS7C [dehydrogenase/reductase (short-chain dehydrogenase/reductase family) member 7C] subfamily. Retinol is the substrate of SRP-35, since its transient overexpression leads to an increased production of all-trans-retinaldehyde. Transfection of C2C12 myotubes with a fusion protein encoding SRP-35-EYFP (enhanced yellow fluorescent protein) causes a decrease of the maximal Ca²⁺ released via RyR (ryanodine receptor) activation induced by KCl or 4-chloro-m-chresol. The latter result could be mimicked by the addition of retinoic acid to the C2C12 cell tissue culture medium, a treatment which caused a significant reduction of RyR1 expression. We propose that in skeletal muscle SRP-35 is involved in the generation of all-trans-retinaldehyde and may play an important role in the generation of intracellular signals linking Ca2+ release (i.e. muscle activity) to metabolism.

Molecular nature of sulfhydryl modification by hydrogen peroxide on type 1 ryanodine receptor1

To elucidate the molecular nature of sulfhydryl modification by hydrogen peroxide on type 1 ryanodine receptor (RyR1). Rabbit skeletal muscle sarcoplasmic reticulum was treated with hydrogen peroxide, then RyR1 complex was isolated. The proteins in the complex were analysed by electrophoresis, Western blot and electron microscopy. (1) Hydrogen peroxide induces inter-subunit cross-linking within the tetrameric RyR1 molecule; (2) in parallel to inter-subunit cross-linking, the RyR1 molecule changes morphology; (3) the chemical and morphological changes are reversible: upon reduction by reducing agents, the RyR1 molecule regains its original state. These findings suggest that the molecular mechanism of RyR1 channel activity in sarcoplasmic reticulum regulated by hydrogen peroxide is through inter-subunit cross-linking within the tetrameric RyR1 molecule, which in turn induces structural changes of RyR1.

Ryanodine Receptor-Mediated Rapid Increase in Intracellular Calcium Induced by 7,8-Benzo(a)Pyrene Quinone in Human and Murine Leukocytes

Benzo(a)pyrene (BaP) is an environmentally prevalent polycyclic aromatic hydrocarbon (PAH) known to produce immunotoxicity in murine and human lymphocytes. Previous studies by our lab have shown that certain BaP metabolites increase intracellular Ca(2+) in human and murine lymphocytes. The mechanism by which these BaP metabolites increase Ca(2+) may involve src kinase activation and mitochondrial oxidative stress. We have implicated a new pathway of Ca(2+) elevation in lymphocytes produced by a novel BaP metabolite, BaP-7,8-dione (7,8-BPQ). This ortho quinone is produced from BaP-7,8-dihydrodiol by aldoketoreductase 1C1 (AKR1C) isoforms in human cells. We have previously shown that 7,8-BPQ increases Ca(2+) levels in an in vitro rabbit skeletal muscle sarcoplasmic reticulum (SR) vesicle model via interaction with ryanodine receptors (RyR). In the present study, we found that 7,8-BPQ produced a RyR-dependent rapid increase in intracellular Ca(2+) in the Daudi human B cell line. However, other BP-diones including 1,6-, 3,6-, and 6,12-BPQs failed to produce a rapid increase in Ca(2+). Instead they produced a late increase in intracellular Ca(2+), presumably via a redox-cycling-dependent loss of Ca(2+) buffering capacity by mitochondria. Functional RyR were detected in Daudi using a (3)H-ryanodine binding assay. The studies were extended to normal human peripheral blood and murine spleen cells, where it was found that 7,8-BPQ rapidly elevated intracellular Ca(2+) in B cells and T cells in both species. The Ca(2+)-elevating effect of 7,8-BPQ was prevented by pretreatment with a high concentration of ryanodine (500 muM). Collectively, these results demonstrate a novel mechanism of Ca(2+) elevation by an environmentally relevant metabolite of BaP in murine and human lymphocytes.

Antioxidants prevented oxidative injury of SR induced by Fe2+/H2O2/ascorbate system but failed to prevent Ca2+‐ATPase activity decrease

Dysfunction of sarcoplasmic reticulum (SR) Ca2+-ATPase induced by oxidative stress may be a contributing factor to the development of serious age related diseases. Incubation of sarcoplasmic reticulum (SR) vesicles of rabbit skeletal muscles with Fe2+/H2O2/ascorbate decreased the SH group content of SR approximately to 35% and Ca2+-ATPase activity to 50% of control not oxidized sample. Protein carbonyls increased twofold, lipid peroxidation was also significantly elevated. The antioxidant effects of trolox, the pyridoindole derivative stobadine and of the standardized extracts from bark of Pinus Pinaster PycnogenolR (Pyc) and from leaves of Ginkgo biloba (EGb 761) were studied on oxidatively injured SR. All antioxidants exerted preventive effects against the oxidized lipids and protein SH groups of SR vesicles. Trolox and stobadine did not influence protein carbonyl formation, while flavonoid extracts prevented carbonyl generation, probably by binding to protein. The preventive effects of the antioxidants studied on lipids and protein SH groups were however not associated with protection of Ca2+-ATPase activity. Stobadine and trolox exerted no effect on enzyme activity, Pyc and EGb 761 enhanced the inhibitory effect of Ca2+-ATPase activity in oxidatively injured SR. Concluding, under the conditions of oxidative stress induced by Fe2+/H2O2/ascorbate against SR of rabbit skeletal muscle, the agents studied demonstrated antioxidant effects yet failed to protect Ca2+-ATPase activity.

Effects of tetrandrine on calcium transport, protein fluorescences and membrane fluidity of sarcoplasmic reticulum.

To understand whether the molecular mechanism of Tetrandrine (Tet)'s pharmacological effects is concerned with sarcoplasmic reticulum calcium transport so as to be involved in myocardial contractility, we observed the effects of Tet on calcium transport and membrane structure of rabbit skeletal muscle sarcoplasmic reticulum vesicles (SR) and rat cardiac sarcoplasmic reticulum vesicles (CSR). Calcium uptake was monitored with a dual-wavelength spectrophotometer. Protein conformation and fluorescence polarization were measured by fluospectrophotometric method and membrane lipids labelled with fluorescence probes for SR, respectively. 128 micromol l(-1) Tet reduced the initial rate of calcium uptake to 59% of control 6 min after reaction. Tet un-competitively inhibited SR Ca(2+), Mg(2+)-ATPase activity, causing the stoichiometric ratio of SR Ca(2+)/ATP to decrease to 1.43 from 2.0 of control. Inhibitory rates on SR Ca(2+),Mg(2+)-ATPase by Tet were reduced from 60% in the absence of phosphate to 50% in the presence of phosphate and reduced from 92% in 1 mmol l(-1) ATP to 60% in 5 mmol l(-1) ATP. Tet markedly reduced SR intrinsic protein fluorescence, while it slightly decreased the thiol(SH)-modified protein fluorescence of SR labelled with N-(3-pyrene)-maleimide. Tet slightly increased fluorescence polarization in the middle and deep layers of SR membrane lipids labelled with 7- or 12-(9-anthroyloxy) stearic acid (AS) probes, whereas it did not change that of SR labelled with 1, 6-diphenyl-1,3,5-hexatrine (DPH). These results revealed that prevention of SR calcium uptake by Tet was due to inhibition of the SR calcium pump Ca(2+),Mg(2+)-ATPase, changes in spatial conformation of the pumps protein molecules and a decrease in the extent of motion of membrane lipid molecules, thus altering the regulation of [Ca(2+)](i) and myocardial contractility.

Modulation of the calmodulin-induced inhibition of sarcoplasmic reticulum calcium release channel (ryanodine receptor) by sulfhydryl oxidation in single channel current recordings and [(3)H]ryanodine binding.

The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl-sulfonic acid (4-CMPS) and 4, 4'-dithiodipyridine (4,4'-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [(3)H]ryanodine binding to HSR vesicles. 0.1 microm CaM reduced the open probability (P(o)) of the calcium release channel at maximally activating calcium concentrations (50-100 microm) from 0.502 +/- 0.02 to 0.137 +/- 0.022 (n = 28), with no effect on unitary conductance. 4-CMPS (10-40 microm) and 4,4'-DTDP (0.1-0.3 mm) induced a concentration dependent increase in P(o) (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4'-DTDP to higher concentrations in single channel recordings and [(3)H]ryanodine binding. 40 microm 4-CMPS induced a near maximal (P(o) > 0.9) and 0.3 mm 4,4'-DTDP a submaximal (P(o) = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4'-DTDP affected Ca-[(125)I]calmodulin binding to HSR. 1 mm MgCl(2) reduced P(o) from 0.53 to 0.075 and 20-40 microm 4-CMPS induced a near maximal channel activation (P(o) > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4'-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel.

Antagonistic effect of ganglioside GM1 and GM3 on the activity and conformation of sarcoplasmic reticulum Ca 2+-ATPase

It was found that rabbit skeletal muscle sarcoplasmic reticulum (SR) contained two main gangliosides: NeuNAcα 2→3 Galβ 1→4 Glcβ 1→1′ceramide (GM3) and Galβ 1→3 GalNAcβ 1→4(NeuNAcα 2→3) Galβ 1→4 Glcβ 1→1′ceramide (GM1), and that the most abundant ganglioside GM3 could positively modulate the SR Ca 2+-ATPase activity. In this paper, the effect of GM1 on Ca 2+-ATPase was further investigated and compared with that of GM3. The study demonstrates that GM1 has an opposite effect with respect to GM3 on the activity of SR Ca 2+-ATPase. Using assays, including intrinsic and time-resolved fluorescence and fluorescence quenching, the conformational changes of SR Ca 2+-ATPase induced by GM1 and GM3 were compared. Obtained results indicate that GM1 could make the Ca 2+-ATPase molecules less compact in the hydrophilic domain but more compact in the hydrophobic domain, while GM3 makes the enzyme more compact in both the hydrophilic and hydrophobic domain. Homogeneous GM1 and GM3 with the same ceramide moiety had similar effects on SR Ca 2+-ATPase activities compared to their natural counterparts, suggesting that the carbohydrate chain may be the key moiety of the ganglioside molecule to be responsible for the difference of the effect on enzyme activity.

GroEL protects the sarcoplasmic reticulum Ca(++)-dependent ATPase from inactivation in vitro.

The molecular chaperone, GroEL, facilitates correct protein folding and inhibits protein aggregation. The function of GroEL is often, though not invariably, dependent on the co-chaperone, GroES, and ATP. In this study it is shown that GroEL alone substantially reduces the inactivation of purified Ca(++)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum. In the absence of GroEL, the enzyme became completely inactive in about 45-60 hours when kept at 25 degrees C, while in the presence of an equimolar amount of GroEL, the enzyme remained approximately 80% active even after 75 hours. Equimolar amounts of BSA or lysozyme were unable to protect the enzyme from inactivation under identical conditions. Analysis by SDS-PAGE showed GroEL was acting by blocking the aggregation of ATPase at 25 degrees C. GroEL was not as effective in protection at -20 degrees C or 4 degrees C. These results are discussed in the context of current models of the GroEL mechanism.

Functional Consequences of Mutations of Conserved, Polar Amino Acids in Transmembrane Sequences of the Ca2+ Release Channel (Ryanodine Receptor) of Rabbit Skeletal Muscle Sarcoplasmic Reticulum

The potential role in Ca2+ release channel function of highly conserved, polar, and small amino acids in predicted transmembrane sequences in the rabbit skeletal muscle ryanodine receptor (RyR1) was investigated through mutagenesis. Acidic amino acids Asp3987, Glu4032, Asp4815, Asp4917, Asp4938, and Asp4969 and amidated residues Asn4034, Asn4037, Asn4574, Asn4805, Asn4806, and Gln4933, and Gly4033 were mutated to Ala, and Ala3988 was mutated to Val. When expressed in HEK-293 cells and challenged with either caffeine or 4-chloro-m-cresol, mutants E4032A, N4806A, D4815A, and D4917A did not respond, indicating that Ca2+ release channel function was impaired. None of these mutants exhibited specific binding of [3H]ryanodine. Mutants N4805A and Q4933A showed a diminished response to both caffeine and 4-chloro-m-cresol, but [3H]ryanodine binding was not altered. Other mutant responses and the responses of mutants E4032D, N4806Q or D, D4815N or E, and D4938N or E were unaltered when compared with RyR1. However, mutants E4032Q, D4917N or E, and Q4933N or E displayed neither caffeine nor 4-chloro-m-cresol response nor [3H]ryanodine binding. Sedimentation assays indicated that the nonfunctional mutants did contain tetrameric complexes, implying that defects in the assembly of a functional channel did not occur with specific mutations in transmembrane sequences. These results support the view that amino acids Glu4032 (M2), Asn4806 (M7), Asp4815 (M7), Asp4917 (M10), and Gln4933 (M10) are involved in channel function and regulation.

Identification of 30 kDa calsequestrin-binding protein, which regulates calcium release from sarcoplasmic reticulum of rabbit skeletal muscle.

In a previous study [Yamaguchi, Kawasaki and Kasai (1995) Biochem. Biophys. Res. Commun. 210, 648-653], we showed that the stilbene derivative 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid activates the Ca2+ channel in the sarcoplasmic reticulum (SR) in rabbit skeletal muscle, and it does not bind to the channel protein itself but to the SR 30 kDa protein. Furthermore, the 30 kDa protein was shown to bind to calsequestrin (CSQ), which is one of the regulators of the Ca2+ release channel in the SR. In the present study, we determined the partial amino acid sequence of the CSQ-binding 30 kDa protein and, consequently, this protein was proved to be highly similar to ADP/ATP translocase (AAT) expressed in the mitochondria in a variety of cells. By Western-blotting analysis, the CSQ-binding 30 kDa protein was recognized by the antibody raised against bovine cardiac AAT and, furthermore, depolarization-induced Ca2+ release monitored in the rabbit skeletal muscle triads was significantly activated by the antibody. As a result of cloning and sequencing of the cDNA encoding AAT of the rabbit skeletal muscle, the amino acid sequence was found to be the same as that of the CSQ-binding 30 kDa protein determined above. Furthermore, the expressed product of the cDNA encoding AAT in Escherichia coli was proved to bind to CSQ. These results suggest that AAT itself is expressed in the rabbit skeletal muscle SR and regulates the Ca2+ release from the SR; that is, excitation-contraction coupling of the skeletal muscle cell.

Biochemical mechanisms of calcium mobilisation induced by mastoparan and chimeric hormonewmastoparan constructs

Ca 2+ efflux, Ca 2+-ATPase, and membrane permeability measurements were used to investigate the biochemical mechanisms of Ca 2+ release induced by mastoparan (MP) and the chimeric hormone-MP constructs incorporating galanin (galparan) or vasopressin antagonist (M375 and M391) moieties. Comparative studies utilised preparations of porcine cerebellar microsomes and rabbit skeletal muscle sarcoplasmic reticulum (SR). MP and chimeric peptides galparan, M375 and M391 induce Ca 2+ release over a range of concentrations from 0.3–10 μM. Comparison of MP and three chimeric, N-terminal extended, constructs indicates that N-terminal extension modifies the biological properties of MP, producing changes in efficacy which are enzyme-isoform-specific. Biochemical studies indicate that the chimeric analogues and MP inhibit Ca 2+-ATPases and directly activate the ryanodine receptor (RyR) to release Ca 2+ from both heavy SR (HSR) and microsomes. The same peptides have no effect on the InsP 3 receptor (InsP 3R). Other actions that include modest changes in membrane permeability may also contribute to the Ca 2+-mobilising action of MP and chimeric constructs.

Crystal structure of calsequestrin from rabbit skeletal muscle sarcoplasmic reticulum.

Calsequestrin, the major Ca2+ storage protein of muscle, coordinately binds and releases 40-50 Ca2+ ions per molecule for each contraction-relaxation cycle by an uncertain mechanism. We have determined the structure of rabbit skeletal muscle calsequestrin. Three very negative thioredoxin-like domains surround a hydrophilic center. Each monomer makes two extensive dimerization contacts, both of which involve the approach of many negative groups. This structure suggests a mechanism by which calsequestrin may achieve high capacity Ca2+ binding. The suggested mechanism involves Ca2+-induced collapse of the three domains and polymerization of calsequestrin monomers arising from three factors: N-terminal arm exchange, helix-helix contacts and Ca2+ cross bridges. This proposed structure-based mechanism accounts for the observed coupling of high capacity Ca2+ binding with protein precipitation.

Single-channel properties of the recombinant skeletal muscle Ca2+ release channel (ryanodine receptor)

We report transient expression of a full-length cDNA encoding the Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum (ryanodine receptor) in HEK-293 cells. The single-channel properties of the 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate-solubilized and sucrose gradient-purified recombinant Ca2+ release channels were investigated by using single-channel recordings in planar lipid bilayers. The recombinant Ca2+ release channel exhibited a K+ conductance of 780 pS when symmetrical 250 mM KCl was used as the conducting ion and a Ca2+ conductance of 116 pS in 50 mM luminal Ca2+. Opening events of the recombinant channels were brief, with an open time constant of approximately 0.22 ms. The recombinant Ca2+ release channel was more permeable to Ca2+ than to K+, with a pCa2+/pK+ ratio of 6.8. The response of the recombinant Ca2+ release channel to various concentrations of Ca2+ was biphasic, with the channel being activated by micromolar Ca2+ and inhibited by millimolar Ca2+. The recombinant channels were activated by ATP and caffeine, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Most recombinant channels were asymmetrically blocked, conducting current unidirectionally from the luminal to the cytoplasmic side of the channel. These data demonstrate that the properties of recombinant Ca2+ release channel expressed in HEK-293 cells are very similar, if not identical, to those of the native channel.

Inositol polyphosphates modify the kinetics of a small chloride channel in skeletal muscle sarcoplasmic reticulum.

The actions of D-myo-inositol 1,4,5-trisphosphate (IP3) and D-myo-inositol 1,3,4,5-tetrakisphospate (IP4) on small chloride (SCl) channels from rabbit skeletal muscle sarcoplasmic reticulum are reported. We find that the inositol polyphosphates (6-40 microM) are potent reversible blockers of SCl channels in lipid bilayers at -40 mV with >10(-5) m cis (cytoplasmic) Ca2+ when added to the cis, but not trans, chamber. IP3 or IP4 at 20 microM reduced the mean open time from 89 +/- 16 msec to 11 +/- 2 msec or to 8.0 +/- 1.0 msec respectively, by abolishing the longest time constant component in the open time distribution. Neither IP3 nor IP4 altered the six single-channel conductance levels. The fraction of low conductance events increased approximately 4-fold and the dwell time at the lower conductance levels increased approximately 3-fold. Channel gating was altered so that most transitions were between the closed level and an open level, in contrast to control channels which remained open for long periods with many transitions between the six open levels. The actions of the inositol polyphosphates were: (1) not prevented by 20 microg/ml cis heparin (an IP3 receptor blocker); (2) mimicked by 10 microM cis synthetic inositol polyphosphates, L-chiro-inositol 1,4,6-trisphosphate and L-chiro-inositol 1,4,6-trisphosphorothionate (which do not bind to IP3 receptors); (3) mimicked by cis additions of the polyanions heparin or hepran (20 microg/ml each) and vanadate (500 microM). The results suggest that an interaction between polyanions and SCl channels would allow the channels to be modulated in vivo by inositol polyphosphates.

P-173 - Analysis of inhibitory effect of Bromostyloguanidine on Ca2+-induced Ca2+ release channels in rabbit skeletal muscle sarcoplasmic reticulum

P-173 - Analysis of inhibitory effect of Bromostyloguanidine on Ca2+-induced Ca2+ release channels in rabbit skeletal muscle sarcoplasmic reticulum

Chloride-induced Ca2+ release from the sarcoplasmic reticulum of chemically skinned rabbit psoas fibers and isolated vesicles of terminal cisternae.

There is increasing evidence that Ca2+ release from sarcoplasmic reticulum (SR) of mammalian skeletal muscle is regulated or modified by several factors including ionic composition of the myoplasm. We have studied the effect of Cl- on the release of Ca2+ from the SR of rabbit skeletal muscle in both skinned psoas fibers and in isolated terminal cisternae vesicles. Ca2+ release from the SR in skinned fibers was inferred from increases in isometric tension and the amount of release was assessed by integrating the area under each tension transient. Ca2+ release from isolated SR was measured by rapid filtration of vesicles passively loaded with 45Ca2+. Ca2+ release from SR was stimulated in both preparations by exposure to a solution containing 191 mm choline-Cl, following pre-equilibration in Ca2+-loading solution that had propionate as the major anion. Controls using saponin (50 microg/ml), indicated that the release of Ca2+ was due to direct action of Cl- on the SR rather than via depolarization of T-tubules. Procaine (10 mM) totally blocked Cl-- and caffeine-elicited tension transients recorded using loading and release solutions having ([Na+] + [K+]) x [Cl-] product of 6487.69 mm2 and 12361.52 mm2, respectively, and blocked 60% of Ca2+ release in isolated SR vesicles. Surprisingly, procaine had only a minor effect on tension transients elicited by Cl- and caffeine together. The data from both preparations suggests that Cl- induces a relatively small amount of Ca2+ release from the SR by activating receptors other than RYR-1. In addition, Cl- may increase the Ca2+ sensitivity of RYR-1, which would then allow the small initial release of Ca2+ to facilitate further release of Ca2+ from the SR by Ca2+-induced Ca2+ release.

Purification and characterization of ryanotoxin, a peptide with actions similar to those of ryanodine

We purified and characterized ryanotoxin, an approximately 11.4-kDa peptide from the venom of the scorpion Buthotus judiacus that induces changes in ryanodine receptors of rabbit skeletal muscle sarcoplasmic reticulum analogous to those induced by the alkaloid ryanodine. Ryanotoxin stimulated Ca2+ release from sarcoplasmic reticulum vesicles and induced a state of reduce unit conductance with a mean duration longer than that of unmodified ryanodine receptor channels. With Cs+ as the current carrier, the slope conductance of the state induced by 1 microM ryanotoxin was 163 +/- 12 pS, that of the state induced by 1 microM ryanodine was 173 +/- 26 pS, and that of control channels was 2.3-fold larger (396 +/- 25 pS). The distribution of substate events induced by 1 microM RyTx was biexponential and was fitted with time constants approximately 10 times shorter than those fitted to the distribution of substates induced by 1 microM ryanodine. Bath-applied 5 microM ryanotoxin had no effect on the excitability of mouse myotubes in culture. When 5 microM ryanotoxin was dialyzed into the cell through the patch pipette in the whole-cell configuration, there was a voltage-dependent increase in the amplitude of intracellular Ca2+ transients elicited by depolarizing potentials in the range of -30 to +50 mV. Ryanotoxin increased the binding affinity of [3H]ryanodine in a reversible manner with a 50% effective dose (ED50) of 0.16 microM without altering the maximum number (Bmax) of [3H]ryanodine-binding sites. This result suggested that binding sites for ryanotoxin and ryanodine were different. Ryanotoxin should prove useful in identifying domains coupling the ryanodine receptor to the voltage sensor, or domains affecting the gating and conductance of the ryanodine receptor channel.

Selective binding of FKBP12.6 by the cardiac ryanodine receptor.

The calcium release channels (CRC)/ryanodine receptors of skeletal (Sk) and cardiac (C) muscle sarcoplasmic reticulum (SR) are hetero-oligomeric complexes with the structural formulas (ryanodine recepter (RyR)1 protomer)4(FKBP12)4 and (RyR2 protomer)4(FKBP12.6)4, respectively, where FKBP12 and FKBP12.6 are isoforms of the 12-kDa receptor for the immunosuppressant drug FK506. The sequence similarity between the RyR protomers and FKBP12 isoforms is 63 and 85%, respectively. Using 35S-labeled FKBP12 and 35S-labeled FKBP12.6 as probes to study the interaction with CRC, we find that: 1) analogous to its action in skeletal muscle sarcoplasmic reticulum (SkMSR), FK506 (or analog FK590) dissociates FKBP12.6 from CSR; 2) both FKBP isoforms bind to FKBP-stripped SkMSR and exchange with endogenously bound FKBP12 of SkMSR; and 3) by contrast, only FKBP12. 6 exchanges with endogenously bound FKBP12.6 or rebinds to FKBP-stripped CSR. This selective binding appears to explain why the cardiac CRC is isolated as a complex with FKBP12.6, whereas the skeletal muscle CRC is isolated as a complex with FKBP12, although only FKBP12 is detectable in the myoplasm of both muscle types. Also, in contrast to the activation of the channel by removal of FKBP from skeletal muscle, no activation is detected in CRC activity in FKBP-stripped CSR. This differential action of FKBP may reflect a fundamental difference in the modulation of excitation-contraction coupling in heart versus skeletal muscle.