FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2059320957> ?p ?o ?g. }

• W2059320957 endingPage "32691" @default.
• W2059320957 startingPage "32680" @default.
• W2059320957 abstract "The effects of ruthenium red (RR) on the skeletal and cardiac muscle ryanodine receptors (RyRs) were studied in vesicle-Ca2+ flux, [3H]ryanodine binding, and single channel measurements. In vesicle-Ca2+flux measurements, RR was more effective in inhibiting RyRs at 0.2 μm than 20 μm free Ca2+. [3H]Ryanodine binding measurements suggested noncompetitive interactions between RR inhibition and Ca2+regulatory sites of RyRs. In symmetric 0.25 m KCl with 10–20 μm cytosolic Ca2+, cytosolic RR decreased single channel activities at positive and negative holding potentials. In close to fully activated skeletal (20 μmCa2+ + 2 mm ATP) and cardiac (200 μm Ca2+) RyRs, cytosolic RR induced a predominant subconductance at a positive but not negative holding potential. Lumenal RR induced a major subconductance in cardiac RyR at negative but not positive holding potentials and several subconductances in skeletal RyR. The RR-related subconductances of cardiac RyR showed a nonlinear voltage dependence, and more than one RR molecule appeared to be involved in their formation. Cytosolic and lumenal RR also induced subconductances in Ca2+-conducting skeletal and cardiac RyRs recorded at 0 mV holding potential. These results suggest that RR inhibits RyRs and induces subconductances by binding to cytosolic and lumenal sites of skeletal and cardiac RyRs. The effects of ruthenium red (RR) on the skeletal and cardiac muscle ryanodine receptors (RyRs) were studied in vesicle-Ca2+ flux, [3H]ryanodine binding, and single channel measurements. In vesicle-Ca2+flux measurements, RR was more effective in inhibiting RyRs at 0.2 μm than 20 μm free Ca2+. [3H]Ryanodine binding measurements suggested noncompetitive interactions between RR inhibition and Ca2+regulatory sites of RyRs. In symmetric 0.25 m KCl with 10–20 μm cytosolic Ca2+, cytosolic RR decreased single channel activities at positive and negative holding potentials. In close to fully activated skeletal (20 μmCa2+ + 2 mm ATP) and cardiac (200 μm Ca2+) RyRs, cytosolic RR induced a predominant subconductance at a positive but not negative holding potential. Lumenal RR induced a major subconductance in cardiac RyR at negative but not positive holding potentials and several subconductances in skeletal RyR. The RR-related subconductances of cardiac RyR showed a nonlinear voltage dependence, and more than one RR molecule appeared to be involved in their formation. Cytosolic and lumenal RR also induced subconductances in Ca2+-conducting skeletal and cardiac RyRs recorded at 0 mV holding potential. These results suggest that RR inhibits RyRs and induces subconductances by binding to cytosolic and lumenal sites of skeletal and cardiac RyRs. sarcoplasmic reticulum ryanodine receptor skeletal muscle RyR cardiac muscle RyR ruthenium red channel open probability in the absence of a substate open probability of full conductance events in the presence of a substate open probability of RR-related subconductances sum of Pfull and Psub adenosine 5′-(β,γ-methylenetriphosphate) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate The release and sequestration of Ca2+ ions by the sarcoplasmic reticulum (SR),1an intracellular membrane compartment, is essential to the process of cardiac and skeletal muscle contraction and relaxation. The rapid release of Ca2+ is mediated by Ca2+ release channels, also known as ryanodine receptors (RyRs), because they bind the plant alkaloid ryanodine with high affinity and specificity (1Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (592) Google Scholar, 2Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 1-51PubMed Google Scholar, 3Sutko J.L. Airey J.A. Physiol. Rev. 1996; 76: 1027-1071Crossref PubMed Scopus (365) Google Scholar, 4Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 5Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (842) Google Scholar, 6McPherson P.S. Campbell K.P. J. Biol. Chem. 1993; 268: 13765-13768Abstract Full Text PDF PubMed Google Scholar). Skeletal and cardiac muscles express two major isoforms of the RyR, RyR1 and RyR2, respectively. In striated muscles, RyRs are concentrated in the junctional SR membrane near transverse tubular, voltage-sensitive l-type Ca2+ channels (dihydropyridine receptors). A muscle action potential initiates dihydropyridine receptor conformational changes that activate the RyRs via a direct physical interaction in skeletal muscle or mediate the influx of Ca2+ in cardiac muscle, leading to the release of Ca2+ from the SR and subsequent muscle contraction. Both RyRs have been isolated as 30 S protein complexes composed of four 560-kDa (RyR polypeptide) and four 12-kDa (FK506-binding protein) subunits (1Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (592) Google Scholar, 2Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 1-51PubMed Google Scholar, 3Sutko J.L. Airey J.A. Physiol. Rev. 1996; 76: 1027-1071Crossref PubMed Scopus (365) Google Scholar, 4Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 5Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (842) Google Scholar). The two channel activities are affected by endogenous and exogenous effectors, such as Ca2+, Mg2+, ATP, caffeine, ryanodine, and ruthenium red. Ruthenium red (RR) is one of the most potent inhibitors of SR Ca2+ release (2Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 1-51PubMed Google Scholar). RR also inhibits mitochondrial Ca2+ uptake. However, this activity was due to a contaminant (7Reed K.C. Bygrave F.L. FEBS Lett. 1974; 46: 109-114Crossref PubMed Scopus (27) Google Scholar) that may be related to an oxygen-bridged R360 complex that, at concentrations as high as 10 μm, was without effect on SR Ca2+ uptake or release (8Matlib A.M. Zhou Z. Knight S. Ahmed S. Choi K.M. Krause-Bauer J. Phillips R. Altschuld R. Katsube Y. Sperelakis N. Bers D.M. J. Biol. Chem. 1998; 273: 10223-10231Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). RR is a polycationic dye with a linear structure consisting of three ruthenium atoms with a net valence of 6. In SR vesicles, RR increased the rate of Ca2+ uptake and decreased the rate of Ca2+release at concentrations ranging from 1 nm to 20 μm (9Ohnishi S.T. J. Biochem. 1979; 86: 1147-1150Crossref PubMed Scopus (81) Google Scholar, 10Miyamoto H. Racker E. FEBS Lett. 1981; 133: 235-238Crossref PubMed Scopus (39) Google Scholar, 11Kim D.H. Ohnishi S.T. Ikemoto N. J. Biol. Chem. 1983; 258: 9662-9668Abstract Full Text PDF PubMed Google Scholar, 12Chamberlain B. Volpe P. Fleischer S. J. Biol. Chem. 1984; 259: 7547-7553Abstract Full Text PDF PubMed Google Scholar, 13Chu A. Volpe P. Costello B. Fleischer S. Biochemistry. 1986; 25: 8315-8324Crossref PubMed Scopus (31) Google Scholar, 14Meissner G. Henderson J.S. J. Biol. Chem. 1987; 262: 3065-3073Abstract Full Text PDF PubMed Google Scholar, 15Palade P. J. Biol. Chem. 1987; 262: 6135-6141Abstract Full Text PDF PubMed Google Scholar, 16Chu A. Diaz-Munoz M. Hawkes M.J. Brush K. Hamilton S.L. Mol Pharm. 1990; 37: 735-741PubMed Google Scholar, 17Calviello G. Chiesi M. Biochemistry. 1989; 28: 1301-1306Crossref PubMed Scopus (30) Google Scholar, 18Zimanyi I. Pessah I.N. J. Pharmacol. Exp. Ther. 1991; 256: 938-946PubMed Google Scholar). In muscle fibers, RR concentrations greater than 20 μm are required to inhibit SR Ca2+release because of RR binding to myoplasmic proteins (19Baylor S.M. Hollingworth S. Marshall M.W. J. Physiol. 1989; 408: 617-635Crossref PubMed Scopus (24) Google Scholar, 20Brunder D.G. Gyorke S. Dettbarn C. Palade P. J. Physiol. 1992; 445: 759-778Crossref PubMed Scopus (13) Google Scholar). In intact single frog twitch muscle fibers, the estimated free RR concentration for half-block of SR Ca2+ release was 2.4 μm, in good agreement with the range reported for SR vesicle preparations (19Baylor S.M. Hollingworth S. Marshall M.W. J. Physiol. 1989; 408: 617-635Crossref PubMed Scopus (24) Google Scholar). In [3H]ryanodine binding measurements, RR decreased the B max value and increased the K D value, with the latter effect being due to a slower association rate (16Chu A. Diaz-Munoz M. Hawkes M.J. Brush K. Hamilton S.L. Mol Pharm. 1990; 37: 735-741PubMed Google Scholar). In single channel measurements, micromolar concentrations of RR decreased the channel open probability of the skeletal (21Smith J.S. Coronado R. Meissner G. Nature. 1985; 316: 446-449Crossref PubMed Scopus (262) Google Scholar) and cardiac (22Rousseau E. Smith J.S. Henderson J.S. Meissner G. Biophys. J. 1996; 50: 1009-1014Abstract Full Text PDF Scopus (172) Google Scholar) RyRs by producing prolonged channel closings. A different effect was observed when ryanodine was used to lock the channels into a permanently open, Ca2+-insensitive subconductance state. RR blocked single ryanodine-modified skeletal RyRs by binding in a voltage-dependent manner to multiple sites located in the conductance pore of the channel (23Ma J. J. Gen. Physiol. 1993; 102: 1031-1056Crossref PubMed Scopus (97) Google Scholar). The present study was undertaken to clarify the effects of RR on ryanodine-unmodified skeletal and cardiac RyRs. The effects of RR on RyRs were investigated in SR Ca2+ uptake, [3H]ryanodine binding, and single channel measurements using SR vesicles and purified cardiac and skeletal muscle RyRs. The results show that RR modifies the gating and conductance of the RyRs by multiple mechanisms, depending on channel activity, membrane potential, and sidedness of RR addition. [3H]Ryanodine was purchased from NEN Life Science Products. Unlabeled ryanodine was obtained from Calbiochem (San Diego, CA), ruthenium red was from Fluka (Ronkonkoma, NY), and phospholipids were from Avanti Polar Lipids (Alabaster, AL). Ryanodine and ruthenium red were prepared as concentrated stock solutions in 0.25 m KCl, 20 mm KHepes, pH 7.4, before their use. All other chemicals were of analytical grade. “Heavy” rabbit skeletal and canine cardiac muscle SR membrane fractions enriched in [3H]ryanodine binding and Ca2+ release channel activities were prepared in the presence of protease inhibitors (100 nm aprotinin, 1 μm leupeptin, 1 μm pepstatin, 1 mm benzamidine, 0.2 mm phenylmethylsulfonyl fluoride) as described (14Meissner G. Henderson J.S. J. Biol. Chem. 1987; 262: 3065-3073Abstract Full Text PDF PubMed Google Scholar, 24Meissner G. J. Biol. Chem. 1984; 259: 2365-2374Abstract Full Text PDF PubMed Google Scholar). The 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-solubilized 30 S RyR complexes were isolated by rate density gradient centrifugation and reconstituted into proteoliposomes by removal of CHAPS by dialysis (25Lee H.-B. Xu L. Meissner G. J. Biol. Chem. 1994; 269: 13305-13312Abstract Full Text PDF PubMed Google Scholar). ATP-dependent45Ca2+ uptake by SR vesicles was determined using a filtration method. Samples were incubated for 1 h at 24 °C in 0.25 m KCl, 20 mm KHepes, pH 7.4, solutions containing 50 μm Ca2+, 0.2 mm Pefabloc, and 20 μm leupeptin in the absence of ryanodine or the presence of 300 μm ryanodine to close the SR Ca2+ release channel (26Jones L.R. Cala S.E. J. Biol. Chem. 1981; 256: 11809-11818Abstract Full Text PDF PubMed Google Scholar, 27Fleischer S. Ogunbunmi E.M. Dixon M.C. Fleer E.A.M. Proc. Natl. Acad Sci. U. S. A. 1995; 82: 7256-7259Crossref Scopus (308) Google Scholar, 28Meissner G. J. Biol. Chem. 1986; 261: 6300-6306Abstract Full Text PDF PubMed Google Scholar, 29Feher J.J. LeBolt W.R. Manson N.H. Circ. Res. 1989; 65: 1400-1408Crossref PubMed Scopus (72) Google Scholar).45Ca2+ uptake was initiated at 24 °C by the addition of 10 volumes of 0.25 m KCl, 20 mmKHepes, pH 7.4, solutions containing 5 mm MgATP, 5 mm NaN3, 0.2 mm EGTA, various RR concentrations, and 45Ca2+ to yield free Ca2+ concentrations of 0.2 and 20 μm. At various times, aliquots of the samples were placed on 0.45 μm Millipore filters under vacuum and rinsed with three 1-ml volumes of a 0.25 m KCl, 5 mm KPipes, pH 6.8, solution containing 0.1 mm EGTA, 10 mm Mg2+, and 10 μm RR. Radioactivity remaining with the vesicles on the filters was counted by liquid scintillation. Unless otherwise indicated, SR vesicles were incubated for 20–24 h at 24 °C in 0.25m KCl, 20 mm KHepes, pH 7.4, solutions containing 0.2 mm Pefabloc, 20 μm leupeptin, 1 nm [3H]ryanodine, and various RR and free Ca2+ concentrations. Nonspecific [3H]ryanodine binding was determined using a 1000-fold excess of unlabeled ryanodine. Aliquots of the samples were diluted with 10 volumes of ice-cold water and placed on Whatman GF/B filters soaked with 2% polyethyleneimine. Filters were washed with three 5-ml volumes of ice-cold 0.1 m KCl, 1 mm KPipes, pH 7.0. Radioactivity remaining with the filters was determined by liquid scintillation counting to obtain bound [3H]ryanodine. Hill constants (K i ) and coefficients (n i ) of [3H]ryanodine binding inhibition by RR were determined using the following equation, B=B0/(1+([RR]/Ki)ni)Equation 1 where B is [3H]ryanodine binding at a given [RR], and B o is the binding maximum in the absence of RR. Single channel measurements were performed by fusing proteoliposomes containing the purified RyRs with Mueller-Rudin type bilayers containing phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine in the ratio 5:3:2 (25 mg of total phospholipid per ml ofn-decane) (30Tripathy A. Resch W. Xu L. Valdivia H.H. Meissner G. J. Gen. Physiol. 1998; 111: 679-690Crossref PubMed Scopus (75) Google Scholar). The side of the bilayer to which the proteoliposomes were added was designated as the cis side. The trans side was defined as ground. Single channels were recorded in symmetric KCl buffer solutions (0.25 m KCl, 10–20 mm KHepes, pH 7.4) with additions as indicated in the text. Unless otherwise indicated, electrical signals were filtered at 2–4 kHz, digitized at 10–20 kHz, and analyzed as described (30Tripathy A. Resch W. Xu L. Valdivia H.H. Meissner G. J. Gen. Physiol. 1998; 111: 679-690Crossref PubMed Scopus (75) Google Scholar). Data acquisition and analysis were performed using a commercially available software package (pClamp 6.0.3, Axon Instruments, Burlingame, CA) and an IBM-compatible computer (Pentium processor) with 12-bit analog/digital-digital/analog converter (Digidata 1200, Axon Instruments). In the absence of RR-related subconductance states, channel open probability in the absence of a substate (P o) was obtained by setting the threshold level at 50% of the current amplitude between the closed (c) and open (o) states. P o values in multichannel recordings were calculated according to the equation P o = Σ iP i /N, where N is the total number of channels, and P i is the ichannel open probability. In some conditions, RR formed a predominant reduced conductance level (see Fig. 9 A, right panel, bottom trace, s). RR formed additional subconductance states, but with some exceptions (see Figs. 6 D and 7 B), these occurred infrequently and were not further analyzed. The concentration and voltage dependence of the RR-related subconductances were obtained by determining the open probability of the full conductance events (P full), RR-related subconductance events (P sub), and the sum ofP full and P sub(P tot) (31Tinker A. Lindsay A.R.G. Williams A.J. Biophys. J. 1992; 61: 1122-1132Abstract Full Text PDF PubMed Scopus (33) Google Scholar). P full was obtained by setting the threshold level at 50% current amplitude between subconductance (s) and open (o) current levels (see Fig. 9 A, right panel, bottom trace). This measurement eliminated inclusion of substates inP full. P tot was obtained by setting the threshold level at 50% of the current between the close (c) and subconductance (s) current levels (see Fig. 9 A, right panel, bottom trace). The open probability of the subconductance state, P sub, was obtained by the equation P sub = P tot− P full.Figure 6Effects of cytosolic and lumenal RR on single RyR1s. Single channel currents were recorded at −40 mV (left panels, downward deflections from closed levels,c) and +40 mV (right panels, upward deflections) in symmetric 0.25 m KCl, 10 mm KHepes, pH 7.4, medium containing the indicated concentrations of free cytosolic Ca2+ and ATP before (A–D, top traces) and after the addition of the indicated concentrations of cytosolic or lumenal RR. Lumenal (contaminant) Ca2+ was ∼4 μm.A and B, effect of cytosolic RR on channel activity of two RyR1s at 20 μm (A) and 200 μm (B) free Ca2+. C,effect of cytosolic RR on channel activity of single RyR1 at 20 μm free Ca2+ and 2 mm ATP. RR induced four substates with conductances of 12.5, 28, 45, and 60% of the control conductance in a voltage-dependent manner. The substate with a conductance of 45% of the control was the predominant one. D, effect of lumenal RR on channel activity of single RyR1 at 20 μm free cytosolic Ca2+. Lumenal RR induced three voltage-dependent substates with conductances of 85, 55, and 25% of the control conductance.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Effects of RR on Ca2+-conducting RyR1 at 0 mV. Single channel currents were recorded at 0 mV (downward deflections from closed levels, c and dotted lines) in symmetric 0.25 m KCl, pH 7.4, solutions containing 10 mm lumenal Ca2+ and the indicated cytosolic Ca2+, Mg2+, and ATP concentrations and cytosolic (A and C) or lumenal (B) RR concentration. Each panel shows one of three or four similar recordings. Single channel currents were filtered at 300 Hz to decrease noise.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the absence of substates, the Hill inhibition constant (K i ) and inhibition coefficient (n i ) of P o were determined by the following equation, P0=P0,max/(1+([RR]/Ki)ni)Equation 2 where P o,max is P oin the absence of RR. In the presence of substates, the Hill association constant (K a ) and coefficient (n a) of P sub state were determined by the following equation. Psub=Ptot/(1+(Ka/[RR])na)Equation 3 Assuming a Boltzman distribution between theP full and P sub states, their voltage dependence was described by the following equation, Psub/Pfull=exp[(ZtotFV−Gi)/RT]Equation 4 where Z tot is the effective gating charge of the reaction of RR with the RyR. The other terms have their usual meanings. Free Ca2+ concentrations ⋝1 μm were determined using a Ca2+ selective electrode (Nico Scientific, Philadelphia, PA). Free Ca2+ concentrations of <1 μm were obtained by including in the solutions the appropriate amounts of Ca2+ and EGTA as determined using the stability constants and the mixed solution program published by Schoenmakers et al. (32Schoenmakers J.M. Visser G.J. Flick G. Theuvene A.P.R. BioTechniques. 1992; 12: 870-879PubMed Google Scholar). The effects of RR on RyR ion channel activity were assessed in SR vesicle-45Ca2+ uptake measurements. Skeletal muscle SR vesicles of high buoyant density (previously designated heavy SR vesicles, Ref. 24Meissner G. J. Biol. Chem. 1984; 259: 2365-2374Abstract Full Text PDF PubMed Google Scholar) were actively loaded in 0.25 m KCl medium containing 0.2 μm (Fig.1 A) and 20 μm(Fig. 1 B) free 45Ca2+. The SR Ca2+ pump is present in these vesicles, but only 70–90% have RyR1 (24Meissner G. J. Biol. Chem. 1984; 259: 2365-2374Abstract Full Text PDF PubMed Google Scholar). To prevent release of sequestered45Ca2+, RyR1 was fully closed prior to45Ca2+ uptake by incubation for 1 h with 300 μm ryanodine (26Jones L.R. Cala S.E. J. Biol. Chem. 1981; 256: 11809-11818Abstract Full Text PDF PubMed Google Scholar, 27Fleischer S. Ogunbunmi E.M. Dixon M.C. Fleer E.A.M. Proc. Natl. Acad Sci. U. S. A. 1995; 82: 7256-7259Crossref Scopus (308) Google Scholar, 28Meissner G. J. Biol. Chem. 1986; 261: 6300-6306Abstract Full Text PDF PubMed Google Scholar, 29Feher J.J. LeBolt W.R. Manson N.H. Circ. Res. 1989; 65: 1400-1408Crossref PubMed Scopus (72) Google Scholar). In the absence of RR, pretreatment with ryanodine increased the levels of45Ca2+ sequestered by the vesicles 3-fold in uptake medium containing 0.2 or 20 μm free Ca2+. In ryanodine-untreated vesicles at 0.2 μm Ca2+, 1 μm RR doubled the amount of 45Ca2+ taken up by the vesicles. 10 μm RR was nearly as effective as pretreatment with ryanodine in increasing 45Ca2+ uptake. By contrast, at 20 μm Ca2+, 50 μmRR was required to achieve Ca2+ uptake levels approaching those observed in ryanodine-treated vesicles. RR did not increase45Ca2+ uptake by vesicles pretreated with ryanodine. For ryanodine-treated vesicles, a small decrease in the45Ca2+ uptake rate was observed in the presence of 50 μm RR, in agreement with the suggestion that high levels of RR inhibit the SR Ca2+ pump (33Kargacin G.J. Ali Z. Kargacin M.E. Pfluegers Arch. 1998; 436: 338-342Crossref PubMed Scopus (26) Google Scholar). The effects of RR on Ca2+ uptake levels of ryanodine-treated and -untreated skeletal and cardiac SR vesicles were compared. 45Ca2+ uptake levels were determined after an uptake period of 2 min. Cardiac SR contained a smaller proportion of vesicles responding to treatment with ryanodine (Fig.2), in accordance with a 4–5-fold lowerB max value of [3H]ryanodine binding for cardiac than skeletal SR vesicles (see Fig. 5). Ryanodine significantly increased 45Ca2+ uptake or cardiac SR vesicles at 20 μm Ca2+(p < 0.05; n = 4) but not at 0.2 μm Ca2+ (p = 0.1). In ryanodine-untreated SR vesicles, RR was more effective at 0.2 μm than at 20 μm Ca2+ in raising 45Ca2+ uptake to levels in the ryanodine-treated vesicles. In agreement with previous vesicle ion flux measurements (9Ohnishi S.T. J. Biochem. 1979; 86: 1147-1150Crossref PubMed Scopus (81) Google Scholar, 10Miyamoto H. Racker E. FEBS Lett. 1981; 133: 235-238Crossref PubMed Scopus (39) Google Scholar, 11Kim D.H. Ohnishi S.T. Ikemoto N. J. Biol. Chem. 1983; 258: 9662-9668Abstract Full Text PDF PubMed Google Scholar, 12Chamberlain B. Volpe P. Fleischer S. J. Biol. Chem. 1984; 259: 7547-7553Abstract Full Text PDF PubMed Google Scholar, 13Chu A. Volpe P. Costello B. Fleischer S. Biochemistry. 1986; 25: 8315-8324Crossref PubMed Scopus (31) Google Scholar, 14Meissner G. Henderson J.S. J. Biol. Chem. 1987; 262: 3065-3073Abstract Full Text PDF PubMed Google Scholar, 15Palade P. J. Biol. Chem. 1987; 262: 6135-6141Abstract Full Text PDF PubMed Google Scholar, 16Chu A. Diaz-Munoz M. Hawkes M.J. Brush K. Hamilton S.L. Mol Pharm. 1990; 37: 735-741PubMed Google Scholar, 17Calviello G. Chiesi M. Biochemistry. 1989; 28: 1301-1306Crossref PubMed Scopus (30) Google Scholar, 18Zimanyi I. Pessah I.N. J. Pharmacol. Exp. Ther. 1991; 256: 938-946PubMed Google Scholar), the results indicate that the skeletal and cardiac muscle RyRs are inhibited by RR. The data in Fig. 2 further show that the extent of inhibition depends on the Ca2+concentration of the uptake medium. Two possible reasons for this Ca2+ dependence are that SR Ca2+ pump activity is dependent on Ca2+ concentration and/or that Ca2+ ions compete with RR for the Ca2+regulatory sites on the RyRs.Figure 5Scatchard analysis of [3H]ryanodine binding to skeletal muscle (A) and cardiac muscle (B) SR vesicles in the presence and absence of RR. Specific [3H]ryanodine binding was determined as described under “Experimental Procedures” in 0.25 m KCl medium containing 0.5–50 nm [3H]ryanodine and the indicated concentrations of RR, AMPPCP, and free Ca2+.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The highly specific plant alkaloid ryanodine was used to obtain information on the mechanism of RR inhibition of RyR1 and RyR2 independent of SR Ca2+ pump activity. [3H]Ryanodine binding is widely used as a probe of channel activity because of its preferential binding to open RyR ion channel states (1Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (592) Google Scholar, 2Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 1-51PubMed Google Scholar, 3Sutko J.L. Airey J.A. Physiol. Rev. 1996; 76: 1027-1071Crossref PubMed Scopus (365) Google Scholar, 4Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 5Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (842) Google Scholar). Fig. 3 compares the Ca2+ dependence of [3H]ryanodine binding to skeletal and cardiac muscle SR vesicles in 0.25 m KCl solutions containing varying concentrations of RR. In the absence of RR, two bell-shaped activation/inactivation curves were obtained. In agreement with previous reports (1Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (592) Google Scholar, 2Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 1-51PubMed Google Scholar, 3Sutko J.L. Airey J.A. Physiol. Rev. 1996; 76: 1027-1071Crossref PubMed Scopus (365) Google Scholar, 4Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 5Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (842) Google Scholar), the binding data indicate that RyR1 and RyR2 are activated by Ca2+ binding to cytosolic high affinity receptor sites and inhibited by Ca2+ binding to low affinity sites, with higher Ca2+ concentrations required to inhibit RyR2. Higher RR concentrations were required to inhibit [3H]ryanodine binding to RyR2 than RyR1 (Fig. 3). RR decreased the amounts of [3H]ryanodine bound without markedly changing the Ca2+ dependence of [3H]ryanodine binding. The effects of RR concentration on [3H]ryanodine binding were determined at 2.5, 15, and 200 μm Ca2+and at 15 μm free Ca2+ in the presence of the nonhydrolyzable ATP analog AMPPCP. We also tested conditions comparable to those in Figs. 1 and 2 by using solutions that contained 5 mm MgAMPPCP and 20 μm or 200 μmfree Ca2+. In the absence of RR, the highest binding of [3H]ryanodine to RyR1 was observed at 15 μmfree Ca2+ and 5 mm AMPPCP (Fig.4 A) and to RyR2 at 200 μm Ca2+ (Fig. 4 B). The results agree with single channel measurements that μmCa2+ and mm ATP optimally activate RyR1, whereas RyR2 is nearly fully activated by μmCa2+ alone (see Figs. 6 C and 9 A). RR inhibited [3H]ryanodine binding to RyR1 and RyR2 in a concentration-dependent manner (Fig. 4). Solid lines in Fig. 4 were obtained by fitting the [3H]ryanodine binding data to Equation 1. The average Hill inhibition constants (K i ) and coefficients (n i ) (TableI) indicate that RR was more effective in inhibiting [3H]ryanodine binding at 2.5 μmCa2+ than at 15 μm or 200 μmCa2+. The binding data could not be fitted when it was assumed that RR inhibited [3H]ryanodine binding by competing with Ca2+ for the Ca2+ activation sites via a competitive mechanism (not shown). RR was least effective in inhibiting the RyRs in 15 μm Ca2+ medium containing 5 mm AMPPCP (Fig. 4 and Table I). Similarly, the presence of 5 mm MgAMPPCP decreased the effectiveness of RR in inhibiting [3H]ryanodine binding to RyR1 and RyR2. Scatchard analysis of two sets of binding data from Fig. 4 indicated that RR inhibited [3H]ryanodine binding by decreasing theB max and K D values (Fig.5). Taken together, the [3H]ryanodine binding data suggest that RR inhibits RyR1 and RyR2 by noncompetitive interactions between RyR Ca2+regulatory and RR inhibition sites.Table IEffects of RR on [ 3 H]ryanodine binding to RyR1 and RyR2ConditionRyR1RyR2K in iK in inmnm2.5 μm Ca2+49 ± 16 (3)1.5 ± 0.3 (3)16 ± 2 (3)1.0 ± 0.1 (3)15 μm Ca2+91 ± 44 (5)1.3 ± 0.2 (5)156 ± 103 (6)1.1 ± 0.2 (6)200 μm Ca2+109 ± 49 (5)1.3 ± 0.2 (5)434 ± 187 (8)1.1 ± 0.3 (8)15 μm Ca2+ + 5 mmAMPPCP2970 ± 1620 (3)1.5 ± 0.2 (3)5920 ± 390 (3)0.7 ± 0.1 (3)20 μm Ca2++ 5 mm MgAMPPCP660 ± 240 (3)1.1 ± 0.2 (3)3420 ± 1000 (3)1.0 ± 0.2 (3)200 μm Ca2+ + 5 mmMgAMPPCP—1-a—, not determined.—4150 ± 1300 (3)0.9 ± 0.2 (3)Hill inhibition constants (K i ) and coefficients (n i ) were obtained as indicated in the legend to Fig. 4. Values are the mean ± S.E. of the number of experiments shown in parentheses.1-a —, not determined. Open table in a new tab Hill inhibition constants (K i ) and coefficients (n i ) were obtained as indicated in the legend to Fig. 4. Values are the mean ± S.E. of the number of experiments shown in parentheses. The kinetics of RR inhibition of the RyRs were further examined in single channel measurements. Proteoliposomes containing purified RyR were fused with planar lipid bilayers. A strong dependence of single channel activities on cis-Ca2+ concentration indicated that the large cytosolic region of the channels faced thecis (cytosolic) chamber in a majority (>98%) of the recordings (34Xu L. Meissner G. Biophys. J. 1998; 75: 2302-2312Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 35Tripathy A. Xu L. Mann G. Meissner G. Biophys. J. 1995; 69: 106-119Abstract Full Text PDF PubMed Scopus (259) Google Scholar). Channels that could not be activated by 1–10 μm Ca2+ were discarded. The majority of channels were recorded in symmetric 0.25 m KCl solutions rather than with a lumenal Ca2+ solution to eliminate large Ca2+ gradients near the cytosolic channel pore sites (34Xu L. Meissner G. Biophys. J. 1998; 75: 2302-23" @default.
• W2059320957 created "2016-06-24" @default.
• W2059320957 creator A5037334396 @default.
• W2059320957 creator A5054088186 @default.
• W2059320957 creator A5080433401 @default.
• W2059320957 creator A5088774939 @default.
• W2059320957 date "1999-11-01" @default.
• W2059320957 modified "2023-10-18" @default.
• W2059320957 title "Ruthenium Red Modifies the Cardiac and Skeletal Muscle Ca2+ Release Channels (Ryanodine Receptors) by Multiple Mechanisms" @default.
• W2059320957 cites W125040153 @default.
• W2059320957 cites W1483833346 @default.
• W2059320957 cites W1490746554 @default.
• W2059320957 cites W1508964468 @default.
• W2059320957 cites W1509170501 @default.
• W2059320957 cites W1558794683 @default.
• W2059320957 cites W1563253655 @default.
• W2059320957 cites W1914131776 @default.
• W2059320957 cites W1965861043 @default.
• W2059320957 cites W1971197209 @default.
• W2059320957 cites W1978095253 @default.
• W2059320957 cites W1979027161 @default.
• W2059320957 cites W1982594839 @default.
• W2059320957 cites W1991753089 @default.
• W2059320957 cites W1997403679 @default.
• W2059320957 cites W2004841523 @default.
• W2059320957 cites W2018067600 @default.
• W2059320957 cites W2018879679 @default.
• W2059320957 cites W2019843818 @default.
• W2059320957 cites W2020372983 @default.
• W2059320957 cites W2020954521 @default.
• W2059320957 cites W2023226631 @default.
• W2059320957 cites W2058336277 @default.
• W2059320957 cites W2060055554 @default.
• W2059320957 cites W206588626 @default.
• W2059320957 cites W2072720045 @default.
• W2059320957 cites W2077397218 @default.
• W2059320957 cites W2104294443 @default.
• W2059320957 cites W2118383537 @default.
• W2059320957 cites W2122005870 @default.
• W2059320957 cites W2122951173 @default.
• W2059320957 cites W2124271343 @default.
• W2059320957 cites W2128497087 @default.
• W2059320957 cites W2128574295 @default.
• W2059320957 cites W2134325626 @default.
• W2059320957 cites W2138310843 @default.
• W2059320957 cites W2138450406 @default.
• W2059320957 cites W2158587870 @default.
• W2059320957 cites W2162977387 @default.
• W2059320957 cites W2320181845 @default.
• W2059320957 cites W71582486 @default.
• W2059320957 doi "https://doi.org/10.1074/jbc.274.46.32680" @default.
• W2059320957 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10551824" @default.
• W2059320957 hasPublicationYear "1999" @default.
• W2059320957 type Work @default.
• W2059320957 sameAs 2059320957 @default.
• W2059320957 citedByCount "104" @default.
• W2059320957 countsByYear W20593209572012 @default.
• W2059320957 countsByYear W20593209572013 @default.
• W2059320957 countsByYear W20593209572014 @default.
• W2059320957 countsByYear W20593209572015 @default.
• W2059320957 countsByYear W20593209572016 @default.
• W2059320957 countsByYear W20593209572017 @default.
• W2059320957 countsByYear W20593209572018 @default.
• W2059320957 countsByYear W20593209572019 @default.
• W2059320957 countsByYear W20593209572020 @default.
• W2059320957 countsByYear W20593209572021 @default.
• W2059320957 countsByYear W20593209572022 @default.
• W2059320957 countsByYear W20593209572023 @default.
• W2059320957 crossrefType "journal-article" @default.
• W2059320957 hasAuthorship W2059320957A5037334396 @default.
• W2059320957 hasAuthorship W2059320957A5054088186 @default.
• W2059320957 hasAuthorship W2059320957A5080433401 @default.
• W2059320957 hasAuthorship W2059320957A5088774939 @default.
• W2059320957 hasBestOaLocation W20593209571 @default.
• W2059320957 hasConcept C113217602 @default.
• W2059320957 hasConcept C12554922 @default.
• W2059320957 hasConcept C126322002 @default.
• W2059320957 hasConcept C134018914 @default.
• W2059320957 hasConcept C170493617 @default.
• W2059320957 hasConcept C178790620 @default.
• W2059320957 hasConcept C185592680 @default.
• W2059320957 hasConcept C190712762 @default.
• W2059320957 hasConcept C2778996579 @default.
• W2059320957 hasConcept C2779657890 @default.
• W2059320957 hasConcept C2779959927 @default.
• W2059320957 hasConcept C519063684 @default.
• W2059320957 hasConcept C55493867 @default.
• W2059320957 hasConcept C71924100 @default.
• W2059320957 hasConcept C86803240 @default.
• W2059320957 hasConcept C95444343 @default.
• W2059320957 hasConceptScore W2059320957C113217602 @default.
• W2059320957 hasConceptScore W2059320957C12554922 @default.
• W2059320957 hasConceptScore W2059320957C126322002 @default.
• W2059320957 hasConceptScore W2059320957C134018914 @default.
• W2059320957 hasConceptScore W2059320957C170493617 @default.
• W2059320957 hasConceptScore W2059320957C178790620 @default.
• W2059320957 hasConceptScore W2059320957C185592680 @default.
• W2059320957 hasConceptScore W2059320957C190712762 @default.

• next