The electrophysiology of the contractile protozoan Actinocoryne contractilis was studied with conventional intracellular recording techniques.
Resting membrane potential (−78 mV, S.D. = 8, A =18) was dependent upon external K+. Rapid action potentials (overshoot up to 50 mV) were evoked either by mechanical stimulation or by current injection. Graded membrane depolarizations induced by graded mechanical stimuli correspond to receptor potentials.
The receptor potential was mainly Na+-dependent; the action potential was also mainly Na+-dependent, but involved a minor Ca2+-dependence. The two components of the action potential could be separated in Ca2+-free solution containing EGTA (1 mmol l−1), in low-Na+ solutions or by the addition of Co2+. The repolarizing phase of the action potential was sensitive to TEA ions and to 4-aminopyridine (4-AP).
Action potentials were followed in 10–20 ms by a rapid all-or-none contraction of the axopods and stalk. Contraction was blocked in Ca2+-free solution containing EGTA and by Co2+, which suggests a requirement of external Ca2+ for this event. Contraction was also abolished by 4-AP.
In different protozoans, external stimuli evoke a variety of responses, including escape or avoidance reactions, light emission and cell contraction (Eckert & Sibaoka, 1968; Naitoh, 1968; Wood, 1982). The remarkable stalked marine heliozoan Actinocoryne contractilis Febvre-Chevalier, which consists of a head, bearing long axopods, attached to an amoeboid base by a single or multiple stalk (Fig. 1A) is very sensitive to mechanical stimuli. Under normal conditions, it reacts to brief mechanical stimuli or to vibrations by a very rapid (50–60 ms) all-or-none contraction of axopods, head and stalk into the base. The axopods appear less sensitive to local mechanical stimuli than the head itself, for example contact of slow-moving prey organisms with an axopod leads to the production of a food vacuole which passes along towards the head; however, extensive stimulation of many axopods elicits contraction of the cell. External electric fields also elicit contraction. The patterns of contraction resulting from electrical and mechanical stimuli have been examined previously (Febvre-Chevalier & Febvre, 1980; Febvre-Chevalier, 1981) using high-speed cinematography (8000 frames s−1). Contraction can also be evoked by current injection, as described in the present paper.
Re-extension of the head and stalk is a relatively slow process, taking 20 min or so (Febvre-Chevalier & Febvre, 1980; Febvre-Chevalier, 1981). Since the head is up to 40 μm in diameter in large individuals, and microelectrodes may be inserted in it without evoking contraction, Actinocoryne offers a peculiarly suitable preparation for examining the correlation of membrane electrical events with contractile activity. In other protozoans studied previously, the relationship between membrane electrical events and contraction is not well understood (see Naitoh, 1982). This paper describes the initiation and ionic basis of the propagated action potential related to contraction.
MATERIALS AND METHODS
Specimens of Actinocoryne were collected from benthic habitats (see Febvre-Chevalier, 1980) in the bay of Villefranche-sur-Mer (in the Mediterranean near Nice). The heliozoans and the small piece of substrate to which they were attached were kept in filtered sea water in Petri dishes, and fed upon oligotrich ciliates. One or several specimens were transferred into solid watchglasses containing about 5 ml of solution for the experiments. Mechanical stimuli were delivered either by simply tapping the mounting of the recording electrode (in conjunction with a transient store with pre-triggering this gave records of potential changes prior to retraction of the cell) or with a blunted glass micropipette attached to a small speaker. Graded mechanical stimuli of 1–5 ms duration given by the latter method were approximately proportional to the speaker coil current, but for various reasons were only semi-quantitative. Electrical stimuli were given via a second electrode inserted in the head (Fig. 1B) after placement of the recording electrode; the current injected was measured across a 10-MΩ resistor in the ground line. Both electrodes were filled with KC1 (3mol l−1) and had resistances between 8 and 10 MΩ. Contractile activity was not monitored, although the delay between the membrane response and the beginning of contraction was approximately indicated by the abrupt return to zero potential as the recording electrode was displaced from the cell by the contraction (Fig. 3B,C).
The reference artificial sea water solution (ASW) had the following composition (in mmol l−1): NaCl, 500; MgCl2, 58; KC1, 10; CaCl2, 10; and was buffered to pH 8·0 with tris (hydroxymethyl)-aminomethane-HCl (Tris-HCl) or tris (hydroxy-methyl)methylaminopropane sulphonic acid (TAPS). Na+-free solutions were made by substituting NaCl with Tris or occasionally with N-methyl glucamine. Trisglycine and choline chloride were not found suitable. Ca2+-free solutions were made by omitting CaClz and adding 0·5–1·0 mmol l−1 ethyleneglycol-bis-(β-amino-ethylether)N,N′-tetra-acetic acid (EGTA). Variations from ASW up to 50mmol l−i were not corrected osmotically.
Co2+, Mn2+, Sr2+ and Ba2+ were used at 40-50 mmol l−1. 4-Aminopyridine (4-AP) was used at concentrations between 1 and 10 mmol l−1. pH of the solution was adjusted to that of sea water prior to experiments. Tetraethylammonium chloride (TEA) was used at 10 mmol l−1.
Solutions were exchanged using a peristaltic pump with a flow rate of 3–4 ml min−1. The animals were incubated in test solutions for a minimum of 10 min prior to experiment. All experiments were performed at room temperature (10–20 °C).
The resting potential was remarkably stable, and did not alter even if some axopods contracted. Maximum and minimum values were −86 and −68 mV (−78 mV, S.D. = 8, N=18). The membrane potential approximated to the behaviour of a Nernst electrode (Fig. 2), although measurements could not be made at high external K+ concentrations as the cells contracted.
A series of mechanical stimuli of increasing strength evoked a series of graded membrane depolarizations, either followed by slower repolarizations (Fig. 3A) or if the threshold value around −20 mV was reached, by an overshooting action potential (Fig. 3B). Subthreshold graded depolarizations summated to produce an action potential provided they were less than 20 ms apart (Fig. 3C). In view of the graded response to increasing stimuli and the way in which they may summate, these depolarizing potentials are regarded as receptor potentials. The receptor potentials were of characteristic form, showing a rapid rise and initial fall, followed by a slower repolarization phase restoring resting potential after some 300 ms. In low-Na+ or Na+-free solutions, receptor potentials large enough to generate action potentials could not be produced by mechanical stimuli that were large enough to evoke action potentials in ASW (Fig. 4A,B); but were seen when external Na+ was increased. The amplitude of the receptor potentials evoked was correlated with external Na+ concentration, but the relationship between values for peak receptor potential and external Na+ has not been determined. Receptor potentials apparently identical to those in ASW were observed when animals were mechanically stimulated in Ca2+-free solutions, but a small contribution of Ca2+ to the receptor potential has not been excluded.
Action potentials were evoked by current injection (Fig. 5). They were identical to action potentials produced by mechanical stimulation (Fig. 3B). The firing threshold of action potentials evoked by current injection was between 58 and 75 mV above the resting potential. Around these values, the input resistance of the cell decreased. The l/V relationship was linear over the range of −80 mV to +60 mV (resting potential — 0) (Fig. 6). Above 60 mV, the slope changed indicating outward rectification, and action potentials could be evoked by an increase in injected current. The apparent input resistance of the cell over the range below action potential threshold was around 100–400 KΩ. The amplitude of electrically evoked action potentials varied between 110 and 134mV (121mV, S.D. =9, N = 18), and overshoots were between 28 and 50mV (42·5 mV, S.D. = 8, N= 18). Action potential duration at half spike amplitude was 0·5 ms, and spike origin to return to resting potential occupied around 10 ms. There was no undershoot.
It was sometimes possible to insert an electrode into the base of an animal which was contracted, in which case identical action potentials were recorded on mechanical stimulation (by tapping the electrode) to those seen in the head of expanded animals (Fig. 7A), except that since there was no further contraction, the electrode remained in the cell. Simultaneous records from electrodes placed in the head and base of specimens attached to glass coverslips showed that mechanical stimuli to the head evoked action potentials that were followed by similar, though somewhat attenuated, potential changes in the base (Fig. 7B). Since normal action potentials could be recorded from the base of contracted specimens, it is probable that the attenuation resulted from the difficulty of recording from the very thin base of the animals attached to coverslips and that the membrane of the entire cell is excitable and propagates action potentials. If this is the case, they are propagated at around 3 ms−1.
The ionic basis of the action potential
Na+-dependence of the action potentials
In SW or ASW solutions containing tetrodotoxin (TTX) at concentrations between 10−9 and 10−6mmol l−1 the action potential was unaffected, and normal rapid contraction followed the action potential.
In Ca2+-free ASW with 1 mmol l−1 EGTA, action potentials resembled those in SW or ASW, but were not followed by contraction (Fig. 8A). In such a Ca2+-free solution, resting membrane potential approached that observed in ASW (−74 instead of −78 mV). In contrast, the firing threshold and the overshoot decreased, −30 mV instead of −20 mV and 27·5 mV instead of 42·5 mV, respectively.
Below 150 mmol l−1 Na+ action potentials could not be evoked in Ca2+-free solutions, even with large depolarizations; above 150 mmol l−1 Na+ the amplitude of the overshoot was related to Na+ concentration, the slope being around 30 mV per decade change in Na+ instead of 58 mV as predicted by the Nernst equation (Fig. 9). Sometimes, in Ca2+-free solutions, rhythmic firing occurred a few seconds after impalement (Fig. 10) following initial large oscillations of membrane potential which led to a series of 10 or more action potentials at 0·5–1·0 Hz.
Contractile activity is evidently critically dependent upon external Ca2+ concentration. In Ca2+-free solutions containing less than 1 mmol l−1 EGTA, rapid contraction followed the action potential, although after prolonged sojourn in such solutions, and several cycles of contraction and re-extension, contractile activity following subsequent action potentials was much slowed or even abolished. With 1 mmol l−1 or more EGTA, contractile activity was completely abolished from the outset in the majority of experiments (Figs 8A, 10). In some cases, however, a slow contraction of the stalk followed one or more action potentials, this kind of slow contraction being entirely different to the normal rapid contraction. If such slow contractions occurred, the stalk and head were subsequently re-extended in the same Ca2+-free solution with EGTA.
Addition of 10-50 mmol l−1 Mn2+ to ASW had no obvious effect upon the action potential, but contraction was either delayed or slowed. Addition of 10–50 mmol l−1 Co2+ to ASW or SW after a few minutes induced initially a slow contraction of the stalk, which either began spontaneously or when the electrodes were inserted. This slow contractile activity was not accompanied by any changes in membrane potential. In a few experiments addition of Co2+ solutions did not cause contraction of the stalk, and in such cases, repetitive action potentials were evoked during current injection. These action potentials were not followed by contraction (Fig. 11).
Ca2 + -dependence of the action potentials
In most experiments with Na+-free solutions, the axopods retracted soon after the solution was introduced, to form a thick, sticky layer which made impalement difficult. Resting potential (− 80 mV) slightly exceeded the mean value in ASW, whatever the Ca2+ content of the solution. Occasionally, long-lasting, irregular oscillations of membrane potential were observed upon impalement; these did not evoke regenerative responses.
No regenerative responses were seen when Na+-free solutions contained less than 10 mmol l− 1 Ca2+. They could be elicited with great difficulty when the Ca2+ content was 10 mmol l− (the level in ASW). In such solutions, the response of the membrane to current injection was often a plateau depolarization whose amplitude and duration increased with that of the injected current. Occasionally, overshooting action potentials were obtained. Unlike those in ASW, however, the spike was followed by a short (5–8 ms) plateau before rapid repolarization (Fig. 8B).
Regenerative membrane responses were obtained more easily in Na+-free solutions, by increasing Ca2+ above its normal level in SW or ASW (up to 200 mmol l−1). In such high-Ca2+ solutions, the spike was not followed by a plateau (Fig. 12A,B) and the firing threshold for the action potential rose with increasing Ca2+. The slope of the variation of the overshooting amplitude in such high-Ca2+, Na+-free solutions (extrapolated for a decade change in external Ca2+ concentration) was some 28 mV, close to the value for a Ca2+ electrode. Action potentials in Na+-free solutions containing 50–200 mmol l−1 Ca2+ were followed by rapid contractions.
Contractile activity in Na+-free solutions had also been observed in preparations incapable of producing regenerative responses (Fig. 12C). Here, the contractions of the stalk were incomplete and slower, and were observed after large depolarizing pulses which took membrane potential close to zero potential. Such contractions have been observed even in solutions containing less than 30 mmol l−1 Ca2+.
In some preliminary experiments with Ba2+ and Sr2+, Na+-free solutions were used when Ca2+ was substituted to avoid Na+-dependent electrogenesis. When Ca2+ was substituted by 50 mmol I-1 Ba2+, resting potential remained unchanged at − 80 mV but regenerative response and contraction were not observed. Solutions containing 50 mmol l−1 Sr2+ evoked contraction as soon as they came into contact with the cell, usually before it could be impaled. With prior treatment in Na+-free solutions without Ca2+ and with 1 mmol l−1 EGTA, the cells remained extended when Sr2+ solution was added. Resting potential was − 90 mV, and as with Ba2+ substitution, no regenerative response was observed. However, in contrast to Ba2+ solutions, contraction followed injection of depolarizing current pulses. As already mentioned, the Ca2+ blocking agents Co2+ and Mn2+ added to SW or ASW produced no obvious effects upon the action potential, but contractions were slowed or abolished.
The repolarizing phase of the action potential
Both TEA and 4-AP (known as blocking agents for voltage-dependent K+ conductances) prolonged the repolarizing phase of the action potential. Addition of 10 mmol l−1 TEA to ASW (Fig. 13A) greatly lengthened the repolarizing phase, and contraction occurred before the resting potential was attained. Similarly, 4-AP at 10 mmol l−1 lengthened the initial repolarizing phase, but this was followed by a more rapid repolarization to resting potential level (Fig. 13B). At l–2 mmol l−1, 4-AP did not obviously affect repolarization, but rhythmic action potentials were evoked, initiated either by impalement or by the first current pulse (Fig. 14).
Much the most striking and unexpected effect of 4-AP was that, at concentrations above 1 mmol l−1, contraction was completely abolished (Fig. 14) and the appearance of the animal changed. The stalk and axopods became rigid, and whereas in SW or ASW, prior to the addition of 4-AP, the axopods could be bent if lightly stroked with a fine probe, after the addition of 4-AP they were so rigid that they could be broken off.
In Na+-free solutions containing 50–100 mmol l−1 Ca2+ and 1–2 mmol l− 1 4-AP, action potentials could be generated by current injection. At 50 mmol l−1 Ca2+, regenerative responses were difficult to obtain, but if evoked, were not followed by contractile activity (Fig. 15A). Above 50 mmol l−1 Ca2+, regenerative responses were easier to obtain and were then followed by rapid contractions (Fig. 15B).
Membrane electrogenesis has been extensively studied in ciliates (reviewed by Naitoh, 1982); Actinocoryne is the first heliozoan examined. In free-swimming ciliates, such as Paramecium and Stylonichia, graded mechanical stimuli to the anterior region evoke graded depolarizing receptor potentials, which reverse ciliary beat (Naitoh & Eckert, 1969; de Peyer & Machemer, 1978). Unlike those of Actinocoryne however, these are Ca2+-dependent (Naitoh, Eckert & Friedman, 1972; de Peyer & Machemer, 1977). Mechanical stimulation of the posterior region evokes hyperpolarizing receptor potentials carried by K+, which accelerate forward ciliary beat (Naitoh & Eckert, 1973; de Peyer & Machemer, 1978), but in Actinocoryne as in Stentor (Wood, 1982) mechanical stimulation of different regions of the cell yields only depolarizing potentials.
In the sessile Vorticella it is not yet clear whether there are regenerative membrane responses (Shiono & Naitoh, 1978), whereas in Zoothamnium (Moreton & Amos, 1979) regenerative responses are found. In free-swimming ciliates, the responses to current injection may be nearly all-or-none (Stylonichia, some Paramecium mutants; Wood, 1982; Hinrichsen & Saimi, 1984) or, more usually, graded according to stimulus intensity (Paramecium, Naitoh & Eckert, 1968). Such graded responses can be experimentally converted into all-or-none action potentials by procedures which alter the Ca2+-dependence or the inactivation of the Ca2+ current (Brehm & Eckert, 1978; Eckert & Brehm, 1979) rather than the Ca2+-activated K+ conductance (Brehm, Dunlap & Eckert, 1978).
In Actinocoryne, rapid all-or-none overshooting action potentials precede contraction in normal conditions. In experimental conditions, the action potentials may be carried by Na+ or by Ca2+, but contraction requires the presence of external Ca2+. In normal SW or ASW the following considerations suggest that whilst there is a small Ca2+ component, the action potential is predominantly carried by Na+. (1) Action potentials in 500 mmol l− 1 Na+, Ca2+-free solutions are similar to those in SW or ASW. (2) In Na+-free solutions they are smaller and at 10 mmol l− 1 Ca2+ only evoked with difficulty by current injection. (3) Co2+ added to SW or ASW does not inhibit membrane electrogenesis, nor does the addition of lmmoll-1 EGTA to 500 mmol l−1 Na+/Ca2+-free solutions, but in both cases action potentials evoked by current injection are not followed by contraction. (4) Rapid contractions following action potentials in Na+-free solutions (see 2), and the absence of contractions following action potentials in solutions containing Co2+ or EGTA (see 3) suggest that in SW or ASW there is an entry of Ca2+ during the action potential.
Action potentials that are carried by Na+ and Ca2+ are, however, known from a variety of excitable tissues (Hagiwara & Byerly, 1981). These are all from metazoans; Actinocoryne is thus the first exception to Hille’s (1984) generalization that sodium-requiring action potentials are unknown outside the metazoan animals, and it is the only protozoan studied which exhibits a mixed Na+/Ca2+ action potential. Although in most ciliates there are Ca2+ spikes (Naitoh et al. 1972; Wood, 1982), a small Na+ conductance is known in some Paramecium mutants (Saimi & Kung, 1980; Hinrichsen & Saimi, 1984), but this is Ca2+-activated, and hence abolished in Ca2+-free solutions.
Although voltage clamp studies would be necessary to establish the different conductances during the action potential, the effects of TEA and 4-AP on the repolarizing phase suggest that it is brought about by a voltage-activated K+ conductance. The increased duration of the action potential seen when Ca2+ entry is blocked by Co2+ and the plateau in 10 mmol l−1 Ca2+/Na+-free solution which disappears when Ca2+ is increased, suggest that a Ca2+-activated K+ conductance may also be involved in repolarization.
In normal SW or ASW, all-or-none rapid contractions always follow action potentials with a delay of some 10 ms and cannot be evoked without a preceding action potential. It therefore seems probable that under normal conditions, contraction is triggered by the action potential. In other contractile protozoa, according to Naitoh (1982), contraction is not clearly linked to membrane electrical activity.
Our results show that external Ca2+ is required for rapid all-or-none contraction, but it is not clear how Ca2+ entry is related to contraction. Contraction evidently requires disassembly of axonemal microtubules, since if they are stabilized with D2O, contraction is inhibited. In contracted specimens, the axonemal microtubules are disassembled and appear in random arrays in the base (Febvre-Chevalier, 1980).
The regulatory role of Ca2+ in microtubule assembly and disassembly is now clear (Schliwa, 1976), and it is known that an increase in internal Ca2+ induces disassembly (see Dustin, 1984). It is thus reasonable to suppose that the microtubular scaffolding of Actinocoryne is disassembled by the transient Ca2+ entry during the action potential.
However, normal contractions are so rapid that they can hardly be brought about by a microtubule sliding mechanism, and it is possible that the microfibrils seen in bundles in the base of contracted specimens may be involved. Whatever the mechanism of contraction may be, entry of external Ca2+ is required to trigger it, although evidently our observations do not exclude a subsequent linked Ca2+ release from intracellular stores, triggered by Ca2+ entry during the action potential. There are numerous small vesicles and dense granules within the cytoplasm which might act as such stores.
Under certain experimental conditions, instead of the rapid all-or-none contractions seen in SW or ASW, slow or incomplete contractions may follow mechanical or electrical stimulation. Thus, for example, normal rapid contractions are seen in Ca2+-free solutions at first, but after prolonged sojourn contractions slow and may finally be abolished. Again, in solutions containing 1 mmol l−1 EGTA, rapid contractions are blocked, but slow contractions may still take place. Since the mechanism of excitation-contraction coupling appears to be Ca2+-dependent, such slow contractions persisting after EGTA or Co2+ treatment may perhaps be attributed to Ca2+ ions linked to the outer surface of the cell membrane, which is covered with a mucous coat. The axopods are not covered with a mucous coat and it is noteworthy that they do not contract under conditions where the stalk shows slow contractions.
We are unable to offer an explanation for the striking effect of 4-AP on contraction. In Actinocoryne, 4-AP blocks contraction in concentrations at which no obvious effects are exerted on the repolarizing phase of the action potential, i.e. at concentrations below which its previously known action as a specific blocker of voltage-dependent K+ conductance (Pelhate & Pichon, 1974) is shown. Regenerative responses are obtained in Na+-free solutions containing 4-AP and Ca2+, so that it does not seem that 4-AP abolishes contraction by blocking Ca2+ entry.
Cell membranes are permeable to 4-AP (Bowman, 1982) so that it presumably acts internally, inhibiting one or more of the intracellular steps involved in contraction; that contraction takes place in the presence of 4-AP in Na+-free solutions containing high levels of Ca2+ may suggest that there is a competitive interaction between 4-AP and internal Ca2+-sensitive sites.
This work was supported by the CNRS [UA 671 and University of Nice (J.F. and C.F-C.), LA 244 (A.B.)], financial support from the Browne fund of the Royal Society (Q.B.) is gratefully acknowledged. TTX solutions were kindly given by Dr G. Romey (Institut de Biochimie, University of Nice).