The Mechanism of Inhibition of Ryanodine Receptor Channels by Imperatoxin I, a Heterodimeric Protein from the Scorpion Pandinus imperator*

(Received for publication, February 6, 1997)

Fernando Z. Zamudio Dagger , Renaud Conde Dagger , Carolina Arévalo §, Baltazar Becerril Dagger , Brian M. Martin §, Hector H. Valdivia §par ** and Lourival D. Possani Dagger par

From the § Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706, the Dagger  Department of Molecular Recognition and Structural Biology, Biotechnology Institute, National Autonomous University of Mexico, Cuernavaca, Morelos 62271, Mexico, and the  National Institute of Mental Health, Unit on Molecular Structures, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

We present an in-depth analysis of the structural and functional properties of Imperatoxin I (IpTxi), an ~15-kDa protein from the venom of the scorpion Pandinus imperator that inhibits Ca2+ release channel/ryanodine receptor (RyR) activity (Valdivia, H. H., Kirby, M. S., Lederer, W. J., and Coronado, R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12185-12189). A cDNA library was prepared from the venomous glands of this scorpion and used to clone the gene encoding IpTxi. From a single continuous messenger RNA, the information coding for the toxin is translated into two mature polypeptide subunits after elimination of a basic pentapeptide. The IpTxi dimer consists of a large subunit (104-amino acid residues) with phospholipase A2 (PLA2) activity covalently linked by a disulfide bond to a smaller (27 amino acid residues), structurally unrelated subunit. Thus, IpTxi is a heterodimeric protein with lipolytic action, a property that is only shared with beta -bungarotoxins, a group of neurotoxins from snake venoms. The enzymatic subunit of IpTxi is highly homologous to PLA2 from bee (Apis mellifera) and lizard (Heloderma horridum) venoms. The small subunit has no significant similarity to any other known peptide, including members of the Kunitz protease inhibitors superfamily that target the lipolytic effect of beta -bungarotoxins. A synthetic peptide with amino acid sequence identical to that of the small subunit failed to inhibit RyR. On the other hand, treatment of IpTxi with p-bromophenacylbromide, a specific inhibitor of PLA2 activity, greatly reduced the capacity of IpTxi to inhibit RyRs. These results suggested that a lipid product of PLA2 activity, more than a direct IpTxi-RyR interaction, was responsible for RyR inhibition.


INTRODUCTION

Scorpion venoms contain families of small basic proteins that modify the gating mechanism of Na+ channels or block with high affinity K+ channels of excitable cells (1, 2). These toxins have been invaluable tools in the identification, purification, structural mapping, and functional characterization of the corresponding ionic channels. The study of other ionic channels have also been aided by toxins from poisonous animals. For instance, snake venoms contain potent neurotoxins that block acetylcholine receptors (3), and snail and spider venoms contain small molecular weight proteins directed against neuronal Ca2+ channels (4, 5). A Cl- channel-specific blocker peptide was also recently isolated from a scorpion venom (6). Thus, there exist a vast array of natural ligands useful for structural and functional characterization of ionic channels.

The Ca2+ release channel of SR1 constitutes the major pathway for Ca2+ release during the process of excitation-contraction coupling in cardiac and skeletal muscle (7). The Ca2+ release channel binds the plant alkaloid ryanodine with nanomolar affinity, hence the name ryanodine receptor (RyR). Ryanodine has been an invaluable tool in the structural and functional characterization of RyR. The alkaloid binds to a conformationally sensitive domain on the RyR protein and may be used in binding assays as an index of the functional state of the channel (8, 9). However, ryanodine displays extremely slow dissociation kinetics that make its effect practically irreversible. Furthermore, certain concentrations of ryanodine may open RyRs while others may block them (10), leading to ambiguous results.

From the venom of the African scorpion Pandinus imperator, we isolated Imperatoxin I (IpTxi), a ~15-kDa protein that inhibited [3H]ryanodine binding to cardiac and skeletal SR by blocking RyR channels (11). At concentrations well above the half-maximal effective concentrations (ED50) exhibited for RyR, IpTxi did not modify the binding of ligands targeted against other transporters and ionic channels of striated muscle (11). IpTxi blocked RyR rapidly and reversibly, and when injected in ventricular cells it decreased twitch amplitude and intracellular Ca2+ transients, suggesting a selective blockade of Ca2+ release from the SR (11).

In this study, we carried out an in-depth analysis of the mechanism of action of IpTxi on RyRs of cardiac and skeletal muscle. We determined the complete amino acid and nucleotide sequence of IpTxi and show that, like beta -bungarotoxins, IpTxi is a heterodimeric protein composed of a high molecular weight subunit with PLA2 activity and a small, structurally unrelated subunit. We also show that free fatty acids, lipid products of IpTxi-PLA2 activity, are involved in the inhibition of RyR. IpTxi thus offers an alternative way to block RyR distinct from ryanodine and other ligands that require a physical interaction with the RyR protein.


EXPERIMENTAL PROCEDURES

Chemicals and Reagents

Protease lysine C (Lys-C) and restriction enzymes were from Boehringer Mannheim. Chemicals and solvents for peptide sequencing were from Millipore Co. Primers for polymerase chain reaction (lambda gt11 forward and reverse; 1218 and 1222) and VentR polymerase were from New England BioLabs. DNA sequencing kit (Sequenase version II), was from U. S. Biochemical Corp. Subcloning and sequencing vector (pBluescript phagemid; pKS), M13 -20, and M13 reverse sequencing primers were from Stratagene. Brain phosphatidylethanolamine and brain phosphatidylserine were from Avanti Polar Lipids. [3H]Ryanodine was from DuPont NEN. p-Bromophenacyl bromide (pBPB) was from Sigma.

Purification of IpTxi

P. imperator venom was obtained by electric stimulation of scorpions maintained alive in the laboratory. Venom (120 mg per batch) was suspended in double distilled water and centrifuged at 15,000 × g for 30 min. The supernatant was applied onto a column (0.9 × 190 cm) of Sephadex G-50 superfine (Pharmacia Biotech Inc.). Fractions were eluted with 20 mM NH3OAc (pH 4.7) at a flow rate of 20 ml/h. Fraction II containing IpTxi was applied to a column (0.9 × 30 cm) of carboxymethyl (CM)-cellulose 32 (Whatman) equilibrated with 20 mM NH3OAc (pH 4.7). Peptides were eluted at a flow rate of 20 ml/h with a linear gradient of 250 ml of 20 mM NH3OAc (pH 4.7) and 250 ml of the same buffer containing 0.55 M NaCl. Peptides displaying capacity to inhibit [3H]ryanodine binding and phospholipase activity were dialyzed against deionized water (3 × 30 min), concentrated by lyophilization, and injected into a C4 reverse-phase HPLC column (Vydac). IpTxi was eluted with a linear gradient of 0-60% acetonitrile in 0.1% trifluoroacetic acid running at 1 ml/min for 60 min. The purity and identity of IpTxi was confirmed by amino acid sequence analysis, as described for Na+ channel-blocking peptides (12). IpTxi was quantified either by amino acid analysis or by absorbance at 280 nm (A280 nm) using an extinction coefficient (epsilon ) = 14,952.

Amino Acid Analysis and Microsequencing of IpTxi

Amino acid analysis was performed on samples hydrolyzed in 6 N HCl with 0.5% phenol at 110 °C in evacuated, sealed tubes as described (13). Reduction of IpTxi with dithiothreitol and alkylation with iodoacetic acid was performed as described (13). Reduced and alkylated IpTxi was cleaved with protease Lys-C in 200 µl of 25 mM Tris-HCl (pH 7.2), and 1.0 mM EDTA, at a enzyme:peptide ratio = 1:100. Microsequence determination of native, reduced, and carboxymethylated toxin, and their peptide fragments was carried out with a 6400/6600 MilliGen/Biosearch Prosequencer, using the peptide adsorbed protocol on CD Immobilon membranes.

Cloning and Sequencing of the cDNA Encoding IpTxi

Two oligonucleotides encoding for two different regions of IpTxi were synthesized as described (13). Oligonucleotide 1 (5'-AC(N)ATGTGGGG(N)AC(N)AA(G/A)TGGTG-3'; where N means any nucleotide) encoded for the first 8 amino acids of the amino terminus of the large subunit of IpTxi. Oligonucleotide 2 (5'-GA(G/A)GC(N)GG(N)TA(T/C)GG(N)GC(N)TGGGC-3') encoded for residues 14-21 of the small subunit. Total RNA was purified from the venomous glands (telsons) of 30 scorpions. The telsons were pulverized in liquid nitrogen and poured into 10 ml of GT buffer (4 M guanidinium isothiocyanate, 0.025 M sodium citrate (pH 7.0), 0.5% N-lauryl sarcosine, and 0.03 M beta -mercaptoethanol), vortexed, and centrifuged at 5,000 × g for 10 min. One ml of 2 M sodium acetate (pH 4) was added to the recovered supernatant. The resulting solution was extracted with 10 ml of water-saturated phenol plus 2 ml of chloroform:isoamyl alcohol (49:1, v:v), allowing to sit on ice for 15 min. After centrifuging at 10,000 × g for 20 min at 4 °C, the aqueous layer was precipitated with 1 volume of isopropyl alcohol, incubated at -20 °C for 1 h, and centrifuged at 10,000 × g for 20 min at 4 °C. The pellet was dissolved in 0.3 ml of GT buffer and 0.3 ml of isopropyl alcohol, incubated at -20 °C for 1 h, and pelleted again. The pellet was washed with 95% ethanol, briefly dried under vacuum, and resuspended in RNase-free water. Messenger RNA was purified following the instructions of the Hybond-mAP protocol (messenger affinity paper; Amersham Corp., RPN.1511). cDNA synthesis was performed as described (13) from 5 µg of mRNA. The cloning of the cDNA library was also performed as described (13). The screening of the cDNA library was performed with oligonucleotides 1 and 2 separately, but the clones detected with oligonucleotide 1 were analyzed first. Inserts of cDNA from positive clones were polymerase chain reaction-amplified using lambda  gt11 forward and reverse primers. Polymerase chain reaction products were purified from agarose gel and ligated into the EcoRV site of pBluescript (pKS) phagemid. The ligation reactions were used to transform Escherichia coli DH5-alpha cells. Plasmid DNA from white colonies was digested with BamHI and HindIII to verify the size of the original inserts. Clones of interest were sequenced using the SequenaseR kit version 2 (U. S. Biochemical Corp.) on both strands. Oligonucleotides lambda  gt11 forward, lambda  gt11 reverse, M13 -20, and M13 reverse were used for sequencing.

Sequence Comparisons

The amino acid sequence of the large and small subunits of IpTxi were compared with those of other proteins deposited in the protein data base of GenBank (Los Alamos National Laboratory, Los Alamos, NM) by computer analysis using the program Blitz version 1.5 (Biocomputing Research Unit, University of Edinburgh, UK).

Determination of Phospholipase A2 Activity

Phospholipase A2 activity was determined by the titration method of Shiloah et al. (14), using dilute egg yolk (1:20 in saline solution) as substrate. Liberation of acid was measured at pH 8.0 and 37 °C by titration with 5 mM NaOH under a constant stream of N2. One unit of enzyme is defined as the amount of enzyme that liberates 1 µmol of free fatty acid/min under the above conditions. Inhibition of phospholipase A2 with pBPB was carried out as described by Díaz et al. (15). One hundred µl of 200 µM IpTxi in 35 mM Tris (pH 8.0) were mixed with 10 µl of 2 mM p-BPB in acetone and incubated for 16 h at room temperature under dim light. The reaction was stopped by filtration in a Sephadex G-10 column. pBPB-treated IpTxi elutes in the void volume of the column, whereas the excess of pBPB is retarded.

Synthesis of the Small Subunit of IpTxi

The small subunit of IpTxi was synthesized by the solid phase method of Merrifield (16), using t-butyloxycarbonyl-amino acids. This subunit contains a Cys residue at position 4. To avoid formation of inter-peptide disulfide bonds, Cys4 was substituted by Ala (peptide A), Met (peptide M), or Cys (peptide C) protected with 3-nitro-2-pyridinesulfonyl (thiol-protecting group that will not allow formation of disulfide bonds). At the end of the synthesis all three peptides were separated in a C18 reverse-phase HPLC column, using the conditions described above. The purity of the synthetic peptides was confirmed by both amino acid analysis and microsequencing, as described above.

[3H]Ryanodine Binding Assays

[3H]Ryanodine binding to pig cardiac and rabbit skeletal SR vesicles was carried out for 90 min at 36 °C in 0.1 ml of 0.2 M KCl, 1 mM Na2EGTA, 0.995 mM CaCl2, 10 mM Na-Pipes (pH 7.2). The calculated free Ca2+ was 10 µM. [3H]Ryanodine (68.4 Ci/mmol) was diluted directly in the incubation medium to a final concentration of 7 nM. Protein concentration was 0.2-0.4 and 0.3-0.5 mg/ml for skeletal and cardiac SR, respectively. Samples were filtered on Whatman GF/B glass fiber filters and washed twice with 5 ml of distilled water. A Brandel M24R cell harvester was used for filtration. Nonspecific binding was determined in the presence of 10 µM unlabeled ryanodine and has been subtracted from each sample.

Planar Bilayer Technique

Recording of single RyR in lipid bilayers was performed as described previously (17). Briefly, a phospholipid bilayer of phosphatidylethanolamine:phosphatidylserine (1:1 dissolved in n-decane to 20 mg/ml) was formed across an aperture of ~300 µm diameter in a delrin cup. The cis chamber (900 µl) was the voltage control side connected to the head stage of a 200 A Axopatch amplifier, and the trans chamber (800 µl) was held at virtual ground. Both chambers were initially filled with 50 mM cesium methane sulfonate and 10 mM Tris/Hepes (pH 7.2). After bilayer formation, cesium methane sulfonate was raised to 300 mM in the cis side, and 100-200 µg of SR vesicles were added. After detection of channel openings, Cs+ in the trans chamber was raised to 300 mM to collapse the chemical gradient. Single channel data were collected at steady voltages (-30 mV) for 2-5 min. Channel activity was recorded with a 16-bit VCR-based acquisition and storage system at a 10-kHz sampling rate. Signals were analyzed after filtering with an 8-pole Bessel filter at a sampling frequency of 1.5-2 kHz. Data acquisition and analysis were done with Axon Instruments software and hardware (pClamp v6.0, Digidata 200 AD/DA interface).


RESULTS

Purification of IpTxi

Purification of IpTxi from P. imperator venom was performed in three chromatographic steps as described under "Experimental Procedures" and shown in Fig. 1. After fractionation of whole venom in Sephadex G-50 (Fig. 1A), five fractions were collected and assayed for effects on [3H]ryanodine binding. Fraction 2 contained polypeptides in the range of 8-16 kDa and was the only fraction that inhibited [3H]ryanodine binding. The second step in the purification of IpTxi consisted of ion-exchange chromatography in CM-cellulose (Fig. 1B). The fraction containing IpTxi (indicated by the arrow) eluted early in the run, when the concentration of NaCl was ~160 mM. Fig. 1C shows the chromatographic profile of IpTxi after elution from a reverse-phase C4 HPLC column. Only a minor contaminant was present (labeled with asterisk), which was discarded for further studies of IpTxi structure. However, amino acid sequence analysis of purified IpTxi yielded two different amino acids per reaction cycle, with an apparent equivalent stoichiometry. This indicated either that a contaminant was still present at the end of our purification procedure or that IpTxi was composed of two different peptide subunits.


Fig. 1. Purification of IpTxi. A, P. imperator soluble venom (120 mg of protein) was applied to a Sephadex G-50 column (0.9 × 190 cm) equilibrated and run with 20 mM ammonium acetate buffer (pH 4.7). 5-ml samples were collected and tested for their capacity to inhibit [3H]ryanodine binding. Only fraction 2 totally inhibited [3H]ryanodine binding. B, fraction 2 was further separated through a CM-cellulose column (0.9 × 30 cm), equilibrated, and run with 20 mM ammonium acetate buffer (pH 4.7). A linear gradient of sodium chloride resolved 5 subfractions, the first of which (indicated by the arrow) contained IpTxi. C, the subfraction from the CM-cellulose column containing IpTxi was lyophilized and injected into a C4 reverse-phase HPLC column and eluted with a 0-100% linear gradient of acetonitrile containing 0.1% trifluoroacetic acid. The peak labeled with an asterisk was not studied further. D, dose-response curve for whole venom and purified components. [3H]Ryanodine (7 nM) was incubated with cardiac SR protein in 0.2 M KCl, 10 µM CaCl2, 10 mM Na-Hepes (pH 7.2) in the absence (control, 100%) and the presence of indicated concentrations of venom components. Nonspecific binding was determined in the presence of 20 µM ryanodine and has been subtracted from this and subsequent results.
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We determined the dose-response relationship for each of the active venom components to assess their potency to inhibit RyRs (Fig. 1D). Whole P. imperator venom and fraction 2 inhibited [3H]ryanodine binding to cardiac SR with a concentration of 20.2 and 4.3 µg/ml, respectively, yielding the half-maximal effect (IC50); IC50 for pure IpTxi was 0.7 µg/ml. Based on this value and the molecular mass of IpTxi (~15 kDa), the estimated apparent dissociation constant (Kd) was 46 nM. Essentially identical results were obtained when skeletal SR was used for displacement studies (not shown).

Electrophoretic Analysis of IpTxi

To elucidate whether IpTxi was composed of two subunits, an aliquot of IpTxi was reduced and alkylated. The modified toxin was then chromatographed in a Bio-Gel P30 column (Fig. 2A) to eliminate reaction by-products and excess of reagents. Three peaks were obtained (Fig. 2A) and analyzed by SDS-PAGE (Fig. 2B). Peak 1 (labeled Red, abbreviation for reduced) had lower molecular mass than native IpTxi (labeled Nat); peak 2 could not be resolved in the same gel (not shown), but direct sequencing indicated that it corresponds to a ~3-kDa peptide (Fig. 3A); peak 3 was not peptidic in nature, indicating that it corresponded to the reducing and alkylating reagents. Fig. 2C shows that the apparent molecular mass of native IpTxi was ~15 kDa and that of the reduced IpTxi was ~12 kDa. Thus, treatment of IpTxi with thiol-reducing agents separates a large (~12 kDa) from a small (~3 kDa) subunit.


Fig. 2. Separation of large and small subunits of IpTxi. A, IpTxi (100 µg) was reduced and alkylated as described under "Experimental Procedures," applied to a Bio-Gel P30 column (0.9 × 27 cm), and eluted with 10% acetic acid. Peak 1 corresponds to a ~12-kDa peptide, peak 2 to a ~3-kDa peptide (determined by direct sequencing), and peak 3 to reaction by-products. B, SDS-PAGE analysis of 3 µg of native IpTxi (Nat) and 3 µg of peak 1 (Red). Gel was 10% polyacrylamide. Proteins were stained with Coomassie Blue. The running gel (R.G.) and the tracking dye (T.D.) positions are indicated by arrows. C, the retention factor (Rf) for various molecular weight markers is plotted as a function of log10 molecular weight (M.W.). The apparent molecular weight for native and reduced IpTxi was extrapolated from a linear regression to the data points.
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Fig. 3. Amino acid and nucleotide sequences of IpTxi. A, the complete amino acid sequence of IpTxi was obtained from direct Edman degradation using peak 1 (large subunit) and peak 2 (small subunit) of Fig. 2A (labeled d) and from proteolytic fragments (lc1, lc2, and lc3) of peak 1. To obtain the proteolytic fragments, peak 1 (100 µg) was incubated for 4 min at 36 °C with 1 µg of Lys-C endopeptidase in Tris buffer as specified in the Boehringer catalog. The reaction products were separated by HPLC, essentially as described in the legend to Fig. 1A. Numbers on top of the amino acid sequence correspond to the amino acid positions in the primary structure. B, nucleotide sequence of the cDNA gene encoding for the entire IpTxi molecule with the deduced amino acid sequence below each base triplet. The putative signal peptide sequence (relative position -31 to -1) is underlined. The pentapeptide (relative position 105-109) is not found in the mature protein and is assumed to be cleaved during processing. Numbers on right refers to the nucleotide and amino acid positions.
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Peptide Sequence Determination

The reduced and alkylated derivatives of IpTxi (peak 1 and 2 of Fig. 2A) were sequenced by direct Edman degradation. As shown in Fig. 3A, we identified the first 27 amino acid residues of the large subunit, and all 28 amino acid residues of the small subunit. To identify the remaining residues, the large subunit was cleaved with Lys-C endopeptidase, which yielded three peptide fragments that eluted with different retention times in an HPLC column (data not shown). The amino acid sequence of each of these peptides, obtained also by Edman degradation, is underlined in Fig. 3A.

Cloning and Sequencing of the cDNA Encoding IpTxi

Two oligodeoxynucleotides synthesized according to the amino acid sequence of stretch 1-8 of the large subunit and stretch 14-21 of the small subunit were used to screen a cDNA library, constructed from the venomous glands of P. imperator scorpions. These oligodeoxynucleotides were used as probes to isolate a full-length IpTxi cDNA clone. Both probes led to the isolation of the same cDNA clone. Its complete nucleotide sequence is shown in Fig. 3B. This sequence contained an open reading frame of 167 amino acids encompassing 1) a putative signal peptide (first underlined 31 amino acids, positions -31 to -1); 2) the mature ~12-kDa, large subunit of IpTxi (104 amino acids, positions 1-104); 3) a putative connector pentapeptide (RRLAR, positions 105-109); and 4) the mature small subunit (27 amino acids; positions 110-136). Thus, a single continuous cDNA clone encoded the two polypeptide subunits of IpTxi. This finding confirmed that IpTxi is a heterodimeric protein. Since treatment of IpTxi with thiol-reducing agents (Fig. 2A) effectively separates both subunits, this suggests that they are covalently linked by a disulfide bridge.

Sequence Homology of IpTxi Subunits

A comparison of the amino acid sequence of the two subunits of IpTxi with sequences available from GenBank revealed intriguing results. First, the small subunit shared no significant similarity with sequences of scorpion peptide blockers of Na+, K+, or Cl- channels, with members of the Kunitz protease inhibitor superfamily, or with any other sequence deposited in the protein data base. Thus, this small subunit constitutes a new class of scorpion peptides. On the other hand, Fig. 4 shows that the large subunit of IpTxi was 38% homologous to PLA2 from honey bee (A. mellifera) venom and 35% homologous to PLA2 from heloderma (H. horridum) venom. These two PLA2s compose group III of secreted PLA2 (18), whose main features are low molecular mass (~14 kDa), high disulfide bond content, and strict dependence on Ca2+ for lipolytic effect. Since the major criterion for classification is sequence homology more than function, IpTxi may be classified within this group with the remarkable difference, however, of possessing an accessory protein.


Fig. 4. Amino acid sequence homology between the large subunit of IpTxi and PLA2s from group III. The amino acid sequence of the large subunit of IpTxi (first row) was aligned with that of bee (A. mellifera, second row) and lizard (H. suspectum, third row) venom PLA2. A few gaps (dashes) were introduced to enhance similarities. Bold letters indicate exact homology for the three PLA2s. Dots indicate end of the sequence, whereas X indicates unknown residues, as indicated in the reference source. Amino acid sequences taken from IpTxi, this work; bee PLA2, GeneBank Accession No. X16709[GenBank]; Heloderma suspectum PLA2, Gomez et al. (32).
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Lack of Inhibition by the Short Subunit of IpTxi

We tested if the small subunit of IpTxi was responsible for blocking RyR. Reduced and carboxymethylated small subunit (peak 2 of Fig. 2A), at concentrations up to 15 µM, had no effect on the binding of [3H]ryanodine to cardiac or skeletal RyR (data not shown). To discard the presence of spurious reagents as being the cause of negative results, we synthesized the small subunit in three distinctive forms as follows: peptide C was similar to the native small subunit, and peptides A and M were also similar to the native small subunit, except that Cys4 was substituted by Ala and Met, respectively. This avoided the formation of interpeptide disulfide bridges caused by oxidation of Cys4. The three peptides were HPLC-purified before testing. Table I shows that none of the purified synthetic peptides had significant effect on the binding of [3H]ryanodine to cardiac RyR or on the binding of [3H]PN200-110 to voltage-dependent Ca2+ channels of sarcolemma (19).

Table I. Effect of synthetic peptide analogs of the small subunit of ipTxi on the binding of [3H]ryanodine and [3H]PN200-110 to cardiac sarcolemma and SR vesicles

Peptide concentration tested was 10 µM. Results are the mean ± S.D. of three independent determinations. Peptide concentration tested was 10 µM. Results are the mean ± S.D. of three independent determinations.
[3H]Ryanodine bindinga [3H]PN200-110 bindingb

% %
Control 100 100
Peptide A 116  ± 8 100  ± 6
Peptide M 110  ± 4 111  ± 12
Peptide C 106  ± 5 111  ± 7

a Binding of [3H]ryanodine to SR vesicles was carried out as described in the text.
b Binding of [3H]PN200-110 to cardiac sarcolemma was performed as described (19).

The Phospholipase A2 Activity of IpTxi Is Important to Inhibit RyR

We next investigated if the large subunit of IpTxi, or its enzymatic activity, was involved in RyR inhibition. The reduced and carboxymethylated form of the large subunit of IpTxi (peak 1) was incapable of inhibiting [3H]ryanodine binding to cardiac or skeletal RyR. However, this was expected given that internal disulfide bridges are essential for preservation of the toxin's tridimensional structure as well as for expression of its enzymatic activity (20). We thus resorted to pBPB, a covalent modifier of His residues that specifically blocks phospholipase A2 activity (15) without affecting disulfide bonds. The rate of fatty acid liberation by 1 mol of IpTxi was 384 ± 26 and 41 ± 12 mol min-1 in control and after incubation with pBPB, respectively (n = 4; data not shown). Thus, treatment of IpTxi with pBPB decreased substantially its PLA2 activity. Fig. 5 shows that pBPB-treated IpTxi decreased dramatically its capacity to inhibit the binding of [3H]ryanodine to cardiac and skeletal RyR (open symbols). This was in contrast to control IpTxi (a batch of IpTxi that underwent the same treatment as pBPB-treated IpTxi, except that pBPB was omitted), which retained its capacity to inhibit cardiac and skeletal RyR with high affinity (filled symbols).


Fig. 5. Treatment with a PLA2 inhibitor lowers the potency of IpTxi to inhibit RyR. IpTxi (200 µM) was incubated with 2 mM pBPB (filled symbols) for 16 h as described under "Experimental Procedures" or with acetone (open symbols), the drug vehicle, as control. Binding of [3H]ryanodine to cardiac (circles) or skeletal (squares) SR was performed in the absence (defined as 100%) or in the presence of indicated concentrations of IpTxi.
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Ca2+ Dependence of IpTxi Effect

The enzymatic activity of PLA2 from group III requires Ca2+ for catalysis (20). Therefore, we expected that if the inhibition of RyRs by IpTxi resided in its enzymatic activity, inhibition should be apparent only in the presence of micromolar [Ca2+]. Fig. 6 shows that Ca2+ was essential for binding of [3H]ryanodine to RyRs and for detection of the IpTxi effect. The left panel shows the Ca2+ dependence of [3H]ryanodine binding to skeletal SR and the effect of IpTxi. Specific binding in control (open circles) had a threshold for detection at 100 nM [Ca2+] (pCa 7) and was optimal at 10-100 µM [Ca2+]. Higher [Ca2+] inhibited binding, giving rise to a bell-shaped curve. In the presence of IpTxi (filled circles), the binding curve was dramatically decreased in absolute values. The percentage of IpTxi inhibition was 5.5, 32, and 71% at pCa 7, 6, and 5, respectively. No further inhibition was observed at higher [Ca2+]. Thus, the degree of IpTxi inhibition increased with [Ca2+].


Fig. 6. Ca2+ dependence of IpTxi inhibition of [3H]ryanodine binding. Rabbit skeletal (0.3 mg/ml) or pig cardiac (0.4 mg/ml) SR vesicles were incubated for 90 min at 36 °C with 7 nM [3H]ryanodine in the absence (open circles) or the presence (filled circles) of 200 nM IpTxi. The incubation medium consisted of 0.2 M KCl, 10 mM Na-Hepes (pH 7.2), 1 mM EGTA, and CaCl2 necessary to bring free [Ca2+] to the desired value. The Ca2+:EGTA ratios were calculated by a computer program using affinity constants given in Fabiato (33).
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In cardiac SR (Fig. 6, right panel), the Ca2+ dependence of [3H]ryanodine binding also had a threshold for detection at pCa 7 and a maximum at pCa 5 (control, open circles). However, unlike skeletal SR, inactivation of binding by high [Ca2+] was not pronounced. Binding decreased only by 30% with respect to maximum in cardiac RyRs, and it decreased by 80% in skeletal RyRs. This distinctive response of RyR to Ca2+ is also displayed by individual RyR reconstituted in lipid bilayers (8, 21), indicating that the binding assay effectively tracks the activity of the receptor. In the presence of IpTxi, the binding curve was markedly decreased. The percentage of inhibition was 10, 38, and 83% at pCa 7, 6, and 5, respectively. Thus, although cardiac and skeletal RyRs respond differently to Ca2+, the inhibitory effect of IpTxi increased equally with [Ca2+] in both isoforms.

Inhibition of RyR by Supernatant of IpTxi-treated SR Vesicles

The membranes of SR vesicles contain different classes of phospholipids that may serve as substrates for the PLA2 activity of IpTxi. We reasoned that if the inhibitory properties of IpTxi were at least partly due to its enzymatic activity, then inhibition should be observed by incubating RyR with supernatant of IpTxi-treated SR vesicles. Control SR vesicles were diluted in binding medium to 1 mg/ml and incubated at 36 °C with 100 nM IpTxi. After 30 min, the IpTxi-treated SR vesicles were pelleted at 32,000 × g for 15 min, and a clear supernatant was obtained. The supernatant was then tested for its capacity to inhibit the binding of [3H]ryanodine to cardiac SR vesicles. A control tube that contained only IpTxi in binding medium without SR vesicles was run in parallel to determine the percentage of inhibition caused by the toxin alone. Fig. 7 shows that increasing concentrations of IpTxi-treated SR supernatant inhibited the binding of [3H]ryanodine dose-dependently (filled circles). This inhibition could not be attributed to IpTxi because the control supernatant reduced binding only marginally (open circles). For instance, in the most extreme case, 10 µl of IpTxi-treated SR supernatant inhibited 100% of specific binding; the estimated concentration of IpTxi in this tube was 10 nM, which inhibited binding only by 22%. Hence, the lipolytic activity of IpTxi releases from SR vesicles a phospholipid product that is capable of inhibiting RyR activity.


Fig. 7. Inhibition of RyR by supernatant of IpTxi-treated SR. SR supernatant was prepared as described in the text. Binding of [3H]ryanodine to cardiac SR vesicles was carried out in the absence (control, 100%) or presence of indicated volumes of SR supernatant (filled symbols). To keep the reaction volume constant, binding medium was added to SR supernatant to a final volume of 10 µl. To determine the inhibition caused by IpTxi alone, control vials containing IpTxi without SR vesicles were spun in parallel. The final concentration of IpTxi in the binding assays and the inhibition associated with it is shown by the open symbols.
[View Larger Version of this Image (13K GIF file)]

Reversibility of IpTxi Effect

To rule out the possibility that inhibition of RyR by IpTxi was caused by an irreversible disruption of the channel protein, we incubated SR vesicles (1 mg/ml) with water (control) or with 1 µM IpTxi (IpTxi-treated SR). After 30 min at 36 °C, an aliquot was taken from each sample. Then, both SR samples were washed twice, pelleted, and prepared for [3H]ryanodine binding. Fig. 8 shows that binding of [3H]ryanodine to IpTxi-treated vesicles was decreased with respect to control after 30 min of incubation. However, these vesicles displayed essentially the same binding as control after washing off IpTxi. Thus, RyRs recover their capacity to bind [3H]ryanodine after treatment with IpTxi. Assuming that the [3H]ryanodine binding assay followed changes in channel gating (8-10, 17, 21), the changes described above suggest that IpTxi released a phospholipid product that bound to RyRs (or a closely associated regulatory protein) and decreased open probability (po); after removal of the phospholipid product, po could recover to control levels.


Fig. 8. Reversibility of IpTxi effect. Prewashed vesicles are cardiac SR vesicles incubated with water (control) or with 1 µM IpTxi (IpTxi-treated SR) for 30 min at 36 °C. Washed vesicles are the same control and IpTxi-treated vesicles after being pelleted and washed twice with binding medium. Binding of [3H]ryanodine was conducted in all vesicles and is expressed in absolute values (pmol/mg).
[View Larger Version of this Image (28K GIF file)]

Single Channel Effects of IpTxi

To test directly the above hypothesis and to gain insight on the manner in which IpTxi inhibits RyRs, we reconstituted swine cardiac RyR in planar lipid bilayers, as described (11, 17). Fig. 9 shows traces from continuous recording at -30 mV holding potential in the absence (control) and the presence of increasing concentrations of IpTxi. Channel activity was monitored over 80 s in each condition, and the mean po was obtained from current histograms. The traces show that IpTxi blocks RyR dose-dependently: at low concentrations (<= 200 nM) IpTxi decreases the lifetime of the open events, converting long openings into brief, frequently unresolved open events; at higher concentrations, both long and brief openings progressively decrease in frequency until they disappear. A quantitative description of this effect is presented in the open time histograms of Fig. 9B. In control, 2,034 events could be fitted with three exponentials with mean open time (tau ) = 0.7 ms (66%), 1.9 ms (29%), and 9.8 ms (5%). In the presence of 500 nM IpTxi, the histogram for 801 openings collected during the same period as control was monoexponential with tau  = 0.8 ms. Both the decrease in the frequency and duration of the open events contributed to the decrease in po. Fig. 9C shows the log dose-response relation of po as a function of IpTxi concentration. The IC50 for IpTxi inhibition was 140 nM. Since this value is in fairly good agreement with that determined in binding experiments (50-80 nM), this indicates that [3H]ryanodine, at the concentrations used, does not interfere with the inhibitory capacity of IpTxi. More importantly, the results show that the lipolytic product of IpTxi is capable of interacting directly with the RyR or with a closely associated protein that regulates channel gating.


Fig. 9. Inhibition of cardiac RyR activity by IpTxi. A, pig cardiac RyR were reconstituted in planar lipid bilayers and recorded in the absence (control) and the presence of indicated concentrations of IpTxi. Holding potential: -30 mV. Recording solution: symmetrical 300 mM cesium methane sulfonate and 10 mM Na-Hepes (pH 7.2). Under these conditions, Cs+ flows from the luminal (trans) to the cytosolic (cis) side of the channel, and openings are represented by downward deflections of the base-line current. B, open time histograms in the absence (open bars) and the presence (filled bars) of 500 nM IpTxi. C, plot of open probability (Po) as a function of [IpTxi]. Smooth line is a fit to data points using the expression po = (po control)/1 + ((IpTxi)/IC50)nH. Where po control is the po in the absence of IpTxi (0.32); IC50 is the concentration of IpTxi that produces half-maximal inhibition (140 nM); and nH is the Hill coefficient (1.22).
[View Larger Version of this Image (33K GIF file)]

Inhibition of RyR by Fatty Acids

PLA2 catalyzes the hydrolysis of the Sn2 fatty acyl bond of phospholipids to release free fatty acids and lysophospholipids (22). We tested the effect of each of these lipids separately on RyR activity. Fig. 10A shows that lysophosphatidylcholine (lyso-PC), at concentrations up to 300 µM, does not modify substantially the open lifetime or the unitary conductance of cardiac RyR. On the other hand, linoleic acid, an 18-carbon unsaturated fatty acid, blocks RyR at lower concentrations and in a manner that is reminiscent of the blockade produced by IpTxi. Addition of 30 µM linoleic acid to the cytosolic face of the channel decreases the lifetime and the frequency of open events; similar to IpTxi, the fatty acid induces the appearance of openings that are too fast to be resolved by our recording bandwidth.


Fig. 10. Selective inhibition of RyR by fatty acids. A, representative traces of cardiac RyR activity in control (top trace) and in the presence of 300 µM lyso-PC (middle trace) or 30 µM linoleic acid (bottom trace). Openings are represented by downward deflections of the base-line current. For the experiment shown plus two additional experiments, mean po was 0.43 ± 0.09 (control), 0.46 ± 0.11 (lyso-PC), and 0.12 ± 0.06 (linoleic acid). B, the binding of 7 nM [3H]ryanodine to cardiac SR vesicles was determined in the presence of methanol (maximal concentration = 1%) and this value defined as 100%, or in the presence of indicated concentrations of lipids dissolved in methanol.
[View Larger Version of this Image (29K GIF file)]

The potency of several fatty acids to inhibit RyRs was determined by their capacity to inhibit [3H]ryanodine binding to cardiac SR vesicles (Fig. 10B). Palmitic acid, a 16-carbon saturated fatty acid, partially inhibited RyR but at concentrations >= 100 µM. Arachidonic acid, linoleic acid, and oleic acid, unsaturated fatty acids abundant in SR membranes of cardiac and skeletal muscle (23), totally inhibited RyR with an IC50 = 25, 55, and 70 µM, respectively. In agreement with single channel results, lyso-PC and lysophosphatidylethanolamine were unable to modify [3H]ryanodine binding. Thus, not all PLA2 products are capable of inhibiting RyRs. Among the fatty acids, the potency to inhibit RyRs increases with the carbon chain length and the number of unsaturations.


DISCUSSION

In the present study, we determined the complete amino acid and nucleotide sequence of IpTxi, a heterodimeric protein from the venom of the scorpion P. imperator, and unraveled the molecular mechanism by which it inhibits RyRs of cardiac and skeletal muscle.

The deduced amino acid sequence from the cDNA encoding IpTxi and the amino acid sequence determined by Edman degradation strongly suggests that IpTxi is synthesized as a precursor of the pre-pro form. Putative signal and connector peptides must be removed to produce mature IpTxi. The proposed signal peptide does have a large content of acidic residues (8 out of the last 18 residues are acidic), a property that is rarely seen in signal peptides. For this reason, we cannot exclude whether the proposed signal peptide codes for another peptide before coding for the large subunit of IpTxi. Regarding the connecting pentapeptide Arg-Arg-Leu-Ala-Arg (positions 105 to 109 in the cDNA sequence of Fig. 3), we gather that several enzymes must be required to eliminate it from the mature protein. Initially, a cleavage must occur at Arg109 (monobasic site), as it occurs in the maturation of prosomatostatin and other prohormones (24). Two characteristics distinguish this cleavage site and both are present in the cDNA of IpTxi: (i) Arg (or other basic residue) is immediately preceded by Ala or Leu (or both); (ii) Arg is located three or five residues upstream the basic amino acid involved in the cleavage. Next, another cleavage must occur at Arg106 (dibasic site) as is the case during the maturation of diverse peptidic hormones (25). A third enzyme with carboxypeptidase activity must intervene to remove Arg105 and Arg106. Because of this complicated pattern of maturation, we assume that the linking of the small and large subunit of IpTxi must be important for the function of IpTxi.

Mature IpTxi is composed of a ~3-kDa peptide covalently linked to a ~12-kDa peptide with PLA2 activity. The large subunit of IpTxi conserves the most important substructures of secreted PLA2 from group III (Fig. 4). The His/Asp pair, essential for Ca2+ binding, is in position 33/34 of IpTxi and is preceded in the three PLA2s by a Cys-Cys-Arg motif; the amino terminus (roughly residues 4-12 of IpTxi), presumably involved in substrate binding, is also highly conserved in the three PLA2s; lastly, the Cys residues, essential for maintaining proper folding through disulfide bonds, may be used in this group as the elemental frame to align homologous sequences and to identify stretches of sequence that have been inserted or deleted through evolution. Only Cys101 of IpTxi (relative position 104 in Fig. 4) does not match with Cys residues present at the carboxyl terminus of the other PLA2 shown. It is likely, therefore, that Cys101 is involved in the disulfide bridging with Cys4 of the small subunit.

The presence in IpTxi of a PLA2 subunit covalently linked to a small, structurally unrelated peptide is reminiscent of the bipartite arrangement of beta -bungarotoxins (26), a group of snake neurotoxins that inhibit neurotransmitter release (27). Each beta -bungarotoxin dimer has a PLA2 subunit covalently bound to a smaller subunit related to Kunitz-type protease inhibitors. Members of the Kunitz superfamily bind to and block voltage-dependent K+ and Ca2+ channels (28). Thus, although the small subunit of IpTxi showed no homology to members of the Kunitz superfamily, its distinctive arrangement within the toxin suggested that it might act as a blocker. For this reason, it was surprising at first to realize that the small subunit was not directly responsible for inhibiting RyRs. Direct addition of the small subunit to our [3H]ryanodine binding assays failed to inhibit RyRs, as did a synthetic peptide with amino acid sequence similar to that of the small subunit and two additional derivatives designed to avoid interpeptide disulfide bridge formation (Table I). Hence, the target site for the small subunit, or its biological function, remains to be determined.

Given our failure to detect inhibition by the small subunit, we turned our attention to the large subunit of IpTxi. In a reduced and carboxymethylated form, the large subunit of IpTxi failed to inhibit RyRs (not shown). However, this was not surprising given that disulfide bridges are essential to maintain the toxin's three-dimensional structure (20). It was the treatment of IpTxi with pBPB, a covalent modifier of His residues that inhibits PLA2 activity without affecting disulfide bridges (15), that decreased substantially the capacity of IpTxi to inhibit RyRs (Fig. 5). This suggested that a lipid product of PLA2 activity was involved in the inhibition of RyRs. Three separate lines of evidence supported this notion. 1) Inhibition of RyR by IpTxi was favored by the presence of Ca2+ (>= 1 µM; Fig. 6), as expected from the Ca2+ dependence of enzymatic activity of PLA2 from group III and several other groups (20). 2) The supernatant of IpTxi-treated SR vesicles could inhibit RyRs even before IpTxi was present in a concentration large enough to do so (Fig. 7); this indicated that RyR inhibition was brought about by a lipid product of IpTxi released from SR vesicles during the incubation period. 3) The kinetics of RyR inhibition by IpTxi were mimicked by direct addition of linoleic acid (but not of lyso-PC) to the cytoplasmic side of the channel (Figs. 9 and 10); other long chain, unsaturated fatty acids could also inhibit RyRs (Fig. 10). Thus, a potential scenario for inhibition of RyR by IpTxi is as follows: in the presence of micromolar [Ca2+], the large subunit of IpTxi catalyzes the hydrolysis of the Sn2 fatty acyl bond of the phospholipids of the SR membrane. The reaction yields free fatty acids and lysophospholipids; the former are released into the incubation medium, and the latter may be liberated or remain embedded into the SR membrane. Free fatty acids bind to RyR or to a closely associated protein that controls gating. At low concentrations, they produce an incomplete block of RyR; higher concentrations gradually block the RyR completely, giving rise to the dose-response relationship of channel activity and [3H]ryanodine binding versus IpTxi concentration of Figs. 1, 5, and 9.

PLA2s are abundant components of snakes, scorpions, and bee venoms and often constitute the main toxic component to mammals. Although all PLA2s induce a variety of pathological symptoms including neurotoxicity and myotoxicity (29), they differ in their mechanism of action and in their molecular targets. For example, the snake neurotoxin crotoxin is composed of a PLA2 subunit and an inhibitory subunit, which keeps the phospholipase inactive until binding to a presynaptic receptor triggers the dissociation of the inhibitory subunit (30). In contrast, notexin from Netechis scutatus scutatus is a PLA2 without an associated subunit that produces muscle paralysis by binding to a specific receptor in the neuromuscular junction (31). IpTxi is the first example of a scorpion toxin in which a PLA2 is found chaperoned by a smaller, structurally unrelated subunit. In a previous study (11), we found that several transporters and ion channels of striated muscle including the inositol trisphosphate receptor, the muscarinic receptor, voltage-sensitive Na+ channels, and the Ca2+-ATPase of SR were insensitive to micromolar concentrations of IpTxi. These results argue against an indiscriminate effect on key molecules of excitation-contraction coupling by the lipolytic action of IpTxi. However, an important question that still remains to be answered concerns the presence of the small subunit of IpTxi. What is its molecular target? What function does it perform to enhance the PLA2 activity or to suppress it while in transit to the specific receptor? Regardless of the primary site affected, a major contribution of this study was to establish the fact that the molecular mechanism involved in the toxicity of IpTxi will most likely involve abnormalities in intracellular Ca2+ mobilization due to blockade of RyRs.


FOOTNOTES

*   This work was supported by Howard Hughes Medical Institute Grant 75191-527104, CONACyT 4734-N, and European Commission CI1*-CT94-0045 (to L. D. P.), and by National Institutes of Health Grant HL55438, and a Grant-in-Aid from the American Heart Association (to H. H. V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence may be addressed. Mexico: Tel.: 011-5273-171209; Fax: 011-5273-172388; E-mail: possani{at}ibt.unam.mx. U. S.: Tel.: 608-265-5960; Fax: 608-265-5512; E-mail: hhvaldiv{at}facstaff.wisc.edu.
**   Recipient of a Minority Scientist Research Award from the American Heart Association.
1   The abbreviations used are: SR, sarcoplasmic reticulum; pBPB, p-bromophenacyl bromide; PLA2, phospholipase A2; RyR, ryanodine receptor; lyso-PC, lysophosphatidylcholine; HPLC, high performance liquid chromatography; Pipes, 1,4-piperazinediethanesulfonic acid.

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