Correspondence to: László Csernoch, Department of Physiology, University of Debrecen, P.O. Box 22, Debrecen, Hungary, H-4012. Fax:36-52-432-289 E-mail:csl{at}phys.dote.hu.
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Abstract |
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The effects of the muscle relaxant dantrolene on steps of excitation-contraction coupling were studied on fast twitch muscles of rodents. To identify the site of action of the drug, single fibers for voltage-clamp measurements, heavy SR vesicles for calcium efflux studies and solubilized SR calcium release channels/RYRs for lipid bilayer studies were isolated. Using the double Vaseline-gap or the silicone-clamp technique, dantrolene was found to suppress the depolarization-induced elevation in intracellular calcium concentration ([Ca2+]i) by inhibiting the release of calcium from the SR. The suppression of [Ca2+]i was dose-dependent, with no effect at or below 1 µM and a 53 ± 8% (mean ± SEM, n = 9, cut fibers) attenuation at 0 mV with 25 µM of extracellularly applied dantrolene. The drug was not found to be more effective if injected than if applied extracellularly. Calculating the SR calcium release revealed an equal suppression of the steady (53 ± 8%) and of the early peak component (46 ± 6%). The drug did not interfere with the activation of the voltage sensor in as much as the voltage dependence of both intramembrane charge movements and the L-type calcium currents (ICa) were left, essentially, unaltered. However, the inactivation of ICa was slowed fourfold, and the conductance was reduced from 200 ± 16 to 143 ± 8 SF-1 (n = 10). Dantrolene was found to inhibit thymol-stimulated calcium efflux from heavy SR vesicles by 44 ± 10% (n = 3) at 12 µM. On the other hand, dantrolene failed to affect the isolated RYR incorporated into lipid bilayers. The channel displayed a constant open probability for as long as 3050 min after the application of the drug. These data locate the binding site for dantrolene to be on the SR membrane, but be distinct from the purified RYR itself.
Key Words: calcium current, intramembrane charge, calcium release, ryanodine receptor, single channel
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INTRODUCTION |
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In susceptible individuals, the inhalation of certain volatile anesthetics triggers a sudden increase in body temperature, which, if left untreated, is usually lethal. The underlying reason for this increase in body temperature (malignant hyperthermia [MH]*) is the abnormal activation of skeletal muscle fibers and the consequent increase in metabolic rate throughout the body. In MH, apparently, the presence of the anesthetic is sufficient to trigger the release of calcium from its internal store, the SR in resting, unstimulated skeletal muscle fibers. To prevent this increase in muscle activity, dantrolene is administered. Dantrolene itself is a hydantoid derivative muscle relaxant that has been shown to decrease contractile force (
During normal excitation of a skeletal muscle fiber the change in transverse (t)-tubular voltage governs the conformational changes of the t-tubular dihydropyridine receptors (DHPRs) acting as voltage sensors. The interaction of the DHPRs with the calcium release channel of the SR, the RYRs, leads to the release of calcium from the SR into the myoplasmic space. In MH, this chain of events is disrupted, and calcium release can occur even in the absence of voltage sensor activation. The effect of dantrolene is, by a yet unknown mechanism, to suppress SR calcium release.
It is currently accepted that dantrolene must, either directly or indirectly, affect the RYR itself ( component (
and calcium release (
Dantrolene or its water-soluble analogue azumolene has been reported to inhibit calcium efflux from heavy SR vesicles (
Despite this close association, the calcium release channel and the dantrolene-binding site in skeletal muscle have not been equated. On the one hand, Fruen and co-workers (
To date, three isoforms of the calcium release channel have been identified in mammals. In spite of the homology between isoforms found, predominantly, in skeletal (RYR1) and cardiac (RYR2) muscles, dantrolene is known to preferentially inhibit calcium release in skeletal, but not in cardiac muscle (
Although dantrolene was shown to suppress the calcium transients in frog skeletal muscle (
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MATERIALS AND METHODS |
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Enzymatic Isolation of Single Fibers
Single skeletal muscle fibers were isolated enzymatically from the extensor digitorum communis or flexor digitorum brevis muscles of rats or mice and mounted into a double Vaseline gap chamber or silicone clamp as described earlier (
For the Vaseline-gap experiments, the selected fiber was transferred into a recording chamber (
For silicone-clamp experiments, the major part of an isolated mouse skeletal muscle fiber was electrically insulated with silicone grease so as to achieve whole-cell voltage clamp on a short portion (50100 µm long) of the fiber extremity (
Optical Set-up and Voltage Clamp
The experimental set-up and the data acquisition have been described in detail in our earlier reports ( > 600 nm) and was also epi-illuminated at 380 nm or at the isosbestic wavelength of Fura-2 using a 75 W Xenon arc lamp (model 60000; Oriel). Using appropriate interference filters and dichroic mirrors, light intensities were simultaneous recorded at 510, 720, and 850 nm for the detection of APIII absorbance and Fura-2 fluorescence. Fibers were voltage-clamped and the holding potential was set to -100 mV. In silicone-clamp experiments, the light from a high pressure mercury bulb (model USH102DH;, USHIO INC.) was used for fiber illumination. Excitation was set at 335 nm and fluorescence was detected simultaneously at 405 and 470 nm by two photo multipliers, using an appropriate set of filters and dichroic mirror. Fibers were voltage-clamped at a holding potential of -80 mV using a patch-clamp amplifier (model RK-400; Bio-Logic) in whole-cell configuration, connected to a microelectrode filled with the "intracellular-like" solution (see above). All experiments were performed at room temperature (2022°C).
Preparation of SR Vesicles and RYR
Heavy and light SR (HSR and LSR, respectively) vesicles and the RYR calcium release channel were isolated from rat skeletal muscle as described earlier (
For preparation of RYR, the HSR vesicles (3 mg/ml) were solubilized for 2 h at 4°C with 1% CHAPS (3[(3-chloramidopropyl)dimethyl-amino]-1-propanesulfonate) in a solution containing 1 M NaCl, 100 µM EGTA, 150 µM CaCl2, 5 mM AMP, 0.45% phosphatidylcholine, 20 mM Na-PIPES, pH 7.2, and protease inhibitors (
Calculation of [Ca2+]i and Rrel
In Vaseline-gap experiments, the changes in myoplasmic free calcium concentration ([Ca2+]i) were calculated from APIII absorbance as described by
The rate of calcium release from the SR (Rrel) was calculated from the calcium transients measured in Vaseline-gap experiments using the procedure described in
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(1) |
where Rrel,i[max] is the maximal release rate, V' is the voltage at half-maximal release rate, and k is the slope factor to the calculated data.
Intramembrane Charge Movement and Calcium Current
Intramembrane charge transfer and calcium current through L-type calcium channels were calculated by measuring membrane currents in response to depolarizing and hyperpolarizing pulses as described in detail previously (
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(2) |
where Qmax is the maximal available charge, and V' and k have their usual meaning.
Calcium currents (ICa) were measured using 800-ms-long depolarizing pulses exploring the -50 to +60 mV voltage range. To enable the subtraction of the linear capacitive component the -20-mV hyperpolarizing pulses were also extended to 800 ms in duration. The external calcium concentration was elevated to 5 mM as detailed above. The peak ICa versus voltage relationship was fitted with:
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(3) |
where ECa is the calcium equilibrium potential, Gmax is the maximal conductance, and all other parameters have their usual meaning. All currents, the amount of charge moved, and the maximal conductance were normalized to fiber capacitance to take the size of the individual fibers into account.
Calcium Efflux from HSR Vesicles
HSR vesicles were actively loaded with calcium, resulting in a 1.20 ± 0.11 µmol/mg protein average calcium content of the vesicles. Calcium efflux was determined by measuring the extravesicular calcium concentration using a Fluoromax (SPEX Inc.) spectrofluorometer modified for absorption measurements by monitoring the transmittance at 710 and 790 nm, and calculating the corrected absorbance change (A710 - A790) as described earlier (
Planar Lipid Bilayer Measurements
CHAPS solubilized RYR molecules were incorporated into planar lipid bilayers (
Chemicals
Fura-2 and indo-1 were purchased from Molecular Probes Inc., APIII was purchased from ICN Biomedicals. Protease inhibitors were purchased from Boehringer, Merck, and from Sigma-Aldrich. Lipids were obtained from Avanti Polar Lipids, [3H]ryanodine was purchased from DUPONT, and all other chemicals were from Sigma-Aldrich.
For calcium efflux and bilayer experiments, a dantrolene stock of 18 mM was prepared in DMSO, and the required aliquot was added directly into the measuring chamber. This stock was diluted in the external solution to determine the molar extinction coefficient of the drug ( = 14,700 cm-1M-1 at 390 nm). In Vaseline-gap or silicone-clamp experiments, dantrolene was dissolved in the applied aqueous solution to an attempted final concentration of 100 µM. Unsolubilized particles were removed by filtering the solution through 0.22-µm filters (Millipore Corp.). Using the molar extinction coefficient, this fully saturated solution was found to be 24.8 µM and will be referred to as 25 µM in RESULTS. Lower concentrations were obtained by diluting this fully saturated stock.
In statistical analyses, all values were expressed as mean ± SEM, statistical significance was calculated with the t test assuming significance for P < 0.05.
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RESULTS |
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Dantrolene Suppresses Calcium Transients
Fig 1 illustrates the effect of extracellular application of 25 µM dantrolene on the calcium transients elicited by short depolarizing pulses on intact mouse skeletal muscle fibers using the silicone-clamp technique. Fig 1 A shows a series of indo-1 calcium transients elicited by 20-ms-long depolarizing pulses to 0 mV applied every 30 s. Application of dantrolene produced an 50% decrease in the maximal amplitude of the calcium transient from 0.88 ± 0.24 µM (n = 4), the effect being complete within <2 min. Fig 1 B shows the time-dependent evolution of the mean maximum change in [Ca2+]i reached in response to a 20-ms duration depolarizing pulse to 0 mV, in control conditions, and upon dantrolene application and washout. Data correspond to the mean values obtained from four fibers. Values of peak change in [Ca2+]i from each fiber were normalized to the peak value measured in response to the first depolarization. In average, dantrolene produced a 58% suppression of the peak calcium transient, the effect being partly and slowly reversible upon dantrolene washout. This effect of dantrolene was not associated to any significant change in resting [Ca2+]i, which, within this series of measurements, exhibited a mean value of 64 ± 13 nM in control and 62 ± 12 nM at the time when dantrolene produced its full effect on the peak [Ca2+]i.
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These measurements were repeated using the double Vaseline-gap system to enable the calculation of SR calcium release. Thus, Fig 2 presents calcium transients and the calculated rate of calcium release from the SR (Rrel; see MATERIALS AND METHODS) at different dantrolene concentrations. Measurements in dantrolene were taken 510 min after the solution exchange. This was, based on Fig 1, believed to be long enough for the drug to reach its binding site and exert its full effects. As will be demonstrated in more detail later (see Fig 4, Fig 6, and Table 1), we were unable to reach substantial recoveries from the suppression caused by dantrolene if the drug was present for several minutes in the bathing solution. Therefore, Fig 2 presents data from three different fibers for the three different dantrolene concentrations (1, 5, and 25 µM) tested. To ease the comparison among different fibers, traces are presented on different scales that were selected to give equal maxima for the transients in control.
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Dantrolene caused a dose-dependent suppression of [Ca2+]i, reaching 65% at the highest concentration. The attenuation was present both in the rate of rise and in the amplitude of the calcium signals. The transients showed a continuous increase in control and after the addition of 1 and 5 µM dantrolene, whereas a definite maximum, followed by a gradual decline was measured if 25 µM dantrolene was present in the external solution. All of these characteristic changes were reflected in the calculated efflux rate of calcium from the SR. Although 1 µM dantrolene did not cause any measurable alteration in Rrel, higher concentrations gradually suppressed both the early peak and the quasi-steady component of SR calcium release.
As demonstrated in Fig 3 for 5 µM dantrolene, the above mentioned effects on [Ca2+]i were present at every membrane potential tested. Dantrolene attenuated the calcium transients by suppressing the rate of increase of both the early, fast elevation and the more gradual increase observed after 30 ms into the depolarizing pulse. On average, the maximal increase in 5 µM dantrolene was found to be 0.9 ± 0.2 µM (n = 7) for a 100-ms-long depolarization to 0 mV as compared with 1.8 ± 0.3 µM measured on the same fibers under control conditions. Although 1 µM dantrolene did not attenuate the maximal [Ca2+]i significantly (from 1.5 ± 0.3 to 1.4 ± 0.4 µM, n = 13), 25 µM of the drug caused a 53 ± 8% suppression (from 1.6 ± 0.3 to 0.7 ± 0.1 µM, n = 9). These data were consistent with those obtained on intact fibers (Fig 1), confirming that fibers retain their full dantrolene sensitivity after mounting them into the double Vaseline-gap system.
Fig 4 goes on to plot the maximal increase in [Ca2+]i in the whole voltage range examined to demonstrate that dantrolene had similar effects at every membrane potential. To ease the comparison between different fibers and drug concentrations, all data from a given fiber were normalized before averaging. For normalization, the maximum of the calcium transient measured under control conditions with a depolarization to 0 mV on the given fiber was used. Furthermore, Fig 4 displays the results, again normalized, from measurements where dantrolene was removed from the extracellular medium as the last intervention during the experiment. In these fibers, the full [Ca2+]i versus voltage excursion was completed three times, and, as is shown in Fig 4 A, the fibers remained fairly stable during such interventions. This observation allowed us to conclude that much of the suppression that remained after the washout of dantrolene was due to the fact that the drug was not completely removed from its binding site within the period of 1520 min necessary to complete the measurements at all voltages. Fig 4 A also demonstrates that increasing the amplitude of the depolarizing pulse beyond 100 mV, i.e., depolarizing the fibers to +10 or +20 mV, did not significantly increase the magnitude of the calcium transients in control. To restrict the number of depolarizing pulses to a minimum necessary to assess the voltage dependence, pulses beyond 0 mV were not included into the experimental protocol for the other two dantrolene concentrations.
Comparing the traces in Fig 3 at -40 mV and the data presented for control conditions and in the presence of dantrolene in Fig 4 (B and C) reveals that the drug did not, significantly, alter the membrane potential at which detectable increase in [Ca2+]i was observed (-51.1 ± 2.5 and -53.3 ± 3.0 mV in control and in the presence of 25 µM dantrolene, respectively, n = 9). These data, taken together, confirm that dantrolene suppresses the depolarization-induced elevation of [Ca2+]i in a generally voltage-independent manner in mammalian skeletal muscle fibers.
SR Permeability Is Attenuated by Dantrolene
To describe the effects of dantrolene on the SR calcium release process, the rate of calcium release was corrected for depletion of calcium in the SR and the obtained transients were normalized to SR contents. This content was found to be fairly stable over the fibers included into the present study (1.8 ± 0.1, 2.0 ± 0.1, and 1.9 ± 0.2 mM for the fibers subjected to 1, 5, and 25 µM dantrolene; n = 13, 7, and 9) and to be unaltered by the treatment with dantrolene. Similarly, dantrolene left the resting [Ca2+]i ([Ca2+]rest) as well as the parameters of the removal model unaltered. As an example, the values of [Ca2+]rest were found to be 109 ± 17 nM in control and 107 ± 12 nM after the addition of 25 µM dantrolene. The corresponding values of the parameters of the removal model, if fitted independently in control and in the presence of the drug, were as follows: koff,M-P = 8.5 ± 2.1 and 5.2 ± 1.6 s-1, PVmax = 3.1 ± 0.5 and 2.5 ± 0.3 µMs-1, koff,C-E = 3.3 ± 0.8 and 3.6 ± 0.8 s-1, and kon,C-E = 2.1 ± 0.4 and 2.0 ± 0.4 µMs-1. Therefore, in the calculations, we used the same values for the removal parameters for control, in the presence of the drug and after washout in most fibers, unless they gave unrealistic Rrel records (e.g., large negative phases after repolarization). In the latter case, separate sets of parameters were used.
Fig 5 presents SR calcium permeabilities calculated from the calcium transients in Fig 3. In this case, the same set of removal parameters was used before and after the addition of the drug. Dantrolene caused a marked suppression of both the early and the sustained steady components of SR permeability at all membrane potentials tested. This observation is presented in Fig 6, where pooled data of the two kinetic components of SR permeability are shown as a function of membrane potential in control in the presence of the drug and after washout. The organization of Fig 6 follows that of Fig 4, individual data points were normalized before averaging. To this end, Equation 1 was fitted to the data (peak or steady level versus voltage) in control for each and every fiber and the obtained maximum (Equation 1, Rrel,i[max]) was used for normalization. Fig 6 reveals a dose-dependent suppression of both the early peak and the steady component of SR permeability reaching >50% in case of 25 µM dantrolene. It also shows that, as expected from the data on the calcium transients, no significant recovery was achieved after the removal of the drug.
To quantify the suppression caused by the different concentrations, Equation 1 was fitted to the data (peak or steady level versus voltage) in the presence and absence of dantrolene and after washout in every fiber. The obtained parameters, Pmax or SLmax (Equation 1, Rrel,i[max] with i = peak or steady level, respectively), the midpoint voltage, and the slope factor, are presented in Table 1 after averaging. The data reveal that 1 µM dantrolene did not have any measurable effects on the depolarization-induced increase in SR permeability. On the other hand, 5 and 25 µM of the drug significantly (P < 0.02) attenuated both the early peak and the steady level. Although the effect on the steady level was somewhat larger than on the peak, 53 ± 8 vs. 46 ± 6% for 25 µM, this difference did not prove to be statistically significant.
Comparing the voltage dependence of the different components as well as the effect of dantrolene on the voltage dependence did not reveal any systematic difference or trend. The only statistically significant change occurred at 25 µM, where a shift toward more positive membrane potentials was detected in the voltage dependence of the peak. The fact that dantrolene had very little effects on the voltage dependence of either component suggested a voltage-independent block of SR permeability by the drug. This was confirmed by directly calculating the suppression in every fiber in the -40 to 0 mV voltage range and averaging these relative data. The largest and the smallest values were, respectively, 0.63 ± 0.14 (at -40 mV) and 0.45 ± 0.09 (at -30 mV) for the peak and 0.65 ± 0.13 (at 0 mV) and 0.55 ± 0.1 (at -30 mV) for the steady level with 5 µM dantrolene. Corresponding values in 25 µM were 0.59 ± 0.06 (at -40 mV) and 0.43 ± 0.08 (at -20 mV) for the peak and 0.51 ± 0.02 (at -40 mV) and 0.39 ± 0.05 (at 0 mV) for the steady level, none of which was found to be significantly different. These data establish that dantrolene attenuates SR permeability in a voltage-independent manner. The extent of suppression is equal for the two kinetic components, peak and steady level, of SR permeability.
Intracellular Application Does Not Alter the Effective Concentration for Dantrolene
The following series of measurements were designed to test whether or not extracellular application of dantrolene could somehow limit the access of the drug to its putative intracellular binding site, since submicromolar dissociation constants have been reported (150 nM;
Under our standard conditions of microinjection, the concentration of the fluorescent dye present in the injected solution is estimated to be diluted by a factor of 510, once fully equilibrated within the entire volume of the cell (
Fig 7 shows the results from a fiber where dantrolene was injected a few minutes before voltage clamping and giving depolarizing pulses. In this fiber, a 20-ms-long pulse to 0 mV was applied every minute. Fig 7 A shows calcium transients measured 13 (trace a), 53 (trace b) and 103 min (trace c) after dantrolene had been injected into the fiber. Each trace is the average of five consecutive calcium transients. Fig 7 B follows the evolution of the change in the peak of [Ca2+]i over the course of the experiment. No significant suppression of the peak [Ca2+]i occurred under these conditions, only toward the end of the experiment. However, note that the mean value for the maximum of the first calcium transients obtained in these injected fibers was 0.76 ± 0.1 µM (n = 8) as compared with 0.88 ± 0.24 µM in control cells (Fig 1).
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To test the possibility that the relatively little, if any, effect of injected dantrolene was due to its fast diffusion, the above experiments were repeated on fibers isolated from rats. In these fibers, the length of diffusion was, on average, twice that in mouse fibers. The inset in Fig 7 displays the evolution of the relative amplitude of the calcium transients from these experiments. As in Fig 1 B, the first calcium transient was used for normalization and the normalized values were averaged over the fibers. The injection of dantrolene caused an 20% decrease in the amplitude of the calcium transients that was complete within 15 min.
Assuming that the first calcium transient (taken 5 min after injection) represents the control situation, these data suggest that intracellularly applied dantrolene has relatively little effect on the micromolar concentration. The magnitude of this effect is comparable to that seen with extracellular application. If, on the other hand, the injected dantrolene exerted the majority of its effects before the first calcium transient was taken, then additional application of external dantrolene should have little, or no, further effect.
Fig 8 tests this hypothesis by showing results from two other fibers that were injected with dantrolene. In Fig 8 (A and B), measurements were taken 30 min after dantrolene had been injected. The fiber was repeatedly depolarized using 20-ms-long pulses to 0 mV, and the effect of a brief, transient extracellular application of 25 µM dantrolene was tested. Results show that, under these conditions, extracellular dantrolene still maintained its potency to partially depress the depolarization-induced increase in [Ca2+]i. This conclusion is further strengthened by the results shown in Fig 8 (C and D), which were obtained from another fiber 1 h after dantrolene had been injected. A 30-ms-long pulse to 0 mV was applied every minute. Traces shown in Fig 8 C correspond to the average of three successive calcium transients measured in control (a), and in the presence of 0.5 (b), 12.5 (c), and 25 µM (d) extracellular dantrolene. In this experiment, the presence of each dantrolene concentration was maintained for 5 min and followed by a 5-min washout period. Under these conditions, dantrolene at 12.5 and 25 µM had an effective, dose-dependent depressing effect on the peak calcium transients. Similar results were obtained on four other fibers with 40 ± 2% and 67 ± 11% suppression at 12.5 and 25 µM dantrolene, respectively. Overall, these results strongly argue against the possibility that dantrolene, when applied intracellularly would suppress calcium transients at a much lower concentration than when applied in the extracellular medium. This implies that the surface membrane does not limit the ability of dantrolene to access its binding site.
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Effects of Dantrolene on the Dihydropyridine Receptor
Results so far suggest an interaction of the drug with a step of excitation-contraction coupling subsequent to the voltage-sensing event. To directly exclude the involvement of the voltage sensor as the putative dantrolene-binding site, its two functional manifestations, intramembrane charge movements and L-type calcium currents, were measured in separate sets of experiments. These experiments also tested the possibility of any "backward" flow of information in the coupling process, i.e., did the suppression of SR calcium release affect the functioning of the DHPRs.
Fig 9 A presents nonlinear capacitive currents representing intramembrane charge movement in the absence and presence of 25 µM dantrolene. The transients were recorded in response to 100-ms-long depolarizing pulses exploring the -80 to 0mV voltage range. The actual currents were normalized to linear capacitance to take into account the differences in fiber size, when comparing different fibers, or any possible change in passive electric properties during the experiments. However, the linear capacitance, on average, did not change significantly during the course of these experiments since it was found to be 13.7 ± 0.7 nF (n = 12) under control conditions and 13.7 ± 0.9 nF on the same cells after the addition of dantrolene. Thus, the fibers were stable not only from the aspect of calcium release, but also electrically.
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To assess the voltage dependence of charge transfer the currents were integrated and Equation 2 was fitted to the charge versus voltage data both before and after the addition of dantrolene. Average values of the parameters from these fits are presented in Table 2. To visualize the effects of dantrolene on the voltage dependence of charge transfer, Fig 9 B plots the normalized charge, amount of charge at a given voltage divided by Qmax determined under the given condition in that fiber, as a function of membrane potential during the test pulse. Although there seemed to be a slight shift of the voltage dependence to the right along the voltage axis, by 5 mV, together with a slight decrease in steepness (an increase in parameter k) these changes did not prove to be statistically significant. Similarly, the elevation of the total amount of available charge, from 23.8 ± 2.4 to 27.6 ± 3.9 nCµF-1 on average (n = 10), remained within the limits of statistical variation.
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Another manifestation of DHPR function, the L-type calcium current was measured in a separate set of experiments using 800-ms-long depolarizing pulses and elevated calcium concentration in the external solution while exploring a wider, from -50 to +60 mV, voltage range. Calcium currents were readily observed under these conditions, and they displayed all the usual characteristics in control (Fig 10 Aa) as described earlier (160 ms into the pulse, currents slowly inactivated with a time constant on the order of 0.20.5 s (0.26 ± 0.02 s at 0 mV; Fig 10 E). After the addition of 25 µM dantrolene, currents became smaller and their inactivation was drastically slowed down (Fig 10 Ab). To describe the voltage dependence of current activation, the maximal current, averaged over the fibers, was plotted as a function of membrane potential in Fig 10 C and fitted with Equation 3. Although the fit revealed a substantial decrease in maximal conductance, from 184 to 111 SF-1, the other parameters describing the voltage dependence remained, essentially, unaltered.
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Since the alteration in calcium current seen in the presence of dantrolene resembled that of current run-down (
Since the maximal conductance was indeed suppressed by dantrolene, it was of interest to see whether some of the alteration in inactivation was also due to the presence of the drug. Thus, Fig 10 E plots the time constants of inactivation, obtained by fitting a single exponential function to the decaying part of the current traces, as a function of the membrane potential. As for Fig 10 D, only those data were included in the figure where the measurements in the given condition, control or dantrolene, comprised the first series of records. As demonstrated in Fig 10 E, dantrolene caused a three- to fourfold increase (from 0.26 ± 0.02 to 0.80 ± 0.18 s at 0 mV) in the time constant of inactivation at every membrane potential tested. It is not clear at present, whether the decrease in the conductance L-type calcium channels and in the rate of inactivation is a direct or an indirect effect of dantrolene on the DHPRs. Nevertheless, the data establish that the drug does not affect the voltage sensor of EC coupling in a way that could explain its effects on SR permeability. Therefore, the putative binding site must lay further downstream in the steps of events leading to the opening SR calcium channels.
Dantrolene Inhibits Calcium Efflux from HSR Vesicles
Previous sections have established that 25 µM dantrolene suppresses SR permeability by 50% without altering the voltage sensor function of the DHPRs. However, they do not exclude a putative binding site to be located in the surface membrane that might influence the interaction of DHPRs and the RYRs. To exclude this possibility, HSR vesicles were prepared, and the effect of dantrolene was tested on the efflux of calcium.
HSR vesicles were actively loaded with calcium in the presence of ATP and, after the ATP was essentially used up, to prevent reuptake during release, calcium efflux was initiated by the addition of 300 µM thymol, a potent activator of RYRs (
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Since thymol could not be removed from the solution during the course of an experiment, Fig 11 compares different experiments for the different dantrolene concentrations. However, it should be noted that the amount of protein and the amount of calcium loaded into the HSR vesicles were identical for the different experimental conditions. The latter was ensured by the addition of known and set amounts of calcium into the extravesicular space, and by continuously monitoring its uptake into the vesicles. The amounts of calcium released, therefore, can be compared directly.
The presence of dantrolene in the extravesicular medium substantially reduced the rate of calcium efflux from HSR vesicles. The attenuation of the maximal release rate reached 44 ± 11% at 12 µM, a value that is comparable with that obtained from intact fibers for the suppression of SR permeability (Table 1), suggesting that the putative dantrolene-binding site was fully preserved during the isolation of the HSR vesicles. These data clearly place the putative binding site to be on a SR-associated molecule.
Dantrolene Does Not Influence the Isolated RYR
Previous experiments (
Fig 12 demonstrates the effects of high (50 µM) dantrolene concentration on the gating of the RYR. The traces are representative (2.2-s long) segments of current records from an experiment that lasted for more than an hour. The incorporation was initiated under control conditions with 50 µM [Ca2+] on either side of the bilayer. Under these conditions (Fig 12 A), the channel displayed an open probability (Po) of 0.175 and a conductance of 555 pS, as expected from earlier studies. However, in contrast to previous results using SR vesicles, the addition of 50 µM dantrolene into the cis chamber influenced neither the open probability nor the conductance of the purified channel. This is shown in Fig 12B and Fig C, at 5 and 30 min after the addition of the drug, respectively. The inset of Fig 12 follows the evolution of the open probability of the channel during the 30-min after the addition of the drug, demonstrating that dantrolene did not have any transient or long-term effects on channel gating.
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Fig 12 goes on to demonstrate that the drug did not interfere with the ability of well-known regulators to modulate channel function. First, the concentration of calcium was reduced in the cis chamber (Fig 12 D), which resulted in a drastic reduction in Po (to 0.001). This was followed by the readministration of calcium (Fig 12 E), and then the addition of 3 mM ATP (Fig 12 F) into the cis chamber. This first restored the Po to its starting value and then resulted, as expected, in an activation of the channel (Po = 0.874). Finally, the addition of ryanodine caused the characteristic transition to a half-conducting state.
Since previous experiments suggested a high affinity, activatory binding site for dantrolene (
These data establish that dantrolene has no effect, either transiently or permanently, on the gating behavior of the purified calcium release channel incorporated into planar lipid bilayers. The fact that the pharmacology of the channel is retained renders the possibility of major modification during the purification, unlikely. Taken together, these data strongly argue against the possibility of the binding site for dantrolene being on the purified ryanodine receptor itself.
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DISCUSSION |
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For the first time, these experiments give a complete account of the effects of dantrolene on steps of excitation-contraction coupling in mammalian skeletal muscle fibers. They explored the voltage sensing function of the DHPRs by measuring both intramembrane charge movements and L-type calcium currents. They also assessed the functioning of the RYRs by comparing calcium release flux from the SR in intact fibers to calcium efflux from HSR vesicles and to alterations in single-channel properties of the calcium release channel. Dantrolene was found not to interfere with the voltage-sensing event, but was found to suppress SR calcium permeability and calcium efflux to a similar extent. On the other hand, the drug failed to alter the single-channel properties and the basic pharmacology of the isolated RYR. These data place the putative binding site to be on a molecule associated with the SR membrane, but exclude the purified calcium release channel itself.
Calcium Release from the SR
Dantrolene has been in use for treating MH for more than three decades (
Due to the lack of data on calcium transients from mammalian skeletal muscle fibers in the presence of dantrolene, no account on the effects of the drug on SR calcium release has been published so far. Not only is this true for mammalian skeletal muscle but, to our knowledge, also for striated muscle cells from other classes of vertebrates or invertebrates. These experiments would be of special interest since lower vertebrates have been shown to express two isoforms of RYR in their skeletal muscle (
In mammalian fibers predominantly expressing the skeletal isoform of RYR, we haven't found any significant difference between the effects on the two kinetic components, peak and steady level, of SR permeability (Table 1). This observation is similar to that reported for low (10 µM) concentrations of tetracaine on the same preparation (
Location of the Putative Dantrolene-binding Site
There is a detailed description in the literature of reduced [3H]ryanodine binding to and reduced calcium efflux from SR vesicles in the presence of dantrolene or its water-soluble analogue azumolene (
Whether the putative dantrolene-binding site could be equated with the RYR itself, or not, has been a long controversy in the literature. Recent biochemical data link the binding site of [3H]azidodantrolene to a 160- or a 172-kD protein (
Effects of Dantrolene of DHPR Function
Previous studies on the effect of dantrolene on intramembrane charge movements in frog skeletal muscle have revealed a slight reduction in total charge (, the delayed component of intramembrane charge (
It has been reported that dantrolene does not influence L-type calcium currents in frog twitch muscle fibers (
To explain the observed voltage dependence and dantrolene action on current inactivation three possibilities should be considered: (1) the effect of reduced calcium current or SR calcium release; (2) direct action of the drug on the DHPR; and (3) retrograde effect from the putative intracellular dantrolene-binding site. The "U-shaped" voltage dependence of the time constant strongly argues in favor of a current and/or calcium-dependent inactivation, which could be due to either of the following: (1) the depletion of calcium in the restricted space of the t-tubular system; (2) a direct effect of the calcium ions passing through the L-type calcium channel itself; or (3) calcium ions released from the SR. Plotting the time constant as a function of the peak current in the control (unpublished data) gave a linear function, further strengthening the above idea. However, a similar plot in the presence of dantrolene gave a straight line with a different slope, indicating that the shift seen in the presence of the drug could not be due, simply, to a reduction in current.
SR calcium release, known to be important in the heart, seems unlikely to be the cause of current inactivation under our experimental conditions. The presence of 20 mM EGTA in the internal solution should have reduced the change in [Ca2+]i even in the relatively restricted compartment of the triadic space (see calculations in
It should be stressed that the properties mentioned above do not, inevitably, rule out the current and/or calcium dependence of inactivation. Nevertheless, they suggest that other possibilities, such as voltage dependence or an interaction between dantrolene or its binding site with the DHPR, should be considered. In this framework, the drug does not influence the normal voltage sensing machinery for activation (compare intramembrane charge movements in the absence and presence of the drug in Fig 9), but interferes with (slows the transition of) those responsible for inactivation.
This brings up the possibility that a retrograde effect from the putative intracellular dantrolene-binding site or from the altered gating of RYR would be responsible for the observed effects. The latter is supported by data obtained on dyspedic myotubes from mice, where L-type calcium currents were reduced and the gating kinetics of the calcium channels was altered (
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Footnotes |
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* Abbreviations used in this paper: APIII, antipyrylazo III; DHPR, dihydropyridine receptor; MH, malignant hyperthermia; Po, open probability; Rrel, rate of calcium release from the SR; t-tubular, transverse tubular.
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Acknowledgements |
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The authors wish to thank Ms. R. Öri, T. Kiss-Tóth, and B. Lukács for skilful technical assistance.
This work was supported by research grants from Hungary (OTKA 030246, 034894, ETT 49/2000, FKFP 0193/2001, and AKP 98-75 3,2/44) and the European Community (CT96-0032). P. Szentesi holds a Bolyai fellowship.
Submitted: 6 March 2001
Revised: 12 July 2001
Accepted: 9 August 2001
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References |
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