Osmotic Tolerance of Ca-Dependent Excitability in the Marine Ciliate Paramecium Calkinsi

Electrically excitable cells may respond to changes in the osmotic or ionic concentration of their environment either with rapid adaptation or with loss of their excitability (which may involve irreversible damage). In unicellular organisms, such as ciliate protozoans, ionic and osmotic regulatory mechanisms are limited exclusively to the cell membrane. Ciliates usually tend to maintain a hyperosmotic cytoplasm as compared to the external medium (see, for example, Prusch, 1977). Electrophysiological work on freshwater ciliates has demonstrated that these cells, like other ‘euryhaline’ freshwater animals, can tolerate relatively large variations in concentration and composition of the external solution without harm to their electrical excitability (Naitoh & Eckert, 1968; de Peyer & Machemer, 1977). Marine ciliates, which are. abundant in the coastal and brackish-water regions, have not yet been investigated electrophysiologically. These cells may encounter a wide salinity range in the sea water, similar to metazoan animals in the same habitat.

The present paper describes a first electrophysiological investigation on a marine ciliate under conditions of varying ionic and osmotic environments.

Paramecium calkinsi was reared in a culture solution consisting of 8 or 20 g/l Tropicmarin (corresponding to approximately 20% and 50% artificial sea water /ASW) as given below), 2 mm-Na₂HPO₄ buffer (pH 7·6–7·8) and 1·25 g/l Cerophyl. To adapt the cells to different salinities (equivalent to 25%, 75% and 100% ASW), the 20 g/l Tropicmarin solution was gradually diluted with distilled water, or concentrated with a 40 g/l Tropicmarin solution, at 24–48 h intervals, to give cultures in 10 g/l and 30 g/l Tropicmarin solution (corresponding to 25% and 75% ASW, respectively) after 1 week, and cultures in 40 g/1 Tropicmarin solution (corresponding to 100% ASW) after 2 weeks. For experimentation cells were selected which had been equilibrated for at least 48 h in 10, 20, 30 or 40 g/l Tropicmarin solution.

Single specimens of Paramecium calkinsi (between no and 140 μm in length, Fig. 1) were impaled with two single glass microelectrodes. One microelectrode was filled with 2 M-K-citrate (50–70 MΩ) and used for current injection, the other microelectrode was filled with 3 M-KCl (40–80 MΩ) for voltage recording. A third microelectrode, filled with 3 M-KCl, was positioned just outside the cell. The membrane potential was measured as the voltage difference recorded by the intracellular and the extracellular KCl-microelectrodes.

The design for the injection of constant current and membrane voltage recording was essentially the same as described by Naitoh & Eckert (1972). For membrane voltage-clamp a high-gain differential amplifier (AD 171 K) was used. The input resting resistance was determined by injecting a small hyperpolarizing constantcurrent pulse (0·2 nA) of 80–150 ms duration. In the voltage-clamp experiments the membrane was held at its resting potential (‘holding potential’). The voltage was changed stepwise for approximately 60 ms by + 5 mV up to + 30 mV and to – 40 mV. Membrane currents were monitored by a current-voltage converter connected to the bath. The maximum ionic currents, i.e. the peak early inward current and the steadystate current, were thus measured. For current-voltage relationships the peak early inward current was corrected for leakage current (about 0·5 nA or less for a ± 10 mV voltage step).

For the experiments, the 100% ASW had the following ionic composition (in mM): NaCl, 434; KCl, 10; CaCl₂, 10; MgCl₂, 53; NaHCO₃, 2·5; Hepes buffer (N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid), 2; the pH being 8·0–8·1. The various dilutions of the ASW (20%–75 %) were prepared from this 100% ASW by adding the appropriate amounts of distilled water. The Na+-free ASW contained choline-Cl for NaCl; in the Ca²⁺-free ASW CaCl₂ was exchanged for the osmotically equivalent amount of NaCl. In one test solution the Ca²⁺ concentration was increased by replacing MgCl₂ with CaCl₂ (thus having 32 mM-CaCl₂ and zero MgCl₂ in 50% ASW). In another test solution all CaCl₂ was exchanged for SrCl₂. If solutions were exchanged within the same experiment, the experimental chamber (volume less than 1·5 ml) was slowly perfused, exchanging at least 10 times the volume of the chamber. All experiments were performed with the experimental chamber held at 17–18 °C.

Specimens of P. calkinsi cultured in 50% ASW were slowly adapted to 25%, 75% and 100% ASW, as described in Methods. After equilibration the cells had a normal shape and size with no apparent signs of osmotic stress. The membrane resting potential was – 8 mV in 75 % ASW and was slightly increased with rising salinity (–10 mV in 100% ASW) or dilution (– 15·6 mV in 25% ASW; see Table 1). The resting input resistance was lowest in 100% ASW (11·7 MΩ) and rose to 23·2 MΩ in a salinity of 25 % ASW (Table 1).

Injections of square, inward going, currents passively hyperpolarized the membrane, revealing membrane time constants between 10 and 15 ms (Fig. 2). Membrane depolarizations following small outward going currents were electrotonic and largely symmetric to hyperpolarizations up to positive shifts of 4–6 mV. With larger depolarizations of the membrane regenerative responses, ‘graded action potentials’, were elicited. The amplitude of these action potentials had peak amplitudes up to 30 mV with overshoots up to 20 mV depending on the resting potential and the stimulus Current strength. During sustained depolarization the action potential was followed by potential oscillations of decreasing amplitude before a steady-state level was achieved after 100–150 ms. The membrane excitability remained largely unaffected in external media containing between 100% and 25% ASW, which corresponds to a range in osmolarity between 1084 and 271 m-osmol (Fig. 2).

Recordings of membrane currents under voltage-clamp conditions at different salinities of the ASW (Fig. 3) agreed with the observations made in the unclamped membrane. The course of the I-V relationship remained essentially unchanged in the different salinities, 25%, 50% and 75% ASW. The relationships were shifted to more positive potentials as the ASW concentration was increased, as a result of the more positive holding (resting) potential in 50% and 75% ASW. The steady-state I-V relationships were almost linear in the hyperpolarizing direction (up to – 30 mV) and exhibited a pronounced delayed rectification beyond 10 mV depolarization.

The early inward currents corrected for ohmic leakage were maximal with voltage steps of 10–15 mV, i.e. at membrane potentials between –5 mV and +5 mV.

Paramecium calkinsi can rapidly adapt to moderate changes in salinity of the environment imposed within minutes on to the cell. Fig. 4 shows a series of typical membrane responses of one cell exposed to diluted ASW and subsequently to a raised concentration of the ASW (25% →20% →25% → 30%). After exchanging the bathing solution over a period of 2–3 min the membrane potential attained a new resting value in the next 3–5 min. Action potentials were evoked by depolarizing current pulses after stabilization of the resting potential. It is seen in Fig. 4 that the action potentials in 20% and 30% ASW were little, if at all, different from those in 25 % ASW. The shifts in resting potential and in input resistance were largely reversible, although, after exchange lasting only 3–5 min, the range of fluctuation in potential was larger than in long-term adapted cells. In the example given in Fig. 4, the resting potential in 25% ASW was –18 mV in the fully equilibrated cell, but – 14 mV after 20% ASW exposure and – 16 mV after re-adaptation from 30% ASW (latter trace not shown). There was, however, a consistent tendency, like in long-term adapted cells, that both resting input resistance and resting membrane potential increased in lower salinity (20% ASW) and decreased in higher salinity (30% ASW).

Replacement of the total Ca²⁺ with isosmotic amounts of Na⁺ (3·8 m,M-NaCl for 2·5 mM-CaCl₂ in 25 % ASW) suppressed the regenerative responses of the membrane (Fig. 5). The graded action potentials and oscillations of the membrane disappeared when the exchange of the solutions had been completed (approximately after 1 min, lower left traces of Fig. 5); at this time, membrane rectification was, however, still perceptible. With continued exposure to the Ca-free solution (lower right traces of Fig. 5) the input resistance strongly decreased to approximately 30% of the control value, and rectification almost disappeared, so that the I-V relationship was linearized (see Fig. 7B). The resting potential was slightly less negative (2–4 mV) than in the normal solution. No signs of membrane excitability were detectable even with depolarizations as large as 25 mV. The generation of action potentials was fully reversible after re-introduction of the normal Ca-containing ASW (Fig. 5, upper right traces). Membrane resting potential and input resistance also returned to their control values (–16 mV, 20 MΩ). The experiments suggest that Ca²⁺ ions are necessary for the production of action potentials, and that Mg²⁺ ions are not involved.

When Sr²⁺ was substituted for Ca²⁺, the electrical excitability of the membrane was essentially unmodified (Fig. 6). The membrane potential tended to be slightly more negative in the Sr²⁺-containing ASW as compared with the normal, Ca²⁺containing ASW. Voltage-clamp experiments showed that the course of the steadystate I-V relationship, including the resting input resistance, exhibited prominent delayed rectification and insignificant inward rectification, and thus resembled that obtained in normal Ca²⁺-containing 50% ASW. We conclude that Sr²⁺ can substitute for Ca²⁺ as a charge carrier across the membrane of Paramecium calkinsi.

To test whether Na⁺ could act as a charge carrier, external Na was replaced by choline (in 25% ASW). The membrane resting potential was up to 15 mV more negative in the Na⁺-free ASW ( – 25 to – 32 mV), and the input resistance rose threefold to approximately 70 MΩ The depolarizing responses of the membrane following constant current stimulation were unaltered apart from an increase in relative threshold potential of up to 5 mV (Fig. 7 A). The I-V relationship, as obtained from steadystate potentials during constant current injection, revealed a strong increase in rectification, in the inward as well as in the outward direction in the Na+-free solution (Fig. 7B). This property, together with the raised input resistance, sharply contrasts to the I-V relationship from cells in Ca-free ASW, where the relationship was almost linear, and the input resistance was decreased with respect to that in the normal solution. The data suggest that Na⁺ ions contribute to the maintenance of the resting potential of this membrane but are not involved in excitation.

The Ca²⁺ concentration of 50% ASW was raised more than sixfold (from 5 to 32 HIM) by substitution of CaCl₂ for MgCl₂. Under these conditions the membrane hyperpolarized by 2–5 mV and the input resistance tended to increase slightly. The regenerative membrane responses were enhanced in the Ca²⁺-rich solution. The peak early inward current following positive voltage-clamp steps increased (Fig. 8) and approximately doubled as compared to that observed in the normal 50% ASW. The peak inward current was observed with 20 mV depolarizing voltage steps as compared to 10–15 mV steps for the peak inward current in the normal solution. Thus, while the steady-state I-V relationship was negatively shifted along the voltage axis in solutions of raised Ca²⁺, the peak early inward current showed a positive shift along the axis.

For these experiments, 50% ASW with high Ca²⁺ contents (32 HM) was used to increase the amplitude of the action potential (see above). Action potentials elicited by 10 ms outward going current pulses reached peak amplitudes of up to 40 mV (Fig. 9 A, B). The maximum rate of rise of the action potentials was up to 10 V/s, and :he maximum rate of repolarization amounted to 12–15 V/s (see middle traces in Fig. 9 A, B). A small negative afterpotential was followed by a prominent and long-lasting positive afterpotential. The amplitude and the duration of this positive late response were related to the size of the preceding action potential; they typically imounted to approximately 5 mV and 150 ms, respectively.

Graded action potentials including the afterpotential were also elicited at the end of large membrane hyperpolarizations ( ⩾50 mV) produced by 100 ms inward current pulses (‘anodal-break excitation’, see Fig. 9C). The amplitude and rate of rise of these action potentials tended to increase with the amplitude of the preceding hyperpolarization. Large ‘anodal-break excitations’ were also followed by a pronounced positive afterpotential.

When identical depolarizing current pulses were given at intervals of less than 300 ms, the amplitude and the rates of rise and fall of the second action potential were reduced (Fig. 10A). This relative refractoriness of the membrane was most conspicuous at stimulus intervals of less than 150 ms. Small active responses following the second current pulse were detectable with stimulus intervals down to 30 ms (B-D). When the second stimulus occurred at intervals of less than 30 ms, the membrane response was only passive, i.e. no active response was elicited (E). These observations suggest that the period of relative refractoriness following an action potential is approximately 30–300 ms. The absolute refractoriness of the membrane falls within 30 ms after an excitation.

The main results of the present paper, for the membrane of P. calkinsi, show that (1) the electrical excitability, represented by graded action potentials, is virtually unchanged in cells adapted to very different salinities and osmolarities of their surrounding medium, and (2) these action potentials are due to an inward membrane current carried by Ca²⁺ ions.

The tolerance of the electrical excitability to a wide range of dilutions of the ASW bears some interesting similarities and differences compared to that of other invertebrate excitable cells. Metazoan invertebrates may regulate their extracellular microenvironment, e.g. by means of a blood-brain barrier, and thus protect the nervous system from large changes in ionic concentration and osmotic stress (see Abbott & Treherne, 1977; Treherne & Schofield, 1979). In some euryhaline osmoconformers, such as the polychaete annelids Mercierella and Sabella, progressive dilution of the blood ionic concentration is compensated by active extrusion of ions from the cells and by membrane hyperpolarization, in order to maintain the electrochemical ionic gradients used for electrical excitation (see e.g. Carlson & Treherne, 1977; Treherne & Pichon, 1978). In the stenohaline osmoconformer Maia (Crustacea) the spike-generating mechanism suffers irreversible damage by relative modest hyposmolarity (Pichon & Treherne, 1976). The marine euryhaline ciliate Miamiensis was found to maintain its cytoplasm hypertonic to the environment at all salinities (20%–100% ASW) by Spelling water by means of contractile vacuoles (Kaneshiro, Dunham & Holz, 1969). In analogy to Miamiensis it is likely that active osmotic and ionic regulation of the cytoplasm also occurs by contractile vacuoles in Paramecium calkinsi. In contrast to invertebrate axons, however, where the spike mechanism is due to a Na+-electrogenesis, the spike mechanism in this marine ciliate, like in freshwater ciliates, involves a Ca²⁺-electrogenesis. Due to the very low intracellular Ca-activity usually found in living cells, presumably 10⁻⁷ M or lower, variation of the external Ca²⁺ concentration induces only comparatively small changes in the total Ca²⁺ gradient across the cell membrane. Thus, with ASW-dilutions from 100% to 20%, little influence on the action potential would be expected by the concomitant decrease in the Ca²⁺ gradient.

The experiments described above present the following evidence for Ca²⁺ ions carrying the inward current during the action potential: (1) Removal of Ca²⁺, but not of Mg²⁺ or Na⁺, from the bathing solution reversibly abolished the electrical excitability. (2) Replacement of Ca²⁺ by Sr²⁺, however, retained the action potential or early inward current. (3) Raising the external Ca²⁺ concentration produced an increase in amplitude and rate of rise of the action potential, and also increased the peak early inward current. The action potentials and membrane currents recorded were very similar to those shown for freshwater ciliates (for references see Machemer & de Peyer, 1977; Eckert & Brehm, 1979). In both groups, freshwater as well as marine ciliates, the fiction potentials appear to be due to an increase in membrane conductance to Ca²⁺ ions, despite the vast difference in the ionic composition of their external environment. The excess Na⁺ in the fluid habitat of marine ciliates is not directly involved in the generation of action potentials, although removal of Na⁺ had some drastic effects on the membrane resistance and rectification. Thus external Na⁺ appears to have some indirect effects on membrane excitation by e.g. changing the I-V relationship, and by influencing the relative threshold for the generation of action potentials. It has recently been reported for the wild-type and paranoiac-mutant of the freshwater ciliate P. tetraurelia that Na⁺ ions can carry a slow inward current, which is absent in the pawn-mutant, where the Ca²⁺-electrogenesis is defect (Saimi & Kung, 1980). The role of external Na⁺ in the marine species should therefore be more carefully investigated at various Na⁺ concentrations and with different Na-substitutes under voltage-clamp conditions.

In conclusion, the present study has shown that the mechanism of electrical excitation in a marine ciliate is very similar to that shown for freshwater ciliates, and relatively unaffected by variations of ionic concentration and osmolarity of the external medium. By tolerating a wide range of external salinity, marine cells allow ample space for experimental manipulation of the external ionic concentrations. A more thorough comparison of the electrical membrane properties of freshwater and marine ciliates might provide further support for the hypothesis (Potts & Parry, 1964; Kaneshiro, Holz & Dunham, 1969) of a freshwater ancestry of brackish-water and marine ciliates.

This work was supported by the Deutsche Forschungsgemeinschaft, SFB 114, TP A5.