Vaseline-gap Chamber Research Articles

The Rapid Repolarization of the Action Potential in Twitch Skeletal Muscle Fibers from Frog is due Almost Entirely to a Calcium-Activated K Current

Action potentials (aps) in cut fibers mounted in a double Vaseline-gap chamber were generated by short current-clamp pulses superimposed on a holding current that maintained the resting potential at −90 mV. Ca2+ release from the sarcoplasmic reticulum (SR) was assessed with either the EGTA/phenol red method or the SR [Ca2+] indicator, tetramethylmurexide. In contrast to the rapid rate of repolarization of the ap with normal SR Ca contents, in the near absence of Ca, the time course of repolarization was approximately exponential with an average exponential time constant of 12.5 ms. Removal of K from the internal and external solutions eliminated the effect of SR Ca2+ release on the ap indicating that the current is carried by K+ ions. The time course of total current during the repolarization was assessed from the rate of change of voltage. Taking into account a small Ca-insensitive background current and the relationship between K current and permeability (PK) on voltage using the Goldman-Hodgkin-Katz equation, the time course of PK is seen to closely match that of the myoplasmic Ca transient. At physiological SR Ca load, the Ca-activated K current, denoted IK(Ca), at its peak was ∼80% of the total current. Since almost all of the background current was associated with recharging the membrane capacitance via the holding current passing through ohmic pathways in the cut-fiber preparation, the peak percentage of current responsible for repolarization in an intact fiber carried by IK(Ca) should be significantly greater than 90%. While results with voltage-clamp stimulation indicate a complex dependence of IK(Ca) on calcium and voltage similar to that displayed by BKCa channels, the IK(Ca) current was insensitive to the BKCa channel inhibitor charybdotoxin.

Deterministic inactivation of calcium release channels in mammalian skeletal muscle.

Enzymatically dissociated fibres from the extensor digitorum communis muscle of rats were mounted into a double Vaseline gap chamber. The rate of calcium release (R(rel)) from the sarcoplasmic reticulum (SR) and changes in SR permeability to Ca2+ (PSR) were calculated from measured changes in intracellular calcium concentration. Calcium release during a prepulse attenuated the inactivating component of PSR of the subsequent test pulse. The suppression was graded, larger release causing greater suppression, as expected from a calcium-dependent inactivation process. However, if the dissociation constant of the putative inhibitory calcium binding site (Kd) was estimated using different test pulses different affinities were obtained: a smaller test pulse yielded a smaller Kd. Comparing the suppression of the inactivatable component of PSR during the test pulse (suppression) with the inactivatable component during the prepulse (pre-inactivation) revealed a linear relationship with a regression coefficient close to unity. Lowering intracellular magnesium by decreasing its concentration to 25 microM in the internal solution altered the time course of PSR. The maximal peak-to-steady-level ratio was increased to 6.3 +/- 0.4 (n = 10, mean +/- s.e.m.) from a control value of 3.0 +/- 0.2 (n = 19). Despite the apparent change in steady-state inactivation, suppression remained equal to that pre-inactivation. Our results support the view that a depolarizing pulse always recruits the same set of calcium release channels and a portion of these channels undergoes a deterministic inactivation process.

Frog skeletal muscle fibers recovering from fatigue have reduced charge movement.

Following prolonged exercise, muscle force production is often impaired. One possible cause of this force deficit is impaired intracellular activation. We have used single skeletal muscle fibers from the lumbrical muscle of Xenopus laevis to study the effects of fatigue on excitation-contraction coupling. Fatigue was induced in 13 intact fibers. Five fibers recovered in normal Ringer only (fatigued-only fibers). The remaining eight fibers were subjected to a brief hypotonic treatment (F-H fibers) that is known to prolong the effects of fatigue. Intramembrane charge movement, changes in intracellular calcium concentration ([Ca2+]i) and force transients were measured in a single Vaseline gap chamber under voltage clamp. In F-H fibers, membrane capacitance was reduced. Confocal microscopy showed that this was not due to closure of the transverse tubules. The amount of normalized intramembrane charge was reduced from 21.0 +/- 2.8 nC/microF (n = 10) in rested fibers to 12.2 +/- 1.1 nC/microF in F-H fibers. However, the voltage dependence of intramembrane charge movement was unchanged. In F-H fibers, force production was virtually abolished. This was the consequence of the greatly reduced [Ca2+]i accompanying a depolarizing pulse. In recovering fatigued-only fibers, while the maximal available charge was not significantly smaller (18.3 +/- 1.1 nC/ microF), both calcium and force were reduced, albeit to a lesser extent than in F-H fibers. The data are consistent with a model where fatigue reduces the number of voltage sensors in the t-tubules and, in addition, alters the coupling between the remaining functional voltage sensors and the calcium channels of the sarcoplasmic reticulum.

Tools for flash-photolysis experiments on voltage-clamped muscle fibre segments.

An experimental set-up is described that allows the combination of rapid transmembrane voltage changes and photometric calcium recording with the fast photochemical turnover of substances applied externally or intracellularly to cut skeletal muscle fibres. It consists of a double-vaseline-gap system, designed for use with a xenon-flash-lamp device and a dual-wavelength microscope photometer. The pools of the vaseline gap chamber that contain the solutions surrounding the cut ends and the voltage-clamped segment of the muscle fibre are closed and have volumes of 20-50 microl. Thin tubes allow rapid solution change or continuous perfusion in the chamber compartments. Accessory tools were constructed to simplify focussing and measuring the flash-light intensity. A pilot light delivered from a red laser diode is used as a guide beam to target the ultraviolet (UV) flash to the preparation. The light distribution in the focal region and the relative changes in flash intensity with increasing numbers of flashes were quantified with an instrument that integrates the photo-current of a UV-sensitive silicon diode. The function of the set-up was demonstrated by measuring the efficiency of Ca2+ release from DM-nitrophen in quartz capillaries using the Ca(2+)-sensitive dye antipyrylazo III and by recording the flash-induced recovery of L-type calcium currents in muscle fibres blocked by the light-sensitive dihydropyridine drug nifedipine.

Involvement of methionine residues in the fast inactivation mechanism of the sodium current from toad skeletal muscle fibers.

The role of methionine residues on the fast inactivation of the sodium channel from toad skeletal muscle fibers was studied with the mild oxidant chloramine-T (CT). Isolated segments of fibers were voltage clamped in a triple Vaseline gap chamber. Sodium current was isolated by replacing potassium ions by tetramethylammonium ions in the external and internal solutions. Externally applied chloramine-CT was found to render noninactivating a large fraction of sodium channels and to slow down the fast inactivation mechanism of the remainder fraction of inactivatable channels. The action of CT appeared to proceed first by slowing and then removing the fast inactivation mechanism. The voltage dependence of the steady-state inactivation of the inactivatable CT-treated currents was shifted +10 mV. CT also had a blocking effect on the sodium current, but was without effect on the activation mechanism. The effects of CT were time and concentration dependent and irreversible. The use of high CT concentrations and/or long exposure times was found to be deleterious to the fiber. This side effect precluded the complete removal of fast inactivation. The effects of CT on the fast inactivation of the sodium current can be explained assuming that at least two methionine residues are critically involved in the mechanism underlying this process.

Local calcium release in mammalian skeletal muscle.

1. Fluo-3 fluorescence associated with Ca2+ release was recorded with confocal microscopy in single muscle fibres mechanically dissected from fast twitch muscle of rats or frogs, voltage clamped in a two Vaseline-gap chamber. 2. Interventions that elicited Ca2+ sparks in frog skeletal muscle (low voltage depolarizations, application of caffeine) generated in rat fibres images consistent with substantial release from triadic regions, but devoid of resolvable discrete events. Ca2+ sparks were never observed in adult rat fibres. In contrast, sparks of standard morphology were abundant in myotubes from embryonic mice. 3. Depolarization-induced gradients of fluorescence between triadic and surrounding regions (which are proportional to Ca2+ release flux) peaked at about 20 ms and then decayed to a steady level. Gradients were greater in frog fibres than in rat fibres. The ratio of peak over steady gradient (R) was steeply voltage dependent in frogs, reaching a maximum of 4.8 at -50 mV (n = 7). In rats, R had an essentially voltage-independent value of 2.3 (n = 5). 4. Ca2+-induced Ca2+ release, resulting in concerted opening of several release channels, is thought to underlie Ca2+ sparks and the peak phase of release in frog skeletal muscle. A diffuse 'small event' release, similar to that observed in these rats, is also present in frogs and believed to be directly activated by voltage. The present results suggest that in these rat fibres there is little contribution by CICR to Ca2+ release triggered by depolarization, and a lack of concerted channel opening.

Effect of sarcoplasmic reticulum (SR) calcium content on SR calcium release elicited by small voltage-clamp depolarizations in frog cut skeletal muscle fibers equilibrated with 20 mM EGTA.

Cut muscle fibers from Rana temporaria (sarcomere length, 3.5-3.9 micro(m); 14-16 degreesC) were mounted in a double Vaseline-gap chamber and equilibrated with an external solution that contained tetraethyl ammonium- gluconate and an internal solution that contained Cs as the principal cation, 20 mM EGTA, and 0 Ca. Fibers were stimulated with a voltage-clamp pulse protocol that consisted of pulses to -70, -65, -60, -45, and -20 mV, each separated by 400-ms periods at -90 mV. The change in total Ca that entered into the myoplasm (Delta[CaT]) and the Ca content of the SR ([CaSR]) were estimated with the EGTA/phenol red method (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259-336). Fibers were stimulated with the pulse protocol, usually every 5 min, so that the resting value of [CaSR] decreased from its initial value of 1,700-2, 300 microM to values near or below 100 microM after 18-30 stimulations. Three main findings for the voltage pulses to -70, -65, and -60 mV are: (a) the depletion-corrected rate of Ca release (release permeability) showed little change when [CaSR] decreased from its highest level (>1,700 microM) to approximately 1,000 microM; (b) as [CaSR] decreased below 1,000 microM, the release permeability increased to a maximum level when [CaSR] was near 300 microM that was on average about sevenfold larger than the values observed for [CaSR] > 1,000 microM; and (c) as [CaSR] decreased from approximately 300 microM to <100 microM, the release permeability decreased, reaching half its maximum value when [CaSR] was approximately 110 microM on average. It was concluded that finding b was likely due to a decrease in Ca inactivation, while finding c was likely due to a decrease in Ca-induced Ca release.

Kinetics of contractile activation in voltage clamped frog skeletal muscle fibers

Excitation-contraction coupling events leading to the onset of contraction were studied in single skeletal frog muscle fibers. This entailed the simultaneous measurement of the changes in intracellular calcium concentration using antipyrylazo III and fura-2, isometric force, and clamp voltage in a modified single vaseline gap chamber for the first time. The calcium transients were incorporated into an analysis of calcium binding to regulatory sites of troponin C (TnC) that permitted both a linear and a cooperative interaction. The analysis assumed that the onset of mechanical activation corresponds with a particular TnC saturation with calcium setting constraints for the calcium binding parameters of the regulatory sites. Using a simple model that successfully reproduced both the time course and the relative amplitudes of the measured isometric force transients over a wide membrane potential range, k(off) of TnC was calculated to be 78 s(-1) for the cooperative model at 10 degrees C. Together with the above constraints this gave a dissociation constant of 8.8 +/- 2.5 microM and a relative TnC saturation at the threshold (Sth) that would cause just detectable movement of 0.17 +/- 0.03 (n = 13; mean +/- SE). The predictions were found to be independent of the history of calcium binding to the regulatory sites. The observed delay between reaching Sth and the onset of fiber movement (8.7 +/- 1.0 ms; mean +/- SE, n = 37; from seven fibers) was independent of the membrane potential giving an upper estimate for the delay in myofilament activation. We thus emerge with quantitative values for the calcium binding to the regulatory sites on TnC under maintained structural conditions close to those in vivo.

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.

The effect of temperature on charge movement repriming in amphibian skeletal muscle fibers.

Cut twitch muscle fibers, mounted in a triple Vaseline-gap chamber, were used to study the effects of temperature on intramembranous charge movement and, in particular, on the repriming of charge 1 (the intramembranous charge that normally moves in the potential range between -100 and +40 mV). Changing the holding potential from -90 to 0 mV modified the voltage distribution of charge movement but not the maximum movable charge. Temperature changes between 16 and 5 degrees C did not modify the fiber linear capacitance, the maximum nonlinear intramembranous charge, or the voltage distribution of charge 1 and charge 2 (the intramembranous charge moving in the membrane potential range between approximately -4 and -160 mV). We used a pulse protocol designed to study the repriming time course of charge 1, with little contamination from charge 2. The time course of charge movement repriming at 15 degrees C is described by a double exponential with time constants of 4.2 and 25 s. Repriming kinetics were found to be highly temperature dependent, with two rate-limiting steps having Q10 (increase in rate of a process by raising temperature 10 degrees C) values of 1.7 and 7.1 above and below 11.5 degrees C, respectively. This is characteristic of processes with a high energy of activation and could be associated with a conformational change of the voltage sensor or with the interaction between the voltage sensor and the calcium release channel.

Ca2+ release from the sarcoplasmic reticulum compared in amphibian and mammalian skeletal muscle.

Puzzled by recent reports of differences in specific ligand binding to muscle Ca2+ channels, we quantitatively compared the flux of Ca2+ release from the sarcoplasmic reticulum (SR) in skeletal muscle fibers of an amphibian (frog) and a mammal (rat), voltage clamped in a double Vaseline gap chamber. The determinations of release flux were carried out by the "removal" method and by measuring the rate of Ca2+ binding to dyes in large excess over other Ca2+ buffers. To have a more meaningful comparison, the effects of stretching the fibers, of rapid changes in temperature, and of changes in the Ca2+ content of the SR were studied in both species. In both frogs and rats, the release flux had an early peak followed by fast relaxation to a lower sustained release. The peak and steady values of release flux, Rp and Rs, were influenced little by stretching. Rp in frogs was 31 mM/s (SEM = 4, n = 24) and in rats 7 +/- 2 mM/s (n = 12). Rs was 9 +/- 1 and 3 +/- 0.7 mM/s in frogs and rats, respectively. Transverse (T) tubule area, estimated from capacitance measurements and normalized to fiber volume, was greater in rats (0.61 +/- 0.04 microns-1) than in frogs (0.48 +/- 0.04 micron-1), as expected from the greater density of T tubuli. Total Ca in the SR was estimated as 3.4 +/- 0.6 and 1.9 +/- 0.3 mmol/liter myoplasmic water in frogs and rats. With the above figures, the steady release flux per unit area of T tubule was found to be fourfold greater in the frog, and the steady permeability of the junctional SR was about threefold greater. The ratio Rp/Rs was approximately 2 in rats at all voltages, whereas it was greater and steeply voltage dependent in frogs, going through a maximum of 6 at -40 mV, then decaying to approximately 3.5 at high voltage. Both Rp and Rs depended strongly on the temperature, but their ratio, and its voltage dependence, did not. Assuming that the peak of Ca2+ release is contributed by release channels not in contact with voltage sensors, or not under their direct control, the greater ratio in frogs may correspond to the relative excess of Ca2+ release channels over voltage sensors apparent in binding measurements. From the marked differences in voltage dependence of the ratio, as well as consideration of Ca(2+)-induced release models, we derive indications of fundamental differences in control mechanisms between mammalian and amphibian muscle.

Ryanodine interferes with charge movement repriming in amphibian skeletal muscle fibers

Cut twitch muscle fibers mounted in a triple Vaseline-gap chamber were used to study the effects of ryanodine on intramembranous charge movement, and in particular on the repriming of charge 1. Charge 1 repriming was measured either under steady-state conditions or by using a pulse protocol designed to study the time course of repriming. This protocol consisted of repolarizing the fibers to -100 mV from a holding potential of 0 mV, and then measuring the reprimed charge moving in the potential range between -40 and +20 mV. Ryanodine at a high concentration (100 microM) did not affect the maximum amount of movable charge 1 and charge 2, or their voltage dependence. This indicates that the alkaloid does not interact with the voltage sensor molecules. However, ryanodine did reduce the amount of reprimed charge 1 by approximately 60% suggesting the possibility of a retrograde interaction between ryanodine receptors and voltage sensors.

A slow component of intramembranous charge movement during sarcoplasmic reticulum calcium release in frog cut muscle fibers.

Cut muscle fibers from Rana temporaria were mounted in a double Vaseline-gap chamber and equilibrated with an end-pool solution that contained 20 mM EGTA and 1.76 mM Ca (sarcomere length, 3.3-3.8 microns; temperature, 14-16 degrees C). Sarcoplasmic reticulum (SR) Ca release, delta[CaT], was estimated from changes in myoplasmic pH (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259-336). The maximal value of delta[CaT] obtained during a depleting depolarization was assumed to equal the SR Ca content before stimulation, [CaSR]R (expressed as myoplasmic concentration). After a depolarization to -55 to -40 mV in fibers with [CaSR]R = 1,000-3,000 microM, currents from intramembranous charge movement, Icm, showed an early I beta component. This was followed by an I gamma hump, which decayed within 50 ms to a small current that was maintained for as long as 500 ms. This slow current was probably a component of Icm because the amount of OFF charge, measured after depolarizations of different durations, increased according to the amount of ON charge. Icm was also measured after the SR had been depleted of most of its Ca, either by a depleting conditioning depolarization or by Ca removal from the end pools followed by a series of depleting depolarizations. The early I beta component was essentially unchanged by Ca depletion, the I gamma hump was increased (for [CaSR]R > 200 microM), the slow component was eliminated, and the total amount of OFF charge was essentially unchanged. These results suggest that the slow component of ON Icm is not movement of a new species of charge but is probably movement of Q gamma that is slowed by SR Ca release or some associated event such as the accompanying increase in myoplasmic free [Ca] that is expected to occur near the Ca release sites. The peak value of the apparent rate constant associated with this current, 2-4%/ms at pulse potentials between -48 and -40 mV, is decreased by half when [CaSR]R approximately equal to 500-1,000 microM, which gives a peak rate of SR Ca release of approximately 5-10 microM/ms.

Effect of sarcoplasmic reticulum calcium depletion on intramembranous charge movement in frog cut muscle fibers.

Cut muscle fibers from Rana temporaria (sarcomere length, 3.3-3.5 microns; temperature, 13-16 degrees C) were mounted in a double Vaseline-gap chamber and equilibrated for at least an hour with an internal solution that contained 20 mM EGTA and phenol red and an external solution that contained predominantly TEA-gluconate; both solutions were nominally Ca-free. The increase in total myoplasmic concentration of Ca (delta[CaT]) produced by sarcoplasmic reticulum (SR) Ca release was estimated from the change in pH produced when the released Ca was complexed by EGTA (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. Journal of General Physiology. 106:259-336). The resting value of SR Ca content, [CaSR]R (expressed as myoplasmic concentration), was taken to be equal to the value of delta[CaT] obtained during a step depolarization (usually to -50 to -40 mV) that was sufficiently long (200-750 ms) to release all of the readily releasable Ca from the SR. In ten fibers, the first depolarization gave [CaSR]R = 839-1,698 microM. Progressively smaller values were obtained with subsequent depolarizations until, after 30-40 depolarizations, the value of [CaSR]R had usually been reduced to < 10 microM. Measurements of intramembranous charge movement, Icm, showed that, as the value of [CaSR]R decreased, ON-OFF charge equality held and the amount of charge moved remained constant. ON Icm showed brief initial I beta components and prominent I gamma "humps", even after the value of [CaSR]R was < 10 microM. Although the amplitude of the hump component decreased during depletion, its duration increased in a manner that preserved the constancy of ON charge. In the depleted state, charge movement was steeply voltage dependent, with a mean value of 7.2 mV for the Boltzmann factor k. These and other results are not consistent with the idea that there is one type of charge, Q beta, and that I gamma is a movement of Q beta caused by SR Ca release, as proposed by Pizarro, Csernoch, Uribe, Rodríguez, and Ríos (1991. Journal of General Physiology. 97:913-947). Rather, our results imply that Q beta and Q gamma represent either two distinct species of charge or two transitions with different properties of a single species of charge, and that SR Ca content or release or some related event alters the kinetics, but not the amount of Q gamma. Many of the properties of Q gamma, as well as the voltage dependence of the rate of SR Ca release for small depolarizations, are consistent with predictions from a simple model in which the voltage sensor for SR Ca release consists of four interacting charge movement particles.

Calcium inactivation of calcium release in frog cut muscle fibers that contain millimolar EGTA or Fura-2.

Cut muscle fibers from Rana temporaria (sarcomere length, 3.4-4.2 microns) were mounted in a double Vaseline-gap chamber (14-15 degrees C) and equilibrated with end-pool solutions that contained 20 mM EGTA and 1.76 mM Ca. Sarcoplasmic reticulum (SR) Ca release was estimated from changes in pH (Pape, P. C., D.-S. Jong, and W.K. Chandler. 1995. Journal of General Physiology. 106:000-000). Although the amplitude and duration of the [Ca] transient, as well as its spatial spread from the release sites, are reduced by EGTA, SR Ca release elicited by either depolarizing voltage-clamp pulses or action potentials behaved in a manner consistent with Ca inactivation of Ca release. After a step depolarization to -20 or 10 mV, the rate of SR Ca release, corrected for SR Ca depletion, reached a peak value within 5-15 ms and then rapidly decreased to a quasi-steady level that was about half the peak value; the time constant of the last half of the decrease was usually 2-4 ms. Immediately after an action potential or a 10-15 ms prepulse to -20 mV, the peak rate of SR Ca release elicited by a second stimulation, as well as the fractional amount of release, were substantially decreased. The rising phase of the rate of release was also reduced, suggesting that at least 0.9 of the ability of the SR to release Ca had been inactivated by the first stimulation. There was little change in intramembranous charge movement, suggesting that the changes in SR Ca release were not caused by changes in its voltage activation. These effects of a first stimulation on the rate of SR Ca release elicited by a second stimulation recovered during repolarization to -90 mV; the time constant of recovery was approximately 25 ms in the action-potential experiments and approximately 50 ms in the voltage-clamp experiments. Fura-2, which is able to bind Ca more rapidly than EGTA and hence reduce the amplitude of the [Ca] transient and its spatial spread from release sites by a greater amount, did not prevent Ca inactivation of Ca release, even at concentrations as large as 6-8 mM. These effects of Ca inactivation of Ca release can be simulated by the three-state, two-step model proposed by Schneider, M. F., and B. J. Simon (1988, Journal of Physiology. 405:727-745), in which SR Ca channels function as a single uniform population of channels. (ABSTRACT TRUNCATED AT 400 WORDS)

Calcium release and its voltage dependence in frog cut muscle fibers equilibrated with 20 mM EGTA.

Sarcoplasmic reticulum (SR) Ca release was studied at 13-16 degrees C in cut fibers (sarcomere length, 3.4-3.9 microns) mounted in a double Vaseline-gap chamber. The amplitude and duration of the action-potential stimulated free [Ca] transient were reduced by equilibration with end-pool solutions that contained 20 mM EGTA with 1.76 mM Ca and 0.63 mM phenol red, a maneuver that appeared to markedly reduce the amount of Ca complexed by troponin. A theoretical analysis shows that, under these conditions, the increase in myoplasmic free [Ca] is expected to be restricted to within a few hundred nanometers of the SR Ca release sites and to have a time course that essentially matches that of release. Furthermore, almost all of the Ca that is released from the SR is expected to be rapidly bound by EGTA and exchanged for protons with a 1:2 stoichiometry. Consequently, the time course of SR Ca release can be estimated by scaling the delta pH signal measured with phenol red by -beta/2. The value of beta, the buffering power of myoplasm, was determined in fibers equilibrated with a combination of EGTA, phenol red, and fura-2; its mean value was 22 mM/pH unit. The Ca content of the SR (expressed as myoplasmic concentration) was estimated from the total amount of Ca released by either a train of action potentials or a depleting voltage step; its mean value was 2,685 microM in the action-potential experiments and 2,544 microM in the voltage-clamp experiments. An action potential released, on average, 0.14 of the SR Ca content with a peak rate of release of approximately 5%/ms. A second action potential, elicited 20 ms later, released only 0.6 times as much Ca (expressed as a fraction of the SR content), probably because Ca inactivation of Ca release was produced by the first action potential. During a depolarizing voltage step to 60 mV, the rate of Ca release rapidly increased to a peak value of approximately 3%/ms and then decreased to a quasi-steady level that was only 0.6 times as large; this decrease was also probably due to Ca inactivation of Ca release. SR Ca release was studied with small step depolarizations that open no more than one SR Ca channel in 7,000 and increase the value of spatially averaged myoplasmic free [Ca] by only 0.2 nM.

An improved double vaseline gap voltage clamp to study electroporated skeletal muscle fibers

An improved voltage clamp with a double vaseline gap chamber was designed to study electroporated skeletal muscle fibers. The new clamp eliminated spike overshock of membrane potential when applying step stimulation occurring in the traditional configuration. It allowed greater consistency in membrane potential distribution. After the intracellular resistances of the fiber segment at the vaseline gap area were compensated, it was possible to change membrane potential more quickly. Using this technique, strong electrical pulses used to mimic the situation of electrical shock can be delivered to the cell membrane by voltage clamp. Transmembrane currents of skeletal muscle cell were simultaneously measured during a high pulsed shock and resolved into different components. Distinct transient changes of the transmembrane current, involving the time courses of the formation of electroporation and their recovery time constants, can be recorded. Because of more even membrane potential distribution and faster response to pulsed membrane potential change, this technique is also suitable for membrane study under physiological conditions.

Perchlorate enhances transmission in skeletal muscle excitation-contraction coupling.

The effects of the anion perchlorate (present extracellularly at 8 mM) were studied on functional skeletal muscle fibers from Rana pipiens, voltage-clamped in a Vaseline gap chamber. Established methods were used to monitor intramembranous charge movement and flux of Ca release from the sarcoplasmic reticulum (SR) during pulse depolarization. Saponin permeabilization of the end portions of the fiber segment (Irving, M., J. Maylie, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:1-41) substantially reduced the amount of charge moving during conventional control pulses, thus minimizing a technical error that plagued our previous studies. Perchlorate prolonged the ON time course of charge movement, especially at low and intermediate voltages. The OFFs were also made slower, the time constant increasing twofold. The hump kinetic component was exaggerated by ClO4- or was made to appear in fibers that did not have it in reference conditions. ClO4- had essentially no kinetic ON effects at high voltages (> or = 10 mV). ClO4- changed the voltage distribution of mobile charge. In single Boltzmann fits, the midpoint potential V was shifted -20 mV and the steepness parameter K was reduced by 4.7 mV (or 1.78-fold), but the maximum charge was unchanged (n = 9). Total Ca content in the SR, estimated using the method of Schneider et al. (Schneider, M. F., B. J. Simon, and G. Szucs. 1987. Journal of Physiology. 392:167-192) for correcting for depletion, stayed constant over tens of minutes in reference conditions but decayed in ClO4- at an average rate of 0.3 mumol/liter myoplasmic water per s. ClO4- changed the kinetics of release flux, reducing the fractional inactivation of release after the peak. ClO4- shifted the voltage dependence of Ca release flux. In particular, the threshold voltage for Ca release was shifted by about -20 mV, and the activation of the steady component of release flux was shifted by > 20 mV in the negative direction. The shift of release activation was greater than that of mobile charge. Thus the threshold charge, defined as the minimum charge moved for eliciting a detectable Ca transient, was reduced from 6 nC/microF (0.55, n = 7) to 3.4 (0.53). The average of the paired differences was 2.8 (0.33, P < 0.01). The effects of ClO4- were then studied in fibers in modified functional situations. Depletion of Ca in the SR, achieved by high frequency pulsing in the presence of intracellular BAPTA and EGTA, simplified but did not eliminate the effects of ClO4-.(ABSTRACT TRUNCATED AT 400 WORDS)

Reduction of calcium inactivation of sarcoplasmic reticulum calcium release by fura-2 in voltage-clamped cut twitch fibers from frog muscle.

Cut fibers from Rana temporaria and Rana pipiens (striation spacing, 3.9-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14 degrees C. The Ca indicator purpurate-3,3' diacetic acid (PDAA) was introduced into the end pools and allowed to diffuse into the optical recording site. When the concentration at the site exceeded 2 mM, step depolarizations to 10 mV were applied and the [Ca] transient measured with PDAA was used to estimate Ca release from the sarcoplasmic reticulum (SR) (Baylor, S. M., W. K. Chandler, and M. W. Marshall. 1983. Journal of Physiology. 344:625-666). With depolarization, the rate of SR Ca release increased to an early peak and then rapidly decreased several-fold to a quasi-steady level. The total amount of Ca released from the SR at the time of peak rate of release appeared to be independent of SR Ca content, consistent with the idea that a single activated channel might pass, on average, a fixed number of ions, independent of the magnitude of the single channel flux. A possible explanation of this property is given in terms of locally induced Ca inactivation of Ca release. The solution in the end pools was then changed to one with PDAA plus fura-2. SR Ca release was estimated from the [Ca] transient, as before, and from the delta [Cafura-2] signal. On average, 2-3 mM fura-2 increased the quasi-steady level of the rate of SR Ca release by factors of 6.6 and 3.8, respectively, in three fibers from Rana temporaria and three fibers from Rana pipiens. The peak rate of release was increased in five of the six fibers but to a lesser extent than the quasi-steady level. In all fibers, the amplitude of the free [Ca] transient was markedly reduced. These increases in the rate of SR Ca release are consistent with the idea that Ca inactivation of Ca release develops during a step depolarization to 10 mV and that 2-3 mM fura-2 is able to reduce this inactivation by complexing Ca and thereby reducing free [Ca]. Once the concentration of fura-2 becomes sufficiently large, a further increase reduces the rate of SR Ca release. On average, 5-6 mM fura-2 increased the quasi-steady rate of release, compared with 0 mM fura-2, by 6.5 and 2.9, respectively, in four fibers from Rana temporaria and three from Rana pipiens.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of fura-2 on action potential-stimulated calcium release in cut twitch fibers from frog muscle.

Cut fibers (striation spacing, 3.6-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14-15 degrees C. One or both of the Ca indicators fura-2 and purpurate-3,3' diacetic acid (PDAA) were introduced into the optical recording site by diffusion from the end pools. Sarcoplasmic reticulum (SR) Ca release was elicited by action potential stimulation. With resting [fura-2] = 0 mM at the optical site, the [Ca] transient measured with PDAA was used to estimate SR Ca release (Baylor, S.M., W.K. Chandler, and M.W. Marshall. 1983. Journal of Physiology. 344:625-666). With resting [fura-2] > 0 mM, the contribution from Ca complexation by fura-2 was added to the estimate. When resting [fura-2] was increased from 0 to 0.5-2 mM, both the amount of SR Ca release and the maximal rate of release were increased by approximately 20%. These results are qualitatively similar to those obtained in intact fibers (Baylor, S.M., and S. Hollingworth. 1988. Journal of Physiology. 403:151-192; Hollingworth, S., A. B. Harkins, N. Kurebayashi, M. Konishi, and S. M. Baylor. 1992. Biophysical Journal. 63:224-234) and are consistent with a reduction of Ca inactivation of SR Ca release produced by 0.5-2 mM fura-2. With resting [fura-2] > or = 2 mM, the PDAA [Ca] transient was reduced to nearly zero and SR Ca release could be estimated from delta [Cafura-2] alone. When resting [fura-2] was increased from 2-4 to 5-6 mM, both the amount of SR Ca release and the maximal rate of release were decreased by approximately half, consistent with a possible reduction of Ca-induced Ca release (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophysical Journal. 60:867-873) or a possible pharmacological effect of fura-2.