Outward Current Conducting ion Channels in Tonoplasts of Vigna unguiculata

J. PlantPhysiol. Vol. 139. pp. 63-69(1991)

Outward Current Conducting ion Channels in Tonoplasts of Vigna unguiculata FRANS

J. M. MAATHUIS and HIDDE B. A. PRINS

University of Groningen, Department of Plant Biology, Ecotrans 1, P.O. Box 14,9750 AA Haren, The Netherlands Received December 29, 1990 . Accepted July 1, 1991

Summary Patch clamp studies were done on tonoplasts of V. unguiculata. Apart from the usually present inward rectifying K+ conducting channels (IRC), vacuoles showed outward rectifying channels (ORC), although less frequently. The ORC are activated at physiological tonoplast potentials, in contrast to IRC, and have lower single channel conductances. Another difference is that ORC have a higher cation anion selectivity (PK+/PCl - = 17-22) and a different Ca+ + dependence. Addition of BaCb to the cytoplasmic side of the tonoplast caused a 50 % decrease in the IRC conductance but did not affect ORC conductance. Outward rectifying channels were observed in root, stem and storage tissue of several other plant species and may form an integral part of higher plant tonoplasts. However, the frequency of appearance in all species was low.

Key words: Vigna unguiculata; ion channel; outward rectifying; patch clamp; tonoplast.

Introduction

In the past few years the patch clamp technique has offered the opportunity to investigate ion channels in higher plant membranes. In the tonoplast, several channel types have been observed: K + conducting, slowly activated channels (SV type) (Colombo et al., 1988; Coyaud et aI., 1987; Hedrich et al., 1986; Hedrich et al., 1988), fast activated channels (FV type) (Hedrich and Neher, 1987) and Ca + + conducting channels (Alexandre et al., 1990). The most abundant type seems to be the slowly activated inward rectifier (IRC) with a conductance ranging from 50 to at least 250 pS. This channel type has been observed in many species and is now fairly well characterized in terms of conductance, inhibition, voltage dependence and selectivity (Hedrich et al., 1988; Hedrich and Kurkdjian, 1988). One of the striking features of IRC is the lack of selectivity and a voltage activation range that is usually out of the physiological tonoplast potential range. Additionally, a high cytoplasmic Ca + + concentration (a pCa of about 6) is necessary to induce maximum activity.

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Ecotrans publication no. 18.

© 1991 by Gustav Fischer Verlag, Stuttgart

Because of this it is difficult to hypothesize a function for IRC channels, since the above described characteristics would imply that, under physiological conditions, the channels are closed. Despite these characteristics it has been proposed that nonselective ion transport involved with turgor regulation, dissipation of the tonoplast potential gradient and transport of photosynthetic compounds, like malate, are served by this class of channels (Coyaud et al., 1987; Hedrich et aI., 1988). IRC may also playa role in Na+ retention in the vacuole by reducing the open probability after growth on NaCI (Maathuis and Prins, 1990). Apart from activity of SV type IRC we occasionally recorded activity at positive tonoplast potentials in Plantago root vacuoles (Maathuis and Prins, 1989). Channel opening occurred at physiological tonoplast potentials and resulted in an outward current. Gating of outward rectifying channels (ORC) was less frequently observed compared with IRC activity. Stem vacuoles of V. unguiculata also showed ORC activity and were investigated to confirm if this kind of channel was generally present and in order to characterize it. In this article results on conductivity, ion selectivity, Ca + + dependence and (voltage dependent) gating kinetics of ORC in V. unguiculata are presented and their possible physiological role is discussed.

FRANS J. M. MAATHUIS and HIDDE B. A. PRINS

64

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Fig. l: Single channel data of inward rectifying and outward rectifying channels present in the same patch: Pipette and bath solution: lOOmM KCl, 10mM Mes/Tris, pH 7.5, 0.05 mM CaCh and 1 mM MgCI2. Potentials are given as pipette potentials at the right side of the graphs. Open level is denoted by <0>, closed level by . (a): V. unguiculata cv. Sativa cell attached patch. IRC conductance is 102 pS (channels open at positive potentials), ORC conductance is 61 pS (channels open at negative potentials). (b): V. unguiculata cv. TN-l inside-out patch. Two IRC channels are present with conductances of 97 pS and 25 pS. One ORC conductance of 60pS can be seen. (c): Current-voltage graph of the (open) channels recorded in Fig. 1 b. (A ): Inward rectifying channel with a conductance of 97 pS, (.): Inward rectifying channel with a conductance of 25 pS and (.): Outward rectifiying channel with a conductance of 60pS.

Outward rectifying ion channels in the tonoplast

Materials and Methods Plant material

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Vigna unguiculata L. cv.'s TN-8863, KN-1 and Sativa were germinated and grown on vermiculite in a green house with the following regime: 14 h light period, 50 % RH and a dayI night temperature of 22/16 oc, Two to three-week-old seedlings were used for experiments.

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Isolation of vacuoles Stems of V. unguiculata with a diameter of 4 - 5 mm were cut in thin slices with a sharp razor blade while immersed in the experimental solution. Vacuoles, ranging from 5 to 50 I'm in diameter, were spontaneously freed from the tissue and transferred to the experimental chamber.

40 msec.

Experimental solutions In most experiments the bath and pipette solution had the following composition: 10 mM MeslTris pH 7.5, 0.05 mM CaCb, 100 mM KCl and 1 mM MgCb. The osmolarity was adjusted with sorbitol to 500 - 550 m Osmol. For determination of cationl anion selectivities the KCl concentration of the pipette solution was changed to 15 mM with an adjusted osmolarity. KCl in the bath was exchanged for NaCI to determine the K + INa + selectivity.

Fig. 2: Double pulse protocol to invoke opening and closure of IRC and subsequent opening of ORC. A holding potential of 20 m V (Pipette potential) was applied to an inside-out patch of V. unguiculata cv. TN-1 in order to open IRC with a conductance of 102 pS. At the onset of a potential step to -70mV (arrow) the IRC that are open (small upward deflection in the graph before the arrow) start to close. After closure of the 3 IRC, opening of ORC with a conductance of 60 pS occurred. Solutions were as in Fig. 1.

Electrophysiology Electrodes were prepared from soft glass capillaries; they were pulled on a two stage puller, coated with Sylgard and heat polished. Resistances varied between 5 and 9 Mohm. Gigaohm seals between electrode and tonoplast were made by gentle suction and appeared within a second. The whole vacuole configuration was made with 5ms pulses of 600-1000mV. Voltage and current clamp measurements were done with a List Electronics EPC 7 amplifier. Capacity compensation and series resistance compensation (whole vacuole configuration) were done with circuitry on the amplifier. Pulses and data were transferred via an AID converter, under control of
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Results

Conductances In most preparations o'nly IRC activity could be recorded; sometimes vacuoles showed only ORC activity. In Fig. 1 a and b raw data of both IRC and ORC activity occurring in the same patch of V. unguiculata stem vacuoles are shown. The related current-voltage graphs are given in Fig. 1 c, revealing 3 conductances of 98, 25 (inward rectifying) and 60 pS (outward rectifying). Bath and pipette solution contained 100 mM KCI in these experiments. Clearly, the currents through ORC are smaller than through IRC for comparable pipette potentials, indicating that ORC are distinct from IRe. This can also be seen after a double pulse experiment in which opening of IRC is followed by closure of IRC and subsequent opening of ORe. Both the different current

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Fig. 3: Whole vacuole recordings of IRC and ORC activity in V. un-

guiculata cv. TN-8863. (a): A vacuole in the whole vacuole configuration was clamped at holding potentials ranging from -50 to 50 m V for 5 seconds and changing 10 m V per trace. The resulting inward (downward deflection) and outward (upward deflection) currents are plotted. Pipette and bath solutions were as in Fig. 1. The excised outside-out patch of this vacuole showed IRC and ORC activities. (b): Current-voltage graph of the (steady state) whole vacuole currents corresponding to Fig. 3 a . Inward currents are much larger than outward currents at comparable membrane potentials.

FRANS J. M. MAATHUIS and HIDDE B. A. PRINS

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amplitude and the lower noise level of the open channel indicate that 2 distinct ion channels are involved here (Fig. 2). The whole vacuole conductance (Fig. 3 a and b) of ORC is much lower than that of IRC This partly originates from a lower single channel conductance (Fig. 1 c) but to a larger extent from a lower probability of being in the open state. This results in a 5 -10 times smaller outward current compared with the inward current at comparable potentials (see also Fig. 7). Outward currents at 50 mV are typically 200-500pA while at -50mV, inward currents may become several nA per vacuole.

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Calcium dependence For maximum gating probability IRC need a high cytoplasmic Ca+ + concentration (approximately 10- 6 M). This calcium dependence may be of regulatory importance. Hedrich and Neher observed 2 vacuolar channels, activatad at different cytoplasmic calcium concentrations (Hedrich and Neher, 1987). ORC in Vigna vacuoles of the cultivars Sativa and KN-1 showed a calcium dependence comparabld do that of dhe iRC, although when the bath solution was titrated with EGTA they tended to close at a somewhat lower calcium concentration than the IRC IRC activity had totally ceased at approximately 50nM free Ca+ + while ORC gating was only completely inhibited at approximately 20 nM (Fig. 4). Variation of vacuolar calcium concentrations did not affect the gating.

SeLectivity Usually the function of ion channels is sought in dissipation of specific ion gradients. This implies a high ionic selectivity. IRC conduct a range of ions (Coyaud et aI., 1987; Hedrich et al., 1988), both cations (K +, N a +, Rb +) and anions (CI- , N0 3 - , malate). ORC of Vigna unguicuLata cv. Sativa and cv. KN-1 were tested for selectivity using KCI, KN0 3 and NaCI solutions. The reversal potential (i.e. the membrane potential at which the membrane current reverses its direction) in 100/15 mM (bath/pipette) KCI or KN0 3 solutions ranged from -38 to -43 m V (Fig. 5). Using ion activities and the Goldman equa-

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Fig. 5: Current-voltage relation of ORC in an inside-out excised patch from V. unguiculata cv. Sativa. Single channel recordings were made using 100 mM KCl in the bath and 15 mM KCl in the pipette. A plot of the current amplitude versus the membrane potential reveals a reversal potential (i.e. the membrane potential at I = 0) that can be used to calculate the K/Cl selectivity.

tion a cation/anion selectivity of 18 - 22 was calculated with a slightly higher conductance for N0 3 - compared with CI- . This is significantly higher than the cation/anion selectivity of IRC with a PK+/P C1 - of 4-6 (Maathuis and Prins, 1990). When KCI was exchanged for NaCI no change in conductance or reversal potential appeared in Vigna vacuoles, indicating a PK+/P Na + of 1.

The inhibitory effect ofBa + + ions IRC and ORC behaved quite differently with respect to Ba + + ions. Addition of 7 mM BaCh to the cytoplasmic side in an outside-out patch configuration resulted in a decreased conductance of only IRC The conductance changed from 103 pS to 47 pS. In contrast, it did not affect the ORC conductance at all, which remained at 59 pS (Fig. 6). Blocking by Ba + + ions may depend on the side of the membrane it is ap-

Fig. 4: Calcium dependence of ORe. An outside-out patch from a V. unguiculata vacuole showed decreasing ORC activity after titration of the bath medium with EGTA. Different free Ca + + concentrations in the bath solution were 500nM (upper trace), 60nM (middle trace) and 20nM (lower trace). The pats;h was clamped at 50mV. Apart from the EGTA/Ca+ + concentrations the solutions were as in Fig. 1.

Outward rectifying ion channels in the tonoplast plied to. It was not tested whether ORC are blocked if Ba + + was added to the vacuolar side of the tonoplast.

Kinetics and gating Open/closed analysis of IRC and ORC reveal a comparable mean open time at - 50 m V (IRC activation) and 50 m V (ORC activation). Fig. 7 a and b shows the open time distributions of IRC and ORe. Both distributions were best

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fitted with a single exponential from which mean open times of 2.6 msec (IRC) and 2.8 msec (ORC) were calculated. Open time distributions at other membrane potentials gave comparable values and it was concluded that the mean open time of both IRC and ORC was voltage independent (results not shown). One of the characterizations of IRC is the slow activation in the whole vacuole configuration (Coyaud et aI., 1987; Hedrich et al., 1988; Maathuis and Prins, 1989). This results

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Fig. 6: Blocking of inward currents by Ba + + . BaCh (7 mM) was added to the bath medium in an outside-out patch configuration of V. unguiculata cv. Sativa. (a): IRC activity without BaCh, showing a single channel conductance of 103 pS. (b): IRC activity in the presence of BaCh at the same membrane potentials as in (a). Clearly, the current amplitudes have decreased and the single channel conductance was reduced to 47 pS. (c): ORC activity of the same patch in the presence of BaCh. The single channel conductance was not affected and remained 59 pS. Solutions were as in Fig. 1.

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FRANS J. M. MAATHUIS and HIDDE B. A. PRINS

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Fig. 8: Whole vacuole activation of IRC and ORC in V. unguiculata. Whole vacuole configuration showing SV type of (slowly activated) IRC and ORC activities. A double pulse protocol was applied: Starting from a holding potential of 0 mV the membrane potential was stepped to -50mV for 5 seconds (arrow 1) and subsequently to 50 mV for 5 seconds (arrow 2). SV type opening of IRC and ORC occurs. For the inward current it takes approximately 2.5 seconds to reach the steady state level, for the outward current approximately 1 second. After the onset of the second potential step (arrow 2) deactivation of the inward current appears prior to activation of the outward current. Experimental solutions were as in Fig. 1.

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Fig.7: Open time distribution of IRC and ORC in an outside-out patch of V. unguicu· lata. A histogram of the different single channel open times was made and fitted with an exponential function to calculate the mean open time. The patch was clamped at -50mV (IRC activation, Fig. 7a) or 50mV (ORC activation, Fig. 7b) for approximately 50 seconds. Data were sampled at 9 kHz and filtered at 2 kHz. The minimum resolution for the open time distribution was 0.5 msec. The mean open time calculated from the single exponential fit was 2.6 msec for the IRC and 2.8 msec for the ORC.

in half times of activation of several hundreds of milliseconds. In Vigna cultivars ORC had activation time constants (thalf of activation is approximately 300 msec. at 50 mV) that were comparable with IRC time constants (Fig. 8). Gating of both IRC and ORC is voltage dependent. The steepness of the voltage dependency (i.e. the change in open time of the channel after a change in the membrane potential) can be taken as a measure of the gating charge (Hille, 1984). Voltage dependent gating can be expressed as Fo = Fo = To/T, * 100 where Fo is the fractional open time (in %), To the total open time and T t the total sampled time. In Fig. 9, Fo at different potentials is given in a particular patch. It is clear that IRC show a much steeper voltage dependence (Fig. 9 a) than ORC (Fig. 9 b). In IRC, a 15-24 mV increase in tonoplast potential causes a 10-fold increase in open time. For ORC the same increase in open time would need 87 -110 mV change of tonoplast potential.

Discussion

Apart from a few reports (Hedrich and Neher, 1987; Maathuis and Prins, 1989; Pantoja et aI., 1989), literature on outward current conducting channels in plant cell vacuoles is

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Fig.9: Voltage dependence of gating propability. Patches of V. unguiculata tonoplasts were analyzed for the total open time of the present channels relative to the total sample time. The pipette potential was plotted versus the fraction open time (see text). (a): Outside-out patch with IRC activity. About 24 mV change in pipette potential causes a 10-fold increase in channel open time. (b): Outside-out patch with ORC activity. A 87 mV change in pipette potential causes a 10-fold change in channel open time. Solutions were as in Fig. 1.

Outward rectifying ion channels in the tonoplast

scarce. Obviously, in most vacuoles these channels are not present or cannot be activated under the experimental conditions. In vacuoles of V. unguiculata species we observed patches with only IRC activity, with only ORC activity (i.e. ion channels that show increasing open probability at positive tonoplast potentials) and patches with IRC and ORC activity. IRC and ORC activity in one patch was also recorded in Plantago root vacuoles (Maathuis and Prins, 1989), in Trifolium pratense stem vacuoles (50 pS ORC conductance) and Allium cepa bulb scale vacuoles (58 pS ORC conductance, results not shown). In spite of the very low frequency of appearance ORC may form a class of channels that is an integral and general part of higher plant tonoplasts. Inhomogeneous cell populations are derived from the plant tissues used (roots from Plantago, stems from Vigna); therefore, the reason these channels are not often observed could be that ORC are only present in specialized cells or at certain life stages of the plant. In Plantago species (Maathuis and Prins, 1989) the frequency of ORC observations was higher using young plants while in Vigna species the opposite was the case. It might also be the case that under the experimental conditions used, ORC activity is hardly induced and that unknown (cellular) factors are needed for gating. Additionally, the actual number of outward rectifying channels may be smaller than that for IRe. The main difference between IRC and ORC is the voltage dependent gating. IRC are normally activated at non-physiological (negative) tonoplast potentials, although some activation may occur at physiological potentials and show a steep voltage dependence. On the other hand, ORC show gating at normal tonoplast potentials (20 to 30 mV) and are less voltage dependent. Figure 9 shows the percentage open time of IRC and ORC relative to the total sampled time. Patches of V. unguiculata with IRC activity show a 6-8 times steeper voltage dependence than patches with ORC activity. Additionally, the differences in conductances and selectivities evidently show that ORC are not IRC, which show occasional gating at positive tonoplast potentials. This is also indicated by the behaviour in the presence of Ba + + ions. Ba + + decreases currents through SV type IRC (Hedrich and Kurkdjian, 1988) and probably blocks channels by occupying binding sites. In this way it reduces fluxes of the permeant ion (Hille, 1984). As Fig. 6 shows, specifically IRC are blocked. Outward currents are not affected; this clearly indicates 2 different channels. Ion channels form pathways for the passive dissipation of ion gradients. In the tonoplast, ion transport serves turgor regulation (K +), storage of metabolic and harmful ions (N 0 3 - , malate, N a + , Cl-), dissipation of charge etc. It seems unlikely that all these tasks are performed by one class of channels although IRC can conduct all these ions. At least three classes of K + conducting ion channels in tonoplasts of higher plants are known at present: SV inward rectifying channels, outward rectifying channels (SV in Vigna species, FV in Plantago species), and a low conductance channel (20-30pS), which is inward rectifying (Maathuis and Prins, 1990) or shows inward and outward currents but has a different Ca + + dependence (Hedrich and Neher, 1987). The latter is of the FV type and has a quit different Ca + + dependence than the outward current conduct-

69

ing channel described in this report (Hedrich and Neher, 1987). ORC may be a specific class of vacuolar ion channels, although less frequently present or active. The higher selectivity of ORC leads to specific ion transport (especially K+) from vacuole to cytoplasm. At Ca + + concentrations normally met in cells, the gating probability should be very low, thereby preventing ion gradients over the tonoplast from dissipation. Transport of K + becomes necessary when K + has to be redistributed between vacuole and cytoplasm for turgor regulation or has to be retranslocated from older to younger parts (Bogemans et aI., 1990), processes that could involve a signal that elevates cytoplasmic Ca + + to increase the opening probability.

References ALEXANDRE, J., J. P. LASSALLES, and R. T. KAno: Opening of Ca + + channels in isolated red beet root vacuole membrane by inositol 1,4,5-triphosphate. Nature 343, 567 - 570 (1990). BOGEMANS, J., J. M. STASSART, and L. NEIRINCKX: Effect of NaCI stress on ion retranslocation in barley. J. Plant Physiol. 135, 753-758 (1990). COLOMBO, R., R. CERANA, P. LADo, and A. PERES: Voltage-dependent channels permeable to K + and Na + in the membrane of Acer pseudoplatanus vacuoles. J. Membr. BioI. 103, 227-236 (1988). COYAUD, L., A. KURKDjIAN, R. KAno, and R. HEDRICH: Ion channels and ATP driven pumps involved in ion transport across the tonoplast of sugarbeet vacuoles. Biochim. Biophys. Acta 902, 263 - 268 (1987). HEDRICH, R., H. BARBIER-BRYGOO, H . FELLE, U. I. FLUGGE, U. LtlTrGE, F. J. M. MAATHUIS, S. MARx, H. B. A. PRINS, K. RASCHKE, H. SCHNABL, J. I. SCHROEDER, 1. STRUVE, L. TAIZ, and P. ZIEGLER: General mechanisms for solute transport across the tonoplast of plant vacuoles: a patch clamp survey of ion channels and proton pumps. Botanica Acta 101, 7 -13 (1988). HEDRICH, R., U. I. FLUGGE, and J. M. FERNANDEZ: Patch-clamp studies of ion transport in isolated plant vacuoles. FEBS 204, 228-232 (1986). HEDRICH, R. and A. KURKDjIAN: Characterization of an anion permeable channel from sugar beet vacuoles: effect of inhibitors. The EMBO J. 7, 3661-3666 (1988). HEDRICH, R. and E. NEHER: Regulation of voltage dependent ion channels in plant vacuoles by cytoplasmic calcium. Nature 329, 833-835 (1987). HILLE, B.: Classical biophysics of the squid giant axon. In: Ionic channels of excitable membranes, 23 - 57. Sinauer Associates Inc., Sunderland (1984). MAATHUIS, F. J. M. and H. B. A. PRINS: Patch clamp studies on root cell membranes of a salt tolerant and a salt sensitive Plantago species. In: DAINTY, J., M. I. DE MlCHEUS, E. MARRE, and F. RAslCALDOGNO (eds.): Plant membrane transport: The current position, 521-524. Elsevier, Amsterdam (1989). -

- Patch clamp studies on root cell vacuoles of a salt tolerant and a salt sensitive Plantago species. Plant Physiol. 92, 23-28 (1990).

PANTOJA, 0., J. DAINTY, and E. BWMWALD: Ion channels in vacuoles from halophytes and glycophytes. FEBS 255, 92-96 (1989).