Ca²⁺-Dependent Photoreceptor Potential in Paramecium Bursaria

Paramecium bursaria is a ciliated protozoan which contains symbiotic green algae, chlorella (Muscatine, Karakashian & Karakashian, 1967; Brown & Nielsen, 1974). It shows an avoiding reaction when it comes to a shaded area (Engelmann, 1882) or when exposed to a step-decrease in light intensity (Saji & Oosawa, 1974). Such a photoresponse is mediated by the presence of the chlorella. In chlorella-free P. bursaria, an avoiding reaction is generally given to lighted areas or in response to a step-increase in light intensity (Iwatsuki & Naitoh, 1981 ; Niess, Reisser & Wiessner, 1981).

In P. caudatum and P. aurelia, swimming behaviour has been shown to be closely related to the membrane potential (Naitoh & Eckert, 1974; Eckert & Brehm, 1979) and a transient change in this potential, termed a receptor potential, is given in response to a mechanical or thermal stimulus (Naitoh & Eckert, 1969; Ogura & Machemer, 1980; Toyotama, 1981; Hennessey, Saimi & Kung, 1983; Nakaoka, Kurotani & Itoh, 1987). P. bursaria has not been studied electrophysiologically.

In this investigation, we show that a step-increase in illumination induces a receptor potential in chlorella-free P. bursaria. The receptor potential is caused by a transient increase in Ca²⁺ conductance of the membrane.

A chlorella-free type of Paramecium bursaria (Mit-C^w, supplied by Dr I. Miwa of Ibaraki University) was cultured in a hay infusion inoculated with Klebsiella pneumoniae. The cultures were maintained on a fixed illumination cycle of 12 h dark and 12 h light (a fluorescent lamp of about 10³lux) at 22°C. Stationary phase Paramecium were collected by low-speed centrifugation and suspended in a control solution containing 0·25 mmol 1^-1 CaC1₂, 2 mmol 1^-1 KCl and 2 mmol 1^-1 Tris-HCl (pH 7·2).

To carry out the experiments in the absence of action potentials, the cells were deciliated by incubation in control solution containing 5 % ethanol for 2 min at 22°C, and returned to control solution (Ogura & Machemer, 1980). Intracellular recordings were started about 30 min after deciliation and continued for 2–3h. Data were obtained only from cells which showed no action potentials.

Methods of intracellular recording were similar to those described by Naitoh & Eckert (1972). The electrodes were filled with 0·1 mol1^-1 KCl, or 0·1 mol1^-1 KCl plus 0·1 mol 1^-1 EGTA neutralized with KOH, and their resistances were 100–130MΩ. The cells were placed in a glass vessel mounted on an inverted microscope, and electrodes were inserted from the upper side (Nakaoka et al. 1987).

The membrane potential was set at various levels by injection of constant current of the order of 10⁻¹⁰ A. Twenty to thirty seconds after the potential had been set, and when it had become relatively stable, photostimulation was commenced.

The light source for stimulation was a 100-W halogen lamp with a variable-supply voltage. An i.r. filter and an interference filter with a half-bandwidth of 9-11 nm were placed in front of the lamp to obtain monochromatic light. Short pulses of light were obtained by using a camera shutter between the lamp and the microscope stage. A photocell was placed between the shutter and the stage to record the light pulse.

Light intensity was measured with a calibrated silicon photodiode. Light stimuli were usually of white light, with an intensity of about 1 mW cm^-2, in the plane of the specimen which elicited a maximum amplitude of receptor potential. To determine the dependence of the receptor potential upon light intensity or wavelength, stimulations were made using monochromatic light.

The light source for observing the cell was a 15-W tungsten lamp with a 700-nm long-pass filter.

A step-increase in illumination produced a transient change in membrane potential (Fig. 1A). In initial experiments, using electrodes filled with 0·1 mol1^-1 KCl, it was found that the amplitude of the potential change was gradually reduced by repetition of the stimulus (Fig. 1A). If electrodes were filled with 0·1 mol1^-1 KCl plus 0·1 mol 1⁻¹ EGTA, then the potential change was not decreased by repetitive stimuli (Fig. IB), so these electrodes were adopted for the following experiments.

When the duration of the light pulse was 10⁻³s, no potential change could be detected amongst the potential fluctuations (Fig. 2). A small potential change was elicited by a light pulse of 2×10⁻³ s. As the duration of the pulse was increased, the potential change grew larger in amplitude and duration, until at pulse lengths greater than 30⁻¹s, no further increase was observed (Fig. 2). The potential change was transient and the membrane potential recovered to almost the original level within 2 s, even when the light was continuously on.

Usually, a step-down in light intensity induced little change in the potential, but in a few cases a step-down induced a transient hyperpolarization.

Where R represents the amplitude with maximum R_max, and I represents the light intensity at half-saturation intensity I_o (Fig. 3B).

To obtain an action spectrum, the wavelength of the stimulating light was changed and the amplitude of the elicited potential change was measured (Fig. 4). Large depolarizations were induced at two wavelengths: approx. 420nm and approx. 560 nm.

The resting potential was shifted by injection of a constant current, and then a light stimulus was applied (Fig. 5A). The elicited depolarization became larger when the membrane potential was made more negative than the resting potential. When the membrane potential was made more positive, the response to the light stimulus became smaller. Further positive shift caused the response to disappear and increased the fluctuation of the membrane potential. The polarity of the receptor potential was not reversed by the positive shift. A negative shift increased the amplitude of the response.

The positively shifted potential at which the response disappeared was largely dependent on the Ca²⁺ concentration in the external medium (Fig. 5B). This potential showed a slope of 27 mV for a 10-fold change in Ca²⁺ concentration. Addition of K⁺ or Mg^24- to the external medium had little effect on the potential at which the response disappeared, but resulted in depolarization of the resting potentials.

The receptor potential obtained in response to successive step-increases in illumination showed a reproducibility when the electrodes contained EGTA, and showed a gradual decrease when the electrodes did not. It is therefore likely that some of the EGTA in the electrode diffuses into the cytoplasm and acts to maintain a low Ca²⁺ concentration, which is a necessary condition for generation of the receptor potential.

The action spectrum in the present study (Fig. 4) was obtained by measurements of receptor potentials. However, this spectrum differs from those of previous reports which were obtained by counting cells accumulating in response to light stimuli. I watsuki & Naitoh (1981) reported that chlorella-free specimens show photodispersal at 560 nm, but no dispersal at around 420nm. Pado (1972) showed that chlorella-containing specimens accumulated at about 420 nm, but did not accumulate at 560 nm. We have observed that, at both 420 nm and 560nm, ciliated and chlorella-free specimens show a photophobic swimming response (unpublished data). Such a difference in the action spectra may come from some differences in the specimens used for the experiments. Accurate determination of the action spectrum is needed for identification of the photoreceptor chromophore.

A positive shift of the membrane potential, produced by current injection, decreased the amplitude of the potential change induced by the light stimulus, and a negative shift increased the amplitude (Fig. 5A). This observation rules out the possibility that the membrane permeability for Na⁺ or K⁺ contributes to the potential change, because the concentrations of these ions inside the cell are higher than those outside (Yamaguchi, 1963; Oka, Nakaoka & Oosawa, 1986) and the equilibrium potentials of these ions are at negative levels below the resting potential in our experimental conditions. The positively shifted potential at which the response disappeared was Ca²⁺-dependent (27 mV/log [Ca²⁺]_o) and was almost independent of K⁺ or Mg^24- concentration (Fig. 5B). Because the Ca²⁺ dependency approaches the theoretical slope of 29 mV for a Ca²⁺ diffusion potential, the transient depolarization elicited by step-up photostimulation is interpreted to be primarily caused by a transient increase in the membrane conductance to Ca²⁺. This interpretation is in agreement with the observation that the polarity of the receptor potential is not reversed by a positive potential shift, because an extremely low concentration of intracellular Ca²⁺ prevents outward flux of Ca²⁺, even when the membrane conductance to Ca²⁺ increases.

In various vertebrate and invertebrate photoreceptors, the receptor potentials are primarily caused by a change in membrane conductance to Na⁺ (Brown, Hagiwara, Koike & Meech, 1970; Yau, MacNaughton & Hodgkin, 1981). The receptor potential in scallop retina is, exceptionally, caused by an increase in membrane conductance to K⁺ (Gorman & McReynolds, 1978).

Where gK and g_ca represent the membrane conductances to K⁺ and Ca²⁺, respectively, E_K and E_ca represent the diffusion potentials of the respective ions, and application of photostimulation changes only g_Ca by Ag_Ca. When E_m is fixed at – 30 mV, the slope of Ca²⁺ dependency roughly reflects (Δg_ca/G)E_ca-Ten-fold changes in external Ca²⁺ concentration change E_ca by 29 mV and the response by 2·2 mV. Then, the ratio Δg_ca/G is 0·07. That is, 7 % of the total conductance is increased by photostimulation.

In this study, measurements were started about 30min after deciliation and continued for 2 h, and it is possible that the cilia had begun to regenerate during this period (Machemer & Ogura, 1979). Therefore, we cannot exclude the possibility that part of the Ca²⁺ conductance increased by photostimulation is located on the ciliary membrane. In a ciliated cell, the small depolarization elicited on the soma membrane can open voltage-sensitive Ca²⁺ channels on the ciliary membrane (Ogura & Takahashi, 1976; Dunlap, 1977). Opening of the voltage-sensitive channel could be accompanied by a large depolarization and an influx of Ca²⁺ into cilia, and this could induce photophobic behaviour.

This work was supported by a Grant-in-Aid for Special Project Research on the Mechanism of Bioelectrical Response (61107006) from the Japanese Ministry of Education, Science and Culture.