Proc. Nati. Acad. Sci. USA Vol. 89, pp. 12185-12189, December 1992 Physiology
Scorpion toxins targeted against the sarcoplasmic reticulum Ca2+-release channel of skeletal and cardiac muscle (ryanodine receptors/Pandinus imperator venom/planar bilayer/ventricular myocytes/Ca2+ indicator)
HECTOR H. VALDIVIA*t, MARK S.
KIRBYf, W. JONATHAN LEDERER*, AND ROBERTO CORONADO*t
*Department of Physiology, University of Wisconsin School of Medicine, Madison, WI 53706; and tDepartment of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201
Communicated by Michael V. L. Bennett, September 21, 1992
ABSTRACT We report the purification of two peptides, called "imperatoxin inhibitor" and "imperatoxin activator," from the venom of the scorpion Pandinus imperator targeted against ryanodine receptor Ca2+-release channels. Imperatoxin inhibitor has a Mr of 10,500, inhibits [3Hjryanodine binding to skeletal and cardiac sarcoplasmic reticulum with an EDso of 10 nM, and blocks openings of skeletal and cardiac Ca2+-release channels incorporated into planar bilayers. In whole-cell recordings of cardiac myocytes, imperatoxin inhibitor decreased twitch amplitude and intracellular Ca2+ transients, suggesting a selective blockade of Ca2+ release from the sarcoplasmic reticulum. Imperatoxin activator has a Mr of -8700, stimulates [3H]ryanodine bind in skeletal but not cardiac sarcoplasmic reticulum with an ED50 of -6 nM, and activates skeletal but not cardiac Ca2+-release channels. These ligands may serve to selectively "turn on" or "turn off" ryanodine receptors in fragmented systems and whole cells. Activation of muscle, neurons, and secretory cells by voltage, neurotransmitters, or hormones can evoke a release of Ca2+ from intracellular Ca2+ stores (1). Two types of Ca2+ channels, namely ryanodine receptors and inositol 1,4,5trisphosphate (InsP3) receptors, have been shown to control the intracellular Ca2+ permeability of many cells. In striated muscle, these channels transduce membrane voltage, sarcolemmal Ca2+ entry, and other external stimuli into an increase in the Ca2+ permeability of the sarcoplasmic reticulum (SR) (2). Elucidation of the mechanism of Ca2+ release from intracellular stores depends critically on the specificity of pharmacological agents to selectively alter a single intracellular Ca2+ channel type. The alkaloid ryanodine is presently the only ligand available to dissect the contribution of ryanodine receptors to intracellular Ca2+ release in situ. However, the usefulness of this compound is limited by the fact that it has an extremely slow association and dissociation kinetics that makes the onset of the pharmacological effect slow and essentially irreversible (3). To accelerate the onset, micromolar instead of nanomolar levels of ryanodine are used, but at micromolar concentrations the alkaloid inhibits other Ca2+ channels (4). Furthermore, certain concentrations of ryanodine may open the Ca2+-release channel while others may block it (5), leading to ambiguous results (6). Alternative ligands that act fast, reversibly, and in a simple manner should be more helpful in establishing the contribution of ryanodine receptors to intracellular Ca2+ signals. Scorpion venoms have traditionally represented an invaluable source of peptide toxins specific for a single channel type (7). In the present report, we screened venom from several genera of scorpions with the hope of finding peptides specific for ryanodine receptors. We purified two peptides from the venom of Pandinus imperator that selectively blocked [im-
peratoxin inhibitor (IpTxi)] or activated [imperatoxin activator (IpTxa)] ryanodine receptors of skeletal and cardiac muscle. Part of these results have been communicated in an abstract form (8). EXPERIMENTAL PROCEDURES Purification of Scorpion Toxins. Lyophilized P. imperator venom was obtained from Latoxan (Rosans, France). Venom (50 mg per batch) was extracted in 2-3 ml of deionized water and chromatographed on a column (1. 5 x 125 cm) of Sephadex G-50 fine. Fractions were eluted with 20 mM NaOAc (pH 4.7) at a flow rate of 10 ml/hr. Fraction II containing IpTxi and fraction III containing IpTxa described in Fig. 1B were applied separately to a column (1 x 25 cm) of carboxymethyl (CM)-cellulose 32 (Pharmacia) equilibrated with 20 mM NaOAc (pH 4.7). Peptides were eluted at a flow rate of 12 ml/hr with a linear gradient of 250 ml of 20 mM NaOAc (pH 4.7) and 250 ml of the same buffer containing 0.5 M NaCl. A peptide from fraction II containing >90% of the inhibitory activity (IpTxi) and a peptide from fraction III containing >90% of the stimulatory activity (IpTxa) were eluted as single symmetric peaks when the NaCl concentration at the top of the CM-cellulose 32 column reached 65 and 340 mM, respectively. IpTxi and IpTx. were dialyzed against deionized water in Spectrapor 3M dialysis membrane (Spectrum Medical Industries), concentrated by vacuum centrifugation, and injected into a C18 reverse-phase HPLC column (,uBondapac, Waters Associates). IpTxi and IpTxa were eluted with a linear gradient of 5-80o acetonitrile in 0.1% trifluoroacetic acid run at 1 ml/min for 60 min. Binding Assays. [3H]Ryanodine [60 mCi/mmol (1 Ci = 37 GBq); DuPont/New England Nuclear] binding was carried out as described (9) for 90 min at 36TC in 0.2 M KCl/1 mM Na2EGTA/0.995 mM CaCl2 (10 AuM free Ca2+; ref. 9)/10 mM sodium Pipes, pH 7.2. Binding of [3H]saxitoxin, [3H]ouabain, and [3H]PN200-110 was carried out in rabbit skeletal muscle transverse tubular membranes as described (10). Binding of [3H]quinuclinidyl benzylate was carried out in bovine cardiac sarcolemma as described (11). Binding of [3H]InsP3 was carried out in rat brain microsomes as described (12). Ca2+ Transients and Twitch Recordings. Adult rat ventricular myocytes were isolated by using a standard collagenase dispersion technique (13). Myocytes were constantly superfused at 350C in a chamber mounted on the stage of an inverted microscope. Intracellular dialysis was achieved by using standard patch-clamp electrodes (2 to 5 Mohms) with the whole-cell configuration, in which the pipette ruptured the cell for insertion of the electrode. Myocyte length was measured with a video-based edge detector. Indo-1 fluoresAbbreviations: SR, sarcoplasmic reticulum; IpTxa and IpTxi, imperatoxin activator and inhibitor, respectively; Ins P3, inositol 1,4,5-
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
trisphosphate. tTo whom reprint requests should be addressed. 12185
12186
Proc. Nati. Acad. Sci. USA 89 (1992)
Physiology: Valdivia et al.
N aC
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FIG. 1. (A) Effect of Pandinus venom on the binding of 7 nM [3H]ryanodine to cardiac (e) and skeletal (o) SR vesicles, which was measured in the absence (control, 100%) and in the presence of the indicated concentrations of Pandinus venom. Binding in control was 1.8 and 0.42 pmol/mg of protein for skeletal and cardiac SR, respectively. For other species results (numbers after the species represent skeletal SR binding activity in pmol/mg SD in the presence of 800 j.g/ml of venom; numbers in parentheses are the percent binding of control) were Androctonus mauritanicus, 1.83 0.32 (102%); Androctonus australis, 1.26 0.40(70%); Buthus arenicola, 1.93 0.44 (107%); Buthus occitanus mardochei, 2.16 0.46 (120%o); Buthus occitanus tunetanus, 2.10 0.28 (118%o); Buthotus hottentota, 6.75 1.22 (375%); Buthotus judaicus, 6.26 1.11 (348%); Leiurus quinquestriatus, 2.46 0.60 (137%); and Leiurus g. hebraeus, 2.09 0.39 (116%). (B) Chromatographic profile of Pandinus venom and effect of venom fractions on PH]ryanodine binding. Pandinus venom was loaded on a Sephadex G-50 column and eluted with 20 mM NaOAc (pH 4.7) as described in text. Fractions (5 ml) were collected and pooled as indicated by the shaded bars. Fractions I through VI (5 ,.g/ml) were assayed for effects on [3H]ryanodine binding to skeletal SR. Elution of molecular weight markers is indicated by the arrowheads. BSA, bovine serum albumin; Cyt C, cytochrome c. ±
±
±
±
±
±
±
±
cence was measured and calibrated at 350C as described (14). The control electrode back-filling solution contained 122 mM KCl, 2.25 mM KH2PO4, 6 mM MgC2, 5 mM K2ATP, 9 mM Hepes, 3.6 mM sodium creatine phosphate, and 0.05 mM K5Indo-1, with pH adjusted to 7.15 at 350C with KOH. IpTxi (1 AM) was always added to the back-filling solution. The superfusing solution contained 118 mM NaCl, 4.75 mM KCI, 0.9 mM KH2PO4, 1.2 mM MgSO4, 5 mM Hepes, 5 mM sodium Hepes, and 10 mM glucose, with pH adjusted to 7.4 at 350C with NaOH. After establishing the whole-cell configuration, there was a small depolarization from -72 + 2.5 mV after 1 min to -69 3.9 mV after 10 min in control cells (n = 6) and a small hyperpolarization from -70.6 3.4 mV after 1 min to -71.3 4.7 mV 5 after 9 min in the presence of toxin (n = 6). These changes were not found to be statistically significant. ±
Others. Skeletal and cardiac SR were prepared from rabbit back and leg white muscle or bovine myocardium in the presence of protease inhibitors as described for skeletal SR (15). Planar bilayer composition and CsCl solutions used in cis and trans chambers have been described (16). Samples for SDS/PAGE (sodium dodecyl sulfate/polyacrylamide gel electrophoresis) were incubated for 15 min at 800C in 2% SDS/2% (vol/vol) 2-mercaptoethanol/1O% (vol/vol) glycerol/10 mM Tris (pH 6.8) and run on a 6-15% linear polyacrylamide gel gradient. Gels were stained with 0.05% Coomassie blue R in 10o acetic acid. Molecularweight standards were from Sigma.
RESULTS concentrations, At nanomolar [3H]ryanodine binds to the tetrameric ryanodine receptor with an apparent dissociation constant of -5 nM (3). Assuming that the high-affinity
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TIME (min) FIG. 2. HPLC profiles of purified toxins. (A) CM-cellulose-eluted toxin from fraction II (IpTx;) and CM-cellulose-eluted toxin from fraction III (IpTxa) were separately injected into a C18 reverse-phase column and eluted with a linear gradient of 5-801% acetonitrile in 0.1% trifluoroacetic acid at 1 ml/min for 60 min. (B) Coomassie brilliant blue stain of SDS/PAGE of venom fractions on a 6-15% polyacrylamide gradient gel. Lanes: 1, 30 tug of whole venom; 2, 20 1Lg of fraction II; 3, 15 jug of fraction III; 4, 2 jxg of IpTxi; 5, 2.5 pLg of IpTxa. Molecular weight standards indicated by the arrowheads were myosin (200,000), frgalactosidase (116,000), phosphorylase b (97,400), bovine serum albumin (68,000), ovalbumin (43,000), and cytochrome c (12,000).
Physiology: Valdivia et al.
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FIG. 3. Effect of toxins on Ca2+-release channel activity. Control Ca2+-release channel activities are from rabbit skeletal SR (A and B) or bovine cardiac SR (C and D). In each experiment, control and test records are from the same channel before (upper two traces) and after (lower two traces) cis addition of 50 nM of IpTxi (Left) or IpTxa (Right). Channel openings are shown as upward deflections at +20 mV (A and B) or 0 mV (C and D). Open probabilities for the entire recording period before and after addition of toxin were 0.118 and 0.021 (A), 0.101 and 0.343 (B), 0.590 and 0.053 (C), and 0.346 and 0.302 (D). Plots correspond to the cumulative sum of the open probability of a single channel, P, multiplied by the number of observable channels, n. The nP product was averaged during inter-
vals of 5 s. t = 0 corresponds to the beginning of the control period (curve labeled C) or to the time immediately after the addition of toxin. Calibration bars are 30 pA and 50 ms.
binding site for ryanodine is formed when the channel is open (9), venoms that changed the binding activity of [3H]ryanodine were considered sources of toxins that could potentially interfere with channel gating. Of 10 scorpion venoms screened in this manner (see Fig. 1 legend), that of P. imperator was the only venom to produce a profound inhibition of [3H]ryanodine binding (Fig. 1A). In a standard assay containing 0.2 M KCl, 10 mM free Ca2+, and 7 nM [3H]ryanodine (pH 7.2), the inhibition produced by whole Pandinus venom in cardiac and skeletal SR was dose dependent with a half-maximal effect at a venom concentration of 10 ug/mi (Fig. 1A). After fractionation on Sephadex G-50 (Fig. 1B), six venom fractions were collected and assayed for effects on [3H]ryanodine binding. Fraction I was composed of proteins in the range of 40-200 kDa eluted in the void volume. Fraction II contained polypeptides in the range of 8-14 kDa. Fractions III and IV contained polypeptides in the range of
Natl. Acad.
Sci. USA 89 (1992)
12187
4-8 kDa, and fractions V and VI contained smaller peptides. As indicated by the bars in Fig.1B, the inhibitory activity was exclusively concentrated in fraction II. Also identified in Fig. 1B is fraction III, a component of Pandinus venom that surprisingly stimulated [3H]ryanodine binding. Fraction III accounted for 20-fold with an ED50 of -6 nM in skeletal SR but had no effect on cardiac SR (Fig. 4B). Thus, IpTxj and IpTxa could modify the gating state of the ryanodine receptor in the absence of ryanodine, and, in addition, IpTxa could distinguish between the skeletal and cardiac isoforms. Toxin specificity was further investigated by the effect of imperatoxins on the binding activity of radiolabeled ligands for various membrane receptors of muscle and brain. Table 1 shows that at a concentration of 100 nM, IpTxa did not interfere with the specific binding of [3H]saxitoxin selective for the voltage-dependent Na+ channel; [3H]ouabain selective for the Na+/K+ pump; [3H]quinuclidinyl benzylate, the selective antagonist of the cardiac muscarinic receptor; or [3H]PN200-110, selective for the high-affinity drug site on the
Physiology: Valdivia et al.
12188
Proc. Natl. Acad. Sci. USA 89 (1992) 2000
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FIG. 4. Dose-response relationship for IpTxi (A) and IpTxa (B). The specific binding of 7 nM [3H]ryanodine was measured in the absence (control, 100%) and the presence of indicated concentrations of IpTxi or IpTxa; 100lo binding (control) corresponds to 1.1 and 0.3 pmol/mg of protein for skeletal (o) and cardiac (e) SR, respectively.
dihydropyridine (DHP) receptor. Results for IpTxi were the same except that it inhibited =40% of the specific binding of
[3H]PN200-110
to skeletal muscle transverse tubules. In
addition, neither IpTxi nor IpTxa nor the whole venom (not shown) interfered with the binding of D-myo-[3H]InsP3 to brain microsomes, which suggested that the toxins were unlikely to interfere with InsP3 receptors. Fig. 5 illustrates the effects of IpTxi in isolated adult rat ventricular myocytes with simultaneous monitoring of intracellular Ca2+ and cell length. A patch electrode was used to maintain the myocyte in the whole-cell current clamp mode and to perfuse into cells the fluorescent Ca2+ indicator Indo-1 in the case of controls (Fig. 5 Right), or Indo-1 plus 1 ILM IpTxj in the case of a test cell (Fig. 5 Left). Fig. 5A shows that in both control and test myocytes, contraction initially declined, illustrating the well-known negative staircase seen in rat ventricle following a train of stimuli. On initiating stimulation, the first twitch was 30.5 6.5% (mean + SD, n = 5) of cell length in control myocytes and 27.5 5.1% (n = 6) of cell length in test myocytes and decreased to 23.3 6.5% and 21.0 5.8%, respectively, after 1 min. Control myocytes showed no further decrease in twitch shortening over the next 9 min. Fig. 5A Right shows that in the cell perfused with IpTxi, there was a much greater decline in twitch shortening. The latter occurred in parallel with the increase in the Indo-1 signal as the contents ofthe pipette diffused into the myocyte. The twitch shortening, 5 to 9 min after establishing whole-cell recording in the IpTxi-treated myocyte, corresponded to 1.8 0.9%o of the cell length or 9%o of the contraction seen in controls after 10 min. Fig. SB shows changes in intracellular Ca2+ transients underlying the changes in cell length shortly after rupturing into the cell (1 min) and after the onset of IpTxi (7 min). In IpTxi-treated cells, peak Ca2+ during the transient 0.34 ,uM at 1 min to was significantly reduced from 1.38 0.39 0.12 ,uM after 7 min (P> 10-4, paired t test) in parallel with the decrease in twitch strength. In contrast, the Ca2+ transient and twitch strength on control cells showed little variation over the same or longer monitoring period. These ±
±
±
±
±
±
results clearly showed that IpTxi inhibited the Ca2+ transient underlying excitation-contraction coupling in adult rat ventricular myocytes.
DISCUSSION Purified IpTxi and IpTxa migrated as single polypeptides with Mr values of 410,500 and -8700, respectively. These values are larger than those of scorpion toxins targeted against the voltage-dependent Na+ channel (=7000) (17), or K+ channel (=4000) (18) but are similar to the latter in specificity and high affinity. The tissue specificity of IpTxa was probably due to a structural difference between cardiac and skeletal ryanodine receptors. A cardiac isoform is expressed in heart and brain, whereas a homologous skeletal muscle isoform is expressed in both fast-twitch and slow-twitch muscle (19). Functional differences between the two isoforms have been found in the conductance of the channel (20), in the Ca2+dependence of activation and inactivation (21), and in the binding of monoclonal antibodies raised against one isoform that do not cross-react with the other isoform (22). IpTxj inhibited Ca2+-release channels of skeletal and cardiac muscle with similar potency, suggesting that the toxin binds to a site present in both isoforms. In contrast, IpTx. activated ryanodine receptors of skeletal muscle but had no effect on cardiac ryanodine receptors nor did it stimulate [3H]ryanodine binding, suggesting that a binding site for IpTxa may not be present in the cardiac isoform. Interestingly, there are peptides in the venom of the scorpion Buthotus hottentota that stimulate [3H]ryanodine binding in both skeletal and cardiac isoforms (16). These stimulatory toxins from Pandinus and Buthotus venoms should help to map the topological differences between the cardiac and skeletal ryanodine receptors.
IpTxj had profound effects on the Ca2+ transient underlying contraction in adult heart ventricular myocytes. At short times (
Scorpion toxins targeted against the sarcoplasmic reticulum Ca2+-release channel of skeletal and cardiac muscle (ryanodine receptors/Pandinus imperator venom/planar bilayer/ventricular myocytes/Ca2+ indicator)
HECTOR H. VALDIVIA*t, MARK S.
KIRBYf, W. JONATHAN LEDERER*, AND ROBERTO CORONADO*t
*Department of Physiology, University of Wisconsin School of Medicine, Madison, WI 53706; and tDepartment of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201
Communicated by Michael V. L. Bennett, September 21, 1992
ABSTRACT We report the purification of two peptides, called "imperatoxin inhibitor" and "imperatoxin activator," from the venom of the scorpion Pandinus imperator targeted against ryanodine receptor Ca2+-release channels. Imperatoxin inhibitor has a Mr of 10,500, inhibits [3Hjryanodine binding to skeletal and cardiac sarcoplasmic reticulum with an EDso of 10 nM, and blocks openings of skeletal and cardiac Ca2+-release channels incorporated into planar bilayers. In whole-cell recordings of cardiac myocytes, imperatoxin inhibitor decreased twitch amplitude and intracellular Ca2+ transients, suggesting a selective blockade of Ca2+ release from the sarcoplasmic reticulum. Imperatoxin activator has a Mr of -8700, stimulates [3H]ryanodine bind in skeletal but not cardiac sarcoplasmic reticulum with an ED50 of -6 nM, and activates skeletal but not cardiac Ca2+-release channels. These ligands may serve to selectively "turn on" or "turn off" ryanodine receptors in fragmented systems and whole cells. Activation of muscle, neurons, and secretory cells by voltage, neurotransmitters, or hormones can evoke a release of Ca2+ from intracellular Ca2+ stores (1). Two types of Ca2+ channels, namely ryanodine receptors and inositol 1,4,5trisphosphate (InsP3) receptors, have been shown to control the intracellular Ca2+ permeability of many cells. In striated muscle, these channels transduce membrane voltage, sarcolemmal Ca2+ entry, and other external stimuli into an increase in the Ca2+ permeability of the sarcoplasmic reticulum (SR) (2). Elucidation of the mechanism of Ca2+ release from intracellular stores depends critically on the specificity of pharmacological agents to selectively alter a single intracellular Ca2+ channel type. The alkaloid ryanodine is presently the only ligand available to dissect the contribution of ryanodine receptors to intracellular Ca2+ release in situ. However, the usefulness of this compound is limited by the fact that it has an extremely slow association and dissociation kinetics that makes the onset of the pharmacological effect slow and essentially irreversible (3). To accelerate the onset, micromolar instead of nanomolar levels of ryanodine are used, but at micromolar concentrations the alkaloid inhibits other Ca2+ channels (4). Furthermore, certain concentrations of ryanodine may open the Ca2+-release channel while others may block it (5), leading to ambiguous results (6). Alternative ligands that act fast, reversibly, and in a simple manner should be more helpful in establishing the contribution of ryanodine receptors to intracellular Ca2+ signals. Scorpion venoms have traditionally represented an invaluable source of peptide toxins specific for a single channel type (7). In the present report, we screened venom from several genera of scorpions with the hope of finding peptides specific for ryanodine receptors. We purified two peptides from the venom of Pandinus imperator that selectively blocked [im-
peratoxin inhibitor (IpTxi)] or activated [imperatoxin activator (IpTxa)] ryanodine receptors of skeletal and cardiac muscle. Part of these results have been communicated in an abstract form (8). EXPERIMENTAL PROCEDURES Purification of Scorpion Toxins. Lyophilized P. imperator venom was obtained from Latoxan (Rosans, France). Venom (50 mg per batch) was extracted in 2-3 ml of deionized water and chromatographed on a column (1. 5 x 125 cm) of Sephadex G-50 fine. Fractions were eluted with 20 mM NaOAc (pH 4.7) at a flow rate of 10 ml/hr. Fraction II containing IpTxi and fraction III containing IpTxa described in Fig. 1B were applied separately to a column (1 x 25 cm) of carboxymethyl (CM)-cellulose 32 (Pharmacia) equilibrated with 20 mM NaOAc (pH 4.7). Peptides were eluted at a flow rate of 12 ml/hr with a linear gradient of 250 ml of 20 mM NaOAc (pH 4.7) and 250 ml of the same buffer containing 0.5 M NaCl. A peptide from fraction II containing >90% of the inhibitory activity (IpTxi) and a peptide from fraction III containing >90% of the stimulatory activity (IpTxa) were eluted as single symmetric peaks when the NaCl concentration at the top of the CM-cellulose 32 column reached 65 and 340 mM, respectively. IpTxi and IpTx. were dialyzed against deionized water in Spectrapor 3M dialysis membrane (Spectrum Medical Industries), concentrated by vacuum centrifugation, and injected into a C18 reverse-phase HPLC column (,uBondapac, Waters Associates). IpTxi and IpTxa were eluted with a linear gradient of 5-80o acetonitrile in 0.1% trifluoroacetic acid run at 1 ml/min for 60 min. Binding Assays. [3H]Ryanodine [60 mCi/mmol (1 Ci = 37 GBq); DuPont/New England Nuclear] binding was carried out as described (9) for 90 min at 36TC in 0.2 M KCl/1 mM Na2EGTA/0.995 mM CaCl2 (10 AuM free Ca2+; ref. 9)/10 mM sodium Pipes, pH 7.2. Binding of [3H]saxitoxin, [3H]ouabain, and [3H]PN200-110 was carried out in rabbit skeletal muscle transverse tubular membranes as described (10). Binding of [3H]quinuclinidyl benzylate was carried out in bovine cardiac sarcolemma as described (11). Binding of [3H]InsP3 was carried out in rat brain microsomes as described (12). Ca2+ Transients and Twitch Recordings. Adult rat ventricular myocytes were isolated by using a standard collagenase dispersion technique (13). Myocytes were constantly superfused at 350C in a chamber mounted on the stage of an inverted microscope. Intracellular dialysis was achieved by using standard patch-clamp electrodes (2 to 5 Mohms) with the whole-cell configuration, in which the pipette ruptured the cell for insertion of the electrode. Myocyte length was measured with a video-based edge detector. Indo-1 fluoresAbbreviations: SR, sarcoplasmic reticulum; IpTxa and IpTxi, imperatoxin activator and inhibitor, respectively; Ins P3, inositol 1,4,5-
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
trisphosphate. tTo whom reprint requests should be addressed. 12185
12186
Proc. Nati. Acad. Sci. USA 89 (1992)
Physiology: Valdivia et al.
N aC
BSA Cyt C Y
y
-
30C
-
20C
B 0.8 +
c C
-0
.0
0.6-
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Elution volume. ml
P. imperator venom, tg/ml
FIG. 1. (A) Effect of Pandinus venom on the binding of 7 nM [3H]ryanodine to cardiac (e) and skeletal (o) SR vesicles, which was measured in the absence (control, 100%) and in the presence of the indicated concentrations of Pandinus venom. Binding in control was 1.8 and 0.42 pmol/mg of protein for skeletal and cardiac SR, respectively. For other species results (numbers after the species represent skeletal SR binding activity in pmol/mg SD in the presence of 800 j.g/ml of venom; numbers in parentheses are the percent binding of control) were Androctonus mauritanicus, 1.83 0.32 (102%); Androctonus australis, 1.26 0.40(70%); Buthus arenicola, 1.93 0.44 (107%); Buthus occitanus mardochei, 2.16 0.46 (120%o); Buthus occitanus tunetanus, 2.10 0.28 (118%o); Buthotus hottentota, 6.75 1.22 (375%); Buthotus judaicus, 6.26 1.11 (348%); Leiurus quinquestriatus, 2.46 0.60 (137%); and Leiurus g. hebraeus, 2.09 0.39 (116%). (B) Chromatographic profile of Pandinus venom and effect of venom fractions on PH]ryanodine binding. Pandinus venom was loaded on a Sephadex G-50 column and eluted with 20 mM NaOAc (pH 4.7) as described in text. Fractions (5 ml) were collected and pooled as indicated by the shaded bars. Fractions I through VI (5 ,.g/ml) were assayed for effects on [3H]ryanodine binding to skeletal SR. Elution of molecular weight markers is indicated by the arrowheads. BSA, bovine serum albumin; Cyt C, cytochrome c. ±
±
±
±
±
±
±
±
cence was measured and calibrated at 350C as described (14). The control electrode back-filling solution contained 122 mM KCl, 2.25 mM KH2PO4, 6 mM MgC2, 5 mM K2ATP, 9 mM Hepes, 3.6 mM sodium creatine phosphate, and 0.05 mM K5Indo-1, with pH adjusted to 7.15 at 350C with KOH. IpTxi (1 AM) was always added to the back-filling solution. The superfusing solution contained 118 mM NaCl, 4.75 mM KCI, 0.9 mM KH2PO4, 1.2 mM MgSO4, 5 mM Hepes, 5 mM sodium Hepes, and 10 mM glucose, with pH adjusted to 7.4 at 350C with NaOH. After establishing the whole-cell configuration, there was a small depolarization from -72 + 2.5 mV after 1 min to -69 3.9 mV after 10 min in control cells (n = 6) and a small hyperpolarization from -70.6 3.4 mV after 1 min to -71.3 4.7 mV 5 after 9 min in the presence of toxin (n = 6). These changes were not found to be statistically significant. ±
Others. Skeletal and cardiac SR were prepared from rabbit back and leg white muscle or bovine myocardium in the presence of protease inhibitors as described for skeletal SR (15). Planar bilayer composition and CsCl solutions used in cis and trans chambers have been described (16). Samples for SDS/PAGE (sodium dodecyl sulfate/polyacrylamide gel electrophoresis) were incubated for 15 min at 800C in 2% SDS/2% (vol/vol) 2-mercaptoethanol/1O% (vol/vol) glycerol/10 mM Tris (pH 6.8) and run on a 6-15% linear polyacrylamide gel gradient. Gels were stained with 0.05% Coomassie blue R in 10o acetic acid. Molecularweight standards were from Sigma.
RESULTS concentrations, At nanomolar [3H]ryanodine binds to the tetrameric ryanodine receptor with an apparent dissociation constant of -5 nM (3). Assuming that the high-affinity
A I.
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TIME (min) FIG. 2. HPLC profiles of purified toxins. (A) CM-cellulose-eluted toxin from fraction II (IpTx;) and CM-cellulose-eluted toxin from fraction III (IpTxa) were separately injected into a C18 reverse-phase column and eluted with a linear gradient of 5-801% acetonitrile in 0.1% trifluoroacetic acid at 1 ml/min for 60 min. (B) Coomassie brilliant blue stain of SDS/PAGE of venom fractions on a 6-15% polyacrylamide gradient gel. Lanes: 1, 30 tug of whole venom; 2, 20 1Lg of fraction II; 3, 15 jug of fraction III; 4, 2 jxg of IpTxi; 5, 2.5 pLg of IpTxa. Molecular weight standards indicated by the arrowheads were myosin (200,000), frgalactosidase (116,000), phosphorylase b (97,400), bovine serum albumin (68,000), ovalbumin (43,000), and cytochrome c (12,000).
Physiology: Valdivia et al.
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FIG. 3. Effect of toxins on Ca2+-release channel activity. Control Ca2+-release channel activities are from rabbit skeletal SR (A and B) or bovine cardiac SR (C and D). In each experiment, control and test records are from the same channel before (upper two traces) and after (lower two traces) cis addition of 50 nM of IpTxi (Left) or IpTxa (Right). Channel openings are shown as upward deflections at +20 mV (A and B) or 0 mV (C and D). Open probabilities for the entire recording period before and after addition of toxin were 0.118 and 0.021 (A), 0.101 and 0.343 (B), 0.590 and 0.053 (C), and 0.346 and 0.302 (D). Plots correspond to the cumulative sum of the open probability of a single channel, P, multiplied by the number of observable channels, n. The nP product was averaged during inter-
vals of 5 s. t = 0 corresponds to the beginning of the control period (curve labeled C) or to the time immediately after the addition of toxin. Calibration bars are 30 pA and 50 ms.
binding site for ryanodine is formed when the channel is open (9), venoms that changed the binding activity of [3H]ryanodine were considered sources of toxins that could potentially interfere with channel gating. Of 10 scorpion venoms screened in this manner (see Fig. 1 legend), that of P. imperator was the only venom to produce a profound inhibition of [3H]ryanodine binding (Fig. 1A). In a standard assay containing 0.2 M KCl, 10 mM free Ca2+, and 7 nM [3H]ryanodine (pH 7.2), the inhibition produced by whole Pandinus venom in cardiac and skeletal SR was dose dependent with a half-maximal effect at a venom concentration of 10 ug/mi (Fig. 1A). After fractionation on Sephadex G-50 (Fig. 1B), six venom fractions were collected and assayed for effects on [3H]ryanodine binding. Fraction I was composed of proteins in the range of 40-200 kDa eluted in the void volume. Fraction II contained polypeptides in the range of 8-14 kDa. Fractions III and IV contained polypeptides in the range of
Natl. Acad.
Sci. USA 89 (1992)
12187
4-8 kDa, and fractions V and VI contained smaller peptides. As indicated by the bars in Fig.1B, the inhibitory activity was exclusively concentrated in fraction II. Also identified in Fig. 1B is fraction III, a component of Pandinus venom that surprisingly stimulated [3H]ryanodine binding. Fraction III accounted for 20-fold with an ED50 of -6 nM in skeletal SR but had no effect on cardiac SR (Fig. 4B). Thus, IpTxj and IpTxa could modify the gating state of the ryanodine receptor in the absence of ryanodine, and, in addition, IpTxa could distinguish between the skeletal and cardiac isoforms. Toxin specificity was further investigated by the effect of imperatoxins on the binding activity of radiolabeled ligands for various membrane receptors of muscle and brain. Table 1 shows that at a concentration of 100 nM, IpTxa did not interfere with the specific binding of [3H]saxitoxin selective for the voltage-dependent Na+ channel; [3H]ouabain selective for the Na+/K+ pump; [3H]quinuclidinyl benzylate, the selective antagonist of the cardiac muscarinic receptor; or [3H]PN200-110, selective for the high-affinity drug site on the
Physiology: Valdivia et al.
12188
Proc. Natl. Acad. Sci. USA 89 (1992) 2000
B c ._
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FIG. 4. Dose-response relationship for IpTxi (A) and IpTxa (B). The specific binding of 7 nM [3H]ryanodine was measured in the absence (control, 100%) and the presence of indicated concentrations of IpTxi or IpTxa; 100lo binding (control) corresponds to 1.1 and 0.3 pmol/mg of protein for skeletal (o) and cardiac (e) SR, respectively.
dihydropyridine (DHP) receptor. Results for IpTxi were the same except that it inhibited =40% of the specific binding of
[3H]PN200-110
to skeletal muscle transverse tubules. In
addition, neither IpTxi nor IpTxa nor the whole venom (not shown) interfered with the binding of D-myo-[3H]InsP3 to brain microsomes, which suggested that the toxins were unlikely to interfere with InsP3 receptors. Fig. 5 illustrates the effects of IpTxi in isolated adult rat ventricular myocytes with simultaneous monitoring of intracellular Ca2+ and cell length. A patch electrode was used to maintain the myocyte in the whole-cell current clamp mode and to perfuse into cells the fluorescent Ca2+ indicator Indo-1 in the case of controls (Fig. 5 Right), or Indo-1 plus 1 ILM IpTxj in the case of a test cell (Fig. 5 Left). Fig. 5A shows that in both control and test myocytes, contraction initially declined, illustrating the well-known negative staircase seen in rat ventricle following a train of stimuli. On initiating stimulation, the first twitch was 30.5 6.5% (mean + SD, n = 5) of cell length in control myocytes and 27.5 5.1% (n = 6) of cell length in test myocytes and decreased to 23.3 6.5% and 21.0 5.8%, respectively, after 1 min. Control myocytes showed no further decrease in twitch shortening over the next 9 min. Fig. 5A Right shows that in the cell perfused with IpTxi, there was a much greater decline in twitch shortening. The latter occurred in parallel with the increase in the Indo-1 signal as the contents ofthe pipette diffused into the myocyte. The twitch shortening, 5 to 9 min after establishing whole-cell recording in the IpTxi-treated myocyte, corresponded to 1.8 0.9%o of the cell length or 9%o of the contraction seen in controls after 10 min. Fig. SB shows changes in intracellular Ca2+ transients underlying the changes in cell length shortly after rupturing into the cell (1 min) and after the onset of IpTxi (7 min). In IpTxi-treated cells, peak Ca2+ during the transient 0.34 ,uM at 1 min to was significantly reduced from 1.38 0.39 0.12 ,uM after 7 min (P> 10-4, paired t test) in parallel with the decrease in twitch strength. In contrast, the Ca2+ transient and twitch strength on control cells showed little variation over the same or longer monitoring period. These ±
±
±
±
±
±
results clearly showed that IpTxi inhibited the Ca2+ transient underlying excitation-contraction coupling in adult rat ventricular myocytes.
DISCUSSION Purified IpTxi and IpTxa migrated as single polypeptides with Mr values of 410,500 and -8700, respectively. These values are larger than those of scorpion toxins targeted against the voltage-dependent Na+ channel (=7000) (17), or K+ channel (=4000) (18) but are similar to the latter in specificity and high affinity. The tissue specificity of IpTxa was probably due to a structural difference between cardiac and skeletal ryanodine receptors. A cardiac isoform is expressed in heart and brain, whereas a homologous skeletal muscle isoform is expressed in both fast-twitch and slow-twitch muscle (19). Functional differences between the two isoforms have been found in the conductance of the channel (20), in the Ca2+dependence of activation and inactivation (21), and in the binding of monoclonal antibodies raised against one isoform that do not cross-react with the other isoform (22). IpTxj inhibited Ca2+-release channels of skeletal and cardiac muscle with similar potency, suggesting that the toxin binds to a site present in both isoforms. In contrast, IpTx. activated ryanodine receptors of skeletal muscle but had no effect on cardiac ryanodine receptors nor did it stimulate [3H]ryanodine binding, suggesting that a binding site for IpTxa may not be present in the cardiac isoform. Interestingly, there are peptides in the venom of the scorpion Buthotus hottentota that stimulate [3H]ryanodine binding in both skeletal and cardiac isoforms (16). These stimulatory toxins from Pandinus and Buthotus venoms should help to map the topological differences between the cardiac and skeletal ryanodine receptors.
IpTxj had profound effects on the Ca2+ transient underlying contraction in adult heart ventricular myocytes. At short times (
