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Originally published In Press as doi:10.1074/jbc.M310466200 on December 29, 2003

J. Biol. Chem., Vol. 279, Issue 12, 11853-11862, March 19, 2004
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Multiple Actions of Imperatoxin A on Ryanodine Receptors

INTERACTIONS WITH THE II-III LOOP "A" FRAGMENT*

Angela F. Dulhunty{ddagger}§, Suzanne M. Curtis{ddagger}, Sarah Watson{ddagger}, Louise Cengia¶, and Marco G. Casarotto{ddagger}

From the {ddagger}Division of Molecular Bioscience, John Curtin School of Medical Research and Research School of Chemistry, Canberra and Biotron Limited, Eggleston Rd., Australian National University, P. O. Box 334, Australian Capital Territory 2601, Australia

Received for publication, September 22, 2003 , and in revised form, December 16, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Imperatoxin A is a high affinity activator of ryanodine receptors. The toxin contains a positively charged surface structure similar to that of the A fragment of skeletal dihydropyridine receptors (peptide A), suggesting that the toxin and peptide could bind to a common site on the ryanodine receptor. However, the question of a common binding site has not been resolved, and the concentration dependence of the actions of the toxin has not been fully explored. We characterize two novel high affinity actions of the toxin on the transient gating of cardiac and skeletal channels, in addition to the well documented lower affinity induction of prolonged substates. Transient activity was (a) enhanced with 0.2-10 nM toxin and (b) depressed by >50 nM toxin. The toxin at >=1 nM enhanced Ca2+ release from SR in a manner consistent with two independent activation processes. The effects of the toxin on transient activity, as well as the toxin-induced substate, were independent of cytoplasmic Ca2+ or Mg2+ concentrations or the presence of adenine nucleotide and were seen in diisothiocyanostilbene-2',2'-disulfonic acid-modified channels. Peptide A activated skeletal and cardiac channels with 100 nM cytoplasmic Ca2+ and competed with Imperatoxin A in the high affinity enhancement of transient channel activity and Ca2+ release from SR. In contrast to transient activity, prolonged substate openings induced by the toxin were not altered in the presence of peptide A. The results suggest that Imperatoxin A has three independent actions on ryanodine receptor channels and competes with peptide A for at least one action.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Excitation-contraction (EC)1 coupling is the process that facilitates Ca2+ release from the sarcoplasmic reticulum (SR) of muscle fibers following depolarization of the surface/transverse (t-) tubule membrane. A protein-protein interaction between the dihydropyridine receptor (DHPR) and ryanodine receptor (RyR) underlies EC coupling in skeletal muscle. The DHPR L-type Ca2+ channel in the t-tubule membrane detects surface depolarization and transmits a signal to the RyR channel in the SR via an interaction between the cytoplasmic domains of the two proteins. The interacting region of the DHPR is located between the second and third transmembrane repeats in the {alpha}1 subunit (II-III loop) (1). The interacting regions of the RyR are less clearly defined but are likely to involve residues 1076-1112 (2) and residues 1837-2168 (3).

The recombinant DHPR II-III loop activates skeletal RyRs (4, 5). The loop has been arbitrarily divided into four segments, A, B, C, and D (6). The C region (residues 720-765) is strongly implicated in EC coupling (7-9), and a random coil peptide corresponding to this region modifies the activity of the skeletal RyR (10, 11). A second region of the II-III loop, the A region (residues 671-690), is of interest because its corresponding peptide fragment induces Ca2+ release from the SR and enhances current flow through RyR channels with high affinity (5, 6, 10, 12-18). Although the A region is not essential for skeletal EC coupling in myocytes (7, 9), it may play a role in the DHPR-RyR interaction (8), and it is a useful probe for assessing RyR function (11, 17, 19).

The ability of peptide A to activate RyRs is highly correlated with its capacity to adopt an {alpha}-helical structure (15, 16, 18) and with the orientation of positively charged residues along one surface of the molecule (18). Curiously, two scorpion toxins, Imperatoxin A and Maurocalcine (having 82% sequence identity), have structural features in common with peptide A. Although the intrinsically disulfide-stabilized {beta}-sheet structure of the toxins is vastly different from the {alpha}-helical structure of the A peptide, the toxins and peptide A share a similar surface orientation of positively charged residues (18, 20). Because of this structural similarity, several studies have examined the possibility that the scorpion toxins and peptide A bind to the same site on RyR1. Two studies have concluded that they bind to the same, or overlapping sites (13, 18), whereas a third study concluded that Maurocalcine/Imperatoxin A and peptide A bind to independent sites (19). These different conclusions could have arisen if more than one binding site exists for either the toxins and for peptide A, and not all sites are common to the two compounds. At least two binding sites for peptide A have been defined, one within the channel pore, which leads to voltage-dependent channel block (12), and a site (or sites) on the cytoplasmic domain of the channel, which support its voltage-independent actions (10, 12). Here we investigate the possibility that Imperatoxin A also has multiple actions on RyR activity and examine interactions of between the toxin and peptide A in modifying RyR channel gating.

We find that there are a least three separate effects of Imperatoxin A on cardiac and skeletal RyR channels that can be distinguished by their affinity and reversibility and by their ability to compete with peptide A in its native and a modified form. The results show that peptide A competes with Imperatoxin A for the high affinity activation of transient channel openings. On the other hand, peptide A does not prevent the characteristic toxin-induced prolonged substate activity. The results suggest that there is at least one common or overlapping binding site for Imperatoxin A and peptide A as well as independent binding sites. These observations raise the questions of which of the Imperatoxin A binding sites has been identified on the RyR (21) and whether this site is the site that also interacts with peptide A.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Peptides—Peptides A and A1(D-R18) were synthesized as in Green et al. (18). Imperatoxin A was synthesized by Auspep Australia and folded using procedures outlined by Fajloun et al. (22)

Vesicle Preparation—Preparation of SR vesicles, Ca2+ release from SR, and single channel techniques have been described previously. Heavy skeletal SR vesicles were prepared from rabbit back and leg muscle (11, 12), whereas cardiac SR vesicles were prepared from sheep heart (23).

Single Channel Techniques—Bilayers of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine (5:3:2 w/w) (Avanti Polar Lipids, Alabaster, AL) were formed across an aperture of ~200-µm diameter in the wall of a 1.0-ml Delrin cup (Cadillac Plastics, Australia). Terminal cisternae vesicles (10 µg/ml) were added to the cis chamber. The cytoplasmic side of channels incorporated into the bilayer faced the cis solution. Bilayer potential was controlled, and single channel currents were recorded, using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Bilayer potential is expressed as Vcis - Vtrans (Vcytoplasm - Vlumen).

Bilayers were formed and vesicles incorporated using cis solutions containing (in millimolar): 230 CsCH3O3S/20 CsCl/5.0 CaCl2/10 TES/500 mM mannitol (pH 7.4) with CsOH and a trans solution containing (in millimolar) 30 CsMS/20 CsCl/1 CaCl2/10 TES (pH 7.4). Following incorporation, (a) the cis solution was replaced with an identical solution, except that the [Ca2+] was between 0.1 and 100 µM and (b) 200 mM CsCH3O3S was added to the trans chamber for symmetry. Drugs were added to the cis chamber and removed by perfusion with 10 ml of cis solution.

Analysis of Channel Activity—Channel activity was analyzed over one to two 30-s periods of continuous activity at +40 mV and then at -40 mV. Slow fluctuations in the baseline were corrected using an in-house baseline correction program (written by Dr. D. R. Laver). Channel activity was measured either as "mean current" (the average of all data points in a record) or as open probability (Po), using a threshold analysis with the program Channel 2, (developed by P. W. Gage and M. Smith, John Curtin School of Medical Research). Measurements of mean current, performed on records from experiments containing 1-4 channels, included all channel activity from the smallest subconductance level to maximum openings. On the other hand, open probability (Po), mean open time (To), and mean closed time (Tc) measurements are restricted to records in which the opening of a single channel only could be detected and exclude openings that fall within the baseline noise. In this case threshold levels for channel opening and closing were set to exclude baseline noise (a) at ~20% of the maximum single channel conductance when examining transient openings in the absence of prolonged substates or (b) at 50% of the maximum conductance or 50% of the substate level to measure Po of maximum or substate conductance respectively, when substates were present.

Channel activity is expressed as relative Po to include data in which activity varied from ~0.0001 to ~0.1 and data from bilayers containing more than one channel. Relative Po was calculated either (a) from I't/I'c, where I't is the mean current under test conditions and I'c is the control mean current, or (b) from Pot/Poc, where Pot is the open probability under test conditions and Poc the control open probability. Because the mean current divided by the maximum current approximates open probability, I't/I'c {equiv} Pot/Poc.

DIDS Modification—Channels modified by the disulfinic stilbene derivative, diisothiocyanostilbene-2',2'-disulfonic acid (DIDS), were used in some cases with a cytoplasmic [Ca2+] of 100 nM to enhance channel activity under control conditions. DIDS modification does not alter the regulation of RyRs by Mg2+, ryanodine, or ruthenium red (24), although it can interact with other properties of RyRs (25-27). Channels were exposed to 100 or 300 µM DIDS in the cis chamber for 4-6 min, and then DIDS was removed by perfusion. Activity increased in the presence of DIDS and then fell with removal of the reversible component of activation. However, activity remained higher than before exposure to DIDS because of covalent bonds formed between isothiocyanate groups and NH2, OH, and aromatic groups on a variety of amino acid residues (28).

Ca2+ Release from SR—Extravesicular Ca2+ was monitored at 710 nm using a Cary 3 spectrophotometer (12). The cuvette solution was stirred continuously, and temperature was controlled at 25 °C. Skeletal SR (100 µg of protein) was added to the cuvette solution (final volume of 2 ml), containing (in millimolar): 100 KH2PO4 (pH 7); 4 MgCl2; 1 Na2ATP; 0.5 antipyrylazo III. Ca2+,Mg2+-ATPase activity was suppressed with thapsigargin (200 nM (29)). The same solutions were used with cardiac SR except that an ATP-regenerating system (phospho(enol)pyruvate (5 mM) and pyruvate kinase (25 µg/ml)) was added, and Ca2+-induced Ca2+ release was triggered by addition of 20 µM Ca2+ to the cuvette solution.

When toxin was examined alone, it was added 2 min after thapsigargin. When peptide was added before toxin, either peptide or an equivalent volume of water (vehicle) was added 2 min after thapsigargin, and then toxin or an equivalent volume of water (vehicle) was added after a further 2 min. Release rates in each experiment were measured 10-20 s after toxin addition. Ca2+ release rate, R, as a function of [toxin] was fitted by a Hill equation, R = Rb + Rmax{1/[1 + (Tx50/Tx)H]}, where Rb is the baseline Ca2+ leak or Ca2+-induced Ca2+ release in thapsigargin, Rmax is the maximum toxin-induced release rate, Tx is [toxin], Tx50 the [toxin] for activation to 50% maximum, and H is the Hill coefficient.

Statistics—Average data are given as mean ± S.E. The significance of the difference between control and test values was tested a using either (a) a Student's t test, either one or two sided and either for independent or paired data, as appropriate or (b) using the non-parametric "sign" test (30). Differences were considered to be significant when p <= 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RyR activity demonstrated two distinct gating modes, a "transient" mode and a maintained "substate" mode. The transient mode comprised all control channel activity and was observed in toxin-modified channels. In this mode, the duration of channel openings varied from ~0.5 to ~1000 ms with brief submaximal openings and longer openings to the maximum conductance. The maintained substate mode was seen only after exposure to Imperatoxin A and was characterized by openings lasting from ~1 to 20 s. The number of transient openings increased with toxin concentrations between 200 pM and 20 nM, then declined with >20 nM toxin. Prolonged substate openings appeared with >100 nM toxin.

Imperatoxin A Alters the Probability of Transient RyR Channel Openings
Increased Transient Activity—Imperatoxin A applied at picomolar concentrations to the cis (cytoplasm) side of RyR channels caused an increase in the frequency of transient openings. The increase occurred rapidly, within the 30-s period of toxin addition and stirring. Prolonged substate openings were not induced with these low toxin concentrations. The activating effect on transient openings was only slowly reversible when the toxin was perfused from the cis chamber (Fig. 1, A-C).



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  		FIG. 1.
High affinity activation of transient RyR activity with Imperatoxin A at cisconcentrations of 500 pM to 20 nM. In A-C, 5-s recordings are shown on the left, and all-points histograms of 30 s of activity are on the right. Records in A and B are from one cardiac RyR at -40 and +40 mV. The first record in each panel was obtained with 10 µM cis Ca2+ and 2 mM cis ATP, the second and third after adding 500 pM and 1 nM toxin. Transient openings increased with the toxin at both potentials. In C, traces from an experiment with a bilayer containing four active cardiac RyRs under control conditions with 10 µM cis Ca2+, and then after adding 10, 20, and 50 nM Imperatoxin A and finally 2 and 6 min after toxin removal. Note the increase in activity with 10 and 20 nM toxin, a decline with 50 nM toxin, and an increase immediately after toxin removal. In this and subsequent channel records, the closed level (c) is indicated by a solid line, and maximum single channel currents (broken lines) are labeled "o." In C, maximum open levels for summation of 2, 3, and 4 (o4) channels are indicated by horizontal arrows. D and E show average data for activation of transient RyR activity (at -40 and +40 mV) under a variety of conditions. Relative open probability is shown as a function of [toxin] between 100 pM and 100 nM. Open symbols indicate skeletal RyRs, and filled symbols indicate cardiac RyRs. {blacktriangleup}, 10 µM cis Ca2+, 2 mM cis ATP (n = 4); {blacktriangledown}, 10 µM cis Ca2+ (n = 6); {blacksquare}, 100 nM cis Ca2+, 100 µM DIDS-modified (n = 5); {circ}, 10 µM cis Ca2+ (n = 5); {square}, 10 µM cis Ca2+, 2 mM cis ATP or AMP-PNP (n = 5); {diamond}, 300 nM cis Ca2+, 2 mM cis MgATP (n = 4).

 
Activity increased in cardiac (n = 20 experiments) and skeletal (n = 19) channels that were either Ca2+-activated (cis Ca2+ of 10 or 100 µM; n = 18) or at sub-activating cis [Ca2+] (100-300 nM) (n = 20), in the absence (n = 10) or presence of ATP (n = 17) (with 2 mM Mg2+, n = 4) or AMP-PNP (n = 5) and in DIDS-modified channels (n = 5) (Fig. 1, D and E). All channels were activated by toxin at concentrations up to 10 nM. At 50-300 nM, some channels were activated while others were inhibited. Channels recorded with low cis [Ca2+] showed average activation at higher toxin concentrations than those recorded with higher activating cis [Ca2+] (Fig. 1, D and E).

The changes in channel gating associated with increased activity were measured in a subset of data from skeletal and cardiac RyR channels (Table I). The increased activity of skeletal RyRs with 1 and 10 nM toxin was due to an increase in channel open time, and a reduction in closed time. The increase in open time was also seen in cardiac channels, but in contrast to skeletal channels, there was no significant change in closed durations at lower toxin concentrations.


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  		TABLE I
Single channel parameters from a subset of experiments in which one channel only was active in the bilayer

The skeletal data were obtained either with 2 mM ATP and 10 µM Ca2+ in the cis chamber (n = 4) or with 300 nM Ca2+ and 2 mM MgATP (n = 3). The cardiac data were obtained with either 10 µM Ca2+ (n = 8) or with 10 µM Ca2+ plus 2 mM ATP (n = 4). The relative changes in channel parameters were similar under all conditions and similar at +40 mV and -40 mV. Therefore data under each condition at each potential are combined and expressed as relative open probability (Po), relative open time (To), and relative closed time (Tc).

 
Inhibition of Transient Activity—Transient channel openings decreased when the toxin was increased to between 50 and 500 nM (see Fig. 1C). Some channels were activated for several minutes after exposure to higher [toxin] and were then inhibited (Fig. 2A). Activity was rapidly restored to an activated (greater than control) level upon removal of the toxin, indicating raid reversibility of the inhibitory action.



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  		FIG. 2.
Inhibition of transient RyR activity by Imperatoxin A at >=100 nM. A, 5-s recordings at +40 mV (left) and 30-s all-points histograms (right) with 10 µM cis Ca2+. Transient activity was initially activated (trace 2) and then inhibited (trace 3) by 100 nM toxin, with further inhibition by 500 nM toxin (trace 4) and some openings to the toxin-induced substate (st). Activity was recovered to an activated level (relative to control) after toxin removal (trace 5). Average data in B were obtained at +40 and -40 mV. Open symbols indicate skeletal RyRs, and filled symbols indicate cardiac RyRs. {blacktriangleup}, 100 nM cis Ca2+, 100 µM DIDS-modified (n = 5); {blacktriangledown}, 10 µM cis Ca2+, 2 mM cis ATP (n = 4); {circ}, 10 µM cis Ca2+ and 2 mM cis ATP or AMP-PNP (n = 4). C, effects of Imperatoxin A on channel activation by Ca2+ and adenine nucleotides -40 and +40 mV. 200 nM toxin was added for 3-4 min before the cis [Ca2+] was increased to 100 µM for 4 min and then 2 mM ATP or AMP-PNP added. Channels were either activated (left, n = 6) or inhibited (right, n = 5) by the toxin. Relative Po is compared under control conditions (c, with 300 nM cis Ca2+), then with toxin (T), cis [Ca2+] of 100 µM (Ca) and then 100 µM plus 2 mM ATP or AMP-PNP (Ad, adenine nucleotide). The y axis is split to display the range of relative Po values.

 
The inhibition of transient activity was independent of prolonged substate openings. Although all channels exhibiting prolonged substates also showed fewer transient openings, the number of transient openings was often substantially reduced in the absence of substate activity (100 nM toxin (Fig. 2A)). Inhibition reduced Po to less than control when the activity was initially high (in channels with activating cis Ca2+ and/or ATP (Fig. 2A)). When activity was low (in the presence of 100-300 nM Ca2+ and/or MgATP), Po fell to less than the toxin-activated level, but often remained higher than control (e.g. the skeletal channels in Table I).

The decline in transient activity was caused by an increase in closed time (Table I). Curiously, the open times in skeletal RyRs continued to increase as toxin concentration increased (Table I), perhaps indicating that the activating effect of the toxin increased with [toxin], but was overwhelmed by an independent inhibitory effect. Persistent inhibition was also seen following perfusion of inhibiting [toxin] from the cis chamber, when removal of inhibition revealed a strong slowly reversible activation (e.g. Fig. 2A). On average, cardiac RyRs showed a significant decline in open times and increase in closed durations when inhibited by 1 µM toxin (Table I). Inhibition was seen in all cardiac (n = 13) and skeletal (n = 9) channels exposed to higher toxin concentrations and was independent of bilayer potential or cis [Ca2+] (n = 9 for <=300 nM Ca2+ or n = 13 for 10 µM Ca2+), and occurred without ATP or Mg2+ (n = 8) or with 2 mM ATP (n = 14) and 2 mM Mg2+ (n = 4), as well as in DIDS-modified channels (n = 5) (Fig. 2B).

Toxin-activated channels were further activated by Ca2+, ATP, or AMP-PNP, whereas inhibited channels could not be activated to the same extent by these ligands (Fig. 2C). In this experiment, with 100 nM cis Ca2+, cardiac RyR channels were either activated or inhibited by 200 nM toxin. The toxin-activated channels were further activated when cis Ca2+ was increased to 100 µM and activated again by either 2 mM ATP or AMP-PNP. In contrast, although activity increased in the toxin-inhibited channels, the increase (relative to control) was significantly less than in the activated channels.

Peptide A Competes with Imperatoxin A for Activation of Transient Opening—It has been suggested that peptide A and Imperatoxin A compete for a single site on the RyR. Cardiac channels were activated by 1 nM toxin and by subsequent additions of peptide A up to 500 nM (Fig. 3A). Activity declined when the peptide was increased to 1 µM. However, the plateau of activation with peptide plus toxin was no greater than that with peptide alone, suggesting that there was no summation of the effects of the two compounds and supporting the concept of the same or overlapping binding sites. Similar results were obtained when channels were exposed to 10 nM toxin, although there was a significant reduction in activity when the [peptide] reached 500 nM. In a similar experiment (Fig. 3B), channels were first exposed to 50 nM peptide A and then to increasing concentrations of toxin. Activity tended to increase with peptide plus toxin up to 600 pM, but activation was significantly less than expected from the summation of two independent processes (Fig. 3B). Channel activity declined when higher concentrations of toxin were added with peptide. The decline in activity with higher concentrations of toxin plus peptide A (or modified peptide A (A1-R18D) (18)) was particularly apparent in skeletal RyR channels (Table II and Fig. 3, C and D). Transient openings declined with 1 nM toxin in the presence of 1 µM peptide A (Fig. 3), even though there was no inhibition of RyRs by either 1 nM toxin or 1 µM peptide A alone.



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  		FIG. 3.
Combined actions of peptide A and Imperatoxin A on transient channel activity. The bins in A and B show average relative open probability for cardiac RyRs with 300 nM cis Ca2+. In A, empty bins are for peptide A alone (n = 7), and the cross-hatched bars and filled bars represent data for peptide A added after Imperatoxin A at 1 nM (n = 5) or 10 nM (n = 3), respectively. Data are shown under control conditions (control), after adding toxin (toxin), and with each concentration of peptide A. The broken line shows expected activity if activation was additive (i.e. A alone at 500 nM (open bar) plus the mean of 1 and 10 nM toxin). B, data under control conditions (control), after adding 50 nM peptide A (peptide), and then Imperatoxin A as indicated (n = 7). The broken line shows expected activity for added effects (A alone at 50 nM (second bar) plus the mean of 1 and 10 nM toxin from A). In A and B, note 1) maximum activation with toxin plus peptide A is no different from that with peptide A alone and 2) a decline in activity with higher concentrations of toxin plus peptide. C and D, exclusive activation of skeletal RyRs by either peptide A or Imperatoxin A: 5-s current records (left) and 30-s all-points histograms (right) with a cis [Ca2+] of 100 nM. C, 10 nM toxin increased channel activity at +40 mV and activity declined with 1 µM peptide A plus toxin. Conversely, D shows an increase in activity at -40 mV with 1 µM peptide A and then a decrease after adding 1 nM toxin.

 


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  		TABLE II
Combined actions of Imperatoxin A (IpTx) and either the native peptide A or modified peptide A (A1-R18D), on the relative probability of transient skeletal RyR channel activity

The concentrations of toxin and peptide used were maximal for activation of transient openings. Data obtained at +40 and -40 mV have been combined, because similar results were obtained at the two potentials. Each of the toxins, peptide A and A1-R18D, increase the relative open probability when added alone. However, subsequent addition of a second compound failed to increase activity further. Some of this data were presented in Green et al. (18) and have been re-evaluated for comparison with data in this report.

 
Prolonged Substate Openings Induced by Imperatoxin A
Imperatoxin A induces prolonged channel openings to substate levels in cardiac and skeletal RyRs (31). In the present experiments, toxin concentrations of 1-10 µM were required to consistently observe substate activity within 2-10 min of exposure to the toxin, although substate activity could occasionally be seen after prolonged exposure to 10-100 nM toxin. Channels demonstrated abrupt transitions between the substate and transient gating modes (Fig. 4A). In the substate mode, the channel was almost continually open with Po close to 1.0 and average open times of ~1-20 s. Substate gating was interrupted by bursts of transient activity with frequent channel openings from the closed to the maximum conductance levels (Fig. 4A).



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  		FIG. 4.
Imperatoxin A induces prolonged substate openings with transitions between the substate and transient gating. A, activity recorded at -40 mV from a cardiac RyR with 10 µM cis Ca2+ and 500 nM Imperatoxin A (left), and all-points histograms (right). The upper trace is a 30-s recording with transitions (i) between the closed state, the dominant toxin-induced substate at ~25% of the maximum conductance, and a lower substate at ~ 12% and (ii) between the substate and transient gating modes. The record was filtered at 100 Hz to show the different substate levels. The lower trace is an expansion of the transitions between substate and transient gating. B, Imperatoxin A-induced substates and gating transitions in a DIDS-modified cardiac RyR channel at -40 and +40 mV, with 100 nM cis Ca2+. In each panel, 5-s current records are to the left, and 30-s all-points histograms are to the right. The first trace in each panel shows DIDS-modified activity, the second and third traces show activity after adding 1 µM toxin then after removal of the toxin. Note (a) the similar dominant substate level, and smaller substate levels (dotted line next to the closed level), at both potentials, (b) the strong inhibition of transient gating in the presence of the toxin, and (c) the rapid recovery of transient activity following removal of the toxin, but maintained substate activity.

 
The currents from the DIDS-modified channel in Fig. 4B, recorded with 100 nM Ca2+, illustrate three important features: 1) the fall in transient activity in the presence of 1 µM toxin; 2) the similarity of the toxin's action (and substate levels) at +40 and -40 mV; and 3) rapid recovery of transient openings after removal of the toxin. The substate activity remained for several minutes after removal of the toxin and restoration of transient activity. Two or three different conductance levels are apparent in the toxin-induced substate activity (Fig. 4). The highest conductance substate dominated and was a similar fraction of the maximum conductance at +40 and -40 mV (Table III). The substate was significantly higher in the skeletal RyRs (~33% maximum) than in cardiac RyRs (29% maximum).


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  		TABLE III
Properties of the prolonged toxin-induced subconductance state in skeletal and cardiac RyR channels

Data shown are averages of that obtained with 500 nM and 1 µM toxin for six cardiac channels or for 100 nM and 1 µM toxin for six skeletal channels.

 
The substate parameters were not obviously affected by the channel type (cardiac or skeletal), voltage across the bilayer, cis Ca2+ or Mg2+ or modification by DIDS. The average combined data in Table III shows that the substate mode occupied ~70% to 80% of channel activity and that, within that mode, the Po was ~1.0, the mean open time was ~2-7 s, and mean closed time was between ~11 and 40 ms.

The Effects of Peptide A on the Toxin-induced Substate—The interaction between peptide A and Imperatoxin A during prolonged substate activity was examined in DIDS-modified channels with a cis [Ca2+] of 300 nM. The RyR in Fig. 5 was activated to a Po of 0.999 by 300 µM DIDS. Activity fell to 0.44 when DIDS was removed ("Materials and Methods"). 1 µM peptide A increased Po of the DIDS-modified channel to 0.977. Subsequent addition of 1 µM toxin resulted in an immediate decline in transient openings (Po = 0.15) and the slower appearance of prolonged substate activity with the usual gating transitions between substate and transient modes. Additional substate levels between 50 and 90% of maximum were apparent with toxin plus peptide in some channels (e.g. Fig. 5). These substate levels were not obvious with 1 µM peptide A alone (Fig. 5, A and B) but were occasionally seen when peptide A was added to normal (not DIDS-modified) channels at concentrations of >5 µM (12).



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  		FIG. 5.
The effects of peptide A on toxin-induced substate activity Ca2+ release from SR vesicles. A and B show 5-s current records (left), and 30-s all-points histograms (right), from a cardiac RyR with 100 nM cis Ca2+, at -40 and +40 mV. The traces show (from the top down): activity after adding 300 µM DIDS to the cis chamber; irreversible DIDS-modified activity (after removal of cis perfusion); strong activation when 1 µM peptide A was added to the cis solution; inhibition of transient channel activity by 1 and 10 µM Imperatoxin A and the toxin induction of substate activity. Note peptide A-induced subconductance openings (arrows in the final traces in A and B) between the toxin-induced substate and the maximum conductance. C-E, average Ca2+ release from SR vesicles in nanomoles/mg/min. C, toxin-induced Ca2+ release from skeletal SR (the inset shows an expanded Ca2+ release by low concentrations of toxin). The solid line shows the sum of two Hill functions fitted to the data with maximum rates of 650 and 5300 nmol/mg/ml, AC50 values of 190 nM and 4.4 µM and Hill coefficients of 1 and 0.95. The broken line shows the best fit of a single Hill equation with a maximum rate of 5900 nmol/mg/ml, an AC50 of 4.3 µM, and a Hill coefficient of 1.1. D,Ca2+-induced Ca2+ release from cardiac SR. The solid line shows the sum of two Hill functions fitted to the data with maximum release rates of 25 and 116 nmol/mg/ml, AC50 values of 25 and 400 nM and Hill coefficients of 1 and 2. The broken line shows the best fit of a single Hill equation with a maximum rate of 140 nmol/mg/ml, an AC50 of 350 nM, and Hill coefficients of 2. E, effect of peptide A on toxin-induced Ca2+ release from skeletal SR. Rates are shown in the absence of toxin or peptide (c), with 1 µM peptide A (A), with 1 nM toxin (t), with 1 µM peptide A plus with 1 nM toxin (A+t), or 1 µM peptide A plus with 5 µM toxin (A+t).

 
The records in Fig. 5 (A and B) reinforce several novel features of the actions of Imperatoxin A and peptide A. 1) Peptide A activates cardiac RyR channels after DIDS modification. 2) 1 µM Imperatoxin A inhibits transient channel gating with minimal substate activity. 3) Imperatoxin A induces its characteristic substate in the presence of peptide A. 4) Peptide A-induced substates, between 50 and 90% s of the maximum conductance, occur in the presence of toxin-induced substate activity.

When 1 or 10 µM peptide A was added to channels after 1 µM toxin in three experiments, no change in Imperatoxin A-induced substate activity was observed, but additional substate activity occasionally appeared, similar to that shown in Fig. 5.

Taken together these results suggest that, as with maurocalcine (19), the effects of peptide A and the ability of Imperatoxin A to induce prolonged substate openings are independent actions, probably due to the compounds binding to separate sites on the RyR.

Effects of Imperatoxin A on Ca2+ Release from SR
The effects of Imperatoxin A on Ca2+ release were examined to see whether the high affinity action of the toxin on RyR channels could be seen in a system that was more intact and physiological than the bilayer.

Concentration Dependence of Effects of Imperatoxin A on Ca2+ Release—Addition of only 1 nM Imperatoxin A significantly enhanced resting Ca2+ release from thapsigargin-blocked skeletal SR vesicles (Fig. 5C). Higher concentrations of toxin resulted in the release of Ca2+ at progressively higher rates, up to >5000 nmol/mg/min with 40 µM toxin (Fig. 5C). The data were best fitted by the sum of two Hill functions, the first with a maximum rate of 650 nmol/mg/min, an AC50 of 190 nM, and Hill coefficient of 0.95. The second had with a maximum of 5300 nmol/mg/min, an AC50 of 4.4 µM, and a Hill coefficient of 1.0. Single Hill functions did not fit the data, particularly at low toxin concentrations (broken line, Fig. 5C). Similar results were obtained with Ca2+-induced Ca2+ release from cardiac SR (Fig. 5D).2 These results are consistent with the single channel data indicating two activation processes, one high affinity, equivalent to the increase in the probability of transient openings, and one with lower affinity, equivalent to prolonged substate openings.

Competition between Imperatoxin A and Peptide A in Ca2+ Release from SR—Exposure of skeletal vesicles to 1 µM peptide A resulted in an increase in the rate of release that was similar to that induced by exposure to 1 nM Imperatoxin A (Fig. 5D). However, the rate of release was not enhanced when 1 nM toxin was added after 1 µM peptide A. Thus there appeared to be no additive effect on release when peptide A was present with a low concentration of toxin. On the other hand, the rate of release of Ca2+ induced by 5 µM toxin tended to be greater when toxin was added to peptide-activated channels. These results are again consistent with the single channel data, which showed interdependent actions of the peptide and the high affinity effect of the toxin, but an additive effect of the peptide and the lower affinity effect of the toxin (i.e. substate openings).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Activation of transient gating of skeletal and cardiac RyR channels by picomolar concentrations of Imperatoxin A is described for the first time. Novel observations with DIDS-modified channels illustrate the robust nature of (a) high affinity activation and lower affinity inhibition of transient activity by Imperatoxin A, (b) toxin-induced substate gating, and (c) activation by peptide A. We show that peptide A competes with Imperatoxin A only for actions on transient channel gating. The toxin-induced substate persists in the presence of peptide A, with additional transient openings to conductance levels peculiar to the peptide. The results raise questions about the functional effects of binding to the site identified for Imperatoxin A (21) and whether peptide A also binds to the site. Finally the multiple actions of the toxin provide an explanation for divergent reports of the existence of a common binding site with peptide A (13, 18, 19).

Multiple Independent Actions of Imperatoxin A—The effects of Imperatoxin A are separated by concentration dependence, reversibility, gating, and interactions with peptide A. The increase in transient activity, with as little as 200 pM toxin, occurred with an increase in open time and decrease in closed times, and was irreversible in the short term. Activated channels were further activated by Ca2+ and ATP (or AMP-PMP). Peptide A competes with the toxin for transient activation. Toxin at >10 nM inhibited transient openings and prolonged closed times. Inhibited channels could not be fully activated by ATP or AMP-PNP. Inhibition was (a) rapidly reversed when the toxin was removed and (b) enhanced by peptide A. The third action of the toxin was the induction of prolonged substates that persisted after recovery of transient gating when the toxin was removed and persisted in the presence of peptide A. The enhancement of transient RyR activity by picomolar concentrations of Imperatoxin A has not been reported, possibly because previous studies have not systematically examined of the concentration dependence of the toxin, but focused on the toxin-induced substate (31) and compared it with peptide A-induced substates (13).

Ca2+ Release from SR—Imperatoxin A at >=1 nM released Ca2+ from SR. A dual activating effect of the toxin was suggested by the need for two Hill functions fit to the data. The fits suggested a small high affinity effect added to a large low affinity effect, corresponding, respectively, to the high affinity activation of transient gating and the lower affinity substate induction. The effects on Ca2+ release were at ~10-fold higher concentrations than effects on channels in bilayers. The reason for this shift to higher [toxin] in Ca2+ release experiments is unclear but is also seen with the DHPR II-III loop and loop peptides (4, 12). Because each of the four RyR subunits are likely to contain toxin binding sites, it is curious that the Hill coefficients were around 1. This could mean that binding of one toxin molecule to the tetramer was sufficient to evoke a maximum effect. However, by analogy with Ca2+ inhibition (23), it is likely that the low coefficients are due to averaging release through many channels with individual variations in responses to the toxin.

The inhibition of transient activity was not reflected in Ca2+ release from SR, possibly because (a) of variations between individual channels in the toxin concentration at which inhibition was apparent, (b) activity may have remained greater than control in some channels, even though it was less than the maximum toxin-activated level, and (c) any inhibitory effect on transient activity would have been swamped by the current flowing through substate openings at higher [toxin]. Under the bilayer conditions, the Po for transient openings with low cis [Ca2+] in the presence of inhibiting toxin concentrations was between 0.001 and 0.0001, and thus the average current flowing through one channel would be between 0.01 and 0.001 pA, assuming there was a maximum single channel current of 10 pA. In contrast, when the channel was open at the substate level at 30% of the maximum conductance for 80% of the time, the average current through the channel would be ~2.5 pA.

Actions of Peptide A—Peptide A also has multiple independent effects. First, it increases transient activity in skeletal RyRs with high affinity (>=10 nM) and releases Ca2+ from skeletal SR (5, 6, 10, 12-18). Ca2+-induced Ca2+ release from cardiac SR is enhanced by the peptide and cardiac RyRs activated at sub-activating cis [Ca2+].3 The second action is a voltage-independent inhibition, seen specifically with the modified A1-R18D at >=10 nM in skeletal and cardiac RyRs with 100 µM cis Ca2+.3 The native peptide A is less active with 100 µM cis Ca2+ than with lower [Ca2+] (12, 17). Because peptide structure is not Ca2+-dependent,3 this inhibition raises the possibility either that there is a separate inhibitory site or the activation site can become inhibitory. The third action is a voltage-dependent block of the skeletal and cardiac RyR pore with >=~5 µM peptide A, leading to transient substate activity, particularly when the current pulls the peptide into the pore (at +40 mV) (10, 12, 32). The substates have conductances of <10-90% of the maximum conductance, with lower levels dominating at positive potentials.

Interactions between Imperatoxin A and Peptide A: Common and Independent Binding Sites—The non-additive nature of activation of transient gating by Imperatoxin A and peptide A is indicative of identical or overlapping sites so that binding of either peptide or toxin precludes binding of the other compound. In contrast, the toxin-induced substate activity persisted in the presence of peptide A suggesting independent binding sites (Fig. 6).



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  		FIG. 6.
A schematic representation of the various Imperatoxin A and peptide A binding sites on a RyR channel in a bilayer situation.

 
The results with inhibition are less easily explained. Addition of peptide plus toxin at activating concentrations for the individual compounds, resulted in a decline in activity at +40 and -40 mV and shift of inhibition to lower [toxin]. Peptide A alone does not cause a voltage-independent inhibition with 100 nM cis Ca2+ (previous section), so that the results could suggest that peptide A binds to a site near the toxin inhibition site to increase the affinity for the toxin. However, an additive effect cannot be ruled out, because the modified A1-R18D can cause voltage-independent inhibition under appropriate conditions. Thus the shift in inhibition to lower toxin concentrations could be explained by the compounds binding to the same or different inhibitory sites. Additive effects could not be determined because inhibition by either compound reduced transient channel activity to very low levels. Overall, these data suggest that there is (a) a common transient activation site for Imperatoxin A and peptide A, (b) independent substate induction sites for the toxin and peptide, and (c) a toxin inhibition site whose relationship to the peptide A site is unclear (Fig. 6).

Similar Actions of the Toxin and Peptide on Skeletal and Cardiac RyR Channels—Imperatoxin A induces similar substate activity in skeletal and cardiac RyR channels (Table III and Ref. 31). Similarly, both skeletal and cardiac II-III loop and II-III loop peptides can interact with the either the skeletal or cardiac RyR (33, 34).3 These results imply that a physical interaction can occur between either skeletal or cardiac RyRs and DHPRs, if the proteins were appropriately targeted (32). This is difficult to test, because DHPRs do not form tetrads and are not aligned with cardiac RyRs (35).

The Imperatoxin A-induced Substate—The toxin-induced substate was ~30% of the maximum conductance at both +40 and -40 mV in cardiac and skeletal RyRs. This differs from reported Imperatoxin A-induced substates of 43% at -40 mV and 28% at +40 mV (31), perhaps because we used Cs+ as the current carrier (as opposed to K+), or native rather than CHAPS-solubilized RyRs. Maurocalcine-induced substates are also voltage-independent (19).

Previous Studies of Imperatoxin A and Peptide A Binding—Findings that peptide A and Imperatoxin A do, or do not, bind to one site are consistent with multiple actions of the compounds. Gurrola et al. (13) showed competition between the toxin and peptide A in Ca2+ release from SR, [3H] ryanodine binding and 125I-Imperatoxin A binding. Because these parameters do not reveal channel gating, the competition could have been for any of the functional sites and was most likely for the transient activation site. Indeed Green et al. (18) found interactions between peptide A and effects of the toxin on transient gating. Chen et al. (19) report independent peptide A and Maurocalcine-induced substates. Thus previous reports are consistent with the present finding that the toxin and peptide A compete for transient activation, but not for substate induction. In addition, the observation that peptide A inhibits the increase in [3H]ryanodine binding induced by the Imperatoxin A (13), even though toxin and peptide individually activate Ca2+ release, is consistent with the fact that transient channel gating is inhibited when toxin and peptide A are added together.

A binding site for Imperatoxin A has been identified on the cytoplasmic domain of the RyR, close to the membrane (21). Because the site is likely to be a high affinity site, toxin binding to it may activate transient openings and thus also be the peptide A binding site. This site is remote from RyR domain 6, which is closest to the t-tubule membrane and the DHPR (36, 37). If the A region of the II-III loop does interact with the RyR under any conditions in vivo, it is likely that the interaction is with a part of the protein that is closer to domain 6, than the toxin binding site. It is thus possible that the inhibitory action of the peptide A and the toxin are caused by binding to a site that differs from the activation site and is located closer to the T-tubule membrane (Fig. 6).

In conclusion, we find that Imperatoxin A has three independent functional effects on cardiac and skeletal RyR channels. The skeletal DHPR II-III loop-derived peptide (peptide A) competes with the toxin for its high affinity activation of transient channel openings. Peptide A modifies a lower affinity toxin-induced inhibition of transient activity and has an independent action on the lowest affinity effect of the toxin in inducing prolonged substate activity. We suggest that there are three functional binding sites for the toxin and that at least one site is shared by peptide A.


   FOOTNOTES
 
* The work was supported by grants form the National Heart Foundation of Australian (Grant G-01C-0296) and the National Health and Medical Research Council (Grant 224235). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed. Tel.: 61-6-125-4491; Fax: 61-6-125-4761; E-mail: angela.dulhunty{at}anu.edu.au

1 The abbreviations used are: EC, excitation-contraction; SR, sarcoplasmic reticulum; DHPR, dihydropyridine receptor; t-tubule, transverse tubule membrane; RyR, ryanodine receptor; TES, N-tris[hyroxymethyl]methyl-2-aminoethanesulfonic acid; DIDS, diisothiocyanostilbene-2',2'-disulfonic acid; AMP-PNP, adenosine 5'-({beta},{gamma}-imino)triphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; AC50, concentration for 50% activation. Back

2 Note that the rates of Ca2+ release from the crude cardiac SR preparation are significantly lower than those from the heavy skeletal SR preparation, largely because of an ~10-fold greater contamination with longitudinal SR vesicles that do not contain Ca2+ release channels. Back

3 A. F. Dulhunty and M. G. Casarotto, unpublished data. Back


   ACKNOWLEDGMENTS
 
We are grateful to Suzy Pace and Joan Stivala for assistance with the preparation and characterization of SR vesicles.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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G. M. Gibbs, M. J. Scanlon, J. Swarbrick, S. Curtis, E. Gallant, A. F. Dulhunty, and M. K. O'Bryan
The Cysteine-rich Secretory Protein Domain of Tpx-1 Is Related to Ion Channel Toxins and Regulates Ryanodine Receptor Ca2+ Signaling
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