Potassium Channels at Chara Plasmalemma

Journal of Experimental Botany, Vol. 36, No. 163, pp. 228-239, February 1985

Potassium Channels at Chara Plasmalemma M. J. BEILBY Botany School, Downing Street, Cambridge CB2 3EA, U.K.

ABSTRACT Exposure to high K + medium transforms Chara plasmalemma into [K + ] o —sensitive state (K + state). The current-voltage (I/V) characteristics under such conditions display a negative conductance region. This feature results from the complex time and voltage dependence of K + channel opening At potentials more negative than a threshold p.d. the channels are closed and the I/V characteristics become linear with a low slope conductance of ~ 0 8 S m 2 and only a weak dependence on [ K + ] o . Such behaviour is usually associated with a non-specific leak current The threshold level for K + channel closing depends on [ K + ] o . In 2-0 mol m~ 3 and 5-0 mol irT 3 K + medium the membrane resting p.d. follows £ K , but hyperpolarizes gradually if the [ K + ] o is lowered. The proton pump thus appears to be non-operative, while the cell is in the K.+ state, and recovers slowly as the cell is returned to a low K.+ medium. Excitation currents decline if the cells are kept in K + state for some hours. Key words: K + channels; Chara corallina, Proton pump; Current/voltage characteristics; Conductance.

INTRODUCTION There is growing evidence for the existence of gated potassium channels in the plasmalemma of the giant celled algae (Smith and Walker, 1981; Sokolik and Yurin, 1981; Keifer and Lucas, 1982; Findlay and Coleman, 1983; Findlay, Tyerman, and Paterson, 1984; Bisson, 1984). In Chara corallina the study of the dynamics of these channels is made difficult by the presence of parallel conductance processes, such as the proton pump, excitation channels and the leak pathway. In the hyperpolarized state the proton pump seems to dominate the membrane potential and conductance, but a [K+]0—sensitive state (referred to as the 'K + state') can be brought about by the removal of Ca 2+ from the outside medium (Hope and Walker, 1961; Spanswick, Stolarek, and Williams, 1967; Keifer and Lucas, 1982), an increase of the K + concentration (Spanswick, 1972; Keifer and Lucas, 1982) or a depolarization of the membrane by an action potential, the use of inhibitors or the removal of ATP (Oda, 1962; Keifer and Spanswick, 1978,1979; Smith and Walker, 1981).TheK+ state is characterized by the resting p.d. responding to changes of [K + ] o in a Nernstian fashion and by a high resting conductance. It is still not clear, however, what proportion of the K + state is controlled by passive membrane permeability (due to K + , Na + , Cl ~and perhaps H + ) and which by the K + specific channels. The status of the proton pump, while the cell is in the K + state, is also not clear. These problems are addressed in the following study. MATERIALS AND METHODS The apparatus and experimental methods have been described extensively on previous occasions (Beilby and Beilby, 1983; Beilby, 1984). Young leaf cells of Chara corallina were cut off the plants and stored in high Na + APW (KC1,01 mol m" 3 , NaCl, 2-0 or 5-0 mol m " 3; CaCl 2 ,0-5 mol m" 3; HEPES,

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Received 1 August 1984

Beilby—Potassium Channels in Chara

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Potential (mV) -200 -100 -300 Fio. 1. // V (A) and G/ V (B) characteristics of the plasmalemma pretreated for ~ 24 h in high Na + APW (20 mol m" 3 or 50 mol m~\ The horizontal barsrepresentdata grouping into intervals of 15 mV, the vertical bars are the standard error for five cells. -400

1-0 mol m~3; NaOH to adjust to pH 7-5, ~0-47 mol m 3) for several days. This pre-treatment was adopted to keep the total cation concentration in the outside medium constant and to avoid possible changes in the cell wall potential (Hope and Walker, 1961). Also [ C r ] 0 remained constant, so that the driving force for excitation was unchanged as K + was substituted for Na + . The // V curves were obtained by the bipolar staircase method (Beilby and Beilby, 1983). However, in high K + APW where the resting potential declined close to the excitation threshold, the advantage of balancing the hyperpolarizing and depolarizing steps was lost (Fig. 7) and the possibility of transport number effects (Barry and Hope, 1969) must be considered. Pulsewidths up to 80 ms were used for the high K+ APW data gathering. Although excitability declined in the K + state (see results), it still limited the voltage interval for the I/V data to levels more negative than 50 mV. Conductance, G, was obtained either by differentiation of the I/V curve or by the sine perturbation method (Beilby and Beilby, 1983).

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FIG. 2. Stability of the K state in a typical cdl: exposure to 50 mol m ~ K. APW for (o) 45 min, (•) 3 h, (A) overnight

To investigate the effect of [K + ] o on the membrane properties the medium was adjusted to 01 mol m " 3 NaCI and 2-0 mol m ~ 3 or 50 mol m~3 K.C1. In some experiments the cells were pretreated in high K + APW overnight. Theflowof APW through the chamber was fast, especially while [K + ] o was being changed. Similarly to observations of Keifer and Lucas (1982), initial exposure to high Na + APW caused only small depolarizations of the resting p.d. (~30 mV), but a recovery after an excitation often took up to 30 min. These effects were transient and disappeared after ~24 h in high Na + APW. The I/V curves displayed a sigmoid shape and the conductance profile showed a maximum typical of the pump state obtained in normal APW (as above, but NaCI = 1-0 mol m~3)—see Fig. 1 and compare it to Fig. 4 of Beilby (1984). The high NaCI concentration, therefore, did not seem to interfere with the proton pump and the cells remained in the pump state. In most experiments the cells were illuminated by fibre optics source Intralux HI50 Volpi, light intensity of 50-100 /iE m~2 s" 1 (10-20 W m~2, when the light frequency was approximated as the middle of the visible spectrum). To study the free running action potential the membrane was clamped to some potential more depolarized than the excitation threshold and the clamp was manually switched off upon rise of the excitable current. The potential-measuring electrode was kept in the cytoplasm in all experiments. The electrical properties of the plasmalemma were exploited to establish the position of the electrode (Beilby, 1984). RESULTS Generation, stability and I/V characteristics of the K+ state In 2 0 mol m~ 3 K + APW the K + state established itself only after several hours of exposure and/or numerous depolarizations through voltage clamping. In 50 mol m ~ 3 K + APW the exposure time necessary was shorter and a single clamp to potentials more depolarized than the excitation threshold was sufficient to bring the cell into K + state. When the K + state was reached and the cell remained in high K + APW, the I/V characteristics were fairly stable (Fig. 2).

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FIG. 3. A typical response of the resting p.d. in the K + state to changes in [K + 1, (A) in light, initial concentration of 2O mol ra ~ 3 K + , (B) in dim light and initial concentration o f 5 O m o l m ~ 3 K + .(c) The IIVcharacteristics recorded at points indicated in (B) by dots, (o) 50 mol m~ 3 K + , (A) 2 0 mol m " 3 K + , (a) 01 mol m~ 3 K + .

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FIG. 4. A typical // V (A) and G/ K (B) characteristics of the plasmalemma before (o, ) and 2 h after K i state (A,——). The K + state was obtained by an exposure to 2-0 mol m " 3 K + APW.

Return to 0 1 mol m 3 K + APW, while in K + state and lit conditions, resulted in a fast increase followed by a gradual repolarization of the resting p.d. (Fig. 3A), but even after several hours the cells did not regain the full resting p.d. of the previous pump state (Fig. 4A). The conductance profile also did not recover (Fig. 4B). The slow component of the p.d. change was largely inhibited in dim light. This behaviour was convenient for the measurement of the I/Vcharacteristics (Fig. 3B, C). A summary of results at three different [K + ] o is shown in Fig. 5. All the data recorded in high K + APW displayed a negative conductance region somewhat similar to that associated with excitation process (Kishimoto, 1972), but at more hyperpolarized potentials.

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