Action potential waveform voltage clamp shows significance of different Ca2+ channel types in developing ascidian muscle

Ca2+ entry plays a key role in translating electrical activity into cellular output. In a given cell, that cellular output changes during development. In mature cells, it takes the form of physiological processes such as transmitter release or contraction. In developing cells, Ca2+ entry triggers activity-dependent developmental programmes, such as those controlling neurite outgrowth, synaptic connectivity, or ion channel maturation (Gu & Spitzer, 1997; Moody, 1998a, b). The forms of electrical activity that underlie Ca2+ entry serving these two functions are often very different. In developing cells, activity most often takes the form of spontaneous, long-duration action potentials, whereas in mature cells, action potentials are most often short in duration and triggered by external inputs (Spitzer, 1991; Moody, 1995). This difference is reflected in the ion channels that mediate activity: the properties of ion channels present at early stages of development are often markedly different from those of their mature counterparts (Moody, 1998a, b). How these properties are optimized to mediate the patterns of Ca2+ entry required at different stages of development is poorly understood. We have studied this question in developing ascidian muscle. Ascidians are marine chordates whose small cell number in the larvae, rapid development, and early commitment of cell fates have made them a classic preparation in embryology. In the species we use, Boltenia villosa, muscle lineage cells in the larva are pigmented, so they can be identified at all stages of development. We have used this property to make an extensive voltage-clamp study of the development of Ca2+ and K+ channels in these cells (larval ascidian muscle has no Na+ current; contraction is triggered by Ca2+ entry) (Davis et al. 1995; Greaves et al. 1996; Dallman et al. 1998). Early in development, just after neurulation and before innervation and contractility appear, Boltenia muscle cells are spontaneously active. Activity takes the form of 2–4 Hz bursts of action potentials lasting about 20 s, separated by 1 min silent periods. Action potentials are long in duration (mean 34 ms) and are mediated by a high-threshold, inactivating Ca2+ current (ICa(i)) and a voltage-gated delayed K+ current which activates slowly (τ= 60 ms). Spontaneous activity lasts about 6 h, starting just after neurulation, and is triggered by the transient absence during that time of the resting conductance of the cells, an inwardly rectifying K+ current. The inward rectifier is present at all stages of development before and after this spontaneous activity occurs. In mature muscle, action potentials occur only in response to motor neuron input, are short in duration (mean 9.4 ms), and can occur at frequencies up to 10–20 Hz during larval swimming. The mature action potential is mediated by a high-threshold, sustained Ca2+ current (ICa(m)) and a Ca2+-activated K+ current that activates rapidly (τ= 8 ms). The development of this Ca2+-activated K+ current, which shortens the action potential, depends completely on the spontaneous activity: blocking this activity prevents its appearance in mature muscle, without affecting the development of any other current (Dallman et al. 1998). These cells therefore exist in two distinct electrophysiological states, each with its stage-specific forms of activity, Ca2+ and K+ channel types, and Ca2+-dependent cellular functions. In this paper, we examine how the immature and mature ion channels are optimized to their stage-specific roles. To do this, we have used action potential waveforms (APWs) recorded from immature and mature cells as voltage-clamp commands (Llinas et al. 1982; Gola et al. 1986; McCobb & Beam, 1991). We find that the slightly more negative activation voltage and the inactivation of ICa(i) serve to spread out Ca2+ entry over the entire waveform of spontaneous activity, including the subthreshold interspike voltage trajectory. ICa(i) inactivation also limits spontaneous activity to relatively low frequencies. As action potential duration shortens during development, total Ca2+ entry per event (spike plus interspike voltage trajectory) does not change, because the increased density of ICa(m) compensates for the shorter spike duration. Ca2+ entry in mature muscle is, however, confined to the action potential waveform itself. This is due to the more positive voltage of activation and the lack of inactivation of ICa(m), properties which also permit high-frequency firing. These results emphasize the significance of differences between immature and mature ion channel types and the complex relationships that exist between them.