Programme Contents

An action potential activated Ca²⁺ current in skeletal muscle

Ca²⁺ entry into cells is a fundamental process that enables cells to regulate cytoplasmic [Ca²⁺], [Ca²⁺] in intracellular stores and many Ca²⁺-dependent intracellular processes from gene expression to muscle contraction. Cardiac cells have an absolute requirement for Ca²⁺ entry via the L-type Ca²⁺ channel upon membrane excitation to induce Ca²⁺ release from the sarcoplasmic reticulum (SR) and consequently activate the contractile apparatus. The L-type Ca²⁺ channel also exists in skeletal muscle but the duration of action potentials (APs) in skeletal fibres is too brief (2-5 ms), compared to that in cardiomyocytes (100-200 ms), to activate the channel to any degree. Instead, the α-subunit of the L-type Ca²⁺ channel in skeletal muscle acts as a voltage sensor, which directly activates Ca²⁺ release from the SR. This is not to say that skeletal muscle L-type Ca²⁺ channels cannot pass Ca²⁺, they simply require a relatively long period of depolarization that does not occur under normal physiological conditions. Yet, there is evidence for Ca²⁺ entry associated with periods of low frequency excitation of skeletal muscle (Gissel & Clausen, 1999), but the pathway of Ca²⁺ entry during normal excitation in skeletal muscle fibres has not been identified due to inherent limitations in the techniques used to record very small Ca²⁺ fluxes during normal excitation. Our aim was to use a recently developed fluorescence technique (Launikonis & Rìos, 2007) to identify whether there is a t-system Ca²⁺ current associated with normal excitation in skeletal muscle.

The Animal Ethics Committee at Rush Medical Centre approved the use of animals in this project. Male rats (3 months old) were killed by asphyxiation and the extensor digitorum longus (EDL) muscles were removed. Intact fibres were exposed to a Na⁺-based physiological solution containing mag-indo-1 salt. Fibres were mechanically skinned, trapping the dye in the t-system, and transferred to a chamber containing a K⁺-repriming solution with rhod-2. Net changes in the finite t-system [Ca²⁺] ([Ca²⁺]_t−sys) of the skinned fibre, d[Ca²⁺]_t-sys/dt, could be equated to t-system Ca²⁺ current (Launikonis & Rìos, 2007). The chamber was equipped with platinum electrodes that ran parallel to the mounted fibre. In other experiments, skinned fibres without dye in the t-system were bathed in a K⁺-repriming solution with indo-5F and rhod-2. The indo analogues with rhod-2 were simultaneously imaged during field stimulation on a Leica SP-2 confocal microscope in linescan mode, with the scanning line positioned parallel to the long axis of the fibre. The group scanning speed of the three lasers used to excite mag-indo-1 (or indo-5F) and rhod-2 was 1.9 ms/line.

Imaging the cytoplasmic Ca²⁺ ([Ca²⁺]_cyto) transient during field stimulation of skinned fibre preparations in the presence of rhod-2 and indo-5F in the bathing solution produced a uniform and rapid (∼ 5 ms) increase in [Ca²⁺]_cyto as indicated by both dyes. This imaging technique allowed calibration of the Ca²⁺ transient in skinned fibres for the first time (with indo-5F) and the parallel imaging of rhod-2 provided a reference for EC coupling viability the next group of experiments. Under our imaging conditions, γK_D,Ca of indo-5F was 2.38 μM and indicated a peak [Ca²⁺]_cyto of 1.1 μM following excitation. Following correction of the raw [Ca²⁺]_cyto calibration for the slow off rate of indo-5F (75 s^-1), a peak [Ca²⁺]_cyto of 4 μM was estimated to be reached in about 2 ms. Thus skinned fibres release Ca²⁺ at a normal rate and magnitude in response to physiological excitation. Simultaneous imaging of cytoplasmic rhod-2 and t-system trapped mag-indo-1 showed that there was indeed an influx of Ca²⁺ into the cell following an AP when [Ca²⁺]_t-sys was 0.2 mM or greater. The current decayed exponentially and lasted approximately 70 ms. Subsequent APs produced no further t-system Ca²⁺ current in the following 200 ms, even though Ca²⁺ was released from sarcoplasmic reticulum, thus defining an inactivation period for this current. When [Ca²⁺]_t-sys was about 0.1 mM, a transient rise in [Ca²⁺]_t-sys was observed almost concurrently with the increase in [Ca²⁺]_cyto following the action potential. The change in direction of Ca²⁺ flux was consistent with changes in driving force for Ca²⁺. This is the first direct demonstration of a marked Ca²⁺ flux that inactivates, associated with an AP in skeletal muscle.

Gissel H & Clausen T. (1999) American Journal of Physiology, 276: R331-9.

Launikonis BS & Rìos E. (2007) Journal of Physiology, 583: 81-97.