Calcium currents during contraction and shortening in enzymatically isolated murine skeletal muscle fibres

Abstract

1. Calcium currents (ICa) were monitored in enzymatically isolated murine toe muscle fibres using the two-microelectrode voltage-clamp technique. ICa was recorded (i) in hypertonic solution to suppress contraction, and (ii) in actively shortening fibres in isotonic solution. 2. In hypertonic solution the threshold potential for ICa was about -30 mV for both 2 and 10 mM external Ca2+ solution. Maximum peak currents measured -12.6 +/- 2.3 nA (mean +/- s.d.; n = 4) in 2 mM Ca2+ and -65 +/- 15 nA (n = 7) in 10 mM Ca2+. The time to peak (TTP) ICa was 96 +/- 22 ms (n = 4) in 2 mM Ca2+ and 132 +/- 13 ms (n = 7) in 10 mM Ca2+. The exponential decay of ICa was similar in 2 and 10 mM Ca2+ with rate constants (tau-1(V)) of 3.7 s-1 (2 mM) and 3.8 s-1 (10 mM) at +10 mV. 3. ICa in isotonic 10 mM Ca2+ solution was recorded by inserting the micropipettes exactly opposite to each other close to the centre of mass of the fibre where negligible contraction-induced movement occurs. 4. In isotonic 10 mM Ca2+ solution ICa had a smaller peak amplitude (-45 +/- 5 nA; n = 7) and faster TTP (82.8 +/- 22.1 ms; n = 7) than in hypertonic solution. The exponential decay of ICa showed a significantly larger tau-1(V) of 6.4 s-1 at +10 mV (P < 0.03). 5. To test for calcium depletion, extracellular Ca2+ was buffered by malic acid in isotonic solution to 9 mM. The decay of ICa had a time constant of 348 +/- 175 ms (n = 14) vs. 107 +/- 24 ms (n = 12; P < 0.001) at 0 mV in unbuffered 10 mM Ca2+ solution. 6. We conclude that calcium depletion from the transverse tubular system contributes significantly to the decay of calcium currents in murine toe muscle fibres under hypertonic as well as isotonic conditions. In the latter, depletion is even more prominent.

Figure 1. Recording chamber

Schematic diagram of the recording chamber and the circuit for the 2-MVC technique using an additional bath clamp (μ, amplifer gain). The fibre is placed in the middle of the chamber allowing the micropipettes to be inserted perpendicular to the fibre axis. ME2, voltage-sensing microelectrode; ME1, current-passing microelectrode; V_c, command voltage; R_b, compensating bath current electrode for bath current I_bath; R_s, voltage-sensing bath electrode for bath voltage V_sense.

Figure 2. Two microelectrodes approaching a single fibre perpendicular to the fibre axis

Scale bar, 50 μm.

Figure 4. Calcium currents in hypertonic and isotonic 10 mM Ca²⁺ solution

Calcium currents were elicited by the activation pulse protocols in the insets. A, currents recorded from a single fibre (length, 602 ± 14 μm; diameter, 56 ± 7 μm) bathed in hypertonic 10 mM Ca²⁺ solution containing 300 mM sucrose. The non-contracting fibres showed inward Ca²⁺ currents which decayed under maintained depolarization with 1.2 s test pulses. B, currents recorded in isotonic 10 mM Ca²⁺ solution (fibre dimensions: length, 595 ± 14 μm; diameter, 42 ± 7 μm). The fibre contracted and shortened with larger depolarizing steps of 600 ms duration. Note that fibre survival declined with longer pulse durations and also the voltage range was smaller in the isotonic case. For easier comparison only the first 600 ms in the hypertonic case are shown.

Figure 8. Decay of I_Ca under maintained depolarization

A, the decay of calcium currents under maintained depolarization is shown for four membrane potentials for two fibres of similar dimensions in 10 mM Ca²⁺ hypertonic (length, 630 μm; diameter, 42 μm; continuous lines) or isotonic solution (length, 616 μm; diameter, 42 μm; dashed lines). The decay was fitted by a single exponential with time constant τ. B, rate constants τ⁻¹(V) in hypertonic (•, n = 7) and isotonic solution (○, n = 5) of similar value were calculated from the fits performed in A and are plotted for different membrane potentials. In the isotonic case, calcium currents showed a significantly faster decay for potentials more positive than 0 mV (P < 0.03, except for +20 mV where P = 0.16; Student's t test) indicating that at least one other mechanism (calcium depletion) is responsible for the decay of calcium currents in addition to voltage-dependent inactivation.

Figure 3. The voltage drop (K) calculated for toe muscle fibres approximated as uniform short cables

A, the voltage drop K at the end of a muscle fibre with length l is shown relative to the varying membrane resistance R_m. B, the effect of percentage shortening of fibre length during contraction is given for an average fibre with initial length l = 525 μm and a radius a = 24 μm. The voltage drop was continuously minimized during shortening with constant fibre volume improving the uniform spread of voltage along the membrane.

Figure 11. Inactivation (f_∞) of calcium currents

Recordings were obtained using the inactivation pulse protocol consisting of a variable prepulse followed by a depolarization to 0 mV. A, records for a single fibre in hypertonic solution (length, 595 μm; diameter, 42 μm). Peak currents were then normalized with respect to the maximum peak inward current measured during the 0 mV test pulse and plotted vs. the prepulse potential. B, averaged f_∞ plots for ten fibres fitted with a Boltzmann function (dotted line, half-inactivation).

Figure 12. Comparison of calcium and barium currents in single fibres in 10 mM Ca²⁺ and 10 mM Ba²⁺ hypertonic solution

A, currents recorded in hypertonic solution containing 10 mM Ca²⁺ (left) and after changing to 10 mM Ba²⁺ (right) from a single fibre (length, 770 μm; diameter, 56 μm) stimulated for each condition with the same pulse protocol. Peak currents were smaller in amplitude when recorded in Ba²⁺. B, corresponding I-V plot for peak currents (•, 10 mM Ca²⁺; ○, 10 mM Ba²⁺). C, the decrease of the mean maximum peak current for Ba²⁺ (n = 6) was calculated and compared with the mean maximum peak current for Ca²⁺, which was set to 100 %.

Figure 5. I-V plot for peak Ca²⁺ currents in hypertonic and isotonic 10 mM Ca²⁺ solution

The I-V plot shows the averaged peak currents (±s.d.) vs. command voltage for seven single fibres in hypertonic (300 mM sucrose added, •) and isotonic solution (normal 10 mM Ca²⁺ saline, ○). In the two cases the threshold potential was the same (−30 mV). Shortening fibres (○) showed significantly smaller amplitudes for peak currents in 10 mM Ca²⁺ compared with non-shortening fibres (•). The maximum inward currents for the isotonic case were shifted by about 5 mV towards more positive potentials compared with the hypertonic case.

Figure 6. Slowly activating calcium currents (I_Ca) in hypertonic solution containing 2 mM Ca²⁺

A, I_Ca in a single fibre bathed in hypertonic test solution containing a physiological concentration of 2 mM Ca²⁺. Pulse protocol is shown in the inset. Fibre dimensions were: length, 495 ± 7 μm; diameter, 49 ± 7 μm. Holding potential, −70 mV. B, the corresponding I-V plot. Peak currents were usually reduced by a factor of approximately 5–7 compared with measurements done in 10 mM Ca²⁺-containing solution (see Figs 4 and 5).

Figure 7. Rise time (10–90 %) of calcium currents in hypertonic and isotonic 10 mM Ca²⁺ solution

Averaged 10–90 % rise times are shown for maximal activation of inward currents of single fibres in hypertonic (•, n = 8) and isotonic (○, n = 6) solution relative to the membrane potential. In the isotonic case the rise times for positive voltage steps were slightly faster than in the hypertonic case.

Figure 9. Actively shortening single fibres bathed in isotonic unbuffered 10 mM Ca²⁺ and buffered 9 mM Ca²⁺ solution

A, the slow I_Ca is shown in a representative fibre bathed in unbuffered isotonic 10 mM Ca²⁺ solution (left plot: length, 490 ± 14 μm; diameter, 70 ± 10 μm), elicited by a test pulse to 0 mV from a holding potential of −70 mV. The decay of the current was fitted by a single exponential with the indicated time constant. The right plot shows the slow I_Ca in another fibre after solution exchange to calcium-buffered isotonic 9 mM Ca²⁺ solution (length, 455 ± 14 μm; diameter, 42 ± 7 μm). The time constant of the decay was increased fourfold. B, I_Ca in a different fibre (length, 490 ± 14 μm; diameter, 70 ± 7 μm) for both unbuffered (left plot) and buffered (right plot) solution. In some fibres (about 40 % of the experiments), a second component of the inward current was detected following the initial peak of the decline. In C the means and s.d. of the time constants for the decay in twelve unbuffered and fourteen buffered fibres are shown. On average, buffered vs. unbuffered fibres showed a ratio of 3 : 1 for the time constant (P < 0.001; Student's t test).

Figure 10. Activation of calcium currents for isotonic and hypertonic 10 mM Ca²⁺ solution

The activation of calcium currents was calculated from the I-V plot for the activation pulse protocol of two single fibres using the equation for the activation d_∞=I_peak,Ca/(g_Ca(V - V_rev)). The I-V plots are given for membrane current density. The maximum slope conductance g_Ca was interpolated from the I-V plot. The activation plot was well fitted by a Boltzmann function. A, hypertonic case. B, isotonic case. Note that half-activation (dotted line) was similar in the two cases (0 mV vs.+1 mV).