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Multiple Actions of Imperatoxin A on Ryanodine Receptors

2004; Elsevier BV; Volume: 279; Issue: 12 Linguagem: Inglês

10.1074/jbc.m310466200

ISSN

1083-351X

Autores

Angela F. Dulhunty, Suzanne M. Curtis, Sarah Watson, Louise H. Cengia, Marco G. Casarotto,

Tópico(s)

Marine Toxins and Detection Methods

Resumo

Imperatoxin A is a high affinity activator of ryanodine receptors. The toxin contains a positively charged surface structure similar to that of the A fragment of skeletal dihydropyridine receptors (peptide A), suggesting that the toxin and peptide could bind to a common site on the ryanodine receptor. However, the question of a common binding site has not been resolved, and the concentration dependence of the actions of the toxin has not been fully explored. We characterize two novel high affinity actions of the toxin on the transient gating of cardiac and skeletal channels, in addition to the well documented lower affinity induction of prolonged substates. Transient activity was (a) enhanced with 0.2-10 nm toxin and (b) depressed by >50 nm toxin. The toxin at ≥1 nm enhanced Ca2+ release from SR in a manner consistent with two independent activation processes. The effects of the toxin on transient activity, as well as the toxin-induced substate, were independent of cytoplasmic Ca2+ or Mg2+ concentrations or the presence of adenine nucleotide and were seen in diisothiocyanostilbene-2′,2′-disulfonic acid-modified channels. Peptide A activated skeletal and cardiac channels with 100 nm cytoplasmic Ca2+ and competed with Imperatoxin A in the high affinity enhancement of transient channel activity and Ca2+ release from SR. In contrast to transient activity, prolonged substate openings induced by the toxin were not altered in the presence of peptide A. The results suggest that Imperatoxin A has three independent actions on ryanodine receptor channels and competes with peptide A for at least one action. Imperatoxin A is a high affinity activator of ryanodine receptors. The toxin contains a positively charged surface structure similar to that of the A fragment of skeletal dihydropyridine receptors (peptide A), suggesting that the toxin and peptide could bind to a common site on the ryanodine receptor. However, the question of a common binding site has not been resolved, and the concentration dependence of the actions of the toxin has not been fully explored. We characterize two novel high affinity actions of the toxin on the transient gating of cardiac and skeletal channels, in addition to the well documented lower affinity induction of prolonged substates. Transient activity was (a) enhanced with 0.2-10 nm toxin and (b) depressed by >50 nm toxin. The toxin at ≥1 nm enhanced Ca2+ release from SR in a manner consistent with two independent activation processes. The effects of the toxin on transient activity, as well as the toxin-induced substate, were independent of cytoplasmic Ca2+ or Mg2+ concentrations or the presence of adenine nucleotide and were seen in diisothiocyanostilbene-2′,2′-disulfonic acid-modified channels. Peptide A activated skeletal and cardiac channels with 100 nm cytoplasmic Ca2+ and competed with Imperatoxin A in the high affinity enhancement of transient channel activity and Ca2+ release from SR. In contrast to transient activity, prolonged substate openings induced by the toxin were not altered in the presence of peptide A. The results suggest that Imperatoxin A has three independent actions on ryanodine receptor channels and competes with peptide A for at least one action. Excitation-contraction (EC) 1The abbreviations used are: EC, excitation-contraction; SR, sarcoplasmic reticulum; DHPR, dihydropyridine receptor; t-tubule, transverse tubule membrane; RyR, ryanodine receptor; TES, N-tris[hyroxymethyl]methyl-2-aminoethanesulfonic acid; DIDS, diisothiocyanostilbene-2′,2′-disulfonic acid; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; AC50, concentration for 50% activation. coupling is the process that facilitates Ca2+ release from the sarcoplasmic reticulum (SR) of muscle fibers following depolarization of the surface/transverse (t-) tubule membrane. A protein-protein interaction between the dihydropyridine receptor (DHPR) and ryanodine receptor (RyR) underlies EC coupling in skeletal muscle. The DHPR L-type Ca2+ channel in the t-tubule membrane detects surface depolarization and transmits a signal to the RyR channel in the SR via an interaction between the cytoplasmic domains of the two proteins. The interacting region of the DHPR is located between the second and third transmembrane repeats in the α1 subunit (II-III loop) (1Tanabe T. Beam K.G. Powell J.A. Numa S. Nature. 1988; 336: 134-139Crossref PubMed Scopus (584) Google Scholar). The interacting regions of the RyR are less clearly defined but are likely to involve residues 1076-1112 (2Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 7791-7794Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and residues 1837-2168 (3Proenza C. O'Brien J. Nakai J. Mukherjee S. Allen P.D. Beam K.G. J. Biol. Chem. 2002; 277: 6530-6535Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The recombinant DHPR II-III loop activates skeletal RyRs (4Lu X. Xu L. Meissner G. J. Biol. Chem. 1994; 269: 6511-6516Abstract Full Text PDF PubMed Google Scholar, 5O'Reilly F.M. Robert M. Jona I. Szegedi C. Albrieux M. Geib S. De Waard M. Villaz M. Ronjat M. Biophys. J. 2002; 82: 145-155Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The loop has been arbitrarily divided into four segments, A, B, C, and D (6El-Hayek R. Antoniu B. Wang J. Hamilton S.L. Ikemoto N. J. Biol. Chem. 1995; 270: 22116-22118Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The C region (residues 720-765) is strongly implicated in EC coupling (7Proenza C. Wilkens C.M. Beam K.G. J. Biol. Chem. 2000; 275: 29935-29937Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 8Ahern C.A. Bhattacharya D. Mortenson L. Coronado R. Biophys. J. 2001; 81: 3294-3307Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 9Wilkens C.M. Kasielke N. Flucher B.E. Beam K.G. Grabner M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5892-5897Crossref PubMed Scopus (67) Google Scholar), and a random coil peptide corresponding to this region modifies the activity of the skeletal RyR (10Stange M. Tripathy A. Meissner G. Biophys. J. 2001; 81: 1419-1429Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 11Haarmann C.C. Green D. Casarotto M.G. Laver D.R. Dulhunty A.F. Biochem. J. 2003; 372: 305-316Crossref PubMed Scopus (39) Google Scholar). A second region of the II-III loop, the A region (residues 671-690), is of interest because its corresponding peptide fragment induces Ca2+ release from the SR and enhances current flow through RyR channels with high affinity (5O'Reilly F.M. Robert M. Jona I. Szegedi C. Albrieux M. Geib S. De Waard M. Villaz M. Ronjat M. Biophys. J. 2002; 82: 145-155Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 6El-Hayek R. Antoniu B. Wang J. Hamilton S.L. Ikemoto N. J. Biol. Chem. 1995; 270: 22116-22118Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 10Stange M. Tripathy A. Meissner G. Biophys. J. 2001; 81: 1419-1429Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 12Dulhunty A.F. Laver D.R. Gallant E.M. Casarotto M.G. Pace S.M. Curtis S. Biophys. J. 1999; 77: 189-203Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 13Gurrola G.B. Arevalo C. Sreekumar R. Lokuta A.J. Walker J.W. Valdivia H.H. J. Biol. Chem. 1999; 274: 7879-7886Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 14Zhu X. Gurrola G. Jiang M.T. Walker J.W. Valdivia H.H. FEBS Lett. 1999; 450: 221-226Crossref PubMed Scopus (28) Google Scholar, 15Casarotto M.G. Gibson F. Pace S.M. Curtis S.M. Mulcair M. Dulhunty A.F. J. Biol. Chem. 2000; 275: 11631-11637Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 16Casarotto M.G. Green D. Pace S.M. Curtis S.M. Dulhunty A.F. Biophys. J. 2001; 80: 2715-2726Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 17Gallant E.M. Curtis S. Pace S.M. Dulhunty A.F. Biophys. J. 2001; 80: 1769-1782Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 18Green D. Pace S. Curtis S.M. Sakowska M. Lamb G.D. Dulhunty A.F. Casarotto M.G. Biochem. J. 2003; 370: 517-527Crossref PubMed Scopus (26) Google Scholar). Although the A region is not essential for skeletal EC coupling in myocytes (7Proenza C. Wilkens C.M. Beam K.G. J. Biol. Chem. 2000; 275: 29935-29937Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 9Wilkens C.M. Kasielke N. Flucher B.E. Beam K.G. Grabner M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5892-5897Crossref PubMed Scopus (67) Google Scholar), it may play a role in the DHPR-RyR interaction (8Ahern C.A. Bhattacharya D. Mortenson L. Coronado R. Biophys. J. 2001; 81: 3294-3307Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), and it is a useful probe for assessing RyR function (11Haarmann C.C. Green D. Casarotto M.G. Laver D.R. Dulhunty A.F. Biochem. J. 2003; 372: 305-316Crossref PubMed Scopus (39) Google Scholar, 17Gallant E.M. Curtis S. Pace S.M. Dulhunty A.F. Biophys. J. 2001; 80: 1769-1782Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 19Chen L. Esteve E. Sabatier J.M. Ronjat M. De Waard M. Allen P.D. Pessah I.N. J. Biol. Chem. 2003; 278: 16095-16106Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The ability of peptide A to activate RyRs is highly correlated with its capacity to adopt an α-helical structure (15Casarotto M.G. Gibson F. Pace S.M. Curtis S.M. Mulcair M. Dulhunty A.F. J. Biol. Chem. 2000; 275: 11631-11637Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 16Casarotto M.G. Green D. Pace S.M. Curtis S.M. Dulhunty A.F. Biophys. J. 2001; 80: 2715-2726Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 18Green D. Pace S. Curtis S.M. Sakowska M. Lamb G.D. Dulhunty A.F. Casarotto M.G. Biochem. J. 2003; 370: 517-527Crossref PubMed Scopus (26) Google Scholar) and with the orientation of positively charged residues along one surface of the molecule (18Green D. Pace S. Curtis S.M. Sakowska M. Lamb G.D. Dulhunty A.F. Casarotto M.G. Biochem. J. 2003; 370: 517-527Crossref PubMed Scopus (26) Google Scholar). Curiously, two scorpion toxins, Imperatoxin A and Maurocalcine (having 82% sequence identity), have structural features in common with peptide A. Although the intrinsically disulfide-stabilized β-sheet structure of the toxins is vastly different from the α-helical structure of the A peptide, the toxins and peptide A share a similar surface orientation of positively charged residues (18Green D. Pace S. Curtis S.M. Sakowska M. Lamb G.D. Dulhunty A.F. Casarotto M.G. Biochem. J. 2003; 370: 517-527Crossref PubMed Scopus (26) Google Scholar, 20Mosbah A. Kharrat R. Fajloun Z. Renisio J.-G. Blanc E. Sabatier J.-M. Al Ayeb M. Dabon H. Proteins: Struct. Funct. Genet. 2000; 40: 436-442Crossref PubMed Scopus (80) Google Scholar). Because of this structural similarity, several studies have examined the possibility that the scorpion toxins and peptide A bind to the same site on RyR1. Two studies have concluded that they bind to the same, or overlapping sites (13Gurrola G.B. Arevalo C. Sreekumar R. Lokuta A.J. Walker J.W. Valdivia H.H. J. Biol. Chem. 1999; 274: 7879-7886Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 18Green D. Pace S. Curtis S.M. Sakowska M. Lamb G.D. Dulhunty A.F. Casarotto M.G. Biochem. J. 2003; 370: 517-527Crossref PubMed Scopus (26) Google Scholar), whereas a third study concluded that Maurocalcine/Imperatoxin A and peptide A bind to independent sites (19Chen L. Esteve E. Sabatier J.M. Ronjat M. De Waard M. Allen P.D. Pessah I.N. J. Biol. Chem. 2003; 278: 16095-16106Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). These different conclusions could have arisen if more than one binding site exists for either the toxins and for peptide A, and not all sites are common to the two compounds. At least two binding sites for peptide A have been defined, one within the channel pore, which leads to voltage-dependent channel block (12Dulhunty A.F. Laver D.R. Gallant E.M. Casarotto M.G. Pace S.M. Curtis S. Biophys. J. 1999; 77: 189-203Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), and a site (or sites) on the cytoplasmic domain of the channel, which support its voltage-independent actions (10Stange M. Tripathy A. Meissner G. Biophys. J. 2001; 81: 1419-1429Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 12Dulhunty A.F. Laver D.R. Gallant E.M. Casarotto M.G. Pace S.M. Curtis S. Biophys. J. 1999; 77: 189-203Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Here we investigate the possibility that Imperatoxin A also has multiple actions on RyR activity and examine interactions of between the toxin and peptide A in modifying RyR channel gating. We find that there are a least three separate effects of Imperatoxin A on cardiac and skeletal RyR channels that can be distinguished by their affinity and reversibility and by their ability to compete with peptide A in its native and a modified form. The results show that peptide A competes with Imperatoxin A for the high affinity activation of transient channel openings. On the other hand, peptide A does not prevent the characteristic toxin-induced prolonged substate activity. The results suggest that there is at least one common or overlapping binding site for Imperatoxin A and peptide A as well as independent binding sites. These observations raise the questions of which of the Imperatoxin A binding sites has been identified on the RyR (21Samso M. Trujillo R. Gurrola G.B. Valdivia H.H. Wagenknecht T. J. Cell Biol. 1999; 146: 493-499Crossref PubMed Scopus (63) Google Scholar) and whether this site is the site that also interacts with peptide A. Peptides—Peptides A and A1(D-R18) were synthesized as in Green et al. (18Green D. Pace S. Curtis S.M. Sakowska M. Lamb G.D. Dulhunty A.F. Casarotto M.G. Biochem. J. 2003; 370: 517-527Crossref PubMed Scopus (26) Google Scholar). Imperatoxin A was synthesized by Auspep Australia and folded using procedures outlined by Fajloun et al. (22Fajloun Z. Kharrat R. Chen L. Lecomte C. Di Luccio E. Bichet D. El Ayeb M. Rochat H. Allen P.D. Pessah I.N. De Waard M. Sabatier J.M. FEBS Lett. 2000; 469: 179-185Crossref PubMed Scopus (93) Google Scholar) Vesicle Preparation—Preparation of SR vesicles, Ca2+ release from SR, and single channel techniques have been described previously. Heavy skeletal SR vesicles were prepared from rabbit back and leg muscle (11Haarmann C.C. Green D. Casarotto M.G. Laver D.R. Dulhunty A.F. Biochem. J. 2003; 372: 305-316Crossref PubMed Scopus (39) Google Scholar, 12Dulhunty A.F. Laver D.R. Gallant E.M. Casarotto M.G. Pace S.M. Curtis S. Biophys. J. 1999; 77: 189-203Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), whereas cardiac SR vesicles were prepared from sheep heart (23Laver D.R. Roden L.D. Ahern G.P. Eager K.R. Junankar P.R. Dulhunty A.F. J. Membr. Biol. 1995; 147: 7-22Crossref PubMed Scopus (152) Google Scholar). Single Channel Techniques—Bilayers of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine (5:3:2 w/w) (Avanti Polar Lipids, Alabaster, AL) were formed across an aperture of ∼200-μm diameter in the wall of a 1.0-ml Delrin cup (Cadillac Plastics, Australia). Terminal cisternae vesicles (10 μg/ml) were added to the cis chamber. The cytoplasmic side of channels incorporated into the bilayer faced the cis solution. Bilayer potential was controlled, and single channel currents were recorded, using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Bilayer potential is expressed as Vcis - Vtrans (Vcytoplasm - Vlumen). Bilayers were formed and vesicles incorporated using cis solutions containing (in millimolar): 230 CsCH3O3S/20 CsCl/5.0 CaCl2/10 TES/500 mm mannitol (pH 7.4) with CsOH and a trans solution containing (in millimolar) 30 CsMS/20 CsCl/1 CaCl2/10 TES (pH 7.4). Following incorporation, (a) the cis solution was replaced with an identical solution, except that the [Ca2+] was between 0.1 and 100 μm and (b) 200 mm CsCH3O3S was added to the trans chamber for symmetry. Drugs were added to the cis chamber and removed by perfusion with 10 ml of cis solution. Analysis of Channel Activity—Channel activity was analyzed over one to two 30-s periods of continuous activity at +40 mV and then at -40 mV. Slow fluctuations in the baseline were corrected using an in-house baseline correction program (written by Dr. D. R. Laver). Channel activity was measured either as "mean current" (the average of all data points in a record) or as open probability (Po), using a threshold analysis with the program Channel 2, (developed by P. W. Gage and M. Smith, John Curtin School of Medical Research). Measurements of mean current, performed on records from experiments containing 1-4 channels, included all channel activity from the smallest subconductance level to maximum openings. On the other hand, open probability (Po), mean open time (To), and mean closed time (Tc) measurements are restricted to records in which the opening of a single channel only could be detected and exclude openings that fall within the baseline noise. In this case threshold levels for channel opening and closing were set to exclude baseline noise (a) at ∼20% of the maximum single channel conductance when examining transient openings in the absence of prolonged substates or (b) at 50% of the maximum conductance or 50% of the substate level to measure Po of maximum or substate conductance respectively, when substates were present. Channel activity is expressed as relative Po to include data in which activity varied from ∼0.0001 to ∼0.1 and data from bilayers containing more than one channel. Relative Po was calculated either (a) from I′t/I′c, where I′t is the mean current under test conditions and I′c is the control mean current, or (b) from Pot/Poc, where Pot is the open probability under test conditions and Poc the control open probability. Because the mean current divided by the maximum current approximates open probability, I′t/I′c ≡ Pot/Poc. DIDS Modification—Channels modified by the disulfinic stilbene derivative, diisothiocyanostilbene-2′,2′-disulfonic acid (DIDS), were used in some cases with a cytoplasmic [Ca2+] of 100 nm to enhance channel activity under control conditions. DIDS modification does not alter the regulation of RyRs by Mg2+, ryanodine, or ruthenium red (24Oba T. Koshita M. Van Helden D.F. Am. J. Physiol. 1996; 271: C819-C824Crossref PubMed Google Scholar), although it can interact with other properties of RyRs (25Hill A.P. Sitsapesan R. Biophys. J. 2002; 82: 3037-3047Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 26Klinger M. Bofill-Cardona E. Mayer B. Nanoff C. Freissmuth M. Hohenegger M. Biochem. J. 2001; 355: 827-833Crossref PubMed Scopus (23) Google Scholar, 27Hirata Y. Nakahata N. Ohkura M. Ohizumi Y. Biochim. Biophys. Acta. 1999; 1451: 132-140Crossref PubMed Scopus (8) Google Scholar). Channels were exposed to 100 or 300 μm DIDS in the cis chamber for 4-6 min, and then DIDS was removed by perfusion. Activity increased in the presence of DIDS and then fell with removal of the reversible component of activation. However, activity remained higher than before exposure to DIDS because of covalent bonds formed between isothiocyanate groups and NH2, OH, and aromatic groups on a variety of amino acid residues (28O'Neill E.R. Sakowska M.M. Laver D.R. Biophys. J. 2003; 84: 1674-1689Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Ca2+ Release from SR—Extravesicular Ca2+ was monitored at 710 nm using a Cary 3 spectrophotometer (12Dulhunty A.F. Laver D.R. Gallant E.M. Casarotto M.G. Pace S.M. Curtis S. Biophys. J. 1999; 77: 189-203Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The cuvette solution was stirred continuously, and temperature was controlled at 25 °C. Skeletal SR (100 μg of protein) was added to the cuvette solution (final volume of 2 ml), containing (in millimolar): 100 KH2PO4 (pH 7); 4 MgCl2; 1 Na2ATP; 0.5 antipyrylazo III. Ca2+,Mg2+-ATPase activity was suppressed with thapsigargin (200 nm (29Sagara Y. Inesi G. J. Biol. Chem. 1991; 266: 13503-13506Abstract Full Text PDF PubMed Google Scholar)). The same solutions were used with cardiac SR except that an ATP-regenerating system (phospho(enol)pyruvate (5 mm) and pyruvate kinase (25 μg/ml)) was added, and Ca2+-induced Ca2+ release was triggered by addition of 20 μm Ca2+ to the cuvette solution. When toxin was examined alone, it was added 2 min after thapsigargin. When peptide was added before toxin, either peptide or an equivalent volume of water (vehicle) was added 2 min after thapsigargin, and then toxin or an equivalent volume of water (vehicle) was added after a further 2 min. Release rates in each experiment were measured 10-20 s after toxin addition. Ca2+ release rate, R, as a function of [toxin] was fitted by a Hill equation, R = Rb + Rmax{1/[1 + (Tx50/Tx)H]}, where Rb is the baseline Ca2+ leak or Ca2+-induced Ca2+ release in thapsigargin, Rmax is the maximum toxin-induced release rate, Tx is [toxin], Tx50 the [toxin] for activation to 50% maximum, and H is the Hill coefficient. Statistics—Average data are given as mean ± S.E. The significance of the difference between control and test values was tested a using either (a) a Student's t test, either one or two sided and either for independent or paired data, as appropriate or (b) using the non-parametric "sign" test (30Minum E.W. King B.M. Bear G. Statistical Reasoning in Psychology and Education. 3rd ed. John Wiley & Sons, y, New York1993: 475-494Google Scholar). Differences were considered to be significant when p ≤ 0.05. RyR activity demonstrated two distinct gating modes, a "transient" mode and a maintained "substate" mode. The transient mode comprised all control channel activity and was observed in toxin-modified channels. In this mode, the duration of channel openings varied from ∼0.5 to ∼1000 ms with brief submaximal openings and longer openings to the maximum conductance. The maintained substate mode was seen only after exposure to Imperatoxin A and was characterized by openings lasting from ∼1 to 20 s. The number of transient openings increased with toxin concentrations between 200 pm and 20 nm, then declined with >20 nm toxin. Prolonged substate openings appeared with >100 nm toxin. Increased Transient Activity—Imperatoxin A applied at picomolar concentrations to the cis (cytoplasm) side of RyR channels caused an increase in the frequency of transient openings. The increase occurred rapidly, within the 30-s period of toxin addition and stirring. Prolonged substate openings were not induced with these low toxin concentrations. The activating effect on transient openings was only slowly reversible when the toxin was perfused from the cis chamber (Fig. 1, A-C). Activity increased in cardiac (n = 20 experiments) and skeletal (n = 19) channels that were either Ca2+-activated (cis Ca2+ of 10 or 100 μm; n = 18) or at sub-activating cis [Ca2+] (100-300 nm) (n = 20), in the absence (n = 10) or presence of ATP (n = 17) (with 2 mm Mg2+, n = 4) or AMP-PNP (n = 5) and in DIDS-modified channels (n = 5) (Fig. 1, D and E). All channels were activated by toxin at concentrations up to 10 nm. At 50-300 nm, some channels were activated while others were inhibited. Channels recorded with low cis [Ca2+] showed average activation at higher toxin concentrations than those recorded with higher activating cis [Ca2+] (Fig. 1, D and E). The changes in channel gating associated with increased activity were measured in a subset of data from skeletal and cardiac RyR channels (Table I). The increased activity of skeletal RyRs with 1 and 10 nm toxin was due to an increase in channel open time, and a reduction in closed time. The increase in open time was also seen in cardiac channels, but in contrast to skeletal channels, there was no significant change in closed durations at lower toxin concentrations.Table ISingle channel parameters from a subset of experiments in which one channel only was active in the bilayerSkeletal RyRToxin concentration1 nm10 nm100 nm1 μm10 μmRelative Po4.30 ± 1.08aA significant change in the open probability.4.28 ± 0.82aA significant change in the open probability.3.74 ± 1.14aA significant change in the open probability.2.17 ± 1.032.30 ± 1.29Relative To1.36 ± 0.451.31 ± 0.15aA significant change in the open probability.1.58 ± 0.05aA significant change in the open probability.1.80 ± 0.34aA significant change in the open probability.1.72 ± 0.04aA significant change in the open probability.Relative Tc0.43 ± 0.07aA significant change in the open probability.0.41 ± 0.12aA significant change in the open probability.1.62 ± 0.683.07 ± 1.42.1 ± 0.08Cardiac RyRToxin concentration500 pm1 nm10 nm100 nm1 μmRelative Po1.89 ± 0.24aA significant change in the open probability.2.00 ± 0.37aA significant change in the open probability.1.59 ± 0.37aA significant change in the open probability.1.31 ± 0.260.20 ± 0.06aA significant change in the open probability.Relative To2.21 ± 1.08aA significant change in the open probability.1.59 ± 0.43aA significant change in the open probability.1.70 ± 0.50aA significant change in the open probability.1.32 ± 0.17aA significant change in the open probability.0.50 ± 0.08aA significant change in the open probability.Relative Tc1.12 ± 0.362.06 ± 1.251.32 ± 0.271.29 ± 0.44.65 ± 2.17aA significant change in the open probability.a A significant change in the open probability. Open table in a new tab Inhibition of Transient Activity—Transient channel openings decreased when the toxin was increased to between 50 and 500 nm (see Fig. 1C). Some channels were activated for several minutes after exposure to higher [toxin] and were then inhibited (Fig. 2A). Activity was rapidly restored to an activated (greater than control) level upon removal of the toxin, indicating raid reversibility of the inhibitory action. The inhibition of transient activity was independent of prolonged substate openings. Although all channels exhibiting prolonged substates also showed fewer transient openings, the number of transient openings was often substantially reduced in the absence of substate activity (100 nm toxin (Fig. 2A)). Inhibition reduced Po to less than control when the activity was initially high (in channels with activating cis Ca2+ and/or ATP (Fig. 2A)). When activity was low (in the presence of 100-300 nm Ca2+ and/or MgATP), Po fell to less than the toxin-activated level, but often remained higher than control (e.g. the skeletal channels in Table I). The decline in transient activity was caused by an increase in closed time (Table I). Curiously, the open times in skeletal RyRs continued to increase as toxin concentration increased (Table I), perhaps indicating that the activating effect of the toxin increased with [toxin], but was overwhelmed by an independent inhibitory effect. Persistent inhibition was also seen following perfusion of inhibiting [toxin] from the cis chamber, when removal of inhibition revealed a strong slowly reversible activation (e.g. Fig. 2A). On average, cardiac RyRs showed a significant decline in open times and increase in closed durations when inhibited by 1 μm toxin (Table I). Inhibition was seen in all cardiac (n = 13) and skeletal (n = 9) channels exposed to higher toxin concentrations and was independent of bilayer potential or cis [Ca2+] (n = 9 for ≤300 nm Ca2+ or n = 13 for 10 μm Ca2+), and occurred without ATP or Mg2+ (n = 8) or with 2 mm ATP (n = 14) and 2 mm Mg2+ (n = 4), as well as in DIDS-modified channels (n = 5) (Fig. 2B). Toxin-activated channels were further activated by Ca2+, ATP, or AMP-PNP, whereas inhibited channels could not be activated to the same extent by these ligands (Fig. 2C). In this experiment, with 100 nm cis Ca2+, cardiac RyR channels were either activated or inhibited by 200 nm toxin. The toxin-activated channels were further activated when cis Ca2+ was increased to 100 μm and activated again by either 2 mm ATP or AMP-PNP. In contrast, although activity increased in the toxin-inhibited channels, the increase (relative to control) was significantly less than in the activated channels. Peptide A Competes with Imperatoxin A for Activation of Transient Opening—It has been suggested that peptide A and Imperatoxin A compete for a single site on the RyR. Cardiac channels were activated by 1 nm toxin and by subsequent additions of peptide A up to 500 nm (Fig. 3A). Activity declined when the peptide was increased to 1 μm. However, the plateau of activation with peptide plus toxin was no greater than that with peptide alone, suggesting that there was no summation of the effects of the two compounds and supporting the concept of the same or overlapping binding sites. Similar results were obtained when channels were exposed to 10 nm toxin, although there was a significant reduction in activity when the [peptide] reached 500 nm. In a similar experiment (Fig. 3B), channels were first exposed to 50 nm peptide A and then to increasing concentrations of toxin. Activity tended to increase with peptide plus toxin up to 600 pm, but activation was significantly less than expected from the summation of two independent processes (Fig. 3B). Channel activity declined when higher concentrations of toxin were added with peptide. The decline in activity with higher concentrations of toxin plus peptide A (or modified peptide A (A1-R18D) (18Green D. Pace S. Curtis S.M. Sakowska M. Lamb G.D. Dulhunty A.F. Casarotto M.G. Biochem. J. 2003; 370: 517-527Crossref PubMed Scopus (26) Google Scholar)) was particularly apparent in skeletal RyR channels (Table II and Fig. 3, C and D). Transient openings declined with 1 nm toxi

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