Effects of dantrolene on steps of excitation-contraction coupling in mammalian skeletal muscle fibers.

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 microM and a 53 +/- 8% (mean +/- SEM, n = 9, cut fibers) attenuation at 0 mV with 25 microM 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 (I(Ca)) were left, essentially, unaltered. However, the inactivation of I(Ca) 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 microM. 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 30-50 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.

Altered inhibition of the rat skeletal ryanodine receptor/calcium release channel by magnesium in the presence of ATP.

Magnesium-induced inhibition of the skeletal ryanodine receptor/calcium-release channel (RyR) was studied in the presence and absence of ATP under isolated conditions and in situ, by examining the RyR incorporated into a planar lipid bilayer and the calcium release flux (Rrel) in isolated single fibres mounted in the double Vaseline gap system. When the incorporated RyR had been activated by calcium (50 microM) in the absence of ATP, the magnesium-induced inhibition showed co-operativity with a Hill coefficient (N) of 1.83 and a half-inhibitory concentration (IC50) of 635 microM. When the open probability was measured in the presence of 5 mM ATP and at a low calcium concentration, the magnesium-induced inhibition was non-cooperative (N=1.1, IC50= 860 microM). In isolated muscle fibres, in the presence of ATP, lowering the intracellular magnesium concentration ([Mg2+]i) increased the maximal Rrel and shifted its voltage dependence to more negative membrane potentials. Increasing [Mg2+]i had the opposite effect. The concentration dependence was described with an IC50 of 174 microM, N=1, under depolarized conditions and showed a tenfold increase in affinity in polarized fibres. At the concentration required for the measurements from isolated fibres, ATP had a full activatory effect on the isolated channel. At a low calcium concentration, the RyR had two ATP-binding sites with half-activatory concentrations of 19 and 350 microM, respectively.

Regulation of the rat sarcoplasmic reticulum calcium release channel by calcium.

The regulation by calcium of the ryanodine receptor/SR calcium release channel (RyR) from rat skeletal muscle was studied under isolated conditions and in situ. RyRs were either solubilized and incorporated into lipid bilayers or single fibres were mounted into a Vaseline gap voltage clamp. Single channel data were compared to parameters determined from the calculated calcium release flux. With K+ (250 mM) being the charge carrier the single channel conductance was 529 pS at 50 microM Ca2+ cis and trans, and decreased with increasing cis [Ca2+]. Open probability showed a bell shaped calcium dependence revealing an activatory and an inhibitory Ca2+ binding site (Hill coefficients of 1.18 and 1.28, respectively) with half activatory and inhibitory concentrations of 9.4 and 298 microM. The parameters of the inhibitory site agreed with the calcium dependence of channel inactivation deduced from the decline in SR calcium release in isolated fibres. Mean open time showed slight [Ca2+] dependence following a single exponential at every Ca2+ concentration tested. Closed time histograms, at high [Ca2+], were fitted with three exponentials, from which the longest was calcium independent, and resembled the recovery time constant of SR inactivation (115+/-15 ms) obtained in isolated fibres. The data are in agreement with a model where calcium binding to the inhibitory site on RyR would be responsible for the calcium dependent inactivation in situ.

Biphasic effect of bimoclomol on calcium handling in mammalian ventricular myocardium.

1. Concentration-dependent effects of bimoclomol, the novel heat shock protein coinducer, on intracellular calcium transients and contractility were studied in Langendorff-perfused guinea-pig hearts loaded with the fluorescent calcium indicator dye Fura-2. Bimoclomol had a biphasic effect on contractility: both peak left ventricular pressure and the rate of force development significantly increased at a concentration of 10 nM or higher. The maximal effect was observed between 0.1 and 1 microM, and the positive inotropic action disappeared by further increasing the concentration of bimoclomol. The drug increased systolic calcium concentration with a similar concentration-dependence. In contrast, diastolic calcium concentration increased monotonically in the presence of bimoclomol. Thus low concentrations of the drug (10 - 100 nM) increased, whereas high concentrations (10 microM) decreased the amplitude of intracellular calcium transients. 2. Effects of bimoclomol on action potential configuration was studied in isolated canine ventricular myocytes. Action potential duration was increased at low (10 nM), unaffected at intermediate (0.1 - 1 microM) and decreased at high (10 - 100 microM) concentrations of the drug. 3. In single canine sarcoplasmic calcium release channels (ryanodine receptor), incorporated into artificial lipid bilayer, bimoclomol significantly increased the open probability of the channel in the concentration range of 1 - 10 microM. The increased open probability was associated with increased mean open time. The effect of bimoclomol was again biphasic: the open probability decreased below the control level in the presence of 1 mM bimoclomol. 4. Bimoclomol (10 microM - 1 mM) had no significant effect on the rate of calcium uptake into sarcoplasmic reticulum vesicles of the dog, indicating that in vivo calcium reuptake might not substantially be affected by the drug. 5. In conclusion, the positive inotropic action of bimoclomol is likely due to the activation of the sarcoplasmic reticulum calcium release channel in mammalian ventricular myocardium.

Effects of tetracaine on sarcoplasmic calcium release in mammalian skeletal muscle fibres.

1. Single muscle fibres were dissociated enzymatically from the extensor digitorum communis muscle of rats. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation-contraction coupling were studied under voltage clamp conditions in the presence and absence of the local anaesthetic tetracaine. 2. Changes in intracellular calcium concentration ([Ca2+]i) were monitored using the calcium sensitive dyes antipyrylazo III and fura-2 and the rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) was calculated. Tetracaine decreased the maximal attained [Ca2+]i and suppressed, in a dose-dependent manner, both the early peak and the steady level of Rrel in the voltage range examined. 3. The concentration dependence of the effects on the two kinetic components of Rrel were almost identical with a half-effective concentration (K50) of 70 and 71 microM and a Hill coefficient (nH) of 2.7 and 2.3 for the peak and the steady level, respectively. Furthermore, the drug did not alter the peak to steady level ratio up to a concentration (50 microM) that caused a 35 +/- 5 % reduction in calcium release. Higher concentrations did suppress the ratio but the degree of suppression was voltage independent. 4. Tetracaine (50 microM) neither influenced the total available intramembrane charge nor altered its membrane potential dependence. It shifted the transfer function, the normalized SR permeability versus normalized charge to the right, indicating that similar charge transfer caused a smaller increase in SR permeability. 5. To explore the site of action of tetracaine further the ryanodine receptor (RyR) calcium release channel of the SR was purified and reconstituted into planar lipid bilayers. The reconstituted channel had a conductance of 511 +/- 14 pS (n = 8) in symmetric 250 mM KCl that was not affected by tetracaine. Tetracaine decreased the open probability of the channel in a concentration-dependent manner with K50 = 68 microM and nH = 1.5. 6. These experiments show that tetracaine suppresses SR calcium release in enzymatic isolated mammalian skeletal muscle fibres. This effect is due, presumably, to the decreased open probability of the RyR in the presence of the drug. Since both the inactivating peak and the steady level of Rrel were equally affected by tetracaine, our observations suggest that there is a tight coupling between these kinetic components of SR calcium release in mammalian skeletal muscle.

Calsequestrin: more than 'only' a luminal Ca2+ buffer inside the sarcoplasmic reticulum.

In striated muscle, the sarcoplasmic reticulum (SR) Ca2+ release/ryanodine receptor (RyR) channel provides the pathway through which stored Ca2+ is released into the myoplasm during excitation-contraction coupling. Various luminal Ca2+-binding proteins are responsible for maintaining the free [Ca2+] at 10(-3)-10(-4) M in the SR lumen; in skeletal-muscle SR, it is mainly calsequestrin. Here we show that, depending on its phosphorylation state, calsequestrin selectively controls the RyR channel activity at 1 mM free luminal [Ca2+]. Calsequestrin exclusively in the dephosphorylated state enhanced the open probability by approx. 5-fold with a Hill coefficient (h) of 3.3, and increased the mean open time by about 2-fold, i.e. solely dephosphorylated calsequestrin regulates Ca2+ release from the SR. Because calsequestrin has been found to occur mainly in the phosphorylated state in the skeletal-muscle SR for the regulation of RyR channel activity, the dephosphorylation of calsequestrin would appear to be a quintessential physiological event.

Concentration-dependent effects of tetracaine on excitation-contraction coupling in frog skeletal muscle fibres.

The effects of low (10-100 microM) concentrations of tetracaine on intermembrane charge movement and on the rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) were studied in cut skeletal muscle fibres of the frog using the voltage clamp technique. The fibres were mounted in a single or double vaseline gap chamber to study the events near the contraction threshold or in a wide membrane potential range. Although the 'hump' component of charge movement (Q gamma) was suppressed to some extent, the voltage dependence and the parameters of the Boltzmann distribution were not modified significantly at tetracaine concentrations below 50 microM. At 50 and 100 microM of tetracaine the midpoint voltage of the Boltzmann distribution was shifted to higher membrane potentials and the steepness was decreased. The total available charge remained the same at all concentrations tested. Using fura-2 to measure calcium transients at 100 microM tetracaine the threshold for calcium release was found to be significantly shifted to more positive membrane potentials. Tetracaine reversibly suppressed both the early inactivating peak and the steady-level of Rrel but the concentration dependence of the effects was markedly different. The inactivation component of calcium release was decreased with a Hill coefficient of approximately 1 and half effective concentration of 11.8 microM while the steady-level was decreased with a Hill coefficient of greater than 2 and a half effective concentration of 47.0 microM. These results favour two sites of action where tetracaine would suppress the calcium release from the SR.

Activation of the skeletal muscle ryanodine receptor by suramin and suramin analogs.

Ca2+ release from skeletal muscle sarcoplasmic reticulum is activated by adenine nucleotides and suramin. Because suramin is known to interact with ATP-binding enzymes and ATP receptors (P2-purinergic receptors), the stimulation by suramin has been postulated to occur via the adenine nucleotide-binding site of the ryanodine receptor/Ca2+-release channel. We tested this hypothesis using suramin and the following suramin analogs: NF037, NF018, NF023, and NF007. The suramin analogs stimulate the binding of [3H]ryanodine binding to sarcoplasmic reticulum membranes with the following rank order of potency: suramin (EC50 = approximately 60 microM) > NF037 (EC50 = approximately 150 microM) > NF018 > NF023 > NF007. The suramin-induced stimulation occurs via a myoplasmic binding site on the ryanodine receptor as confirmed by binding experiments and single-channel recordings with the purified protein. This binding site is different than that for ATP, a conclusion that is supported by the following observations: (i) Suramin stimulates the association rate and inhibits the dissociation rate of [3H]ryanodine, whereas ATP analogs increase only the on-rate. (ii) In the presence of suramin but not of ATP analogs, [3H]ryanodine binding is resistant to the inhibitory effect of millimolar Mg2+ and Ca2+. (iii) ATP analogs and suramin have an additive effect on [3H]ryanodine binding. (iv) Affinity labeling of the purified ryanodine receptor with 2',3'-dialdehyde [alpha-32P]ATP or after in situ oxidation of [gamma-32P]ATP is not affected by suramin. Thus, our results show that suramin acts as a direct and potent stimulator of the ryanodine receptor but that this action is mediated via a binding site different from that for adenine nucleotides.

Effects of cardiac glycosides on excitation-contraction coupling in frog skeletal muscle fibres.

1. The effects of digoxin and ouabain on the calcium release flux from the sarcoplasmic reticulum (SR), isometric tension and intramembrane charge movement were studied in voltage clamped skeletal muscle fibres of the frog. 2. Both cardiac glycosides increased both calcium transients and simultaneously recorded tension at all membrane potentials, showing different effects on the peak and on the steady components of the calcium release flux. These effects were attained at an extracellular digoxin concentration of 5 nM and an estimated intracellular ouabain concentration of 1-2 nM. Digoxin and ouabain thus exerted their effects at the same concentration on calcium release in skeletal muscle as previously observed in isolated cardiac-type ryanodine receptor (RyR) calcium release channels. 3. The peak of SR calcium release increased at all voltages, with the largest potentiation at intermediate membrane potentials. This increase in calcium release flux was attained despite an unchanged SR calcium content. The attenuated release rate therefore reflected an increased number of open RyR channels rather than increased SR loading. 4. These effects could be attributed to an increase in calcium release activation and not a decrease in the rate of inactivation. Rather, the rate of inactivation was enhanced at all voltages as expected from the increased calcium concentration in the triadic junction. 5. In contrast, CMA (17 alpha-acetoxy-6-chloro-4, 6-pregnadiene-3,20-dione; 5 microM), a Na(+)-K(+)-ATPase inhibitor with no positive inotropic effects on the heart, neither influenced SR calcium release nor antagonized the effects of ouabain. 6. Both digoxin and ouabain preserved total intramembrane charge apart from a small negative shift in the mid-point voltage and increase in slope factor. 7. Both digoxin and ouabain induced calcium release from heavy SR vesicles at rates comparable to that induced by ryanodine or caffeine. 8. It is concluded that at least part of the inactivating component of SR calcium release involves distinct RyR calcium release channels that resemble the cardiac RyR isoform in its specific sensitivity to cardiac glycosides.

4-Chloro-m-cresol, a potent and specific activator of the skeletal muscle ryanodine receptor.

The aim of the present study was to determine the effects of 4-chloro-m-cresol (4-CmC), a preservative often added to drugs intravenously administered, on the skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release channel/ryanodine receptor. In heavy SR vesicles obtained from rabbit back muscles, 4-CmC stimulated (Ca2+)-activated [3H]ryanodine binding with an EC50 of about 100 microM. In the same concentration range, 4-CmC directly activated the isolated Ca2+ release channel reconstituted into planar lipid bilayers. The sensitivity to 4-CmC was found to be higher when applied to the luminal side of the channel suggesting binding site(s) different from those of nucleotides and caffeine. In skeletal muscle fibre bundles obtained from biopsies of patients susceptible to malignant hyperthermia, a skeletal muscle disease caused by point mutations in the ryanodine receptor, 4-CmC evoked caffeine-like contractures. Contrary to caffeine which induces contractures in millimolar concentrations, the threshold concentration for 4-CmC was 25 microM compared to 75 microM for non-mutated control fibres. Since these data strongly indicate that 4-CmC specifically activates SR Ca2+ release also in intact cell systems, this substance might become a powerful tool to investigate ryanodine receptor-mediated Ca2+ release in muscle and non-muscle tissue.