The anticalmodulin drug, W-7 [N--(6 aminohexyl)-5-chloro-l-naph-thalenesulphonamide] specifically inhibits the voltage-dependent Ca-current of Paramecium as well as the behavioural consequence of Ca2+ influx, backward swimming. The dechlorinated analogue, W-5, is four to five times less effective. Analysis of membrane currents under voltage clamp shows that W-7 not only reversibly inhibits the voltage-dependent Ca-current but also shifts the voltage sensitivity of this Ca-current towards less negative voltages in a concentration-dependent manner. We suggest that Paramecium can be used as a system to screen behaviourally for other Ca-channel blockers as well as to study the mechanism of action of these drugs.
A Paramecium swims by beating the thousands of cilia which cover its body. Depolarizing stimuli can cause backward swimming by activating a voltagedependent Ca2+ action current. This current increases intracellular Ca2+ level to cause reversal of the ciliary beat direction and subsequent backward swimming (for review see Kung & Saimi, 1982). The duration of stimulated backward swimming upon transferring the Paramecium into a high concentration of K+ (20 mm) has been used as an estimate of the voltage-dependent Ca-channel activity (Haga, Forte, Saimi & Kung, 1982).
The calmodulin antagonist W-7 [N-(6 aminohexyl)-5-chloro-l-naphthalene-sulphonamide] inhibits backward swimming in Paramecium. The concentration of drug which inhibits 50% of the bovine calmodulin-dependent phosphodiesterase activity, the ID50, is about 30 μm for W-7 and about 240 μm for its dechlorinated analogue, W-5 (Nelson, Andrews & Karnovsky, 1983). W-7 reduces the backward swimming elicited by Ba2+ by 50 % at a concentration of 40/IM, while W-5 is ineffective at this concentration (Otter, Satir & Satir, 1983). Although this correlation suggests a calmodulin involvement in the swimming behaviour of Paramecium it should be noted that W-7 also has effects on several calmodulin-independent functions (Hirata, Suematsu & Koga, 1982; Luthra, 1982; Tanaka, Ohmura & Hidaka, 1982; Wise et al. 1982; Schatzman, Raynor & Kuo, 1983).
Since no pharmacological agent has been previously described which can block the Paramecium Ca-current (Eckert & Brehm, 1979), a genetic approach has been used to isolate the Ca-current (Oertel, Schein & Kung, 1977) and other membrane currents (Kung & Saimi, 1982). We now report that W-7 is a suitable pharmacological agent which can selectively and reversibly inhibit the voltage-dependent Ca-current of Paramecium.
MATERIALS AND METHODS
Stocks and cultures
Wild type Paramecium caudatum, stock G3, and a mutant which lacks a voltagedependent calcium current, cnrA (16A712) (Takahashi & Naitoh, 1978), were cultured at 25 °C in Cerophyl medium supplemented with 0·03 % proteose peptone (Difco) and 5 mg l−1 stigmasterol (Sigma). These stocks were kindly provided by Drs M. Takahashi and Y. Naitoh. Cultures were innoculated with Enterobacter aerogenes 24 h before introducing the paramecia (Sonneborn, 1970).
The standard recording solution (Ca-K solution) contained 4mm-KCl, 1 mm-CaCl2, 1 mm-MOPS (morpholinopropanesulphonic acid) and was buffered to pH 7-0 with Tris-base [Tris(hydroxymethyl)aminomethane]. To isolate the inward Ca-current, the K-currents were inhibited by adding 10mm-TEA-Cl (tetraethylammonium chloride) to the Ca-K solution and using 2M-CsCl in the microelectrodes. This procedure blocked 90–95 % of the voltage-dependent K-current (Eckert & Brehm, 1979 ; Hinrichsen & Saimi, 1984). The behavioural testing solution contained 20mm-KCl in Dryl’s solution as described by Haga et al. (1982). W-7 and W-5 (Rikaken Co., Ltd) were dissolved in DMSO (dimethylsulphoxide) or ethanol and added to the recording and behavioural testing solutions with a concentration of organic solvent not exceeding 1·5 % (v/v).
The techniques for voltage clamp and current injection and methods of recording were similar to those described by Satow & Kung (1979). The 2M-KC1 or 2M-CSC1 microelectrodes used for voltage clamp and current injection experiments had resistances of 10–40 MΩ. The relatively high resistance electrodes were used to assure optimal cell condition during the multiple perfusion experiments. The perfusion rate was about 10 ml min−1 and recording was begun within 1 min after perfusion. The recording chamber volume was 1 ml. The membrane was held at −40 mV and the currents were recorded following step depolarizations and hyperpolarizations from this level. Current measurements presented in Figs 2, 4, 5 and 7 were background subtracted by determining the difference between the measured value and the leakage current, which was linearly extrapolated from the measured leakages near the holding level. The membrane resistance, Rm, was determined from the slope of the extrapolated leakage-current line.
W-7 inhibits the duration of backward swimming in a concentration-dependent manner in Paramecium. About 40–50 cells were added to 2 ml of the Ca-K solution and left at room temperature (22 ± 1°C) for 15 min without drug. Individual cells were behaviourally tested by transferring them to 2 ml of 20mm-KCl in Dryl’s solution with drug and timing the duration of backward swimming as described by Haga et al. (1982). This solution depolarizes the cells due to the high external K+ concentration. With no drug in the test solution the duration of backward swimming was 56·0 ± 8·0 s (N = 8). This was reduced by 50 % by addition of about 20 μm W-7 or 150 μm W-5 to the test solution (Fig. 1). When the test solution contained 150 μm. W-7, the cells showed about 1 s backward swimming followed by forward swimming. Since W-7 was dissolved in ethanol the ethanol concentration was as high as 0·8 % in the final test solution. Addition of up to 1·5 % ethanol to the test solution without drug did not affect the duration of backward swimming. Note that at the concentration at which W-7 is 50% effective (20 μm), W-5 was ineffective (Fig. 1).
The behavioural effects of W-7 were rapidly reversible and were not time dependent. After a 5 min incubation of cells in the Ca-K solution with 20μm W-7, the cells were either tested or transferred to the Ca-K solution without drug. The suppression of backward swimming was dependent only upon the presence of the drug in the test solution, showing that the drug was rapidly effective and almost immediately reversible (Table 1). Exposure to 20 μm W-7 for up to 1 h did not change the responsiveness of the cells to 20μm W-7 or its reversibility (data not shown).
Effects of W-7 on membrane currents
The only voltage-dependent membrane electrophysiological property which was observed to change following perfusion with 20 μm W-7 was the transient inward current. The experimental protocol was as follows: (1) a cell was voltage clamped at −40 mV in the Ca-K solution and membrane currents recorded following various hyperpolarizing and depolarizing voltage steps, (2) the recording chamber was perfused with 20 μm W-7 in the same solution and the voltage steps repeated, and (3) the cell was returned by perfusion to the Ca-K solution without drug and the recordings repeated. Recording began as soon as the perfusions were completed. At 20 μm, W-7 had no effect on the voltage-dependent K-current or the hyperpolarization-dependent currents (both measured at 15 ms) but the transient depolarization-dependent inward current (largely the Ca-current, see below) was decreased by about 40% (Fig. 2). 20 μm W-5 had no effect on any of these currents.
The effects on the transient inward current were largely reversible. Perfusion of a cell with the Ca-K solution following recording in 20 μm W-7 caused the maximal inward current (Imw) to return to near the original level (Fig. 2, Table 2). A possible shift in the voltage at which the Imax was seen (Vmax) was suggested in the presence of 20 μm W-7 (Fig. 2, Table 2). This was studied more directly with the isolated Ca-current|
Recordings from cells with free running membrane potentials showed that there was also no change in the resting membrane potential, membrane resistance, or the height of the action potential triggered by injected current when cells were bathed in 20 μm W-7. The membrane potential was unaffected by concentrations of W-7 as high 150 μm (data not shown).
Isolation of the voltage-dependent Ca-current
The cnrA mutant, which is defective in its voltage-dependent Ca-channel (Takahashi & Naitoh, 1978), was used to determine the effectiveness of 2M-CSC1 electrodes and external TEA-Cl in blocking the voltage-dependent outward K-current. 150 μmW-7 was added to ensure that the outward K-current (measured at 15 ms) was not contaminated with any Ca2+ currents. This early outward K-current seen in the Ca-K solution (using 2 M-KCl electrodes) was unaffected by 150 μm W-7 but was eliminated by using 2M-CSC1 electrodes and 10 mm external TEA-Cl, revealing a small sustained inward current similar to that described by Eckert & Brehm (1979). Addition of 150 μm W-7, along with 2M-CSC1 electrodes and 10 mm external TEA-Cl, eliminated the sustained inward current as well (Fig. 4). Under these conditions it was determined that the voltage-dependent outward K-current was inhibited by more than 95 % at all voltages tested.
As with cnrA, wild type showed no outward current (other than the background leakage current) in the presence of 2M-CSC1 electrodes and 10 mm external TEA-Cl. The unmasked sustained inward current had an Imax of 1·4 ± 0·7 nA (N = 5) with a Vmax of about 10’0 mV. Addition of either 150 μm W-7 or 400 μm W-5 eliminated this sustained inward current as well (data not shown).
Concentration effects of W-7 and W-5
Both the Imax and the Vmax of the isolated voltage-dependent Ca2+ inward current changed in a concentration-dependent manner following perfusion with W-7. Addition of 150 μm W-7 decreased the Imax to about 10 % and shifted the Vmax of the remaining current by 10–15 mV less negative. Smaller changes were seen at 40 and 100fiM W-7 (Fig. 5). Ethanol alone, at the same concentration as was present with 150 μm W-7, did not produce such changes.
W-5 had the same electrophysiological effects as W-7 but a higher concentration was required. W-5 was ineffective at 40 μm, the concentration at which W-7 inhibited 40 % of the Ca-current (Fig. 6A, B). At 150 μm W-5 inhibited 40% of the current are W-7 inhibited >90% of that current (Fig. 6C, D). 150 μm W-5 also shifted the Vmax 5-10 mV less negative (data not shown).
The estimated concentration for 50 % inhibition of the isolated voltage-dependent Ca2+ inward current was about 50 μm for W-7 and about 200 μm for W-5 (see Fig. 7). The W-7 effects were about 90 % reversible up to 40 μm but at higher concentrations reversibility was not as reliable. In one case 80 % of the inward current recovered after treatment with 150 μm W-7 while in most cases the recovery was only 30% of the original Imax when 150 μm W-7 was used.
W-7 inhibits both the duration of backward swimming in response to 20 mm-K+ and the voltage-dependent inward Ca-current in a concentration-dependent and reversible manner in Paramecium. W-5 has the same effects as W-7 but higher concentrations of W-5 are needed. W-7 and W-5 are about 90% effective at concentrations of about 150 μm and 400 μm respectively, but at 20 μm, the concentration where W-7 is 50 % effective at inhibiting backward swimming and about 30 % effective at inhibiting the voltage-dependent Ca-current, W-5 is ineffective. Thus W-5 can serve as a control for non-specific effects of W-7 at 20 μm.
The electrophysiological effects of W-7 appear to be specific for the voltagedependent Ca-current. Both the hyperpolarization-induced currents and the voltagedependent K-current are unaffected by up to 150 μm W-7. At the effective concentration of W-7, 20 μm, there was no effect on either the time to peak or the relaxation of the transient inward Ca-current. The membrane potential, membrane resistance and height of the action potential also did not change in 20 μm W-7. Secondary effects on other Ca2+-dependent functions, such as the Ca2+-dependent K-current, are expected sequences of the reduced Ca2+ influx in the presence of W-7. This Ca2+-dependent K-current of Paramecium is also reduced in mutants with decreased inward Ca-current (Satow & Kung, 1980a), by EGTA iontophoresis (Saimi, Hinrichsen, Forte & Kung, 1983), and by the addition of an antibody which inhibits the inward Ca-current (Ramanathan et al. 1983). The reduced Ca2+-dependent K-current does not affect the voltage-dependent K-current since the two currents are temporally distinct (Satow & Kung, 1980b). We cannot rule out direct effects of W-7 on the Ca2+-dependent K-current, however, since it has been suggested that calmodulin antagonists can directly inhibit this current (Lackington & Orrego, 1981).
The voltage-dependent Ca-current was isolated for study by blocking the outward voltage-dependent K-current with internal CsCl (delivered by 2m-CSC1 electrodes) and external 10mm-TEA-Cl. This procedure was greater than 95% effective in blocking the outward currents without greatly affecting the inward Ca-current. The sustained inward current which was revealed under these conditions has been ascribed to the remainder of the Ca-current after the major inactivation and presumably uses the same channel as the transient inward Ca-current (Eckert & Brehm, 1979). Therefore the decrease in both the transient and sustained inward currents in the presence of W-7 may be due to inhibition of Ca-channel activity.
The behavioural and electrophysiological reversibility is seen at low concentrations of W-7 (see Tables 1 and 2) but the effects of higher concentrations are not as readily reversible. As seen in Fig. 3, the effects of 100 μm W-7 are not completely reversible. This may be due either to some irreversible damage at high drug concentration or to accumulation of intracellular W-7 (Kanamori, Naka, Asano & Hidaka, 1981). The anticalmodulin effects of W-7 could affect viability. W-7 binds to calmodulin (Hidaka et al. 1980) which is present in Paramecium (Walter & Schultz, 1981 ; Maihle et al. 1981; Rauh & Nelson, 1981) and serves important functions (Satir, Garofalo, Gilligan & Maihle, 1980; Garofalo, Gilligan & Satir, 1983; Rauh, Levin & Nelson, 1980). Although cells are viable in 20W-7 for hours, 150 μm W-7 kills cells in 5-10 min.
W-7 has many properties which could be involved in the mechanism of action of its Ca-current inhibition: (1) W-7 is a potent anticalmodulin drug (Hidaka, Naka & Yamaki, 1979; Hidaka, Yamaki, Totsuka & Asano, 1979; Hidaka et al. 1978, 1980, 1981b ; Hidaka Asano & Tanaka, 1981a; Kanamori et al. 1981 ; Niki, Niki & Hidaka, 1981; Tanaka et al. 1982) and calmodulin may be involved in membrane excitation (Carp, Aronstam, Witkop & Albuquerque, 1983; Takahashi, Ogura & Maruyama, 1983). (2) W-7 inhibits calmodulin-independent enzymes such as phospholipidsensitive protein kinase (Tanaka et al. 1982; Schatzman et al. 1983) and Na+,K+-ATPase and Mg2+-ATPase (Luthra, 1982). Such enzymes could be involved either directly or indirectly in Ca2+ channel regulation. (3) W-7 inhibits Ca2+ uptake by mitochondria (Hirata et al. 1982) and Ca2+ binding to membranes (Tanaka et al. 1982). This could affect internal Ca2+ concentration which could change the driving force for Ca2+ influx. Since EGTA injections did not change the W-7 effect in Paramecium (unpublished observation) we feel that these effects alone cannot account for the decreased Ca2+ inward current. (4) W-7 can partition into membrane lipids and possibly disrupt the physical properties of the excitable membrane. (5) W-7 is positively charged at neutral pH, a property which may affect membrane surface charge and surface potential (see Satow & Kung, 1981). (6) W-7 has an affinty forhydrophobicareasofproteins(Tanaka et al. 1982; Schatzmane et al. 1983). Since the Ca-channel must have hydrophobic areas, the drug may cause an allosteric inhibition. W-7 could also block the Ca-channel in a manner similar to the action of D600 and verapamil in other systems (Lee & Tsien, 1983). It is also possible that the Ca-channel of Paramecium is simply very sensitive to general membrane changes (Ramanathan et al. 1983). Thus the mechanism of action of W-7 remains an area for future study.
The inhibition of a voltage-dependent Ca-current has also been shown with another anticalmodulin drug, TI233, in pheochromocytoma cells (Takahashi et al. 1983). TI233 inhibited high K+-stimulated norepinephrine release due to a decreased Ca2+ influx. This is analogous to the decreased high K+-stimulated backward swimming and decreased Ca2+ current by W-7 in Paramecium. Although a calmodulin involvement has been suggested for TI233 action (Takahashi et al. 1983), Hidaka et al. (1981a)have suggested that the blockage of norepinephrine release in the thoracic aorta by W-7 is independent of calmodulin. A clear warning from this work and our work with W-7 is that if anticalmodulin drugs are used to study some cell function it must be remembered that anticalmodulin drugs might inhibit Ca2+ influx, a property which could affect Ca2+-dependent cellular processes.
We suggest that Paramecium can be used as a behavioural screen for other drugs which may inhibit Ca2+ inward current as well as providing a system for analysing the mechanism of action of such drugs.
We thank M. Takahashi for the kind gifts of the cnrA mutant and for critical reading of the manuscript. We also thank Y. Saimi for consultation, development of the use of caesium electrodes, and critical reading of the manuscript. This work was supported in parts by NIH 1-F32-NS-06950-01 to TMH and NSF BNS-7918554 and PHS GM-22714 to CK.