(Received for publication, February 6, 1997)
From the § Department of Physiology, University of
Wisconsin Medical School, Madison, Wisconsin 53706, the
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
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 -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
-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.
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 -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.
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.
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 () = 14,952.
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 IpTxiTwo 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
-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
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-
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
gt11 forward,
gt11 reverse, M13 -20, and M13 reverse were used for
sequencing.
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 ActivityPhospholipase 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 IpTxiThe 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 TechniqueRecording 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).
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.
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 IpTxiTo 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.
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 IpTxiTwo 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.
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.
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).
|
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
min1 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).
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+].
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 VesiclesThe 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.
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.
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 (
) = 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
= 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.
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.
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.
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 -bungarotoxins (26), a
group of snake neurotoxins that inhibit neurotransmitter release (27).
Each
-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.