Activation of Ryanodine Receptors by Imperatoxin A and a Peptide Segment of the II-III Loop of the Dihydropyridine Receptor
1999; Elsevier BV; Volume: 274; Issue: 12 Linguagem: Inglês
10.1074/jbc.274.12.7879
ISSN1083-351X
AutoresGeorgina B. Gurrola, Carolina Arévalo, R. Sreekumar, Andrew J. Lokuta, Jeffery W. Walker, Héctor H. Valdivia,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoExcitation-contraction coupling in skeletal muscle is believed to be triggered by direct protein-protein interactions between the sarcolemmal dihydropyridine-sensitive Ca2+ channel and the Ca2+ release channel/ryanodine receptor (RyR) of sarcoplasmic reticulum. A 138-amino acid cytoplasmic loop between repeats II and III of the α1 subunit of the skeletal dihydropyridine receptor (the II-III loop) interacts with a region of the RyR to elicit Ca2+ release. In addition, small segments (10–20 amino acid residues) of the II-III loop retain the capacity to activate Ca2+ release. Imperatoxin A, a 33-amino acid peptide from the scorpion Pandinus imperator, binds directly to the RyR and displays structural and functional homology with an activating segment of the II-III loop (Glu666-Leu690). Mutations in a structural motif composed of a cluster of basic amino acids followed by Ser or Thr dramatically reduce or completely abolish the capacity of the peptides to activate RyRs. Thus, the Imperatoxin A-RyR interaction mimics critical molecular characteristics of the II-III loop-RyR interaction and may be a useful tool to elucidate the molecular mechanism that couples membrane depolarization to sarcoplasmic reticulum Ca2+ release in vivo. Excitation-contraction coupling in skeletal muscle is believed to be triggered by direct protein-protein interactions between the sarcolemmal dihydropyridine-sensitive Ca2+ channel and the Ca2+ release channel/ryanodine receptor (RyR) of sarcoplasmic reticulum. A 138-amino acid cytoplasmic loop between repeats II and III of the α1 subunit of the skeletal dihydropyridine receptor (the II-III loop) interacts with a region of the RyR to elicit Ca2+ release. In addition, small segments (10–20 amino acid residues) of the II-III loop retain the capacity to activate Ca2+ release. Imperatoxin A, a 33-amino acid peptide from the scorpion Pandinus imperator, binds directly to the RyR and displays structural and functional homology with an activating segment of the II-III loop (Glu666-Leu690). Mutations in a structural motif composed of a cluster of basic amino acids followed by Ser or Thr dramatically reduce or completely abolish the capacity of the peptides to activate RyRs. Thus, the Imperatoxin A-RyR interaction mimics critical molecular characteristics of the II-III loop-RyR interaction and may be a useful tool to elucidate the molecular mechanism that couples membrane depolarization to sarcoplasmic reticulum Ca2+ release in vivo. dihydropyridine receptor ryanodine receptor sarcoplasmic reticulum Imperatoxin A excitation-contraction Chinese hamster ovary high pressure liquid chromatography 4-morpholine propanesulfonic acid In cardiac and skeletal muscle, the dihydropyridine receptor (DHPR)1 of the external membrane and the Ca2+ release channel/ryanodine receptor (RyR) of sarcoplasmic reticulum (SR) are key components of excitation-contraction (E-C) coupling, the series of events that link an electrical stimulus (depolarization) to a mechanical contraction (1Bers D. Excitation Contraction Coupling and Cardiac Contractile Force. Kluwer Academic Publishers, The Netherlands1991Google Scholar). Skeletal and cardiac muscle express different subtypes of DHPR and RyR, which account for different E-C coupling mechanisms. In the heart, a small influx of Ca2+ through DHPRs triggers the opening of RyRs (2Fabiato A. J. Gen. Physiol. 1985; 85: 247-290Crossref PubMed Scopus (685) Google Scholar). In skeletal muscle, however, external Ca2+ is not required for Ca2+ release (3Armstrong C.M. Bezanilla F. Horowicz P. Biochim. Biophys. Acta. 1972; 267: 605-612Crossref PubMed Scopus (299) Google Scholar). Contractions are instead triggered by membrane depolarizations, and because Ca2+release may be arrested immediately upon repolarization, a mechanical coupling between the DHPR and the RyR is thought to mediate E-C coupling (4Chandler W.K. Rakowski R.F. Schneider M.F. J. Physiol. (Lond.). 1976; 254: 285-316Crossref Scopus (165) Google Scholar, 5Rı́os E. Pizarro G. Physiol. Rev. 1992; 71: 849-908Crossref Scopus (496) Google Scholar). Compelling evidence indicates that the skeletal DHPR subtype is indispensable to elicit a Ca2+-independent (skeletal-type) contraction (6Tanabe T. Beam K.G. Adams B.A. Niidome T. Numa S. Nature. 1990; 346: 567-569Crossref PubMed Scopus (492) Google Scholar, 7Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 344: 451-453Crossref PubMed Scopus (193) Google Scholar) and that the 138-amino acid cytoplasmic loop between repeats II and III of the α1 subunit participates in this process (6Tanabe T. Beam K.G. Adams B.A. Niidome T. Numa S. Nature. 1990; 346: 567-569Crossref PubMed Scopus (492) Google Scholar, 8Nakai J. Tanabe T. Konno T. Adams B. Beam K.G. J. Biol. Chem. 1998; 273: 24983-24986Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). In experiments with isolated peptides, the II-III loop activates purified RyRs (9Lu X. Xu L. Meissner G. J. Biol. Chem. 1994; 269: 6511-6516Abstract Full Text PDF PubMed Google Scholar), and a small fragment of the II-III loop (Thr671-Leu690) induces Ca2+release from SR vesicles (10El-Hayek R. Antoniu B. Wang J. Hamilton S.L. Ikemoto N. J. Biol. Chem. 1995; 270: 22116-22118Crossref PubMed Scopus (127) Google Scholar). In dysgenic myotubes, skeletal-type E-C coupling is partially restored by a chimeric DHPR that is entirely cardiac except for a short segment of skeletal II-III loop (Phe725-Pro742) (8Nakai J. Tanabe T. Konno T. Adams B. Beam K.G. J. Biol. Chem. 1998; 273: 24983-24986Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Although apparently contradictory in the identity of the activating region, these results suggest that specific domains of the II-III loop directly interact with the RyR to change its conformational state and produce Ca2+release. Therefore, in skeletal muscle, the II-III loop stands as the strongest candidate among regions of the DHPR to bind to RyRs. However, the precise amino acid residues of the II-III loop that trigger Ca2+ release remain unknown. Furthermore, other DHPR segments (11Slavik K.J. Wang J.P. Aghdasi B. Zhang J.Z. Mandel F. Malouf N. Hamilton S.L. Am. J. Physiol. 1997; 272: C1475-C1481Crossref PubMed Google Scholar) or subunits (12Beurg M. Ahern C.A. Sukhareva M. Perez-Reyes E. Powers P.A. Gregg R.G. Coronado R. Biophys. J. 1998; 74: A235Google Scholar) have not been discarded as points of contact. We have previously shown that Imperatoxin A (IpTxa), a 33-amino acid peptide from the scorpion Pandinus imperator, is a high-affinity activator of RyRs (13El-Hayek R. Lokuta A.J. Arévalo C. Valdivia H.H. J. Biol. Chem. 1995; 270: 28696-28704Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 14Zamudio F.Z. Gurrola G.B. Arévalo C. Sreekumar R. Walker J.W. Valdivia H.H. Possani L.D. FEBS Lett. 1997; 405: 385-389Crossref PubMed Scopus (62) Google Scholar). The biological significance of IpTxa is unknown, because the apparent target for this membrane-impermeable peptide is located intracellularly. Because some peptide toxins activate intracellular signaling pathways by mimicking surface receptors (15Higashijima T. Uzu S. Nakajima T. Ross E.M. J. Biol. Chem. 1988; 263: 6491-6494Abstract Full Text PDF PubMed Google Scholar, 16Ménez A. Bontems F. Roumestand C. Gilquin B. Toma F. Proc. R. Soc. Edinb. Sect. B (Biol.). 1992; 99B: 83-103Crossref Google Scholar), we tested the hypothesis that IpTxa activates RyRs by mimicking a domain of the DHPR that is critical to trigger Ca2+release. We found that IpTxa and a synthetic peptide with an amino acid sequence corresponding to a segment of the II-III loop (Glu666-Leu690) (10El-Hayek R. Antoniu B. Wang J. Hamilton S.L. Ikemoto N. J. Biol. Chem. 1995; 270: 22116-22118Crossref PubMed Scopus (127) Google Scholar) activate RyRs in a similar manner and appear to compete for a common binding site on the channel protein. Both peptides bind to RyRs via a structural domain consisting of a cluster of basic amino acids (Arg681-Lys685 of the II-III loop and Lys19-Arg24 of IpTxa) followed by a hydroxylated amino acid (Ser687 of the II-III loop and Thr27 of IpTxa). Thus, IpTxapresents an interesting case of toxin mimicry of effector proteins that may be used to identify regions of the RyR that trigger Ca2+ release. If the peptide segment emulated by IpTxa is an actual participant in the DHPR/RyR interaction, IpTxa may also be exploited to identify regions of the RyR involved in E-C coupling. [3H]Ryanodine (60–80 Ci/mmol) was from NEN Life Science Products, agelenin and Tx2-9 were from The Peptide Institute, Inc. (Osaka, Japan), bovine brain phosphatidylethanolamine and phosphatidylserine were from Avanti Polar Lipids (Birmingham, AL), and Fmoc-amino acids were from Applied Biosystems. Polyclonal skeletal RyR antibody was from Upstate Biotechnology. Peroxidase-conjugated secondary antibody and ω-conotoxin were from Calbiochem. The chemiluminescence detection kit was from Boehringer Mannheim. Pre-cast linear gradient polyacrylamide gels were from Bio-Rad. All other reagents were of high-purity reagent grade. [3H]Ryanodine (7 nm) was incubated for 90 min at 36 °C with 40–50 μg of rabbit skeletal SR vesicles in medium containing 0.2 m KCl, 10 μm CaCl2, and 10 mm Na-Hepes (pH 7.2) in the absence and presence of peptides. Free ligand and bound ligand were separated by rapid filtration on Whatman GF/B glass fiber filters, as described previously (13El-Hayek R. Lokuta A.J. Arévalo C. Valdivia H.H. J. Biol. Chem. 1995; 270: 28696-28704Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 14Zamudio F.Z. Gurrola G.B. Arévalo C. Sreekumar R. Walker J.W. Valdivia H.H. Possani L.D. FEBS Lett. 1997; 405: 385-389Crossref PubMed Scopus (62) Google Scholar). Native IpTxa(10-μg batches) was purified according to established procedures (13El-Hayek R. Lokuta A.J. Arévalo C. Valdivia H.H. J. Biol. Chem. 1995; 270: 28696-28704Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar,14Zamudio F.Z. Gurrola G.B. Arévalo C. Sreekumar R. Walker J.W. Valdivia H.H. Possani L.D. FEBS Lett. 1997; 405: 385-389Crossref PubMed Scopus (62) Google Scholar) and iodinated to a specific activity of 60–80 Ci/mmol with the Bolton-Hunter© method following the specifications of the manufacturer (New England Nuclear). The binding of125I-IpTxa to skeletal SR and Chinese hamster ovary (CHO) cell homogenates was performed under conditions identical to those described for [3H]ryanodine, except that the protein concentration was 0.1–0.2 mg/ml in the case of CHO cells.B max and K D of the125I-IpTxa-receptor complex were obtained by fitting data points with the following equation: B =B max ×125I-IpTxa/(K D +125I-IpTxa), where B is the specific binding of 125I-IpTxa. CHO cells were transfected by lipofection with plasmid pRRS11, the rabbit skeletal muscle RyR (RyR1), as described previously (17Bhat M.B. Zhao J. Zang W. Balke C.W. Takeshima H. Wier W.G. Ma J. J. Gen. Physiol. 1997; 110: 749-762Crossref PubMed Scopus (83) Google Scholar). Expression of the RyR was confirmed by immunoblot analysis using monoclonal antibodies against the skeletal RyR and by [3H]ryanodine binding. Control and transfected cells were homogenized in 500 mmsucrose, 1 mm EGTA, and 10 mm Hepes-Tris (pH 7.4) and spun at 44,000 × g for 30 min (17Bhat M.B. Zhao J. Zang W. Balke C.W. Takeshima H. Wier W.G. Ma J. J. Gen. Physiol. 1997; 110: 749-762Crossref PubMed Scopus (83) Google Scholar). The pellet was recovered and used for [3H]ryanodine binding experiments. Linear analogs of IpTxaand the II-III loop were synthesized by the solid-phase methodology with Fmoc amino acids in an automated peptide synthesizer and subjected to the same cyclization and HPLC purification method as described previously (14Zamudio F.Z. Gurrola G.B. Arévalo C. Sreekumar R. Walker J.W. Valdivia H.H. Possani L.D. FEBS Lett. 1997; 405: 385-389Crossref PubMed Scopus (62) Google Scholar). Analytical HPLC, amino acid analysis, and mass spectrometry confirmed the structure and the purity of the synthetic peptides. Photoactivatable IpTxa was prepared by insertingp-benzoyl-phenylalanine, a photoactivatable cross-linker (18Wilson C.J. Husain S.S. Stimson E.R. Dangott L.J. Miller K.W. Maggio J.E. Biochemistry. 1997; 36: 4542-4551Crossref PubMed Scopus (22) Google Scholar), in place of Leu7 during the synthesis of IpTxa. Photoactivatable IpTxa was subjected to the same cyclization and HPLC purification method as described for synthetic IpTxa (14Zamudio F.Z. Gurrola G.B. Arévalo C. Sreekumar R. Walker J.W. Valdivia H.H. Possani L.D. FEBS Lett. 1997; 405: 385-389Crossref PubMed Scopus (62) Google Scholar). Recording of single RyR in lipid bilayers was performed as described previously (13El-Hayek R. Lokuta A.J. Arévalo C. Valdivia H.H. J. Biol. Chem. 1995; 270: 28696-28704Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 19Xiao R.-P. Valdivia H.H. Bogdanov K. Valdivia C. Lakatta E.G. Cheng H. J. Physiol. 1997; 500: 343-354Crossref PubMed Scopus (164) Google Scholar). Single channel data were collected at steady voltages (+30 mV) for 2–5 min in symmetrical 300 mm cesium methanesulfonate, 10 μm CaCl2, and 10 mm Na-Hepes (pH 7.2). IpTxa and the II-III loop peptide were added to thecis chamber, which corresponded to the cytosolic side of the channel (13El-Hayek R. Lokuta A.J. Arévalo C. Valdivia H.H. J. Biol. Chem. 1995; 270: 28696-28704Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 19Xiao R.-P. Valdivia H.H. Bogdanov K. Valdivia C. Lakatta E.G. Cheng H. J. Physiol. 1997; 500: 343-354Crossref PubMed Scopus (164) Google Scholar). The addition of the peptides to the trans(luminal) side of the channel was without effect. In some experiments, we added 10 mm CaCl2 to the transsolution. At 0 mV, Ca2+ was the only charge carrier in these experiments, and both peptides were effective in inducing a subconductance state of about one-fourth of the full conductance level. However, the low signal:noise ratio obtained under these conditions made the analysis of the kinetic effect difficult. For the experiments presented here, we omitted Ca2+ in the transsolution. Signals were filtered with an 8-pole low pass Bessel filter at 2 kHz and digitized at 5 kHz. Data acquisition and analysis were done with Axon Instruments software and hardware (pClamp v6.0.2, Digidata 200 AD/DA interface), as described previously (13El-Hayek R. Lokuta A.J. Arévalo C. Valdivia H.H. J. Biol. Chem. 1995; 270: 28696-28704Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 19Xiao R.-P. Valdivia H.H. Bogdanov K. Valdivia C. Lakatta E.G. Cheng H. J. Physiol. 1997; 500: 343-354Crossref PubMed Scopus (164) Google Scholar). The current values for the full and subconductance states were obtained from Gaussian fits to the all point amplitude histograms, as described previously (19Xiao R.-P. Valdivia H.H. Bogdanov K. Valdivia C. Lakatta E.G. Cheng H. J. Physiol. 1997; 500: 343-354Crossref PubMed Scopus (164) Google Scholar). SR vesicles were purified from rabbit white fast skeletal muscle, as described previously (13El-Hayek R. Lokuta A.J. Arévalo C. Valdivia H.H. J. Biol. Chem. 1995; 270: 28696-28704Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 19Xiao R.-P. Valdivia H.H. Bogdanov K. Valdivia C. Lakatta E.G. Cheng H. J. Physiol. 1997; 500: 343-354Crossref PubMed Scopus (164) Google Scholar). Maximal [3H]ryanodine binding site density was typically 3–5 pmol/mg protein. Ca2+ release from SR vesicles was measured by the method of Palade (20Palade P. J. Biol. Chem. 1987; 262: 6135-6141Abstract Full Text PDF PubMed Google Scholar), with slight modifications. Briefly, SR vesicles (60 μg of protein in 20 μl of reaction medium) were placed in a cuvette containing 980 μl of 95 mm KCl, 20 mm K-MOPS (pH 7.0), 7.5 mm sodium pyrophosphate, 250 μm Antipyrylazo III, 1.5 mm MgATP, 25 μg of creatine phosphokinase, and 5 mm phosphocreatine. The mixture was allowed to equilibrate for 3 min at 37 °C under constant stirring. Free Ca2+was monitored by measuring A 710–790 nm using a diode array spectrophotometer (Hewlett-Packard Model 8452A). Vesicles were actively filled by three to five consecutive additions of 10 nmol of CaCl2 before the addition of IpTxa or the II-III loop peptide. The total amount of Ca2+ loaded was quantified by the addition of 5 μm of the Ca2+ ionophore A23187 at the end of each experiment. SR microsomes (0.4 mg/ml) were incubated with 30 nm photoactivatable IpTxa (see above) in buffer containing 10 μm free Ca2+, 200 mm KCl, and 10 mm Na-Hepes (pH 7.2) in the absence and the presence of 50 μm IpTxa. After 60 min at 36 °C, 1-ml aliquots were spread over 1-cm-diameter plastic wells and irradiated at short range with ultraviolet light (360 nm) for 30 min. Samples were washed twice with incubation buffer by centrifugation in a table-top minifuge at 12,000 rpm. Pellets were then resuspended in Laemmli buffer (0.25 m Tris, pH 6.8, 0.4m dithiothreitol, 8% SDS, 40% glycerol, and 0.04% bromphenol blue) and subjected to SDS-polyacrylamide gel electrophoresis on two identical linear gradient acrylamide gels (4–12%). Proteins contained in one gel were stained with Coomassie Blue, whereas proteins in the other gel were transferred to nitrocellulose membranes for Western blot analysis. Blots were probed first with a rabbit polyclonal RyR1 antibody (dilution, 1:3,000) and then with an anti-rabbit peroxidase-conjugated secondary antibody. Dried gels were then exposed to x-ray film for 2 days. The amino acid sequence of IpTxa (14Zamudio F.Z. Gurrola G.B. Arévalo C. Sreekumar R. Walker J.W. Valdivia H.H. Possani L.D. FEBS Lett. 1997; 405: 385-389Crossref PubMed Scopus (62) Google Scholar) exhibits no significant homology with the well-characterized Na+ and K+channel scorpion toxins (data not shown). However, IpTxadoes share 45% and 42% sequence identity with agelenin (21Hagiwara K. Sakai T. Miwa A. Kawai N. Nakajima T. Biomed. Res. 1990; 1: 181-186Crossref Scopus (30) Google Scholar) and Tx2-9 (22Cordeiro M.doN. Diniz C.R. Valentim A.doC. von Eickstedt V.R. Gilroy J. Richardson M. FEBS Lett. 1992; 310: 153-156Crossref PubMed Scopus (86) Google Scholar), respectively, two spider toxins that block presynaptic (P-type) Ca2+ channels (Fig.1 A). The Cys residues, which stabilize the three-dimensional structure by forming disulfide bridges (16Ménez A. Bontems F. Roumestand C. Gilquin B. Toma F. Proc. R. Soc. Edinb. Sect. B (Biol.). 1992; 99B: 83-103Crossref Google Scholar), are similarly arranged in these three peptides (gray boxes). Indeed, they may be used as a frame to align the amino acid sequence of ω-conotoxin MVIIC, a snail peptide that blocks P-type Ca2+ channels (23Hillyard D.R. Monje V.R. Mintz I.M. Bean B.P. Nadasdi L. Ramachandran J. Miljanich G. Azimi-Zoonooz A. McIntosh J.M. Cruz J.L. Neuron. 1992; 9: 69-77Abstract Full Text PDF PubMed Scopus (441) Google Scholar), and to reveal regions of homology (open boxes). Fig. 1 B shows that, despite the demonstrated structural kinship among these peptides, only IpTxa is capable of enhancing [3H]ryanodine binding. ED50, the concentration of IpTxarequired to produce a half-maximal effect (6.4 ± 3.1 nm, mean ± S.D.; n = 18), is only slightly higher than that exhibited by [3H]ryanodine among ligands of RyRs (24Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 1-51PubMed Google Scholar). This selective and high-affinity effect suggests that IpTxa possesses a unique structural motif that activates RyRs, which is not present even in structurally related peptides. To test whether IpTxa may be used independently as a high-affinity, specific ligand for RyRs, we radiolabeled IpTxa and conducted binding experiments in the absence of ryanodine. Fig.2 A shows that the radiolabeled derivative of IpTxa retained high affinity (K D = 11 ± 3 nm) and bound to skeletal SR with a maximal receptor site density (B max) of 16.1 ± 1.9 pmol/mg protein (n = 3). In the same tissue, theB max for [3H]ryanodine was 3.7 ± 0.6 pmol/mg protein. Thus, assuming all125I-IpTxa binding occurs to the RyR, the125I-IpTxa:[3H]ryanodine binding site stoichiometry is 4.3:1. Because one [3H]ryanodine molecule binds with high affinity to the tetrameric RyR (24Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 1-51PubMed Google Scholar), this ratio suggests that about four IpTxa molecules bind to every RyR tetramer. In CHO cells transfected with the skeletal RyR (Fig. 2 B, + RyR1), the125I-IpTxa:[3H]ryanodine binding site stoichiometry is 4.6:1 (n = 2). In naı̈ve CHO cells, there is neither 125I-IpTxa (Fig.2 B, Untransfected) nor [3H]ryanodine binding (data not shown). To confirm that IpTxa physically interacts with the RyR monomer in skeletal SR, we prepared photoactivatable IpTxa, a synthetic derivative of IpTxa in which Leu7was replaced by the light-sensitive cross-linkerp-benzoyl-phenylalanine (18Wilson C.J. Husain S.S. Stimson E.R. Dangott L.J. Miller K.W. Maggio J.E. Biochemistry. 1997; 36: 4542-4551Crossref PubMed Scopus (22) Google Scholar). The photoreactive derivative retained high affinity for the RyR (K D = 12 ± 4 nm; n = 3; data not shown). Fig.3 A shows a SDS-polyacrylamide gel electrophoresis profile of SR proteins that were radiated with ultraviolet light after incubation with 30 nmphotoactivatable 125I-IpTxa in the absence (lane 1) and the presence (lane 2) of 50 μm unlabeled IpTxa. An immunoblot analysis using a skeletal RyR polyclonal antibody recognized only the high molecular weight band of SR proteins (Fig. 3 B). The autoradiogram of the SDS-gel (Fig. 3 C) shows clear labeling of the band corresponding to the RyR (lane 1). Other bands are also labeled, most likely from a nonspecific interaction with the toxin, because labeling persists in the presence of excess IpTxa (lane 2). Together with data from Fig. 2, these results indicate that IpTxa makes direct protein-protein interactions with the RyR with a stoichiometry of four IpTxa molecules per single RyR channel. The functional effect of the IpTxa-RyR interaction was tested in planar lipid bilayer and Ca2+release experiments. Fig. 4 Ashows that 50 nm IpTxa added to the cytoplasmic (cis) side of the skeletal RyR induced the appearance of a subconductance state corresponding to ∼25% of the full conductance as previously shown (25Trypathy A. Resch W. Xu L. Valdivia H.H. Meissner G. J. Gen. Physiol. 1998; 111: 679-690Crossref PubMed Scopus (76) Google Scholar). Although of small amplitude, the subconductance state displayed a mean open time that was >100-fold longer than that of unmodified channels. Ion flow would therefore be expected to be greater for an IpTxa-modified channel, despite its lower conductance. Fig. 4 B shows that IpTxa elicited Ca2+ release from actively loaded SR vesicles in a dose-dependent manner. The effect of IpTxa was blocked by ruthenium red, consistent with Ca2+ release occurring through RyRs. Strikingly similar results were observed with a 25-amino acid synthetic peptide with primary sequence (Glu666-Leu690; Fig.5 A) overlapping that of peptide A (Thr671-Leu690), a segment of the II-III loop that activates RyRs (10El-Hayek R. Antoniu B. Wang J. Hamilton S.L. Ikemoto N. J. Biol. Chem. 1995; 270: 22116-22118Crossref PubMed Scopus (127) Google Scholar). The 25-amino acid segment of the II-III loop (henceforth termed "the II-III loop peptide"), like IpTxa, induced the appearance of a small-amplitude and long-lifetime subconductance state (Fig. 4 C) and elicited Ca2+ release from SR (Fig. 4 D). Thus, albeit with different affinity, the two apparently unrelated peptides exhibit similar functional effects on RyRs.Figure 5Amino acid sequence and structural domains of IpTxa and the II-III loop peptide. A, complete amino acid sequence of IpTxa and the segment of the II-III loop used in this study. No regions of homology were observed, except for a cluster of basic amino acids (boxes) followed by Ser or Thr (ovals). B, simplified scheme showing the proposed structural analogy between IpTxa and the II-III loop. Vertical lines represent regions of the peptides without significant homology. Rectangles correspond to the cluster of basic amino acids (Lys19-Arg24 and Arg681-Lys685, respectively), andovals correspond to the hydroxylated amino acid (Thr26 and Ser687, respectively).View Large Image Figure ViewerDownload (PPT) A one-to-one residue alignment between IpTxaand the II-III loop peptide does not reveal significant homology in their amino acid sequence (Fig. 5 A). However, both IpTxa and the II-III loop peptide display a structural motif consisting of a cluster of basic amino acids (boxes, Lys19-Arg24 and Arg681-Lys685, respectively) followed by Thr or Ser, two hydroxylated amino acids (ovals, Thr26and Ser687, respectively). In IpTxa, the cluster of basic amino acids is interrupted by Cys21 and encompasses the sequence KCK, which is also found in agelenin and Tx2-9 (Fig. 1 A). Therefore, it is likely that the KCK sequence alone does not suffice to activate RyRs and that Cys21stabilizes the peptide structure without intervening in protein-protein interactions with the RyR. Hydroxylated amino acids in a position close to Thr26 of IpTxa are also found in agelenin (Ser28) and in Tx2-9 (Thr23 and Thr24), but none is preceded by a cluster of basic amino acids. Likewise, the motif RRG, which appears in IpTxa and ω-conotoxin MVIIC, is only followed by a hydroxylated amino acid in the former peptide. Indeed, similar to Ser687 of the II-III loop (27Lu X. Xu L. Meissner G. J. Biol. Chem. 1995; 270: 18459-18464Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), the distinctive arrangement of Thr26 of IpTxa with the preceding residues produces a phosphorylation consensus for several protein kinases (28Kemp B.E. Pearson R.B. Trends Biol. Sci. 1990; 15: 342-346Abstract Full Text PDF PubMed Scopus (807) Google Scholar). As presented in Fig. 5 B, the structural motif consisting of a cluster of positively charged amino acids followed by a hydroxylated residue is not found in other peptide toxins or in other regions of the DHPR, including the β-subunit. Thus, IpTxa and the II-III loop peptide share a specific arrangement of amino acid residues that may be responsible for their similar functional effect on RyR channels. To test whether the analogous functional effects produced by IpTxa and the II-III loop peptide (Fig. 4) result from activation of the same modulatory site on the RyR, we carried out competition experiments between the two peptides. Fig.6 A shows that the II-III loop peptide incrementally decreases the capacity of IpTxa to activate RyRs. The ED50 for II-III loop-inhibition of the IpTxa effect was 1.3 ± 0.7 μm(n = 3), in agreement with the value calculated from direct activation of [3H]ryanodine binding by the II-III loop peptide (Fig. 7 B). In contrast, a scrambled II-III loop (a synthetic peptide with amino acid composition identical to the II-III loop peptide but in random sequence) was incapable of stimulating [3H]ryanodine binding (data not shown) or of abolishing the IpTxa effect (Fig. 6 B). Thus, the effects of the II-III loop peptide require a defined amino acid sequence and are unrelated to peptide mass or electrical charge. In other competition studies, the II-III loop peptide displaced the binding of 125I-IpTxa to SR vesicles with an ED50 of 36 ± 4 μm(Fig. 5 C). This reduced affinity may be due to displacement of 125I-IpTxa from sites of nonequivalent affinity, or it may result from positive allosteric interaction between the II-III loop peptide and 125I-IpTxa. Nevertheless, the II-III loop-125I-IpTxacompetition appears to be specific, because the scrambled II-III loop had no significant effect at concentrations up to 300 μm(Fig. 6 C).Figure 7Parallel mutations in IpTxa and the II-III loop peptide lead to analogous effects. A andB, effect of substituting Thr or Ser. Specific binding of [3H]ryanodine to skeletal SR was measured in the absence (100%) or in the presence of (A) native or synthetic IpTxa (○ and ●, respectively) or derivatives T26A or T26E or (B) the II-III loop peptide (B,Control) or derivatives S687A and S687E. Results are the mean ± S.D. of n = 4–6 independent experiments.View Large Image Figure ViewerDownload (PPT) If, by analogy with other peptide toxin-ion channel associations (29Hidalgo P. MacKinnon R. Science. 1995; 268: 307-310Crossref PubMed Scopus (425) Google Scholar, 30Dudley S.C. Todt H. Lipkind G. Fozzard H.A. Biophys. J. 1995; 69: 1657-1665Abstract Full Text PDF PubMed Scopus (100) Google Scholar), the high-affinity IpTxa-RyR interaction is mediated by electrostatic forces, then mutations in the binding domain of IpTxa should alter its electrostatic potential and the measured affinity constant of the IpTxa-RyR complex. Likewise, if IpTxa and the II-III loop peptide bind to the skeletal RyR via a common structural motif, then corresponding mutations should evoke parallel changes in affinity for both peptides. Fig. 7 A shows that synthetic IpTxa (a synthetic peptide with an amino acid sequence identical to that of native IpTxa; Ref. 14Zamudio F.Z. Gurrola G.B. Arévalo C. Sreekumar R. Walker J.W. Valdivia H.H. Possani L.D. FEBS Lett. 1997; 405: 385-389Crossref PubMed Scopus (62) Google Scholar) activates [3H]ryanodine binding to skeletal SR with potency and affinity identical to native IpTxa (5 ± 3 nm; n = 6). Other synthetic derivatives of IpTxa in which Thr26 was replaced with Ala (T26A) or with the negatively charged residue Glu (T26E) increased [3H]ryanodine binding with an affinity 12- and 160-fold lower (ED50 = 60 ± 5 and 800 ± 78 nm nm, respectively). Mutations to the II-III loop peptide elicited qualitatively similar results (Fig.7
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