Activation of Ryanodine Receptors by Imperatoxin A and a Peptide Segment of the II-III Loop of the Dihydropyridine Receptor*

Georgina B. Gurrola, Carolina Arévalo, Raghava Sreekumar, Andrew J. Lokuta, Jeffery W. Walker, and Hector H. ValdiviaDagger

From the Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

Excitation-contraction coupling in skeletal muscle is believed to be triggered by direct protein-protein interactions between the sarcolemmal dihydropyridine-sensitive Ca2+ channel and the Ca2+ release channel/ryanodine receptor (RyR) of sarcoplasmic reticulum. A 138-amino acid cytoplasmic loop between repeats II and III of the alpha 1 subunit of the skeletal dihydropyridine receptor (the II-III loop) interacts with a region of the RyR to elicit Ca2+ release. In addition, small segments (10-20 amino acid residues) of the II-III loop retain the capacity to activate Ca2+ release. Imperatoxin A, a 33-amino acid peptide from the scorpion Pandinus imperator, binds directly to the RyR and displays structural and functional homology with an activating segment of the II-III loop (Glu666-Leu690). Mutations in a structural motif composed of a cluster of basic amino acids followed by Ser or Thr dramatically reduce or completely abolish the capacity of the peptides to activate RyRs. Thus, the Imperatoxin A-RyR interaction mimics critical molecular characteristics of the II-III loop-RyR interaction and may be a useful tool to elucidate the molecular mechanism that couples membrane depolarization to sarcoplasmic reticulum Ca2+ release in vivo.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

In cardiac and skeletal muscle, the dihydropyridine receptor (DHPR)1 of the external membrane and the Ca2+ release channel/ryanodine receptor (RyR) of sarcoplasmic reticulum (SR) are key components of excitation-contraction (E-C) coupling, the series of events that link an electrical stimulus (depolarization) to a mechanical contraction (1). Skeletal and cardiac muscle express different subtypes of DHPR and RyR, which account for different E-C coupling mechanisms. In the heart, a small influx of Ca2+ through DHPRs triggers the opening of RyRs (2). In skeletal muscle, however, external Ca2+ is not required for Ca2+ release (3). Contractions are instead triggered by membrane depolarizations, and because Ca2+ release may be arrested immediately upon repolarization, a mechanical coupling between the DHPR and the RyR is thought to mediate E-C coupling (4, 5).

Compelling evidence indicates that the skeletal DHPR subtype is indispensable to elicit a Ca2+-independent (skeletal-type) contraction (6, 7) and that the 138-amino acid cytoplasmic loop between repeats II and III of the alpha 1 subunit participates in this process (6, 8). In experiments with isolated peptides, the II-III loop activates purified RyRs (9), and a small fragment of the II-III loop (Thr671-Leu690) induces Ca2+ release from SR vesicles (10). In dysgenic myotubes, skeletal-type E-C coupling is partially restored by a chimeric DHPR that is entirely cardiac except for a short segment of skeletal II-III loop (Phe725-Pro742) (8). Although apparently contradictory in the identity of the activating region, these results suggest that specific domains of the II-III loop directly interact with the RyR to change its conformational state and produce Ca2+ release. Therefore, in skeletal muscle, the II-III loop stands as the strongest candidate among regions of the DHPR to bind to RyRs. However, the precise amino acid residues of the II-III loop that trigger Ca2+ release remain unknown. Furthermore, other DHPR segments (11) or subunits (12) have not been discarded as points of contact.

We have previously shown that Imperatoxin A (IpTxa), a 33-amino acid peptide from the scorpion Pandinus imperator, is a high-affinity activator of RyRs (13, 14). The biological significance of IpTxa is unknown, because the apparent target for this membrane-impermeable peptide is located intracellularly. Because some peptide toxins activate intracellular signaling pathways by mimicking surface receptors (15, 16), we tested the hypothesis that IpTxa activates RyRs by mimicking a domain of the DHPR that is critical to trigger Ca2+ release. We found that IpTxa and a synthetic peptide with an amino acid sequence corresponding to a segment of the II-III loop (Glu666-Leu690) (10) activate RyRs in a similar manner and appear to compete for a common binding site on the channel protein. Both peptides bind to RyRs via a structural domain consisting of a cluster of basic amino acids (Arg681-Lys685 of the II-III loop and Lys19-Arg24 of IpTxa) followed by a hydroxylated amino acid (Ser687 of the II-III loop and Thr27 of IpTxa). Thus, IpTxa presents an interesting case of toxin mimicry of effector proteins that may be used to identify regions of the RyR that trigger Ca2+ release. If the peptide segment emulated by IpTxa is an actual participant in the DHPR/RyR interaction, IpTxa may also be exploited to identify regions of the RyR involved in E-C coupling.

    EXPERIMENTAL PROCEDURES

Materials-- [3H]Ryanodine (60-80 Ci/mmol) was from NEN Life Science Products, agelenin and Tx2-9 were from The Peptide Institute, Inc. (Osaka, Japan), bovine brain phosphatidylethanolamine and phosphatidylserine were from Avanti Polar Lipids (Birmingham, AL), and Fmoc-amino acids were from Applied Biosystems. Polyclonal skeletal RyR antibody was from Upstate Biotechnology. Peroxidase-conjugated secondary antibody and omega -conotoxin were from Calbiochem. The chemiluminescence detection kit was from Boehringer Mannheim. Pre-cast linear gradient polyacrylamide gels were from Bio-Rad. All other reagents were of high-purity reagent grade.

[3H]Ryanodine and 125I-IpTxa Binding Assay-- [3H]Ryanodine (7 nM) was incubated for 90 min at 36 °C with 40-50 µg of rabbit skeletal SR vesicles in medium containing 0.2 M KCl, 10 µM CaCl2, and 10 mM Na-Hepes (pH 7.2) in the absence and presence of peptides. Free ligand and bound ligand were separated by rapid filtration on Whatman GF/B glass fiber filters, as described previously (13, 14). Native IpTxa (10-µg batches) was purified according to established procedures (13, 14) and iodinated to a specific activity of 60-80 Ci/mmol with the Bolton-Hunter© method following the specifications of the manufacturer (New England Nuclear). The binding of 125I-IpTxa to skeletal SR and Chinese hamster ovary (CHO) cell homogenates was performed under conditions identical to those described for [3H]ryanodine, except that the protein concentration was 0.1-0.2 mg/ml in the case of CHO cells. Bmax and KD of the 125I-IpTxa-receptor complex were obtained by fitting data points with the following equation: B = Bmax × 125I-IpTxa/(KD + 125I-IpTxa), where B is the specific binding of 125I-IpTxa.

Transfection of CHO Cells with RyR-- CHO cells were transfected by lipofection with plasmid pRRS11, the rabbit skeletal muscle RyR (RyR1), as described previously (17). Expression of the RyR was confirmed by immunoblot analysis using monoclonal antibodies against the skeletal RyR and by [3H]ryanodine binding. Control and transfected cells were homogenized in 500 mM sucrose, 1 mM EGTA, and 10 mM Hepes-Tris (pH 7.4) and spun at 44,000 × g for 30 min (17). The pellet was recovered and used for [3H]ryanodine binding experiments.

Synthesis of Peptides-- Linear analogs of IpTxa and the II-III loop were synthesized by the solid-phase methodology with Fmoc amino acids in an automated peptide synthesizer and subjected to the same cyclization and HPLC purification method as described previously (14). Analytical HPLC, amino acid analysis, and mass spectrometry confirmed the structure and the purity of the synthetic peptides. Photoactivatable IpTxa was prepared by inserting p-benzoyl-phenylalanine, a photoactivatable cross-linker (18), in place of Leu7 during the synthesis of IpTxa. Photoactivatable IpTxa was subjected to the same cyclization and HPLC purification method as described for synthetic IpTxa (14).

Planar Bilayer Recording of RyRs-- Recording of single RyR in lipid bilayers was performed as described previously (13, 19). Single channel data were collected at steady voltages (+30 mV) for 2-5 min in symmetrical 300 mM cesium methanesulfonate, 10 µM CaCl2, and 10 mM Na-Hepes (pH 7.2). IpTxa and the II-III loop peptide were added to the cis chamber, which corresponded to the cytosolic side of the channel (13, 19). The addition of the peptides to the trans (luminal) side of the channel was without effect. In some experiments, we added 10 mM CaCl2 to the trans solution. At 0 mV, Ca2+ was the only charge carrier in these experiments, and both peptides were effective in inducing a subconductance state of about one-fourth of the full conductance level. However, the low signal:noise ratio obtained under these conditions made the analysis of the kinetic effect difficult. For the experiments presented here, we omitted Ca2+ in the trans solution. Signals were filtered with an 8-pole low pass Bessel filter at 2 kHz and digitized at 5 kHz. Data acquisition and analysis were done with Axon Instruments software and hardware (pClamp v6.0.2, Digidata 200 AD/DA interface), as described previously (13, 19). The current values for the full and subconductance states were obtained from Gaussian fits to the all point amplitude histograms, as described previously (19).

Purification of SR Vesicles and Determination of Ca2+ Release-- SR vesicles were purified from rabbit white fast skeletal muscle, as described previously (13, 19). Maximal [3H]ryanodine binding site density was typically 3-5 pmol/mg protein. Ca2+ release from SR vesicles was measured by the method of Palade (20), with slight modifications. Briefly, SR vesicles (60 µg of protein in 20 µl of reaction medium) were placed in a cuvette containing 980 µl of 95 mM KCl, 20 mM K-MOPS (pH 7.0), 7.5 mM sodium pyrophosphate, 250 µM Antipyrylazo III, 1.5 mM MgATP, 25 µg of creatine phosphokinase, and 5 mM phosphocreatine. The mixture was allowed to equilibrate for 3 min at 37 °C under constant stirring. Free Ca2+ was monitored by measuring A710-790 nm using a diode array spectrophotometer (Hewlett-Packard Model 8452A). Vesicles were actively filled by three to five consecutive additions of 10 nmol of CaCl2 before the addition of IpTxa or the II-III loop peptide. The total amount of Ca2+ loaded was quantified by the addition of 5 µM of the Ca2+ ionophore A23187 at the end of each experiment.

Cross-Linking of Photoactivatable IpTxa, SDS-Polyacrylamide Gel Electrophoresis, and Western Blot Analysis of RyR-- SR microsomes (0.4 mg/ml) were incubated with 30 nM photoactivatable IpTxa (see above) in buffer containing 10 µM free Ca2+, 200 mM KCl, and 10 mM Na-Hepes (pH 7.2) in the absence and the presence of 50 µM IpTxa. After 60 min at 36 °C, 1-ml aliquots were spread over 1-cm-diameter plastic wells and irradiated at short range with ultraviolet light (360 nm) for 30 min. Samples were washed twice with incubation buffer by centrifugation in a table-top minifuge at 12,000 rpm. Pellets were then resuspended in Laemmli buffer (0.25 M Tris, pH 6.8, 0.4 M dithiothreitol, 8% SDS, 40% glycerol, and 0.04% bromphenol blue) and subjected to SDS-polyacrylamide gel electrophoresis on two identical linear gradient acrylamide gels (4-12%). Proteins contained in one gel were stained with Coomassie Blue, whereas proteins in the other gel were transferred to nitrocellulose membranes for Western blot analysis. Blots were probed first with a rabbit polyclonal RyR1 antibody (dilution, 1:3,000) and then with an anti-rabbit peroxidase-conjugated secondary antibody. Dried gels were then exposed to x-ray film for 2 days.

    RESULTS

Selective Activation of [3H]Ryanodine Binding by IpTxa among Ca2+ Channel Toxins-- The amino acid sequence of IpTxa (14) exhibits no significant homology with the well-characterized Na+ and K+ channel scorpion toxins (data not shown). However, IpTxa does share 45% and 42% sequence identity with agelenin (21) and Tx2-9 (22), respectively, two spider toxins that block presynaptic (P-type) Ca2+ channels (Fig. 1A). The Cys residues, which stabilize the three-dimensional structure by forming disulfide bridges (16), are similarly arranged in these three peptides (gray boxes). Indeed, they may be used as a frame to align the amino acid sequence of omega -conotoxin MVIIC, a snail peptide that blocks P-type Ca2+ channels (23), and to reveal regions of homology (open boxes). Fig. 1B shows that, despite the demonstrated structural kinship among these peptides, only IpTxa is capable of enhancing [3H]ryanodine binding. ED50, the concentration of IpTxa required to produce a half-maximal effect (6.4 ± 3.1 nM, mean ± S.D.; n = 18), is only slightly higher than that exhibited by [3H]ryanodine among ligands of RyRs (24). This selective and high-affinity effect suggests that IpTxa possesses a unique structural motif that activates RyRs, which is not present even in structurally related peptides.


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Fig. 1.   A, regions of homology between IpTxa and other Ca2+ channel-blocking peptides. The amino acid sequences of omega -conotoxin MVIIC (23), agelenin (21), IpTxa (14), and Tx2-9 (22) were aligned using the Cys residues, which are critical to maintain the three-dimensional structure of the peptides. Gaps (-) have been introduced to maximize homology. B, selective effect of IpTxa on [3H]ryanodine binding. The binding of [3H]ryanodine (7 nM) to skeletal SR vesicles (30-40 µg) was conducted in 0.2 M KCl, 10 µM CaCl2, and 10 mM Na-Hepes (pH 7.2) for 90 min at 36 °C. Control binding (100%) was defined as the specific binding of [3H]ryanodine in the absence of peptide toxins and corresponded to 0.71 ± 0.06 pmol/mg protein. Data are the mean ± S.D. of n = 16 (IpTxa) and an average of n = 3 (other peptides).

Physical Interaction of IpTxa with RyRs-- To test whether IpTxa may be used independently as a high-affinity, specific ligand for RyRs, we radiolabeled IpTxa and conducted binding experiments in the absence of ryanodine. Fig. 2A shows that the radiolabeled derivative of IpTxa retained high affinity (KD = 11 ± 3 nM) and bound to skeletal SR with a maximal receptor site density (Bmax) of 16.1 ± 1.9 pmol/mg protein (n = 3). In the same tissue, the Bmax for [3H]ryanodine was 3.7 ± 0.6 pmol/mg protein. Thus, assuming all 125I-IpTxa binding occurs to the RyR, the 125I-IpTxa:[3H]ryanodine binding site stoichiometry is 4.3:1. Because one [3H]ryanodine molecule binds with high affinity to the tetrameric RyR (24), this ratio suggests that about four IpTxa molecules bind to every RyR tetramer. In CHO cells transfected with the skeletal RyR (Fig. 2B, + RyR1), the 125I-IpTxa:[3H]ryanodine binding site stoichiometry is 4.6:1 (n = 2). In naïve CHO cells, there is neither 125I-IpTxa (Fig. 2B, Untransfected) nor [3H]ryanodine binding (data not shown).


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Fig. 2.   IpTxa binds directly and with high affinity to skeletal RyR. Specific binding of 125I-IpTxa to (A) skeletal SR or to (B) CHO cells transfected with the skeletal RyR (+ RyR1) or to CHO cells without gene transfection (Untransfected). The binding of 125I-IpTxa to skeletal SR was carried out exactly as described for [3H]ryanodine. In the case of CHO cells, protein concentration was 10-20 µg. Results are from one of three (skeletal SR) or two (CHO cells) similar experiments performed in duplicate. Experimental error was <= 10%.

To confirm that IpTxa physically interacts with the RyR monomer in skeletal SR, we prepared photoactivatable IpTxa, a synthetic derivative of IpTxa in which Leu7 was replaced by the light-sensitive cross-linker p-benzoyl-phenylalanine (18). The photoreactive derivative retained high affinity for the RyR (KD = 12 ± 4 nM; n = 3; data not shown). Fig. 3A shows a SDS-polyacrylamide gel electrophoresis profile of SR proteins that were radiated with ultraviolet light after incubation with 30 nM photoactivatable 125I-IpTxa in the absence (lane 1) and the presence (lane 2) of 50 µM unlabeled IpTxa. An immunoblot analysis using a skeletal RyR polyclonal antibody recognized only the high molecular weight band of SR proteins (Fig. 3B). The autoradiogram of the SDS-gel (Fig. 3C) shows clear labeling of the band corresponding to the RyR (lane 1). Other bands are also labeled, most likely from a nonspecific interaction with the toxin, because labeling persists in the presence of excess IpTxa (lane 2). Together with data from Fig. 2, these results indicate that IpTxa makes direct protein-protein interactions with the RyR with a stoichiometry of four IpTxa molecules per single RyR channel.


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Fig. 3.   Covalent labeling of the 125I-IpTxa receptor. A, photoactivatable 125I-IpTxa (30 nM) was incubated with 50 µg of SR vesicles for 60 min at 36 °C in the absence (lane 1) and the presence (lane 2) of 50 µM IpTxa. Vesicles were illuminated at short range with ultraviolet light (360 nm) for 30 min, washed twice by centrifugation, and resuspended in sample buffer for SDS-polyacrylamide gel electrophoresis in a 4-12% polyacrylamide gel. B, the identity of the RyR was investigated by Western blot analysis using a rabbit polyclonal antibody against the skeletal RyR (Upstate Biotechnology). Unlabeled SR proteins were run in parallel to those shown in A and transferred to nitrocellulose membranes as described under "Experimental Procedures." C, autoradiography of the gel in A. T.D., tracking dye. Results are representative of n = 3 experiments (cross-linking) or n = 4 experiments (Western blots).

Functional Effect of IpTxa and a Short Segment of the II-III Loop-- The functional effect of the IpTxa-RyR interaction was tested in planar lipid bilayer and Ca2+ release experiments. Fig. 4A shows that 50 nM IpTxa added to the cytoplasmic (cis) side of the skeletal RyR induced the appearance of a subconductance state corresponding to ~25% of the full conductance as previously shown (25). Although of small amplitude, the subconductance state displayed a mean open time that was >100-fold longer than that of unmodified channels. Ion flow would therefore be expected to be greater for an IpTxa-modified channel, despite its lower conductance. Fig. 4B shows that IpTxa elicited Ca2+ release from actively loaded SR vesicles in a dose-dependent manner. The effect of IpTxa was blocked by ruthenium red, consistent with Ca2+ release occurring through RyRs. Strikingly similar results were observed with a 25-amino acid synthetic peptide with primary sequence (Glu666-Leu690; Fig. 5A) overlapping that of peptide A (Thr671-Leu690), a segment of the II-III loop that activates RyRs (10). The 25-amino acid segment of the II-III loop (henceforth termed "the II-III loop peptide"), like IpTxa, induced the appearance of a small-amplitude and long-lifetime subconductance state (Fig. 4C) and elicited Ca2+ release from SR (Fig. 4D). Thus, albeit with different affinity, the two apparently unrelated peptides exhibit similar functional effects on RyRs.


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Fig. 4.   Functional effect of IpTxa and the II-III loop on skeletal RyRs. Rabbit skeletal RyR channels were reconstituted in planar lipid bilayers (11) and activated by 10 µM (cis) cytosolic Ca2+ in the absence (Control) and ~1 min after the addition of 50 nM IpTxa (A) or 3 µM II-III loop (B) to the cis side. The peptides induced the appearance of subconductance states of long lifetime (arrows). Mean open times (tau ), which were calculated from open time histograms, were 0.7 ± 0.1 ms (83%) and 4.2 ± 1.4 ms (17%) in control; 0.8 ± 0.1 ms (63%), 3.8 ± 1.4 ms (21%), and 319 ± 58 ms (17%, substate) after IpTxa (n = 3); and 0.9 ± 0.2 ms (73%), 3.6 ± 1.5 ms (19%), and 243 ± 34 ms (8%, substate) after II-III loop peptide (n = 3). The addition of peptides to the (trans) luminal side of the channel was without effect. Holding potential was +30 mV for all traces. c, closed; o, open. Ca2+ release from actively loaded SR vesicles by IpTxa (B) or the II-III loop peptide (D) was measured with the spectrophotometric Ca2+ indicator Antipyrylazo III, as described previously (20). Arrows indicate the addition of 10 nmol of Ca2+ to the 1-ml reaction medium. Thapsigargin (1 µM) was added simultaneously with the peptides to block Ca2+ uptake by the SR. When equilibrium was reached, the Ca2+ ionophore A23187 (5 µM) was added to assess SR Ca2+ content (asterisks). Calibration bars, 60 s (horizontal) and 5 µM Ca2+ (vertical).


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Fig. 5.   Amino acid sequence and structural domains of IpTxa and the II-III loop peptide. A, complete amino acid sequence of IpTxa and the segment of the II-III loop used in this study. No regions of homology were observed, except for a cluster of basic amino acids (boxes) followed by Ser or Thr (ovals). B, simplified scheme showing the proposed structural analogy between IpTxa and the II-III loop. Vertical lines represent regions of the peptides without significant homology. Rectangles correspond to the cluster of basic amino acids (Lys19-Arg24 and Arg681-Lys685, respectively), and ovals correspond to the hydroxylated amino acid (Thr26 and Ser687, respectively).

Structural Analogy between IpTxa and the II-III Loop Peptide-- A one-to-one residue alignment between IpTxa and the II-III loop peptide does not reveal significant homology in their amino acid sequence (Fig. 5A). However, both IpTxa and the II-III loop peptide display a structural motif consisting of a cluster of basic amino acids (boxes, Lys19-Arg24 and Arg681-Lys685, respectively) followed by Thr or Ser, two hydroxylated amino acids (ovals, Thr26 and Ser687, respectively). In IpTxa, the cluster of basic amino acids is interrupted by Cys21 and encompasses the sequence KCK, which is also found in agelenin and Tx2-9 (Fig. 1A). Therefore, it is likely that the KCK sequence alone does not suffice to activate RyRs and that Cys21 stabilizes the peptide structure without intervening in protein-protein interactions with the RyR. Hydroxylated amino acids in a position close to Thr26 of IpTxa are also found in agelenin (Ser28) and in Tx2-9 (Thr23 and Thr24), but none is preceded by a cluster of basic amino acids. Likewise, the motif RRG, which appears in IpTxa and omega -conotoxin MVIIC, is only followed by a hydroxylated amino acid in the former peptide. Indeed, similar to Ser687 of the II-III loop (27), the distinctive arrangement of Thr26 of IpTxa with the preceding residues produces a phosphorylation consensus for several protein kinases (28). As presented in Fig. 5B, the structural motif consisting of a cluster of positively charged amino acids followed by a hydroxylated residue is not found in other peptide toxins or in other regions of the DHPR, including the beta -subunit. Thus, IpTxa and the II-III loop peptide share a specific arrangement of amino acid residues that may be responsible for their similar functional effect on RyR channels.

Competition Experiments between IpTxa and the II-III Loop Peptide-- To test whether the analogous functional effects produced by IpTxa and the II-III loop peptide (Fig. 4) result from activation of the same modulatory site on the RyR, we carried out competition experiments between the two peptides. Fig. 6A shows that the II-III loop peptide incrementally decreases the capacity of IpTxa to activate RyRs. The ED50 for II-III loop-inhibition of the IpTxa effect was 1.3 ± 0.7 µM (n = 3), in agreement with the value calculated from direct activation of [3H]ryanodine binding by the II-III loop peptide (Fig. 7B). In contrast, a scrambled II-III loop (a synthetic peptide with amino acid composition identical to the II-III loop peptide but in random sequence) was incapable of stimulating [3H]ryanodine binding (data not shown) or of abolishing the IpTxa effect (Fig. 6B). Thus, the effects of the II-III loop peptide require a defined amino acid sequence and are unrelated to peptide mass or electrical charge. In other competition studies, the II-III loop peptide displaced the binding of 125I-IpTxa to SR vesicles with an ED50 of 36 ± 4 µM (Fig. 5C). This reduced affinity may be due to displacement of 125I-IpTxa from sites of nonequivalent affinity, or it may result from positive allosteric interaction between the II-III loop peptide and 125I-IpTxa. Nevertheless, the II-III loop-125I-IpTxa competition appears to be specific, because the scrambled II-III loop had no significant effect at concentrations up to 300 µM (Fig. 6C).


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Fig. 6.   Activation of RyRs by IpTxa and competition with the II-III loop peptide. A, inhibition of IpTxa effect by the II-III loop peptide. [3H]Ryanodine (7 nM) was incubated with skeletal SR in the presence of IpTxa alone (Control) or IpTxa plus the indicated concentrations of the II-III loop. For each curve, 100% represents the specific binding of [3H]ryanodine in the absence of IpTxa. Data points were fitted with the following equation: B = Bmax/1 + (KD/[IpTxa]). B, binding of [3H]ryanodine was measured in the presence of IpTxa alone (Control) or IpTxa plus the indicated concentrations of the peptide RLKSQKMRATGKAERARSEK (scrambled II-III loop). C, displacement of 125I-IpTxa binding by the II-III loop. The binding of 125I-IpTxa (30 nM) to skeletal SR was carried out exactly as described for [3H]ryanodine binding. Nonspecific binding was determined in the presence of 50 µM IpTxa and subtracted from each data point. All data points are the mean of three independent determinations performed in duplicate. Experimental error was <= 5% (A and B) and <= 10% (C).


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Fig. 7.   Parallel mutations in IpTxa and the II-III loop peptide lead to analogous effects. A and B, effect of substituting Thr or Ser. Specific binding of [3H]ryanodine to skeletal SR was measured in the absence (100%) or in the presence of (A) native or synthetic IpTxa (open circle  and , respectively) or derivatives T26A or T26E or (B) the II-III loop peptide (B, Control) or derivatives S687A and S687E. Results are the mean ± S.D. of n = 4-6 independent experiments.

Effect of Mutations in IpTxa and the II-III Loop Peptide-- If, by analogy with other peptide toxin-ion channel associations (29, 30), the high-affinity IpTxa-RyR interaction is mediated by electrostatic forces, then mutations in the binding domain of IpTxa should alter its electrostatic potential and the measured affinity constant of the IpTxa-RyR complex. Likewise, if IpTxa and the II-III loop peptide bind to the skeletal RyR via a common structural motif, then corresponding mutations should evoke parallel changes in affinity for both peptides. Fig. 7A shows that synthetic IpTxa (a synthetic peptide with an amino acid sequence identical to that of native IpTxa; Ref. 14) activates [3H]ryanodine binding to skeletal SR with potency and affinity identical to native IpTxa (5 ± 3 nM; n = 6). Other synthetic derivatives of IpTxa in which Thr26 was replaced with Ala (T26A) or with the negatively charged residue Glu (T26E) increased [3H]ryanodine binding with an affinity 12- and 160-fold lower (ED50 = 60 ± 5 and 800 ± 78 nM nM, respectively). Mutations to the II-III loop peptide elicited qualitatively similar results (Fig. 7B). The replacement of Ser687 with Ala (S687A) or with Glu (S687E) decreased the affinity of the II-III loop peptide from 0.81 ± 0.2 µM (control) to 4.2 ± 1.1 and 22 ± 4 µM, respectively. Therefore, substituting Thr or Ser of IpTxa and the II-III loop peptide with nonpolar or charged amino acids has a substantial impact on the ability of both peptides to activate RyRs. However, neither mutation results in a complete loss of peptide activity.

Mutations within the cluster of basic amino acids elicit more dramatic effects. In IpTxa, replacing Arg23 with Glu (R23E) abolished IpTxa stimulation of [3H]ryanodine binding (Fig. 8A). The corresponding substitution in the II-III loop peptide, Arg684 to Glu (R684E), also abolished its effect on [3H]ryanodine binding (Fig. 8B). In contrast, replacing either Lys8 of IpTxa or the comparable amino acid Lys675 of the II-III loop peptide with Glu (K8E and K675E, respectively) yielded ED50 values of 19 ± 4 nM and 1.5 ± 0.3 µM, respectively. Thus, the dramatic effect of mutations within the cluster of basic amino acids cannot be solely attributed to a change of electrical charge because mutations distant to the cluster but producing the same electrical change have relatively minor effects.


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Fig. 8.   Effect of substitutions within the cluster of basic amino acids. Specific [3H]ryanodine binding to skeletal SR was measured in the absence (100%) or the presence of IpTxa derivatives K8E and R23E (A) or the II-III loop derivatives K675E and R684E (B). Results are the mean ± S.D. of n = 4 independent experiments.


    DISCUSSION

The molecular mechanism by which depolarization of the skeletal T-tubule membrane induces Ca2+ release from the SR remains an outstanding problem in E-C coupling, with a physical DHPR/RyR interaction being the most plausible hypothesis. Whereas the identification of the RyR region(s) involved in this interaction is in progress (31, 32), there is already substantial evidence to invoke the participation of the II-III loop of the alpha 1 subunit of the DHPR (6, 8-10). As discussed below, there is still controversy regarding the precise structural domain(s) of the II-III loop involved in this interaction. In this study, we identified a unique structural motif involved in activation of RyRs in IpTxa and in a short segment of the II-III loop. Although the participation of this structural motif in E-C coupling awaits further testing, our results provide a structural framework for a mechanical model in which specific amino acids of the II-III loop are capable of interacting with RyRs and triggering Ca2+ release.

Analysis of the amino acid sequence of IpTxa and the effect of mutations strongly suggest that the structural motif encompassing Lys19-Arg24, the cluster of basic amino acids, followed by Thr26, the hydroxylated amino acid, is involved in IpTxa binding to the RyR. Our results, however, do not allow us to ascertain the weight of individual amino acids within this structural domain for participation in binding. For instance, agelenin, Tx2-9, and omega -conotoxin, three structurally related peptides that are incapable of increasing [3H]ryanodine binding (Fig. 1), bear resemblance to IpTxa in several regions, except in the proposed structural domain. However, agelenin and Tx2-9 do maintain the KCK motif, and thus the actual involvement of these residues in the binding of IpTxa remains undetermined. Either the KCK motif is independent of the binding site or, in analogy to other peptide toxins (29, 30), it may be involved in IpTxa docking without participating in activation. The pair of Arg residues following the KCK motif is clearly unique to IpTxa. As shown in Fig. 8, replacing Arg23 with Glu (R23E) totally abolished the capacity of IpTxa to activate RyRs. Because a similar substitution in a region distant to this cluster (K8E; Fig. 8) was without major functional consequences, the lack of effect of R23E was probably the result of local changes in the electrostatic potential of the toxin's binding site, rather than global conformational changes. Lastly, the involvement of Thr26 was demonstrated by hydrophobic (T26A) and charged (T26E) substitutions (Fig. 5). Although the mutated peptides retained their ability to activate RyRs, there was a significant loss of affinity for both peptides.

Replacing equivalent amino acids in the II-III loop peptide produced results that were qualitatively similar to those obtained with IpTxa mutants (Figs. 7 and 8). These data, plus the competition experiments in which the II-III loop peptide inhibited the effect of IpTxa (Fig. 6A) and displaced the binding of 125I-IpTxa (Fig. 6C), strongly suggest that both peptides activate RyRs by a physical interaction with the channel protein via the aforementioned structural domain. If this structural domain of the II-III loop is important for the activation of RyR in vivo, then IpTxa is a peptide mimetic of an effector protein, and its structure-activity information may have direct implications for the mechanism of E-C coupling in skeletal muscle. In RyRs reconstituted in lipid bilayers, IpTxa and the II-III loop peptide induced the appearance of a subconductance state, again supporting the notion that both peptides lead to the same conformational changes in the channel protein.

The small-amplitude, long-lifetime subconductance state produced by IpTxa and the II-III loop peptide was seen in Cs+-conducting (Figs. 4 and 5) and in Ca2+-conducting RyRs (data not shown; see Ref. 25) and is clearly different from that produced by FK506 and ryanodine. FK506 and other immunosupressants that strip RyRs of the accessory protein FKBP12 produce rapidly fluctuating subconductance states that represent approximately one-half and one-fourth of the full conductance state (33, 19). Ryanodine irreversibly locks the channel in a subconductance state of variable amplitude, depending on the charge carrier (34). However, the effects of IpTxa and the II-III loop peptide are also different from those produced by the whole 138-amino acid II-III loop, which activates RyRs by increasing the frequency of full openings without inducing the appearance of a subconductance state (9, 27). These results argue against the II-III loop fragment studied here being an exact functional equivalent of the whole II-III loop. They suggest instead that the II-III loop-RyR interaction is more complex, perhaps encompassing several points of contact that, when acting concertedly, modify the RyR kinetics in a way that is more similar to the activation produced in vivo. In skeletal muscle cells, the DHPR-RyR interaction involves orthograde (DHPRright-arrowRyR) as well as retrograde (RyRright-arrowDHPR) signals (32). Thus, providing that the II-III loop peptide studied here is an actual participant in the E-C coupling mechanism, this fragment and IpTxa may be regarded as partial effectors of the DHPR-RyR interaction whose critical task is to relay the signal that initiates Ca2+ release.

Using synthetic peptides corresponding to small segments of the II-III loop, El-Hayek et al. (10) found that only the amino-terminal portion (peptide A, Thr671-Leu690) was capable of activating RyRs. More recently, the same group (35) reduced the length of the activator peptide to 10 residues (Arg681-Leu690) that encompassed the structural domain studied here. Interestingly, a pentapeptide corresponding to the cluster of basic amino acids was insufficient to activate RyRs (35), and our own results indicate that a scrambled II-III loop peptide is also without effect (data not shown). Therefore, the distinctive distribution of the positively charged residues and their relation to the hydroxylated amino acid are critical determinants of the structural domain that binds and activates RyRs.

Lu et al. (27) found that phosphorylation of Ser687 of the skeletal II-III loop (or substitution by Ala) abolished the capacity of the whole loop to activate RyRs. Leong and MacLennan (36) used the II-III loop in an affinity column and found that replacement of Lys677 or Lys682 with Glu decreased the capacity of the II-III loop to interact with peptide fragments of the skeletal RyR. These results are in line with the hypothesis that the amino-terminal region of the II-III loop binds to and activates the skeletal RyR. However, expressing chimeras of cardiac and skeletal DHPR in dysgenic myotubes, Nakai et al. (8) found that Phe725-Pro742 was the most important region of the skeletal DHPR to elicit a skeletal-type contraction. This region is located almost at the middle of the II-III loop, more than 30 residues downstream from peptide A. Although the results with DHPR chimeras are apparently contradictory to those obtained with isolated peptides, it is still possible that the structural domain of peptide A postulated here as being important for activation of RyRs participates in the transmission signal from DHPR to RyR. A potential scenario would be that Phe725-Pro742, which determines skeletal-type E-C coupling (8), binds to the skeletal RyR at rest to inhibit Ca2+ release and that depolarization of the T-membrane rearranges the II-III loop such that peptide A interacts with and activates the RyR. In agreement with this hypothesis, El-Hayek et al. (10) found that the activating effect of peptide A was antagonized by Glu724-Pro760, a fragment of the skeletal II-III loop encompassing the amino acid sequence identified by Nakai et al. (8). However, even in this simplified scenario, outstanding issues remain unsolved. For example, skeletal-type contractions do not require entry of external Ca2+, whereas activation of RyRs by peptide A (10), IpTxa (13), or the whole II-III loop (9) requires Ca2+, at least at suboptimal levels. Again, this may reflect the fact that these peptides are only partial effectors of the transmission signal, with other segments of the DHPR conferring Ca2+ independence to the DHPR-RyR interaction.

Toxin mimicry of surface receptors is not unprecedented. Mastoparan, a peptide from wasp venom, is a potent secretagogue that mimics an intracellular loop of G protein-coupled receptors (15). As previously shown (16), the capacity of foreign peptides to activate intracellular signals may yield insights into the molecular mechanisms of signal transduction in the target cells. In the case of IpTxa, the high affinity of 125I-IpTxa demonstrates a direct protein-protein interaction with the RyR and reveals a ~4:1 stoichiometry of IpTxa/RyR binding sites (Fig. 2). Assuming that IpTxa is a surrogate high-affinity ligand for the II-III domain that triggers Ca2+ release, these results suggest that in the intact muscle, up to four II-III loops directly gate one RyR. This would be consistent with the current structural model of E-C coupling in skeletal muscle (26), where every other foot protein (RyR) faces a tetrad of T-tubule particles representing four DHPR molecules. Another inference based on the above assumption is that the II-III loop but not the carboxyl terminus of the alpha 1 subunit (11) or the beta -subunit (12) is structurally endowed to trigger Ca2+ release. This is because the structural domain identified as being responsible for activating RyRs (Fig. 4) is contained within the II-III loop sequence only. However, as mentioned above, our results do not rule out the possibility of multiple sites of interaction between the DHPR and the RyR because inhibitory sites of contact have been detected in other segments of the alpha 1 subunit (11) and even within the II-III loop itself (10).

Regardless of its potential use as a peptide probe to study the molecular determinants of E-C coupling, it is important to keep in mind that IpTxa triggers conformational changes in the channel protein that ultimately lead to Ca2+ release (Fig. 4). The specificity, high affinity, and reversibility of the IpTxa-RyR interaction may thus be exploited to identify amino acids of the RyR directly responsible for channel opening.

    ACKNOWLEDGEMENTS

We thank Hiroshi Takeshima and Jianjie Ma for providing CHO cells transfected with the RyR, Fernando Zamudio for help with bilayer experiments, and Joel Armstrong for iodination of IpTxa.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL55438 and PO1 HL47053 (to H. H. V. and J. W. W.).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.

Dagger An Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Physiology, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706. Tel.: 608-265-5960; Fax: 608-265-5512; E-mail: valdivia{at}physiology.wisc.edu.

    ABBREVIATIONS

The abbreviations used are: DHPR, dihydropyridine receptor; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; IpTxa, Imperatoxin A; E-C, excitation-contraction; CHO, Chinese hamster ovary; HPLC, high pressure liquid chromatography; K-MOPS, 4-morpholine propanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
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