István Jóna

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.

Surface plasmon resonance studies prove the interaction of skeletal muscle sarcoplasmic reticular Ca2+ release channel/ryanodine receptor with calsequestrin

A high affinity molecular interaction is demonstrated between calsequestrin and the sarcoplasmic reticular Ca2+ release channel/ryanodine receptor (RyR) by surface plasmon resonance. K D values of 92 nM and 102 nM for the phosphorylated and dephosphorylated calsequestrin have been determined, respectively. Phosphorylation of calsequestrin seems not to influence this high affinity interaction, i.e. calsequestrin might always be bound to RyR. However, the phosphorylation state of calsequestrin determines the amount of Ca2+ released from the lumen. Dephosphorylation of approximately 1% of the phosphorylated calsequestrin could be enough to activate the RyR channel half‐maximally, as we have shown previously [Szegedi et al., Biochem. J. 337 (1999) 19].

Critical Amino Acid Residues Determine the Binding Affinity and the Ca2+ Release Efficacy of Maurocalcine in Skeletal Muscle Cells

Maurocalcine (MCa) is a 33 amino acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. MCa and mutated analogues were chemically synthesized, and their interaction with the skeletal muscle ryanodine receptor (RyR1) was studied on purified RyR1, sarcoplasmic reticulum (SR) vesicles, and cultured myotubes. MCa strongly potentiates [3H]ryanodine binding on SR vesicles (7-fold at pCa 5) with an apparent EC50 of 12 nm. MCa decreases the sensitivity of [3H]ryanodine binding to inhibitory high Ca2+ concentrations and increases it to the stimulatory low Ca2+ concentrations. In the presence of MCa, purified RyR1 channels show long-lasting openings characterized by a conductance equivalent to 60% of the full conductance. This effect correlates with a global increase in Ca2+ efflux as demonstrated by MCa effects on Ca2+ release from SR vesicles. In addition, we show for the first time that external application of MCa to cultured myotubes produces a cytosolic Ca2+ increase due to Ca2+ release from 4-chloro-m-cresol-sensitive intracellular stores. Using various MCa mutants, we identified a critical role of Arg24 for MCa binding onto RyR1. All of the other MCa mutants are still able to modify [3H]ryanodine binding although with a decreased EC50 and a lower stimulation efficacy. All of the active mutants produce both the appearance of a subconductance state and Ca2+ release from SR vesicles. Overall, these data identify some amino acid residues of MCa that support the effect of this toxin on ryanodine binding, RyR1 biophysical properties, and Ca2+ release from SR.

FKBP12 modulation of the binding of the skeletal ryanodine receptor onto the II-III loop of the dihydropyridine receptor

In skeletal muscle, excitation-contraction coupling involves a functional interaction between the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR). The domain corresponding to Thr(671)-Leu(690) of the II-III loop of the skeletal DHPR alpha(1)-subunit is able to regulate RyR properties and calcium release from sarcoplasmic reticulum, whereas the domain corresponding to Glu(724)-Pro(760) antagonizes this effect. Two peptides, covering these sequences (peptide A(Sk) and C(Sk), respectively) were immobilized on polystyrene beads. We demonstrate that peptide A(Sk) binds to the skeletal isoform of RyR (RyR1) whereas peptide C(Sk) does not. Using surface plasmon resonance detection, we show that 1) domain Thr(671)-Leu(690) is the only sequence of the II-III loop binding with RyR1 and 2) the interaction of peptide A(Sk) with RyR1 is not modulated by Ca(2+) (pCa 9-2) nor by Mg(2+) (up to 10 mM). In contrast, this interaction is strongly potentiated by the immunophilin FKBP12 (EC(50) = 10 nM) and inhibited by both rapamycin (IC(50) = 5 nM) and FK506. Peptide A(Sk) induces a 300% increase of the opening probability of the RyR1 incorporated in lipid bilayer. Removal of FKBP12 from RyR1 completely abolishes this effect of domain A(Sk) on RyR1 channel behavior. These results demonstrate a direct interaction of the RyR1 with the discrete domain of skeletal DHPR alpha(1)-subunit corresponding to Thr(671)-Leu(690) and show that the association of FKBP12 with RyR1 specifically modulates this interaction.

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 μm 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 μm) 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 μm) 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 μm 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.

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.

Altered elementary calcium release events and enhanced calcium release by thymol in rat skeletal muscle.

The effects of thymol on steps of excitation-contraction coupling were studied on fast-twitch muscles of rodents. Thymol was found to increase the depolarization-induced release of calcium from the sarcoplasmic reticulum, which could not be attributed to a decreased calcium-dependent inactivation of calcium release channels/ryanodine receptors or altered intramembrane charge movement, but rather to a more efficient coupling of depolarization to channel opening. Thymol increased ryanodine binding to heavy sarcoplasmic reticulum vesicles, with a half-activating concentration of 144 micro M and a Hill coefficient of 1.89, and the open probability of the isolated and reconstituted ryanodine receptors, from 0.09 +/- 0.03 to 0.22 +/- 0.04 at 30 micro M. At higher concentrations the drug induced long-lasting open events on a full conducting state. Elementary calcium release events imaged using laser scanning confocal microscopy in the line-scan mode were reduced in size, 0.92 +/- 0.01 vs. 0.70 +/- 0.01, but increased in duration, 56 +/- 1 vs. 79 +/- 1 ms, by 30 micro M thymol, with an increase in the relative proportion of lone embers. Higher concentrations favored long events, resembling embers in control, with duration often exceeding 500 ms. These findings provide direct experimental evidence that the opening of a single release channel will generate an ember, rather than a spark, in mammalian skeletal muscle.

The Na+/Ca2+ exchange blocker SEA0400 fails to enhance cytosolic Ca2+ transient and contractility in canine ventricular cardiomyocytes

AIMS This study was designed to evaluate the effects of the Na(+)/Ca(2+) exchange (NCX) inhibitor SEA0400 on Ca(2+) handling in isolated canine ventricular myocytes. METHODS AND RESULTS Intracellular Ca(2+) ([Ca(2+)](i)) transients, induced by either field stimulation or caffeine flush, were monitored using Ca(2+) indicator dyes. [Ca(2+)](i)-dependent modulation of the inhibitory effect of SEA0400 on NCX was characterized by the changes in Ni(2+)-sensitive current in voltage-clamped myocytes. Sarcoplasmic reticulum (SR) Ca(2+) release and uptake were studied in SR membrane vesicles. Gating properties of single-ryanodine receptors were analysed in lipid bilayers. Ca(2+) sensitivity of the contractile machinery was evaluated in chemically skinned myocytes. In myocytes paced at 1 Hz, neither diastolic [Ca(2+)](i) nor the amplitude of [Ca(2+)](i) transients was significantly altered by SEA0400 up to the concentration of 1 microM, which was shown to inhibit the exchange current. The blocking effect of SEA0400 on NCX decreased with increasing [Ca(2+)](i), and it was more pronounced in reverse than in forward mode operation at every [Ca(2+)](i) examined. The rate of decay of the caffeine-induced [Ca(2+)](i) transients was decreased significantly by 1 microM SEA0400; however, this effect was only a fraction of that observed with 10 mM NiCl(2). Neither SR Ca(2+) release and uptake nor cell shortening and Ca(2+) sensitivity of the contractile proteins were influenced by SEA0400. CONCLUSION The lack of any major SEA0400-induced shift in Ca(2+) transients or contractility of myocytes can well be explained by its limited inhibitory effect on NCX (further attenuated by elevated [Ca(2+)](i) levels) and a concomitant reduction in Ca(2+) influx due to the predominantly reverse mode blockade of NCX and suppression of L-type Ca(2+) current.

Charged surface area of maurocalcine determines its interaction with the skeletal ryanodine receptor.

The 33 amino acid scorpion toxin maurocalcine (MCa) has been shown to modify the gating of the skeletal-type ryanodine receptor (RyR1). Here we explored the effects of MCa and its mutants ([Ala(8)]MCa, [Ala(19)]MCa, [Ala(20)]MCa, [Ala(22)]MCa, [Ala(23)]MCa, and [Ala(24)]MCa) on RyR1 incorporated into artificial lipid bilayers and on elementary calcium release events (ECRE) in rat and frog skeletal muscle fibers. The peptides induced long-lasting subconductance states (LLSS) on RyR1 that lasted for several seconds. However, their average length and frequency were decreased if the mutation was placed farther away in the 3D structure from the critical (24)Arg residue. The effect was strongly dependent on the direction of the current through the channel. If the direction was similar to that followed by calcium during release, the peptides were 8- to 10-fold less effective. In fibers long-lasting calcium release events were observed after the addition of the peptides. The average length of these events correlated well with the duration of LLSS. These data suggest that the effect of the peptide is governed by the large charged surface formed by residues Lys(20), Lys(22), Arg(23), Arg(24), and Lys(8). Our observations also indicate that the results from bilayer experiments mimic the in situ effects of MCa on RyR1.

Skeletal and cardiac ryanodine receptors bind to the Ca2+-sensor region of dihydropyridine receptor α1C subunit

In striated muscles, excitation–contraction coupling is mediated by the functional interplay between dihydropyridine receptor L‐type calcium channels (DHPR) and ryanodine receptor calcium‐release channel (RyR). Although significantly different molecular mechanisms are involved in skeletal and cardiac muscles, bidirectional cross‐talk between the two channels has been described in both tissues. In the present study using surface plasmon resonance spectroscopy, we demonstrate that both RyR1 and RyR2 can bind to structural elements of the C‐terminal cytoplasmic domain of α1C. The interaction is restricted to the CB and IQ motifs involved in the calmodulin‐mediated Ca2+‐dependent inactivation of the DHPR, suggesting functional interactions between the two channels.