Frog Twitch Muscle Fibres Research Articles

Analysis of Calcium (Ca) Transients during Excitation-Contraction Coupling in Frog Twitch Muscle Fibers

Singly-dissected fibers were micro-injected with furaptra, a rapidly-responding Ca indicator. Resting fluorescence (FR) and action-potential evoked changes (ΔF) were measured at 16 oC; ΔF/FR was scaled to units of ΔfCaD, the change in fraction of furaptra in the Ca-bound form. Measured ΔfCaD was compared with ΔfCaD simulated with a kinetic model of the underlying myoplasmic Ca movements. During the period 30-200 ms after an action potential, simulated ΔfCaD decayed toward baseline more slowly than measured ΔfCaD. If ΔfCaD was simulated with a modified model that incorporated competition between Mg and Ca for occupancy of the regulatory sites on troponin (assumed dissociation constant of Mg's reaction with the regulatory sites, 2.2 mM; assumed myoplasmic free [Mg], 1 mM), good agreement with measured ΔfCaD was observed. The results support the conclusion that Mg, at physiological levels, competes with Ca for occupancy of the regulatory sites, as indicated in some experiments from fragmented preparations, including tension-pCa measurements in skinned fibers and biochemical studies of isolated troponin molecules. ΔfCaD in frog was also compared with our previous measurements and simulations of ΔfCaD in mouse fibers (reviewed in J. Gen. Physiol., 2012). In frog, the SR Ca release flux elicited by an action potential appears to be the sum of two components. The time course of the first component is similar to that of the entire flux waveform in both fast-and slow-twitch mouse fibers, while that of the second is several-fold slower; the fractional release amounts are ∼0.85 (first component) and ∼0.15 (second component). An anatomical basis for two release components in frog is the presence of both junctional and para-junctional Ca release channels, whereas the mouse fibers have only junctional channels (Felder and Franzini-Armstrong, 2002). Supported by NIH (GM 086167)

Effects of 17beta-estradiol on tension responses and fatigue in the skeletal twitch muscle fibers of frog.

The effects of 17beta-estradiol (10(-5) M), an active estrogen, on the tension and fatigue responses of single fiber or fiber bundle prepared from frog skeletal muscle were investigated. The administration of 17beta-estradiol caused a transient potentiation of tetanus tension by field stimulation at every minute. This potentiation was not affected by the presence of nicardipine, suggesting that the action of 17beta-estradiol would place the excitation-contraction (E-C) coupling beyond T-tubule depolarization. Fatigue was produced by repeated tetanic stimulation every second until tension declined to approximately 40% of the initial level. Fibers were then allowed to recover by having tetani given to them every minute. In the normal Ringer solution, the time to 50% of the initial tetanus tension was 41.7 s. With the presence of 17beta-estradiol, the time to 50% tension was faster than that of control. The presence of 17alpha-estradiol, a stereoisomer, caused no potentiation of tetanic tension to be stimulated every minute, and the rate of decline of fatigued response was almost the same as that of control, suggesting the existence of specific estrogen receptors in the frog muscle. In fatigued muscle with or without estrogen, the tension to field stimulation was transient and not sustained. When the fatigued muscle was again treated with field stimulation at every minute after the more-frequent stimulation, the recovery rate was increased in 17beta-estradiol. A prompt reduction in temperature to 5 degree C, from 20 degree C, in the presence of caffeine elicited the tension response, a rapid cooling contracture (RCC). The presence of 17beta-estradiol inhibited peak tension and maximum rate of rise of the RCC only after the repetitive electrical stimuli. These results suggest that the potentiation of contraction upon the electrical stimulation by 17beta-estradiol was induced by the increase of myoplasmic-free Ca(2+) concentration via an activation of some E-C coupling process. The 17beta-estradiol-induced facilitation of fatigue response to repetitive tetanus stimuli with high frequency may be due to an increase in the imbalance of Ca(2+) turnover in the cytoplasm.

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 micromol/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 micromol/l, 4-alpha-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 micromol/l PDBu could be antagonized by the PKC inhibitors 10 micromol/l chelerythrine chloride or 10 micromol/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.

Effect of activation of protein kinase C on excitation-contraction coupling in frog twitch muscle fibres.

Intracellular Ca2+ transients were recorded from frog twitch muscle fibres in response to voltage-clamp depolarizing pulses, using arsenazo III as an intracellular Ca2+ indicator. The effect of the activation of protein kinase C (PKC) on the Ca2+ transients was studied. With 1 microM phorbol 12,13-dibutyrate (PDBu), a PKC activator, the peak of the Ca2+ transients increased to about 120% of control during the first 0.5 h, and then decreased gradually to a plateau of 44% of control within the following 2 h. This effect of PDBu could be alleviated significantly by PKC inhibitors, 10 microM polymyxin B (PMB) or 30 microM 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7). Moreover, PDBu caused an upward shift of the strength/duration curve. In Li(+)-loaded muscle fibres the Ca2+ transients could not fully recover after 80 mM K+ exposure for 15 min, while the post-K+ Ca2- transients could be completely restored in the fibres not loaded with Li+. In the presence of 10 microM PMB or 30 microM H-7, a full restoration of the post-K+ Ca2+ transients was seen in Li(+)-loaded fibres. PMB supplemented after high-K+ exposure also could result in a complete recovery of the post-K+ Ca2+ transients in Li(+)-loaded fibres. The role of PKC in modulating excitation-contraction coupling in frog twitch muscle fibres is clearly indicated, but the mechanism(s) and physiological significance remain to be established.

Inward barium current and excitation-contraction coupling in frog twitch muscle fibres

The role of barium ions in excitation-contraction coupling was studied in single isolated frog semitendinosus fibres. Simultaneous recordings of membrane currents and contraction under voltage-clamp conditions in a sucrose-vaseline gap device show that barium ions have a reversible inhibiting effect on contraction. This inhibiting action was correlated to the entry of barium ions via the DHP-sensitive tubular calcium channel. Cytological observations and X-ray microanalysis performed on the fibres used in the electrophysiological experiments indicate that barium ions do not accumulate in the junctional sarcoplasmic reticulum; they can freely diffuse in the intermyofibrillar space and they accumulate in mitochondria. Calcium release experiments performed on isolated sarcoplasmic reticulum vesicles show that barium ions are not able to induce calcium release from calcium-loaded vesicles, they behave as calcium release inhibitors. These results are discussed in relation with the possible role of the slow Ca current in excitation-contraction coupling.

Depression of calcium transients after exposure to high K+ solution in Li(+)-loaded frog twitch muscle fibres and its reversal by exogenous myo-inositol.

Using Arsenazo III as a myoplasmic calcium indicator, we have studied the calcium transients evoked by voltage-clamp depolarizing pulses in frog twitch muscle fibres which had been temporarily depolarized by 80 mmol/L K+ in the absence or presence of myoplasmic Li+. After the high K+ exposure, for either a short (15 min) or long (1 h) time, the post-K+ calcium transients could gradually be restored to the level of the pre-K+ ones, if the fibres were not loaded with Li+. In contrast, the post-K+ calcium transients of Li(+)-loaded fibres could not fully recover, and were depressed in a Li+ concentration-dependent manner. The mean amplitude of the post-K+ responses recorded more than 3.5 h after 15 min high K+ exposure was reduced to 56% of pre-K+ control in the fibres which had been loaded with Li+ in 20 mmol/L Li+ Ringer's solution. This depression could be prevented or partially reversed by exogenous myo-inositol. More depression could be induced by 1 h high K+ exposure, but the presence of exogenous myo-inositol could not clearly prevent the post-K+ calcium transients from reduction. Assuming that high K+ exposure caused a depletion of myo-inositol and probably other changes in the metabolism of inositol phospholipids in Li(+)-loaded fibres, we conclude that some metabolites of phosphoinositides may play modulation roles in excitation-contraction coupling in frog twitch muscle fibres.

Intracellular diffusion in the presence of mobile buffers. Application to proton movement in muscle

Junge and McLaughlin (1987) derived an expression for the apparent diffusion constant of protons in the presence of both mobile and immobile buffers. Their derivation applies only to cases in which the values of pH are considerably greater than the largest pK of the individual buffers, a condition that is not expected to hold in skeletal muscle or many other cell types. Here we show that, if the pH gradients are small, the same expression for the apparent diffusion constant of protons can be derived without such constraints on the values of the pK's. The derivation is general and can be used to estimate the apparent diffusion constant of any substance that diffuses in the presence of both mobile and immobile buffers. The apparent diffusion constant of protons is estimated to be 1-2 x 10(-6) cm2/s at 18 degrees C inside intact frog twitch muscle fibers. It may be smaller inside cut fibers, owing to a reduction in the concentration of mobile myoplasmic buffers, so that in this preparation a pH gradient, if established within a sarcomere following action potential stimulation, could last 10 ms or longer after stimulation ceased.

Effect of prolonged in vitro lithium treatment on calcium transients in frog twitch muscle fibres and its reversal by exogenous myo-inositol

Using arsenazo III as an intracellular indicator to monitor the calcium transients elicited by voltage-clamp depolarizing pulse, the effect of prolonged in vitro lithium treatment on excitation—contraction coupling in frog twitch muscle fibres was investigated. Incubation in 10 mM Li + Ringer's solution for 2 days caused a 46% increase in the amplitude of the calcium transients, while treatment with 30 mM Li + for 2 days produced a depression of 44%. Shortening the bathing time to 1 day, the decrease of calcium transients caused by 30 mM Li + was reversed to a small increase. For the 2-day incubation, both the increase in the amplitude with 10 mM and the decrease with 30 mM Li + were abolished by the presence of 1 mM myo-inositol in the bathing medium. These results imply that the turnover of inositol phospholipids is involved in regulating excitation—contraction coupling in skeletal muscle fibres.

Effect of barium ions on excitation-contraction coupling of frog twitch muscle fibres.

The mechanism for the Ba2(+)-induced potentiation of the twitch tension in frog skeletal muscle was investigated. No significant difference in the peak amplitude of the calcium transients evoked by voltage-clamp depolarizing pulse was found between the fibres bathed in Ca2+ (control) and Ba2+ Ringer's solution, whereas the calcium transients evoked by the action potentials from the fibres in Ba2+ Ringer's solution were increased by about 64%, compared with control. In comparison with control, the action potentials of the fibres in Ba2+ Ringer's solution had a similar overshoot but a significantly longer time course. Our results suggest that excitation-contraction coupling in frog twitch muscle fibres is not altered by replacing Ca2+ with Ba2+. The Ba2(+)-induced potentiation of contraction may be accounted for by broadening of the action potentials.

Changes in Na channel properties of frog and rat skeletal muscles induced by the AaH II toxin from the scorpion Androctonus australis

The effects of the mammal toxin II isolated from the venom of the scorpion Androctonus australis Hector (AaH II) were studied under current and voltage clamp conditions in frog (semitendinosus) and rat (fast e.d.l. and slow soleus) skeletal twitch muscle fibres. In both species, AaH II induced a dose-dependent prolongation of the action potential (AP) leading at saturating concentration to APs with long plateaus of about 1.5 s in frog and 5 s in rat e.d.l. and soleus fibres. The concentrations to induce 50% of the maximal effect (K0.5) were 9.1 x 10(-9) M in the frog and 1.4 x 10(-9) M in the rat. AaH II increased the time constants of inactivation of the peak Na current and induced a maintained Na current that was greater in rat e.d.l. and soleus (31.6% of peak current amplitude at -30 mV; K0.5 = 0.8 x 10(-9) M) than in frog (16.5%; K0.5 = 15.5 x 10(-9) M) muscles. Peak and maintained Na currents were TTX-sensitive and had identical threshold and reversal potentials. The half-maximum maintained permeability occurred at a potential 20 mV more positive than the peak permeability. Recovery from inactivation and steady-state inactivation of the inactivating Na current remained unchanged. The maintained current deactivated with normal fast kinetics. The action of the toxin reversed poorly on washout but could be largely removed by conditioning depolarizations more positive than the reversal potential of the Na current. Our results suggest that, in vertebrate skeletal muscle fibres, AaH II affects all the Na channels and are consistent with the hypothesis that the maintained current originates from a reopening of previously inactivated Na channels.

Effects of hypertonic solutions on calcium transients in frog twitch muscle fibres.

1. The effects of hypertonic solutions on excitation-contraction (e.-c.) coupling in frog skeletal muscle fibres were investigated using Arsenazo III to monitor intracellular calcium transients in voltage-clamped fibres. 2. In solutions made hypertonic with sucrose or sodium chloride, the size of the Arsenazo signal evoked by a 5 ms depolarization to 0 mV was little altered by increases in tonicity up to about twice normal, but declined in higher tonicities, and was almost completely suppressed at 4 times normal tonicity. 3. The latency to onset of the Arsenazo signal was increased in hypertonic solutions (2.3 and 3.1 times normal tonicity), but the decay time constant of the signal was little changed with tonicities up to 2.3 times normal. 4. The rheobase potential for a just-detectable Arsenazo signal was shifted about 4 mV more negative by increases in tonicity up to 2.3 times normal, but further increases reversed the direction of the shift, and in 3.95 times normal tonicity the rheobase was 10 mV more positive than in normal Ringer solution. 5. With short (less than 10 ms) pulse durations the depolarization needed to elicit a threshold Arsenazo signal increased steeply with increasing tonicity. Changes in the strength-duration curve could be accounted for by an increase in the time constant for build-up of a hypothetical coupler in the e.-c. coupling process. 6. Solutions of about twice normal tonicity are commonly used to suppress muscle contraction. Since the size of the Arsenazo signal was only slightly reduced by this tonicity, the main effect is presumably on the contractile proteins.

Histochemical study of calcium on T-tubule membranes and in the sarcoplasmic reticulum, in frog twitch muscle fibres at rest and during activity.

The trigger calcium hypothesis of signal transmission between T-tubules and terminal cisternae (TC) of the sarcoplasmic reticulum (SR) in twitch muscle fibres implies the presence of calcium along T-tubule membranes at rest and its release upon excitation. To test this hypothesis, calcium was immobilised using a fixing and precipitating solution of glutaraldehyde in phosphate buffer at pH 8.0 and the calcium was substituted for by lead. Simultaneous tension recordings revealed the occurrence of contractions or a burst of twitches upon perfusion with the fixative. Procaine or tetrodotoxin (TTX) was used to inhibit this activity. In fibres without fixative-induced activity, precipitates were observed along T-tubules and in adjoining parts of TC. In activated fibres, tubular and TC precipitates were absent. These results are consistent with the trigger calcium hypothesis. In fibres activated by depolarisation, calcium returned to TC after passing successively through different parts of the SR.

Minimal latency of calcium release in frog twitch muscle fibres.

Intracellular release of calcium in frog skeletal muscle fibres was monitored by the use of arsenazo III, in response to voltage clamped depolarizing pulses. A latency of a few milliseconds was evident between the onset of depolarization and the first detectable rise in the arsenazo-calcium signal, and this decreased logarithmically as the depolarization was increased. The minimal latency with strong depolarization (to +20 to +100 mV) was about 2 ms at 5 degrees C. This delay appears to be sufficiently long to be compatible with a chemically mediated coupling mechanism between depolarization and calcium release from the sarcoplasmic reticulum.

New optical spike in frog twitch muscle fibers injected with a potential probe dye NK2935.

Absorbance responses posterior to action potentials were recorded from frog twitch fibers loaded by an impermeant potential probe dye NK2935 that was chemically identical with WW781. Extracellularly loaded NK2935-related responses (EOS) increased in absorbance around 640nm wavelengths and were like action potential in waveform but were slightly preceded by action potential. Model analysis showed that EOS consisted of two components, namely 39% non-delay component reflected with sarcolemma action potential and 61% first-order delay component reflected with transverse tubule (T) action potential (delay-constant tau 1 = 0.48 ms). The T-disrupted fibers showed only the non-delayed response. Intracellularly injected NK2935-related responses (IOS) were also composed of a single spike of the same direction swing as in the EOS case described by the term "ipsidirection," but were clearly preceded by EOS and followed by Ca transient, birefringence, transparency and latency relaxation responses. The T-disrupted fibers showed a small "sidedness" response characterized by both opposite direction swing, namely "contradirection" and non-delay against EOS. Model analysis showed that IOS consisted of two main components, namely second-order delay component with ipsidirection (tau 2 = 0.8 ms) and the further high-order delay component with contradirection. Based on spatio-temporal heterogeneity of NK2935 binding inside fibers and potential-absorbance relation in sarcolemma, the former origin was thought to be in junctional sarcoplasmic reticulum (jSR) responding to T excitation with a transient "positive polarization" estimated roughly 20 mV. Similarly, the latter was possibly in free SR where a slow "negative polarization" estimated -0.9 mV might appear associated with Ca release.

Regeneration of frog twitch and slow muscle fibers after mincing.

Iliofibularis muscles of Rana temporaria were minced and allowed to regenerate in the iliofibularis or the sartorius bed of the same frog. Regenerated muscles were examined for the presence of slow muscle fibers using electrophysiologic, histochemical, and contractile parameters. Muscle regeneration from sartorius mince was also studied. Regeneration was more successful from iliofibularis than from sartorius mince, and the iliofibularis bed was more favorable for regeneration than the sartorius bed for both types of muscle. Twitch fibers regenerated within a few months, but slow fibers could not be identified earlier than 14 months after muscle destruction. Slow muscle fibers regenerated only from iliofibularis mince, both orthotopically and heterotopically. All regenerates capable of maintaining a K-contracture contained histochemically identified slow fibers; the membrane properties of electrophysiologically identified slow fibers were normal. It is concluded that slow muscle fibers regenerate only from the remnants of a muscle that contains slow fibers. The results are discussed with respect to the role of innervating nerve fibers.

Electrical properties of the myotendon region of frog twitch muscle fibers measured in the frequency domain

The electrical properties of the end of a muscle fiber were determined using three microelectrodes, one passing sinusoidal current, the other two recording the resulting voltages. An electrical model was constructed from the morphology of the fiber, including the resistance of the extracellular space between cells; the parameters of this model were determined by fitting the model to the observed voltage responses. Our results, analyzed directly or by curve fits, show that the end of muscle fibers contains a large capacitance resulting from the extensive membrane folds at the myotendon junction. Analysis and simulations show that the extra capacitance at the myotendon junction has substantial effects on measurements of linear properties, in particular on estimates of the capacitance of the membranes. There is little qualitative effect on classical measurements of nonlinear charge movement (provided they were made with one set of electrode locations) if the linear components have been subtracted. Quantitative estimates of nonlinear charge movement and ionic currents are significantly affected, however, because these estimates are customarily normalized with respect to the linear capacitance.

Calcium influx in contracting and paralyzed frog twitch muscle fibers.

Calcium uptake produced by a potassium contracture in isolated frog twitch fibers was 6.7 +/- 0.8 pmol in 0.7 cm of fiber (mean +/- SEM, 21 observations) in the presence of 30 microM D600. When potassium was applied to fibers paralyzed by the combination of 30 microM D600, cold, and a prior contracture, the calcium uptake fell to 3.0 +/- 0.7 pmol (11): the fibers were soaked in 45Ca in sodium Ringer for 3 min before 45Ca, in a potassium solution, was added for 2 min; each estimate of uptake was corrected for 5 min of resting influx, measured from the same fiber (average = 2.3 +/- 0.3 pmol). The calcium influx into paralyzed fibers is unrelated to contraction. This voltage-sensitive, slowly inactivating influx, which can be blocked by 4 mM nickel, has properties similar to the calcium current described by several laboratories. The paired difference in calcium uptake between contracting and paralyzed fibers, 2.9 +/- 0.8 pmol (16), is a component of influx related to contraction. Its size varies with contracture size and it occurs after tension production: 45Ca applied immediately after contracture is taken up in essentially the same amounts as 45Ca added before contraction. This delayed uptake is probably a "reflux" refilling a binding site on the cytoplasmic side of the T membrane, which had been emptied during the prior contracture, perhaps to initiate it. We detect no component of calcium uptake related to excitation-contraction coupling occurring before or during a contracture.

Extracellular ions and excitation-contraction coupling in frog twitch muscle fibres.

Intracellular calcium transients were recorded from voltage-clamped frog twitch muscle fibres using Arsenazo III. The possible role of extracellular ions in excitation-contraction (e.-c.) coupling was examined using ion substitutions and blocking drugs in the bathing medium. Parameters measured included the Arsenazo response size to a standard depolarizing pulse (5 ms, 0 mV) and the strength-duration curve for threshold Arsenazo signal. Addition of tetrodotoxin (TTX) decreased the response size to small (-30 mV, 5 ms), but not large (+30 mV, 10 ms) depolarizations, probably because of poor voltage clamp of the tubular membrane in the absence of TTX. Clamping TTX-treated fibres with the wave form of a recorded action potential gave an Arsenazo response similar to that elicited by the normal action potential (at 10 degrees C). Complete substitution of sodium (by choline, lithium or Tris) or chloride (by methyl sulphate or maleate) in the bathing solution gave no appreciable changes in the size of the Arsenazo response. Reduction of extracellular free [Ca2+] to low levels using EGTA caused a slight reduction in the calcium signal elicited by the standard depolarization (to 74% after a few hours, and to 62% after 2 days; temperature 5-10 degrees C). The strength-duration curve was unchanged. Arsenazo responses about 75% of the control size could be elicited in high potassium solution (42 mM-K2SO4) by strong (+80 mV, 20 ms) depolarizations, after re-polarizing the fibres to -90 mV for a few minutes. The voltage dependence of activation was shifted to more positive potentials in this solution. Tetraethylammonium (TEA) bromide at a concentration of 20 mM did not alter the Arsenazo signal, whilst 120 mM-TEA reduced the response by 25%. 3,4-diaminopyridine (DAP) reduced the size of the Arsenazo signal at a concentration of 5 mM, and caused spontaneous release of calcium from the sarcoplasmic reticulum (s.r.) in the absence of membrane potential changes. The Arsenazo signal elicited by an action potential was enhanced by 1 mM-DAP, because of prolongation of the action potential, but was depressed by higher concentrations. We conclude that e.-c. coupling does not involve the influx of any external ions into the muscle fibre. If a current flow between the T-tubules and the s.r. is involved in e.-c. coupling, then this is probably carried by an efflux of potassium ions.

A comparison of the local anaesthetic effects of cationic, anionic, and neutral amphipathic agents in frog skeletal muscle

Impermeant alkyl amphipathic agents reduced the excitability of frog twitch muscle fibers, indicating that local anaesthesia can be produced by perturbations within the external lamina of the sarcolemma. Cationic (n-alkyl trimethylammonium) and anionic (n-alkyl sulfonate) as well as permeant neutral (n-alkanol) agents have been compared with regard to their local anaesthetic potency. Small impermeant agents (fewer than six carbon atoms) alone were ineffective. Within each series the concentration required to reduce excitability was proportional to the length (hydrophobicity) of the hydrocarbon chain attached to the polar group. However, corresponding members of these three series of compounds differed considerably in their local anaethetic potency. Hence, charged groups as well as apolar moieties can influence local anaesthetic efficacy. The supra-additive character of the local anaesthesia produced by combining impermeant alkyl anions and cations indicates that these two types of amphipaths may produce their similar effects by perturbations at different membrane sites.

Changes in threshold for calcium transients in frog skeletal muscle fibres owing to calcium depletion in the T-tubules.

Strength-duration curves were measured for voltage-clamp depolarizations required to elicit a just detectable rise in intracellular calcium, as monitored using arsenazo III, in frog twitch muscle fibres. In normal Ringer solution, the threshold for a 5 sec duration depolarization was about 5 mV more negative than for a 200 msec duration pulse. The shift in threshold comparing 200 msec and 5 sec pulses was almost abolished in bathing solutions including magnesium or nickel (4 mM), or where the free calcium concentration was buffered. The shift in threshold was little changed by substitution of barium for calcium. These results can be explained by supposing that the 5 sec depolarization activates an inward calcium flux across the T-tubule membrane, which decreases the calcium concentration in the tubules, and hence alters the threshold for activation of excitation-contraction (e.-c.) coupling because of surface charge effects.