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Journal of Physiology (2001), 537.1, pp. 45-56
© Copyright 2001 The Physiological Society
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ABSTRACT |
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INTRODUCTION |
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The intramembrane charge movement in amphibian skeletal muscle normally shows two major components, Q
and Q
(Adrian & Peres, 1979; Huang, 1982, 1988; Hui, 1983; Ríos & Pizarro, 1991). In addition, a charge species with a significantly more positive threshold, Qh, has been described and correlated with L-type calcium channel gating (Shirokova et al. 1995). Unlike this relatively clear-cut situation in amphibian muscle, the separation of charge movement into different components is not so clearly established in mammalian muscle. While a number of previous reports have consistently described one charging component in mammalian fibres (Hollingworth & Marshall, 1981; Dulhunty & Gage, 1983; Delbono et al. 1991; Delbono, 1992; Wang et al. 1999), other works have reported two components of Q, one dihydropyridine-sensitive and the other dihydropyridine-insensitive (Lamb, 1986), and a very small Q component (Simon & Beam, 1985a,b; Lamb, 1986).
The aim of the present work was to perform a detailed examination of both the kinetic and the steady-state properties of mammalian charge movements. We intended to test whether charge movements in mammalian muscle could be resolved into a similar number of components to those established in amphibian muscle under appropriate conditions regarding external solution and voltage clamping. Accordingly, we performed a detailed analysis of the time course of intramembrane charge movement, IICM, over a wide voltage range (from -85 to 30 mV). Currents were recorded using a double Vaseline-gap voltage-clamp technique with an overall rapid clamping time of 150 µs (Francini et al. 1996d) and with a short sampling interval (50 µs). Charge movement kinetics and the steady-state quantity of charge moved were evaluated under two different experimental conditions, with and without Cd2+ in the external solution. The primary goal of omitting Cd2+ from the external solution was to determine which components of the charge movement best correlated with L-type Ca2+ channel gating.
Some aspects of this work have already been reported in abstract form (Francini et al. 1996a,b,c; Bencini et al. 1997; Squecco et al. 1997).
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METHODS |
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Dissection and voltage-clamp recordings
Voltage-clamp recordings were performed, using the double Vaseline-gap technique, on cut segments of single fast twitch fibres from extensor digitorum longus muscle dissected from adult male Wistar rats (400-500 g) killed by decapitation after a 10 min exposure to an ether overdose. All experiments followed the official guidelines laid down by the European Community Council (directive 86/609/EEC) incorporated into Italian Government Legislation. The mean fibre diameter was about 50 µm (range 40-70 µm). The double Vaseline-gap voltage-clamp technique was the same as described in detail in Francini & Stefani (1989) and Francini et al. (1996d). To reduce contamination of the internal and external pools by KCl, four agar-bridges provided electrical connections with the KCl pools containing the Ag-AgCl electrodes. The four agar-bridges were filled with suitable solutions similar to those used in the three-compartments of the chamber. Two agar-bridges were filled with a solution similar to the internal one but without MgATP and glucose. The two others were filled with a solution similar to the external one but without divalent cations. All experiments were performed at 16 °C. Temperature was measured with a thermistor and controlled by a Peltier system.
Solutions
Solutions were similar to those used by Delbono et al. (1991) and were as follows: (1) Ringer-Krebs solution (mM): 145 NaCl, 5 KCl, 2.5 CaCl2, 5.5 MgCl2, 10 glucose, 10 Na-TES; (2) dissecting solution (mM): 95 K2SO4, 0.4 CaCl2, 10 MgCl2 and 10 K-TES; (3) relaxing solution (mM): 2 MgCl2, 150 potassium glutamate, 10 K-TES and 1 K2-EGTA; (4) external solution with Cd2+ (mM): 145 TEA-methanesulphonate, 2 CaCl2, 0.8 CdCl2, 2 MgCl2, 5 TES, 2.5 Rb2SO4, 1 
10-3 TTX, 1 3,4-diaminopyridine and 1 9-anthracene carboxylic acid; (5) external solution without Cd2+ (mM): as the external solution with Cd2+ but without CdCl2; (6) internal solution (mM): 120 sodium glutamate, 10 Na2-EGTA, 10 glucose, 2.29 Mg-ATP, 10 Na-TES and 5 Di-Tris phosphocreatine. The specific blockers were added to the external solution to minimise ionic currents. L-type Ca2+ channels were blocked by Cd2+; Na+ channels by TTX; K+ channels by TEA+, 3,4-diaminopyridine and Rb+; Cl- channels by 9-anthracene carboxylic acid. Chloride currents were also minimised by chloride replacement using methanesulphonic acid and SO42-. Internal solution was Ca2+-buffered with EGTA to prevent muscle contraction and to block any calcium-dependent channel. pH was titrated to 7.4 with TEA-OH and NaOH for external and internal solutions, respectively. The composition of the internal solution was calculated by a computer program developed by Fabiato (1988) using published stability constants (Martell & Smith, 1977). The calculated concentrations were: free Ca2+ 10-10 M (pCa 10), free Mg2+ 1 mM, MgATP complex 2 mM and free ATP2- 0.29 mM. All the solutions were designed to have approximately the same osmolarity. All drugs were obtained from Sigma (St Louis, MO, USA), except for TEA-OH and 9-anthracene carboxylic acid which were from Aldrich-Chemie (Steinheim, Germany).
Stimulation and recording
An IBM compatible personal computer was used. Digital-to-analog and analog-to-digital conversions were carried out by a Digidata 1200 computer interface (Axon Instruments, Inc., Burlingame, CA, USA). Stimulation protocols, data acquisition and recordings were performed by the pCLAMP programs, version 6.02 (Axon Instruments, Inc.). An eight pole Bessel filter with a cut-off frequency of 10 kHz filtered the current records. Both 'on' and 'off' currents were sampled. Due to the fast electronics of our device the overall settling time of potential was about 150 µs (Francini et al. 1996d).
A full exploration of IICM at closely spaced voltage values over a wide voltage range required a large number of recorded sweeps and long lasting stability of fibres. In the present experiments the stability of fibres lasted from 150 to 240 min. Test pulses were applied from a holding potential of -90 mV to the desired potential in increments of 10 mV. To achieve a more detailed analysis in the range between -65 and 10 mV over which the different IICM components show their steepest voltage dependence, two pulse protocols of stimulation were applied. In the first protocol we used test voltages between -85 and 5 mV and in the second protocol we used test voltages between -60 and 30 mV. The pulse duration was 153 ms, long enough to include the full relaxation of the IICM and to determine the time onset of the L-type Ca2+ current, ICa, in external solution without Cd2+. The sampling interval was 50 µs for the whole 'on' phase and the first 100 ms of the 'off' current and 200 µs for the subsequent 360 ms.
IICM and ICa were evaluated after subtracting linear capacitive and leak currents, using properly scaled records derived from currents obtained in response to hyperpolarising control pulses. Currents from five control pulses were averaged. The pulse duration and sampling interval were equal to those of the corresponding test pulses. In general, the control transients were obtained from 20 mV hyperpolarising pulses applied from a holding potential of -90 mV. These voltages used in the control pulses were selected as rat skeletal muscle fibres did not tolerate holding potentials more negative than -100 mV nor voltage steps more negative than -120 mV. Since scaling the control current would constitute a source of noise in the test-minus-control traces, the control records were constructed by fitting a two-exponential function plus a constant (accounting for the leak current) to the 'on' current of the controls, and without such a constant to the related 'off' currents. Each test protocol was followed by a control protocol. Successive voltage test steps were separated by intervals of 100 s. Control steps instead were separated by intervals of 60 s.
The derived Q-V plots were analysed by an approach similar to that employed in amphibian muscle, using the sum of one, two or three Boltzmann terms (Melzer et al. 1986; Hui & Chandler, 1990; Hui & Chandler, 1991; Shirokova et al. 1995) given by the expression:
| (1) |
where Qj,max represents the maximum moveable charge for any kind of charge, j is the number related to the Boltzmann term,Vj is the transition voltage and kj is the steepness factor. Equation (1) was constrained to be zero at the holding potential (-90 mV).
Currents and their time integrals were normalised by the linear capacitance measured by control pulses. Data fitting used a non-linear curve fitting procedure based on the Marquardt-Levenberg algorithm (Sigmaplot 4 and Table Curve 3.10 by Jandel Scientific, CA, USA, and Clampfit 6.02 by Axon Instruments). The sum of exponential or Boltzmann functions consisting of a different number of terms was fitted to the data. The best fit was chosen by means of a test based on the value of the likelihood ratio statistic, LRS, with the same formalism reported in Hui & Chandler (1990). The improvement of the fit was evaluated by 
2 statistics. The number of degrees of freedom by using one, two or three Boltzmann terms was three, six or nine. We used 2 statistics with three degrees of freedom (equal to the difference of number of parameters when two Boltzmann terms or one Boltzmann term were used, as well as in the case of three and two Boltzmann terms). The improvement of the fit was statistically significant (P < 0.05) if the 2 statistics exceeded 7.8. Data were reduced to means ± S.E.M.; n is the number of experiments.
Subsequent data analysis included linear cable analysis of the control records, which yielded information about myoplasmic resistance (ri), membrane resistance (rm), membrane capacitance (cm) and the gap factor of the Vaseline seal defined by re/(re + ri), where re is the external resistance underneath the Vaseline seal (Irving et al. 1987; Hui & Chandler, 1990; Delbono et al. 1991). The electrical constants rm, ri and re were calculated for each muscle fibre according to Irving et al. (1987). The condition of the fibre was tracked by monitoring the holding current and calculating the cable constants' parameters. Moreover, to test the rundown of ICa, a long (1700 ms) depolarising pulse to 10 mV from the holding potential was applied every 20-40 min. The fibre was rejected if there was a change in these parameters larger than 1 % from their initial values.
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RESULTS |
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Passive electrical properties
The values of re/(re + ri) were similar in external solutions without and with Cd2+, being 0.987 ± 0.091 and 0.986 ± 0.082, respectively. They were close to unity, corroborating the reliability of our current and voltage measurements from mammalian muscle fibres in our double Vaseline-gap chamber. The respective values of Rm of 6440 ± 700 and 6400 ± 600 
cm2 and Ri of 380 ± 18 and 395 ± 25 cm at 16 °C corresponded well to those reported for rat skeletal muscle in similar solutions at 17 °C by Delbono et al. (1991) (Rm = 6355 cm2 and Ri = 367 cm). The input capacitances were 9.0 ± 0.3 and 9.1 ± 0.3 nF, respectively, and specific capacitances were 10.6 ± 0.8 and 10.7 ± 0.7 µF cm-2. Consequently, the presence or absence of Cd2+ in the external solution did not significantly change the Vaseline seal or any passive electrical properties.
IICM in fibres bathed in external solution with Cd2+
In the initial experiments IICM was examined with a Cd2+ concentration in the external solution that effectively blocked the ICa. In six fibres this aim was achieved by adding 0.8 mM Cd2+ to the external solution containing 2 mM Ca2+. Concerning two other fibres where ICa was relatively large, 0.8 mM Cd2+ failed to completely block ICa. However, increasing [Cd2+] to 1.5-5 mM markedly increased the leakage current, thus precluding further study. Figure 1A shows representative results obtained by using 0.8 mM Cd2+. The fibre was subjected to progressively larger depolarising voltage steps from the holding potential of -90 mV. The 'on' current traces (IICM,on) regained the baseline for all test voltages applied, indicating an absence of significant ionic currents including ICa. The 'on' current showed a complex time course. The smallest depolarising steps (from -80 to -60 mV) elicited a very early charge movement, Ia, rapidly peaking at about 0.1-0.2 ms, and then decaying almost exponentially to a minimum, at about 1-2 ms. Ia was followed by another charge movement, Ib, with a rising phase that reached a maximum at about 4 ms and then decayed monotonically. With further depolarisations the dip between Ia and Ib became progressively less evident, being replaced by a plateau (from -30 to -20 mV) and finally by a shoulder (from -10 to 30 mV). Delayed hump currents (Q), as in frog skeletal muscle fibres, were not observed. However, larger depolarisations to test potentials around -30 mV resulted in prolongation of the IICM,on decays. With voltage steps up to -40 mV, IICM,on regained the baseline in about 20-30 ms whereas from -30 mV this time increased sharply to 40-60 ms. The occurrence of the 'on' current prolongation around the voltage threshold of -30 mV was accompanied by a prolongation of the duration of the corresponding 'off' tail current (IICM,off). With steps to voltages up to -40 mV, IICM,off regained the baseline in about 15-20 ms whereas from -30 mV this time increased sharply to about 30-35 ms. This prolongation of both IICM,on and IICM,off indicated the movement of at least another slower charging component.
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Figure 1. Charge movements in a fibre bathed in external solution with Cd2+ (0.8 mM) A, test pulses (153 ms long) ranging from -80 to 30 mV applied from a holding potential of -90 mV in increments of 10 mV. Potentials are indicated (in mV) next to the traces. Time zero is the 'on' onset. The 'on' current traces were interrupted between 63 and 150 ms to better observe the first 63 ms section. Only the first 40 ms of the 'off' currents are reported. Lines superimposed on the 'on' traces result from fitting of one (from -80 to -40 mV) or two (from -30 mV) exponential functions, starting from the time at which the current was 50 % of the peak or shoulder. B, steady-state Q-V plot of the 'on' (QICM,on; | ||
To further investigate this possibility we measured the time integrals of 'on' and 'off' current traces. Data from a representative experiment are reported in Fig. 1B. The time integrals demonstrated an equality of 'on' and 'off' charges indicating that ionic current contributions were absent in the current traces even at strong depolarisations. Notably, both the QICM,on-V and QICM,off-V plots showed three clear changes in steepness (Fig. 1B). The two plots were best fitted by eqn (1) as the sum of three Boltzmann functions, Q1, Q2 and Q3. Figure 4A shows QICM data obtained by averaging QICM,on and QICM,off values of the six fibres investigated. Data are reported as means ± S.E.M. (
). The three-Boltzmann fit (eqn (1)) is superimposed as a continuous line. The improvement of the fit with 2 statistics was significant, exceeding 7.8. Indeed, the LRS value using one or to two Boltzmann terms was 82.4 ± 14.1 (range 25-119) and using two or three terms 23 ± 4 (range 11-47).
Thus, the time course of the currents and the Q-V relations identified two major groups of charges. The first group moved even with small depolarising steps and corresponded to two charge components that we named Qa and Qb. These were clearly recognised by their very different time course (see Fig. 1A), but only one Boltzmann term, Q1, described the steady-state amount of charge moved as a whole. The second group corresponded to the prolonged charge movement components with more positive voltage thresholds that were designated as Qc and Qd. They were described by the Q2 and Q3 Boltzmann terms.
The contribution of Qa to Q1,max was estimated by means of the time integral of the 'on' current traces in the first 2 ms when most Ia moved. The values at -50 and 30 mV were 1.2 and 4 nC µF-1, whereas the values of Q1,max at the same voltages were 10 and 26 nC µF-1. Consequently, it could be inferred that Qa was only a small fraction of Q1, and that Q1 mostly represented Qb. On the other hand, the charge moved at voltages positive to -30 mV was actually due to two charge components, Qc (Q2) and Qd (Q3), but they were not resolvable in the time course of IICM as distinct charge components because of their similar kinetics (see below and Fig. 5). In conclusion, Qb was smoothly voltage dependent and its transition voltage occurred at lower voltage depolarisations, whereas Qc and Qd were steeply voltage dependent and their transition voltages occurred at larger depolarisations. Moreover, as shown in Fig. 4A, the bulk of Qc moved in the voltage range between -40 and -10 mV and Qd moved mostly at voltages positive to -10 mV upwards.
Table 1 (column 2) summarises the best fit parameters from all experiments. The parameters of Q2 (Qc) showed the smallest S.E.M. The calculated curves corresponding to each Boltzmann term are reported in Fig. 4A as continuous lines. They are labelled as Qb, Qc and Qd to make the correspondence with Q1, Q2 and Q3 clear. Interestingly, there is a striking shape resemblance between the charge versus voltage curves up to 0 mV found here, and the earlier findings from both cut (Hui & Chandler, 1990) and intact skeletal muscle fibres of the frog (Huang, 1994) that were described as being due to the movement of Q and Q charges. Furthermore, the Boltzmann term Qd (Q3) parallels the Qh charge elicited at positive potentials described in cut fibres of the frog (Shirokova et al. 1995).
Identification of charge components in external solution without Cd2+
To investigate which component of charge movements best correlated with the gating of ICa, the remaining experiments omitted Cd2+ from the external solution. Depolarising voltage pulses made to a voltage positive to -35 mV (Fig. 2) elicited a slow inward ICa characterised by a downward deflection in the 'on' current trace. These records showed noticeable changes in IICM,on kinetics (Fig. 2). By small depolarising steps to test voltages in the -85 to -65 mV range, the dip between Ia and Ib was replaced by a plateau lasting 10-20 ms, which was followed by a monotonic decay. This charging current, Ib, regained the baseline at about 70-90 ms following imposition of the pulse. Larger voltage steps gave rise to a delayed hump-like waveform, rather than a plateau, with a peak at about 13.5 ms at -35 mV. This hump-like current had a prolonged decay, regaining the baseline at 120 ms. From -25 mV upwards the hump became more and more prominent, well separated from Ib by a clear dip. The dip and the peak time progressively decreased by increasing depolarisation from -25 to 5 mV, the former from 5.5 to 2.5 ms, the latter from 13.5 to 7 ms. This hump charge component showed analogy with the delayed Q transients as reported in frog skeletal muscle fibres. The appearance of the hump in 'on' currents around a voltage threshold of -35 mV was accompanied by a prolongation of the corresponding 'off' tail currents. The baseline was regained at about 60-70 ms below the voltage threshold and at about 120 ms for larger depolarisations. Notably, the IICM time course was prolonged in external solution without Cd2+.
Data analysis
In the external solution without Cd2+ inward ICa overlapped with IICM when the applied test-voltage pulses were suprathreshold for ICa activation. Consequently, the 'on' time course of IICM was extracted after removing ICa time course, evaluated by a multiexponential fit, from the original current traces (see: Fig. 1 and Fig. 2C in Francini et al. 1996d).
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Figure 2. IICM and ICa in a fibre bathed in external solution without Cd2+ A, test pulses (153 ms long) ranging from -85 to 5 mV applied from a holding potential of -90 mV in increments of 10 mV. Full length 'on' current traces. Only the first 60 ms of the 'off' currents are reported. Potentials are indicated (in mV) next to the traces. The continuous lines superimposed on the 'on' traces are two-exponential fits to ICa time course. The time constant values are: 42 and 65 ms for -35 mV; 46 and 72 ms for -25 mV; 32 and 78 ms for -15 mV, 27 and 111 ms for -5 mV; 27 and 75 ms for 5 mV. B, Q-V plots for Qon and Qoff; data at 20 and 30 mV were obtained from a second protocol applied to the same fibre. The continuous line through Qoff data is the best fit of the sum of three Boltzmann functions. A and B, horizontal lines indicate the zero level. From the beginning to the end of the experiment, the holding current changed from -31 to -33 nA and re/(re + ri) changed from 0.987 to 0.986. Fibre diameter 50 µm, linear capacitance 6.1 nF or 12.9 µF cm-2 of apparent lateral fibre surface area. Data are normalised to linear capacitance. | ||
The values of the time integral of 'on' and 'off' currents, Qon and Qoff, showed a clear 'on' and 'off' equality between -85 to -40 mV (Fig. 2B and Fig. 4B) in parallel with results obtained in fibres studied in the presence of Cd2+, suggesting that they accounted for intramembrane charge. From about -35 mV the curves diverged. By comparing the present results with those obtained in the presence of Cd2+, this divergence was attributed to ICa activation. Therefore, the voltage value where this divergence occurred was assumed to be the voltage threshold for ICa which was -38.1 ± 2.4 mV (n = 12).
Time course of IICM,on components in the presence of ICa and voltage threshold for ICa
To study the time course of IICM,on components in the presence of ICa we had to consider that the two currents overlapped. Consequently, to extract the IICM time course we must remove the overlapping ICa after having determined its time course. IICM, once determined, could be subtracted from the current records. To deal with this, we first tested for the presence of a lag in ICa activation by means of I-V plots. I-V plots were evaluated at different fixed times from the pulse onset. The I-V plot at 148 ms was U-shaped from -40 mV, indicating ICa activation. The amplitude of this U-shaped curve progressively reduced, decreasing the evaluation time. Its presence was detectable up to about 7 ms. This was an indication that the lag for ICa was shorter than 7 ms. Secondly, we attempted to determine the time course of ICa by a fitting procedure. The fit started between 80 and 100 ms after the pulse onset, a time at which IICM was considered fully relaxed. The best fit (continuous lines, Fig. 2A) was obtained using the following equation:
ICa(t ) = -ICa(1 + ICa1exp(-(t - tCa)/ Ca1) - ICa2 exp(-(t - tCa)/Ca2)),
| (2) |
where Ca1 and Ca2 are the time constants related to ICa1 and ICa2 with Ca1 < Ca2, and tCa is the tubular lag. All the terms in eqn (2) were left as free parameters with the constraint that tCa < 10 ms, based on the features of the I-V plots. The value of tCa resulting from the fit was 1.8-3.8 ms, a lag comparable to that found in frog skeletal muscle (Francini et al. 1996d). The time constants Ca1 and Ca2 were slow: they were 32 and 78 ms for the trace at -15 mV, and 27 and 75 ms for the trace at 5 mV. Thirdly, to better evaluate these slow time constant values we fitted eqn (2) to ICa(t ) elicited by long voltage steps of 1.7 s. To determine the ICa(t ) we initially evaluated the time course of ICa inactivation and then subtracted it from the total time course of ICa according to the procedure reported in Francini et al. (1996d). The Ca1 and Ca2 values found by this procedure were not statistically different from those obtained by shorter voltage steps. These approaches together justified the evaluation of IICM,on by subtracting the calculated ICa(t ) from the original current traces, allowing a selective isolation of charge movement. ICa(t) was set to zero at times below tCa. The result of the subtraction related to the experiment of Fig. 2 is shown in Fig. 3. Comparing Fig. 2 with Fig. 3 gave evidence that the presence of ICa did not significantly affect IICM,on up to about 50 ms since its activation was slow. Moreover, it is worth noting that in the absence of Cd2+ there was a strong change in the IICM time course: a pronounced delayed hump component became clearly evident. Notably, a sharp increase of the current was observed from about -40 mV in the I-V plots in the 5 to 82 ms range. This increase indicated the voltage threshold for Ic (-41.1 ± 2.0 mV), which was very similar to that for ICa, suggesting a possible correlation between the two events (Fig. 4C).
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Figure 3. Time course of IICM,on after removing ICa A, IICM,on time course obtained after subtracting the calculated ICa time course from the original current traces of Fig. 2A. ** indicate the delayed hump charge observed from -35 to 5 mV. The continuous lines superimposed on each recording result from the fit of a single exponential function starting from 22-30 ms. These times correspond to 50 % of the current values at the plateau, or shoulder or delayed hump peak. The time constant values are: 18.2 ms for -85 mV, 18.7 ms for -75 mV, 19.2 ms for -65 mV, 22.3 ms for -55 mV, 19.0 ms for -45 mV, 30.2 ms for -35 mV, 20.0 ms for -25 mV, 26.1 ms for -15 mV, 21.1 ms for -5 mV, 18.9 ms for 5 mV. B, steady-state QICM,on-V plot from A data. The continuous line through the data is the best fit of the sum of three Boltzmann functions. | ||
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Figure 4. Q -V and I-V relationships in fibres bathed in external solution with or without Cd2+ A, steady-state voltage distribution of the amount of charge moving in fibres with (QICM, | ||
Steady-state voltage distribution of QICM,on components determined after removing ICa
The previous extraction procedure for IICM allowed an evaluation of QICM,on as a function of voltage. The QICM,on-V plots showed a complex form for the presence of different slopes paralleling those observed in external solution with Cd2+ (Fig. 3B and Fig. 4B, 
). Once again, the best fit was achieved by a three-Boltzmann-term equation (eqn (1)) reported as a continuous line superimposed on the QICM,on data in Fig. 3B and Fig. 4B. The improvement of the fit with 2 statistics was significant, exceeding 7.8. Indeed, the LRS value using one or two Boltzmann terms was 76.4 ± 15 (range 23-110) and 20 ± 4 (range 9-47; it was 6.1 for one fibre, not statistically significant) using two or three Boltzmann terms. The parameters of the functions are listed in column 3 of Table 1. Again, the parameters of the second Boltzmann term showed the smallest S.E.M. In comparison with the results obtained from experiments on fibres bathed in external solution with Cd2+, the omission of Cd2+ did not significantly influence the total maximum moveable charge or the single charge components, or the steepness factors. On the contrary, it shifted the transition voltages by about 6-8 mV towards negative potentials. To make the comparison easier, data (open circles) and fits (associated dashed lines) related to the experiments carried out in external solution without Cd2+ were shifted in a positive direction in Fig. 4A by 7.1 mV. From this comparison, it is clear that the steady-state quantity of charge moved obtained after subtracting ICa was comparable to that obtained in external solution with Cd2+. In conclusion, these results demonstrate that the lack of delayed shoulders or humps in external solution with Cd2+ was not primarily due to the lack of some kind of charge components but rather to a change in their kinetics.
The predicted QICM and QICM,on Boltzmann functions show the presence of an intramembrane charge of about 1.4-2 nC µF-1 at -110 mV (Fig. 4A and B). Hence, in the pulse going from -90 to -110 mV, used as control, there could be some residual charge that could lead to an underestimation of QICM. Most fibres did not support more negative potentials but we succeeded in applying 20 mV hyperpolarising control pulses from a 100 ms long prepulse at -110 mV in three further fibres. The prepulse was applied from a holding potential of -90 mV. In these three fibres a comparison of the QICM values obtained with these two kinds of controls showed small differences: with the usual control protocol an underestimation of about 3-5 nC µF-1 resulted and the changes of the Boltzmann parameters were less than the 0.8 %.
Kinetic evaluation of Qa, Qb, Qc and Qd charge components
The two major groups of charge movements described above were identified by different approaches. The first group, Qa and Qb, was identified by the time course analysis of the current and the second group, Qc and Qd, by the Boltzmann fit. In order to further corroborate this charge separation we first used I-V plots evaluated for times shorter than 7 ms. Figure 4C shows plots from experiments carried out in external solution with Cd2+ (open symbols) and without Cd2+ (filled symbols). In the time range from 2.5 to 7 ms the plots showed two shoulders that separated three charging currents revealing the presence of Ib, Ic and Id (see 6, 5 and 3 ms plots). In contrast, for evaluation times shorter than 2 ms the plots did not show any shoulder (see 1 and 0.8 ms plots) and they appeared to constitute a single charge component. This charge mostly accounted for Ia, that at this time was largely predominating over the other charge components (Fig. 1A and Fig. 2A). In conclusion, the I-V plot analysis produced interesting findings such as the lack of a lag for Ia while a lag of about 2 ms could be estimated for Ib, Ic and Id. A comparable lag was estimated for ICa.
A second approach sought to identify Ib, Ic and Id by means of kinetic analysis. To achieve this, we used an empirical exponential fitting procedure to test different kinetic contributions to current decay, without assumptions of any specific time course. Due to the complex time course of the early 'on' currents, Ia and the following shoulders or humps, we fitted exponential functions to late current decays that mostly represented the decay of Ib, Ic and Id. The fits started from the time at which the currents were 50 % of the shoulders or the peak of the humps: 4-6 ms and 22-30 ms from the 'on' edge for fibres bathed in external solution with and without Cd2+, respectively. In the absence of Cd2+ the best fits were obtained by one exponential function at any voltage. The same results were achieved in the presence of Cd2+ for voltage steps up to -40 mV whilst for larger voltage steps the best fits were obtained by two decaying exponential functions. The time constant values, found in all fibres bathed in external solution with and without Cd2+, are plotted in Fig. 5A and B, respectively. In both panels three maxima of -V plots are observed. The contribution of the charge movement components to each overall time constant, j (V), could be quantified, according to Simon & Beam (1985b) and Huang (1997), using a simple constant field model for a two-state charge distribution, from which, we have:
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Figure 5. Voltage dependence of the time constants of the 'on' IICM decay Voltage dependence of the time constants obtained by fitting one (B, | ||
| (3) |
where -j is the value of j at (V - Vj)/kj = 0. The values of kj and Vj obtained from eqn (1) constrain the voltage dependence of the time constants for charge distribution, but not their absolute size, -j. This parameter was left free and determined by fitting the experimental values at Vj.
The kj and Vj values of the three-term Boltzmann fit listed in Table 1, columns 2 and 3, appropriately described the b-V, c-V and d-V plots (lines in Fig. 5). This further corroborated the presence of at least three charge movement components (Qb, Qc and Qd) each mainly moving over a given voltage range either with or without Cd2+. It is important to observe that in experiments with Cd2+ b values were relatively smaller than c and d, while without Cd2+ the values were comparable. Consequently, this justifies the approximate representation of d-V curve in the absence of Cd2+ since the fitting procedure could not separate the overall time constant values of b from d. However, the -V plots of Fig. 5 were very similar in the two experimental conditions and corroborated to some extent the identification of Qb, Qc and Qd charge components in mammalian skeletal muscle fibres.
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DISCUSSION |
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In contrast to the relatively clear-cut situation in amphibian skeletal muscle, the separation of charge movement into components has not been well established in mammalian skeletal muscle. Firstly, the present study examined whether a separation of the charge movement into components similar to those accepted in amphibian muscle might also apply to mammalian muscle. Accordingly, intramembrane charge components were investigated in normally polarised mammalian skeletal muscle fibres in much greater detail than on earlier occasions, due to the wide voltage range, the overall rapid clamping time and the short sampling interval used. Ionic currents were eliminated by using suitable blockers of ionic channels. This more thorough study clearly demonstrated four distinct charge components in mammalian muscle (Qa, Qb, Qc and Qd). Secondly, this study aimed to elucidate which charge component was related to Ca2+ channel gating. Experiments carried out without Cd2+ in the external solution demonstrated that Qc and Qd charges were correlated with Ca2+ channel gating and, surprisingly, the presence of a pronounced delayed hump charge component. This pronounced delayed hump charge component was consistently observed and was large in size in contrast to the very small size of the hump component reported by Simon & Beam (1985a) and Lamb (1986). In conclusion, charge movement in mammalian skeletal muscle was found to be very similar to that of amphibian skeletal muscle. In contrast with previous findings (Hollingworth & Marshall, 1981; Simon & Beam, 1985a; Lamb, 1986; Delbono et al. 1991; Delbono, 1992; Szentesi et al. 1997; Wang et al. 1999) this work established important correlations between mammalian and amphibian skeletal muscle. It is important to note that the differences found in experiments with and without Cd2+ were not due to modifications of the passive properties of the fibres because they were very similar in the two experimental conditions.
Correspondence with charge movement components reported in the literature
The significance of our Qb, Qc and Qd charge components can be understood by comparing their properties with those reported in the literature for twitch skeletal muscle of the frog, particularly using inferences based on the steady-state data (Huang, 1982). The steady-state fits obtained from the sum of the three Boltzmann terms are straightforward: Qb is the charge component having the most negative transition voltage and shallow voltage dependence. The two other terms, Qc and Qd, are steeply voltage dependent through less negative membrane potentials. These findings are very similar to those available for frog skeletal muscle, where there is general agreement on the presence of three charge components, Q, Q and Qh, both in cut and intact fibres. In our study, the shallow and steep voltage dependences of Qb and Qc closely resemble those of Q and Q in both cut (Hui & Chandler, 1990) and intact skeletal muscle fibres of the frog (Huang, 1994, 1997). The parameters of the Boltzmann term for Qd show a marked similarity to those related to Qh (Shirokova et al. 1995). These results therefore contribute to the understanding of charge movements in mammalian skeletal muscle fibres because they demonstrate a striking resemblance of the steady-state charge versus voltage curve to earlier findings in amphibian skeletal muscle fibres.
In this work the total amount of charge moved (QICM,max) was greater than that reported in previous works on mammalian muscle fibres investigated using the Vaseline-gap method. We found 48.5 ± 3.3 (range 42.2-73.5) nC µF-1 (Table 1, column 2) in contrast to about 15 nC µF-1 in other reports (Lamb, 1986; Delbono et al. 1991; Delbono, 1992; Szentesi et al. 1997). However, using the microelectrode technique, larger values of about 25 (Dulhunty & Gage, 1983) and 46 nC µF-1 (Hollingworth & Marshall, 1981) have been reported. This discrepancy could be due to our more detailed analysis, the wider test voltage range explored (-85 to 30 mV), or the more effective minimisation of ionic currents than was achieved in previous reports. These conditions allowed the separation of a larger number of charge movement components. Kinetic analysis also corroborated the above conclusions. The time constant values for Qb found in experiments with Cd2+ were very similar to those reported in the literature at similar temperatures, 12-20 °C (Hollingworth & Marshall, 1981; Dulhunty & Gage, 1983; Lamb, 1986; Delbono et al. 1991; Delbono, 1992; Szentesi et al. 1997). The failure of previous studies to observe the slow Ic and Id could be due to the small size of the currents, which could be masked by outward ionic currents and thus eliminated by the sloping line used for evaluating QICM.
Separation of charge movement components
In the present experiments two main groups of charge components were identified. The first group, made up of Qa and Qb, moved in response to relatively small depolarising steps, whereas the second group, Qc and Qd, was transferred from a more positive voltage threshold: -35.6 ± 2.0 (n = 6) and -41.2 ± 2.0 mV (n = 12) in experiments performed in external solution with and without Cd2+, respectively.
Qa charge movement was rapid and complete within the first 2 ms of IICM,on (Fig. 1-3 and Fig. 4C). A rough evaluation of Ia 'on' decay, obtained by fitting a single exponential function to the time course, yielded time constant values, a, of 0.6-1.5 ms and 0.8-2 ms, in external solution with and without Cd2+, respectively. Since Qa did not show any lag it could be regarded as a component of charge originating in the surface membrane, and it could correspond either to the Q charge arising in the surface membrane (Huang & Peachey, 1992) or to the gating processes of other voltage-dependent channels. For example, a fast charge supposed to be the gating charge of Na+ channel was described in fibres with no functional tubules. Interestingly, the values of a were of the same order as those of the gating charge of the Na+ channel evaluated at temperatures close to those used in our experiments (12-15 °C; Collins et al. 1982; Campbell, 1983).
On the other hand, the finding of a lag of about 2 ms for the Qb, Qc and Qd components, comparable to the lag shown by ICa (1.8-3.8 ms), was consistent with the occurrence of such charges in the T-tubules or with a delay intrinsic to such charges (see Simon & Beam, 1985b; Huang & Peachey, 1992). These components were identified by means of the steady-state voltage dependence analysis (Fig. 4A) and the identification was corroborated by kinetic analysis of IICM,on decay (Fig. 5). In addition, the presence of Qc and Qd was confirmed by the properties of the 'off' currents, given the more prolonged IICM decay starting from the voltage threshold for their mobilisation (Fig. 1 and Fig. 2). According to Delbono et al. (1991) and Delbono (1992) the ICa deactivation, evaluated at 17 °C (a temperature close to that used in our experiments: 16 °C), and at the same holding potential (-90 mV), was fast, having a time constant with a value of 2-3 ms. Hence, even in the absence of Cd2+ the slower decay of 'off' currents above the voltage threshold was due to mere charge movements. Prolonged 'off' components have also been reported in both cut (Hui & Chandler, 1991) and intact amphibian muscle fibres (Huang, 1987) and identified with transitions in the Q system.
Interestingly, in external solutions without Cd2+ Qc and Qd appeared somehow related to ICa since they moved from the same voltage threshold. This voltage threshold was crucial, setting the boundary between a voltage range in which only Qa and Qb occurred and a more positive voltage range where Qc, Qd and ICa arose. The Qoff-V plot showed a comparable form to that observed for QICM (Fig. 2B and 4B) in experiments with and without Cd2+. The main difference was the larger value of Qoff in the same voltage range in which Qd moved. The improvement in the fit with 2 statistics was significant, exceeding 7.8. Indeed, the LRS value using one or two Boltzmann terms was 36.4 ± 4 (range 18-64) and using two or three terms it was 15 ± 4 (range 8-37). The best-fit parameters are reported in Table 1 (column 4). The kj and Qj,max values of the Q1 and Q2 terms of the Qoff-V plot were not statistically different from those found for the QICM-V and QICM,on-V plots. In contrast, the values of Q3 were statistically different, Q3,max being 32.0 nC µF-1 for Qoff and 10.8 and 9.7 nC µF-1 for QICM and QICM,on, respectively (Table 1, columns 2 and 3, respectively). On the other hand, Q3 for Qoff showed slight variation in the steepness factor. This strongly supports the hypothesis that the parameters for Qd are comparable to those of ICa activation and that Qd therefore corresponds to the Qh charge described by Shirokova et al. (1994) in skeletal muscle of the frog.
Charge movements in external solution with Cd2+ showed an equality of 'on' and 'off' charges and a steady-state QICM voltage dependence that could be best fitted by the sum of three Boltzmann terms. The absence of external Cd2+ produced a slightly negative 6-8 mV shift in the transition voltage value of all the three Boltzmann functions, but it preserved the total maximum amount of charge, Qmax, and that of each component, Qj,max. Moreover, each of the three Boltzmann terms retained the same value of steepness factor indicating persistent voltage-dependent contributions of Qb, Qc and Qd.
Kinetic changes of charge components in external solution without Cd2+
The omission of Cd2+ produced significant modifications in QICM kinetics. Qb was slowed down while a noticeable delayed hump charge component appeared (Figs 1-3). This is the first time that a pronounced hump-shaped charge has been consistently observed in the 'on' phase for fast skeletal muscle fibres of mammals. Only a small-sized bump or hump was reported in previous works (Simon & Beam, 1985a,b). Thus, the absence of Cd2+ specifically alters Qb, Qc and Qd kinetics while preserving the separate identities of their steady-state charge. A similar modification was also reported by Huang (1996 and 1998) in frog skeletal muscle fibres where the ryanodine receptor antagonists ryanodine and daunorubicin and agonists such as caffeine specifically altered Q kinetics while preserving the separate identities of the steady-state Q and Q. The addition of inorganic ions to minimise ICa has been extensively reported in the literature. Dulhunty & Gage (1983) found that 10 mM Co2+ added to the external solution suppressed ICa, but this caused a shift to positive potentials in the Q-V curve that was attributable to an action on surface charge. Hui (1991) found that on adding 0.5-1 mM external Cd2+ the voltage dependence of Q and Q charge diverged, Q towards negative potentials and Q towards positive potentials. Such an effect could not be attributed to a screening of surface charges. Consequently, it may be speculated that these changes in kinetics might reflect the complex and possibly multi-step mechanisms that have been proposed for delayed Q hump (Huang, 1982; Pizarro et al. 1991; Huang & Peachey, 1992; Ríos et al. 1993; Shirokova et al. 1994; Jong et al. 1995). Such models involve a parametric description of charge transfers whose values can be altered under different external solutions, such as with and without Cd2+. In fact, the value of some parameters is critical for the appearance of pronounced delayed hump currents. Interestingly, the steady-state parameters of each charge component moved with and without Cd2+ showed very small variations. In contrast, the fit of eqn (3) to the data for (V) (Fig. 5) indicated a marked change in kinetics which was mainly due to changes in -j.
In summary, these findings provide strong evidence of a clear separation into four components of intramembrane charge movement in rat skeletal muscle. This supports the statement that there are no fundamental differences with respect to charge movement components between amphibian and mammalian twitch muscle. Notably, experiments carried out in external solution without Cd2+ demonstrate that two charge components, Qc and Qd, correlate well with Ca2+ channel gating.
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Acknowledgements
The authors are most grateful to Dr C. L.-H. Huang for his helpful and stimulating discussions and for his invaluable advice at all stages of manuscript preparation. They also wish to thank Dr G. Piazzesi for helpful comments on the manuscript. The financial support of Telethon-Italy (grant n o. 945) and Cofinanziamento MURST 1999 are gratefully acknowledged.
Corresponding author
F. Francini: Department of Physiological Sciences, University of Florence, Viale G.B. Morgagni, 63, 50134 Florence, Italy.
Email: fabio.francini{at}unifi.it
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) and 'off' (QICM,off; 
) and QICM,on (
), 32 ± 3 (
), 82 ± 3 (
) and 148 ± 5 (
). Open and filled symbols are from experiments with and without Cd2+, respectively. Data points are mean values ± S.E.M., n = 6 or 12 for experiments performed with and without Cd2+, respectively. To make the comparison easier the corresponding S.E.M. bars are indicated without and with a 'cap'.