Suramin Interacts with the Calmodulin Binding Site on the Ryanodine Receptor, RYR1*

Apocalmodulin and Ca2+calmodulin bind to overlapping sites on the ryanodine receptor skeletal form, RYR1, but have opposite functional effects on channel activity. Suramin, a polysulfonated napthylurea, displaces both forms of calmodulin, leading to an inhibition of activity at low Ca2+ and an enhancement of activity at high Ca2+. Calmodulin binding motifs on RYR1 are also able to directly interact with the carboxy-terminal tail of the transverse tubule dihydropyridine receptor (DHPR) (Sencer, S., Papineni, R. V., Halling, D. B., Pate, P., Krol, J., Zhang, J. Z., and Hamilton, S. L. (2001) J. Biol. Chem. 276, 38237–38241). Suramin binds directly to a peptide that corresponds to the calmodulin binding site of RYR1 (amino acids 3609–3643) and blocks the interaction of this peptide with both calmodulin and the carboxyl-terminal tail of the DHPR α1-subunit. Suramin, added to the internal solution of voltage-clamped skeletal myotubes, produces a concentration-dependent increase in the maximal magnitude of voltage-gated Ca2+ transients without significantly altering L-channel Ca2+ channel conducting activity. Together, these results suggest that an interaction between the carboxyl-terminal tail of the DHPR α1-subunit with the calmodulin binding region of RYR1 serves to limit sarcoplasmic reticulum Ca2+ release during excitation-contraction coupling and that suramin-induced potentiation of voltage-gated Ca2+ release involves a relief of this inhibitory interaction.

The skeletal muscle ryanodine receptor (RYR1) functions as a sarcoplasmic reticulum (SR) 1 Ca 2ϩ release channel that plays a central role in excitation-contraction coupling. Two distinct mechanisms are postulated to contribute to the release of Ca 2ϩ from the SR in skeletal muscle. After sarcolemmal depolarization, SR Ca 2ϩ release channels are initially activated via mechanical coupling with DHPRs (or L-type Ca 2ϩ channels) located in the surface membrane (2). However, morphological data indicate that only every other SR Ca 2ϩ release channel is directly coupled with sarcolemmal DHPRs (3). Adjacent, non-DHPR-coupled release channels (4) are thought to be activated either by Ca 2ϩ released via the mechanically coupled channels (3) or by a coordinated or "coupled-gating" mechanism of activation (5). RYR1 and DHPR proteins bind CaM in both its Ca 2ϩ -bound and Ca 2ϩ -free forms (6 -9). Overlapping binding sites for apoCaM and Ca 2ϩ -CaM are located between amino acids 3614 and 3643 of RYR1 (10,11). The carboxyl-terminal tail of the DHPR ␣ 1 -subunit (12,13) appears to have binding sites for both forms of CaM. C3635, located within the putative CaM binding region of RYR1, has been postulated to contribute to oxidation-induced intersubunit cross-linking (14) and was recently demonstrated to be the site of CaM-dependent NO modulation of RYR1 (15).
Studies of the interaction of both the DHPR and RYR1 with apoCaM and Ca 2ϩ CaM have been primarily carried out with uncoupled channels, raising the question of whether CaM interacts with either channel when the two proteins are mechanically coupled in intact skeletal muscle. Slavik et al. (12) provided evidence that a sequence within the carboxyl terminus of the DHPR ␣ 1 -subunit interacts strongly with RYR1. This sequence was later shown to also be a CaM binding motif (13). More recently, Sencer et al. (1) demonstrated that the CaM binding site on RYR1 binds directly to the carboxyl-terminal tail of the DHPR ␣ 1 -subunit. These findings suggest that the CaM binding motifs on the DHPR and RYR1 proteins may actually function as protein-protein interaction motifs rather than strictly as CaM binding domains. However, the functionally relevant binding partners for these motifs have not yet been identified. If the DHPR and RYR1 proteins utilize CaM binding motifs for binding to one another, then CaM could potentially uncouple the mechanical link formed between the CaM binding site of RYR1 and the carboxyl-terminal tail of the DHPR ␣ 1 -subunit. However, the functional consequences of such CaM-mediated uncoupling have yet to be evaluated.
Suramin, a polysulfonated napthylurea is a potent, reversible activator of the RYR, increasing both conductance and P 0 of the channel (16). Klinger et al. (17) found that suramin inhibits the RYR1-CaM interaction, possibly by competing for the CaM binding domain on RYR1. Suko et al. (18) studied the effects of suramin on the single channel behavior of RYR1 and on [ 3 H]ryanodine binding and found that RYR1 channels reconstituted in planar lipid bilayers are activated by high concentrations (0.3-0.9 mM) of suramin. This effect appeared to be due to an increased affinity of the Ca 2ϩ activating site on RYR1 for Ca 2ϩ . These authors suggested that the complex functional effects of suramin arise from an allosteric regulation of the channel and not from alterations in the binding of endogenous ligands (e.g. CaM) involved in channel gating. In the current study, we demonstrate that suramin binds directly to a peptide corresponding to the CaM binding motif on RYR1 and that suramin blocks the interaction of this peptide with the carboxyl-terminal region of the DHPR ␣ 1 -subunit. Moreover, suramin potentiates voltage-gated SR Ca 2ϩ release in whole cell voltageclamped skeletal myotubes, consistent with the idea that suramin disrupts an intrinsic inhibitory interaction between the DHPR carboxyl terminus and the CaM binding domain on RYR1.

EXPERIMENTAL PROCEDURES
Materials-Bovine brain CaM, suramin, and dithiothreitol were obtained from Sigma. Tran 35 S-label (Ͼ1000 Ci/mmol) was purchased from ICN Biomedicals, Inc. (Irvine, CA). [ 3 H]Ryanodine was purchased from Amersham Biosciences, and unlabeled ryanodine was purchased from Calbiochem. A fluorescent derivative of CaM, Alexa Fluor 594-conjugated CaM (Alexa CaM), was purchased from Molecular Probes (Eugene, OR). The peptides were synthesized in the core facility at Baylor College of Medicine (Houston, TX).
SR Membrane Preparation-SR and transverse tubule-enriched membranes were prepared from rabbit hind leg and back-strap skeletal muscle and purified using sucrose gradient centrifugation (19). Protein concentrations were estimated by the method of Lowry using bovine serum albumin as the standard (20).
[ 35 S]Methionine Labeling of CaM-The mammalian CaM was generously provided by Dr. Ruth Altschuld. Metabolic labeling of calmodulin with Tran 35 S-label was performed according to a procedure described previously (7).
Equilibrium [ 35 S]CaM Binding Assay-35 S-Labeled CaM binding to SR membranes (10 g) was determined as described previously (7). The binding buffer contained 50 mM MOPS (pH 7.4), 300 mM NaCl, 0.1% CHAPS, and 1 mM EGTA with 1.2 mM CaCl 2 (high Ca 2ϩ binding buffer, 200 M free Ca 2ϩ ) or 1 mM EGTA alone (low Ca 2ϩ binding buffer, 10 nM free Ca 2ϩ ). Samples were incubated for 2 h at room temperature. Bound radioligand was separated from free by filtration through Whatman GF/F filters and washing 5 times with 3 ml of the ice-cold binding buffer.
Equilibrium [ 3 H]Ryanodine Binding Assay-20-g SR membranes per assay were incubated with 5 nM [ 3 H]ryanodine at room temperature for 17 h in binding buffer (300 mM NaCl, 50 mM MOPS (pH 7.4), 0.1% CHAPS, and either 1 mM EGTA or 1.2 mM CaCl 2 ). Nonspecific binding was defined in the presence of unlabeled ryanodine (final concentration of 10 M). Bound [ 3 H]ryanodine was separated from free by filtering through Whatman GF/F glass fiber filters and washing 5 times with 3 ml of ice-cold binding buffer. The radioactivity was quantified by scintillation counting.
Fluorescence Spectroscopy-The interaction of suramin and R3609 -43 was determined by monitoring the changes in intrinsic tryp-tophan fluorescence of R3609 -43. The fluorescence was recorded on a ISS PC1 photon counting spectrophotometer (Champaign, IL). R3609 -43 in the presence of different concentrations of suramin was excited at 295 nm (UV-solar pass filter), and emission spectra (320 -450 mM) were recorded with a "Cut-on" filter of 309 nm.
Suramin inhibition of Alexa-CaM-R3609 -43 binding was determined by monitoring the changes in emission of Alexa CaM recorded on an ISS PC1 photon-counting spectrophotometer. Samples of Alexa CaM Nondenaturing Gel Electrophoresis-The electrophoretic mobility of CaM was evaluated by nondenaturing polyacrylamide gel electrophoresis under discontinuous conditions as a modified technique described by Laemmli (21). Nondenaturing gels (20% polyacrylamide) were separated at 30 mA under high Ca 2ϩ conditions (200 M CaCl 2 in the gel buffers).
DHPR-RYR Interaction-Pull-down assays were performed using a biotinylated R3609 -43 peptide and streptavidin beads. Briefly, transverse tubule-enriched membranes (1 mg/ml in 150 mM KCl, 25 mM NaCl, 100 M CaCl 2 , 50 mM MOPS (pH7.4)) were solubilized with 1% CHAPS. Aliquots (50 l) of the solubilized membranes were added to streptavidin beads (50 l) that had been preincubated for 1 h with 40 M R3609 -43. After gently mixing for 1 h at room temperature the beads were pelleted for 1 min in a low speed centrifuge, and the supernatant was removed. An additional 300 l of buffer was then added, the sample was vortexed, and the beads were once again pelleted. After the addition of 100 l of H 2 O, beads were extracted with SDS and electrophoresed on a 7.5% SDS-polyacrylamide gels and either stained or Western-blotted with the indicated antibodies.

R3609 -43-D1393-1527 Interaction by Nickel Chelate Plate Assay-
Analysis of the interaction between R3609 -43 and the recombinantly expressed carboxyl-terminal fragment of DHPR ␣ 1 -subunit was determined as follows. The cDNA encoding the skeletal DHPR amino acids ␣ 1 -1393-1527 (D1393-1527) was used to express His-tagged recombinant protein as described by Sencer et al. (1). The His-tagged protein was coupled to nickel chelate assay plates as recommended by the manufacturer (BD Biosciences). 1 M biotinylated R3609 -43 (150 mM KCl, 25 mM NaCl, 100 M CaCl 2 , 50 mM MOPS (pH7.4)) in the presence of various concentrations of suramin was added to the wells, and the plates were mixed at 100 rpm for 1 h at room temperature. The bound biotinylated R3609 -43 was calorimetric-assayed using avidin-conjugated alkaline phosphate enzyme (1:1000), and p-nitrophenyl phosphate as a substrate/chromogen. The reaction was measured at 405 nm on a SpectraMax microplate spectrophotometer (Molecular Devices Corp., Sunnyvale, CA).

Whole Cell Patch Clamp Measurements of Voltage-gated L-currents and Intracellular Calcium Transients in Mouse Myotubes-Myotubes
were prepared from the skeletal muscle of newborn mice as previously described (22)(23)(24). Voltage-gated Ca 2ϩ currents and Ca 2ϩ transients were recorded using the whole cell patch clamp technique. The pipette solution for all experiments was 145 mM cesium aspartate, 0.1 mM Peak L-currents were normalized to cell capacitance (pA/picofarads), plotted as a function of test potential and fitted according to the equation, where G max is the maximal L-channel conductance, V m is test potential, V rev is the extrapolated reversal potential, V G1/2 is the potential for half-maximal activation of G max and, k G is a slope factor. Relative changes in cytosolic Ca 2ϩ were measured using the Ca 2ϩ indicator K 5 -Fluo-3 as described previously (24). Fluorescence traces were analog-filtered ( ϭ 0.5 ms) before digitization and expressed as ⌬F/F 0 , where F 0 is the base-line fluorescence immediately before depolarization, and ⌬F represents the fluorescence change from base line. Fluorescence amplitudes at the end of each test pulse are plotted as a function of test potential and fitted according to the equation, where ⌬F/F max is the calculated maximal change in fluorescence for each test potential (V m ), V F1/2 is the potential for half-maximal activation of ⌬F/F max , and k F is a slope factor. Data Analysis-Inhibition of 35 S-labeled CaM binding to SR membranes was analyzed by non-linear regression (Sigma Plot 2000; Jandel Scientific, San Rafael, CA) using the equation, where B max is the number of binding sites, K d is the apparent dissociation constant for 35 S-labeled CaM binding, [L] is the concentration of 35 S-CaM, y is the concentration of suramin, x is the amount bound, and K i is the apparent inhibitory constant for suramin.
In gel shift assays, densitometry was performed on the peptidebound CaM band. Optical density data obtained in the presence of suramin were normalized to the optical density of the peptide-CaM band alone and plotted as a function of suramin concentration. The data described are the mean Ϯ S.E. for at least three independent determinations.

Suramin Inhibits Ca 2 -bound and Ca 2 -free CaM
Binding to RYR1-Both RYR1 and CaM are Ca 2ϩ -binding proteins. Our studies comparing the interactions of a Ca 2ϩ binding site mutant of CaM and wild type CaM with RYR1 demonstrated that the affinities of both forms of CaM for RYR1 are greater at M than at nM Ca 2ϩ concentrations, indicating that Ca 2ϩ binding to RYR1 increases the affinity for CaM (7). Suramin inhibits CaM binding to RYR1 (17). To determine the affinity of the interaction of suramin with RYR1 we analyzed the concentration dependence for suramin inhibition of 35 S-labeled CaM binding to SR membranes at both nM and M Ca 2ϩ concentrations (Fig. 1). K i values for suramin inhibition of CaM binding to RYR1 at 200 M and Ͻ10 nM free Ca 2ϩ concentrations were 1.7 Ϯ 0.1 (n ϭ 3) and 1.3 Ϯ 0.14 M (n ϭ 3), respectively. These results indicate that suramin inhibits the interaction of both apoCaM and Ca 2ϩ -CaM with RYR1 in a manner that is not altered by Ca 2ϩ binding to RYR1. The complete inhibition of 35 S-labeled CaM binding suggests that suramin binding to RYR1 is competitive with CaM. This is further supported by our finding that the rate of dissociation of 35 S-labeled CaM from RYR1 is not altered by suramin (data not shown). The measured dissociation rate constants for 35 S-labeled CaM bound to SR membranes are 0.33 Ϯ 0.001 min Ϫ1 (n ϭ 3) and 0.35 Ϯ 0.002 min Ϫ1 (n ϭ 3) in the absence and presence of suramin, respectively.
Suramin Binds to Peptide R3609 -43 and Inhibits Its Interaction with CaM-The studies of Klinger et al. (17) indicated that suramin binds competitively to the CaM binding domain on the RYR. However, Suko et al. (18) suggested that suramin effects on RYR channel function involve an allosteric mechanism that occurs in the absence of direct effects on endogenous ligands involved in channel gating. To address whether the suramin interaction with RYR1 is competitive or noncompetitive, we examined the ability of suramin to inhibit the interaction of a peptide corresponding to the CaM binding domain of RYR1 (R3609 -3643) with CaM at both high and low Ca 2ϩ concentrations (Fig. 2). At 200 M and Ͻ10 nM free Ca 2ϩ concentrations, suramin displaces CaM from R3609 -3643. The inhibition was assessed by the changes in the fluorescence of Alexa CaM in the presence or absence of the peptide (R3609 -3643). These results suggest a competitive interaction between suramin and CaM with the R3609 -43 region of RYR1.
Competitive inhibition by suramin of the interaction of CaM with R3609 -43 was confirmed by nondenaturing polyacrylamide gel electrophoresis. As shown in Fig. 3, the ability of R3609 -3643 to bind CaM on non-denaturing gels is completely inhibited at suramin concentrations above 10 M. CaM binding to a peptide (D1665-1685) corresponding to the CaM binding domain of the carboxyl-terminal region of the DHPR ␣ 1 -subunit (13) is not affected by suramin (Fig. 3). These results indicate that suramin inhibition of the binding of CaM is relatively selective to the CaM recognition sequence found in RYR1.
To demonstrate direct binding between suramin and R3609 -3643, we analyzed changes in intrinsic tryptophan fluorescence (excitation at 295 nm) of R3609 -3643 in the presence and absence of 10 M suramin (Fig. 4). The addition of suramin to R3609 -43 peptide (Fig. 4A) induced a shift in tryptophan emis- sion spectra (from a peak at ϳ350 nm in the absence of suramin to ϳ420 nm in the presence of suramin). The suramin-induced shift in emission spectra arises from fluorescence resonance energy transfer (FRET) between the single tryptophan residue of peptide R3609 -43, acting as donor, and the naphthalene rings of suramin as acceptor. The addition of either free tryptophan (data not shown) or a control peptide representing amino acids 135-160 of RYR1 (R135-160) (Fig. 4B) (Fig. 5A), whereas apoCaM enhanced [ 3 H]ryanodine binding to RYR1 (Fig. 5B). These effects of CaM were overcome by increasing concentrations of suramin (from 0.1 to 10 M), suggesting that suramin competes with CaM for a binding site on RYR1 over this concentration range. Higher suramin concentrations (Ͼ100 M) resulted in both the displacement of endogenous FKBP12 and enhanced [ 3 H]ryanodine binding to RYR independent of the presence of CaM (data not shown). Suramin at concentrations greater than 1 M increases [ 3 H]ryanodine binding to RYR1 even in the absence of added CaM (Fig. 5A). The increase in [ 3 H]ryanodine binding is likely due to displacement of endogenous calmodulin in these membranes by suramin. Consistent with this, Western blotting of these membranes with an antibody to calmodulin shows the presence of endogenous calmodulin that is partially displaced by 10 M suramin (Fig. 6A). Endogenous CaM bound to RYR was, however, completely displaced by 10 M suramin (Fig. 6B).
This was inferred from the amounts of CaM detected in the proteins extracted from the filters routinely used for binding assays. Moreover, the suramin-induced changes in [ 3 H]ryanodine binding in the absence of added CaM was not observed when SR membranes were pre-washed extensively (Fig. 6C).
Modulation of DHPR-R3609 -43 Interaction by Suramin-The CaM binding motif of RYR1 physically interacts with the carboxyl terminus of the DHPR ␣ 1 -subunit (1). We analyzed the effect of suramin on this interaction by assessing the ability of R3609 -43 to pull down the DHPR from detergent-solubilized T-tubule membranes. In these experiments a biotinylated derivative of R3609 -3643 (R3609 -3643-biotin) and streptavidin beads was used to pull down DHPRs. Western blotting with anti-␣ 1 -DHPR antibodies of the pull-down fractions demonstrated that 50 M suramin blocked the pull down of the DHPR ␣ 1 -subunit (Fig. 7A). The effect of suramin on the interaction of R3609 -43 and a recombinant protein fragment of carboxyl terminus of the DHPR ␣ 1 -subunit (His-tagged D1393-1527) expressed in Escherichia coli was also analyzed. The interaction was completely inhibited by 10 M suramin (Fig. 7B).
The carboxyl terminus of the DHPR ␣ 1 -subunit (D1393-1527) has been shown to inhibit ryanodine binding to RYR1 (1). D1393-1527 was isolated using nickel-chelated beads after thrombin cleavage to determine whether suramin could prevent inhibition of ryanodine binding. The D1393-1527 preparation contained His tag as a contaminant. 10 M suramin completely attenuated the inhibition of [ 3 H]ryanodine binding by D1393-1527 (Fig. 7C). The His tag by itself has no effect on the [ 3 H]ryanodine binding to RYR1 (data not shown). Together these results indicate that suramin effectively uncouples the physical interaction between the carboxyl terminus of the DHPR ␣ 1 -subunit and a peptide corresponding to the CaM binding site of RYR1.
Effect of Suramin on Excitation-Contraction Coupling in Mouse Myotubes-The results reported here demonstrate that suramin disrupts binding of both CaM and the carboxyl terminus of the DHPR ␣ 1 -subunit to a peptide corresponding to the CaM binding domain of RYR1. To determine whether these effects alter excitation-contraction coupling in intact skeletal muscle cells, we measured L-type Ca 2ϩ channel currents and voltage-gated SR Ca 2ϩ release in normal myotubes in the presence and absence suramin. Because suramin is not membranepermeant, 50 M suramin was included in the patch pipette internal solution (with buffer alone used as control), and recordings were made 5 min after establishing the whole cell configuration. For these experiments, 50 M suramin was used because this concentration of suramin was found in biochemical experiments to maximally inhibit CaM binding to RYR1 (Figs. 1 and 3) and would, therefore, be likely to overcome potential limitations with regard to suramin dialysis and accessibility to junctional RYR1 proteins in patch-clamped myotubes. Maximal L-type Ca 2ϩ channel conductance (G max ) and the voltage required for half-maximal activation of G max (V G1/2 ) were similar in the presence and absence of 50 M internal suramin ( Fig. 8A and Table I). The lack of an effect of suramin on L-type Ca 2ϩ channel activity is consistent with the finding reported here that suramin does not interact with the CaM binding region of the skeletal muscle DHPR. However, 50 M suramin caused a significant increase (67.9 Ϯ 0.2%, n ϭ 13; p Ͻ 0.05) in the maximal ⌬F/F 0 without altering V F1/2 , the voltage required for half-maximal release (Fig. 8B and Table I). The effects of suramin on maximal voltage-gated SR Ca 2ϩ release were concentration-dependent since a smaller increase in maximal ⌬F/F 0 was observed at a lower concentration (5 M suramin: 39.7 Ϯ 0.2%, n ϭ 16; p Ͻ 0.05). The increase in maximal voltage-gated Ca 2ϩ release could arise from either a direct stimulatory effect of suramin on activated SR Ca 2ϩ release channels or via a suramin-mediated disruption of an inhibitory interaction between the carboxyl-terminal region of the DHPR ␣ 1 -subunit and the CaM binding domain of RYR1 (see "Discussion"). DISCUSSION Our results indicate that suramin inhibits both Ca 2ϩ -CaM and apoCaM binding to RYR1 with nearly identical K i values (ϳ1-2 M), suggesting that inhibition by suramin is independent of Ca 2ϩ binding to both CaM and RYR1. A recent study (17) reported that a suramin IC 50 for inhibition of Ca 2ϩ -CaM binding to RYR1 be ϳ10-fold higher than we are reporting. The reason for the different apparent affinity of suramin for RYR1 is not clear but may be related to the use of 125 I-labeled CaM in their study versus 35 S-labeled CaM in the current study. We have previously demonstrated that the iodination of CaM using the Bolton-Hunter reagent can produce substantial artifacts in the analysis of CaM binding to RYR1 (10).
Suramin was found to bind to and competitively inhibit CaM binding to a peptide (R3609 -43) that corresponds to the CaM binding region of RYR1. Therefore, suramin would be expected to compete with CaM for binding to RYR1 proteins that are not interacting with the DHPR (e.g. purified RYR1 proteins or adjacent non-coupled RYR1 proteins present within SR-T tubule junctions  (Fig. 5). Suramin is apparently selective for certain types of CaM binding motifs since it does not inhibit CaM binding to the CaM binding domain of the carboxylterminal region of the DHPR ␣ 1 -subunit (Fig. 3). Because there is considerable difference in the primary sequence of the puta-  Table I (I-V (Table I, F-V) (control: ⌬F/F max ϭ 2.7, V F1/2 ϭ Ϫ2.8 mV, k ϭ 6.2 mV; suramin: ⌬F/F max ϭ 4.2, V F1/2 ϭ 3.0 mV, k ϭ 5.6 mV). In A and B, peak currents and transients were elicited by 200-ms depolarizations to the indicated test potentials. Representative Lcurrents (C) and Ca 2ϩ transients (D) for control and suramin dialyzed-myotubes are shown. Currents and transients for each condition are from the same cell. tive CaM binding domains of these two proteins, it seems likely that these differences form the basis of suramin RYR1 selectivity. This observation is also in agreement with the report by Klinger et al. (17) who found that suramin discriminates among different CaM binding motifs. Our data demonstrate that suramin competes effectively for binding to the CaM binding site on RYR1. We have previously suggested that the CaM binding motif on RYR1 not only binds CaM but also interacts strongly with the carboxyl-terminal tail of the DHPR ␣ 1 -subunit (1). In addition, the DHPR carboxyl terminus significantly inhibits RYR1 activity as assessed by effects on [ 3 H]ryanodine binding to SR membranes (1) and on the activity of Ca 2ϩ release channels reconstituted into planar lipid bilayers (12). We found that the pull down of the detergent-solubilized DHPRs using biotinylated R3609 -43 peptide and streptavidin beads is blocked by suramin. Also, suramin relieves the RYR1 inhibition by the carboxy-terminal tail of the DHPR ␣ 1 -subunit (Fig. 7C). Combined with our previous findings, these data suggest that suramin may have very different effects on DHPR-coupled and non-DHPR-coupled RYR1s. As outlined above, suramin should block the interaction of uncoupled channels with CaM, resulting in inhibition of activity at low Ca 2ϩ levels and enhancement at high Ca 2ϩ . However, because the carboxyl-terminal tail of the DHPR ␣ 1 -subunit inhibits RYR1 activity at both high and low Ca 2ϩ levels, then suramin would be expected to increase the activity of DHPRcoupled RYR1 channels by relieving inhibition mediated by the DHPR carboxyl terminus. Consistent with this model, suramin significantly augmented maximal voltage-gated SR Ca 2ϩ release in whole cell voltage-clamped mouse myotubes in a manner that occurred in the absence of effects on the Ca 2ϩ -conducting activity of the DHPR. This finding supports the model in which suramin acts specifically on modulating RYR1 activity. Two possible explanations exist for suramin potentiation of voltage-gated SR Ca 2ϩ release. First of all, the DHPR carboxyl terminus-RYR1 interaction may act to stabilize a closed/inactivated state of the release channel after membrane depolarization, thus limiting SR Ca 2ϩ release during excitation-contraction coupling. Suramin competition at this site would act to counter this inhibitory interaction and result in increased Ca 2ϩ release channel open probability and a potentiation of SR Ca 2ϩ release. Alternatively, our data cannot rule out a possible direct stimulatory effect of suramin on release channel activity after voltage activation of DHPR-coupled release channels. However, evidence from previous studies using in vitro assays (18,25) indicate that very high levels (0.3-1.0 mM) of suramin are required to activate RYR1, concentrations that are 6 -20 times greater than that found to alter voltage-gated Ca 2ϩ release in intact skeletal myotubes. Our observation of a potentiation of voltagegated Ca 2ϩ release by a relatively low concentration of suramin is even more striking considering potential limitations with regard to junctional accessibility of suramin introduced into myotubes via internal dialysis of patch-clamped myotubes.
Suramin at concentrations higher than 100 M displaces FKBP12 bound to RYR1 (data not shown), which may lead to an increase in release channel activity via a mechanism distinct from what has been suggested here.
In summary, our studies indicate that suramin inhibits both Ca 2ϩ -CaM and apoCaM binding/regulation of RYR1 by competing for the CaM binding sequence on RYR1 (encoded by RYR1 residues 3609 -3643). In addition, suramin increases the magnitude of the voltage-gated Ca 2ϩ release in skeletal myotubes, possibly by disrupting an inhibitory interaction between the carboxyl terminus of the DHPR ␣ 1 -subunit and the CaM binding region of RYR1. Future analysis of the effects of suramin analogs and their binding/regulation of RYR1 is likely to provide better tools for probing the role of CaM in the regulation of RYR1 and for investigating the functional role of the DHPR carboxyl terminus in the regulation of skeletal muscle excitation-contraction coupling.