The Ionic Basis of the Depolarizing Mechanoreceptor Potential of Paramecium Tetraurelia

Mechanical stimulation of the anterior end of Paramecium caudatum triggers a depolarization (Eckert, Naitoh & Friedman, 1972) whereas posterior stimulation triggers a hyperpolarization (Naitoh & Eckert, 1973). The depolarizing response consists of a receptor potential and a regenerative Ca²⁺-based action potential (Eckert et al. 1972). Through the use of deciliated paramecia whose voltage-sensitive Ca-channels were removed with the cilia, the receptor current has been found to be largely carried by Ca²⁺ (Ogura & Machemer, 1980). In Stylonychia, a hypotrichous ciliate, Mg²⁺, Sr²⁺ and Ba²⁺ can substitute for Ca²⁺ in carrying the receptor current (de Peyer & Machemer, 1978; de Peyer & Deitmer, 1980).

Typical pawn mutants have no action potential (Kung & Eckert, 1972; Schein, Bennett & Katz, 1976; Takahashi & Naitoh, 1978; Satow & Kung, 1980a) and have no transient Ca-inward current upon depolarization under voltage clamp (Oertel, Schein & Kung, 1977; Satow & Kung, 1980a). However, a depolarizing receptor potential induced by the mechanical stimulation at the anterior end remain (Takahashi & Naitoh, 1978; Kung, 1979). We now use the pawn mutants to study the ionic basis of this receptor potential.

Three stocks of P. tetraurelia : 51s (wild type), d4-500 (pawn of pwA complementation group) and d4-95 (pawn of thepwBgroup) (Kung, 1971 ; Change et al. 1974) were cultured by standard methods (Sonneborn, 1970). Only robust cells in the logarithmic phase of growth were used and the experiments were performed at room temperature (22 ± 1 °C).

The Ca²⁺ solution in the experiments of Fig. 2 was 1 mM-Ca(OH)₂, 0·5 mM-CaCl₂, 1 mM-citric acid adjusted to pH 7·2 with 1·2–1·5 mM-Trisma base (Sigma). The Ca²⁺-TEA⁺ solution in the experiments in Figs 1, 2 and 3 was the Ca²⁺ solution with the addition of 4 mm freshly dissolved TEA-CI (tetraethylammonium chloride monohydrate, Aldrich). For the rest of the experiments, chlorides were dissolved in a buffer containing 4 mM-TEA⁺ (fresh), 1 mM-citric acid and 3·5 mM-Trisma base at pH 7·2. Different amounts of CaCl₂ were added to this buffer in other cases and the free Ca²⁺ concentrations were calculated as by Ling & Kung (1980). 1 mm-CaCl₂, SrCl₂, BaCh, MgCl₂, MnCl₂ or 8 mm-KCl, NaCl or LiCl was added to the buffer for the experiments in Figs 5 and 6. All chemicals were of reagent grade.

The techniques used to record intracellularly from Paramecium were conventional (Naitoh & Eckert, 1972; Satow & Kung, 1976a). The microelectrodes were filled with 0·5 M-KCI with resistance of 80-100 MQ. Mechanical stimulation was delivered by a glass stylus (tip diameter 5–10 μm) mounted on a phonograph cartridge (Naitoh & Eckert, 1972; Satow & Kung, 1977). Before stimulation, the stylus was micromanipulated to approach and touch the lower surface of the paramecium which was held stationary by the electrodes. The excursion of the stylus and the indentation of the cell surface could be observed under the microscope when the cartridge was piezoelectrically driven by a 2 ms d.c. pulse delivered from an isolated stimulator (Devices, type 2533). The excursion was not measured, since only maximal responses were compared, except in cases where the graded nature was to be demonstrated (Fig. 3). The paramecia were first immersed in the test solution for 5–10min before they were captured and penetrated with microelectrodes. The combined results of mechanical prodding, electrode penetration, and the bathing of specimens in solutions containing only one, often nonphysiological, metal-cation species proved deleterious to the specimens. Thus, experiments displayed in Figs 5 and 6 were limited to durations of 5–10 min after electrode penetration.

Both the wild type and the pawn have a resting potential of about −20 mV in the Ca²⁺-TEA solution. TEA was incorporated to suppress touch-induced hyperpolarization (see below). In the wild type, mechanical stimulation triggers a nearly all-or-none action potential (Fig. 1A) peaking some 40 mV from the resting level (Satow & Kung, 1976b). A similar stimulation (submaximal, see below) triggers a much smaller response in the pawn (Fig. 1B). The upstroke of the wild-type response clearly has two components: a small depolarization (the receptor potential) followed by a regenerative depolarization (the action potential), as evident in both the voltage are the d V/dt traces examined at fast sweep speed (Fig. 1C). The mutant response consists only of the receptor potential (Fig. ID). The receptor potential of the pawn has a slow (hundreds of ms) biphasic fall, especially when the depolarization is large (Figs 1B, 3). The results may be caused by the several K-conductances in the Paramecium membrane (reviewed by Kung & Saimi, 1982), all of which are left intact by the pawn mutations (Kung & Eckert, 1972; Satow & Kung, 19806). These K-conductances apparently have differential sensitivity to TEA, as is evidenced by the spontaneous action potentials of wild type bathed in TEA solutions (Satow & Kung, 1976b).

Posterior stimulation produces a hyperpolarizing response, known to be due to K⁺ efflux (Deitmer, 1981), which can be completely suppressed by the addition of the K-channel blocker, TEA⁺, to the external bath (Naitoh & Eckert, 1973; Deitmer, 1982). In the solution containing 4 mm-TEA, a depolarization is observed regardless of which part of the cell is stimulated (Fig. 2). Our experiments were therefore carried out in this solution. In most cases, the paramecia were prodded near their mid-sections.

The receptor potential was triggered by dimpling the cell surface. The magnitude of the depolarization increased with the extent of the probe excursion until further movement produced no further depolarization (Fig. 3). The probe excursion was proportional to the voltage which drove it and was monitored, though not measured, through the microscope. The rate of depolarization also increased with the strength of the stimulation. In the Ca²⁺-TEA solution, the maximal depolarization induced by mechanical stimulation was from a resting level of −21 ·6 ±7 ·3 mV to −6 ·6 ± 2 ·5 mV (mean ± S.D., N= 4).

A paramecium can survive for hours and show behavioural and electrical responses to mechanical stimulation in solutions containing Ca²⁺ as the sole metal cation. That the mechanosensory depolarization is exhibited in such solutions strongly suggests that Ca²⁺ can serve as a carrier through the ion channel opened by the mechanical stimulation. To test further the dependence of the receptor potential on Ca²⁺, we varied the Ca²⁺ concentration and examined the maximal responses of the pawns (Fig. 4A). Increasing the external free Ca²⁺ concentration from 0 ·13 to 1 ·33 mm increased the maximal response to mechanical stimulation from −25 ·3 ± 6·9 mV to +8·9 ± 0·3mV (mean ± S.D., N = 4). Increasing the external Ca²⁺ concentration also polarized the resting membrane (Naitoh & Eckert, 1968; Satow & Kung,1979). This apparent change in the resting level is now viewed as a response to the change in surface-charge pattern and not a genuine Nernst effect (Eckert & Brehm, 1979; Satow & Kung, 1979, 1981). Discounting the change in resting potential, the mechanically induced response increased by about 20 mV per decade increase in Ca²⁺ (Fig. 4B). These results further substantiate the view that Ca²⁺ normally carries most of the charges in the depolarizing mechanosensory response.

In order to see which metal cations could carry the current for the depolarizing receptor potential, several cations were applied individually in the TEA buffer. The maximal response to mechanical stimulation recorded in the TEA buffer was small (5 mV at most, Fig. 5A) and did not follow the time course of the normal physiological response (Fig. 1). Addition of 8mm-Na⁺, Li⁺ or K⁺, did not significantly change the response (Fig. 5B, C, D). The usual time course was restored by the addition of lmmCa²⁺, Sr²⁺, Ba²⁺ or Mn²⁺ to the TEA buffer (Fig. 5, E-I). Because the specimens deteriorated rapidly in these solutions, comparison of results from different cells and different solutions can only be semi-quantitative. Nonetheless, the magnitude of the maximal response increased 5-to 20-fold after the Ca²⁺-TEA Sr²⁺-TEA solution replaced the TEA buffer. The responses in Ba²⁺, Mg²⁺ and Mn²⁺ were smaller and slower than those in Ca²⁺ or Sr²⁺. These results indicate that all the divalent cations tested can permeate the conductance for the depolarizing mechanosensory response but the monovalent cations cannot.

The above experiments use the pawn mutant of the pwA complementation group (d4-500). Similar results were obtained for the key experiments with a second pawn mutant of the pwB group (d4-95). This pawn also gave graded mechanoreceptor potentials without action potentials in the Ca²⁺-TEA solution. The receptor potential was also present in the Mg²⁺-TEA solution but absent in the Na⁺-TEA solution.

To compare the ion selectivity of the mechanosensory Ca-conductance with that of the voltage-sensitive Ca-conductance, we examined the action potential of the wildtype P. tetraurelia bathed in these single-metal-ion solutions (Fig. 6). Judging from the potential recording or the dV/dt traces, Sr²* and Ba²⁺ can substitute for Ca²⁺ in the generation of action potentials whereas Mg²⁺, Mn²⁺ and Na⁺ cannot. The Bagactivation is small and slow, though significant. Prolonged plateau depolarizations following the action potentials are recorded from the wild type bathed in the Sr²⁺-or the Ba²⁺-TEA solutions. In the latter solution, the plateau lasts for several seconds.

Our results and the recent voltage-clamp results of Saimi & Kung (1982) with these solutions of single metal-ion species showed that Sr²⁺ and Ba²⁺, as well as Ca²⁺, can the carry current through the voltage-sensitive Ca-conductance. The results confirm dose of other workers using solutions of mixed metal ions (Eckert & Brehm, 1979; Naitoh & Eckert, 1972).

Using a mutation to erase the action potential and thereby unmask the receptor potential, we confirmed the previous finding that Ca²⁺ is the usual ion which carries the receptor current for the depolarization response to mechanical stimulation (Ogura & Machemer, 1980). We also found that other divalent cations, but not the monovalent cations, can carry that current in P. tetraurelia as in Stylonychia (de Peyer & Deitmer, 1980).

Mg²⁺ is a candidate for being a physiological carrier of the depolarizing receptor potential. However, the internal concentration of Mg²⁺ is expected to be several orders of magnitude higher than that of Ca²⁺ (Nakaoka & Toyotama, 1979), and therefore the electrochemical gradient is less favourable for Mg²⁺ as a physiological receptor current carrier. As shown in Fig. 4, Mg²⁺ is less effective in that role than Ca²⁺. We found that wild-type P. caudatum showed little electrical response to mechanical stimulation in a solution of 0·01 mm-Ca²⁺ and 0·99mm-Mg²⁺ (data not shown), but both the receptor potential and the action potential returned when the bath was refilled with a 1 mm-Ca²⁺ solution. Our results in P. tetraurelia are similar to those for Stylonychia (de Peyer & Machemer, 1978; de Peyer & Deitmer, 1980) which showed that the depolarizing receptor potential is normally dependent on Ca²⁺, but Mg²⁺ Ba²⁺ and Sr²⁺ could also carry the receptor current in this hypotrichous ciliate. Unlike our finding in Paramecium, however, Mn²⁺ failed to carry that current in Stylonychia. Although systematic study of the membrane resistance in these solutions was difficult in our experiments because of the rapid deterioration of the specimens, we found that the membrane resistance was low when no metal cation was in the bath. Addition of a mono-or divalent cation increases the resistance to 20 MΩ or above. While there are differences in membrane resistance of cells bathed in solutions of different cations these differences alone cannot fully account for the presence of a receptor potential in divalent cation solutions and its absence in monovalent cation solutions (Fig. 4). It is interesting that the mechanoreceptor channels in the hair cells of the inner ear are also relatively nonselective (Corey & Hudspeth, 1979; Edwards, Ottoson, Rydqvist & Swerup, 1981) compared to voltagesensitive channels. In the case of the hair cell, both monovalent and divalent cations permeate the receptor channel.

The conductance for the depolarizing mechanosensory receptor of Paramecium tetraurelia is apparently not very permeable to Na⁺, Li⁺ or K⁺, and is not blocked by TEA⁺ (Fig. 4). Since the equilibrium potential of K⁺ is usually tens of millivolts more negative than the resting level (Oertel, Schein & Kung, 1978; Satow & Kung, 1980a; Eckert, Naitoh & Machemer, 1976) membrane hyperpolarization is to be expected upon mechanical stimulation, if K⁺ permeates the receptor conductance in question. Our results show that no such hyperpolarization occurs even when the bath is completely devoid of K⁺. This result cannot be attributed to the presence of SEA⁺ since TEA⁺ does not block the depolarizing receptor conductance as shown by the receptor potentials when permeant ions are provided. Under natural conditions, without TEA⁺, stimulation at the midsection activates both the Ca²⁺-based depolarising and the K⁺-based hyperpolarizing conductance as seen in the biphasic response of Fig. 2. From their elegant experiments with deciliated, voltage-clamped P. caudatum, Ogura & Machemer (1980) concluded that the receptor current induced by mechanical stimulation of the cell’s anterior is the sum of a K⁺ and a Ca²⁺ current. Stimulation of the cell’s posterior can induce depolarization as revealed when TEA⁺ is included in the bath (Fig. 2). These results mean that the receptors with the Ca²⁺ channels and those with the K⁺ channels can both be activated by stimulations delivered to any part of the paramecium. There are at least two possible explanations of the natural responses: depolarization dominates the response to anterior stimulation and hyperpolarization dominates the response to posterior stimulation. The two populations of receptors may be mingled and distributed along two overlapping gradients over the entire surface of the paramecium (Ogura & Machemer, 1980). Alternatively, the two kinds of receptors, with unspecified distributions, may have different sensitivity. It is known that a smaller mechanical stimulation is needed to elicit a response from the ‘posterior receptor’ (considered to be the same as the K⁺-based hyperpolarizing mechanoreceptor here) than the ‘anterior receptor’ (the Ca²⁺-based depolarizing mechanoreceptor) (see Material and Methods of this paper and Naitoh & Eckert, 1973; Naitoh, 1974). Because the depolarizing response is nearly the same regardless of where the stimulation is delivered, when TEA⁺ is included in bath (Fig. 2, bottom), it is possible that the Ca-based depolarizing receptors are not direction specific but detect impact from any direction. To account for the different responses in the normal, TEA⁺-free, solution (Fig. 2, top), one may speculate that the K⁺-based hyperpolarizing receptors may be much more easily activated and prefer posterior impact to anterior impact. This alternative hypothesis allows us to rationalize Ogura & Machemer’s observation (1980) that anterior stimulation induces both Ca²⁺ and K⁺ currents, and the previously unexplained result of Naitoh & Eckert (1973) that TEA⁺ converts the hyperpolarizing response to a depolarizing one.

While the voltage-sensitive conductance for the action potential and the mechanosensory conductance for the depolarizing receptor potential both use Ca²⁺ as their major natural current carrier the two conductances are quite different. First, they differ in their triggering mechanisms (Eckert et al. 1972). Second, the voltagesensitive Ca-conductance is present exclusively on the ciliary membrane while the mechanosensory Ca-conductance is on the soma membrane (Ogura & Takahashi, 1976; Machemer & Ogura, 1979). Third, the two conductances clearly differ in their ion specificities. Finally, pawn and CNR mutations eliminate the function of the voltage-sensitive Ca-conductance but not the mechanosensory Ca-conductance. These results, taken together, indicate that these two conductances not only serve different functions but most likely also have different molecular components.

This work was supported by NSF grant BNS 79-18554 and PHS grant GM22714 to C.K. ; and PHS postdoctoral fellowship GM06940 to A.D.M.