Raising the intracellular level of inositol 1, 4, 5-trisphosphate changes ...

The EMBO Journal vol.9 no.6 pp. 1 737 - 1741, 1990

Raising the intracellular level of inositol 1,4,5-trisphosphate changes plasma membrane ion transport in characean algae G.Thiel, E.A.C.MacRobbie and D.E.Hanke

1989) has been observed. However, a direct link between

Botany School, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK Communicated by D H. Northcote

increase in the intracellular level of InsP3 and the regulation of membrane transport has yet to be demonstrated. We have examined a plausible role for InsP3 in controlling membrane properties in characean algae. We show that a

Inositol 1,4,5-trisphosphate (InsP3) was introduced into the cytoplasm of characean algae in two different ways: (i) by iontophoretic injection into cytoplasm-enriched fragments from Chara and (ii) by adding InsP3 to the permeabilization medium of locally permeabilized cells of Nitella. In both systems this operation induced a depolarization of the membrane potential, ranging from a few mV to sequences of action potentials. The effect of InsP3 on locally permeabilized Nitella cells was abolished when InsP3 was added together with 30 mM EGTA. When inositol 1,4-bisphosphate or myo-inositol were substituted for InsP3 in this system, there was no change in the membrane potential. On the other hand, increasing the free Ca2+ concentration in the permeabilization medium induced, in a similar fashion to InsP3, action potentials. Similarities between InsP3 and Ca2+ action were also observed upon injection into Chara fragments. Both injections increased an inward current. In the first few seconds after injection the current/voltage characteristics of the InsP3-induced current resembled those of the Ca2+-sensitive current. Subsequently, differences between the InsP3- and Ca2'-induced phenomena became apparent in that the InsP3-induced current continued to increase while the Ca2+-induced current declined, returning to the resting level. Our results suggest that these plant cells contain an InsP3 sensitive system that, under experimental conditions, is able to affect membrane transport via an increase in cytoplasmic free Ca2+. Key words: Ca + / characean algae / InsP3 / membrane transport

Introduction Inositol 1,4,5-trisphosphate (InsP3) mobilizes Ca2+ from internal stores in animal cells (Berridge, 1987). The resultant elevated cytoplasmic calcium activity affects the properties of transport systems across the plasma membrane (Oron et al., 1985; Berridge, 1988). In plant cells the level of free calcium in the cytosol regulates many membrane transport properties (Shiina and Tazawa, 1987; Thaler et al., 1989; Schroder and Hagiwara, 1989) and there is indirect evidence that an exchange with calcium from internal stores also contributes to the regulation of the cytoplasmic calcium activity (Beilby, 1984). For plants an InsP3-induced release of Ca + from isolated vacuoles (Ranjeva et al., 1988), tonoplast vesicles (Schumaker and Sze, 1987) and microsomes (Dr0bak and Ferguson, 1985; Canut et al., Oxford University Press

an

rise in the cytoplasmic InsP3 concentration in Chara and Nitella cells mimics the effects of an increase in cytoplasmic free Ca2+ concentration.

Results myo-Inositol 1,4,5-trisphosphate (InsP3) was introduced into the cytoplasm of characean cells in two different ways: (i) by iontophoresis into the cytoplasm of Chara corallina (Beilby and Blatt, 1986) or (ii) by addition of InsP3 to the permeabilized (Shimmen and Tazawa, 1983) end of a locally permeabilized (Figure 2) internodal cell of Nitella translucens. In both systems these operations gave rise to distinct patterns of depolarization. lontophoretic injection of InsP3 into cytoplasm-enriched fragments of Chara or into the cytoplasm of Chara leaf cells caused either a small transient depolarization (Em going positive) of 13 :i1 6.4 mV (12 cells) with a slow time constant ( - 2-10 min) or a single transient depolarization with a pronounced peak of a few seconds duration, followed by a slow repolarization or a sequence of such events. The time course of the latter change resembled that of an action potential and, like the electrically elicited action potential, transiently halted cytoplasmic streaming. Action potentials were observed in a total of 10 leaf cells and seven cytoplasmenriched fragments. The response to injection of comparable pulses of InsP3 varied considerably in both leaf cells and cytoplasm-enriched fragments. An InsP3 pulse of -500 nA for 60 s into fragments of a similar size could result in a sequence of action potentials in one fragment while a similar fragment exhibited only a small depolarization (Figure lA). However, for the same cell, increasing the total charge (and hence the amount of InsP3) injected induced an increasing response of the membrane potential (Figure 1B). InsP3 (100,uM) was added to the permeabilization medium (cis) of a Nitella cell made permeable over a localized area of the plasma membrane. In the majority of cells visibly permeable to the fluorescent dye Lucifer Yellow, InsP3 induced either a depolarization (AEn, = 21 L 12.7 mV, seven cells, data not shown) or action potential(s) across the non-permeabilized membrane (trans) (14 cells). One permeable cell showed no obvious change in the membrane potential following addition of InsP3 to the cis side. Figure 2A shows that the delay after the addition of InsP3 and

before the voltage response was of the same order as the lag before the first appearance of fluorescent dye in the nonpermeabilized section of the internodal cell (Figure 2A). Cells not permeable to Lucifer Yellow remained unaffected by InsP3 (data not shown). The following control experiments gave results which 1737

G.Thiel, E.A.C.MacRobbie and D.E.Hanke

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Fig. 1. Membrane potential of different cytoplasm-enriched fragments (A) and of single leaf cells of the giant alga C.corallina (B) following iontophoretic injection of InsP3. InsP3 was injected at times indicated by bars: 1: -30 nA for 15 s; 2: -50 nA for 15 s; 3: -100 nA for 15 s; 4, 5 and 6: -500 nA for 60 s. Injections 2, 4,5 and 6 were carried out in conditions of free-running membrane potential. During the injections 1 and 3, fragments were voltage-clamped at the resting potential. Numbers on traces denote recorded voltage (mV). Calibration bars: vertical, 50 mV; horizontal, 60 s. The diagrams (not to scale) indicate the positioning of the potential measuring electrode (Ep) and injection electrode (El) in the vacuole (v) and the cytoplasm (c) respectively.

indicate that the membrane depolarizations and action potentials are a specific consequence of an increase in the activity of InsP3 in the cytoplasm. Neither repeated injection of KCl only into cytoplasm-enriched fragments, nor injection of InsP3 into the vacuole of Chara leaf cells had an effect on the membrane potential (data not shown). Similarly, neither perfusing the permeabilized end of a Nitella cell with medium containing 100 1sM myo-inositol (four cells), nor 100 isM myo-inositol 1,4-bisphosphate (five cells) had a detectable effect on the membrane voltage (data not shown). Increasing the free Ca2+ concentration in the permeabilization medium of Nitella cells by addition of Ca(NO3)2 caused, in a similar fashion to InsP3, action potentials. A representative response from 10 cells is shown in Figure 2B. Addition of Mg(NO3)2, on the other hand, was without an effect on the membrane voltage (Figure 2B). InsP3 (100 IAM) was added to the permeabilization medium of a Nitella cell (permeable to Lucifer Yellow) together with a high concentration of EGTA (30 mM). This operation had no effect on the membrane potential (four cells, data not shown). The injection of Ca2' into Chara fragments caused no detectable membrane depolarization, but current/voltage

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Fig. 2. Membrane voltage of locally perneabilized Nitella translucens internodal cells following an increase in the concentration of InsP3 (A) and Ca2+ (B) in the permeabilized end of the cell, (A) After a permeabilized cell had regenerated a negative potential and vigorous cytoplasmic streaming (35 14m/s), the permeabilized end (cis) was perfused with permeabilization medium containing 1 mM Lucifer Yellow (at the time indicated by shaded area) and the movement of the dye observed by fluorescence microsocpy. With a delay of 260 s (solid arrow) after addition of the dye, yellow fluorescence was observed to enter the trans region. After Lucifer Yellow was withdrawn from the medium, InsP3 was added to give a final concentration of 100 1M. (B) After a permeabilized cell had attained a stable membrane voltage and vigorous cytoplasmic streaming (38 /sm/s) Ca(NO3)2 solution was added to the cis side to increase the Ca2+ concentration to 2 mM. After withdrawing Ca(NO3)2 from the medium, Mg(NO3)2 (2 mM) was added. The modifications of the cis medium are indicated in the bars. The numbers on the traces denote the recorded voltage (mV). Calibration lines: vertical, 100 mV; horizontal, 240 s. The diagram (not to scale) indicates the positioning of the potential measuring electrode (Ep) and reference electrode (Er). The locations of the cytoplasm (c) and the vacuole (v) are indicated. The dashed line marks the portion of the plasma membrane permeabilized. The permeabilized portion of the intemodal cell (cis) is electrically separated from the non-permeabilized portion (trans) by silicone grease (g).

(I/V) profiles showed a shift of the current voltage characteristics with a bias towards negative currents (Figure 3A) as well as a conductance increase over the limited voltage range tested (data not shown). The shift in the profile was transient and I/V profiles recorded 5-6 min after the injection were identical with the I/V characteristics before injection. Injection of EGTA to reduce cytoplasmic Ca2+ (Tillotson and Gorman, 1980) produced a reduction in the same portion of the I/V profile as was increased by Ca 2+ injection (Figure 3B). An injection of Mg2+ or Cl- was without effect on the I/V profile (data not shown). Subtracting the post-treatment I/V profile from the control I/V profile obtained before the injection gives the current difference curve (Al/V), which characterizes the properties of the calcium-generated current (Figure 4A); this current

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--200 Fig. 3. Effect of iontophoretic injection of Ca2+, EGTA and InsP3 into cytoplasm-enriched fragments of C. corallina on the current/voltage (I/V) characteristics. During the injections, fragments were voltageclamped to the resting potential. The open circles in each graph represent stable I/V profiles before the treatments. Subsequent I/V curves were recorded at: (A) 5 s (0) after Ca2+ injection (+500 nA, 60 s), (B) 5 s (0) after EGTA injection (-500 nA, 300 s), and (C) 5s (*, 60 s (A) and 180 s (0) after injection of InsP3 (-500 nA, 60 s). The curves in A and B present the statistical means of five and four experiments respectively. The curves in C are representative of the observations obtained from seven cells. Assuming that all the current injected was carried by InsP3 or Ca2+ respectively, the total load into the cytoplasm-enriched fragments per pulse would be 0.15 nmol Ca2+ or 52 pmol InsP3.

is predominantly inward. The small outward current, which does not increase over the limited span of voltages positive of the reversal potential, was statistically not significant, but was observed in all cells tested. The AT/V data from five cells showed a reversal potential at -159 + 7 mV. I/V profiles recorded after injecting InsP3 into cytoplasmenriched fragments of Chara revealed a shift of the current/voltage characteristics similar to that detected following calcium injection (Figure 3C). The InsP3-generated current was more variable than the calcium-induced current in that the I/V profile changed in a complex pattern with time after injection of the InsP3 (Figures 3C, 4B and C). Figure 4C shows that 5 s after the injection the InsP3-generated current had the same properties as the calcium-related current increase. Both injections altered the current profile across the voltage range tested [represented by the current at -300 mV (1L300) and -120 mV (I- 120)] in a comparable ratio. With time the InsP3-related inward current increased over the whole voltage range (Figure 4C) with the result that the InsP3-generated current reversed at more positive values. This shift in the ratio of evoked currents, IL300: L1120, implies that the response is the consequence of InsP3 activating more than one current. By contrast, if only one current had been activated a constant ratio would have been expected.

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Fig. 4. Current/voltage difference curves (AI/V) (A, B) and single Al values at -300 and -200 mV (C) following injection of Ca2+ or InsP3 into cytoplasm-enriched fragments. The curves represent a polynomial fit to data obtained from subtraction of post-treatment I/V values from the control. (A) A comparsion of the Al/V profile obtained 5 s after injection of Ca2+ (dashed line; data from Figure 2A) with AI/V curves observed 5 s after injection of InsP3 (traces 1 and 2; data from two individual fragments). For evidence of the variability of the InsP3-generated current see also 3B, trace 1. (B) Al/V curves of a single cell (data from Figure 2C) at 5 s (trace 1), 60 s (trace 2) and 180 s (trace 3) after injection of InsP3 (-500 nA, 60 s; data from Figure 2C). (C) The current increase at -120 mV and at -300 mV after injection of Ca2+ (+ 500 nA, 60 s) or InsP3 (-500 nA, 60 s) into cytoplasm-enriched fragments of Chara. The current difference obtained 5 s after injection of calcium or 5, 60, 180, 360 s and 3600 s after injection of InsP3. The numbers on the columns denote the ratio of AI_300: Al120 (± s.d.; n: number of samples). The arrowheads on the current axis denote the standard deviation of the current at -120 mV (4) and -300 mV (1) for subsequent I/V profiles obtained prior to treatment.

It should be stressed that in the first 10 min after injection, all Chara cells tested showed a tendency to revert to the control I/V characteristics but afterwards exhibited an increased conductance over the whole time of recording (60 min) (Figure 4C). This observation was not the consequence of InsP3 leaking from the electrode; it was also observed when the injection electrode was withdrawn after injection. Interpreting long term measurements is confounded by the plant's tendency to exclude the microelectrode tip so that the Em-measuring electrode had to be re-inserted periodically (approximately every 20-30 min). I/V profiles appeared unaffected following re-impalements; nevertheless it cannot be excluded that repeated impalements contributed to the long term conductance rise.

Discussion An intoduction of InsP3 into the cytoplasm of characean cells by two independent methods affected the electrical 1739

G.Thiel, E.A.C.MacRobbie and D.E.Hanke

properties of the membranes of characean cells. This observation suggests that plant cells, like animal cells, contain a mechanism by which an increase in the cytoplasmic InsP3 concentration ultimately modulates properties of transport across the plasma membrane. In animal cells the effect of InsP3 on membrane transport systems is mimicked by an increase in cytoplasmic free Ca2+ (Oron et al., 1985; Parker and Miledi, 1986). This suggests that InsP3 affects membrane transport via its ability to catalyse Ca2+ release from internal stores. The data from the present study suggest that a similar system is involved in the InsP3 effect on transport properties in characean cells. A strong indication for a Ca2+-mediated InsP3 effect (versus a direct interaction with plasma membrane transport systems) is the observation that the membrane depolarization was abolished when InsP3 was added together with a higher concentration of the Ca2+ buffer EGTA. Furthermore an increase in the level of cytoplasmic Ca2 + is known to be an early event in the action potential of characean cells (Lunevsky et al., 1983). The role of cytoplasmic free Ca2+ in triggering action potentials is supported by the effect observed upon increasing the free Ca2+ concentration in the permeabilization medium of locally permeabilized Nitella cells (Figure 2B). Hence, an InsP3-generated action potential in Chara and Nitella (Figures 1 and 2A) could be explained in the same manner by the rise in cytoplasmic calcium activity. The sudden increase in Ca2+ is thought to trigger the activation of Clchannels which in turn contribute to depolarization during action potentials (Beilby, 1984; Sanders et al., 1986). The possiblity that InsP3 activates Ca2+-sensitive Cl channels known from the plasma membrane of characean cells (Lunevksy et al., 1983; Shiina and Tazawa, 1987, 1988; Tsutsui et al., 1986) is consistent with the observed InsP3-induced shift in the I/V curve of Chara cells. For a Cl- activity of 2 mM in the cytoplasm (Coleman, 1986) the reversal potential of this ion is -5 mV. Thus, an InsP3-generated activation of Cl- channels mediated by a rise in the cytoplasmic calcium activity would be consistent with the current/voltage offset toward negative currents (Figure 4B). In what was only a preliminary study it was suggested that plasma membrane Cl- channels become activated after addition of InsP3 to the external medium of intact Nitella (Zherelova, 1989a). The fact that Ca2+ injection into Chara fragments did not affect the transmembrane voltage may be related to the high Ca2+ buffer capacity of the cytoplasm (Brinley et al., 1977; Williamson and Ashley, 1982). Assuming that all the current injected into the cytoplasm-enriched fragments was carried by Ca2 +, the total load into the cytoplasm-enriched fragments per pulse would be 0.15 nmol Ca2+. Chara cells were observed to buffer the calcium rise during an action potential to 6% of the peak value almost instantaneously (Williamson and Ashley, 1982). From this estimate of buffer capacity, a 60 s Ca2+ pulse (500 nA) into an average cytoplasmic volume of 4 Al would increase the resting concentration of cytoplasmic free calcium from 0.2 /tM (Williamson and Ashley, 1982; Miller and Sanders, 1987) to 2.4 ItM. The real increase is likely to be even smaller, because an injection current is not carried by Ca2+ only (Purves, 1981). Thus, the increase in free cytoplasmic calcium generated by an injection may well have remained below the threshold concentration for Cl- channel -

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activation and resultant depolarization, as observed in perfused tonoplast-free Chara (Luhring and Tazawa, 1985; Sanders et al., 1986). The I/V analysis of the changes following Ca2+ (and EGTA) injection, on the other hand, revealed a Ca2+sensitive transport system in addition the Ca2+-sensitive Cl- channels (Figures 3A, B and 4). The Ca2+-sensitive current had a reversal potential of - 159 mV. For a cytoplasmic K+ activity of -110 mM (Beilby and Blatt, 1986) the Nernst potential of K+ is -144 mV. The similarity of the values makes it likely that an increase in cytoplasmic Ca2+ in Chara enhances an inward current carried by K+ ions. The properties of the calcium-induced current suggest that the charge is conducted by the inward rectifying K+ channels known from characean cells (Sokolik and Yurin, 1986; Tester, 1990). Recently, Ca2+ sensitivity has also been suggested for the K+ channels of the plasma membrane of Nitella (Vanselow and Hansen, 1989) and of Eremosphera (Thaler et al., 1989). Comparison of the initial shift in the I/V curve following InsP3 injection with the Ca2+-induced shift in the I/V profile (Figure 4) reveals considerable similarity between the activated currents. The time-dependent shift in the ratio of evoked currents (I-300: L120) suggests that in the short term the InsP3-evoked current is comprised of the timedependent activation of two currents: a K+ current, and a second current with a reversal potential which lies well positive of EK-the proposed Cl- current. The origin of the InsP3-mobilized Ca2 + remains uncertain. In plant cells it has been demonstrated that the tonoplast contains InsP3-activated Ca2 + channels (Alexandre et al., 1989). The idea that the vacuole is an InsP3-sensitive store of internal Ca2+ was further supported by investigations showing that InsP3, but not related substances (like InsP2 or myo-inositol), can release calcium from vacuolar vesicles (Schumaker and Sze, 1987). The responses in the membrane potential of locally permeabiized Nitella cells showed the same pattern of specificity for InsP3. It therefore seems likely, though unproven, that in characean cells the mediation of the effects of exogenous InsP3 by Ca2+ involved its release from internal stores such as the vacuole. However, in the course of an action potential it is also possible that the rise in concentration of cytoplasmic free Ca2+ will be further amplified by an influx across the plasma membrane mediated by the activation of voltagesensitive Ca2+ channels (Zherelova, 1989b). The observed action potentials therefore need not be dependent solely on Ca2+ release from internal stores. In conclusion, our data support the hypothesis that plant cells contain a system by which an increase in the cytoplasmic level of InsP3 would regulate different membrane transport functions. The short term response can be interpreted in terms of a rise in the cytoplasmic Ca2+ activity. However, we do not know if, or to what extent, any external effector can increase cytoplasmic InsP3 in vivo. Thus, the depolarization and action potential can only be interpreted as an indirect assay for the ability of InsP3 to increase the cytoplasmic free Ca2+ concentration. This mechanism may not be of relevance under physiological concentration changes. Therefore it is not yet possible to evaluate the significance, or specify the function, of inositol phosphates in plant membrane transport in vivo.

lnsP3 effect on membrane transport in giant algae

Materials and methods Preparation of cytoplasm-enriched fragments from Chara internodal cells

Cytoplasm-enriched fragments (Beilby and Blatt, 1986) were prepared by centrifugation of long internodal cells of C.corallina for 10 min at a low speed. When exposed to the air, the cells wilted and the lower end containing the cytoplasm was tied off in such a way that the fragment contained cytoplasm and vacuole in a ratio of approximately 1: 1. Local permeabilization of Nitella internodal cells The plasma membrane of N. translucens internodal cells was locally permeabilized by treating only one end of an internodal cell with a modified permeabilization medium (Shimmen and Tazawa, 1983; Tester et al., 1987). A young Nitella internodal cell was partitioned into two regions in a perspex chamber. A long portion (-5-10 mm) (trans) and a short one (-5 mm) (cis) were electrically isolated by a 5 mm long silicone gap (g) (Figure 2). The permeabilization of the cis region was achieved by periodically perfising it with ice cold permeabilization medium (Tester et al., 1987) (in mM: initially 5, later 0.1 EGTA, 30 N-hydroxyethylpiperazine-N'-2-ethanesulphonic acid, 90 2[N-morpholino]ethanesulphonic acid, 105 KOH, 2 MgCl2, 9 KCI, 2 NaCl, 1 K-ATP, -60-70 sorbitol, pH 7.6) for 10 min. The amount of sorbitol added depended on the osmotic pressure of the batch of cells used. The trans portion of the internodal cell was kept in artificial pond water with the appropriate addition of sorbitol to achieve isoosmotic conditions across the two pools. The EGTA concentration in the cis region was subsequently lowered to 0.1 mM and the cell allowed to recover in medium at room temperature. Cells repolarized and after an initial halt, cytoplasmic streaming soon restarted, although the rate was usually lower than in untreated cells. Cells in which the fluorescent dye Lucifer Yellow (mol. wt: 457 daltons), added to the cis side, appeared as a result of cytoplasmic streaming in the trans side were assumed to be also permeable to the slightly smaller molecule of InsP3 (mol. wt 417 daltons). However, no quantitative estimate of the uptake of substances into the cytoplasm was made.

Electrical measurements Chara fragments and leaf cells. After one day in experimental solution [artificial pond water (APW); in mM; 0.54 KCI, 1 NaCl, 0.5 CaC12, 2 mM N-tris [hydroxymethyl]methyl-2-aminoethanesulphonic acid/NaOH at pH 7.5] non-damaged cytoplasm-enriched fragments/leaf cells were impaled with conventional glass microelectrodes (Figure 1). Compounds were injected with appropriate polarity by a constant current generator (WPI 773, USA). The injection electrode was filled with either 1 M CaC12, 1 M MgCl2, 200 mM ethylene glycol bis-(,B-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), 200 mM KCI or 1 mM inositol-1,4,5-trisphosphate in 200 mM KCI. Iontophoretic injection was carried out either in fragments/leaf cells with a free running potential or in fragments clamped at their resting potential. In the latter case fragments were space-clamped and current/voltage profiles were obtained by a bipolar staircase of voltage commands (Beilby and Beilby, 1983).

Brinley,F.J., Tiffert,T., Scarpa,A. and Mullins,L.J. (1977) J. Gen. Physiol., 70, 355-384. Canut,H., Carrasco,A., Graziana,A., Boudet,A.M. and Ranjeva,R. (1989) FEBS Lett., 253, 173-177. Coleman,H.A. (1986) J. Memb. Biol., 93, 55-61. Dr0bak,B.K. and Fergusion,I.B. (1985) Biochem. Biophys. Res. Commun., 130, 1241-1246. Luhring,H. and Tazawa,M. (1985) Plant Cell Physiol., 26, 635-646. Lunevsky,V.Z., Zherelova,O.M., Vostrikov,I.Y. and Berestovsky,G.N. (1983) J. Membrane Biol., 72, 43-58. Miller,A.J. and Sanders,D. (1987) Nature, 326, 397-400. Oron,Y., Dascal,E., Nadler,E. and Lupu,M. (1985) Nature, 313, 141-143. Parker,I. and Miledi,F.R.S. (1986) Proc. R. Soc., 228, 307-315. Purves,R.D. (1981) Microelectrode Methods for Intracellular Recording and lonophoresis. Academic Press, New York. Ranjeva,R., Carrasco,A. and Boudet,A.M. (1988) FEBS Lett., 230, 137-141. Sanders,D. Hansen,U.-P. and Gradmann,D. (1986) In Trawavas,T. (ed.), Molecular and Cellular Aspects of Calcium in Plant Development, Plenum Press, New York, pp. 415-416. Schroder,J. and Hagiwara,S. (1989) Nature, 338, 427-430. Schumaker,K.S. and Sze,H. (1987) J. Biol. Chem., 262, 3944-3946. Shiina,T. and Tazawa,M. (1987) J. Membrane Biol., 96, 263-276. Shiina,T. and Tazawa,M. (1988) J. Membrane Biol., 106, 135-139. Shimmen,T. and Tazawa,M. (1983) Protoplasma, 115, 18-24. Sokolik,A.I. and Yurin,V.M. (1986) J. Memnbrane Biol., 89, 9-22. Tester,M. (1990) New Phytologist, 114, 305-340. Tester,M., Beilby,M.J. and Shimmen,T. (1987) Plant Cell Physiol., 28, 1555-1568. Thaler,M., Steigner,W., Forster,B., Kohler,K., Simonis,W. and Urbach,W. (1989) J. Exp. Bot., 40, 1195-1203. Tillotson,D. and Gorman,A.L.F. (1980) Nature, 286, 816-817. Tsutsui,I., Ohkawa,T., Nagai,R. and Kishimoto,U. (1986) Plant Cell Physiol., 27, 1197-1200. Vanselow,K.H. and Hansen,U.-P. (1989) J. Membrane Biol., 110, 175-187. Williamson,R.E. and Ashley,C.C. (1982) Nature, 296, 647-651. Zherelova,O.M. (1989a) FEBS Lett., 249, 105-107. Zherelova,O.M. (1989b) Comp. Biochem. Phyisol., 94A., 141-145.

Received on February 20, 1990

Perneabilized Nitella. Assuming that permeabilization reduces the resistance of the plasma membrane close to zero on the cis side, the potential across the two pools was measured with external electrodes and considered to be proportional to the membrane potential across the membrane on the trans side (Figure 2).

Acknowledgements We would like to thank Dr Robin Irvine for the kind gifts of InsP3 and InsP2 and Drs Mike Blatt and Mark Tester for constructive discussions. This research was funded by the SERC, whose support is gratefully

acknowledged.

References Alexandre,J., Lassalles,J.-P. and Kado,R.T. (1989) In Dainty,J., DeMichaelis,M.I., Marre,E., Rasi-Caldogno,F. (eds), Plant Membrane Transport: The Current Position. Elsevier, Amsterdam, pp. 249 -254. Beilby,M. (1984) Plant Cell Environ., 7, 415-42 1. Beilby,M.J. and Beilby,B.N. (1983) J. Membrane Biol., 74, 229-245. Beilby,M.J. and Blatt,M.R. (1986) Plant Physiol., 82, 417-422.

Berridge,M.J. (1987) Annu. Rev. Biochem., 56, 159-193. Berridge,M.J. (1988) J. Phyisol., 403, 589-599.

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