Activation of the Ryanodine Receptor Ca2’ Release Channel of Sarcoplasmic Reticulum by a Novel Scorpion Venom*

We identified a peptide fraction from the venom of the scorpion Buthotus hottentota that stimulated binding of [‘Hlryanodine to ryanodine receptors of skeletal and cardiac sarcoplasmic reticulum and brain microsomes in a highly specific manner. Activity was con- centrated in a peptide fraction of M, 5,000-8,000. Assuming a single active peptide in this fraction, we estimated a dissociation constant of 20-30 nM for the interaction of the peptide with the ryanodine receptor. The whole venom and the purified fraction activated skeletal ryanodine receptor Ca2+ release channels in-corporated into planar lipid bilayers. The venom pro- duced a 10-fold increase in the mean open time and induced the appearance of a long lasting subconduct- ance state not seen in controls. Changes were reversi-ble and could be induced by the partially purified venom fraction. This novel scorpion venom should be helpful in establishing the role of ryanodine receptors in the initiation of intracellular Ca2+ release in striated muscle and in nonmuscle cells containing functional ryanodine receptors such as neurons and secretory cells.

presence of this protein in the brain suggests that it may also mediate the caffeine-sensitive release of Ca2+ from intracellular stores of neurons (9, 10). Single channel recordings and whole cell studies have shown that ryanodine opens the Ca2+ release channel (2, 3). When this happens inside a cell, there is a depletion of Ca2+ from intracellular stores (11). Since ryanodine only affects the Ca2+ permeability of the SR, this compound has been used to dissect the contribution of intracellular Ca2+ stores to excitation-contraction coupling (12, 13). However, there are several limitations to the use of ryanodine as a probe of SR function. Ryanodine has extremely slow association and dissociation kinetics (14) that make the onset of the pharmacological effect slow and essentially irreversible. In addition, certain concentrations of ryanodine may open, or others may block, the Ca2+ release channel (15) leading to a controversy about its mechanism of action (16,17). We report a novel ligand of the ryanodine receptor that interacts in a simple manner and may thus contribute to the understanding of the functional role of this protein. In the venom of the scorpion Buthotus hottentota we found high affinity peptide component(s) that selectively increased the binding of [3H]ryanodine to the receptor and opened the Ca2+ release channel. The peptide(s) seemed to act in a simple manner, activating ryanodine receptors rapidly and reversibly. Part of these results have been published in an abstract form (18).

EXPERIMENTAL PROCEDURES
Scorpion Venom-Venom from the African scorpion B. hottentota was obtained from Latoxan (Rosans, France). Approximately 100 mg of lyophilized venom were resuspended in 3 ml of deionized water and centrifuged at 10,000 X g for 15 min to remove mucoid material. The supernatant was saved, and the pellet was extracted twice in 5 ml of deionized water. The combined supernatants were filtered through a nitrocellulose filter with a pore size of 0.66 pm (Millipore, Bedford, MA) and lyophilized until use.
Preparation of SR and Brain Membranes-Heavy SR was prepared from rabbit back and leg white muscle, and separately from bovine ventricle, as described elsewhere (19). SR was stored frozen at -80 "C in 0.32 M sucrose, 0.1 M KCl, and 5 mM Na-Pipes, pH 7.0. Brain microsomes were prepared from rat brain cortex. Five cortices of Sprague-Dawley rats were removed and homogenized in ice-cold 0.32 M sucrose with five strokes of a motor-driven Teflon/glass homogenizer. The resulting homogenate was sedimented at 1,000 X g for 15 min, and the supernatant was used in binding experiments. Homogenizations of muscle and brain tissues were carried out in the presence of the following protease inhibitors: pepstatin A (1 p~) , iodoacetamide (1 mM), phenylmethylsulfonyl fluoride (0.1 mM), leupeptin (1 p M ) , and benzamidine (1 mM).

BindingA~say-[~H]
Ryanodine binding was carried out for 90 min at 36 "C in 0.1 ml of 0.2 M KC1, 1 mM Na2EGTA, 0.995 mM CaCI2, 10 mM Na-Pipes, pH 7.2. The calculated free Ca2+ was 10 p~. Other concentrations of Ca2+ and KC1 are indicated in the text and figure legends. Free Ca2+ concentrations were calculated with a computer program using affinity constants of Fabiato (20).
[3H]Ryanodine (60 mCi/mmol) was purchased from Du Pont-New England Nuclear and was diluted directly in the incubation medium to achieve concentrations in the saturable range of 1-30 nM. During incubation, skeletal SR (0.2-0.3 mg/ml), cardiac SR (0.3-0.5 mg/ml), or brain microsomes (0.6-0.8 mg/ml) were the last reagent to be added to the medium. Samples were filtered on Whatman GF/B glass fiber filters and washed twice with 5 ml of distilled water. A Brandel cell harvester (Gaithersburg, MD) was used for filtration. The nonspecific binding was measured in the presence of 10 p~ unlabeled ryanodine and was subtracted from each sample. In order to establish if [3H]ryanodine binding was affected by proteolytic degradation that may have oc-

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This is an Open Access article under the CC BY license. curred during the binding assay, control incubations were carried out in the presence or absence of the protease inhibitors pepstatin A (1 pM), iodoacetamide (1 mM), phenylmethylsulfonyl fluoride (0.1 mM), leupeptin (1 p~) , and benzamidine (1 mM). Addition of the protease inhibitor mixture did not result in a significant change in the site density or in the affinity of [3H]ryanodine binding.
Planar Bilayer Recording-Planar bilayers were composed of brain phosphatidylethanolamine and brain phosphatidylserine at a 1:l weight ratio (Avanti Polar Lipids, Birmingham, AL) dissolved in decane (Aldrich). Recordings were filtered through a low-pass Bessel filter (Frequency Devices, Haverhill, MA) at 2 kHz and digitized at 4 kHz for analysis (19).
Others-SDS-polyacrylamide gel electrophoresis was performed according to the Laemmli method. Samples were incubated for 15 min at 80 "C in 2% SDS, 2% 8-mercaptoethanol, 10% glycerol, and 10 mM Tris (pH 6.8) and run on a 6-15% linear polyacrylamide gradient. Gels were stained with 0.05% Coomassie Blue R in 10% acetic acid. Molecular weight standards were: myosin, M, 200 (3,21,22). Thus, for the initial screening of various scorpion venoms we used a standard [3H]ryanodine binding assay based on the assumption that changes in binding activity produced by a given venom were likely to reflect activation or inhibition of Ca2+ release channel activity.  Alternatively, there could be a tissuespecific interaction of the venom with one of the two isoforms described for the ryanodine receptor. A cardiac isoform is expressed in heart and brain, whereas the skeletal muscle isoform is expressed in both fast-and slow-twitch skeletal muscle (23). In such a case, the preferential effect of Buthotus venom on the skeletal receptor could be an indication of a structural difference between the venom binding sites in the two isoforms.
Since caffeine is a known agonist of ryanodine receptors (22), we compared the effect of caffeine and Buthotus venom. Fig. 2 shows the biphasic Ca2+ dependence of [3H]ryanodine binding consisting of an increase in the range of pCa 9-5 and a decrease in the range of pCa 4-2. This dual effect of Ca2+ gave rise to a bell-shaped curve which was similar to those described previously (3,19). In the absence of caffeine (open circles), binding had a threshold for detection at pCa 7 and peaked at pCa 5. Caffeine, at a concentration of 10 mM, reduced the threshold for Ca2+-dependent stimulation to pCa 8 and, in addition, shifted the ascending part of the curve toward lower Ca2+ by approximately a factor of 6. The latter is in agreement with previous reports that describe caffeine as a positive modulator at submicromolar Ca2+ (24). The agonist effect of Buthotus venom was markedly different from that of caffeine. Venom, at a concentration of 100 pg/ml (open triangles), increased binding in both the ascending and de-

Skeletal
wending limbs of the pCa curve. The combined effect of caffeine and Buthotus venom (filled triangles) was equally interesting since there was a dramatic synergism. For example, binding at pCa 8 increased from 0.5 pmol/mg for each compound alone to 4.2 pmol/mg for both combined. No synergism was observed, however, at pCa 4 or lower, which is consistent with the idea that caffeine is ineffective at high Ca2+ (24). Ca2+ binding to a high affinity site increases the density of sites available for [3H]ryanodine binding whereas Ca2+ binding to a low affinity site decreases the number of binding sites (24). Within this scheme, the major effect of Buthotus venom could be explained by an action of the venom on both high and low affinity sites for Ca2+. The direct interaction of Buthotus venom with the Ca2+ release channel was investigated in planar bilayer recordings in the absence of ryanodine (19). To identify the component(s) responsible for channel activation we fractionated the venom peptides by gel filtration (26). Fig. 4A shows the chromatographic profile of Buthotus venom in a column of Sephadex G-50. The elution volume of BSA (68 kDa), cytochrome c (12 kDa), and NaCl (58.4 Da) are indicated on the top. Five fractions were pooled as indicated by the horizontal bars and assayed for activation of [3H] ryanodine binding. The bulk of the stimulatory activity (>65%) was concentrated in Fraction I11 although some stimulation was also observed in Fractions I and IV but none in Fraction V. The stimulation by Fraction I was discarded since it was not always present. The inset of Fig. 4A shows that fraction I11 was composed of peptides of M , 5,000-8,000, which was consistent with the active component being a peptide with a size similar to that of other channel-specific scorpion toxins (26). However, unlike other scorpion toxins (26), Fraction I11 could be inactivated by boiling for 2 min (not shown). and skeletal muscle Na+/K+ pump and dihydropyridine receptor, respectively (not shown). These results revealed that Fraction I11 had a high degree of specificity for the SR Ca2+ release channel. Although information on the structural and molecular properties of ryanodine receptors has accumulated rapidly (23,27,28), the functional role of this protein in excitation-contraction coupling remains unknown. Both the reversibility and high specificity of Buthotus venom clearly makes it more advantageous than ryanodine as a chemical probe of excitation-contraction coupling.