Voltage-gated Ca(2+) influx through L-type channels contributes to sarcoplasmic reticulum Ca(2+) loading in skeletal muscle

Abstract

Muscle contraction is triggered by Ca(2+) ions released from the sarcoplasmic reticulum (SR) in response to depolarization of skeletal muscle fibres. Muscle activation is also associated with a voltage-activated trans-sarcolemmal Ca(2+) influx early identified as a current flowing through L-type Ca(2+) channels. Because removal of external Ca(2+) does not impede fibres from contracting, a negligible role was given to this voltage-activated Ca(2+) entry, although the decline of Ca(2+) release is more pronounced in the absence of Ca(2+) during long-lasting activation. Furthermore, it is not clearly established whether Ca(2+) exclusively flows through L-type channels or in addition through a parallel voltage-activated pathway distinct from L-type channels. Here, by monitoring the quenching of fura-2 fluorescence resulting from Mn(2+) influx in voltage-controlled mouse and zebrafish isolated muscle fibres, we show that the L-type current is the only contributor to Ca(2+) influx during long-lasting depolarizations in skeletal muscle. Calibration of the Mn(2+) quenching signal allowed us to estimate a mean Mn(2+) current of 0.31 ± 0.06 A F(-1) flowing through L-type channels during a train of action potentials. Measurements of SR Ca(2+) changes with fluo-5N in response to depolarization revealed that an elevated voltage-activated Ca(2+) current potentiated SR Ca(2+) loading and addition of external Mn(2+) produced quenching of fluo-5N in the SR, indicating that voltage-activated Ca(2+) /Mn(2+) influx contributes to SR Ca(2+) /Mn(2+) loading.

Figure 1. Voltage dependence of Mn²⁺ influx

A, simultaneous recordings of changes in fura‐2 fluorescence (upper traces) and membrane currents (lower traces) in the same fibre in response to depolarizing pulses of 10 s duration to the voltages indicated above the fluorescence traces. Middle traces correspond to derivatives of the fluorescence traces. Fluorescence images were captured at 2 Hz. B, superimposition of the traces of the current, the fura‐2 fluorescence change and its derivative on a fast time scale in response to the indicated voltage protocol. C, relationship between the mean changes in fura‐2 fluorescence and membrane potential (n = 8). D, relationships between the mean peak values of rate of changes in fura‐2 fluorescence (open symbols), corresponding to the derivative of the fluorescence signal, and membrane potential and between the mean Mn²⁺ current (closed symbols) and membrane potential (n = 7).

Figure 2. Conversion of the rate of change in fura‐2 fluorescence induced by Mn²⁺ influx into current density

A, superimposition of the changes in fura‐2 fluorescence induced by a 10 s voltage pulse given to +20 mV (lower trace in B) in the absence of Cd²⁺ (closed circles) and in the presence of Cd²⁺ (closed triangles) and of the difference between the two signals corresponding to the Cd²⁺‐sensitive component of the fluorescence changes (open circles) in the same fibre. Fluorescence images were captured at 1 Hz. B, rate of the Cd²⁺‐sensitive fluorescence change obtained in E. C, membrane currents obtained in the absence of Cd²⁺ (middle trace) and in the presence of Cd²⁺ (upper trace) and current difference corresponding to the Cd²⁺‐sensitive component of the current (lower trace) associated with the changes in fluorescence in A. D, relationship between the rate of the Cd²⁺‐sensitive fura‐2 fluorescence changes and the Cd²⁺‐sensitive component of Mn²⁺ current. Data points were fitted using a linear regression with a slope of 2.8.

Figure 3. Voltage dependence of Mn²⁺ influx in the presence of Cd²⁺

A, simultaneous recordings of changes in fura‐2 fluorescence (upper traces) and membrane currents (lower traces) in the same fibre in response to depolarizing pulses of 10 s duration to the voltages indicated above the fluorescence traces. Middle traces correspond to derivatives of the fluorescence traces. Fluorescence images were captured at 2 Hz. B, relationships between the mean change in fura‐2 fluorescence and membrane potential in the absence (closed symbols) (n = 7) and in the presence of Cd²⁺ (open symbols) (n = 8). C, relationships between the mean rate of changes in fura‐2 fluorescence and membrane potential in the absence (closed symbols) and in the presence of Cd²⁺ (open symbols). D, relationships between the mean calculated Mn²⁺ current density and membrane potential in the absence (closed symbols) and in the presence of Cd²⁺ (open symbols).

Figure 4. Effect of increasing concentrations of Cd²⁺ on voltage‐activated Mn²⁺ influx

A, simultaneous recordings of Mn²⁺ quenching signal, quenching signal derivatives and membrane currents in the same fibre (from top to bottom) in response to a depolarizing pulse of 10 s duration to +20 mV in control (left panel), in the presence of 0.5 mm Cd²⁺ (middle panel) and in the presence of 2 mm Cd²⁺ (right panel). Fluorescence images were captured at 1 Hz. B, mean relative amplitudes of the voltage‐activated quenching signal and their rates in control, and in the presence of 0.5 and 2 mm Cd²⁺.

Figure 5. Recovery from inactivation of voltage‐activated Mn²⁺ influx in the absence and in the presence of external Cd²⁺

A, simultaneous recordings of changes in fura‐2 fluorescence and membrane currents in the same fibre in response to two successive depolarizing pulses of 5 s duration to +20 mV separated by a pulse interval to −80 mV of 1 s (left panel) and 2 s duration (right panel) in the presence of a control external solution. Fluorescence images were captured at 1 Hz. B, simultaneous recordings of changes in fura‐2 fluorescence and membrane currents in the same fibre as in A in response to two successive depolarizing pulses of 5 s duration to +20 mV separated by a pulse interval to −80 mV of 1 s (left panel) and 2 s duration (right panel) in the presence of an external solution containing 0.5 mm Cd²⁺. Fluorescence images were captured at a frequency of 1 Hz. C, mean and SEM normalized changes in fura‐2 fluorescence induced by two successive depolarizing pulses of 5 s duration to +20 mV separated by a pulse interval to −80 mV of 1 s (left panel) and 2 s duration (right panel) in the presence of a control external solution (upper traces) and in the presence of an external solution containing 0.5 mm Cd²⁺ (lower traces).

Figure 6. Absence of voltage‐activated Mn²⁺ influx in isolated fibres from zebrafish larvae

A, transmitted light image of an isolated muscle fibre from zebrafish larvae. B, membrane currents (upper traces) elicited by voltage pulses given to −50, −30, −10, +10, +30 and +50 mV (lower traces). C, changes in fura‐2 fluorescence induced by a 3 s voltage pulse given to +20 mV upon excitation of fura‐2 at 380 nm (first panel from left), and upon excitation of fura‐2 at 360 nm (second panel from left), then induced by replacement of external Ca²⁺ by 10 mm Mn²⁺ at −80 mV upon excitation of fura‐2 at 360 nm (third panel from left), and in response of a 10 s voltage pulse given to +20 mV upon excitation of fura‐2 at 360 nm in the continuous presence of 10 mm Mn²⁺ (fourth panel from left) in the same muscle fibre. Fluorescence images were captured at 1 Hz.

Figure 7. Mn²⁺ influx and calculated Mn²⁺ current density induced by trains of action potentials

A, train of action potentials (50 Hz, 0.5 ms suprathreshold current pulses) (lower trace) and associated fura‐2 fluorescence change (upper trace). B, fura‐2 fluorescence changes in the absence (closed symbols) and in the presence of Cd²⁺ (open symbols) and Cd²⁺‐sensitive current trace (lower trace) induced by a 10 s voltage pulse to +30 mV in the same fibre as in A. Fluorescence images were captured at 2 Hz. C, corresponding rates of fura‐2 fluorescence changes obtained in A in response to the train of action potentials and in B in response to the voltage pulse in the presence of Cd²⁺ and corresponding rate of the Cd²⁺‐sensitive change of fura‐2 fluorescence. D, relationships between the rate of the Cd²⁺‐sensitive fura‐2 fluorescence changes obtained in response to a voltage pulse to +30 mV (closed symbols) and the Cd²⁺‐sensitive component of Mn²⁺ current and between rates of fluorescence changes induced by trains of action potentials (open symbol) and current density. Data points were fitted using a linear regression with a slope of 4.97.

Figure 8. Effect of Cd²⁺ on Mn²⁺ influx induced by trains of action potentials

A, fura‐2 fluorescence changes (upper traces) and corresponding rates of fura‐2 fluorescence changes (middle traces) in response to trains of action potentials (lower traces) in the absence and in the presence of Cd²⁺, in the same fibre. Fluorescence images were captured at 2 Hz. B, mean rates of fura‐2 fluorescence changes induced by trains of action potentials in the absence and in the presence of Cd²⁺ (n = 6). C, superimposition of the first action potential of the train recorded in the absence and in the presence of Cd²⁺ in the same fibre. D, mean durations of the action potentials between the maximal depolarization value and return to −60 mV and between the maximal depolarization and return to 0 mV in the absence and in the presence of Cd²⁺ (n = 6). Data were compared using a paired test.

Figure 9. Effect of TEA and of the Ca²⁺ channel agonist BayK 8644 on Mn²⁺ influx induced by trains of action potentials

A, fura‐2 fluorescence changes (upper traces) and corresponding rates of fura‐2 fluorescence changes (middle traces) in response to trains of action potentials of 10 s duration (lower traces) in the absence and in the presence of 10 mm TEA, in the same fibre. Fluorescence images were captured at 2 Hz. B, mean rates of fura‐2 fluorescence changes induced by trains of action potentials in the absence and in the presence of TEA (n = 8). C, superimposition of the first action potential of a train recorded in the absence and in the presence of TEA in the same fibre. D, mean durations of the action potentials between the maximal depolarization value and return to −60 mV and between the maximal depolarization and return to 0 mV in the absence and in the presence of TEA (n = 8). E, fura‐2 fluorescence changes (upper traces) and corresponding rates of fura‐2 fluorescence changes (middle traces) in response to trains of action potentials of 10 s duration (lower traces) in the absence and in the presence of 5 μm Bay K in the same fibre. Fluorescence images were captured at 2 Hz. F, mean rates of fura‐2 fluorescence changes induced by trains of action potentials in the absence and in the presence of Bay K (n = 6). G, superimposition of the first action potentials of the trains recorded in the absence and in the presence of Bay K in E. Data were compared using a paired test.

Figure 10. Effect of an increase in external [Ca²⁺] and of replacement of Mg²⁺ by Mn²⁺ on voltage‐induced SR Ca²⁺ signals

A, simultaneous recordings of membrane currents (upper traces) and fluo‐5N fluorescence changes (middle traces) in response to a 10 s voltage pulse (lower trace) in the presence of 2.5 and 5 mm external Ca²⁺. Note the different time scales used for the current and the fluorescence traces. Fluorescence images were captured at 1 Hz. Before normalization, the absolute mean values of F/F ₀ were 0.99 and 0.98 at basal level and 0.17 and 0.18 at peak in control and in the presence of 5 mm Ca²⁺, respectively. B, mean values of fluo‐5N fluorescence (upper traces) in response to a voltage pulse (lower trace), at the end of the depolarization and 5, 10, 20 and 50 s after the end of the depolarization in the presence of 2.5 and 5 mm external Ca²⁺ (n = 7). C, simultaneous recordings of membrane currents (upper traces) and fluo‐5N fluorescence changes (middle traces) in response to a 10 s voltage pulse (lower trace) in the presence of 10 mm Mg²⁺ and 10 mm Mn²⁺. Note the different time scales used for the current and the fluorescence traces. Fluorescence images were captured at 1 Hz and then at 0.2 Hz. Before normalization, the absolute mean values of F/F ₀ were 1.00 and 0.95 at basal level and 0.28 and 0.30 at peak in control and in the presence of 10 mm Mn²⁺, respectively. D, mean values of fluo‐5N fluorescence (upper traces) in response to a voltage pulse (lower trace), at the end of the depolarization and 5, 10, 20 and 50 s after the end of the depolarization in the presence of 10 mm Mg²⁺ and 10 mm Mn²⁺ (n = 5) (ns, not significant).