When Paramecium are transferred from the solution to which they have been adapted to a solution of a different K+ concentration, they initially change their swimming behaviour and then gradually recover normal swimming. This adaptation to the new solution takes 2–3 h. We found that in the process of adaptation there was a change in membrane potential: transfer of the cells to a lower K+ concentration caused depolarization of the potential, whereas transfer to a higher concentration produced a hyperpolarization. In both cases, the change in potential was followed by a gradual repolarization to the original value in the control solution. During adaptation, the intracellular concentration of K+ did not change much. It is probable that the cells change their membrane potential towards a constant level by controlling the membrane conductance for K+.
A ciliated protozoan, Paramecium, can alter its swimming behaviour in response to a change in the ionic concentration of the environment (Jennings, 1906). When the K+ concentration of the surrounding medium is lowered, the cells initially accelerate forward swimming and then gradually recover normal swimming (Nakaoka et al. 1983). However, when they are transferred to a solution of a higher K+ concentration, they show transient backward swimming and then recover (Mast & Nadler, 1926). Within a limited range of K+ concentration they can adapt to the new environment.
A close relationship between swimming behaviour and membrane potential has been found in Paramecium (Machemer, 1974; Machemer & Eckert, 1975; Naitoh & Eckert, 1974). Hyperpolarization of the membrane potential causes an increase in the beating frequency of cilia and depolarization causes ciliary reversal. We have therefore measured the change in potential produced by a change in K+ concentration and examined the potential during adaptation.
MATERIALS AND METHODS
Paramecium caudatum (syngen 3, mating type V, stock Ksyl) was cultured at 25°C in a hay infusion inoculated with Klebsiella pneumoniae. Paramecium, at a stationary growth phase, were collected by low speed centrifugation and incubated for about 3 h in various concentrations of KCl, in 0·25 mmol l−1 CaCl2 and 1 mmol l−1 Tris-HCl, pH 7·2.
Determination of swimming velocity
Paramecium were photographed while swimming in a rectangular glass vessel mounted on a copper plate whose temperature was controlled at 25 °C by water flow beneath it (Nakaoka & Oosawa, 1977). Swimming velocity was measured by averaging about 50 tracks on a photograph exposed for 2 s under dark-field illumination of the vessel.
Membrane potential was measured by the method of Naitoh & Eckert (1972). Paramecium were placed in a small glass vessel mounted on the stage of an inverted microscope (Olympus, CK), and microelectrodes were inserted into the cell from above. The recording and current-passing electrodes were filled with 1 mol l−1 KCl: their resistances were between 30 and 50MΩ. A similar electrode was placed just outside the cell. The membrane potential was measured as the voltage difference between the internal and external electrodes after nulling the tip potentials in the bath. The potential was obtained 5–10 min after the experimental chamber had been filled with a test solution. The test solution usually contained various concentrations of KCl, 0·25 mmol l−1 CaCl2 and 1 mmol l−1 Tris-HCl, pH 7·2.
Intracellular K+ concentration
Intracellular concentration of K+ was measured by atomic absorption. After the cells had been adapted to a test solution for about 3 h, they were collected by centrifugation and washed twice with a solution containing 0·25 mmol l−1 CaCl2 and 1 mmol l−1 Tris-HCl, pH 7·2. Part of the cell suspension was diluted 10 or 100 times and the cell density was counted by recording a video image at low magnification. This diluted suspension was then concentrated to 105 cells ml−1 and heated in a boiling water bath for 4 min. Disrupted cells were removed by centrifugation and the supernatant was used for the measurement. Cell volume was assumed to be 5·6×l0−7ml (Fortner, 1925).
Behavioural adaptation to various K+ concentrations
The time taken for adaptation to different concentrations of K+ was determined by measuring swimming velocity at intervals in solutions of various K+ concentrations after initial adaptation to 4 mmol l−1 K+. When external K+ concentration was lowered, the velocity increased between two- and three-fold, and then decreased to a stationary value which was nearly equal to that before the change. This adaptation took 3–4 h (Fig. 1). However, when the cells were transferred to solutions of higher K+ concentration, the swimming velocity did not change, but initially there was a short period of backward swimming.
Membrane potential during adaptation to various K+ concentrations
Membrane potential was measured at intervals after transfer of adapted cells into solutions of different K+ concentrations. Cells transferred to 2 mmol l−1 K+ after adaptation to 8 mmol l−1 K+ showed a membrane potential that gradually increased from its initial low value (Fig. 2). In cells adapted to 2 mmol l−1 K+ and then transferred to 8 mmol l−1 K+, the membrane potential (measured during brief intervals in 2 mmol l−1 K+) gradually decreased to the same value as that of the cells adapted to 8 mmol l−1 K+ (Fig. 2).
Membrane potential after adaptation to various K+ concentrations
The dependence of membrane potential upon external K+ concentration was measured after 3h adaptation to various K+ concentrations (Fig. 3). The potential was proportional to K+ concentration over the range 0·5–16 mmol l−1. More negative potentials were obtained for a given K+ concentration after adaptation to higher K+ concentrations.
Membrane resistance of the cells adapted to various K+ concentrations
Membrane resistance was measured by injecting a small hyperpolarizing current through the recording microelectrode, at K+ concentrations of 2 and 8 mmol l−1, after adaptation to either 2 or 8 mmol l−1 K+ (Table 1). Resistance was reduced both when measured at 8 mmol l−1 K+ and in cells adapted to 8 mmol l−1 K+.
Intracellular K+ concentration
Intracellular K+ concentration, measured by atomic absorption, was found to be 21 ± 3 mmol 1−1 (mean of three different samples) in cells adapted to 2 mmol l−1 K+ for 3h, and 23 ±2 mmol l−1 (mean of three different samples) after adaptation to 8mmol l−1 K+.
When Paramecium were transferred to media of different K+ concentrations, they changed swimming behaviour and then recovered (Fig. 1). During the period of adaptation, they changed the resting potential to make it almost independent of the new K+ concentration (Figs 2, 3).
In the process of adaptation, the K+ concentration inside the cell changed only slightly, but membrane resistance showed significant changes (Table 1). According to the data in Fig. 3, the increase of the membrane potential (Em) with increasing K+ concentration (dEm/dlog[K+]0) was raised by an increase in the K+ concentration of the adaptation medium. Therefore, it is probable that the K+ conductance of the membrane changed during adaptation.
Table 2 summarizes the pk/pCa values and the membrane conductances of cells adapted to 2 and 8 mmol l − 1 K+. Since the K+ conductance (gK) is much larger than the Ca2+ conductance (gca) in the present experimental conditions (Naitoh & Eckert, 1974), the membrane conductances in Table 2 almost correspond to gK- The pk/pCa values in the two groups of cells agree well with the ratio of membrane conductances, supporting the hypothesis that the change in membrane potential is mainly caused by the change in gK.
Thus, it is very probable that, during adaptation, Paramecium change the membrane conductance or permeability to K+, resulting in maintenance of the resting potential at a constant level. It is not known, however, whether the change of conductance is due to a change in the number of channels or a change in the permeability of single channels.