Introduction

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The myoplasmic free Ca²⁺ concentration is regulated tightly by uptake into and release from intracellular Ca²⁺ stores. These Ca²⁺ fluxes are the basis for skeletal muscle relaxation and contraction (Fleischer and Inui, 1989). Efflux of Ca²⁺ occurs via the Ca²⁺-release channel of the sarcoplasmic reticulum, also referred to as ryanodine receptor-1 (RyR₁). Two additional isoforms have been identified (reviewed in Franzini-Armstrong and Protasi, 1997), namely the RyR₂ (cardiac isoform, but also widely expressed in brain) and RyR₃ (thought to be widely expressed at low levels). All isoforms bind ryanodine (as well as related alkaloids from Ryania speciosa) with high affinity, and it was this ligand that made the identification of the channel protein possible (Meissner, 1994). The ryanodine receptor/Ca²⁺-release channel is a homotetramer and resides on the terminal cisternae of the sarcoplasmic reticulum and is closely associated with L-type calcium channel of the sarcolemmal T-tubular system (Melzer et al., 1995). The propagation of the action potential along the surface of the muscle is sensed by the L-type calcium channel, which, upon voltage-dependent activation, provides the triggering signal for gating of the ryanodine receptor.

The ryanodine receptor/Ca²⁺-release channel is unusually large (molecular mass of one subunit = 565 kDa), and most of its size is accounted for by a large hydrophilic segment at the amino terminus (Meissner, 1994). In skeletal muscle, this domain presumably is involved in the direct interaction with the L-type channel and with additional proteins that maintain the highly organized topology of the triad. The carboxy terminus contains the pore-forming hydrophobic segments (Bhat et al., 1997). In addition, the ryanodine receptor contains multiple binding sites for calmodulin and for Ca²⁺, the precise number and location of which are still a matter of debate (Franzini-Armstrong and Protasi, 1997). The action of both calmodulin and Ca²⁺ is dualistic; at concentrations in the low-micromolar range, Ca²⁺ promotes channel opening, whereas submillimolar to millimolar concentrations deactivate the channel, an effect that is less pronounced in RyR₂ and that presumably is absent in RyR₃ (Franzini-Armstrong and Protasi, 1997). Similarly, in its Ca²⁺-free form calmodulin increases the open probability of the channel and promotes [³H]ryanodine binding, but Ca²⁺-liganded calmodulin is a potent inhibitor of the receptor (Meissner, 1986; Plank et al., 1988; Smith et al., 1989); Ca²⁺/calmodulin directly interacts with the channel protein in a 1:1 stoichiometry (Wagenknecht et al., 1997). The binding sites for ryanodine and ATP have been mapped to the carboxy-terminal fragment (Shoshan-Barmatz and Zarka, 1988; Zarka and Shoshan-Barmatz, 1993). By analogy with other ATP binding proteins, the trypanocidal drug suramin initially was proposed to interact with the ATP binding site of the receptor (Emmick et al., 1994). However, this assignment was not supported by a subsequent analysis. Although both suramin and adenine nucleotides stimulate the ryanodine receptor directly from the cytoplasmic side, they exert different effects on the gating properties of the channel (Hohenegger et al., 1996; Sitsapesan and Williams, 1996); most importantly, suramin does not block the covalent incorporation of an ATP analog into the ryanodine receptor (Hohenegger et al., 1996).

To understand the mechanism by which suramin activates the ryanodine receptor, we have searched for a more potent suramin analog. In the present work, we used stimulation of [³H]ryanodine binding as a screening procedure with a reasonably high throughput. This approach led to the identification of NF307, the affinity of which exceeds that of suramin; in addition, NF307 is substantially more efficacious than suramin in enhancing the Ca²⁺ sensitivity of the ion channel. Finally, our experiments show that NF307 and suramin compete with calmodulin for binding to the ryanodine receptor.

	Materials and Methods

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Materials. Leupeptin, pepstatin, phenylmethylsulfonyl fluoride, antipain, CsCl (ultrapure), ruthenium red, calmodulin, and low-molecular-mass protein standards were purchased from Sigma (St. Louis, MO); phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine were purchased from Avanti Polar Lipids (Alabaster, AL); suramin and unlabeled ryanodine were purchased from Calbiochem (San Diego, CA); and Pefabloc was from Boehringer Mannheim (Mannheim, Germany). [³H]Ryanodine was from New England Nuclear (Boston, MA), and reagents for enhanced chemiluminescence and horseradish peroxidase-linked anti-mouse IgG were from Amersham Buchler (Buckinghamshire, UK). The materials for SDS-polyacrylamide gel electrophoresis were obtained from Bio-Rad (Hercules, CA), Calmodulin-Sepharose 4B and molecular mass standards for electrophoresis were obtained from Pharmacia LKB (Uppsala, Sweden). A mouse monoclonal antibody directed against the ryanodine receptor (Airey et al., 1990) was from Biomol (Munich, Germany). Aprotinin was a generous gift from Bayer AG (Wuppertal, Germany). All other reagents were of analytical grade.

Chemical Synthesis. The compounds NF299, NF301, and NF307 are analogs of suramin, in which the central urea bridge of suramin has been displaced by a 1.4-bis(carbamoyl)-piperazine group.

Membrane Preparation and Purification of the Ca²⁺-Release Channel/Ryanodine Receptor. Heavy sarcoplasmic reticulum (HSR) from rabbit white muscle was prepared according to Wyskovsky et al. (1990). The ryanodine receptor was purified as described previously (Suko et al., 1993) with the following modifications (Suko and Hellmann, 1998): HSR (15 mg/ml) was solubilized with 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS) in a buffer containing 40 mM 3-(N-morpholino) ethane sulfonic acid (MOPS)-Tris (pH 7.0), 1 M NaCl, 2 mM dithiothreitol (DTT), 1% (w/v) CHAPS, 0.25% (w/v) phosphatidylcholine, and a cocktail of protease inhibitors (0.5 µg/ml leupeptin, 1.4 µg/ml aprotinin, 1 µg/ml antipain, 1 µM pepstatin, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine) for 1 h. The soluble proteins were separated from the insoluble material by centrifugation (103,000g for 35 min) and subsequently applied onto a linear sucrose gradient from 7.5 to 20% in buffer [40 mM MOPS-Tris, pH 7.0, 300 mM NaCl, 2 mM DTT, 0.5% (w/v) CHAPS, 0.25% (w/v) phosphatidylcholine] and the cocktail of protease inhibitors listed above. After centrifugation (Beckman SW28 rotor; 24,000 rpm, 14 h) the gradient was separated in fractions that were screened by SDS-polyacrylamide gel electrophoresis for the ryanodine receptor. The fractions containing the purified ryanodine receptor were pooled and dialyzed (22-24 h) against a buffer containing 40 mM MOPS-Tris (pH 7.0), 100 mM NaCl, 2 mM DTT, 0.15 mM CaCl₂, 0.1 mM EGTA, and the protease inhibitor cocktail. Proteoliposomes were stored in the presence of 200 mM sucrose at 80°C. All steps of the preparation were carried out at 4°C.

[³H]Ryanodine Binding. Sarcoplasmic reticulum membranes (50 µg) were incubated in 50 µl containing 20 mM HEPES (pH 7.4), 20 nM [³H]ryanodine, 1 µM aprotinin, 1 µM leupeptin, 100 µM Pefabloc, and the concentrations of suramin analogs, caffeine, calmodulin, and Ca²⁺ as indicated in the figure legends. Additionally, two salt concentrations were used, either 750 mM KCl or the combination of 200 mM KCl and 10 mM NaCl (referred throughout the text as 0.75 M KCl and 0.2 M KCl buffer). The free Ca²⁺ concentration was adjusted by altering the ratio of EGTA and CaCl₂. The incubation was carried out at 30°C for 40 min. For kinetic experiments the incubation time was varied from 3 to 180 min. In saturation experiments, the incubation time was 180 min. The reactions were terminated by filtration over glass-fiber filters (presoaked in 1% polyethylenimine) using a Skatron vacuum filtration device. The filters were rinsed with 10 ml of ice-cold 10 mM Tris-HCl (pH 7.4), 700 mM NaCl, 0.17 mM CaCl₂, and 0.2 mM EGTA. Nonspecific binding was determined in the presence of 1000-fold excess of unlabeled ryanodine, which had been added to the incubation mixture before the labeled ligand. None of the compounds investigated affected nonspecific binding. If not otherwise indicated, experiments were carried out in duplicate and each experiment was reproduced at least twice with different protein preparations.

Single-Channel Recordings. Single-channel recordings were carried out after the incorporation of the purified Ca²⁺-release channel/ryanodine receptor into planar lipid bilayers essentially according to Coronado et al. (1992) and as described previously (Suko and Hellmann, 1998). Briefly, the bilayer was formed from a lipid mixture (1:1) of phosphatidylserine and phosphatidylethanolamine dissolved at a concentration of 10 mg/ml each in decane. The cis- and trans-chambers were filled with 0.7 ml and 1.3 ml, respectively. The lipid solution was spread over a 200-µm-diameter aperture in a delrin cup (Warner Instruments Corp., Hamden, CT) separating two aqueous compartments. The cis-bath (0.7 ml) and the trans-bath (1.3 ml) were connected to the head-stage input of a model EPC-9 amplifier (Heka Elektronik, Lambrecht, Germany); the trans-bath was held on virtual ground. Cs⁺ was used as a charge carrier to increase the conductance of the channel (Coronado et al., 1992). The cis- and trans-baths contained 480 mM and 50 mM CsCl, respectively. The buffer composition was 10 mM HEPES-Tris, pH 7.4, 100 µM CaCl₂, 80 µM EGTA (i.e., free Ca²⁺  20 µM); at low free Ca²⁺ the cis solution contained 0.5 mM EGTA and 0.42 mM CaCl₂ (resulting in 0.6 µM calculated free Ca²⁺). Purified Ca²⁺-release channel/ryanodine receptor (1.5-2 µg) and other reagents were added to the cis-chamber. Recordings were filtered at 4 kHz through a low-pass Bessel filter, digitized at 40 kHz, and subsequently stored on a Macintosh PC. Single-channel events were analyzed with TAC V2.5 software (Skalar Instruments, Inc., Seattle, WA). Mean open probability (P_o) of channels and the corresponding lifetimes () of the open and closed events were identified by a 50% threshold analysis and calculated from data segments of 30- to 90-s duration. NF307 was dissolved at 50 mM in DMSO, carryover of which did not exceed a final concentration of 0.2% in the cis-buffer. The holding potentials given in the figure legends were applied with reference to the trans-chamber. All experiments were carried out at 22 to 24°C.

Affinity Chromatography and Gel Electrophoresis. The purified ryanodine receptor (2-3 µg) was diluted in 100 µl binding buffer of the following composition: 20 mM HEPES-NaOH (pH 7.4), 200 mM KCl, 10 mM NaCl, 1 mM EGTA, 1.2 mM CaCl₂, 0.68% CHAPS, and 0.5% phosphatidylcholine. Alternatively, the free Ca²⁺ concentration in the incubation was reduced by adding only 0.85 mM CaCl₂ to the buffer of otherwise identical composition. Preequilibrated calmodulin-Sepharose (40 µl of a 50% slurry) was added. After an incubation period of 60 min at 4°C, the suspension was centrifuged for 5 min at 500g; the supernatant was removed, and the sedimented calmodulin-Sepharose was resuspended in 90 µl binding buffer and recentrifuged. This wash step was repeated twice. Subsequently, the ryanodine receptor was eluted batchwise in four steps with 90 µl binding buffer supplemented with 20 µM calmodulin, 300 µM suramin, or 100 µM NF307. As a control, mock elutions were done in parallel with 90 µl binding buffer. The supernatants of the washes and elutions were mixed with Laemmli sample buffer (supplemented with mercaptoethanol and SDS to yield final concentrations of 0.5% and 2.5%, respectively), and the samples were heated to 95°C for 5 min. Similarly, after the last elution, the calmodulin-Sepharose matrix was boiled in Laemmli sample buffer and centrifuged. Aliquots (corresponding to 30% of the of the individual samples) were applied onto discontinuous SDS-polyacrylamide gels (3% stacking and 7% separating gel). Molecular mass standards were myosin (212 kDa), ₂-macroglobulin (170 kDa), -galactosidase (116 kDa), transferrin (76 kDa), and glutamic dehydrogenase (53 kDa). The resolved proteins were visualized by silver staining. For immunoblotting, nitrocellulose blots were incubated with a mouse monoclonal antibody raised against the skeletal muscle isoform of the ryanodine receptor, and the immunoreactive bands were visualized by enhanced chemiluminescence detection.

Miscellaneous Procedures. Protein concentration was measured by staining with amido black or with the bicinchonic acid assay (Micro-BCA, Pierce, Rockford, IL) using BSA as the standard. Free Ca²⁺ concentrations were calculated by a computer program using binding constants published by Schoenmakers et al. (1992); we note that these constants and the algorithm yield estimates differ to some extent, in particular at low Ca²⁺ concentrations, from those obtained with the program of Fabiato (1988) that we have used previously. Data were fitted by nonlinear least-squares regression to the appropriate equations describing mono-, bi-, or triexponential decay and association as well as saturation isotherms using the Gauss-Newton or Marquardt-Levenberg algorithm.

	Results

Top Summary Introduction Materials and Methods Results Discussion References

[³H]Ryanodine Binding in the Presence of Suramin Analogs. It is generally appreciated that all compounds and manipulations that promote channel opening accelerate the rate of high-affinity [³H]ryanodine binding; this close correlation indicates that ryanodine interacts with the channel in the open conformation. We therefore have used the initial rate of [³H]ryanodine binding to search for ryanodine receptor activators that are structurally related to suramin. In these screening experiments, the free Ca²⁺ concentration was set below the threshold level required for Ca²⁺-dependent high-affinity binding, because this biases the search for compounds that are also potentially capable of sensitizing the ryanodine receptor for Ca²⁺. Suramin is a rigid, symmetric molecule in which two polysulfonated naphtylbenzamide ring systems are connected by a central urea bridge. This is replaced by a piperazine ring in one class of suramin analogs (of several examined), in which we have found a potent activator, NF307. The structures of suramin, NF307, and the two related molecules are shown in Fig. 1. NF307 was found to efficiently promote [³H]ryanodine binding even at the subthreshold Ca²⁺ concentration of 0.45 µM with a calculated EC₅₀ of 69 ± 14 µM (Fig. 2A). Under these assay conditions, NF301, suramin, and NF299 (Fig. 2A) were equipotent and promoted [³H]ryanodine binding with comparable efficacy. However, they were clearly much less active than NF307. It is evident from Fig. 1 that the variations in the structure of the compounds are modest. This is true, in particular, for NF301 and NF307, which differ by the position of a single pair of sulfonic acid residues (meta versus para). We have also tested NF449 and NF503, two analogs that were found to be substantially more selective than suramin in inhibiting individual G protein -subunits (Freissmuth et al., 1996; Hohenegger et al., 1998). Both, NF449 and NF503 (Fig. 2A) as well as related compounds (not shown) were inactive up to 1 mM.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Chemical structures of suramin, NF299, NF301, and NF307.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Stimulation of [ 3H]ryanodine binding to sarcoplasmic reticulum membranes by suramin and suramin analogs. A, sarcoplasmic reticulum membranes (50 µg) were incubated with 20 nM [3H]ryanodine for 45 min at 30°C in absence () or presence of indicated concentrations of NF307 (), suramin (), NF299 (), NF301 (), NF449 (), and NF503 () at a ratio of 0.85 mM Ca2+/1 mM EGTA, yielding a calculated free Ca2+ concentration of 0.45 µM in presence of 0.75 M KCl. Data are means ± S.D. of three independent experiments carried out in duplicate. B, concentration-response curve for NF307 was determined in parallel in presence of 0.75 M () or of 0.2 M KCl () and at a ratio of 0.85 mM Ca2+/1 mM EGTA as described for A; however, lowering ionic strength increases calculated free concentration to 0.61 µM. Data points are means of duplicate determinations in a representative experiment that was repeated twice.

Saturation Isotherms and Kinetic Analysis in the Presence of NF307. The barely detectable levels of basal binding that are observed at submicromolar concentrations of free Ca²⁺ are due to low-affinity binding (K_D = 185 ± 29 nM; Fig. 3A). Addition of 200 µM NF307 promoted the appearance of high-affinity binding sites for [³H]ryanodine (K_D = 25 ± 9, Fig. 3A); this was seen at low (Fig. 3A) as well as at high ionic strength (not shown). We attribute the pronounced increase in binding to an acceleration of the association rate based on the following observations. In the presence of 0.2 M KCl and of 0.6 µM Ca²⁺, the basal apparent association rate k_app was very low (Fig. 3B); because of the essentially linear relation between time and [³H]ryanodine binding, it was not possible to reliably calculate the rate, but we estimated k_app to be below 0.001 min¹. In contrast, upon addition of 200 µM NF307, the binding reaction was described adequately assuming a pseudo-first-order process yielding apparent rate constants of 0.012 ± 0.005 min¹ (Fig. 3B). A similar acceleration of [³H]ryanodine binding was seen in the presence of 10 mM caffeine, which as used as a control (k_app = 0.012 ± 0.002 min¹, Fig. 3B). Because basal binding at 0.6 µM free Ca²⁺ was too low to assess dissociation kinetics, we have also determined the dissociation rate of [³H]ryanodine binding that was supported by 10 µM free Ca²⁺; under these conditions, addition of either caffeine or NF307 to the medium use for 100-fold dilution of the assay volume had only modest effects on the dissociation rate (k_off = 4.3 ± 0.9, 4.3 ± 0.8, and 6.6 ± 1.2 × 10³ min¹, in the absence and presence of 10 mM caffeine and of 100 µM NF307, respectively).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Saturation isotherms for (A) and association of [3H]ryanodine binding (B) to sarcoplasmic reticulum membranes. A, sarcoplasmic reticulum membranes (50 µg) were incubated in 0.2 M KCl buffer containing 0.69 µM free Ca2+ and the indicated concentrations of [3H]ryanodine in absence () and presence of 200 µM NF307 (). Incubation time was 180 min. Lines were drawn by fitting data to an equation describing interaction of radioligand with a single class of binding sites; a two-site model was also tested but did not significantly imporve fit (F-test based on extra sum of squares principle). B, assay conditions were as in A; concentration of [3H]ryanodine was 20 nM. Incubation was done in absence () and presence of 200 µM NF307 () or 10 mM caffeine (). At time points indicated, reaction was terminated by rapid filtration. Data points represent mean of duplicate in representative experiments that were repeated twice.

Single-Channel Recordings of the Purified Ryanodine Receptor in the Presence of NF307. To prove that NF307 directly affected the channel properties of the ryanodine receptor, the purified protein was incorporated into artificial planar lipid bilayers and investigated by single-channel recordings. An example of the effect of NF307 on the purified Ca²⁺-release channel at a low free calcium concentration is illustrated in Fig. 4. Channel P_o of the control at 20 µM Ca²⁺ was 0.46 (Fig. 4A). When the free calcium concentration on the cis side of the bilayer was reduced to 0.6 µM by the addition of EGTA, the Ca²⁺-release channel was predominantly closed (P_o = 0.01; Fig. 4B). The addition of 100 µM NF307 to the solution on the cis side (the cytoplasmic side) increased the open probability (P_o = 0.62, Fig. 4C) as well as the open time constants (Fig. 4, D and E). The NF307 modified Ca²⁺-release channel was blocked by ruthenium red (not shown). Table 1 summarizes the mean values of P_o, current amplitudes, and the distribution of the open and closed lifetimes of controls and of the same channels modified by 100 µM NF307 in five experiments. The increase in the open probability in the presence of low free calcium of 0.6 µM from 0.02 ± 0.006 (control) to 0.53 ± 0.07 (100 µM NF307; n = 5; means ± S.E.M.) mainly is due to an increase in the frequency of channel openings. The open probability in controls of the same channels in the presence of 20 µM Ca²⁺ was 0.48 ± 0.08 (n = 5; means ± S.E.M.), i.e., the NF307-induced increase in P_o was close to values obtained with 20 µM activating Ca²⁺ in four of five experiments. This corresponds to the range of P_o that has been observed in more than 30 purified Ca²⁺-release channel preparations with maximally activating Ca²⁺ concentrations (20-100 µM free Ca²⁺). Moreover, NF307 also increased the open lifetimes at 0.6 µM Ca²⁺, i.e., in three of five experiments a better fit of the open times was obtained by a three-exponential fit (Fig. 4E). In the other two experiments, the parameters for the third exponential component could not be estimated reliably; hence, Table 1 lists only the estimates derived from the equation describing the sum of two exponential processes.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effect of NF307 on a single purified skeletal muscle calcium-release channel at low free calcium (0.6 µM). Single-channel currents, shown as upward deflections, were recorded at 0-mV holding potential with 480 mM/50 mM CsCl (cis/trans). Baselines are indicated by solid lines. Control and test records (A-C) are from same channel. A, control (20 µM Ca2+), Po = 0.46. B, control (0.6 µM Ca2+), Po = 0.01. C, activation of channel by 100 µM NF307 added to the cis side (0.6 µM Ca2+), Po = 0.62. Calibration bars represent 20 pA and 5 ms or 50 ms. Lifetime histograms of open times and channel-opening time constants (o) of control and NF307-modified channel are displayed in D and E, respectively. Channel open probabilities (Po) and o were calculated from 22,825 events (control, 20 µM Ca), 2,530 events (control, 0.6 µM Ca2+), and 29,160 events (100 µM NF307).

View this table: [in this window] [in a new window]   TABLE 1 Means of Po, current amplitudes, and mean open and closed channel lifetimes of controls and stimulation by 100 µM NF307 at 0.6 µM Ca2+ Channel open probabilities (Po), cumulative mean open and closed channel time constants (o, c), and values of channel represented by a time constant (as percentage) for the purified Ca2+-release channel/ryanodine receptor activated by 0.6 µM free cis Ca2+ in absence or presence of 100 µM NF307. Values given are means ± S.E.M. from five channels included in analysis and were calculated from data segments of 30- to 90-s duration. Calcium release channels were recorded at 0-mV holding potential with 480 mM/50 mM CsCl (cis/trans).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effect of 100 µM NF307 on a single purified skeletal muscle calcium-release channel at 20 µM activating Ca2+. Single-channel currents, shown as upward deflections, were recorded at +20 mV holding potential with 480 mM/50 mM CsCl (cis/trans). Baselines are indicated by solid lines. Control and test records are from same channel. A, control: 20 µM Ca2+, Po = 0.21. B, activation of channel by 100 µM NF307 added to cis side (20 µM Ca2+), Po = 0.84. Slope conductances were 529 pS (control) and 518 pS (NF307). Lifetime histograms of open times and channel-opening time constants (o) of control and NF307-modified channel are displayed in C and D, respectively. Solid lines represent fit according to two or three exponentials. Channel open probabilities (Po) and o were calculated from 18,100 events (control) and 27,950 events (100 µM NF307), respectively. Calibration bars represent 50 pA and 5 ms.

View this table: [in this window] [in a new window]   TABLE 2 Mean open and closed channel lifetimes in presence of 20 µM free Ca2+ and after stimulation by 100 µM NF304 at 20 µM Channel open probabilities (Po), cumulative mean open and closed channel time constants (o, c), and values of percentage of the channel represented by a time constant for purified Ca2+-release channel/ryanodine receptor activated by 20 µM cis Ca2+ in absence or presence of 100 µM NF307. Values given are means ± S.E.M. from six channels included in analysis and were calculated from data segments of 30- to 90-s duration. Ca2+-release channels were recorded at 0-mV holding potential with 480 mM/50 mM CsCl (cis/trans).

Ca²⁺ Dependence of the NF307 Effect. At concentrations exceeding the threshold concentration, i.e., in the low micromolar range, Ca²⁺ promotes high-affinity [³H]ryanodine binding and the concentration-response relation is very steep; in contrast, above 100 µM free Ca²⁺, [³H]ryanodine binding to RyR₁ is inhibited, resulting in a bell-shaped curve (Franzini-Armstrong and Protasi 1997; see also Fig. 6). This inhibition was also seen in the presence of 10 mM caffeine and 100 µM NF307 (Fig. 6). In accordance with our previous observations (Hohenegger et al., 1996), the suramin-induced stimulation of the ryanodine receptor was only modestly inhibited by high Ca²⁺ concentrations (Fig. 6A). More importantly, NF307 and caffeine shifted the stimulatory limb of the Ca²⁺ curve to the left irrespective of whether the experiment was carried out in a 0.75 M (Fig. 6A) or in 0.2 M KCl buffer (Fig. 6B). However, basal binding, i.e., binding supported by the sole addition of micromolar concentrations of Ca²⁺, was enhanced at high ionic strength (EC₅₀ for Ca²⁺ = 1.20 ± 0.20 and 0.72 ± 0.08 µM at 0.75 and at 0.2 M KCl, respectively); hence, the shift induced by NF307 appeared more pronounced at 0.2 M KCl (cf. Fig. 6, A and B). Nevertheless, in the presence of NF307 the EC₅₀ values for Ca²⁺ were comparable at low (0.12 ± 0.01 µM) and high ionic strength (0.11 ± 0.02 µM).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Ca2+-dependent [3H]ryanodine binding in presence of NF307 and caffeine. A and B, [3H]ryanodine binding was determined in 0.75 M KCl buffer (A) or 0.2 M KCl buffer (B) in absence (O) and presence of 100 µM NF307 (), 10 mM caffeine (), or 300 µM suramin ( in A). Incubation conditions were as outlined in legend to Fig. 2. Data represent mean of three experiments carried out in duplicate; error bars indicate S.D. C, increasing concentrations of NF307 (,) or suramin (,) were added to sarcoplasmic reticulum membranes incubated in presence of 0.75 M KCl buffer; free Ca2+ concentration was adjusted to 0.19 µM (,) or to 0.82 µM (,). Data points are means of duplicate determinations in a representative experiment that was repeated twice.

NF307 and Suramin Interact with a Calmodulin-Binding Site. Caffeine has long been known to sensitize the ryanodine receptor for Ca²⁺ (Sitsapesan and Williams, 1990; see also Fig. 6, A and B); because NF307 exerted a similar effect, NF307 and caffeine may share a common binding site on the protein. This conjecture appeared unlikely, because the structures of NF307 and caffeine have little in common. An alternative candidate site of action may be the site(s) by which Ca²⁺-free calmodulin stimulates the ryanodine receptor (Buratti et al., 1995; Tripathy et al., 1995). Calmodulin is acidic, and the polyanionic nature of NF307 is evident from Fig. 1. To test these two hypotheses, binding experiments were performed in a nominally Ca²⁺-free medium (2 mM EGTA, no added Ca²⁺). As expected, the apparent affinity of NF307 is reduced under these conditions (EC₅₀ ~ 400 µM; Fig. 7A). However, if 10 mM caffeine was combined with increasing amounts of NF307, the stimulation induced by the combination was strictly additive over the entire range of the NF307 concentration-response curve (Fig. 7A); this finding is incompatible with an action of the two compounds via a common site. In contrast, addition of 2 µM calmodulin blunted the effect of NF307 (Fig. 7A). Because of the large effect of NF307, the y-axis in Fig. 7A covers a range that is too wide to illustrate the smaller stimulatory effect that calmodulin exerted per se. The latter is more readily appreciated in Fig. 7B. More importantly, Fig. 7B also shows that the combination of caffeine and calmodulin resulted in overadditive stimulation. The experiments summarized in Fig. 7, A and B were done at high ionic strength. Analogous results were obtained if the incubation medium contained 0.2 M KCl (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Stimulation and inhibition of [3H]ryanodine by the combined addition of NF307, calmodulin, and caffeine. A, sarcoplasmic reticulum membranes (50-70 µg) were incubated in nominally Ca2+-free buffer (2 mM EGTA in absence of added CaCl2) containing 0.75 M KCl, 20 nM [3H]ryanodine, and increasing concentrations of NF307 in the absence () and presence of 10 mM caffeine () or of 2 µM calmodulin (). B, [3H]ryanodine binding was determined under same ionic conditions as in A in absence (control) and presence of 2 µM calmodulin, 10 mM caffeine, or combination of calmodulin and caffeine. C, assay conditions were similar to those in A, but free Ca2+ concentration was raised to 100 µM. Sarcoplasmic reticulum membranes were incubated with increasing concentrations of calmodulin in absence () and presence of 10 µM (), 30 µM (), and 100 µM () NF307 or 300 µM suramin (). To avoid a reduction in free Ca2+ concentration, calmodulin stock solution was prepared in presence of a 4-fold molar excess of CaCl2 and serial dilutions made therefrom. Binding observed in absence of calmodulin was set to 100% to normalize for interassay variation and stimulatory effect of NF307 and suramin (~1.3-fold at highest concentration of NF307; see also Fig. 6A). Data are means of three to five independent experiments carried out in duplicate; error bars represent S.D.

View larger version (36K): [in this window] [in a new window]   Fig. 8.   Elution of the purified ryanodine receptor from a calmodulin-Sepharose matrix by calmodulin NF307 and suramin. Purified ryanodine receptor (1-2 µg/incubation) was incubated with appropriately equilibrated calmodulin-Sepharose in presence of 200 µM (A) or 0.6 µM free Ca2+ (B) as described under Materials and Methods. Affinity matrix was washed four times with incubation buffer. Subsequently, ryanodine receptor was eluted batchwise with buffer containing 20 µM calmodulin, 300 µM suramin, or 200 µM NF307 as indicated. Mock elution was done with buffer alone (lanes labeled "control"); residual protein trapped in matrix was released by boiling in Laemmli sample buffer. Aliquots corresponding to one-third of input (lanes labeled "i"), of last wash step (lanes labeled "w"), of three successive elution steps (lanes labeled "e1-e3"), and of material released by denaturation of calmodulin-Sepharose matrix (lanes labeled "m") were applied onto SDS-polyacrylamide gels. Proteins were visualized by silver staining. Position of molecular mass standard is indicated (lanes labeled "std"). Two additional experiments have similar results. C, after electrophoresis, purified ryanodine receptor preparation used in A and B was visualized by staining with Coomassie blue (lane Cb) or transferred to nitrocellulose and detected by immunoblotting (lane Wb).

	Discussion

Top Summary Introduction Materials and Methods Results Discussion References

The Ca²⁺-binding protein calmodulin regulates the activity of the ryanodine receptor by at least three mechanisms, i.e., direct activation, direct inhibition, and indirect modulation. The latter is achieved via phosphorylation of the channel protein by Ca²⁺/calmodulin-dependent kinase II (Witcher et al., 1991; Hohenegger and Suko, 1993; Suko et al., 1993). Calmodulin binds directly to the ryanodine receptor; in the absence of Ca²⁺, this interaction results in increased channel opening (Buratti et al., 1995; Tripathy et al., 1995); in contrast, Ca²⁺-liganded calmodulin suppresses the activity of the channel (Meissner, 1986; Plank et al., 1988; Smith et al., 1989). In the present work, we show that the suramin analog NF307 is a potent and direct activator of the ryanodine receptor and that its effect results from an interaction with a calmodulin-binding domain. This conclusion is supported by the mutually antagonistic effects that NF307 and calmodulin exert on [³H]ryanodine binding and by the ability of NF307 to elute the channel protein from a calmodulin-based affinity matrix. Multiple potential calmodulin-binding sites have been predicted based on the deduced primary sequence of the skeletal muscle ryanodine receptor RyR₁; however, the precise number is still a matter of debate (Franzini-Armstrong and Protasi, 1997) and evidence for up to 24 sites/tetrameric receptor has been reported (Yang et al., 1994). At least three binding sites have been identified by overlaying heterologously expressed portions of RyR₁, which had been fused to carrier proteins, with radiolabeled calmodulin (Chen and McLennan, 1994; Menegazzi et al., 1994; Buratti et al., 1995); the relation of these sites to the regions to which calmodulin may bind to the intact protein has been questioned (Tripathy et al., 1995). After reconstitution of the purified ryanodine receptor into phospholipid vesicles, calmodulin was observed to bind with a stoichiometry of three to four molecules and one molecule per monomer in the absence and presence of Ca²⁺, respectively (Tripathy et al., 1995). Kinetic arguments suggest that the site that is occupied by calmodulin in the absence of Ca²⁺ also binds calmodulin in the presence of Ca²⁺ (Tripathy et al., 1995). It presumably is this high-affinity binding site for Ca²⁺/calmodulin that has been visualized by electron cryomicroscopy (Wagenknecht et al., 1997); however, at present it is difficult to gain mechanistic insights from these three-dimensional reconstructions, because the four molecules of Ca²⁺/calmodulin bound per tetramer are located at a 10-nm distance from the putative ion pore (Wagenknecht et al., 1997).

The concentration-response curves for NF307 are steep, the Hill-coefficient being consistently >1 irrespective of the free Ca²⁺ concentration. Given the tetrameric nature of the channel, the steep slope may reflect either cooperativity between several binding sites on one monomer or the cooperative activation of the tetramer via a single binding site on each monomer, and it currently is not possible to differentiate between the two possibilities. Although it is conceivable that binding of NF307 allosterically inhibits the interaction of calmodulin with the ryanodine receptor, the available evidence is consistent with a simpler model of mutual competition for a common site on the ryanodine receptor; this interpretation is supported by the observation that the concentration-response curve of calmodulin is progressively shifted to the right by increasing the concentration of NF307. In contrast to calmodulin, NF307 elicits only stimulatory effects on the ryanodine receptor. The basis for this discrepancy is unknown. Calmodulin is a bipartite, pseudosymmetric molecule; the globular Ca²⁺-binding domains at the amino and at the carboxy terminus are connected by an -helix. Genetic evidence suggests that the two lobes subserve nonequivalent functions in ion-channel regulation. Mutations in the calmodulin gene of Paramecium that alter the carboxy terminus disrupt regulation of one class of ion channels, whereas those affecting the amino terminus abrogate modulation of the other class (for review, see Saimi and Kung, 1994). Given that one binding site on the ryanodine receptor monomer is occupied by calmodulin both in the absence and presence of Ca²⁺ (Tripathy et al., 1995), it is attractive to speculate that the dual actions of calmodulin on channel gating is mediated by different parts of the molecule and that NF307 only mimics the portion of calmodulin that activates the ryanodine receptor.

Apart from its trypanocidal and anthelminthic effects, suramin has multiple pharmacological actions in mammalian organisms (Voogd et al., 1993). However, our experiments show that structural determinants, which go beyond merely providing a rigid backbone for multiple negative charges, are important for the potency and efficacy of suramin analogs at the ryanodine receptor. This is exemplified by the finding that closely related analogs are less active or entirely inactive. Furthermore, suramin analogs that we have reported recently to be more selective G protein antagonists than suramin (Freissmuth et al., 1996; Hohenegger et al., 1998) are inactive when tested on the ryanodine receptor. Finally, apart from the distinct potencies, NF307 and suramin differ in their mode of action at the ryanodine receptor; suramin, but not NF307, blunts the inhibition of the ryanodine receptor that is elicited by millimolar concentrations of Ca²⁺. In contrast, NF307 is considerably a more efficient Ca²⁺-sensitizer than suramin, and the magnitude of its effect is comparable to that of caffeine.

The ryanodine receptor is notorious for its role in side effects occurring in clinical pharmacotherapy (for review, see Zucchi and Ronca-Testoni, 1997). In malignant hyperthermia, halogenated volatile anesthetics (e.g., halothane) alone or in combination with depolarizing neuromuscular-blocking agents (e.g., succinylcholine) trigger uncontrolled Ca²⁺ release from the sarcoplasmic reticulum, muscle contracture, and a hypermetabolic state that precipitates a life-threatening elevation in body temperature. In many cases (presumably 50%), the underlying disturbance is associated with point mutations in the ryanodine receptor (McLennan and Philipps, 1992). The mechanism by which these point mutations cause susceptibility to malignant hyperthermia is not known; however, an analogous disease exists in domestic pigs (referred to as porcine stress syndrome). The ryanodine receptor of animals homozygous for the mutation is more susceptible to the stimulatory effect of calmodulin (O'Driscoll et al., 1996). Similarly, oxygen radicals activate the cardiac ryanodine receptor by relieving the inhibition imposed by calmodulin (Kawakami and Okabe, 1998); this may account for Ca²⁺ overload in cardiac myocytes as a result of oxidative stress that occurs during pathophysiological conditions such as ischemia and reperfusion. Hence, compounds that act at the calmodulin-binding site(s) of the ryanodine receptor are clearly of interest for understanding the mechanism of ryanodine receptor regulation by calmodulin. Ultimately, these ligands also may be useful therapeutically.