Introduction

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Calcium release in skeletal muscle occurs via the calcium release channel (ryanodine receptor) located in the terminal cisternae of the sarcoplasmic reticulum (Imagawa et al., 1987; Inui et al., 1987; Lai et al., 1988; Smith et al., 1988). The skeletal muscle calcium release channel (RyR-1) is a homotetramer with a molecular mass of 2260 kDa (Takeshima et al., 1989; Zorzato et al., 1990). Calcium release and the gating properties of the calcium release channel at a single channel level are regulated by endogenous effectors including calcium, magnesium, adenine nucleotides, the calcium binding proteins calmodulin and sorcin, the immunophillin FK506-binding protein, phosphorylation by protein kinases, sulfhydryl oxidation by nitric oxide, and various exogenous effectors (see reviews: Coronado et al., 1994; Meissner, 1994; Melzer et al., 1995; Zucchi and Ronca-Testoni, 1997).

The trypanocidal drug suramin, a polysulfonated napthylurea, is a potent activator of the ligand-gated calcium release channel of sarcoplasmic reticulum. Suramin released calcium from passively loaded skeletal muscle sarcoplasmic reticulum vesicles (Emmick et al., 1994) as well as from cells containing the RyR-3 isoform, such as Jurkat T-lymphocytes (Hohenegger et al., 1999). The gating of the calcium release channel in skeletal (RyR-1) and cardiac (RyR-2) muscle is markedly influenced by suramin or suramin analogs. Characteristic for the activating effect of suramin in single-channel current recordings was an increase in longer open states, especially with the cardiac calcium release channel and the requirement of lower suramin concentrations to increase the open probability of the cardiac calcium release channel (Sitsapesan and Williams, 1996; Sitsapesan, 1999) compared with that from the skeletal muscle channel (Hohenegger et al., 1996; Sitsapesan and Williams, 1996; Klinger et al., 1999). Furthermore, [³H]ryanodine binding to the calcium release channel of heavy sarcoplasmic reticulum (HSR) of skeletal muscle was activated by suramin, but only marginally inhibited by very large concentrations of calcium or magnesium (Emmick et al., 1994; Hohenegger et al., 1996; Klinger et al., 1999). Suramin released the RyR-1 bound to a calmodulin Sepharose (Klinger et al., 1999) and inhibited [¹²⁵I]calmodulin binding to HSR (Suko et al., 2000).

Because of the effects of suramin on the skeletal muscle calcium release channel found in ligand binding studies to HSR (reduction in calmodulin binding, marginal inhibition of [³H]ryanodine binding by high concentrations of magnesium or calcium), the present investigation was carried out to determine the effect of the inhibitory endogenous ligands (calcium-calmodulin, magnesium, and millimolar calcium) on the suramin-induced activation at a single channel level. Ryanodine is known as a very valuable probe to monitor functional states of the calcium release channel and a good correlation between open probability in single-channel current recordings and the amount of [³H]ryanodine bound to the channel proteins was usually observed (Coronado et al., 1994; Meissner, 1994). Recent studies showed that a single point mutation of the mouse RyR-2 (alanine 4812 to glycine), the suggested pore-forming region of the calcium release channel (Zhao et al., 1999), or mutation of amino acids in the luminal loop linking the M3/M4 region of the RyR-1 (Gao et al., 1999) reduced single-channel current fluctuation and [³H]ryanodine binding (Gao et al., 1999; Zhao et al., 1999). For the above reason, [³H]ryanodine binding to HSR was also carried out in the absence or presence of suramin under similar conditions as used in single-channel current recordings.

The results demonstrate both short and long-term functional changes in single-channel current recordings, alterations in the gating of the calcium release channel by calcium and magnesium, and an apparent dissociation between single-channel current recordings and [³H]ryanodine binding in the presence of suramin.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. Suramin, calmodulin, 4-(chloromercuri)phenyl-sulfonic acid (4-CMPS), MOPS, HEPES, Tris, histidine, CsCl (ultra pure), NaCl (ultra pure), ruthenium red, leupeptin, pepstatin, antipain, phenylmethylsulfonyl fluoride, tetracaine, and neomycin were purchased from Sigma-Aldrich GmbH (Vienna, Austria); [³H]ryanodine was purchased from DuPont-New England Nuclear (Boston, MA); ryanodine was from Agrisystems International (Wind Gap, PA); phosphatidyl serine, phosphatidylethanolamine, and phosphatidylcholine were from Avanti Polar Lipids, Inc. (Alabaster, AL); Delrin bilayer chambers (CD22-200; CD13-200) were from Warner Instrument Corp. (Hamden, NJ). Aprotinin was a generous gift from Bayer Austria AG (Vienna, Austria). All reagents and agents (suramin, calmodulin, 4-CMPS, [³H]ryanodine) were dissolved in MilliQ deionized water.

Preparation of Sarcoplasmic Reticulum. Heavy sarcoplasmic reticulum vesicles (HSR) from rabbit skeletal muscle were prepared as described previously (Suko and Hellmann, 1998). Briefly, white back muscle (fast twitch muscle) was homogenized in a Waring Blender for 1.5 min in a medium containing 10 mM histidine buffer, pH 7.0, and 100 mM NaCl, and centrifuged for 35 min at 4,000g. The supernatant was filtered through cheese cloth and centrifuged for 30 min at 30,000g. The pellet was resuspended in 10 mM histidine buffer, pH 7.0, 0.6 M KCl, and 250 mM sucrose and centrifuged for 35 min at 100,000g. The pellet was washed once in a medium containing 10 mM histidine buffer, pH 7.0, 100 mM NaCl, and 200 mM sucrose, centrifuged again for 35 min at 100,000g and stored at 80°C or used immediately for the purification of the ryanodine receptor-calcium release channel. All buffers used for the preparation and resuspension of HSR contained 0.5 µg/ml leupeptin, 1 µg/ml antipain, 1.4 µg/ml aprotinin, 1 µM pepstatin, 0.1 mM PMSF, 1 mM benzamidine.

Preparation of Calcium Release Channel (Ryanodine Receptor). The calcium-release channel of the terminal cisternae of sarcoplasmic reticulum vesicles was prepared as described previously (Suko and Hellmann, 1998), which was a slight modification of the preparation used before that (Suko et al., 1993). Briefly, heavy sarcoplasmic reticulum vesicles from rabbit skeletal muscle (prepared as above) were solubilized with CHAPS (medium, 40 mM Mops/Tris, pH 7.0, 1 M NaCl, 2 mM DTT, 1% CHAPS, 0.25% or 0.5% phosphatidylcholine, 0.5 µg/ml leupeptin, 1 µg/ml antipain, 1.4 µg/ml aprotinin, 1 µM pepstatin, 0.1 mM PMSF, 1 mM benzamidine, 15 mg/ml HSR; incubation, 60 min at 3-4°C), followed by centrifugation twice for 35 min at 103,000g (Beckman 65 rotor). The supernatant was centrifuged through a linear 7.5 to 20% sucrose gradient equilibrated in 40 mM Mops/Tris, pH 7.0, 300 mM NaCl, 2 mM DTT, 0.5% CHAPS, 0.25% or 0.5% phosphatidyl-choline, 0.5 µg/ml leupeptin, 1 µg/ml antipain, 1.4 µg/ml aprotinin, 1 µM pepstatin, 0.1 mM PMSF, and 1 mM benzamidine for 14 h at 2°C (Beckman SW28 rotor; 38 ml tubes). Fractions containing the ryanodine receptor (determined by SDS-PAGE) were pooled and dialysed for 24 h in a medium containing 20 mM Mops/Tris, pH 7.0, 100 mM NaCl, 2 mM DTT, 0.15 mM CaCl₂, 0.1 mM EGTA, 0.5 µg/ml leupeptin, 1 µg/ml antipain, 1.4 µg/ml aprotinin, 1 µM pepstatin, 0.1 mM PMSF, and 1 mM benzamidine. Sucrose (200 mM final concentration) was added to the proteoliposomes before storage at 78°C. Preparation and dialysis were carried out at 2 to 4°C.

SDS-Polyacrylamide Gel Electrophoresis (PAGE). SDS-PAGE was performed in 5% polyacrylamide gels (0.75 mm thickness) with 3% stacking gels as described previously (Suko et al., 1993). Sucrose gradient fractions were added to a medium containing 10 mM Tris/HCl, pH 6.8, 2% SDS, 2% mercaptoethanol, and 10% glycerol and boiled for 2 min. Gels were stained with 0.05% Coomassie blue in 10% acetic acid. Molecular mass standards were run on two separate lanes of the same gel: Ferritin (440 kDa), thyroglobulin (330 kDa), and myosin (212 kDa). Gradient fractions with the highest content of ryanodine receptor were pooled and used for the preparation of proteoliposomes.

Single Channel Recordings. Single-channel recordings were carried out after incorporation of purified calcium release channels (ryanodine receptors) into planar lipid bilayers, essentially as described previously (Suko and Hellmann, 1998). Planar lipid bilayers were formed from phosphatidylserine (10 mg/ml) and phosphatidylethanolamine (10 mg/ml) in decane (Avanti Polar Lipids). The lipid solution was spread over a 200-µm diameter aperture in a Delrin cup (Warner Instrument Corp.) separating two aqueous compartments. The cis bath solution (2.6 ml) and the trans bath solution (4 ml) were connected to the head stage input of a model EPC-9 amplifier (Heka Elektronik, Lambrecht, Germany) via Ag/AgCl electrodes and CsCl-agar bridges. The trans bath was held at virtual ground. Cs⁺ was used as the charge carrier through the calcium release channel to increase the conductance of the channel (Coronado et al., 1992). The cis solution was composed of 10 mM HEPES/Tris, pH 7.4, 480 mM CsCl, and 50 to 100 µM CaCl₂ or 100 µM CaCl₂ plus 80 µM EGTA (free calcium, 20 µM). The trans solution was composed of 10 mM HEPES/Tris, pH 7.4, and 50 mM CsCl without added calcium or plus calcium in concentrations as used in the cis bath. Unless stated otherwise, purified calcium release channels and other reagents were added to the cis chamber. Recordings were filtered at 4 kHz with a low-pass Bessel filter, digitized at 40 kHz (sampling rate 25 µs) and stored on the hard disc of a Apple Macintosh (Apple, Cupertino, CA). Single channel events were identified using TAC software (ver 2.5; Skalar Instruments, Inc., Seattle, WA). Mean open probability (P_o) of channels were identified by a 50% threshold analysis. The life times of open and closed events were determined by the method of maximum likelihood (TACFit software; Skalar Instruments).

[³H]Ryanodine Binding. [³H]Ryanodine binding was measured under equilibrium conditions as described previously (Suko and Hellmann, 1998). Unless stated otherwise, controls and test samples were assayed in duplicate or triplicate for 90 min at 37°C in 0.2 ml of solution containing 40 mM Mops/Tris, pH 7.0, 0.5 M CsCl (or 1 M NaCl), 0.1 mg HSR, 0.5 µg/ml leupeptin, 1.4 µg/ml aprotinin, 0.1 mM PMSF, and 10 nM [³H]ryanodine, 100 µM Ca²⁺ without or with suramin (0.1 to 10 mM). In a few experiments, the above control medium contained, in addition, 5 to 10 mM EGTA, 10 to 20 µM ruthenium red, 1 mM tetracaine, or 1 mM neomycin. In the calcium dependence experiments, free calcium was varied between 20 nM and 50 mM. In the magnesium inhibition experiments, magnesium was varied between 0.25 and 50 mM MgCl₂. Nonspecific [³H]ryanodine binding was measured in the presence of 100 µM unlabeled ryanodine. Samples were filtered on glass-fiber filters (presoaked in 1% polyethylene imine) and washed with 10 ml of 20 mM Mops/Tris, pH 7.0, 1 M NaCl.

Protein Assay. Protein was measured by the Folin method and in the presence of detergents plus phosphatidylcholine, according to Kaplan and Pedersen (1985), standardized against bovine serum albumin.

Calculations. Curve fitting was carried out using the standard Maquart-Levenberg algorithm provided by Sigma plot 2 (Jandel, San Rafael, CA). Statistical analysis was carried out by t test using Sigmastat 2 software (Jandel, San Rafael, CA) and for multiple comparisons by ANOVA and Scheffé post hoc comparisons. Averaged results are presented as means ± S.E.M.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Single-Channel Current Recordings. In single-channel experiments, current fluctuations of a single purified and reconstituted skeletal muscle calcium release channel were recorded at 20 to 100 µM free calcium in a 50 to 480 mM CsCl gradient and 0 mV or +20 mV holding potential. Cs⁺ was used as current carrier to increase the conductance of the calcium release channel. Calmodulin and the agents suramin and 4-CMPS were added to the cis chamber, which corresponded to the cytosolic face of the calcium release channel.

Effect of Suramin at Activating, Subactivating, and Inhibitory Ca²⁺. The activation of the calcium release channel by suramin was strongly dependent on the concentration of activating free calcium. An example of the activation of a purified calcium release channel by suramin at 100 µM activating Ca²⁺ and 0 mV holding potential is shown in Fig. 1. Suramin (0.3 mM, 0.6 mM and 0.9 mM) increased the open probability in a concentration-dependent manner. In nine experiments, the mean open probability of purified calcium release channels increased from 0.47 to 0.72, 0.82, and 0.87 on addition of 0.3, 0.6, and 0.9 mM suramin, respectively, to the cis side (Table 1A). The channel activation by suramin was maximal at about 5 min after the addition of the compound and reversed by a reduction of free Ca²⁺ to subactivating concentrations. Suramin had no effect on the current amplitude, slope conductance, or reversal potential of the calcium release channel. The slope conductance of single calcium release channels with Cs⁺ as permeant ion was 542 ± 16 pS and 529 ± 8 pS in the presence of 0.3 to 0.9 mM suramin (n = 6; means ± S.E.M.).

View larger version (61K): [in this window] [in a new window]   Fig. 1.   Concentration dependence of activation of a single purified skeletal muscle calcium-release channel by suramin. Single-channel currents, shown as upward deflections, were recorded at 0 mV holding potential with 480 mM/50 mM CsCl (cis/trans). The solid lines indicate the baselines. Ca2+ and suramin were added to the cis side. Left, 400-ms recordings; right, the first 40 ms of each 400-ms recording. A, control, 100 µM Ca2+. B, activation of the channel by 0.3 mM suramin. C, activation of the channel by 0.6 mM suramin. D, activation of the channel by 0.9 mM suramin. E, open lifetime histograms, cumulative mean open channel time constants (o) and percentage areas for control and 0.9 mM suramin. Calibration bars represent 30 pA and 50 ms or 5 ms. Channel open probabilities (Po) and o were calculated from 56,000 events (control), 58,000 events (0.3 mM suramin), 51,000 events (0.6 mM suramin), 48,000 events (0.9 mM suramin), respectively.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Activation of a single purified skeletal muscle calcium-release channel by suramin at subactivating free calcium. Single-channel currents, shown as upward deflections, were recorded at +20 mV holding potential with 480 mM/50 mM CsCl (cis/trans). The solid lines indicate the baselines. Control and test records are from the same channel. Ca2+, EGTA, suramin, and 4-CMPS were added sequentially to the cis side. A, control, 100 µM Ca2+. B, control (0.05 µM Ca2+). C, activation of the channel by 1.2 mM suramin. D, activation of the channel by 80 µM 4-CMPS in the presence of 1.2 mM suramin. Calibration bars represent 50 pA and 5 ms (A) or 50 ms (A-D). Channel open probabilities (Po) were calculated from 42,000 events (control, 100 µM Ca), 1,200 events (control, 0.05 µM Ca), 9,600 events (suramin), and 19,000 events (suramin + 4-CMPS), respectively.

View larger version (51K): [in this window] [in a new window]   Fig. 3.   Activation of a single purified skeletal muscle calcium-release channel by suramin at 50 µM and 1 mM Ca2+. Single-channel currents, shown as upward deflections, were recorded at +20 mV holding potential with 480 mM/50 mM CsCl (cis/trans). The solid lines indicate the baselines. Control and test records are from the same channel. Left, 40- and 400-ms recordings. Suramin and 1 mM Ca2+ were added sequentially to the cis side. A, control, 50 µM Ca2+. B, activation of the channel by 0.9 mM suramin. C, activation by addition of 1 mM Ca2+. Calibration bars represent 50 pA and 50 ms (A-C) or 5 ms (A). Right, open lifetime histograms, cumulative mean open channel time constants (o), and percentage areas. The solid lines represent a fit according to two (control) and four exponential values (suramin). Channel open probabilities (Po) and o were calculated from 28,000 events (control), 45,000 events (0.9 mM suramin), 21,000 events (0.9 mM suramin plus 1 mM Ca2+), respectively.

Effect of Suramin on the Calcium-Calmodulin Induced Inhibition. Calcium-CaM inhibited the open probability of a purified calcium release channel in single-channel current recordings (Fuentes et al., 1994; Tripathy et al., 1995; Suko et al., 2000). The effects of suramin on the calmodulin-induced inhibition of the purified calcium release channel were determined at low (0.1 µM) and high (1-5 µM) calmodulin concentrations (Fig. 4). An example for the Ca-calmodulin induced inhibition and reactivation by suramin in the presence of 1 µM calmodulin is shown in Fig. 5. The percentage inhibition of the calcium release channel by 0.1 or 1 to 5 µM CaM was practically the same (about 27% of the controls). CaM (0.1 µM) significantly reduced the mean open duration (T_o), from 0.40 ± 0.03 ms to 0.18 ± 0.02 ms (n = 6); 1 to 5 µM CaM reduced the T_o from 0.39 ± 0.03 ms to 0.17 ± 0.01 ms (n = 5; means ± S.E.M). The distribution of the open and closed lifetimes (Fig. 5) were similar in low and high calmodulin concentrations and identical to those reported previously (Suko et al., 2000).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Inhibition of a single purified skeletal muscle calcium-release channel by 0.1 to 5 µM calmodulin and reactivation by suramin. Single-channel currents were recorded at 0 mV holding potential with 480 mM/50 mM CsCl (cis/trans) at 50 to 100 µM Ca2+. CaM (0.1 µM CaM, 1 µM or 5 µM) and suramin (0.3, 0.6, and 0.9 mM) were added sequentially to the cis side. Control and test recordings are from the same channel. Values are means ± S.E.M. from four to six experiments in each group. Channel open probabilities (Po) were calculated from 30,000 to 77,000 events. Significantly different (P = 0.05): control versus 0.1 µM CaM; 0.1 µM CaM versus 0.3, 0.6, and 0.9 mM suramin; 0.1 µM CaM + 0.3 mM suramin versus 0.1 µMCaM + 0.9 mM suramin. Control versus 1 to 5 µM CaM; 1 to 5 µM CaM versus 0.3, 0.6, and 0.9 mM suramin; 1 to 5 µM CaM + 0.3 mM suramin versus 1 to 5 µM CaM + 0.6 mM or 0.9 mM suramin (ANOVA).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Inhibition of a single purified skeletal muscle calcium-release channel by 1 µM calmodulin and reactivation by suramin. Single-channel currents, shown as upward deflections, were recorded at 0 mV holding potential with 480 mM/50 mM CsCl (cis/trans). The solid lines indicate the baselines. Ca2+ (50 µM) and 1 µM CaM and suramin were added sequentially to the cis side. Left, 40-ms recordings. Right, open lifetime histograms, cumulative mean open channel time constants (o) and percentage areas. The solid lines represent a fit according to two to four exponential values. A, control, 50 µM Ca2+. B, inhibition of the channel by 1 µM CaM. C, activation of the channel by 0.3 mM suramin. D, activation of the channel by 0.6 mM suramin. E, activation of the channel by 0.9 mM suramin. F, addition of EGTA (free Ca2+ 0.05 µM). Calibration bars represent 30 pA and 5 ms. Channel open probabilities (Po) and o were calculated from 41,000 events (control), 42,000 (CaM), 77,000 events (0.3 mM suramin), 44,000 events (0.6 mM suramin), 42,000 events (0.9 mM suramin), respectively. G, single-channel current-voltage relationship for control and CaM plus suramin. Slope conductance: control, 527 pS (reversal potential, 45 mV); 1 µM CaM plus 0.6 mM suramin (), 530 pS (reversal potential, 44.4 mV); 1 µM CaM plus 0.9 mM suramin (), 524 pS (reversal potential, 44.6 mV).

Effect of Magnesium in the Absence and Presence of Suramin. The inhibitory effect of magnesium on a purified calcium release channel activated by 40 to 100 µM Ca²⁺ (in the absence of suramin) is shown in Fig. 6. Half-maximum inhibition of the open probability was obtained at 0.38 mM Mg²⁺. Mean channel open and closed duration and open and closed time constants in the presence of 1 mM magnesium are shown in Table 2.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Inhibition of the open probability of single purified skeletal muscle calcium-release channels by Mg2+ in the absence and presence of suramin. Single-channel currents were recorded at +20 mV holding potential with 480 mM/50 mM CsCl (cis/trans). Open probabilities for inhibition of the suramin-activated channels by Mg2+ include data from Table 3, A to C. Magnesium inhibition of controls includes data derived in experiments given in Table 3C. Also see text. Data points are means ± S.E.M. The mean values for controls and suramin in the absence and presence of 0.5, 1, and 2 mM Mg2+ were significantly different (P < 0.05). The solid lines represent a fit according to a single exponential decay of Po. Half-maximal inhibition of Po by magnesium occurred at 0.38 mM (control) and 0.83 mM (suramin).

View this table: [in this window] [in a new window]   TABLE 2 Mean open probability, mean current amplitude, and mean open and closed lifetimes in controls and in the presence of 1 mM Mg2+ Single-channel currents were recorded at +20 mV voltage holding potential with 480 mM/50 mM CsCl (cis/trans). Control and test records are from the same channel. Channel open probabilities (Po), mean current amplitude (pA), mean channel open (To) and closed (Tc) duration (ms), cumulative mean open and closed channel time constants (o, c) and percentage of area represented by a time constant for purified calcium release channels activated by 50 to 100 µM cis Ca2+ and in the presence of 1 mM Mg2+ added to the cis chamber. Values are presented as means ± S.E.M. for the indicated number (n) of channels included in the analysis.

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Inhibition of a suramin-activated single purified skeletal muscle calcium-release channel by 4 and 10 mM Mg2+. Single-channel currents, shown as upward deflections, were recorded at +20 mV holding potential with 480 mM/50 mM CsCl (cis/trans). The solid lines indicate the baselines. Controls and test records are from the same channel. Ca2+ (50 µM), 0.9 mM suramin, 4 and 10 mM Mg2+ were added sequentially to the cis side; 40-ms (control, suramin) and 400-ms (magnesium) recordings. A, control, 50 µM Ca2+. B, activation of the channel by 0.9 mM suramin. C, inhibition of the channel by 4 mM Mg2+. D, inhibition by 10 mM Mg2+ (Po was 0.013 with 20 mM Mg2+, not shown). Calibration bars represent 50 pA and 5 ms (A, B) or 50 ms (C, D). Channel open probabilities (Po) were calculated from 42,000 events (control), 33,000 events (suramin), and 12,000 to 33,000 events (magnesium), respectively.

View larger version (32K): [in this window] [in a new window]   Fig. 8.   Inhibition of a suramin-activated single purified skeletal muscle calcium-release channel by 1 mM Mg2+, reactivation by 2.4 mM suramin and inhibition by 4 mM Mg2+. Single-channel currents, shown as upward deflections, were recorded at +20 mV holding potential with 480 mM/50 mM CsCl (cis/trans). The solid lines indicate the baselines. Control and test records are from the same channel. 400-ms recordings. Ca2+ (20 µM), 0.9 mM suramin, 1 mM Mg2+, 2.4 mM suramin (total), and 4 mM Mg2+ (total) were added sequentially to the cis side. A, control, 20 µM Ca2+. B, activation of the channel by 0.9 mM suramin. C, inhibition by 1 mM Mg2+. D, reactivation of the channel by 2.4 mM suramin (total). E, inhibition of the channel by 4 mM Mg2+. Calibration bars represent 50 pA and 5 ms. Channel open probabilities (Po) were calculated from 40,000 events (control), 24,000 to 37,000 (suramin), 39,000 to 61,000 events (magnesium), respectively.

View this table: [in this window] [in a new window]   TABLE 4 Mean open probability, mean current amplitude, and mean open and closed lifetimes of controls, 0.9 mM suramin, and 0.9 mM suramin plus 1, 4, and 10 mM Mg2+ Single-channel currents were recorded at +20 mV voltage holding potential with 480 mM/50 mM CsCl cis/trans. Channel open probabilities (Po), mean current amplitude (pA), mean channel open (To) and closed (Tc) duration (ms), cumulative mean open and closed channel time constants (o, c) and percentage of area represented by a time constant for purified calcium release channels activated by 50 to 100 µM cis Ca2+ and 0.9 mM suramin and inhibited by 1, 4, and 10 mM Mg2+ (cis). Values are presented as means ± S.E.M. Values in parentheses are area %.

[³H]Ryanodine Binding. [³H]Ryanodine binding to HSR vesicles was carried out at equilibrium conditions in 90-min assays at 37°C in the presence of 40 mM Mops/Tris, pH 7.0, 1 M NaCl or 0.5 M CsCl, 10 nM [³H]ryanodine, 100 µM Ca²⁺, and protease inhibitors or at free calcium concentrations ranging from 0.02 µM to 50 mM or in the presence of 0.001 to 50 mM Mg²⁺. Cs⁺ (0.5 M) was used in the calcium dependence and magnesium inhibition assays to keep the conditions in [³H]ryanodine binding as close as possible to the conditions used in single-channel current experiments (0.48 M Cs⁺ cis).

View larger version (23K): [in this window] [in a new window]   Fig. 9.   Concentration dependence of suramin on specific [3H]ryanodine binding to HSR in the presence of 1 M NaCl at 100 µM, 4 µM, and 0.5 µM Ca2+. Specific [3H]ryanodine binding was performed with 40 mM Mops/Tris, pH 7.0, 10 nM [3H]ryanodine, 1 M NaCl and protease inhibitors (see Experimental Procedures) at 37°C for 90 min in the presence of the indicated concentrations of suramin and 100 µM Ca2+ (), 4 µM Ca2+ (), or 0.5 µM Ca2+ (). Data points are means of duplicate determinations of a representative experiment that was repeated twice. The solid line represents a fit of the data according to the Hill equation. The calculated parameters are means ± S.E.M. from three experiments: Bmax = maximum [3H]ryanodine binding; EC50 = Ca2+ concentration giving half-maximum activation; nH = Hill coefficient. Suramin independent binding (pmol/mg HSR): 2.23 ± 0.35 (100 µM Ca2+), 0.40 ± 0.16 (4 µM Ca2+), 0.006 ± 0.005 (0.5 µM Ca2+). Suramin-dependent binding: Bmax = 3.61 ± 0.78 pmol/mg, EC50 = 0.11 ± 0.04 mM, nH = 2.15 ± 0.52 (100 µM Ca2+). Bmax = 6.06 ± 1.29 pmol/mg, EC50 = 0.24 ± 0.03 mM, nH = 1.37 ± 0.13 (4 µM Ca2+). Bmax = 2.74 ± 1.18 pmol/mg, EC50 = 2.22 ± 1.32 mM, nH = 1.12 ± 0.22 (0.5 µM Ca2+).

View larger version (19K): [in this window] [in a new window]   Fig. 10.   Inhibition of specific [3H]ryanodine binding to HSR by magnesium in the absence and presence of 1 mM suramin. Specific [3H]ryanodine binding was performed in the absence () or presence of 1 mM suramin () with 40 mM Mops/Tris, pH 7.0, 10 nM [3H]ryanodine, 0.5 M CsCl, and protease inhibitors (see Experimental Procedures) at 37°C for 90 min. Data points are means from three experiments in the absence or presence of 1 mM suramin. Solid lines represent a fit of the data according to the Hill equation. Calculated parameters are means ± S.E.M. (n = 3); Bmax = (maximum [3H]ryanodine binding); control, 3.80 ± 1.19 pmol/mg HSR; suramin, 6.92 ± 0.93 pmol/mg HSR. Mg2+ concentration giving half-maximum inhibition: control, 0.30 ± 0.12 mM; suramin, 83.4 ± 31.7 mM; the Hill coefficients were 0.81 ± 0.09 (control) and 0.52 ± 0.03 (suramin).

View larger version (19K): [in this window] [in a new window]   Fig. 11.   Calcium dependence of control and suramin-stimulated [3H]ryanodine binding to HSR in the presence of 0.5 M CsCl. Specific [3H]ryanodine binding was performed in the absence () or presence of 1 mM suramin () with 40 mM Mops/Tris, pH 7.0, 10 nM [3H]ryanodine, 0.5 M CsCl, and protease inhibitors (see Experimental Procedures) at 37°C for 90 min. Data points are means from three control experiments (absence of suramin) and three experiments in the presence of 1 mM suramin. The solid lines represent a fit of the data according to the sum of two Hill equations as described previously (Meissner et al., 1997; Suko and Hellmann, 1998). Calculated parameters are means ± S.E.M. (control, n = 4; suramin, n = 3): Bmax = maximum [3H]ryanodine binding; Ka, Ca2+ concentration giving half-maximum activation; KI, Ca2+ concentration giving half-maximum inhibition; na and ni, number of activating or inhibiting calcium species; c_res, calcium-independent binding. Control, Bmax = 3.16 ± 0.24 pmol/mg HSR; Ka = 1.60 ± 0.61 µM; na = 2.86 ± 0.48; Ki = 1175 ± 145 µM, ni = 2.86 ± 0.48, c_res = 0.025 ± 0.006. Suramin, 1 mM, Bmax = 6.60 ± 0.32 pmol/mg HSR; Ka = 0.46 ± 0.03 µM; na = 3.76 ± 0.64; Ki = 48153 ± 10719 µM; ni = 0.51 ± 0.08; c_res = 1.78 ± 0.01.

Long-Term Effect of Suramin. Long-term effects of suramin (0.9 mM or 2.4 mM) were determined in the presence of high concentrations of magnesium or calcium (10 to 20 mM) as illustrated in Figs. 12 to 14. Conditions were similar to those in Fig. 7; alternatively, the calcium-activated calcium release channel was inhibited by 10 mM Mg²⁺, reactivated by suramin about 3 min later, and the time course of activation was observed over a period of 30 to 60 min (Fig. 12). These latter conditions were similar to those used in [³H]ryanodine binding, when magnesium and suramin were simultaneously present at the start of the incubation with HSR.

View larger version (27K): [in this window] [in a new window]   Fig. 12.   Long-term effect of suramin on a single purified skeletal muscle calcium-release channel in the presence of 10 Mg2+. Single-channel currents, shown as upward deflections, were recorded at +20 mV holding potential with 480 mM/50 mM CsCl (cis/trans). The solid lines indicate the baselines. Control and test records are from the same channel; 400-ms recordings. Mg2+ and suramin were added sequentially to the cis side. A, control, 50 µM Ca2+. B, inhibition of the channel by 10 mM Mg2+. C, suramin (2.4 mM; 3 min). D, suramin (2.4 mM; 10 min). E, suramin (2.4 mM; 30 min). Calibration bars represent 50 pA and 50 ms. Channel open probabilities (Po) were calculated from 2250 events (10 mM Mg2+), 24,000 events (10 mM Mg2+ plus 2.4 mM suramin), respectively.

View larger version (27K): [in this window] [in a new window]   Fig. 13.   Long-term effect of suramin on a single purified skeletal muscle calcium-release channel in the presence of 20 mM Mg2+. Single-channel currents, shown as upward deflections, were recorded at +20 mV holding potential with 480 mM/50 mM CsCl (cis/trans). The solid lines indicate the baselines. Control and test records are from the same channel; 40-ms recordings (A, B), 400-ms recordings (C-E). Ca2+, suramin and Mg2+ were added sequentially to the cis side. A, control, 50 µM Ca2+. Activation of the channel by 0.9 mM suramin. C, suramin (2.4 mM) plus 10 mM Mg2+ (5 min). D, suramin (2.4 mM) plus 20 mM Mg2+ (35 min); note the appearance of long open states. E, ruthenium red (7 µM), closed the channel. Calibration bars represent 50 pA and 5 ms (A, B) or 50 ms (C-E). F, open lifetime histograms, cumulative mean open channel time constants (o) and percentage areas in the presence of 20 mM Mg2+ plus 2.4 mM suramin (D). The solid lines represent a fit according to four exponential values. Channel open probabilities (Po) and (o) were calculated from 40,200 events (control), 30,000 events (0.9 mM suramin), 2,300 events (2.4 mM suramin plus 10 mM Mg2+), and 4,542 events (2.4 mM suramin plus 20 mM Mg2+; evaluated from 200 400-ms recordings), respectively.

View larger version (31K): [in this window] [in a new window]   Fig. 14.   Long-term effect of suramin on a single purified skeletal muscle calcium-release channel in the presence of 10 to 15 mM Ca2+. Single-channel currents, shown as upward deflections, were recorded at +20 mV holding potential with 480 mM/50 mM CsCl (cis/trans). The solid lines indicate the baselines. Control and test records are from the same channel; 400-ms recordings. A, control, 100 µM Ca2+. B, activation with 0.9 mM suramin plus 1 mM Ca2+ (5 min). C, inhibition of the channel by 2 mM Ca2+ (28 min). D, inhibition of the channel by 6 mM Ca2+ (7 min). E, activation of the channel by 10 mM Ca2+(15 min) and 15 mM Ca2+ (8 min). o are given for 0.9 mM suramin plus 15 mM Ca2+. Calibration bars represent 50 pA and 50 ms. Channel open probabilities (Po) at 15 mM Ca2+ were calculated from 13,000 events.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The main points of the suramin-induced functional alterations of the skeletal muscle calcium release channel shown in the present study (and not reported in previous studies with suramin) are: the slowly developing channel activation in the presence of inhibiting concentrations of magnesium and calcium (`long-term effect' of suramin); the apparent dissociation of inhibition of the open probability in single-channel current recordings and [³H]ryanodine binding to HSR in the presence millimolar concentrations of magnesium or calcium; the reversal of the calcium-calmodulin induced inhibition by suramin in single-channel current recordings; and the additive activation of P_o by suramin and sulfhydryl oxidation.

Short- and Long-Term Suramin-Induced Channel Activation. At calcium concentrations that cause maximal channel activation (20-100 µM Ca²⁺) or in the presence of 1 mM Ca²⁺ (which inhibits the calcium release channel in the absence of suramin), 0.9 to 1.0 mM suramin caused a marked activation of the purified skeletal muscle calcium release channel (P_o up to 0.9) or a nearly full open state of the channel (P_o = 0.95) within a few minutes after addition of suramin. Characteristic of these increases in P_o was a 3.5-fold increase in the mean open duration (T_o = 0.41 ms versus 1.46 ms) with a shift to _o3/_o4 open states (Table 4). At a P_o of 0.95 (1 mM Ca²⁺), the percentage of _o3/_o4 states increased (Fig. 3). Inhibition of the suramin-activated channels with 10 mM magnesium reduced P_o, T_o, and eliminated _o3/_o4 open states (Table 4). The activating effect of suramin on the skeletal and cardiac calcium release channel of HSR was shown to be reversed when suramin was removed from the cis chamber by perfusion (Sitsapesan and Williams, 1996).

Apparent Dissociation between Single-Channel Current Fluctuation and [³H]Ryanodine Binding to HSR in the Presence of Suramin. Ryanodine binding is thought to occur only to the open calcium release channel (Pessah et al., 1987; Coronado et al., 1994; Meissner, 1994; Hasselbach and Migala, 1998). This interpretation is based on its dependence on channel-activating calcium and complete inhibition by ruthenium red (Table 5). Activation or inhibition of single-channel current fluctuations of the purified calcium release channel and [³H]ryanodine binding to HSR are usually in good agreement; i.e., an increase or decrease in P_o associated with an increase or decrease in [³H]ryanodine binding was shown with calmodulin (Tripathy et al., 1995, Suko et al., 2000), after oxidation of sulfhydryls of cysteines of the calcium release channel (Suko and Hellmann, 1998; Suko et al., 2000) and for suramin or the suramin analog NF307 (present study; Hohenegger et al., 1996; Klinger et al., 1999).