Single-channel analysis of fast transient outward K+ currents in frog skeletal muscle

 The patch-clamp method was employed to examine the voltage-dependent gating mechanism of A-type K⁺ channels, which generate the transient outward K⁺ currents described previously in a study of vesicles derived from the sarcolemma of frog skeletal muscle. Channels were activated by depolarizing pulses. There is evidence for non-random grouping of records with channel openings and blank records when depolarizations were repeated at brief intervals, suggesting a slow process similar to slow inactivation. Binomial analysis was consistent with independent behaviour of the channels. Ensemble average currents obtained from multichannel patches had kinetics similar to those of the macroscopic A-type K⁺ current, I _A. The rate of activation, fitted to n ⁴ kinetics, was fast and voltage dependent. The rate of inactivation had an exponential time course with a voltage-independent time constant. The mean open time and the probability of a channel being open increased with depolarization. The histograms of latency to first opening revealed the presence of more than two voltage-dependent closed states. Channel openings occurred in bursts and the closed-time histograms could be fitted by the sum of two or three exponentials. These results suggest a gating scheme with at least three closed states, probably two open states, and two inactivated states. Received: 4 November 1997 / Received after revision: 9 January 1998 / Accepted: 12 January 1998     

Regulation of resting ionic conductances in frog skeletal muscle

The membrane electrical properties and resting ionic conductances of frog semitendinosus muscle fibres were studied in vitro at 25° C with the two-microelectrode cable technique, in the presence of an activator or inhibitor of protein kinase C (PKC) or in the presence of an activator of adenylate cyclase. The PKC activator, 4-phorbol 12,13-dibutyrate (4-PDB), reduced chloride conductance (G _Cl) at concentrations greater than 1 M and did not affect potassium conductance (G _K). At 150 M, the maximum concentration of 4-PDB tested, G _Cl was reduced by 42%. The inactive phorbol ester 4-phorbol 12,13-dibutyrate did not affect G _Cl or G _K. The inhibitory effect of 4-PDB on G _Cl was prevented by pretreatment of the muscle preparation with the PKC inhibitor staurosporine. The adenylate cyclase activator forskolin (1.5–8 M) significantly increased the G _K of the fibres, without affecting G _Cl. Thus, we conclude that frog skeletal muscle G _Cl, unlike rat muscle G _Cl, is relatively insensitive to activators of PKC. Moreover, in frog muscle, protein kinase A is a likely modulator of G _K, but not G _Cl.     

Voltage-dependent K+ channels in the sarcolemma of mouse skeletal muscle

The voltage-dependent K⁺ channels of the mammalian sarcolemma were studied with the patch-clamp technique in intact, enzymatically dissociated fibres from the toe muscle of the mouse. With a physiological solution (containing 2.5 mM K⁺) in the pipette, depolarizing pulses imposed on a cell-attached membrane patch activated K⁺ channels with a conductance of about 17 pS. No channel activity was observed when the pipette solution contained 2mM tetraethylammonium (TEA), or 2 mM 4-aminopyridine (4-AP). Whole cell recordings from these very small muscle fibres showed the well-known delayed rectifier K⁺ outward current with a threshold of about -40mV. The whole-cell current was completely blocked by 2 mM TEA in the bath, suggesting that the TEA-sensitive channels in the patch were also delayed rectifier channels. The inactivation properties of the channels were studied in the cell-attached mode. Averaged single-channel traces showed at least two types of channels discernible by their inactivation time course at a test potential of 60 mV. The fast type inactivated with a time constant of about 150ms, the slow type with a time constant of about 400 ms. A little channel activity always remained during pulses lasting several minutes, indicating either the presence of a very slowly inactivating third type of K⁺ channel, or the tendency of the fast inactivating channels to re-open at constant voltage. No difference was seen in the single-channel amplitudes of the different types of K⁺ channels. The well characterized adenosine-5-triphosphate-(ATP)-sensitive and Ca²⁺-dependent K⁺ channels, although present, were not active under the conditions used. The results suggest that in mouse skeletal muscle the delayed rectifier channels to not only carry the outward current during excitation but are also responsible for the resting K⁺ conductance.     

Effect of mitochondria poisoning by FCCP on Ca2+ signaling in mouse skeletal muscle fibers

We have studied the effects of mitochondria poisoning by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) on Ca²⁺ signaling in enzymatically dissociated mouse flexor digitorum brevis (FDB) muscle fibers. We used Fura-2AM to measure resting [Ca²⁺]_i and MagFluo-4AM to measure Ca²⁺ transients. Exposure to FCCP (2 μM, 2 min) caused a continuous increase in [Ca²⁺]_i at a rate of 0.60 nM/s and a drastic reduction of electrically elicited Ca²⁺ transients without much effect on their decay phase. Half of the maximal effect occurred at [Ca²⁺]_i = 220 nM. This effect was partially reversible after long recuperation and was not diminished by Tiron, a reactive oxygen species (ROS) scavenger. FCCP had no effects on fiber excitability as shown by the generation of action potentials. 4CmC, an agonist of ryanodine receptors, induced a massive Ca²⁺ release. FCCP diminished the rate but not the amount of Ca²⁺ released, indicating that depletion of Ca²⁺ stores did not cause the decrease in Ca²⁺ transient amplitude. Ca²⁺ transient amplitude could also be diminished, but to a lesser degree, by increases in [Ca²⁺]_i induced by repetitive stimulation of fibers treated with ciclopiazonic acid. This suggests an important role for Ca²⁺ in the FCCP effect on transient amplitude.     

Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle

Thapsigargin has been reported to inhibit ATP-dependent Ca²⁺ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca²⁺ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca²⁺ content of the SR. The SR was first depleted of Ca²⁺ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 M was required to inhibit Ca²⁺ loading by 50%. In contrast, another SR Ca²⁺ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 M and total inhibition at 50 M. When cyclopiazonic acid (100 M) was applied after, rather than during, Ca²⁺ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 M), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca²⁺ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.     

Effects of phorbol esters on excitation-contraction coupling and protein kinase C activity of frog twitch muscle fibers

 By recording the calcium transients evoked by voltage-clamp depolarizing pulse with arsenazo III as a calcium indicator, it has been shown that 1 μmol/l phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) agonist, causes a transient potentiation and then a depression of the calcium transients of twitch muscle fibers in frogs. PDBu also produces an initial translocation and activation of PKC, which is followed by a down-regulation. To find out whether the effect of PDBu on the calcium transients depends on PKC, a correlated study of the effect of phorbol esters on calcium transients and PKC activity was performed. The calcium transients and PKC activity were similarly affected by PDBu in ordinary and cold-accommodated frogs, but the effects occurred more quickly in the latter. However, they still changed in parallel as in ordinary frogs. 1 or 10 μmol/l, 4-α-phorbol, a PKC-inactive analogue of phorbol ester, caused a partial depression of the calcium transients in cold-accommodated frogs, while PKC activity was not affected. Moreover, the transient potentiation of the calcium transients induced by 1 μmol/l PDBu could be antagonized by the PKC inhibitors 10 μmol/l chelerythrine chloride or 10 μmol/l polymyxin B (PMB). All these results suggest that: (1) the transient potentiation of calcium transients induced by PDBu is caused by activation of PKC; (2) phorbol ester can depress the calcium transients by a mechanism that is independent of PKC. Received: 25 September 1998 / Received after revision: 14 December 1998 / Accepted: 19 January 1999     

Caffeine contractures in denervated frog muscle

Caffeine contracture tension, effect of caffeine on the resting membrane potential, and caffeine influx in normal and denervated frog sartorius muscle have been investigated. Peak caffeine contracture tension is increased after denervation at all caffeine concentrations. The percentage increases in tension are highest for lower caffeine concentrations. The caffeine concentration required for half maximum tension is decreased from about 3.6 mM in control muscles to 2.6 mM in denervated muscles. Caffeine at 3.5 mM produces a depolarization of about 6 mV in control muscles and 16 mV in denervated muscles. The large contracture tensions observed in denervated muscles are not due to the greater depolarization produced by the drug in denervated muscles since innervated muscles depolarized to the same level by external K⁺ do not enhance caffeine contracture tension. Both control and denervated muscles are highly permeable to caffeine. The increases in sarcoplasmic reticulum development (Moscatello et al. 1965) and calcium content (Picken and Kirby 1976) promoted by denervation may explain the larger tension elicited by caffeine in denervated muscles.     

The effect of phosphate on the relaxation of frog skeletal muscle

 The effect of phosphate on the relaxation of isometrically contracting single skinned fibres from the semitendinosus muscle of the frog Rana temporaria has been investigated using laser pulse photolysis of the photolabile caged calcium-chelator diazo-2 to rapidly reduce the Ca²⁺ (<2 ms) within the fibre and produce >90% relaxation of force. Relaxation occurred in two phases – an initial linear shoulder which lasted approximately 20 ms followed by a double-exponential phase which gave two rate constants, k ₁ (43.4±1.8 s^–1, mean ±SEM, n=14) and k ₂ (15.6±0.3 s^–1, mean ±SEM, n=14) at 12°C. Increased phosphate concentrations did not affect the linear phase, but slowed the double-exponential phase following photolysis of diazo-2 in a dose-dependent fashion (k ₅₀= 0.9 mM for k ₁, 1.15 mM for k ₂). Reducing the concentration of contaminating phosphate (from 640 μM to 100 μM) led to an increase in the rate of the double-exponential phase (k ₁=67.1±4.4 s^–1, k ₂=19.7±0.6 s^–1, mean ±SEM, n=12). Time-resolved measurements of sarcomere length during relaxation, both in control fibres and in the presence of a raised phosphate concentration, reveal a <2% change throughout the whole relaxation transient, and less than 0.1% at the end of the linear phase. This finding implies that gross changes in sarcomere length do not contribute to the decay of the relaxation transient seen upon diazo-2 photolysis. Our results suggest that cross-bridges in states prior to phosphate release are already committed to force generation and must relax by releasing phosphate, rather than by a reversal of the force-generating step to a weakly bound, low-force phosphate-bound state. These findings also indicate that an increase in the phosphate concentration within muscle fibres plays an important part in the slowing of relaxation observed in skeletal muscle fatigue and that the relaxation transients observed upon diazo-2 photolysis represent a disengagement of the cross-bridges. Received: 14 September 1998 / Received after revision and accepted: 20 October 1998     

Insulin modulation of ATP-sensitive K+ channel of rat skeletal muscle is impaired in the hypokalaemic state

 In the present work, we examined the effects of in vivo administration of insulin to rats made hypokalaemic by feeding a K⁺-free diet. The i.p. injection of insulin in the hypokalaemic rats provoked muscle paralysis within 3–5 h. Consistent with this observation, the skeletal muscle fibres of the paralysed rats were depolarized. In contrast, in the normokalaemic animals, insulin neither provoked paralysis nor produced significant fibre hyperpolarization. In the hypokalaemic rats, insulin almost completely abolished the sarcolemma adenosine triphosphate (ATP)-sensitive K⁺ currents without altering the sensitivity of the channels to ATP or glibenclamide. In contrast, in the normokalaemic rats, insulin enhanced ATP-sensitive K⁺ currents that became also resistant to ATP and glibenclamide. Our experiments indicate that the modulation of the sarcolemma ATP-sensitive K⁺ channels by insulin is impaired in the hypokalaemic state. This phenomenon appears to be related to the fibre depolarization and paralysis observed in the same animals. Received: 21 July 1998 / Received after revision: 17 September 1998 / Accepted: 25 September 1998     

A novel method of extraction of TnC from skeletal muscle myofibrils

 Incubation of mechanically skinned barnacle myofibrillar bundles in 10 mM orthovanadate (pH 6.6) results in the loss of Ca²⁺-dependent force generation, which reduces to 0.98±0.006% (mean ±SEM, n=25) of control levels. Analysis of myofibrillar bundles by gel electrophoresis showed that tension loss is primarily due to the extraction of troponin C (TnC) (65.4±5.04% mean ±SEM, n=5). This is a novel finding, since treating cardiac fibres with orthovanadate results in the removal of both TnC and troponin I (TnI) (28). Ca²⁺ dependence was restored to the myofibrillar bundles following reconstitution with either native isoform of barnacle TnC (BTnC₁: 78.72±12.8%, n=9, BTnC₂: 82.73±20.3%, n=3). The reversible loss of Ca²⁺-dependent tension generation following the removal and replacement of TnC indicates that the regulation of contraction in the barnacle is controlled by thin-filament regulatory proteins. Received: 30 September 1998 / Accepted: 16 December 1998     

Effects of cyclopiazonic acid on Ca2+ regulation by the sarcoplasmic reticulum in saponin-permeabilized skeletal muscle fibres

 The effects of the sarcoplasmic reticulum (SR) Ca²⁺ pump inhibitor cyclopiazonic acid (CPA) were studied in saponin-permeabilized frog skeletal muscle fibres. Release of Ca²⁺ from the SR was triggered by brief (2 s) applications of 40 mM caffeine at 2-min intervals. Changes in [Ca²⁺] within the fibre were monitored continuously using Fura-2 fluorescence. At a bathing [Ca²⁺] of 100 nM, introduction of 20 μM CPA induced a slow release of Ca²⁺ from the SR. The following one to two caffeine-induced Ca²⁺ transients were markedly increased in amplitude and duration. Thereafter, the caffeine-induced Ca²⁺ transients decreased progressively and were barely detectable 6–7 min after introduction of CPA. However, increasing the bathing [Ca²⁺] or increasing the Ca²⁺ loading period resulted in a partial recovery of the caffeine-induced Ca²⁺ transients, suggesting that pump inhibition is incomplete, even in the presence of 100 μM CPA. The slow Ca²⁺ efflux induced by CPA was insensitive to ryanodine, but absent following abolition of SR Ca²⁺ pump activity by ATP withdrawal. These results suggest that the caffeine-induced Ca²⁺ transient reflects a balance between efflux via the SR Ca²⁺ channel and reuptake by the Ca pump. Ca²⁺ release upon addition of CPA may result from inhibition of SR Ca²⁺ uptake, which reveals a tonic Ca²⁺ efflux that is independent of the Ca²⁺ release channels. Received: 26 November 1997 / Received after revision: 12 January 1998 / Accepted: 13 January 1998     

Free calcium in sheep cardiac tissue and frog skeletal muscle measured with Ca2+-selective microelectrodes

Microelectrodes filled with neutral carrier selective to Ca²⁺ were used to measure the free intracellular Ca²⁺ concentration ([Ca²⁺]_i) in sheep cardiac tissue and frog skeletal muscle. Calibration of the electrodes was performed in the presence of a solution resembling the cationic composition of the cytoplasm. [Ca²⁺]_i at rest in normal physiological saline (20–22° C) was 240 nM in Purkinje fibres, 270 nM in ventricular muscle, and 52 nM in skeletal muscle. In Purkinje fibres, elevation of [Ca²⁺]_o from 1.8 mM to 5.4 mM produced a 1.7-fold increase in [Ca²⁺]_i. Elevation of [Ca²⁺]_o from 1.8 mM to 18 mM induced a 2.6-fold increase in [Ca²⁺]_i. Exposure to Na⁺-free solution (Li⁺-substituted) gave rise to elevation of [Ca²⁺]_i by factors of 5.8 and 14 in ventricular muscle and Purkinje fibres, respectively. These latter changes in [Ca²⁺]_i were associated with the development of contractures which reached 34% and 172% of the corresponding twitch tension.     

RISC (Repolarization-induced stop of caffeine-contracture) is not due to store depletion in cultured murine skeletal muscle

 We have combined the patch-clamp technique with Fura-2 measurements to investigate whether RISC (repolarization-induced stop of caffeine-contracture) is a consequence of store depletion in cultured skeletal muscles of rats and mice. Weak depolarizations (–45 to –40 mV) of long duration induced a barely detectable Ca²⁺ transient. Even under these conditions, caffeine-activated Ca²⁺transients (CafTs) were terminated upon membrane repolarization (–70 mV) at all stages of CafT. Following the peak of the CafT, massive Ca²⁺ release was elicited by either flash-photolysis of caged Ca²⁺ or further depolarization to 0 mV, demonstrating the lack of store depletion. Thus, RISC is not due primarily to store depletion but to closure of the Ca²⁺ release channels possibly through a mechanical interaction with voltage sensors. RISC was not present in rat heart muscle, further supporting a role of direct interaction in skeletal muscle. Received: 23 April 1996 / Accepted: 12 July 1996     

The cation selectivity of the sarcoball Ca2+ channel in frog muscle fibres

 We have measured single-channel currents from sarcoplasmic reticulum (SR) blebs (sarcoballs) of frog skeletal muscle fibres using conventional patch-clamp electrodes with excised patches. With both the pipette and bath solutions containing 50 mM Ca(gluconate)₂ the slope conductance of the single channels was 39.2 pS for the most commonly seen state, with a reversal potential of –0.4 mV. The cation selectivity of this channel was investigated by replacing the bathing solution with either gluconate or HEPES salts of selected cations. The Goldman permeability ratios, calculated from the reversal potentials, were found to be P(Ca²⁺)/P(K⁺)=2.4, P(Ca²⁺)/ P(Na⁺)=2.7, P(Ca²⁺)/P(Tris⁺)=3.1, P(Ca²⁺)/P(Mg²⁺)=1.0 and P(Ca²⁺)/P(Ba²⁺)=1.1. Each value for the monovalent ions was found to be less than the corresponding value reported for the SR ryanodine receptor channel from skeletal and cardiac muscle. Single-channel activity could be recorded when the preparation was bathed in symmetrical 50 mM Mg(gluconate)₂ solutions, and these channels had a similar conductance and open probability to that measured when the preparation was bathed in symmetrical Ca(gluconate)₂ solution. The channel activity in symmetrical 50 mM Ca(gluconate)₂ solution was insensitive to bath-applied caffeine (5 mM) and ryanodine (10 μM). The results are in agreement with the conclusion that the sarcoball Ca²⁺ channel is not the ryanodine receptor release channel, but possibly a form of the SR Ca²⁺-ATPase which is uncoupled from the catalytic events of the pump and acts as a passive ion channel. Received: 13 February 1998 / Received after revision: 6 April 1998 / Accepted: 7 April 1998     

Effects of nitric oxide on force-generating proteins of skeletal muscle

 Nitric oxide (NO) has recently been identified as a physiologically important intracellular messenger modulating the contractile activity of skeletal muscle [Kobzik L, Reid MB, Bredt DS, Stamler JS (1994) Nature 372: 546–548]. However, the mechanism of action of NO is not yet known. We used skinned (demembranated) muscle fibres to investigate the mechanism of NO function in muscle contraction. Maximally Ca²⁺-activated single fibres of rat skeletal muscle were exposed to physiologically relevant NO concentrations by adding NO donor molecules into the bath solution. Donor application caused a decline both in the contractile properties and in the myofibrillar adenosine triphosphatase (ATPase) activity. These results reveal a novel molecular mechanism of NO action: a direct inhibition of the force-generating proteins in skeletal muscle.     

Detection of skeletal muscle fatigue using an accelerometer in dynamic cardiomyoplasty

 An animal experiment was done using six mongrel dogs that weighed 28 ± 3 kg to show that an accelerometer could detect skeletal muscle fatigue in dynamic cardiomyoplasty. Through left-side thoracotomy, the heart was exposed and an electrode to sense the heartbeat was positioned on the left ventricle. A left latissimus dorsi muscle flap (LDMF) was inserted into the left chest cavity and rolled around the heart. An accelerometer was put on the rolled LDMF to sense the ventricular acceleration by contraction of the LDMF and the heart. The LDMF was stimulated under these settings: pulse width, 210 μs; stimulation output, 6 V; burst frequency, 30 Hz; burst duration, 200 ms; synchronous ratio, 1 : 4; and synchronous delay, 66 ms. Output voltage from the accelerometer was recorded 1, 3, 5, 10, and 15 min after the beginning of stimulation. Percentages of the amplitude in all dogs after 3, 5, 10, and 15 min were 81 ± 10%, 63 ± 12%, 48 ± 11%, and 45 ± 14% of the values after 1 min, respectively. Significant differences were found between the values after 1 min and those after 3 min, between the values after 3 min and those after 5 min, and between the values after 5 min and those after 10 min. This study suggests that muscle fatigue is detectable with an accelerometer in actual dynamic cardiomyoplasty. Received: May 11, 2001 / Accepted: September 10, 2002 Acknowledgments This work was financially supported in a part by a Grant in Aid for Scientific Research (05671113) from the Ministry of Education, Science, and Culture of Japan. Correspondence to:H. Kuroda     

Electrophysiological study on the high K+ sensitivity of rat glomerulosa cells

 Elevation of extracellular potassium concentration by as little as some tenth of mM activates rat adrenal glomerulosa cells. In the present study some factors responsible for this high K⁺ sensitivity were examined. Using whole-cell voltage-clamp technique we found that both T-type and L-type voltage-dependent Ca²⁺ channels have very low threshold potential (–71 and –58 mV, resp.). By means of patch-clamp technique combined with single-cell fluorimetry we also provided evidence that the activation of I_gl, a K⁺-activated inward rectifying current is associated with Ca²⁺ influx. Both the low activation threshold of voltage-dependent Ca²⁺ channels and the function of I_gl contribute to the exceptional K⁺ sensitivity of the glomerulosa cells. Received: 30 September 1997 / Accepted: 4 November 1997     

Acetylcholine reduces the slow calcium current in embryonic skeletal muscle cells in culture

Xenopus skeletal muscle cells when grown in culture develop a slow inward calcium current that is sensitive to dihydropyridines. Acetylcholine (ACh, 10 M) applied through a puffer pipette caused a large inward current in these cells (at the holding potential) through the nicotinic receptor channels and reduced the inward calcium current (during a step depolarization to 0 mV). After the ACh application was discontinued the holding current rapidly returned to pre-ACh levels (20 s) whereas the calcium current showed a slow, partial recovery to pre-ACh levels. Outward potassium current was also reduced during the application of ACh but recovered completely after ACh was discontinued. The effect of ACh on the calcium current was not mimicked by muscarine (100 M) and was absent when 10 g/ml -bungarotoxin was added to the bath suggesting that the decrease in calcium current was mediated by current through the nicotinic receptor.     

Functional significance of Ca2+ in long-lasting fatigue of skeletal muscle

Repeated activation of skeletal muscle causes fatigue, which involves a reduced ability to produce force and slowed contraction regarding both the speed of shortening and relaxation. One important component in skeletal muscle fatigue is a reduced sarcoplasmic reticulum (SR) Ca²⁺ release. In the present review we will describe different types of fatigue-induced inhibition of SR Ca²⁺ release. We will focus on a type of long-lasting failure of SR Ca²⁺ release which is called low-frequency fatigue, because this type of fatigue may be involved in the muscle dysfunction and chronic pain experienced by computer workers. Paradoxically it appears that the Ca²⁺ released from the SR, which is required for contraction, may actually be responsible for the failure of SR Ca²⁺ release during low-frequency fatigue. We will also discuss the relationship between gross morphological changes in muscle fibres and long-lasting failure of SR Ca²⁺ release. Finally, a model linking muscle cell dysfunction and muscle pain is proposed. Accepted: 6 June 2000     

The effect of ionic strength on the kinetics of rigor development in skinned fast-twitch skeletal muscle fibres

 Recent atomic 3-D reconstructions of the acto-myosin interface suggest that electrostatic interactions are important in the initial phase of cross-bridge formation. Earlier biochemical studies had also given strong evidence for the ionic strength dependence of this step in the cross-bridge cycle. We have probed these interactions by altering the ionic strength (Γ/2) of the medium mainly with K⁺, imidazole⁺ and EGTA^2– to vary charge shielding. We examined the effect of ionic strength on the kinetics of rigor development at low Ca²⁺ (experimental temperature 18–22°C) in chemically skinned single fast-twitch fibres of mouse extensor digitorum longus (EDL) muscle. On average the delay before rigor onset was 10 times longer, the maximum rate of rigor tension development was 10 times slower, the steady-state rigor tension was 3 times lower and the in-phase stiffness was 2 times lower at high (230 mM) compared to low (60 mM) ionic strength. These results were modelled by calculating ATP depletion in the fibre due to diffusional loss of ATP and acto-myosin Mg.ATPase activity. The difference in delay before rigor onset at low and high ionic strength could be explained in our model by assuming a 15 times higher Mg.ATPase activity and a threefold increase in K _m in relaxing conditions at low ionic strength. Activation by Ca²⁺ induced at different time points before and during onset of rigor confirmed the calculated time course of ATP depletion. We have also investigated ionic strength effects on rigor development with the activated troponin/tropomyosin complex. ATP withdrawl at maximum activation by Ca²⁺ induced force transients which led into a ”high rigor” state. The peak forces of these force transients were very similar at low and high ionic strength. The subsequent decrease in tension was only 10% slower and steady-state ”high rigor” tension was reduced by only 27% at high compared to low ionic strength. Addition of 10 mM phosphate to lower cross-bridge attachment strongly suppressed the transient increases in force at high ionic strength and reduced the steady-state rigor tension by 17%. A qualitatively similar but smaller effect of phosphate was observed at low ionic strength where steady-state rigor force was reduced by 10%. The data presented in this study show a very strong effect of ionic strength on rigor development in relaxed fibres whereas the ionic strength dependence of rigor development after thin filament activation was much less. The data confirm the importance of electrostatic interactions in cross-bridge attachment and cross-bridge-attachment-induced activation of thin filaments. Received: 3 September 1997 / Received after revision and accepted: 12 December 1997