HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 539/3/869    most recent 2001.013095v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chawla, S. Articles by Huang, C. L.-H. Search for Related Content PubMed PubMed Citation Articles by Chawla, S. Articles by Huang, C. L.-H.

Journal of Physiology (2002), 539.3, pp. 869-882
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.013095

Differential effects of sarcoplasmic reticular Ca²⁺-ATPase inhibition on charge movements and calcium transients in intact amphibian skeletal muscle fibres

Sangeeta Chawla, Jeremy N. Skepper * and Christopher L.-H. Huang

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

A hypothesis in which intramembrane charge reflects a voltage sensing process allosterically coupled to transitions in ryanodine receptor (RyR)-Ca²⁺ release channels as opposed to one driven by release of intracellularly stored Ca²⁺ would predict that such charging phenomena should persist in skeletal muscle fibres unable to release stored Ca²⁺. Charge movement components were accordingly investigated in intact voltage-clamped amphibian fibres treated with known sarcoplasmic reticular (SR) Ca²⁺-ATPase inhibitors. Cyclopiazonic acid (CPA) pretreatment abolished Ca²⁺ transients in fluo-3-loaded fibres following even prolonged applications of caffeine (10 mM) or K⁺ (122 mM). Both CPA and thapsigargin (TG) transformed charge movements that included delayed (q) 'hump' components into simpler decays. However, steady-state charge-voltage characteristics were conserved to values (maximum charge, Q_max 20-25 nC µF^-1; transition voltage, V* -40 to-50 mV; steepness factor, k 6-9 mV; holding voltage -90 mV) indicating persistent q charge. The features of charge inactivation similarly suggested persistent q and q charge contributions in CPA-treated fibres. Perchlorate (8.0 mM) restored the delayed kinetics shown by 'on' q charge movements, prolonged their 'off' decays, conserved both Q_max and k, yet failed to restore the capacity of such CPA-treated fibres for Ca²⁺ release. Introduction of perchlorate (8.0 mM) or caffeine (0.2 mM) to tetracaine (2.0 mM)-treated fibres, also known to restore q charge, similarly failed to restore Ca²⁺ transients. Steady-state intramembrane q charge thus persists with modified kinetics that can be restored to its normally complex waveform by perchlorate, even in intact muscle fibres unable to release Ca²⁺. It is thus unlikely that q charge movement is a consequence of SR Ca²⁺ release rather than changes in tubular membrane potential.

(Received 2 August 2001; accepted after revision 19 December 2001)
Corresponding author C. L.-H. Huang: Physiological Laboratory, Downing Street, Cambridge CB2 3EG, UK. Email: clh11{at}cus.cam.ac.uk

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

Capacity transients in voltage-clamped amphibian skeletal muscle contain at least two major intramembrane charge components. Small voltage excursions in fully polarized fibres elicit simple exponential q decays consistent with first-order dielectric transitions in a single charge species (Adrian & Peres, 1979; Huang, 1981, 1982). Larger voltage steps that reach or exceed the threshold for release of intracellularly stored Ca²⁺ additionally produce delayed q transients that may be both distinct from and independent of the earlier q transitions (Adrian & Huang, 1984; Huang, 1987; Hui & Chandler, 1991). The q charge was selectively localized to the transverse tubular rather than the surface membranes (Huang & Peachey, 1989). Its pharmacological and steady-state features closely parallel corresponding properties shown by excitation-contraction coupling (Huang, 1982; Vergara & Caputo, 1983; Hui, 1983; Adrian & Huang, 1984; for a review see Huang, 1993). These observations suggested that q currents reflect conformational changes within dihydropyridine receptors (DHPRs) in their function as voltage sensors driving configurational changes in ryanodine-receptor (RyR)-Ca²⁺ release channels during contractile activation (Rios & Brum, 1987; Huang, 1990; Chen & Hui, 1991). Certainly, pharmacological modifications of the RyR reciprocally and reversibly altered the kinetics, but not the total availability of q charge (Huang, 1996, 1998a,b). Biochemical evidence similarly suggests allosteric linkages between DHPRs and the RyR-1 or RyR- isoforms in mammalian and amphibian skeletal muscle triads but not RyR-2 isoforms in cardiac muscle (Anderson & Meissner, 1995; for reviews see Meissner, 1994; Franzini-Armstrong & Jorgensen, 1994). Such distinctions in turn are compatible with the observation of complex q charge movements specifically in skeletal muscle but not in other contractile systems that are triggered indirectly by Ca²⁺-induced Ca²⁺ release (Huang, 1993).

A direct allosteric coupling model would predict that the relevant charge movement features should persist in experimental systems whose capacity for Ca²⁺ release is significantly compromised by depletion of its intracellular stores. Thus, intramembrane charge was conserved, albeit with modified kinetics, in Vaseline gap preparations whose sarcoplasmic reticular (SR) Ca²⁺ was depleted by introduction of intracellular EGTA (Jong et al. 1995; Pape et al. 1996), as expected for capacitative transitions driven primarily by tubular voltage. In contrast, Csernoch et al. (1991) proposed that changes in cytosolic [Ca²⁺] rather than tubular voltage provide the immediate driving force for q charge transfer on the basis of electrophysiological and pharmacological manoeuvres applied to cut fibres. Such [Ca²⁺] changes might initially result from a movement of voltage-dependent q charge. Together, such mechanisms could potentially contribute to a positive feedback enhancement of intramembrane charge transfer (Pizarro et al. 1991).

The present experiments used cyclopiazonic acid (CPA) and thapsigargin (TG), known to deplete SR-Ca²⁺ by inhibiting SR-Ca²⁺-ATPase, in intact fibres (Seidler et al. 1989; Lytton et al. 1991; Sagara & Inesi, 1991; Sagara et al. 1992). The remaining conditions of pulse procedure, external solutions, osmolarity and temperature were comparable to those in previous explorations for reciprocal interactions between the RyR and intramembrane charge (Huang, 1996, 1998a,b). Furthermore, the q system appears to account for most of the intramembrane charge under such conditions (Hui & Chen, 1992, 1995; Jong et al. 1995; Pape et al. 1996). The capacity for such fibres to release Ca²⁺ following either applied depolarization or caffeine treatment was assessed using an assay introduced for intact, fluo-3-loaded muscle fibres by Caputo & Bolanos (1994). These results were compared with alterations in the intramembrane charge and the extent to which delayed q charge movements could be recovered in such preparations.

	METHODS

Top Abstract Introduction Methods Results Discussion References

Sartorius muscles were dissected in cold (4 °C) Ringer solution from cold-adapted frogs (Rana temporaria: Blades Biological, Kent, UK) previously killed by concussion followed by decapitation and pithing (UK Schedule 1 Home Office regulations). They were mounted in a temperature-controlled recording chamber and stretched to give centre fibre sarcomere lengths of 2.2-2.4 µM as measured using a Zeiss 40 water immersion objective and an eyepiece graticule. The Ringer solution was then replaced with the following isotonic solution at the same temperature: 120 mM tetraethylammonium gluconate, 2.0 mM MgCl₂, 2.5 mM RbCl, 800 µM CaCl₂, 1.0 mM 3,4-diaminopyridine, 2 10^-7 M tetrodotoxin and 3 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffered to pH 7.0. This was replaced after 15 min by a similar hypertonic solution containing 500 mM sucrose. The final test solutions that further included the adopted test agents at the concentrations indicated in the results were introduced 15 min prior to beginning electrophysiological studies. These studies were conducted under similarly cooled (2-4 °C) conditions. The pharmacological agents used in the experiments were cyclopiazonic acid (CPA: 0.5, 5.0 or 50 µM), thapsigargin (TG: 30 µM) and perchlorate (8.0 mM).

A three-microelectrode voltage clamp used conventional 3-5 M glass electrodes penetrating the pelvic ends of the superficial muscle fibres directly exposed to the bathing solutions. The voltage-recording electrodes were filled with 3 M KCl and positioned at distances of l = 375 µm (voltage control electrode, V₁) and 2l = 750 µm (second voltage electrode, V₂) from the fibre end respectively (Huang, 2001). The shielded current injection electrode I₀ was filled with 2 M potassium citrate and inserted at 5l/2 = 940 µm. Electrical signals that represented the clamp voltage V₁, the voltage difference (V₁ - V₂), and the injected current I₀(t), were filtered through 3-pole Butterworth filters set to a 1.0 kHz cut-off frequency. Sampling involved a 12-bit analog to digital conversion at a 200 µs sampling interval. Five sweeps, spaced by intervals of 20 s, were averaged into each test or control record.

Test transients were obtained in response to voltage steps of duration 124 ms made to different membrane potentials. The control pulses regularly bracketed successive sets of four to six such test acquisitions. They superimposed +50 mV steps at a 300 ms interval following the introduction of prepulse steps of duration 610 ms to a level of -140 mV. Steady-state values of V₁(t), V₂(t) and injected current I₀(t) were all attained well before the end of such control steps. These were accordingly determined directly from the experimental records without the sloping baseline corrections hitherto required for both control and test responses in some cut fibre preparations. Fibre length constants, , internal longitudinal resistances, r_i, and membrane resistances of unit fibre length, r_m were computed on-line from each individual averaged record. Calculations of fibre diameters, d, and specific membrane resistances, R_m, assumed an internal sarcoplasmic resistivity, R_i of 391 cm in 2.5 times hypertonic solution at 2 °C, and a temperature coefficient of 0.73. The Fig. legends list the calculated cable constants; comparison of their values before and after each experimental run made it possible to assess for significant systematic changes in fibre stability and condition over time. The membrane current through unit fibre surface area I_m(t) was calculated from the equation:

I_m(t) = [V₁(t) - V₂(t)]d/(6l²R_i).

Charge movements were derived by subtracting test traces from traces obtained from control records constituted from the depolarizing and hyperpolarizing responses not only to the control steps but also their associated prepulses. This doubled the effective level of signal averaging and thus further enhanced signal to noise ratio. The derived control traces were then scaled to the ratio of the respective amplitudes of test and control voltage steps. Similar scalings and subtractions were also applied to the averaged records of the test and the control voltage (V₁) steps; the latter further verified that the derived charge movements indeed reflected non-linear contributions to fibre electrical properties. In addition, any small change in linear membrane properties that might have taken place over the course of each set of test voltage steps was also corrected for. Thus, the final control records with which test/control comparisons were made were constituted from a weighted mean of the two bracketing control records, each weighted using the position of the relevant test average within the bracketed test sequence.

The inactivation properties of the intramembrane charge were also studied in CPA-treated fibres. Again the control pulse procedures superimposed +50 mV steps at an interval of 300 ms following each prepulse step to -140 mV. They were imposed at least 30 s after the fibre was returned to a fully polarized holding potential of -90 mV and both preceded and bracketed successive sets of test runs. The test voltage steps were applied at least 30 s following the shift to the specific holding potential, V_H, investigated. They returned the membrane potential to a fixed prepulse level of -90 mV, then applied a fixed test step to -10 mV. The membrane potential was then returned to the chosen holding level following the 'off' portion of the current response. Both test and control records were made up from the averaged results from five such sweeps.

All the results of the steady-state determinations are expressed as means ± standard errors of the mean (S.E.M.). The steady-state charge-voltage data were described using a single two-state Boltzmann relationship between the steady-state charge movement, Q(V), the maximum charge, Q_max, the transition voltage, V*, and the steepness factor, k:

Q(V) = Q_max/{1 + exp[-(V - V*)/k]}.

Some of the data were fitted to the sum of two rather than one Boltzmann functions as described by the differential sensitivities, QV) and Q(V), of q and q charge to sustained depolarization to a holding potential of -50 mV (Huang, 1994b, 1996):

Q(V) = Q(V) + Q(V).

The inactivation functions were also empirically described by two-state Boltzmann-type functions and made use of the values of the charge Q_-10(V_H) transferred by a voltage step from -90 to -10 mV:

Q_-10(V_H) = Q_max,-10{1 - 1/{1 + exp[-(V_H - V _H *)/k]}}.

A least-squares, Levenberg-Marquadt algorithm (Origin: OriginLab Corp., Northampton, MA, USA) performed a weighted fit of these functions to the mean and standard errors of the data points.

For fluorescence measurements, intact lumbricalis V muscle fibres were loaded with the acetoxymethyl (AM) ester of fluo-3 (Molecular Probes) by incubating with 5 µM fluo-3 AM in frog Ringer solution at 25 °C for 25 min (see Caputo & Bolanos, 1994). Muscles were subsequently washed in Ringer solution and mounted close to their resting lengths (sarcomere length 1.9-2.0 µm) between two glass coverslips held together with vacuum grease in a 300 µl perfusion chamber. The chamber was perfused using cooled solutions (4-10 °C). Fluo-3 fluorescence emission, excited with a 488 nm argon laser, was measured at 505-550 nm using a Leica TCS-SP-MP laser scanning confocal system with a 20 water immersion objective (NA 0.7) on a Leica DMIRBE inverted microscope. Muscles were initially bathed in isotonic Ringer solution; this was replaced by 500 mM sucrose-Ringer for 10 min before the application of the experimental solutions, all of which also contained 500 mM sucrose in order to suppress mechanical activity. At such concentrations, sucrose has been shown to suppress mechanical activity (Caputo, 1968) whilst permitting both transitions in intramembrane charge (Huang, 1992) and cytosolic Ca²⁺ transients in response to applied depolarization (Parker & Zhu, 1987) as was also apparent in the present experiments.

Measurements were made from fibres adjacent to the muscle edge showing optimal dye loading. They were obtained over fibre lengths (100-350 µm; and diameters 25-65 µm) that fell clearly within the plane of visualization permitted by a maximum pinhole size that gave a ~ 40 µm slice thickness. The observed changes in fluo-3 fluorescence following introduction of the test solutions proved homogeneous over such regions of interest: they closely agreed with records obtained from smaller ~ 15 µm by 15 µm pixel areas except that the latter showed predictably lower ratios of signal to noise. This agreement was apparent even with the presence of occasional nuclei within such regions of interest. Frame scans were obtained before, during and following perfusion with cooled stimulating solutions. The latter were made up of 10 mM caffeine-sucrose-Ringer or were sucrose-Ringer solutions in which the NaCl and KCl were replaced by 122 mM KCl to which the pharmacological agents under test were also added. Arrival of the stimulating solution in the bath was monitored by fluorescence from sulforhodamine B (63 µg ml^-1) added to the perfusing solution captured between 580 and 620 nm after excitation using a 568 nm laserline. A series of 200 frames sampled at 830 ms per frame (512 512 pixels per frame) monitored fluorescence changes over time. Confocal aperture was at its maximum. Images were analysed using a modified version of the public domain NIH Image program (National Institutes of Health, Bethesda, MD, USA). Fluorescence measurements (F) were made within regions of interest made up of those portions of each fibre within view. These were normalized to resting fluorescence (F₀) values.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Ca²⁺-ATPase inhibitors remove obvious delayed charging phases normally identified with transitions in q charge

The experiments used two chemically distinct agents, cyclopiazonic acid (CPA) and thapsigargin (TG), known to inhibit SR-Ca²⁺-ATPase (Seidler et al. 1989; Lytton et al. 1991; Sagara & Inesi, 1991; Sagara et al. 1992). Both CPA (~2.5 µM) and TG (~ 0.5 µM) inhibit Ca²⁺-ATPase-mediated transport in isolated amphibian muscle SR vesicles. Skinned but otherwise intact frog skeletal muscle fibres require higher concentrations. CPA is then the more effective and specific agent, particularly in amphibian fibres, inhibiting Ca²⁺-ATPase by 50-100 % at concentrations of 7-50 µM in contrast to a 50 % inhibition at a TG concentration > 130 µM. Furthermore, CPA (100 µM) but not TG (300 µM) completely inhibits the Ca²⁺ loading subsequent to caffeine contractures in fibres whose SR was initially loaded with Ca²⁺ (Du, 1996; Du et al. 1994, 1996). The present experiments nevertheless investigated the effects of both reagents with CPA applied more extensively through a wider range of concentrations (0.5, 5.0 and 50 µM).

The experiments first investigated the effects of the Ca²⁺-ATPase inhibitors upon both intramembrane transients and the steady-state distribution of the nonlinear charge obtained following depolarizing steps made to a wide range of test voltages in muscle fibres held at a membrane potential of -90 mV. Intact fibres were thus studied under voltage clamp configurations and conditions of pulse procedure, external solutions, osmolarity and temperature comparable to those adopted in recent studies. The latter both assessed and confirmed charge conservation through the 'on' and 'off' parts of imposed voltage steps (Huang, 1994a), and went on to explore for properties attributable to allosteric coupling between RyRs and the intramembrane charge (Huang, 1996, 1998a,b). Figure 1A illustrates charge movements obtained in control fibres held at a fixed, -90 mV membrane potential and subjected to applied voltage steps to a series of progressively depolarized test levels, V. These test levels were incremented in 5 mV intervals where it was advantageous to investigate for the existence of and changes in steeply voltage-sensitive delayed q charge movements. Thus voltage steps to -50 mV gave rise to the simple exponential current decays that have been previously attributed to q charge (Huang, 1982) and that were followed by the delayed transients identified with q charge transfer (Adrian & Peres, 1979; Huang, 1982; Hui, 1983). These 'on' currents were small but prolonged around their threshold voltages (Fig. 1A, horizontal bars beneath traces). Nevertheless, they were accompanied by a marked increase in the initial amplitudes of their corresponding 'off' tail currents. The detailed time courses of the 'on' q transients were sharply voltage sensitive: even small further depolarizations to levels around -45 mV gave rise to more rapid and prominent current decays clearly distinguishable from the earlier q transitions. As reported on earlier occasions, the two component transients merged at voltages close or positive to -35 mV to leave monotonic decays in which the two charge movement components could not be distinguished.

		View larger version [in this window] [in a new window]
Figure 1. Charge movements in control fibres and comparisons between 'on' and 'off' charge following treatment with CPA A, typical charge movements obtained from a control fibre studied in the absence of Ca2+-ATPase inhibitors showing delayed (q) charging phases as reported on earlier occasions. Fibre a89. Temperature = 2.7 °C, Ri = 427.3 cm. Cable constants: = 2.16 mm, ri = 7904.3 k cm-1, d = 83.0 µm, rm = 367.8 k cm, Rm = 9.59 k cm2, Cm = 6.5 µF cm-2. B and C, plots of 'on' against 'off' intramembrane charge following the addition of 0.5 (B; 5 fibres) and 50 µM CPA (C; 6 fibres: see legend to Fig. 4 for details).

The subsequent test electrophysiological studies were completed within 60 min of adding CPA or TG to the extracellular solutions. Their resulting 'on' charging currents similarly all decayed to time-invariant baselines and accordingly only required simple direct current corrections based on the final 20 ms of such 'on' records. Intervening time-dependent inward current phases reported elsewhere (Csernoch et al. 1991) were nowhere observed in the present study (cf. Hui & Chen, 1994). Gradually developing 'on' outward currents only appeared in some of the responses to the strongest depolarizing steps that extended to test voltages around 0 mV. Values for the integrated 'on' and 'off' charges in CPA-treated fibres closely fell to the line of equality (for 'on' charge = m 'off' charge, m = 0.895 ± 0.024 (0.5 µM CPA; Fig. 1B); 0.967 ± 0.032 (5 µM CPA) and 0.921 ± 0.053 (50 µM CPA; Fig. 1C), respectively. This agreed with earlier findings in fibres studied in the absence of CPA but under otherwise similar conditions (m = 0.959 ± 0.0119: Huang, 1994a; see also Huang, 1996, 1998a,b).

Figure 2 displays typical charge movements from voltage-clamped fibres studied in the presence of 0.5 (A) 5.0 (B) and 50 µM CPA (C) respectively. Voltage steps were made to a series of progressively depolarized test levels, V, from a fixed holding potential of -90 mV. The test steps were closely incremented in order to optimize detection of the existence of, or changes in, the steeply voltage-sensitive delayed q charge movement components. The charging records additionally are displayed at high gain to emphasise contributions of such smaller or delayed current components to the overall waveforms. As reported earlier, the smaller voltage steps transferred little charge. However, depolarizing steps made to test levels close to or beyond -60 mV gave rise to the small monotonic decays hitherto identified with q charge movements (Huang, 1982); these progressively increased in amplitude with increasing depolarization. Some of the fibres in 0.5 µM CPA additionally showed small but discernible delayed components that could be attributed to q currents (Fig. 2A). However, these were considerably less prominent and were only just distinguishable from the early, larger decays in the experimental records that were obtained at membrane potentials around -35 mV and -40 mV (cf. Fig. 1A).

		View larger version [in this window] [in a new window]
Figure 2. Treatment with cyclopiazonic acid (CPA) removes delayed charging phases normally identified with transitions in q charge Typical charge movements obtained from voltage-clamped fibres studied in the presence of 0.5 (A) 5.0 (B) and 50 µM CPA (C). Voltage steps were made to a series of progressively depolarized test levels, V, from a fixed holding potential of -90 mV. Display was at high gain to emphasise contributions of smaller or delayed current components, which can just be discerned in (A) but not in (B) or (C). A, fibre a60 in 0.5 µM CPA. Temperature = 4.5 °C, Ri = 403.8 cm. Cable constants: = 2.12 mm, ri = 8339.1 k cm-1, d = 78.5 µm, rm = 374.09 k cm, Rm = 9.23 k cm2, Cm = 11.4 µF cm-2. B, fibre a61 in 5.0 µM CPA. Temperature = 5.0 °C, Ri = 397.5 cm. Cable constants: = 1.81 mm, ri = 7862.2 k cm-1, d = 80.2 µm, rm = 257.05 k cm, Rm = 6.47 k cm2, Cm = 8.9 µF cm-2. C, fibre z79 in 50 µM CPA. Temperature = 4.3 °C, Ri = 406 cm. Cable constants: = 1.41 mm, ri = 8698 k cm-1, d = 77.1 µm, rm = 172 k cm, Rm = 4.17 k cm2, Cm = 8.3 µF cm-2.

Figure 2B and C demonstrate that higher (5.0 or 50 µM) CPA concentrations further reduced even this limited evidence of delayed q charging transients. Significant contributions from delayed q charge movements were absent within the explored potential range of test voltages even at high display magnifications. The initial amplitude of the currents increased to an extent that would be expected for more rapid transfers of an otherwise conserved available charge. These amplitudes increased with depolarization until charge saturation. Thapsigargin (30 µM) exerted similar effects to CPA upon the time course of the intramembrane charge movement (Fig. 3A). Thus both reagents resulted in charge movements similar in form to those observed following pharmacological manipulations of the RyR in intact fibres (Huang, 1996, 1998b)

		View larger version [in this window] [in a new window]
Figure 3. Charge movements following different manoeuvres to demonstrate steeply voltage-dependent charge movement in fibres exposed to Ca2+-ATPase inhibitors A, charging transients from a fibre treated with the alternative Ca2+ pump blocker thapsigargin (30 µM) independently shows alterations to the charging transients similar to those following treatment with CPA (Fig. 2). B, charge movements following large voltage steps made from a prepulse level of -90 mV to a fixed test voltage of -10 mV in a CPA-treated fibre held at different holding potentials, VH. C, charge movements following test voltage steps made from a prepulse level of -90 mV to a range of test voltages, V, in a CPA-treated fibre following a shift in holding voltage from -90 to -50 mV. A, fibre a26 in thapsigargin. Temperature = 6.1 °C, Ri = 383.9 cm. Cable constants: = 1.67 mm, ri = 5207.9 k cm-1, d = 96.9 µm, rm = 144.75 k cm, Rm = 4.41 k cm2, Cm = 15.4 µF cm-2. Inactivation study (B): fibre a77 in 50 µM CPA. Temperature = 6.0 °C, Ri = 385.14 cm. Cable constants: = 1.56 mm, ri = 8877.8 k cm-1, d = 74.3 µm, rm = 214.8 k cm, Rm = 5.02 k cm2, Cm = 13.6 µF cm-2. Effect of fixed shift in holding voltage studied in fibre a52 (C). Temperature = 4.0 °C, Ri = 410.1 cm. Cable constants: = 1.50 mm, ri = 10070 k cm-1, d = 72.0 µm, rm = 227.69 k cm, Rm = 5.15 k cm2, Cm = 14.66 µF cm-2.

Both cyclopiazonic acid and thapsigargin conserve the steady-state dependence of intramembrane charge upon test potential in fully polarised fibres

Figure 4 plots charge-voltage data obtained in control fibres in the absence of Ca²⁺-ATPase blockers (A, squares), and from fibres studied in the presence of 0.5 µM (B, triangles), 5.0 µM (C, inverted triangles) and 50 µM CPA (D, circles) as well as 30 µM TG (E, diamonds; all plotted as means ± S.E.M.). All such pharmacological interventions preserved both the steady-state charge and the steepness of its voltage dependence. The control results (Fig. 4A) confirm earlier reports of an available charge that increased with progressive depolarization from the -90 mV holding level. This crossed an inflexion at test potentials close to -50 mV before increasing to a maximum value around 20 nC µF^-1 between test voltages of -30 to 0 mV (Huang, 1996). Figure 4B-E confirms similar charge-voltage plots despite treatment with either CPA (B-D) or TG (E).

		View larger version [in this window] [in a new window]
Figure 4. Conservation of steady-state charge-voltage properties in fibres exposed to Ca2+-ATPase inhibitors Charge-voltage curves obtained in the presence of cyclopiazonic acid (CPA) at concentrations of 0.0 (A, ), 0.5 (B, ), 5.0 (C, ) and 50 µM (D, ) and thapsigargin (TG) (30 µM: E, ). Fibres at a -90 mV holding potential. Six fibres studied in 0.0 µM CPA (A): Temperature = 6.2 ± 0.1 °C, Ri = 383 ± 1.6 cm. Cable constants: = 2.0 ± 0.15 mm, ri = 9111.0 ± 713.5 k cm-1, d = 74.5 ± 3.1 µm, rm = 364.1 ± 39.9 k cm, Rm = 8.5 ± 1.09 k cm2, Cm = 6.3 ± 0.47 µF cm-2. Five fibres studied in 0.5 µM CPA (B). Temperature = 5.1 ± 0.17 °C, Ri = 395.9 ± 2.14 cm. Cable constants: = 1.9 ± 0.18 mm, ri = 6565.5 ± 972.26 k cm-1, d = 91.2 ± 7.90 µm, rm = 251.74 ± 51.84 k cm, Rm = 6.79 ± 1.27 k cm2, Cm = 14.5 ± 1.89 µF cm-2. Four fibres studied in 5.0 µM CPA (C). Temperature = 6.4 ± 0.09 °C, Ri = 379.4 ± 1.11 cm. Cable constants: = 1.51 ± 0.23 mm, ri = 6473.25 ± 1492.9 k cm-1, d = 92.9 ± 12.65 µm, rm = 156.39 ± 48.02 k cm, Rm = 4.23 ± 1.29 k cm2, Cm = 9.7 ± 2.51 µF cm-2. Six fibres studied in 50 µM CPA (D). Temperature = 4.75 ± 0.40 °C, Ri = 400.59 ± 5.01 cm. Cable constants: = 1.77 ± 0.18 mm, ri = 9884.8 ± 1992.8 k cm-1, d = 76.1 ± 6.01 µm, rm = 280.5 ± 27.98 k cm, Rm = 6.73 ± 0.95 k cm2, Cm = 7.7 ± 1.10 µF cm-2. Seven fibres studied in 30 µM TG (E). Temperature = 6.5 ± 0.02 °C, Ri = 379.6 ± 0.31 cm. Cable constants: = 2.4 ± 0.23 mm, ri = 4818.94 ± 933.77 k cm-1, d = 116.1 ± 6.17 µm, rm = 244.33 ± 45.31 k cm, Rm = 8.04 ± 1.29 k cm2, Cm = 19.7 ± 3.00 µF cm-2.

Table 1 summarizes results of the least-squares minimizations of the steady-state charge-voltage data obtained at each CPA or TG concentration to an equation for a single two-state Boltzmann system. It confirms that such interventions conserved the steady-state values of the maximum charge, Q_max, and steepness factor k, apart from small shifts in the transition potential V* with the highest concentration of CPA. Thus, introduction of CPA preserved the total charge Q_max at values of 20-23 nC µF^-1; if anything TG slightly increased the maximum charge. Similarly both CPA and TG left the steepness factors, k, at around 6-9 mV. These findings agreed with earlier reports from fibres in both Vaseline gap or microelectrode voltage clamp preparations studied in the absence of CPA or TG (Huang, 1994a,b; 1996; Jong et al. 1995). This strongly suggests that the q charge contribution is conserved in the gluconate-containing extracellular solutions used in the present experiments following addition of either CPA or TG.

The features of charge inactivation suggest that q charge persists following CPA treatment

Figs. 3B and C show typical charge movements from CPA-treated fibres under different conditions of holding potential. They also suggest that q continues to contribute to intramembrane charge in fibres exposed to 50 µM CPA. Thus, Fig. 3B shows charge movements in response to large voltage steps made to a fixed test potential of -10 mV that were imposed 300 ms after a prepulse to -90 mV from a range of holding potentials, V_H. The latter were altered in 10 mV increments in the range -90 to -10 mV with the test procedures applied at least 30 s after each shift in V_H. The experimental records were based on averages of five such sweeps. Groups of five such test manoeuvres were bracketed by sets of control steps from a -140 mV prepulse level to a membrane voltage of -90 mV; these were similarly imposed 30 s after V_H was returned to -90 mV. Such test steps would be expected to transfer both q and q charge.

Such charge movements were consistently monotonic in waveform as expected following large test excursions to strongly depolarized potentials (Fig. 3B). However, their amplitude declined with successively positive shifts in holding potential particularly between the holding voltages of -50 to -40 mV as expected from previous reports on the steeply voltage-dependent inactivation of q charge (Huang, 1994b, 1996). The corresponding steady-state dependence of maximum charge, Q_max(V_H), against holding voltage V_H (Fig. 5A, open circles) confirmed a maximum charge around 25 nC µF^-1 in fully polarized fibres, in agreement with earlier reports (Huang, 1994b, 1996). Small holding potential shifts from -90 to -70 mV did not appreciably influence this total available charge but larger potential changes to between -50 and -60 mV produced a sharp inactivation. The latter fitted a two-state Boltzmann function with values of maximum charge, Q_max = 25.1 ± 1.19 nC µF^-1, transition potential, V*_H = -54.7 ± 1.68 mV and steepness factor, k = 8.9 ± 1.33 mV consistent with a persistent q charge (Huang, 1996).

		View larger version [in this window] [in a new window]
Figure 5. Inactivation properties shown by steady-state intramembrane charge in CPA-containing solutions A, inactivation curves (mean ± S.E.M.; 7 fibres) obtained by determination of the intramembrane charge movement (Qmax(VH)) following voltage steps from -90 mV to -10 mV at different holding potentials, VH (). Temperature = 5.6 ± 0.21 °C, Ri = 389.8 ± 2.61 cm. Cable constants: = 1.50 ± 0.13 mm, ri = 9781.7 ± 1768.2 k cm-1, d = 77.09 ± 7.45 µm, rm = 213.33 ± 34.91 k cm, Rm = 4.87 ± 0.79 k cm2, Cm = 14.1 ± 2.18 µF cm-2. B, the dependence of the steady-state charge (Q(V)) upon test voltage (mean ± S.E.M.; 4 fibres) studied at a holding potential (VH) shifted from -90 to -50 mV (). Temperature = 5.1 ± 0.35 °C, Ri = 396.5 ± 4.53 cm. Cable constants: = 1.60 ± 0.16 mm, ri = 10850.13 ± 1875.2 k cm-1, d = 71.1 ± 5.59 µm, rm = 257.20 ± 14.88 k cm, Rm = 5.76 ± 0.70 k cm2, Cm = 13.1 ± 1.40 µF cm-2.

Figure 3C shows charge movements in response to test steps to potentials, V, between -80 and 0 mV imposed 300 ms after returning the fibre to a prepulse level of -90 mV from a shifted holding potential of -50 mV. The latter produced a selective charge inactivation leaving simple monotonic transients whose amplitudes were significantly reduced and whose voltage dependence reflected considerably shallower charge-voltage curves and a maximum charge reduced to around 10 nC µF^-1 (Fig. 5B, filled circles). Shifts in holding voltage in CPA-treated fibres thus produced a charge separation in close agreement with earlier reports of the selective inactivation of q as opposed to q charge (Huang, 1981, 1994b). Such a separation made it possible to minimize the data displayed in Fig. 4 to the sum of two Boltzmann functions (Hui & Chandler, 1990; Hui & Chen, 1992) using parameters for q charge as obtained at the shifted, -50 mV, holding potential (Fig. 5B; see Huang, 1996; cf. Hui & Chandler, 1990; Hui & Chen, 1992). Table 2 summarizes the resulting values of Q_max, V* and k that were thus extracted for the q system. These gave values of maximum q charge, Q_max, of 10-15 nC µF^-1 and steepness factors, k, between 3-6 mV, in all the cases (a)-(e). The transition potentials similarly showed no systematic variations with CPA treatment. These findings closely agree with earlier isolations of q charge in fibres studied in otherwise similar conditions in the absence of Ca²⁺-ATPase antagonists (Table 2a).

Perchlorate restores delayed kinetics to q charge movement even in Ca²⁺ depleted fibres

The chaotropic ion perchlorate is known to potentiate excitation-contraction coupling in amphibian skeletal muscle (Gomolla et al. 1983; Luttgau et al. 1983; Huang, 1986; Gonzalez & Rios, 1993). It may do so through preferentially shifting the threshold and increasing the prominence of both delayed 'on' q currents and slow 'off' q recovery tails while conserving the corresponding steady-state charge and its steep voltage dependence. This influence on q charge has been attributed to the action of perchlorate upon the RyR (Ma et al. 1993)which in turn is in reciprocal allosteric contact with intramembrane DHPR-voltage sensors (Huang, 1998a). Figure 6 displays charge movements elicited by depolarizing voltage-clamp steps imposed from the -90 mV holding level in CPA-treated muscle fibres studied in the presence of 8.0 mM perchlorate. The potentiating effects of perchlorate persisted with a restoration of delayed q 'hump' currents following their initial suppression by Ca²⁺-ATPase inhibitors. Significant intramembrane charge movements were absent in response to small potential steps to -70 and -80 mV. However, slightly larger depolarizing steps to -60 and -55 mV elicited marked q 'hump' currents consistent with a -10 to -20 mV negative shift in their threshold (Huang, 1994a,b). Such slow 'on' q charge movements thus took place even when appreciable prior q decays were absent: this is incompatible with such q decays causing the delayed movements of q charge (cf. Hui & Chen, 1994). The q decays still appeared at more positive membrane potentials around and beyond -45 mV when prominent q hump waveforms were already well established. Such distinct q 'hump' charge movements were thus readily demonstrable over a wide (-60 to -30 mV) voltage range. The perchlorate treated fibres also showed prolonged 'off' currents that have previously been attributed to recovery of a conserved q charge (Huang, 1987).

		View larger version [in this window] [in a new window]
Figure 6. Effects of perchlorate on charging kinetics in CPA-treated fibres Charge movements were elicited by depolarizing test steps that were varied from the -90 mV holding potential in CPA (50 µM)-treated muscle fibres in the presence of 8.0 mM perchlorate. Note the restoration of 'on' 'hump' currents characteristic of q charge that took place in the absence of any appreciable prior q decay at some membrane potentials and prolonged 'off' recovery tails. Fibre a13. Temperature = 6.0 °C, Ri = 385.1 cm. Cable constants: = 2.59 mm, ri = 6287 k cm-1, d = 88.31 µm, rm = 420.48 k cm, Rm = 11.66 k cm2, Cm = 10.6 µF cm-2.

Perchlorate continued to conserve both intramembrane charge and its steady-state properties in CPA-treated fibres. Figure 7 compares the charge-voltage curves that were obtained from CPA-treated fibres studied before (A, circles) and after treatment with 8 mM perchlorate (B, diamonds). Perchlorate influenced steady-state charge as reported earlier (Huang, 1998a). Thus both the foot of the charge voltage relationship and its inflection at around -60 mV (Fig. 7B) occurs at substantially more negative test voltages (by 15-20 mV) as described on previous occasions (Huang, 1998a) in parallel with the appearance of q charge movements. Nevertheless the charge saturated at similar, or only slightly higher, maximum values. However, there was otherwise no significant change in either the overall form or steepness of the charge-voltage relationship.

		View larger version [in this window] [in a new window]
Figure 7. The effects of perchlorate on steady-state features of the intramembrane charge in CPA-treated fibres Charge-voltage curves in the absence (A, data points as in Fig. 4; ) and the presence of perchlorate (8.0 mM) (B, ) in CPA (50 µM)-treated fibres. Six fibres studied in the presence of both CPA and perchlorate (B). Temperature = 6.1 ± 0.23 °C, Ri = 383.56 ± 2.73 cm. Cable constants: = 2.13 ± 0.17 mm, ri = 6562.77 ± 737 k cm-1, d = 88.3 ± 5.02 µm, rm = 286.16 ± 30.12 k cm, Rm = 7.96 ± 0.99 k cm2, Cm = 10.0 ± 1.43 µF cm-2.

Table 1f summarizes results of least-squares curve-fits of maximum charge, Q_max, transition voltage, V*, and steepness factor, k to single two-state Boltzmann systems in perchlorate-treated fibres exposed to 50 µM CPA. Perchlorate shifted V* to around -57 mV in common with its effects previously reported under other conditions (Huang, 1986, 1998a, Gonzalez & Rios, 1993). Q_max was conserved or slightly increased to around 24-26 nC µF^-1 and k remained similar at around 7-8 mV. Finally, Table 2f confirms appreciable contributions from q charge (Q_max 14-16 nC µF^-1) with steepness factors (k 5-6 mV) as expected from both the controls (Table 2a) and earlier characterizations of q charge (Hui, 1983; Hui & Chandler 1990; Huang & Peachey, 1989; Huang, 1996).

CPA treatment reduces cytosolic Ca²⁺ transients in response to application of K⁺ or caffeine

The final experiments assessed the effect of CPA on the capacity of intact fibres to produce Ca²⁺ transients in response to application of high extracellular [K⁺] (122 mM) and caffeine (10 mM: Luttgau & Oetliker, 1968; Caputo, 1976) using a method modified from Caputo & Bolanos (1994) that employed hypertonic solutions. Fluorescence measurements were made in intact fibres close to the edge of lumbricalis muscles, which showed optimal fluo-3 loading. Figure 8A(a) and B(a) show typical fluorescence traces, normalized to the prestimulus baseline, in single selected fibres in response to application of either 122 mM-[K⁺]-500 mM-sucrose-Ringer solution (A) or 10 mM-caffeine-500 mM sucrose-Ringer solution (B). Replacement by perfusing solution was monitored by the appearance of fluorescence from sulforhodamine B that was added to the test solutions and is indicated by the vertical arrows. Solution changes were complete within 4 s of beginning each perfusion. Both K⁺ (Aa) and caffeine (Ba) produced marked positive deflections in the fluorescence traces. Addition of K⁺ gave peak F/F₀ values of 2.26 ± 0.25 (6 fibres; 3 independent experiments) in agreement with the earlier report (Caputo & Bolanos, 1994) which measured similar transients in response to application of 30-190 mM [K⁺]. Caffeine (10 mM) produced maximum deflections of 1.87 ± 0.2 (6 fibres, 4 independent experiments). CPA (50 µM) pretreatment for 20 min sharply reduced these responses to 1.18 ± 0.04 (Fig. 8Ab; 4 fibres, 3 independent experiments; K⁺ application) and 1.04 ± 0.02 (Fig. 8Bb; 3 fibres, 2 independent experiments; caffeine application), respectively, consistent with the expected effect of CPA in depleting intracellular Ca²⁺ stores. Thapsigargin (30 µM) similarly attenuated such changes in F/F₀ levels by ~ 70 % and was thus less effective than CPA (~85 % reduction) in accordance with earlier reports (Du, 1996; Du et al. 1994, 1996).

		View larger version [in this window] [in a new window]
Figure 8. Effect of CPA on K+ and caffeine induced calcium transients A, normalized fluo-3 fluorescence signals from a muscle fibre in response to 122 mM [K+] in control (a) and 50 µM CPA-pretreated (b) muscles. B, normalized fluo-3 signals in response to 10 mM caffeine in control (a) and 50 µM CPA-pretreated (b) muscle fibres. C, normalized fluo-3 fluorescence signals from muscle fibres stimulated with 122 mM [K+] in the presence of 8 mM perchlorate in control (a) or in CPA-pretreated fibres (b). Arrival of the stimulating solution is indicated in each case by a continuous vertical arrow in the control and a dashed vertical arrow in the CPA-pretreated muscle.

In contrast to its effect on delayed charge movements, perchlorate did not influence the effect of CPA treatment on Ca²⁺ release. Thus, Fig. 8Ca displays typical Ca²⁺ transients following application of 122 mM [K⁺] in perchlorate (8 mM)-treated muscle fibres (peak F/F₀ 2.34 ± 0.22: 5 fibres; 2 independent experiments). However, perchlorate did not influence the consequences of CPA treatment upon the fibre capacity for Ca²⁺ release following application of 122 mM [K⁺] (peak F/F₀ 1.19 ± 0.11; 4 fibres; 2 independent experiments; Fig. 8Cb).

Recent reports describe similar situations in which perchlorate (Huang, 1998a), caffeine (Huang, 1998b) and ouabain (Huang, 2001) all restored delayed q charge movement following its abolition by tetracaine (2 mM: Caputo, 1976; Vergara & Caputo, 1983). Additional experiments, performed here, along the lines described above showed that: (i) in parallel with its effect on the charge movement, tetracaine (2 mM) similarly inhibited Ca²⁺ transients normally triggered by the application of 122 mM [K⁺]. However, (ii) perchlorate (8 mM), caffeine (0.2 mM) and ouabain (500 nM) all similarly failed to restore the capacity for Ca²⁺ release in such tetracaine-treated fibres (data not shown). Such experiments therefore provide at least four examples in which q charge movement can be observed in the face of a markedly reduced capacity for Ca²⁺ release.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

A number of recent reports suggest that the q component of intramembrane charge in skeletal muscle constitutes the electrical signature for conformational transitions in intramembrane DHPR voltage sensors driven by changes in tubular membrane potential (Huang, 1990; Chen & Hui, 1991). Their complex delayed kinetics observed at some voltages have been attributed to allosteric interactions with RyR-Ca²⁺ channels that in turn released Ca²⁺ from intracellular stores during excitation contraction coupling (Huang, 1983, 1996, 1998a,b). Thus, both the steady-state and kinetic properties of the q charge depend steeply and uniquely upon test voltage independently of charging history (Huang, 1994b) and are sensitive to DHPR-specific agents (Huang, 1990; Hui & Chen, 1992). Conversely, both agonist and antagonist modifications of the RyR in intact, voltage-clamped muscle fibres have been shown to reciprocally but reversibly influence the kinetic but not the steady-state properties of q charge, findings compatible with a direct coupling between the voltage sensors and RyRs located outside the tubular membrane field (Huang, 1996, 1998a, b, 2001. Such a tight coupling scheme would predict that the relevant charging phenomenon should persist following imposition of voltage changes even in experimental systems whose intracellular Ca²⁺ stores were sufficiently depleted to compromise their capacity to release Ca²⁺. Thus, Jong et al. (1995) and Pape et al. (1996) reported that intramembrane charge persisted, albeit with modified kinetics, in Vaseline gap preparations whose SR Ca²⁺ was altered by using (20 mM) EGTA-containing solutions including either depleting (0.0 mM) or loading (1.76 mM) Ca²⁺ concentrations. However, Csernoch et al. (1991) proposed that the q current was primarily driven by changes in cytosolic [Ca²⁺] rather than tubular voltage on the basis of physical and pharmacological manipulations that appeared to affect both intramembrane charge and the release of Ca²⁺.

The present experiments tested particular predictions of such schemes in intact fibre preparations under conditions of extracellular solution, tonicity and temperature that closely paralleled those consistently adopted in the previous manipulations of the RyR (Huang, 1996, 1998a,b). They applied two chemically distinct pharmacological agents, CPA and TG, known to inhibit SR Ca²⁺-ATPase and thereby deplete SR Ca²⁺ if applied at effective concentrations (Seidler et al. 1989; Lytton, Westlin & Hanley, 1991; Sagara & Inesi, 1991; Sagara et al. 1992). The experiments studied the effects of CPA in greater detail as this is known to be more effective and specific than TG under such conditions (Du, 1996; Du et al. 1994, 1996).

Such a pharmacological approach complemented previous studies of muscle fibres within double Vaseline gaps which altered SR Ca²⁺ by equilibrating the intracellular pools with (20 mM) EGTA-containing solutions that included either SR-depleting (0.0 mM) or SR-loading (1.76 mM) Ca²⁺ concentrations (Jong et al. 1995; Pape et al. 1996). Applied depolarization to test voltages of -40 or -55 mV in Ca²⁺-loaded fibres then produced rapid and approximately exponential q decays that transferred steady-state charge with a shallow voltage dependence. In contrast the q charge generated a delayed transient followed by a prolonged decay extending over 500 ms (cf. Adrian & Huang, 1984). However, progressive depletion of SR Ca²⁺ selectively reduced the prominence of the latter slow current. Further Ca²⁺ depletion brought about by the application of repeated depolarizing steps to fibres equilibrated with 0 mM [Ca²⁺]/20 mM EGTA-containing intracellular solutions then reduced the prominence of q humps that nevertheless always persisted in charging records (Fig. 2 and Fig. 4 of Jong et al. 1995).

The present pharmacological manipulations of Ca²⁺-ATPase-dependent transport in intact fibres complemented such results. Prior exposure to CPA (50 µM) suppressed Ca²⁺ transients in fluo-3-loaded fibres following application of either caffeine (10 mM) or K⁺ (122 mM) more effectively than exposure to TG (30 µM). This confirmed that the capacity of intact fibres to release intracellularly stored Ca²⁺ was significantly compromised, and that CPA was more effective than TG as reported earlier in such situations (see above). The lower applied CPA concentrations (0.5 and 5 µM) similarly reduced the prominence of delayed q charge movements. This paralleled the earlier results of preceding the test steps by large conditioning depolarizations designed to deplete SR Ca²⁺ (Fig. 5C of Pape et al. 1996) with Ca²⁺ withdrawn from the endpools (Fig. 9B of Pape et al. 1996). However, the higher CPA concentration (50 µM) and TG (30 µM) produced a more extreme situation in which the charging records consist entirely of decaying transients.

Nevertheless the manipulations using either CPA or TG preserved both overall steady-state charge and its voltage dependence in agreement with the earlier reports (Jong et al. 1995; Pape et al. 1996). Such charge-voltage relationships were effectively described by single Boltzmann functions. This offered a useful approach to describe and compare the present results with other data from fibres studied in similar, gluconate-containing, media (Jong et al. 1995; Pape et al. 1996; Huang, 1996, 1998a,b). In any case in such solutions, most intramembrane charge is accounted for by the q component and the voltage dependence of both q and q centre about similar transition voltages, V* (Jong et al. 1995). This contrasts with the situation in methanesulfonate- or sulfate-containing external solutions in which charge-voltage curves were best described in terms of two distinct Boltzmann contributions (Hui & Chen, 1992; Jong et al. 1995). With the exception of a small shift in the transition voltage V* with the highest adopted CPA concentration, both CPA and TG preserved the magnitude of the maximum charge Q_max and steepness factor, k, despite their producing marked kinetic changes. Thus the steeply voltage-sensitive q charge contributions to steady-state charge-voltage properties persisted in such Ca²⁺-depleted fibres in both the present and the earlier experiments (Pape et al. 1996). Furthermore, charge movements showed steady-state inactivation characteristics following CPA treatment that were consistent with continued contributions from steeply voltage-sensitive q charge. Finally, shifts in holding voltage from -90 to -50 mV yielded an isolation of q and q contributions in close agreement with earlier separations (Huang & Peachey, 1989; Huang, 1996, 1998a,b).

In addition, perchlorate restored such delayed waveforms whilst conserving steady-state charge, thus obliterating the effects of CPA on charging kinetics. Yet perchlorate (8.0 mM) did not similarly restore Ca²⁺ release in such CPA-treated fibres. This contrasts with the earlier experiments that restored such charging kinetics by restoring the capacity of the fibre to release Ca²⁺ (Jong et al. 1995; Pape et al. 1996). Further explorations were accordingly made of situations in which perchlorate (Huang, 1998a), caffeine (Huang, 1998b) and ouabain (Huang, 2001) restore q charge following its initial abolition by tetracaine (2.0 mM). All these manoeuvres similarly failed to restore the capacity of such tetracaine-treated fibres to release intracellularly stored Ca²⁺.

In conclusion, q capacitative charge can persist in fibres despite a significantly compromised capacity for Ca²⁺ release both in pharmacologically manipulated intact fibres or in fibres studied between Vaseline seals whose Ca²⁺ stores were manipulated using EGTA (Pape et al. 1996; Jong et al. 1995). Taken together such findings make it unlikely that q charge movement is a consequence of SR Ca²⁺ release.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

ADRIAN, R. H. & HUANG, C. L.-H. (1984). Charge movements near the mechanical threshold in skeletal muscle of Rana temporaria. Journal of Physiology 349, 483-500	[Abstract]
ADRIAN, R. H. & PERES, A. (1979). Charge movement and membrane capacity in frog muscle. Journal of Physiology 289, 83-97	[Abstract]
ANDERSON, K. & MEISSNER, G. (1995). T tubule depolarization-induced SR Ca2+ release is controlled by dihydropyridine receptor- and Ca2+-dependent mechanisms in cell homogenates from rabbit skeletal muscle. Journal of General Physiology 105, 363-383	[Abstract]
CAPUTO, C. (1968). Volume and twitch tension changes in single muscle fibres in hypertonic solutions. Journal of General Physiology 52, 793-809	[Medline]
CAPUTO, C. (1976). The effect of caffeine and tetracaine on the time course of potassium contractures of single muscle fibres. Journal of Physiology 255, 191-207	[Abstract]
CAPUTO, C. & BOLANOS, P. (1994). Fluo-3 signals associated with potassium contractures in single amphibian muscle fibres. Journal of Physiology 481, 119-128	[Abstract]
CHEN, W. & HUI, C. S. (1991). Differential block of charge movement components in frog cut twitch fibres by nifedipine. Journal of Physiology 444, 579-603	[Abstract]
CSERNOCH, L., PIZARRO, G., URIBE I., RODRIGUEZ, M. & RIOS, E. (1991). Interfering with calcium release suppresses I, the hump component of intramembranous charge movement in skeletal muscle. Journal of General Physiology 97, 845-884	[Abstract]
DU, G. G. (1996). The role of the sarcoplasmic reticulum Ca2+ ATPase in the regulation of intracellular Ca2+. D.Phil. Thesis, University of Oxford
DU, G. G., ASHLEY, C. C. & LEA, T. J. (1994). Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle. Pflügers Archiv 429, 169-175	[Medline]
DU, G. G., ASHLEY, C. C. & LEA, T. J. (1996). Ca2+ effluxes from the sarcoplasmic reticulum vesicles of frog muscle: effects of cyclopiazonic acid and thapsigargin. Cell Calcium 20, 355-359	[Medline]
FRANZINI-ARMSTRONG, C. & JORGENSEN, A. O. (1994). Structure and development of excitation-contraction coupling units in skeletal muscle. Annual Reviews of Physiology 56, 509-534	[Medline]
GOMOLLA, M., GOTTSCHALK, G. & LUTTGAU, H. C. (1983). Perchlorate-induced alterations in electrical and mechanical parameters of frog skeletal muscle fibres. Journal of Physiology 343, 197-214	[Abstract]
GONZALEZ, A. & RIOS, E. (1993). Perchlorate enhances transmission in skeletal muscle excitation-contraction coupling. Journal of General Physiology 102, 373-421	[Abstract]
HUANG, C. L.-H. (1981). Dielectric components of charge movements in skeletal muscle. Journal of Physiology 313, 187-205	[Abstract]
HUANG, C. L.-H. (1982). Pharmacological separation of charge movement components in frog skeletal muscle. Journal of Physiology 324, 375-387	[Medline]
HUANG, C. L-H. (1983). Time domain spectroscopy of the membrane capacitance in frog skeletal muscle. Journal of Physiology 341, 1-24	[Abstract]
HUANG, C. L.-H. (1986). The differential effects of twitch potentiators on charge movements in frog skeletal muscle. Journal of Physiology 380, 17-33	[Abstract]
HUANG, C. L.-H. (1987). 'Off' tails of intramembrane charge movements in frog skeletal muscle in perchlorate-containing solutions. Journal of Physiology 384, 491-509	[Abstract]
HUANG, C. L.-H. (1990). Voltage-dependent block of charge movement components by nifedipine in frog skeletal muscle. Journal of General Physiology 96, 535-558	[Abstract]
HUANG, C. L-H. (1992). Intramembrane charge movements in frog skeletal muscle in strongly hypertonic solutions. Journal of General Physiology. 99, 531-544	[Medline]
HUANG, C. L. -H. (1993). Intramembrane Charge Movements in Striated Muscle. Monographs of the Physiological Society, no. 44. Clarendon Press, Oxford
HUANG, C. L.-H. (1994a). Charge conservation in intact frog skeletal muscle fibres in gluconate-containing solutions. Journal of Physiology 474, 161-171	[Abstract]
HUANG, C. L.-H. (1994b). Kinetic separation of charge movement components in intact amphibian skeletal muscle. Journal of Physiology 481, 357-369	[Abstract]
HUANG, C. L-H. (1996). Kinetic isoforms of intramembrane charge in intact amphibian striated muscle. Journal of General Physiology 107, 515-534	[Abstract]
HUANG, C. L.-H. (1998a). The influence of perchlorate ions on complex charging transients in amphibian striated muscle. Journal of Physiology 506, 699-714	[Abstract/Full Text]
HUANG, C. L.-H. (1998b). The influence of caffeine on intramembrane charge movements in intact frog striated muscle. Journal of Physiology 512, 707-721	[Abstract/Full Text]
HUANG, C. L.-H. (2001). Charge movements in intact amphibian skeletal muscle fibres in the presence of cardiac glycosides. Journal of Physiology 532, 509-523	[Abstract/Full Text]
HUANG, C. L.-H. & PEACHEY, L. D. (1989). The anatomical distribution of voltage-dependent membrane capacitance in frog skeletal muscle fibers. Journal of General Physiology 93, 565-584	[Abstract]
HUI, C. S. (1983). Differential properties of two charge components in frog skeletal muscle. Journal of Physiology 337, 531-552	[Abstract]
HUI, C. S. & CHANDLER, W. K. (1990). Intramembranous charge movement in frog cut twitch fibers mounted in a double vaseline-gap chamber. Journal of General Physiology 96, 257-297	[Abstract]
HUI, C. S. & CHANDLER, W. K. (1991). Q and Q components of intramembranous charge movements in frog cut twitch fibers. Journal of General Physiology 98, 429-464	[Abstract]
HUI, C. S. & CHEN, W. (1992). Separation of Q and Q charge components in frog cut twitch fibers with tetracaine. Critical comparison with other methods. Journal of General Physiology 99, 985-1016	[Abstract]
HUI, C. S. & CHEN, W. (1994). Evidence for the non-existence of a negative phase in the hump charge movement component (I) in Rana temporaria. Journal of Physiology 474, 275-282	[Abstract]
HUI, C. S. & CHEN, W. (1995). Differential suppression of charge movement components by gluconate in cut twitch fibres of Rana temporaria. Journal of Physiology 489, 511-517	[Abstract]
JONG, D.-S., PAPE, P. C & CHANDLER, W. K. (1995). Effect of sarcoplasmic reticulum calcium depletion on intramembrane charge movement in frog cut muscle fibers. Journal of General Physiology 106, 659-704	[Abstract]
LUTTGAU, H. C., GOTTSCHALK, L., KOVACS, L. & FUXREITER, M. (1983). How perchlorate improves excitation-contraction coupling in skeletal muscle fibers. Biophysical Journal 43, 247-249	[Abstract]
LUTTGAU, H. C. & OETLIKER, H. (1968). The action of caffeine on the activation of the contractile mechanism in striated muscle fibres. Journal of Physiology 194, 51-74
LYTTON, J., WESTLIN, M. & HANLEY, M. R. (1991). Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca2+-ATPase family of calcium pumps. Journal of Biological Chemistry 266, 17067-17071	[Abstract]
MA, J., ANDERSON, K., SHIROKOV, R., LEVIS, R., GONZALEZ, A., KARHANEK, M., HOSEY, M. M., MEISSNER, G. & RIOS, E. (1993). Effects of perchlorate on the molecules of excitation-contraction coupling of skeletal and cardiac muscle. Journal of General Physiology 102, 423-448	[Abstract]
MEISSNER, G. (1994). Ryanodine receptor/Ca2+ release channels and their regulation by endogenous effectors. Annual Review of Physiology 56, 485-508	[Medline]
PAPE, P. C., JONG, D. S. & CHANDLER, W. K. (1996). A slow component of intramembranous charge movement during sarcoplasmic reticulum calcium release in frog cut muscle fibres. Journal of General Physiology 107, 79-101	[Abstract]
PARKER, I. & ZHU, P. H. (1987). Effects of hypertonic solutions on calcium transients in frog twitch muscle fibres. Journal of Physiology 383, 615-627	[Abstract]
PIZARRO, G., CSERNOCH, L., URIBE, I., RODRIGUEZ, M. & RIOS, E. (1991). The relationship between Q and Ca release from the sarcoplasmic reticulum in skeletal muscle. Journal of General Physiology 97, 913-947	[Abstract]
RIOS, E. & BRUM, G. (1987). Involvement of dihydropyridine receptors in excitation-contraction coupling. Nature 325, 717-720	[Medline]
SAGARA, Y., FERNANDEZ-BELDA, F., DE-MEISS, L. & INESI, G. (1992). Characterization of the inhibition of intracellular Ca2+ transport ATPase by thapsigargin. Journal of Biological Chemistry 267, 12606-12613	[Abstract]
SAGARA, Y. & INESI, G. (1991). Inhibition of the sarcoplasmic reticulum Ca2+ transport ATPase by thapsigargin at subnanomolar concentrations. Journal of Biological Chemistry 266, 13503-13506	[Abstract]
SEIDLER, N. W., JONA, I., VEGH, M. & MARTONOSI, A. (1989). Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 264, 17816-17823	[Abstract]
VERGARA, J. & CAPUTO, C. (1983). Effects of tetracaine on charge movements and calcium signals in frog skeletal muscle fibres. Proceedings of the National Academy of Sciences of the USA 80, 1477-1481	[Medline]

Acknowledgements

The authors thank the Medical Research Council for project support (G9900365) and Calcium Homeostasis Co-operative Group Grants (G9900182) and the Wellcome Trust Joint Research Equipment Initiative (JREI: 055203/Z/98/Z/ST/RC) for equipment funding support. C.L.-H.H. also thanks the Leverhulme Trust for support and S.C. thanks Lucy Cavendish College for a research fellowship and funding support. They also thank Professor Roger Thomas for useful discussions and Mr Brian Secker, M. Swann and Tony Burgess for skilled assistance. Tim Bergel of Cambridge Electronic Design (Science Park, Cambridge) kindly provided useful advice and test versions of experimental software.

This article has been cited by other articles:

				C. S. Hui Association of the I{gamma} and I{delta} Charge Movement with Calcium Release in Frog Skeletal Muscle Biophys. J., February 1, 2005; 88(2): 1030 - 1045. [Abstract] [Full Text] [PDF]