Planta

Planta 149, 213-218 (1980)

9 by Springer-Verlag 1980

Dependence of the Membrane Potential of Chara Cells on External pH in the Presence or Absence of Internal Adenosinetriphosphate Gouta Kawamural, Teruo Shimmen 2 * and Masashi Tazawa 2, 1 Department of Biophysical Engineering, Faculty of Engineering Science, and z Department of Biology, Faculty of Science, Osaka University, Toyonaka 560, Japan

Abstract. The dependence of the membrane potential (Era) and the membrane resistance (Rm) of Chara australis R. Brown on the pH of the external medium (pH0) was studied by controlling the activity of the plasmamembrane H + pump under both light and dark conditions. The activity of the pump was controlled by regulating the internal ATP or Mg 2+ concentration in tonoplast-free cells prepared by vacuolar perfusion. In these cells, which contained Mg.ATP (MgATP cells), Em and Rm were very sensitive to pHo, as in normal cells. Em was more negative in light than in the dark at all pH0 values tested. Tonoplast-free cells with very low [ATP]~ ( - A T P cells) or [Mg2+]i ( - M g cells) showed very weak dependence of Em and Rm on pHo. Thus, the active and not the passive component of Em w a s sensitive to pHo. At the same time, the high permeability of the plasma membrane to H + was questioned. In both - ATP cells and - Mg cells, Em was scarcely affected and R m markedly decreased on illumination. Key words: ATP and membrane potential - Chara Electrogenic pump Light and membrane potential Membrane potential- pH and membrane potential - Proton permeability.

Introduction The membrane potential (Em) of some organisms has two components: the so-called diffusion potential, and the potential which is linked to energy metabolism. The existence of the latter has been suggested * P r e s e n t address." Department of Botany, Faculty of Science, University of Tokyo, Hongo, Bunkyo-kn, Tokyo 113, Japan

CyDTA = 1,2-cyclohexanediamine-N,N'-tetraacetic acid; EGTA = ethyleneglycol-bis-(fl-aminoethylether)N,N'-tetraacetic acid; HK = hexokinase

Abbreviations :

on the basis of experiments in which metabolism was blocked with inhibitors or by low temperature. Slayman et al. (1973) showed that both Em and [ATP]i decreased concomitantly when Neurospora hyphae were treated with azide, CN or N2. Shimmen and Tazawa (1977) succeeded in controlling Em by directly regulating intracellular concentration of ATP ([ATP]I) or Mg 2+ ([Mg2+]i) in Chara cells whose tonoplasts had been removed. They showed that the Mg 2+-dependent ATPase was responsible for the maintenance of E m and therefore was assumed to be involved in the electrogenic ion pump. In Characean cells, the ion species extruded by the electrogenic pump is thought to be H + (Kitasato 1968). Slayman (1965, 1970) showed that the H + efflux of Neurospora hyphae decreased rapidly in parallel with rapid membrane depolarization during treatment with metabolic inhibitors. Tazawa and Shimmen (1977, 1980b) demonstrated the presence of the ATP-dependent H + efflux by directly controlling [ATP]i. In plant cells, Em is normally strongly dependent on the external pH (pHo), while it is only weakly dependent in cells with their metabolism blocked by inhibitors or low temperature (Saito and Senda 1973; Bentrup et al. 1973; Richards and Hope 1974). The membrane potential is more sensitive to pH o in light than in the dark (Saito and Senda 1973; Felle and Bentrup 1976). The membrane of green cells is generally hyperpolarized on illumination. It has been assumed that light activates the putative H + pump which acts electrogen'ically. A stronger dependence of Em on pt-Io in light than in the dark may be related to the activity of the electrogenic H + pump (Saito and Senda 1973; Spanswick 1972). However, inhibitors which lower the pH sensitivity of the membrane may affect not only the activity of the pump itself but also other physiological properties of the plasma membrane. In the present work, the pump activity was directly controlled by regulating [ATP]~ or [Mg2+]i and the

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214

G. Kawamura et al. : pH, ATP and Membrane Potential in Chara

dependence of E m orl pH o was investigated both under light and dark conditions. To control [ATP]i, the tonoplast of Chara cells was removed by replacing the cell sap with a medium containing ethyleneglycolbis-(fl-aminoethylether)N,N'-tetraacetic acid (EGTA) or 1,2-cyclohexanediamine-N,N'-tetraacetic acid (CyDTA), as reported previously (Tazawa et al. 1976; Shimmen and Tazawa 1977). The pH sensitivity of E m w a s found to be dependent on Mg. ATP.

Each medium contained 0.1 mM each of KC1, NaC1 and CaC12. Unbuffered artificial pond water which contained 0.1 mM each of KC1, NaCI and CaC12 and had a pH of about 5.6, was also used. The membrane potential was measured by the ordinary microelectrode method as described in Shimmen et al. (1976). The cell was illuminated with a microscope incandescent lamp (TB-1 ; Olympus, Tokyo, Japan). The fluence rate at the cell was about 30 W m 2. Measurements were done at room temperature (20-25 ~C).

Results Material and Methods The alga Chara australis R. Brown wascultured outdoors in 40-1 pots containing tap water and soil at the bottom. Adjacent internodes were isolated and stored for at least one night in a Petri dish (diameter, 15 cm) filled with pond water. Before the experiment, the vacuole was stained with neutral red. Replacement of the cell sap with an artificial medium was done by cutting both cell ends and perfusing the vacuole with the medium, using a pressure difference between both cell ends (Tazawa 1964). After confirmation that the red-colored cell sap was pushed away entirely from the vacuole, both opened cell ends were closed by ligation with strips of polyester sewing thread (Tetron No. 50; Gunze Company, Osaka). To remove the tonoplast, the cell sap was replaced with a medium containing a Ca 2+ chelator, EGTA or CyDTA (Tazawa et al. 1976; Shimmen and Tazawa 1977). Three kinds of internal perfusion media were used: Mg.ATP medium, HK (hexokinase) medium and CyDTA medium (Table 1). After loss of the tonoplast, the [ATP]I of cells perfused with the HK medium was about 1 pM (Kikuyama et al. 1979). The [Mg2+]i of cells perfused with the CyDTA medium should have been very low after loss of the tonoplast, because CyDTA strongly binds cytoplasmic Mg 2+ (Shimmen and Tazawa 1977). For convenience, celis perfused with Mg.ATP, HK and CyDTA media are designated, in this order, as MgATP, - A T P and - M g cells. The pH of the external medium was buffered with 1 mM DMGA (fl,fl-dimethylglutaric acid; pH 4.4, 5.4), 1 mM MES (2(N-morpholino)ethanesulfonic acid ; pH 6.4), 2 mM HEPES (N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH7.4) and 2 mM Tricine (N-tris(hydroxymethyl)methylglyciue; pH 8.4). The pH was adjusted to each value with NaOH. The final Na + concentrations of the media were adjusted to 1.5 mM by adding NazSO4.

Normal Cells. Figure 1 shows the dependence of Em and Rm of normal cells on pH o under light and dark conditions. For the dark treatments, the cells were stored for 2 or 3 h in the dark in the artificial pond water before use. Irrespective of the light-dark conditions, E m changed greatly in the positive direction with the change in pH 0 from 4.4 to 7.4, and slightly in the positive direction with the change from 7.4 to 8.4. This type of pH 0 dependence of Em has been reported for other species of Characeae, namely Nitella clavata (Kitasato 1968), N. axilliformis (Saito and Senda 1973) and Chara corallina (Richards and Hope 1974). Membrane resistance increased with an increase in pH 0 both under light and dark conditions. Both Em and R m in light were approximately the same as those in the dark, indicating that in normal cells light hardly affected the response of the membrane tO pHo. This finding differs from results obtained in other species in which the pH 0 dependence of Em is, to a greater or lesser extent, modified upon illumination (Saito and Senda 1973; Richards and Hope 1974), and a light-induced potential change has even been observed in Chara australis (Tazawa et al. 1979). Normal cells 250

~

Em

200

Table 1. Composition of internal perfusion media (mM) 150

Medium MgATP

HK

CyDTA

E

4

i,iE 100 I

ATP Hexokinase Glucose MgC12 EGTA CyDTA PIPES Sorbitol K Na pH

1 0 0 6 5 0 30 200 66 2 7.0

0 1 mg/ml 5 6 5 0 30 200 67 0 7.0

1 0 0 0 0 5 30 215 64 4 7.0

3

2

50

~ v

0

I

I

I

I

I

9

8

7

6

5

4

0

pHo

Fig. 1. Dependence of Em and R m of normal Chara australis cells upon external pH (pH0) under light (L) and dark (D) conditions. Values are averages of 5-7 cells with _+standard error of the mean (SE)

G. K a w a m u r a et al. : pH, A T P and M e m b r a n e Potential in Chara

215

- A T P cells

M g A T P cells 250

150

200

~1oo

Em v

E

E h, 5 0

150

Rm ~"~,~

I

El00

4

1

x~ 3

~ g sIDST~''''l} 4~

0

A

2

50

-~

2

v

0

I

I

I

l

I

9

8

7

6

5

i

4

0

pHo

Fig. 2. Dependence of E m and Rra of M g A T P cells on external pH (pHo) under light (L) and dark (D) conditions. M g A T P cells were prepared by internal perfusion with M g A T P m e d i u m (Table 1). Values are averages of 5 9 cells with + SE

We have used Chara australis for several years and found that the cells sometimes do show distinct lightinduced potential changes but none at other times. These changes in the response of the organism to light may be the consequence of seasonal variation of the material.

MgATP Cells. When the cell sap was replaced with the M g . A T P medium, the tonoplast disintegrated within 0.5 h. Active cytoplasmic streaming continues however even after loss of the tonoplast (Tazawa et al. 1976). In the dark experiments, cells after internal perfusion were kept in artificial pond water for 2-3 h in the dark, as had been done with normal cells. In the light experiments, electric measurements were done soon after disintegration of the tonoplast. Figure 2 shows the dependence of Em and R m on pH o under both light and dark conditions. The Em was more negative under light than under dark at all pH0's tested. The E m of tonoplast-free cells during incubation in the dark tended to shift gradually in the depolarizing direction. When such cells were illuminated, E m shifted in the more negative direction as had already been reported by Tazawa et al. (1979). Figure 2 clearly shows that in light, Em was strongly dependent on pH o. The dependence on pH o was as high as that of normal cells. In the dark, the Em response to changes of pH o was smaller than that in light. Membrane resistance in the dark was larger than that in light. It increased with an increase in pH o in both light and dark. - A TP Cells and - M g Cells. When the cell sap was replaced with the H K medium, the tonoplast disappeared within 0.5 h as with the M g . A T P medium

i

I

i

~

i

I

9

8

7

6

5

4

0

PHo Fig. 3. Dependence of Era and R m of - A T P cells of Chara australis on external pH (pHo) under light (L) and dark (D) conditions - A T P cells were prepared by internal perfusion with H K medium (Table 1). Values are averages of 5-7 cells with + SE

and the cytoplasmic streaming soon came to a halt. Cessation of the cytoplasmic streaming is a good indication of the depletion of internal ATP since the streaming is fueled by ATP (Williamson 1975; Tazawa et al. 1976; Shimmen 1978). The E m w a s measured after cessation of the cytoplasmic streaming. The E m of - A T P cells was less negative (Fig. 3) than that of M g A T P cells (Fig. 2), a fact indicating that the potential sustained by the electrogenic pump had disappeared (Shimmen and Tazawa 1977). The response of Em to pH o in - A T P ceils was obviously smaller than that of normal cells or MgATP cells (Figs. 1, 2). The E m in light was slightly more negative than that in the dark at all pH 0 except 4.4. Rm was larger in the dark than in the light, and was insensitive to pH o under both light and dark conditions (Fig. 3). Under both light and dark conditions, the Rm of - ATP cells was markedly larger than that of MgATP cells. Since not only ATP but also Mg 2+ is essential for the electrogenic pump in Chara australis (Shimmen and Tazawa 1977), removal of Mg 2+ from the cell interior even in the presence of enough ATP should stop the pump. Thus, Mg 2 +-depleted cells are expected to behave like ATP-depleted cells in response to pHo. Figure 4 shows the dependence of E m and Rm on pH o under both light and dark conditions when the cell sap was replaced with the C y D T A medium. The E m of - - M g cells was depolarized to a level which was nearly equal to that of - A T P cells (Fig. 3). This indicates that Em sustained by the electrogenic pump disappeared upon removal of Mg 2+. As in - A T P cells, both E m and R m were almost insensitive to pHo under both light and dark

G. Kawamura et al. : pH, ATP and Membrane Potential in Chara

216

-Mg cells

OUT

150 IO0

l!

Em R ~ i J ~ - - m ~ . ~ L =

E

~5o t13 I

D Rm

llgD

IO

"'+-+'+--5

5

L I

I

I

I

I

9

8

7

6

5

i 4

~

0

PHo Fig. 4. Dependence of Em and Rm of - M g cells of Chara australis on external pH (pHo) under light (L) and dark (D) conditions. - M g cells were prepared by internal perfusion with CyDTA medium (Table 1). Values are averages of 6-10 cells with + SE

conditions; R m was markedly larger than that of M g A T P cells; Rm in the dark was larger than that in the light at all pHo's.

Discussion

Relationship between pHo Sensitivity of the Membrane and the Activity of the Electrogenic Pump. Our experiments demonstrate that both m e m b r a n e potential and m e m b r a n e resistance of Chara cells become insensitive to pH0 by depletion of A T P or Mg 2+ in the cell interior. Since this depletion stops the electrogenic p u m p (Shimmen and Tazawa 1977), it is reasonable to assume that the observed loss of p H 0 sensitivity of E m and Rm is closely related to inactivation of the electrogenic pump. In other words, the component of Em which is sensitive to pHo is not the passive but the active one, supported by M g . ATP. Saito and Senda (1973) have also maintained that the metabolically sensitive component of Em is very sensitive to pHo. Electrogenic Pump in Relation to the Electrical-Equivalent Circuit Model. To analyze our results, the electrical-equivalent circuit model of the plasma m e m b r a n e shown in Fig. 5 was considered. The symbols ED, Ee, [e, gD and gp represent, in this order, the diffusion potential, the potential sustained by the pump, the p u m p current, and conductances of the passive iondiffusion channels and the pump. The positive and negative currents represent outward and inward currents, respectively. In Fig. 5, E m and gm (the m e m b r a n e conductance) can be expressed as:

IN Fig. 5. Equivalent circuit model of the Chara membrane. Ep, electromotive force generated by the electrogenic pump; ED, passive diffusion potential; gp, conductance of the pump channel; gD, conductance of the diffusion channel gm=gD+gp,

(1)

E m= gg~D m ED + gram ge Ep.

(2)

Under the assumption that ED and gD of M g A T P cells are equal to Em and gm of - - A T P cells (Fig. 3), ge and Ep under light conditions can be calculated using eqns. (1) and (2), and are shown in Table 2. In the p H o range between 6.4 and 8.4 gp is about 1/2 to 1/3 of gD- In C. corallina Keifer and Spanswick (1978) found that both Em and gm decreased greatly on application of the m e m b r a n e ATPase inhibitors N,N'-dichlorohexylcarbodiimide and diethylstilbestrol. When Em attained the most depolarized level, gm was smaller than 1/10 of the normal value. Thus, gp is assumed to be more than ten times as large as gD. This is in striking contrast to the result of our analysis on C. austral#. The difference m a y be caused either by the difference of materials or by the means of stopping the electrogenic pump. The electromotive force Eo of the H + p u m p can be related to the energy of hydrolysis of A T P in the following manner (Mitchell 1966) : Ep=

n

l o g NAT P

[ATPII + 2.3pRTlog [AD~P~]i [Pi]j! + E. =

(El + E2) + E .

(3)

where n is the number of H § transported in consequence of the hydrolysis of one molecule of A T P ; KATe is the equilibrium constant of A T P hydrolysis;

G. Kawamura et al. : pH, ATP and Membrane Potential in Chara

217

Table 2. Diffusion conductance (gD), pump conductance (gp) and pump electromotive force (Ep)

brahe of Chara is not as permeable to H + as postulated by Kitasato (1968) since the apparent strong dependence of E m o n pH 0 is attributed mostly to the part of E m supported by Mg-ATP. Under the assumption that E m of - A T P cells or - Mg cells is based solely on the diffusion potential (ED), relative ionic permeabilities can be calculated by the well-known Goldman equation:

pHo

gm (S/m 2)

gD (S/m a)

gt' (S/m 2)

E~ (mV)

ED (mV)

Ep (mV)

4.4 5.4 6.4 7.4 8.4

0.91 0.53 0.43 0.42 0.36

0,31 0,29 0.32 0.28 0,28

0.60 0.24 0.11 0.14 0,08

-88 -153 -195 -217 -230

-58 -80 -100 -95 -92

-102 -240 -472 -462 -711

gD and ED are equal to g~ and E m of - A T P and Ev were calculated by eqns. (1) and (2)

cells (Fig. 3). gv

E H is the equilibrium potential of H + across the plasma membrane, and F, R and T have the usual meanings. Since the standard free energy change of ATP hydrolysis ( - R T log KATP) is --35.73 kJ/mol (Slayman et al. 1973), E1 is calculated to be - 3 7 0 inV. Since we have no data on how [ATP]I changes with time after disintegration of the tonoplast, we can only estimate the value of E2 by assuming a proper rate of ATP hydrolysis. When 10% of 1 mM of ATP is hydrolyzed to form ADP and Pi, E 2 is calculated to be - 2 8 7 mV. For 90% hydrolysis E2 is calculated to be - 1 2 0 mV. Since pHi was measured to be 6.9 (Fujii etal, 1979), E H at pH o 6.4 or 7.4 is 30 or - 30 mV. Then,

1

Ep= n [ - 3 7 0 - (280~ 120)]+ 30 mV _- 1 [(_ 680 ~ - 620) ~ ( - 520 ~ - 490)] mV. n

(4)

Since Ev calculated from the experimental data is at pH o 6.4 or 7.4 around - 5 0 0 mV (Table 2), it is reasonable to assume unity for n. Gradmann et al. (1978) proposed 1H+/1ATP for the Neurospora H + pump. There is however a reservation in assuming that go of + A T P cells is equal to gm of - A T P cells. According to eqn. (3) Ev should increase with the slope of 58 mV for the change of one pH unit. However, change in Ev for one pH unit is larger than 58 mV, especially in the pH range below 6.4 (Table 2). To have the same slope of Ev against pH 0 as that of EH, gD at lower pH o should be larger than the value in Table 2. It is then suggested that gi) of + A T P cells is not always equal to gm of --ATP cells. There is evidence supporting this. When ATP or Mg z§ was removed from the cell inside, Em depolarized immediately, while gm sometimes decreased gradually with time (Tazawa and Shimmen 1980a). Passive Membrane Potential and Ionic Permeabilities. Our results strongly indicate that the plasma mem-

, [K +]o +~[Na +1o + 13[H+]0 + y[C1-]i Et) = 58 log~K +]j + cr +l i +/~[H +]~+ y[C1-]o

(5)

where ~, t3 and 7 represent relative permeabilities of Na + (PNa/PK), H + (Pn/PK) and C1- (Pc]PK) with respect to that of K +. In both - A T P and - M g cells [K+]o, [Na+]o and [H+]i are 0.1, 1.5 and 1 0 - 4 m M , respectively. [K+], [Na +] and [C1 ] in the cytoplasm of Chara australis are 112, 3 and 21 mM, respectively (Tazawa et al. 1974), and those in the HK and CyDTA medium are 67, 0 and 12 mM, and 64, 4 mM and 0 m M , respectively (Table 1). Assuming that these ions are dispersed homogenously in the cell after disintegration of the tonoplast, and also assuming that the volume of the cytoplasm is 1/10 the whole cell volume, [K+]i, [Na+]i, and [Cl-]i of - A T P and - M g cells after disintegration of the tonoplast are calculated to be 72, 0.3 and 12.9 mM, and 69, 4 and 2.1 mM, respectively. Since 7 is in the order of 10-3 (Lannoye et al. 1970; Richards and Hope 1974), the term for C1- in eqn. (5) can be omitted. In - A T P and - M g cells, E m at pH o 6.4~8.4 was - 9 5 ~ - - l 1 0 m V . In this pH 0 range, the terms for H + in eqn. (5) can be neglected. In order to fit the observed E m values, ct for - A T P and - M g cells was calculated to be 1 and 0.5, respectively. At pH o 4.4, 5.4 and 6.4, Em in light of - A T P cells was - 5 8 , - 8 0 and - 1 0 0 mV, and that of - M g cells was - 9 0 , - 9 7 and - 1 0 5 mV, respectively. From the difference of E,, between pHo 4.4 and 5.4, 13 is calculated to be 110 for - A T P cells and 40 for - M g cells. In the same way 13 calculated from the difference of E,~ between 5.4 and 6.4 is 420 for - A T P cells and 90 for - M g cells. Thus the ratio of PH/P~ is significantly smaller than that (10,000) estimated by Kitasato (1968), who postulated that the dependence of Em on pH o of normal cells having electrogenic activity is not different from that of ED [(Em) 0 in his paper]. In Chara corallina, Richards and Hope (1974) also estimated PH/PK to be 25. The authors wish to express their grateful acknowledgement to Professors N. Kamiya (National Institute for Basic Biology, Okazaki, Japan) and F. Oosawa (Osaka University) for their interest and discussions, This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

218

G. Kawamura et al. : pH, ATP and Membrane Potential in Chara

References

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Bentrup, F.W., Gratz, H.J., Unbehauen, H. (1973) The membrane potential of Vallisneria leaf cells; evidence for light-induced proton permeability changes. In: Ion transport in plants, p. 171-182, Anderson, W.P., ed. Academic Press, London New York Felle, H., Bentrup, F.W. (1976) Effect of light upon membrane potential, conductance and ion fluxes in Riccia fluitans. J. Membr. Biol. 27, 153-170 Fujii, S., Shimmen, T., Tazawa, M. (1979) Effect of intracellular pH on the light-induced potential change and electrogenic activity in tonoplast-free cells of Chara australis. Plant Cell Physiol. 20, 1315 1328 Gradmann, D., Hansen, U., Long, W.S., Slayman, C.L., Warncke, J. (1978) Current-voltage relationships for the plasma membrane and its principal electrogenic pump in Neurospora crassa. 1. Steady-state conditions. J. Membr. Biol. 39, 333-367 Keifer, D.W., Spanswick, R.M. (1978) Activity of the electrogenic pump in Chara corallina as inferred from measurements of membrane potential, conductance and potassium permeability. Plant Physiol. 62, 653-661 Kikuyama, M., Hayama, T., Fujii, S., Tazawa, M. (1979) Relationship between light-induced potential change and internal ATP concentration in tonoplast-free Chara cells. Plant Cell Physiol. 20, 993 1002 Kitasato, H. (1968) The influence ofH + on the membrane potential and ion "fluxes of Nitella. J. Gem Physiol. 52, 60 87 Lannoye, R.J., Tarr, S.E., Dainty, J. (1970) The effects of pH on the ionic and electrical properties of the internodal cells of Chara austral&. J. Expl Bot. 21,' 543 551 Mitchell, P. (1966) Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biol. Rev. 41,445 502 Richards, J.L., Hope, A.B. (1974) The role of protons in determining membrane electrical characteristics in Chara corallina. J. Membr. Biol. 16, 121-144 Saito, K., Senda, M. (1973) The effect of external pH on the membrane potential of Nitella and its linkage to metabolism. Plant Cell Physiol. 14, 1045-1052 Shimmen, T. (1978) Dependency of cytoplasmic streaming on intracellular ATP and Mg 2+ concentrations. Cell Struct. Funct. 3, 113-121 Shimmen, T., Kikuyama, M., Tazawa, M. (1976) Demonstration

Received 3 August 1979; accepted 27 May 1980