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Journal of Physiology (1992), 457, pp. 195-210 With 11 figures Printed in Great Britain

CALCIUM-INDUCED CALCIUM RELEASE IN CRAYFISH SKELETAL MUSCLE

BY SANDOR GYORKE AND PHILIP PALADE From the Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77550, USA

(Received 11 November 1991) SUMMARY

1. Cut crayfish skeletal muscle fibres were mounted in a triple Vaseline-gap voltage clamp with the Ca2+-sensing dye Rhod-2 allowed to diffuse in via the cut ends. Ca2+ currents across the surface/T-tubule membranes (ICa) were recorded simultaneously with changes in myoplasmic Ca2+ concentration (Ca2+ transients). 2. Excitation-contraction coupling in crayfish skeletal muscle fibres is abolished when calcium in the extracellular solution is replaced by Mg2+. 3. The amplitude of the Ca2+ transients elicited by voltage clamp pulses closely followed the amplitude of the peak calcium currents recorded simultaneously across the surface/T-tubule membranes. This included decreases in both parameters as the pulse potential approached Eca (reversal potential for Ca2+), as well as secondary Ca2+ transients accompanying large tail calcium currents occurring upon repolarization from very large depolarizations. 4. A large contribution of sarcoplasmic reticulum (SR) Ca2+ release to the Ca2+ transients was revealed by a large decrease in the transient caused by the calciuminduced calcium release (CICR) blockers procaine and tetracaine. 5. Short pulses which interrupted the calcium current while SR Ca2+ release was in progress at high rates caused the Ca2+ transient to stop rising nearly immediately after the end of the pulse in most fibres. In about 15% of the fibres the Ca2+ transients continued to rise, albeit at a slower rate, for 10-20 ms after the end of the pulse, as if released Ca2+ was able to elicit some further Ca2+ release from the SR for a while. 6. Even with fibres displaying little sign of continued release after termination of short pulses under control conditions, procaine accelerated the decay of Ca2+ transients elicited by short pulses, indicating that continued release was taking place even as the transient was declining. 7. These results suggest that CICR in crayfish fibres is more closely controlled by a small entry of Ca2+ via surface/T-tubule membrane Ca2+ current than by a larger amount of Ca2+ released from the SR. The limited positive feedback of released Ca2+ on further Ca2+ release allows CICR to remain graded (according to ICa) rather than all-or-none.

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S. GYORKE AND P. PALADE INTRODUCTION

Excitation-contraction (E-C) coupling in invertebrate skeletal muscle requires extracellular calcium (Zacharova & Zachar, 1967; Hidalgo, Luxoro & Rojas, 1979). In this regard it resembles cardiac muscle more than vertebrate skeletal muscle. At the same time, it is clear that arthropod muscle, like most invertebrate skeletal muscle, contains an abundance of SR (Uhrik, Novotova & Zachar, 1980; FranziniArmstrong, Eastwood & Peachey, 1986) that can release large quantities of calcium in response to application of caffeine (Chiarandini, Reuben, Brandt & Grundfest, 1970; Huddart & Oates, 1970) or photolysis of caged Ca2+ (Lea & Ashley, 1990). Calcium-induced calcium release (CICR) has been proposed as the mechanism of E-C coupling in barnacle muscle (Ashley, 1967; Vergara & Verdugo, 1988). Crayfish skeletal muscle contracts when intracellular Ca2+ is elevated (Reuben, Wood, Zollman & Brandt, 1975; Matsumura & Mashima, 1976). Furthermore, crayfish muscle has an abundance of SR, foot-containing diadic couplings between Ttubules and SR (Eastwood, Franzini-Armstrong & Peracchi, 1982; Uhrik, Novotova & Zacharova, 1984), and propagated Ca2+-dependent action potentials when outward currents are reduced sufficiently (Takeda, 1967; Suarez-Kurtz, Reuben, Brandt & Grundfest, 1972). Small diameter crayfish fibres can be conveniently dissected, mounted, cut and voltage clamped in Vaseline-gap chambers (Zahradnik & Zachar, 1987), providing an advantage over the more cumbersome axial wire voltage clamp arrangements required for larger diameter barnacle fibres. CICR has been extensively studied in isolated cardiac myocytes (Beuckelmann & Wier, 1988; Nabauer, Callewaert, Cleemann & Morad, 1989; Niggli & Lederer, 1990; Cleemann & Morad, 1991), but the use of Ca2+-sensitive indicators inside small cardiac myocytes permits only small fluorescence changes to be measured while a relatively large amount of preparation is required to isolate the myocytes. Crayfish skeletal muscle fibres represent a much easier model system for studying the CICR process than other preparations currently available. METHODS

Electrical recording and solutions Fibre segments were mounted in a triple Vaseline-gap voltage clamp chamber (Hille & Campbell, 115 mM NaCH3SO3, 10 mm Ca(CH3SO3)2, 1976), with an external saline (115 mm TEACH 5 mM MOPS (3-(N-morpholino)propanesulphonic acid), pH 7 2) in the 'A' pool, where the electrical and optical recordings are made. The other pools contained an internal solution: 230 mm caesium aspartate, 5 mm sodium phosphocreatine, 3 mm MgATP, 0'5 mm EGTA and 5 mm MOPS, pH 7-2. Linear capacity and leak currents were subtracted from all records. Fibre preparation Small diameter fibres (approximately 150 ,um diameter) were selected from the musculus extensor carpopoditi of the crayfish Procambarus clarkii (Carolina Biological Supply, Burlington, NC, USA) or Cambarus (Nasco, Ft Atkinson, WI, USA). The claws were surgically removed from the animals under tricaine anaesthesia. Fibre segments of 3-4 mm were mounted in the chamber with the 'A' and 'B' pool widths set at approximately 200-300 ,um. After permeabilization of the fibre segment in the 'E' pool with saponin (0-01 % for 1 min), 200-400 /M Rhod-2 (Minta, Kao & Tsien, 1989) was added. After allowing 1 h for equilibration in Rhod-2 in the 'A' pool portion of the fibre, Ca2+ transients were elicited upon depolarization from a holding potential of -90 mV.

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Optical nmeasurements The fibre segment in the 'A' pool was illuminated from above at 45 deg using a fibre optic transmitting the light from a 150 WA tungsten-halogen source passed through a 550 nm broadband filter (40 nm half-bandwidth, Ditric Optics, Hudson, MA, USA). Fluorescence of the fibre fragment 60 50 a)

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[Ca 2] (,uM) Fig. 1. Rhod-2 fluorescence as a function of [Ca2]. EGTA-buffered solutions described in Methods were loaded into capillary tubing together with 50/M Rhod-2 and 1 mM MgCl2. The fluorescence of these solutions was then determined on the same optical set-up utilized for muscle fibre experiments. As seen, fluorescence increases remain quite linear until the free [Ca2+] exceeds 05 jum. Maximal (1000%) fluorescence was determined with 1 mam CaCl2 added to an unbuffered solution, and a 73 % relative fluorescence reading was obtained at 38-9 UM free Ca2+ (not shown). was collected through a long working distance 32 x objective (Leitz MK-40), then passed through a long-pass barrier filter (OG 590, Omega Optical, Brattleboro, VT, USA) onto a photovoltaic cell (EG&G' 440 UV) mounted on the microscope trinocular. Optical signals are normalized to the resting fluorescence. While the Ca2+ transients presented here are not calibrated in terms of [Ca2+], we have performed separate in vitro calibrations on the optical set-up employed for the muscle fibre experiments. Rhod-2 (50 yM) and MgCl2 (1 mM) were added to a solution, one-third of which was one of a series of buffered Ca2+ standards (No. C-3009, Molecular Probes, Eugene, OR, USA), two-thirds of which was 100 mM KCI, 10 mm MOPS, pH 7-1. These solutions were then placed in capillary tubes on the microscope stage for the fluorescence determinations shown in Fig. 1. If we assume a resting [Ca'+] of 100 nm, the peak AF/F of 4 5 observed in our experiments would suggest a value of 550 nm free Ca2+ at the peak of the Ca2+ transient, within the range of linearity of the indicator. These values also fall within the range of Fura-2 determinations reported in isolated cardiac myocytes, another muscle preparation displaying Ca 2+-induced Ca2+ release (Beuckelmann & Wier, 1988; Bers, Lederer & Berlin, 1990; Cleemann & Morad, 1991).

In vitro measurements of Rhod-2 fluorescence In order to correct for direct effects of any of the pharmacological agents employed here (procaine. tetracaine, and caffeine) on Ca2+_dependent changes in Rhod-2 fluorescence, cuvette determinations of Rhod-2 fluorescence were performed in a Fluoro IV spectrofluorometer (Gilford/Corning, Oberlin, OH, USA). The excitation wavelength was set at 547 nm and the fluorescence emission at 90 deg was scanned from 550 to 650nm. Rhod-2 was used at a concentration of 5 m, in a total of 1 5 ml of the same buffered solutions described above. After a

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control scan was performed, different concentrations of procaine, tetracaine or caffeine were added from concentration stocks and the cuvette rescanned. At least four different Ca2+ concentrations were used to determine that the Ca2+ sensitivity had not altered and that the Rhod-2 fluorescence was reduced equally at all [Ca2+] values. The effects noted are summarized in Table 1.

Curve fitting Dose-response relations were fitted using NFIT (Island Products, Galveston, TX, USA). TABLE 1. Effects of selected modifiers of Ca2+-induced Ca2+ release on Rhod-2 fluorescence measured in vitro Fluorescence Concentration decrease (%) (mM) Compound 18 Procaine 3-5 42 100 7 03 Tetracaine 0-6 9 2 Caffeine 05 RESULTS

Dependence of intracellular Caa2+ transients on extracellular [Ca2+] Ca2+ transients in crayfish fibres are absolutely dependent upon the presence of Ca2+ in the extracellular medium. As seen in Fig. 2, a large inward current is elicited by voltage clamp depolarization in the presence of extracellular Ca2+, and this current is accompanied by a large increase in Rhod-2 fluorescence, indicative of a rise in intracellular [Ca2+]. In contrast, when the extracellular Ca2+ is replaced by Mg2+, a much smaller inward current is seen, and there is no change in Rhod-2 fluorescence. A contraction is observed in the presence of extracellular Ca2+, but not in its absence (not shown, Zacharova & Zachar, 1967). These changes are fully reversible, as shown in the right panel in Fig. 2.

A Control

B 0 Ca2+, 10 Mg2+ C Wash

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Fig. 2. Myoplasmic Ca2+ elevations in crayfish fibres in response to depolarization require extracellular Ca2 . Intracellular Ca2+ transients monitored by changes in Rhod-2 fluorescence are shown in the upper trace in each pair. Leak- and capacity-compensated membrane currents are shown in the lower trace in each pair. Each pair of records was obtained in response to a pulse to -10 mV delivered from a holding potential of -90 mV. A, control recording with 10 mm calcium in the external solution. B, substitution of 10 mm magnesium for the calcium in the external solution reduces the inward current and eliminates the Ca2+ transient. C, replacement of calcium in the external solution indicates that the effects of Ca2+ removal were reversible.

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Voltage dependence of the intracellular Ca2+ transients If the membrane is depolarized to different potentials in the presence of Ca2+ in the extracellular solution, little inward current is seen at potentials negative to -40 mV, and there is little increase in Rhod-2 fluorescence (Fig. 3). As the membrane is depolarized further, a significant inward current develops together with an increase in Rhod-2 fluorescence (the Ca2+ transient). On further depolarization (-10 mV) both inward current and Ca21 transient grow in amplitude.

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Fig. 3. Myoplasmic Ca2+ elevations in crayfish fibres display a close correspondence to calcium currents across the surface/T-tubule membranes. In each pair intracellular Ca2+ transients are shown above and leak- and capacity-compensated membrane currents below. Depolarizing voltage clamp pulses were delivered to the potentials indicated from a holding potential of -90 mV. The external solution contained 10 mm calcium. Note that the transients during the pulse are smaller and slower at high positive potentials approaching Eca and that Ca2+ tail currents elicited upon repolarization from those same steps elicit secondary rapid increases in the Ca2+ transients.

When the membrane potential is stepped to positive values, the peak inward current progressively declines in amplitude. This decline is due to the decrease in the driving force on Ca2+ as Em (membrane potential) approaches Eca. The decrease in the inward current is accompanied by a decrease in the rate of rise of Rhod-2 fluorescence and the amplitude it achieves early during the pulse. At very positive potentials, the amplitude of the fluorescence change achieved even late during the pulse is small, but repolarization elicits a large, rapidly deactivating inward tail current and a concurrent very rapid secondary increase in the Rhod-2 fluorescence. The voltage dependence of peak inward current and tail current amplitude are shown in the lower portion of Fig. 4. Plotted above are the Rhod-2 fluorescence values at 20 ms and the peak value obtained upon repolarization. It can be seen that the Rhod-2 fluorescence at 20 ms correlates well with the peak inward current values

(0). At sufficiently high pulse potentials the relatively small Ica remaining at the end of the pulse is followed by a much larger inward tail current due to the increased

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driving force upon repolarization. The tail currents and peak Rhod-2 fluorescence changes occurring shortly after repolarization are plotted with filled circles in Fig. 4. Contribution of SR Ca2' release to intracellular Ca2+ transients To assess the contribution of Ca2' release from the SR to the intracellular Ca21 transients, an examination was made of the effects of the local anaesthetics procaine and tetracaine on both ICa and the Ca21 transients. Procaine and tetracaine have 25

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