A slow calcium-dependent component of charge movement in Rana temporaria cut twitch fibres

Abstract

1. Charge movement was studied in highly stretched frog cut twitch fibres in a double Vaseline-gap voltage-clamp chamber, with the internal solution containing either 0.1 mM EGTA or 20 mM EGTA plus 1. 8 mM total Ca2+. 2. Fibres were stimulated with TEST pulses lasting 100-400 ms. Replacement of the external Cl- with an 'impermeant' anion, such as SO42-, CH3SO3-, gluconate or glutamate, greatly reduced the calcium-dependent Cl- current in the ON segment and generated a slowly decaying inward OFF current in charge movement traces. 3. Application of 20 mM EGTA to the internal solution abolished the slow inward OFF current, implying that the activation of the current depended on the presence of Ca2+ in the myoplasm. The possibility that the slow inward OFF current was carried by cations flowing inwards or anions flowing outwards was studied and determined to be unlikely. 4. During a long (2000 ms) TEST pulse, a slowly decaying ON current was also observed. When the slow ON and OFF currents were included as parts of the total charge movement, ON-OFF charge equality was preserved. This slow capacitive current is named Idelta. 5. When Cl- was the major anion in the external solution, the OFF Idelta was mostly cancelled by a slow outward current carried by the inflow of Cl-. 6. The OFF Idelta component showed a rising phase. The average values of the rising time constants in CH3SO3- and SO42- were similar and about half of that in gluconate. 7. The OFF Idelta component in CH3SO3- had a larger magnitude and longer time course than that in SO42-. The maximum amount of Qdelta in CH3SO3- was about three times as much as that in SO42-, whereas the voltage dependence of Qdelta was similar in the two solutions. 8. Since the existence of Qdelta depends on the presence of Ca2+ in the myoplasm, it is speculated that Qdelta could be a function of intracellular calcium release.

Figure 5. Effect of TEST pulse duration on the OFF transient of a TEST - CONTROL current trace in Ca²⁺-free TEA-CH₃SO₃ external solution

The centre-pool contained solution F and the end-pools contained solution D. From the beginning to the end of the experiment, the holding current changed from -30 to -44 nA and r_e/(r_e+r_i) changed from 0.983 to 0.980. A, traces elicited, from the 213th to the 240th minute, by TEST pulses to -35 mV. The durations of the pulses were (from traces 1-9): 100, 200, 300, 500, 750, 1000, 1250, 1500 and 2000 ms. The thin straight lines in the OFF segments mark the zero-current axes. The half-widths of the OFF transients were: 17.6, 21.6, 26.5, 37.3, 41.7, 42.0, 46.0, 44.0 and 43.3 ms. The OFF charge in each trace was estimated by fitting a sum of two exponential decays and a straight line to the OFF segment and using the straight line for baseline correction. The first 10 points in the OFF segment, representing the I_β transient, were excluded from the fit. The amounts of OFF charge so obtained were: 42, 54, 70, 93, 100, 105, 108, 105 and 112 nC μF⁻¹ for traces 1-9, respectively. The ON charge estimated from trace 9 after subtracting a baseline fitted to the last 500 ms of the ON segment was 109 nC μF⁻¹. B, comparison of the OFF transients of traces 1 and 9 in A after the negative pedestals in the OFF segments had been removed. Fibre diameter, 90 μm (fibre 6).

Figure 1. TEST - CONTROL currents from a cut fibre containing 0.1 mM EGTA

The end-pools contained solution B. Initially, the centre-pool contained a TEA-Cl external solution (solution E). From the beginning to the end of the experiment, the holding current changed from -5 to -8 nA and r_e/(r_e+r_i) remained stable at 0.994. A, representative traces taken from the 59th to the 79th minute. At the 87th minute, the centre-pool solution was changed to a (TEA)₂SO₄ external solution (solution G). B, representative traces taken from the 102nd to the 122nd minute. At the 129th minute, the centre-pool solution was changed back to solution E. C, representative traces taken from the 139th to the 159th minute. In all panels, the numbers to the right of the traces show the potentials during the TEST pulses. The thin straight lines in the OFF segments mark the zero-current axes. Fibre diameter, 96 μm (fibre 1).

Figure 2. Abolition of the slow inward OFF current by 20 mM internal EGTA

The centre-pool contained solution G. Initially, the end-pools contained solution B. From the beginning to the end of the experiment, the holding current changed from -31 to -38 nA and r_e/(r_e+r_i) changed from 0.986 to 0.985. A, representative TEST - CONTROL current traces taken from the 57th to the 77th minute. At the 84th minute, the solution in the end-pools was changed to solution C. B, representative TEST - CONTROL current traces taken from the 117th to the 137th minute. In both panels, the numbers to the right of the traces show the potentials during the TEST pulses. The thin straight lines in the OFF segments mark the zero-current axes. Fibre diameter, 118 μm (fibre 2).

Figure 3. Indifference of the slow inward OFF current to the presence of external frusemide

The end-pools contained solution B. Initially, the centre-pool contained solution G. From the beginning to the end of the experiment, the holding current remained stable at -17 nA and r_e/(r_e+r_i) remained unchanged at 0.987. A, representative TEST - CONTROL current traces taken from the 56th to the 76th minute. At the 82nd minute, the centre-pool solution was changed to a (TEA)₂SO₄ external solution containing 5 mM frusemide (solution G with 3.3 mM SO₄^2- replaced by 5 mM frusemide). B, representative TEST - CONTROL current traces taken from the 89th to the 109th minute. In both panels, the numbers to the right of the traces show the potentials during the TEST pulses. The thin straight lines in the OFF segments mark the zero-current axes. Fibre diameter, 89 μm (fibre 3).

Figure 4. Existence of the slow inward OFF current in the presence of external 3,4-DAP or Cd²⁺

In both experiments, the end-pools contained solution B. A, the centre-pool contained solution G plus 2 mM 3,4-DAP. From the beginning to the end of the experiment, the holding current changed from -29 to -33 nA and r_e/(r_e+r_i) changed from 0.977 to 0.974. TEST - CONTROL current traces were taken from the 90th to the 110th minute. Only representative traces are shown. Fibre diameter, 89 μm (fibre 4). B, the centre-pool contained solution G plus 2 mM Cd²⁺. From the beginning to the end of the experiment, the holding current changed from -30 to -31 nA and r_e/(r_e+r_i) changed from 0.976 to 0.975. TEST - CONTROL current traces were taken from the 135th to the 161st minute. Only representative traces are shown. Fibre diameter, 109 μm (fibre 5). In both panels, the numbers to the right of the traces show the potentials during the TEST pulses. The thin straight lines in the OFF segments mark the zero-current axes.

Figure 7. Waveform of OFF I_δ in Ca²⁺-free TEA-CH₃SO₃ external solution

The centre-pool contained solution F and the end-pools contained solution D. From the beginning to the end of the experiment, the holding current changed from -49 to -53 nA and r_e/(r_e+r_i) changed from 0.979 to 0.978. A, TEST - CONTROL current traces elicited, from the 205th to the 223rd minute, by TEST pulses of different durations to -40 mV. B, OFF segments of difference traces. Traces 1-7 were obtained by subtracting the OFF segment of trace 0 in A from those of traces 1-7 in A. The values of the rise time τ_r, obtained by fitting expression (1) to the rising phases of the segments, are: 2.7, 4.9, 4.7, 5.3, 4.5, 5.1 and 5.2 ms for traces 1-7, respectively. C, OFF segments of difference traces. Traces 1-4 were obtained by subtracting the OFF segments of traces 0-3 in A from that of trace 7 in A. The first trace is the same as the last trace in B. The values of the rise time τ_r, obtained by fitting expression (1) to the rising phases of the segments, are: 5.2, 6.1, 6.2 and 6.6 ms for traces 1-4, respectively. In all panels, the thin straight lines in the OFF segments mark the zero-current axes. Fibre diameter, 104 μm (fibre 8).

Figure 9. Comparison of charge movement components in calcium-free TEA-CH₃SO₃ and Ca²⁺-free (TEA)₂SO₄ external solutions

The end-pools contained solution D. Initially, the centre-pool contained solution F. From the beginning to the end of the experiment, the holding current changed from -31 to -47 nA and r_e/(r_e+r_i) changed from 0.979 to 0.969. A, representative TEST - CONTROL current traces taken from the 175th to the 204th minute. At the 212th minute, the centre-pool solution was changed to solution H. B, representative TEST - CONTROL current traces taken from the 227th to the 251st minute. In A and B, the numbers at the right ends of the traces show the potentials during the TEST pulses. The thin straight lines in the OFF segments mark the zero-current axes. C and E(or D and F), steady-state voltage distributions of fast (Q_β and Q_γ) and slow (Q_δ) OFF charges, respectively, obtained from the time integrals of OFF transients in TEST - CONTROL current traces, some of which are shown in Fig. 9A (or B). In C and D, ▵ and ♦ were obtained by the separation procedure illustrated by curves 1 and 2, respectively, in Fig. 8C. The timeconstants used for computing the rising phases of curve 2 were 9.9 ms in C and 9.1 ms in D. For comparison, the fast ON charges obtained after the baseline correction illustrated in Fig. 8B are shown as ⋄ in D. The pairs of ⋄ and ♦ overlap each other at -60, -50, -25 and -20 mV. In E and F, ♦ represents OFF Q_δ corrected for the rising phase of I_δ. In each of C-F, the smooth curve represents the least-squares fit of eqn (2) to the data (♦) and the best-fit parameters are listed in columns 4-6 and 8-10 of fibre 13 in Table 2. Fibre diameter, 85 μm.

Figure 6. Cancellation of OFF I_δ by slow tail Cl⁻ current in a fibre bathed in TEA-Cl external solution

The centre-pool contained solution E. Initially, the end-pools contained solution B. From the beginning to the end of the experiment, the holding current changed from -13 to -15 nA and r_e/(r_e+ r_i) changed from 0.989 to 0.985. The TEST - CONTROL current traces were elicited by TEST pulses with durations ranging from 400 ms at -70 mV to 100 ms at 0 mV. Only OFF segments of the traces are shown in all panels. A, representative traces taken from the 59th to the 79th minute. At the 85th minute, the centre-pool solution was changed to a modified solution B with 4 mM caesium glutamate replaced by CsCl. B, representative traces taken from the 101st to the 121st minute. At the 127th minute, the centre-pool solution was changed back to solution B without CsCl. C, representative traces taken from the 141st to the 161st minute. In all panels, the numbers at the right ends of the traces show the potentials during the TEST pulses. The thin straight lines in the OFF segments mark the zero-current axes. Fibre diameter, 81 μm (fibre 7).

Figure 8. Separation of I_δ from I_β and I_γ

The centre-pool contained a Ca²⁺-free (TEA)₂SO₄ external solution (solution H) and the end-pools contained solution D. From the beginning to the end of the experiment, the holding current changed from -28 to -34 nA and r_e/(r_e+r_i) changed from 0.983 to 0.980. A, representative TEST - CONTROL current traces taken from the 145th to the 161st minute. The numbers at the right ends of the traces show the potentials during the TEST pulses. The thin straight lines in the OFF segments mark the zero-current axes. B, ON transient of the 4th trace in A. Only the early part of the ON segment is shown on expanded scales. The straight line represents the baseline obtained by fitting a sum of an exponential and a straight line to the points between the tick marks. C, OFF transient of the 4th trace in A. Only the early part of the OFF segment is shown on expanded scales. Curve 1 represents the slow exponential decay obtained by fitting a sum of two exponentials and a straight line to the points between the tick mark and the end of the complete OFF segment (not the end of the part shown). Curve 2 was obtained by multiplying curve 1 by (1 - exp(-t/9.57)). Fibre diameter, 81 μm (fibre 9).