Vacuolar Ion Channels of Higher Plants

Vacuolar Ion Channels of Higher Plants

G. J. ALLEN and D. SANDERS

The Plant Laboratory, Biology Department, University of York, PO Box 373, York YO1 5YW, U K

I.

11.

111.

IV. V. VI.

Introduction .............................................................................. 218 ......................... A . Vacuoles as Ion Stores B. Electrochemical Potential Differences for Ions Across the Vacuolar Membrane ........ .......................................................... 219 otential and Ionic Currents at the C. Polarity of Me ........................................................ 221 Vacuolar Mem D . General Prope Channels, and Some Definitions ......... 221 E. Experimental Characterization of Ion Channels in Vacuoles ........ 222 Cation Channels ......................................................................... A . SV Channels ........ ....... ......................... B . FV Channels ....................................................................... C. Vacuolar K + (VK) Channels ............................ D. Other Inward-rectifying K + Channels ................. E. Hydrostatic and Osmotic Pres F. Vacuolar Voltage-gated Ca2+ G. Inositol 1,4,5-Trisphosphate-ga H . Ryanodine Receptor Homologues Anion Channels ... A . Malate (VMAL B. Chloride (VCI) Channels

226 226 230

....

Summary of Individual Channel Characteristics ...............................

243

Integration of Vacuolar Channel Activity

243

.......................................

Conclusions ............... ........................................... Acknowledgements ..... ............................. References ..................................... .....

Advancc, in Botanical Research VoI 25 incorporating Advances in Plan1 Pathology ISBN 0-12-(XIS9?5-X

Copyright 0 1997 Academic Pre% Imiited All rights of rsproduction in any form reserved

218

G. J . ALLEN and D. SANDERS

I. INTRODUCTION A . VACUOLES AS ION STORES

The accumulation of ions in the vacuolar lumen is a central attribute in many of the principal functions of vacuoles. Most obviously, as storage compartments, vacuoles are used as a repository for nutrient ions when these are in ample supply. Well-studied examples include NO3- (Zhen et al., 1991), K+ (Walker et al., 1996), Pi (Bieleski, 1968) and S042- (Cram, 1983a). In addition, involvement of the vacuole in intracellular signalling arises from its ability to sequester Ca2+ reversibly (Sanders et al., 1995). Furthermore, the maximization of cytosolic surface-to-volume ratio by the vacuole (see Raven, this volume) is most effectively achieved in energetic terms if the osmotic pressure of the vacuolar lumen - which must balance that of the cytosol - is generated by simple inorganic salts absorbed from the external medium. Thus, the energetic cost of accumulating a given ion by H+-coupled transport across both the plasma and vacuolar membranes is normally only one o r two ATP equivalents at each membrane. By contrast, an alternative strategy involving de n o w synthesis of an organic compound to serve solely as an osmoticum is inevitably more costly, even at the level of carbon fixation alone. The utility of ions in generating osmotic pressure within the vacuolar lumen - and with it a storage capacity to enhance water use efficiency - is most clearly visualized in bona fide halophytes, where NaCl can be accumulated to concentrations of several hundred millimolar (Flowers et al., 1977). Finally, in the case of crassulacean acid metabolism (CAM) plants, the vacuole is used to store malate ions which are generated by cytosolic C 0 2 fixation at night when stomata are open (Smith and Bryce, 1992). In all of these cases, the accumulation of ions in the vacuolar lumen must be visualized as representing a dynamic steady state, rather than an irreversible accumulation. Thus, with respect to inorganic nutrients such as NO3- or K + , mobilization occurs in the event that extracellular supply of the nutrient becomes depleted (Zhen and Leigh, 1990; Walker et al., 1996). The ions so released can either be used to bolster cytosolic levels, or can even be exported to rapidly growing tissues where nutritional demand is high. Similarly, rapid release of vacuolar Ca2+ can elevate cytosolic free Ca2+ ([Ca”],) and hence generate intracellular signals via Ca2+/calmodulindomain protein kinases or Ca2+-dependent ion channels (Bush, 1995). Furthermore, cellular release of ions, for example during control of cell turgor in halophytes in response to hypotonic conditions, or, in the case of guard cells, stomata1 closing stimuli, will inevitably involve ion mobilization from the vacuole if cytosolic volume is to be sustained (Cram, 1976, 1980; MacRobbie, 1995). In CAM plants. the raison d’etre of night-time malate storage is to facilitate release of C 0 2 for reductive assimilation once vacuolar mobilization of malate has occurred during the day.

VACUOLAR ION CHANNELS

219

The balance between net vacuolar accumulation and release for a given ion will be determined by the relative activities of two classes of transport system. In general, carriers energize transport of ions (other than H + ) by coupling the flow of ions to that of protons thermodynamically downhill into the cytosol (see Blumwald and Gelli, this volume). Transport in the opposite direction can occur passively via ion channels. The activities of ion channels, which are energetically dissipative, must be tightly regulated to prevent the occurrence of futile cycles. B. ELECTROCHEMICAL POTENTIAL DIFFERENCES FOR IONS ACROSS THE VACUOLARMEMBRANE

The feasibility of channel-mediated transport for a given ion in a particular direction will depend on its electrochemical potential difference (Afi,on) across the vacuolar membrane. In general, the chemical potential differences for each of the major inorganic ions Kf,Ca2+, CI-, NO3- and S042- have been reasonably well established with at least one of a number of techniques, including ion-selective electrodes (Felle, 1993), compartmental flux analysis (Cram, 1983b) and whole tissue assays (which reflect dominantly the vacuolar composition) combined with specific cytosolic assays made, for example, with optical probes (Reid et al., 1993). A range of values reported in various conditions for a range of cell types is shown in Table I. However, the other component of the electrochemical potential difference - the membrane potential - has been much more difficult to quantify. Ideally, measurement of vacuolar membrane potential should be made through simultaneous impalement of a single cell with one electrode located in the cytosol and another in the vacuolar lumen, and the investigator able to distinguish between the respective intracellular locations. This has been achieved to date only in the giant internodal cells of charophyte algae (Spanswick and Williams, 1964; Findlay and Hope, 1964), where the different excitatory properties of the vacuolar and plasma membranes have enabled estimation of a value in the region of -20 mV (cytosol with respect to lumen: see below). A negative polarity is consistent with the electrogenic pumping of H + from the cytosol to the vacuolar lumen by the V-type H+-ATPase and the H+-pyrophosphatase at this membrane (see Davies and Zhen et al., this volume). In higher plant tissue, independent impalement of electrodes into a single cell is not generally feasible, and early conclusions regarding electrode location were based on the criterion that vacuolar impalements would be associated with higher input resistance than cytosolic impalements, where intercellular pathways for current spread should result in a large effective membrane surface in comparison with vacuolar impalements (Bates et al., 1982). Grouping the potential measurements into two ranges of highand low-input resistance resulted in an estimate of vacuolar membrane

TABLE I Electrochemical potential differences for inorganic ions across the vacuolar membrane

K+

Ca2+

c1NOS

so2-

Luminal [ion] (mM)

Cytosolic [ion] (mM)

(H mol-')a

124 10 69 182 400

81 (replete) 45 (starved) 72 182 80d

+0.9 +5.7 +2.0 +1.9 -2.1

AILion

Species

Methodb

Hordeum vulgare (root) Hordeum vulgare (root) Hordeum vulgare (root) Eremosphaera viridis Commelina communis (open guard cell)

TBME TBME TBME DBME CFA , DBME

Reference'

1.6 x 10-4 1.5 x 10-4 2 x 10-4 3x

-13.8 -20.0 -22.1 -16.2

Eremosphaera viridis Riccia fluitans (rhizoids) Zea mays (roots) Nitellopsis obtusa

DBME DBME DBME DBME

3 5 5 6

30 6.2

7 0.4 2.2

- 12.6

-6.5

-4.5

Conocephalum conicum Daucus carota Eremosphaera viridis

DBME CFA DBME

7 8 3

3 35

0.6 4

-5.9 -7.3

Conocephalum conicum Hordeum vulgare

DBME DBME

7 9

0.3 0.7

-7.5 -3.0

Lemna minor Daucus carota

CFA CFA

8 8

0.2

2.3 1.5 1.o

44

1.3 0.5

aElectrochemical gradients estimated assuming a vacuolar membrane potential of -20 mV. bKey to methods: DBME, double-barrelled microelectrode; TBME, triple-barrelled microelectrode; CFA, compartmental flux analysis. 'Key to references: 1, Walker et al. (1996); 2, Walker et al. (1995); 3, Bethmann et al. (1995); 4, MacRobbie and Lettau (1980); 5. Felle (1988); 6, Miller and Sanders (1987); 7, Trebacz et al. (1994); 8, Cram (1983a); 9, Zhen et al. (1992). dConcentrations recalculated using estimate of the osmotic volume of a guard cell.

VACUOLAR ION CHANNELS

22 1

potential in the region of -50 mV. More recent independent impalements with multibarrel electrodes incorporating a p H sensor to determine intracelM a r location ( > p H 7, assumed to be cytosol) revealed no significant difference in voltage between luminal and cytosolic impalements (Walker et al., 1995). and a similar conclusion has been reached from studies with ion-selective electrodes on the alga Erernosphaeru (Bethmann et al., 1995). In no case have systematic and continuous estimates of vacuolar membrane potential been made in response to nutritional or other environmental conditions, so the possible existence of subtle transients in membrane potential remains a matter of conjecture. For the purposes of defining overall driving force, we have assumed that a membrane potential of -20 mV is a reasonable reflection of the physiological steady-state value. The resultant driving forces on a number of ions for which chemical potential differences can be estimated are shown in Table I. It is clear that for Ca2+ and the inorganic anions listed, entry into the cytosol can be channel-mediated (i.e. Apionis negative). For K + , the absence of a clear driving force in one direction or the other means that channelmediated transport of the ion either into or out of the cytosol is possible. Indeed, it seems possible that the polarity of the electrochemical potential difference could be determined principally by nutritional conditions, or in the case of guard cells, their state of turgidity since vacuolar K + concentration falls approximately to that of the cytosol when stomata close (MacRobbie and Lettau, 1980; cf. Table I). C . POLARITY OF MEMBRANE POTENTIAL AND IONIC CURRENTS AT THE VACUOLARMEMBRANE

To unify the description of electrical events at the plasma and vacuolar membranes, the accepted convention is to refer potentials and ionic currents to the extracytosolic compartment (Bert1 et ul., 1992a). Thus, a flux of cations from the lumen to the cytosol or of anions from the cytosol to the lumen will comprise an inward current. Note that in many papers published prior to the acceptance of this convention in 1992, a reverse polarity convention was common. D . GENERAL PROPERTIES OF ION CHANNELS, AND SOME DEFINITIONS

In contrast to carriers, ion channels can catalyse very rapid fluxes of ions of the order lo6 to logs-’. These high turnover numbers translate to electrical currents of between 0.2 and 2 0 p A for a monovalent ion, which enables electrical currents through single channels to be resolved with the patch clamp technique (see below). Channels exhibit ionic selectivity. This can be simply for cations over

-

222

G . J. ALLEN and D. SANDERS

anions or vice versa, but more typically, a distinct preference is shown for a single ionic species. The strict requirement for control of channel activity to prevent futile cycles is reflected in additional regulatory properties. All ion channels switch stochastically between closed (non-conducting) and open (conducting) states in a process known as gating. The equilibrium between these states is normally determined by membrane potential and/or by the concentration of some ligand, and such channels are referred to respectively as being voltageor ligand-gated. In addition, longer-term controls, such as phosphorylation level, can result in changes in channel activation state. Thus, when rendered inactive, this higher level of control results in non-responsiveness to gating factors. E. EXPERIMENTAL CHARACTERIZATION OF ION CHANNELS IN VACUOLES

Ionic channels are most effectively studied with electrophysiological techniques which allow a detailed assessment of the channel-mediated currents, particularly as these are influenced by membrane voltage. While radiometric studies in vesicles can provide useful and rapid information on the pharmacological properties of channels - especially those gated by ligands - it is possible to gain only relatively crude information on the voltage dependence of channels from such studies, and no information at all on channel selectivity. It is fair to say that the patch clamp technique has revolutionized the study of channels at the vacuolar membrane as at no other membrane. Thus, unlike the plasma membranes of plant and animal cells, the vacuolar membrane is not susceptible to conventional microelectrode impalement. On the other hand, intact vacuoles are relatively easily isolated - either by mechanical slicing of tissue (Leigh and Branton, 1976) or by gentle osmotic lysis of protoplasts (Raschke and Hedrich, 1989) - and these large organelles provide ideal material for the formation of the gigaohm seals which are a prerequisite for patch clamp recording. Vacuoles lend themselves to all four patch clamp recording modes described originally by Hamill et al. (1981). In the vacuole-attached mode (analogous to the cell-attached mode), the activities of single ion channels in the membrane patch can be detected, although this mode is of limited use because there is no experimental control of the luminal solution, and the transmembrane voltage is not known. However, if the membrane patch is excised from the vacuole, channel activity can be recorded in inside-out patch mode, which enables control of solution composition on both sides of the membrane and of membrane potential. Alternatively, the membrane patch can be broken - either with a voltage pulse or with suction - and electrical access is then gained to the luminal side. In this whole-vacuole mode, the

VACUOLAR ION CHANNELS

223

activities of an ensemble of ion channels in the membrane are recorded. Currents can be normalized on the basis of membrane surface area by expressing them in relation to membrane capacitance (which is proportional to surface area), resulting in units of picoamps per picofarad (PA pF-'). Selection can, of course, be made for a particular class of ion channel, for example by recording in the absence and presence of a specific gating ligand. The advantage of monitoring these so-called macroscopic currents is that an overview of gating and activation behaviour is achieved readily - albeit without the level of detail possible from single-channel recordings. Finally, pipette withdrawal after attainment of the whole-vacuole mode will occasionally result in refolding of the membrane around the tip of the pipette, and hence recordings in outside-out patch mode. This recording mode is particularly important for vacuoles because it is required for studying the effects of cytosolic regulators on single channels. Figure 1 shows examples of the whole-vacuole and outside-out patch recording modes as they relate to two of the dominant channel types in plant vacuoles. (The properties of the channels are described in more detail in subsequent sections: the recordings in Fig. 1 are intended to illustrate the methodological approaches.) Whole-vacuole currents at low [Ca2'], are shown in Fig. 1A and are typical of fast-activating vacuolar (FV) channel activity. The membrane is clamped successively through a series of voltages, and the resultant current traces are overlaid. The currents are timeindependent - that is, they flow instantaneously after the application of a voltage pulse. The derived steady-state current-voltage ( I - V ) relationship on the right shows that for the recording conditions in which KCI was present at equal concentrations on both sides of the membrane, the dominant direction of the current is outward (i.e. into the lumen). This indicates rectification of the channel, in which current passes more readily in one direction than the other, even in essentially symmetrical ionic conditions. Figure 1B shows typical traces recorded at much higher [Ca"],. Here the voltage pulse elicits a time-dependent current which is characteristic of slowly activating vacuolar (SV) channels. It is immediately clear - both from the current traces and the I-V relationship - that the current is very strongly rectifying over positive potentials. The question then arises concerning the ionic identity of the currents. This is commonly assessed by using the zero-current voltage (or reversal potential, E,,,) as a guide. No current will flow through a channel when the electrical driving force is just balanced by the chemical driving force provided by the permeant ion. Each ion will have its own reversal potential (designated E K , Eel, and so on) which can be calculated from the Nernst equation, and the extent to which Ere, for the channel mirrors changes in a particular Eion as the ionic gradients are changed can be used to identify the most permeant ion. An additional problem arises in the case of strongly rectifying channels such as the SV channel: Ere, is poorly defined by the steady-state I-V

224

G . J. ALLEN and D. SANDERS

Instantaneous

31

Instantaneous (FV)

+lo0 mV

3

-

0 mV -100 mV

.,J

B

-10 ~

1 sec

-v

500 p~ Ca"

(svl.

Time deoendent .

41 3

Time dependent 3,

+lo0 mV

E l

L

3

0

-100-80-60-40-20

0 mV 100 mV

J -1

.1

0 mV

-

O

0 mV

1 sec

-60

4

20 40 60 80 100 Voltage (mV)

o-60 -40 - 0

+120 mV

m

-

20 40 60 80 100 Voltage (mv)

V

-60

T

J

VACUOLAR ION CHANNELS

225

D 64 mV

c+ 0,+ 40 mV

c+

OmV

C +

I

Fig. 1 , (A) Whole-vacuole currents and the current-voltage ( I - V ) relationship from a Vicia faba guard cell vacuole under low [Ca'+], conditions where the current is dominated by the instantaneous fast vacuolar (FV) current. Currents were recorded following 2 0 m V steps positive or negative from a holding potential of OmV. The pipette solution contained 200 mM KCI, 5 mM Mes-Tris (pH 5 . 5 ) and sorbitol to 485 mosmol I-'. The bath solution contained 200 mM KCl, 25 mM Tris-Mes (pH 7 . 5 ) , sorbitol to 485 mosmol I-', 5 mM EGTA and CaCI,, to give a [Ca2+Ifreeof 10 nM. (B) Whole-vacuole currents and I-V relationship from a Viciufubu guard cell vacuole under high [Ca2'Ic conditions where the current is dominated by the time-dependent slow vacuolar (SV) current. Currents were recorded following 20 mV steps positive or negative from a holding potential of 0 mV. The pipette solution contained 200 mM KCI, 5 mM Mes-Tris (pH 5.5) and sorbitol to 485 mosmoll-'. T h e bath solution contained 200 mM KCI, 25 mM Tris-Mes (pH 7 . 3 , sorbitol to 485 mosmol I-', 5 mM EGTA and CaCI2, to give a [Ca2+Ifreeof 500 p M . (C) Whole-vacuole SV tail currents recorded in the same conditions as (B) but with the bath KC1 concentration reduced to 20 mM KCI in the second case. Tail currents were recorded by pulsing to + 120 mV, followed by a second pulse to a value between +60 and -60 mV in 15 m V steps. (D) Single-channel events recorded from an outside-out patch pulled from a Vicia faba vacuole under the same conditions as in (B), and therefore corresponding to SV channel activity. Single-channel openings can be seen as distinct current steps from the baseline. Openings are only observed at positive potentials, consistent with the whole-vacuole currents seen in (B).

relationship because the channel is shut at these potentials. In such cases, the reversal potential is assessed from the so-called tail currents, as shown in Fig. 1C. After applying a permissive voltage of +120 mV to allow channel opening, a rapid switch of the holding potential to values between -60 and t-60 mV at 15 mV increments captures current through the channels before

226

G. J . ALLEN and D. SANDERS

they have had time to close fully. These are the tail currents. They clearly reverse at OmV for the symmetrical conditions in the left-hand panel, but reverse at +47 mV in the presence of a ten-fold cytosol-inward KC1 gradient across the membrane. Since E K is +59 mV, the current is at least dominantly carried by K + . However, both Ecl and Eca are negative, and the lack of perfect agreement between EK and Ere, might imply a finite permeability to either CI- or Ca2+. Such possibilities can only be assessed through exploration of the effects of further ionic conditions. However, it should be noted that this widely used reversal potential analysis assumes independent movement of single ions through a pore, and that in cases of multi-ion pores the ionic permeability ratios which emerge from the analysis become concentration dependent and lacking in physical meaning (Hille, 1992). Examples of single-channel activity in an outside-out membrane patch are shown in Fig. 1D. The single-channel events were recorded at high [Ca2'],, and indicate SV channel activity. Current steps from the baseline ( C ) where all channels are closed can clearly be seen. Again, the channel can be seen to rectify, with opening events common only at positive potentials. The electrical conductance of a single channel is derived as the slope of the I-V relationship for the open channel (not shown here), and is expressed in picosiemens (pS).

11. CATION CHANNELS A.

SVCHANNELS

These channels were originally reported in vacuoles from the storage root of beet (Coyaud et al., 1987; Hedrich and Neher, 1987), but have subsequently been described in a wide range of species and tissue types, which indicates probable ubiquitous distribution (Hedrich et al., 1988, and references below). Nevertheless, widely disparate estimates of single-channel conductance have been reported for different species (between 50 and 250 pS in 100 mM KCI: Hedrich et al., 1988). Similarly, the magnitude of SV channel-mediated currents varies between different cell types, with whole-vacuole currents amounting to between 10 and 100pA pF-' in red beet storage root and 100-500 pA pF-' in guard cells at a permissive potential of 100 mV.

+

1. Gating We have already seen (Fig. 1) that the SV channel is strongly rectifying, with the whole-vacuole time-dependent I-V relationship exhibiting non-linear characteristics and the channel activating at non-resting (positive) values of membrane potential. The time-dependence appears to have two kinetic components which depend on the frequency of stimulation, suggesting that the channel might reside in more than one closed state (Gambale etal., 1993). An outstanding problem in research on SV channels relates to establishing

VACUOLAR ION CHANNELS

227

conditions required to bring the activation potential to within the normal range of slightly negative membrane potentials, or to defining conditions in which the membrane potential will move transiently into the range of positive activation potentials for SV channels described so far (Hedrich and Neher, 1987; Bethke and Jones, 1994; Ward and Schroder, 1994; Allen and Sanders, 1996). A striking feature of the SV channel is its activation by [Ca”],. Careful titration of [Ca’+], revealed activation of SV activity in beet at 300 nM (Hedrich and Neher, 1987), and subsequent studies have confirmed activation over the low-to-mid-nanomolar range for barley aleurone cells (Bethke and Jones, 1994), Chenopodium suspension cells (Reifarth et al., 1994) and Viciu guard cells (Schulz-Lessdorf and Hedrich, 1995; Allen and Sanders, 1996). Activation by Ca2+ interacts with that by membrane potential through a Ca*+-induced lowering of the voltage threshold for activation (Hedrich and Neher, 1987; Linz and Kohler, 1994; Reifarth et al., 1994; Schulz-Lessdorf and Hedrich, 1995). The effects of [Ca2+], are mediated via calmodulin. Thus, SV currents are inhibited by the calmodulin antagonists W-7, W-5, trifluoperazine and R 24571 (Weiser e f a / . , 1991; Bethke and Jones, 1994; Schulz-Lessdorf and Hedrich, 1995). Inhibition can be reversed and Ca*+-dependence reinstated by addition of plant (but not bovine brain) calmodulin (Weiser et al., 1991). Among other divalent cations, Ba*+ is ineffective in activating the channel in Vicia guard cells (Schulz-Lessdorf and Hedrich, 1995) and Mg*+ is ineffective in Arubidopsis cultured cells (Colombo et al., 1996), although, curiously, Mg’+ appears to be able to substitute for Ca’+ both in beet storage root and Vicia guard cells (Davies and Sanders, 1995; Allen and Sanders, 1996). This clear dependence on [Ca2’], over a range in which [@+I, is known to fluctuate during intracellular signalling (Bush, 1995) is reasonable prima facie evidence that activation of SV channels might play a role in signal transduction. This notion is further strengthened by the finding that SV channels from guard cells are progressively opened as the p H on both sides of the membrane becomes more alkaline (Schulz-Lessdorf and Hedrich, 1995). The physiological significance of this observation is that the range of cytosolic pH over which activity titrates (6.0-8.0) encompasses the pH range over which abscisic acid (ABA) induces elevation of guard cell pH during stomatal closure (Irving et al., 1992; Blatt and Armstrong, 1993). The differential response of the SV channel t o cytosolic H + and Ca2+ therefore mirrors the general directions in the concentration changes of each of these ions during stomatal closure.

2. Selectivity The SV channel was originally characterized as a monovalent cation-selective channel, with permeability not only to K + but also to Na+ (Coyaud e f al.,

228

G. J. ALLEN and D. SANDERS

1987; Colombo et al., 1988; Pantoja et al., 1989; Maathuis and Prins, 1990, 1991). Additionally, however, a significant anion permeability was mooted (Hedrich et al., 1986; Coyaud et al., 1987; Hedrich et af., 1988). This latter proposal was the result of whole-vacuole and single-channel determinations of Ere, in the presence of a transmembrane KCI gradient. There, Ere, was observed to move towards - but did not reach - EK,and the offset was taken to indicate a significant permeability to C1- . However, later studies involving C1- substitution by large organic anions (Colombo et af., 1988; Lado et af., 1989; Amodeo et af., 1994; Allen and Sanders, 1996) or the setting of Ecl to a value very different from that of all other ionic equilibrium potentials (Ward et af., 1995) failed to discern an anion permeability through a shift in E,,,. Indeed, single-channel measurements demonstrated no change in conductance on removal of CI-, although effects on channel gating were recorded (Pantoja et af., 1992a; Amodeo et al., 1994). Clearly, permeability to another ion in the solutions must account for the failure of Ere, to coincide with EK,and it seems likely that Ca2+ could fulfil that role since, in order to saturate SV channel opening, Ca2+ has usually been retained at 1 mM on the cytosolic or both sides of the membrane. This results in a value of Eca lying on the same (negative) side of E K as Ecl in the presence of an inward KCI gradient. Pantoja el al. (1992b), working with beet vacuoles, reported vacuolar Ba2+ currents with many of the same characteristics as SV-mediated currents, including outward rectification and slow (>1 s) voltage activation times. The question of Ca2+ permeation was addressed directly by Ward and Schoeder (1994), who derived a permeability ratio Pc,:PK = 3:l based on measurement of reversal potentials. The notion of Ca2+ permeation was supported in similar subsequent studies (Allen and Sanders, 1995; Schulz-Lessdorf and Hedrich, 1995), although the issue of anion permeation remains contentious. Thus, disparity between measured values of E,,, and those calculated on the basis of a channel permeable only to Ca2+ and K+ has been ascribed to anion permeation (Schulz-Lessdorf and Hedrich, 1995). Alternatively this disparity can be attributed to Mg2+ permeation, which then obviates the requirement to invoke anion permeation (Allen and Sanders, 1996). The estimates of Pca:PKvary with the specific proportions of the two ions present on either side of the membrane (Allen and Sanders, 1996), and this behaviour is characteristic of multi-ion pores where movement of ions through the channel is non-independent. This then undermines the basic approach for calculation of ionic permeability ratios by measurement of reversal potentials since the assumption of independent ion movement is violated (Hille, 1992). Thus, quantitative considerations of permeation properties are not possible for the SV channel using reversal potential methods. Nevertheless, the clear shifts in Ere, with Eta, taken together with the measurements of SV channel-mediated currents in simple CaC12 solutions, are strongly indicative of Ca2+ permeation (Ward and Schroeder, 1994;

VACUOLAR ION CHANNELS

229

Schulz-Lessdorf and Hedrich, 1995; Allen and Sanders, 1996). Furthermore, SV channel-mediated whole-vacuole and single-channel currents decrease with a rise in free Ca2+ (Ward and Schroeder, 1994; Allen and Sanders, 1995, 1996), which suggests that Ca2+ permeation and selectivity are facilitated through high-affinity binding of the ion within an otherwise non-selective pore, as is the case for animal plasma membrane Ca2+ channels. Several characteristics of SV channels, including widely variable ionic permeability ratios in mixtures of CaZ+ and K+ and the existence of negative apparent permeability ratios calculated for some ionic conditions (Allen and Sanders, 1996), have been predicted by an eight-state model for catalysis of ion permeation in which K + and Ca2+ compete for a common binding site (Gradmann, 1996). Noting the inappropriate permeability ratios calculated when independent electrodiffusion is assumed, this class of model might turn out to provide a better description of Ca2+ and K + permeation through SV channels. However, whatever the mechanism of its permeation, Ca2+ will move via SV channels info the cytosol, driven by the very large electrochemical potential difference for Ca2+, and despite the outward voltage rectification of the SV channel. These Ca2+ activation and Ca2+ permeability properties of SV channels suggest that the channels could participate in Ca2+-induced Ca2+ release from the vacuole. In many respects (Ca2+ permeability, activation by Ca2+ and high pH) SV channels bear remarkable similarities to the YVCCl channel of yeast (Bert1 et al., 1992b). However, the voltage-dependence of the yeast channel favours openings at cytosol-negative potentials, and in this respect the two channel types differ. 3. Pharmacology A wide range of compounds has been reported to inhibit currents through SV channels, including tetraethylammonium (TEA), 9-aminoacridine, quinacrine and quinine (Weiser and Bentrup, 1993). Surprisingly, the acetylcholine receptor antagonist (+)-tubocurarine and the K+ channel blocker charybdotoxin are also effective (Weiser and Bentrup, 1990, 1991, 1993). A number of inhibitors of anion transport, including 4,4’-diisothiocyanatostilbene-2,2’-disulfonicacid (DIDS), 4-acetamido-4‘isothiocyanatostilbene-2,2’-disulfonicacid (SITS) and Zn2+ also inhibit SV channels (Hedrich and Kurkdjian, 1988), and this observation might have reinforced the early notion that the channel is significantly permeable to anions. However, pharmacological profiles are poor guides to selectivity, as indicated, for example, by the general reactivity of DIDS with the &-amino groups of lysine residues. 4.

Function The gating and permeability properties of SV channels indicate that they could participate in Ca2+-induced K + and Ca’+ release from the vacuole,

230

G . J . ALLEN and D . SANDERS

or even in K+ uptake into the lumen, depending on the prevailing electrochemical potential difference for K+ . The way in which the gating properties of SV channels, as well as those relating to phosphorylation state (Allen and Sanders, 1995), might be integrated into coordinated responses of vacuolar channels during signalling is discussed in section V of this chapter.

B. FV CHANNELS

A channel that activates instantaneously in response to voltage changes was first reported in beet vacuoles and named the fast vacuolar (FV) channel, to designate its rapid kinetics and to distinguish it from the SV channel (Hedrich and Neher, 1987). Currents characteristic of FV channels have also been recorded from broad bean guard cell vacuoles (Allen and Sanders, 1996) and barley mesophyll vacuoles (Schonknecht et a f . , 1996). 1 . Gating FV channels are voltage-dependent. Currents carried by FV channels are outwardly rectifying: whole-vacuole recordings show larger currents at positive potentials in symmetrical conditions (Allen and Sanders, 1996; see also Fig. 1A), whereas in the physiological range of negative membrane potentials (0 to -40mV) there is a limited conductance. Thereafter, FV channels activate progressively with negative-going voltage. These voltagedependent gating characteristics give whole vacuole FV channel currents a characteristic shape (see Fig. 1A). Intriguingly, FV channels show an opposite Ca2+ dependence to that exhibited by SV channels, with inhibition of activity at [Ca2'], >300 nM in beet and >100nM in guard cells (Hedrich and Neher, 1987; Allen and Sanders, 1996). In addition, vacuolar Ca2+ inhibits FV-mediated currents, especially in the inward direction (J. M. Ward and J. I. Schroeder, personal communication; Schonknecht et al., 1996), and the currents are very sensitive to cytosolic pH, with an optimum at pH 7.3 (G. J. Allen, unpublished observations).

2. Permeation and selectivity Studies of whole vacuole currents indicate that FV channels are cationselective (Allen and Sanders, 1996; Schonknecht et al., 1996), and selectivity for K+ over C1- is confirmed by single-channel studies (Hedrich and Neher, 1987). Single channels have a conductance of 30pS in symmetric 200mM KCl (Hedrich and Neher, 1987). Nothing is known regarding the pharmacology of FV channels.

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3. Functions While a definite demonstration of the function of FV channels is lacking, some proposals have been made on the basis of the known properties. One possibility is that the channel facilitates K+ release from guard cell vacuoles during stomata1 closure, since currents are fivefold greater in the presence of a tenfold cytosol-directed Kf gradient in the presence of 10 nM [Ca*+], (Allen and Sanders, 1996). An alternative possibility is that FV channel function relates to provision of a shunt conductance for the vacuolar electrogenic H+ pumps. This proposal arose from observations on beet vacuoles that instantaneous currents measured in whole-vacuole mode, and possibly FV channelmediated, are activated by both ATP and increased cytosolic pH (Davies and Sanders, 1995). In normal conditions, therefore, this channel could provide a pathway for return current flow, facilitating luminal acidification in the absence of an opposing membrane potential. Conversely, in metabolically restricted conditions (for example, anoxia), with cytosolic pH low and H + ATPase activity decreased through a drop in ATP level, the channel-mediated K+ leak from the vacuole would be reduced. To date, however, the ATP dependence of FV channels has not been studied at the single-channel level. C. VACUOLAR K' (VK) CHANNELS

VK channels comprise a second, but discrete, type of instantaneously activating K+ channel. They have been described in most detail in vacuoles of broad bean guard cells (Ward and Schroeder, 1994; Allen and Sanders, 1996). 1. Gating One feature which distinguishes VK from FV channels is the voltage independence of VK channels. VK channels also show an opposing dependence on [Ca2+Ic:they are fully activated at S p M Ca2+ (Ward and Schroeder, 1994), although detailed titration studies have demonstrated that activation begins at concentrations above 100 nM (Allen and Sanders, 1996). Finally, VK channels are activated at low cytosolic pH (Ward and Schroeder, 1994): activity reaches a maximum at pH (7.5, and declines progressively over the pH range 7.0-8.0.

2. Perrrieation and selectivity Single-channel studies in symmetric 100 mM KCI have revealed that VK channels have a single-channel conductance of 70 pS (Ward and Schroeder, 1994). They are very highly selective for K f , again in contrast to FV channels. Thus, although VK channels will conduct Rb' and NH4+ to some extent,

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neither Cs+, Na+ nor Li+ permeate detectably (Ward and Schroeder, 1994). Nothing is known regarding the pharmacology of VK channels.

3. Function VK channels have so far been described only in guard cells, where they are thought to have a role in facilitating release of vacuolar K+ which is required for stomatal closure. The [Ca2+], dependence of VK channels is in accord with this proposal, since guard cell [Ca2+], normally rises during stomatal closure (Gilroy et al., 1990). However, the observation that cytosolic pH rises slowly in response to the closing stimulus ABA (Irving et al., 1992; Blatt and Armstrong, 1993), coupled with the substantial decrease in VK channel activity above pH 7.5, suggests that other pathways are available for sustained K+ release during closure. One such pathway might be through the SV channel (Ward and Schroeder, 1994). An additional role for VK channels has also been proposed by Ward and Schroeder (1994). This relates to the capacity of VK channels, on opening, to activate voltage-gated ion channels through membrane depolarization towards E K . The proposal is considered in the context of integration of ion channel activities towards the end of this review. 4.

Distribution Although VK channels have not been firmly identified in cell types other than guard cells, there is some evidence for their presence in at least one other cell type where they are likely to co-reside with FV-like channels. In the freshwater alga Eremosphaera viridis, there are two components to the instantaneous current (Linz and Kohler, 1994). The first component is linear with voltage and is carried by a channel with a unitary conductance of 75 pS (in symmetric 100mM KCl). In some respects, this channel resembles the VK channel, although its activity is decreased by a rise in [Ca2'],. The second component is active principally at positive potentials (i.e. strongly outwardly rectifying) and is carried by a channel with a unitary conductance of 35 pS. These features are similar to those of FV channels, as is the decrease of channel activity at low cytosolic pH. It appears that evolutionary prototypes of VK and FV channels might occur in this alga. D. OTHER INWARD-RECTIFYING K+ CHANNELS

Other channels that are K+-selective and active at negative potentials (i.e. opposite to that of SV channels) have been described in vacuoles from a number of species, including beet (Pantoja et al., 1992c), tobacco (Ping et a f . , 1992b) and Vigna unguicufata (runner bean: Maathuis and Prins, 1991). These channels exhibit a slow time-dependence in the whole-vacuole mode, = 20-5O:l). Although they might be and are markedly K+-selective (PK:PCI similar to FV channels with respect to their voltage dependence, they are

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distinguished by their dependence on [Ca2'], (Maathuis and Prins, 1991). The pharmacological and functional properties of these channels are not known. E.

HYDROSTATIC AND OSMOTIC PRESSURE (HOP)-ACTIVATED CHANNELS

A class of ion channel which is activated by pressure in the lumen of beet vacuoles, or in the presence of an osmotic gradient, has been described by Alexandre and Lassalles (1991). The channel is inward rectifying and slightly selective for Kf over C1-. Although the function of the channel is not known, it seems possible that it could be geared to regulate vacuolar volume during osmotic stress and hence - more importantly - cytosolic volume. F. VACUOLAR VOLTAGE-GATED Ca'

'

(VVCa) CHANNELS

In addition to SV channels, which are Ca"-permeant and depolarization ativated, Ca2+ channels which are activated on membrane hyperpolarization have been reported in vacuoles of beet (Johannes et al., 1992a; Gelli and Blumwald, 1993) and guard cells (Allen and Sanders, 1994a). 1. Gating The channels activate at negative potentials, and have been studied both at the single-channel (Johannes et a l . , 1992a; Allen and Sanders, 1994a) and whole-vacuole (Gelli and Blumwald, 1993) levels. Currents through VVCa channels show some time dependence in whole-vacuole recordings, but single-channel currents are activated instantaneously. The channels are strongly rectifying: they open only rarely at positive potentials. Typically, activation is over the voltage range (-20 to -50 mV) normally associated with the steady-state vacuolar membrane potential. Activity of VVCa channels is either insensitive to the prevailing [Ca2'], (Johannes etal., 1992a; Allen and Sanders, 1994a), or inhibited when [Ca2'], exceeds values of 1 p M (Gelli and Blumwald, 1993). However, all studies report activation as luminal Ca'+ increasos over the millimolar range: the half-maximal activation constant in beet at 0 niV is 1.4 mM Ca2+ (Johannes and Sanders, 199Sa). The dcpendenco of activation on Ca2+ concentration predicts that binding of two Ca'+ ions is required to open the channel (Johannes and Sanders, 199Sa). The effect of luminal Ca2+ is to shift the threshold for voltage activation to less negative potentials, thereby leading to an increase in open state probability over the physiological range of membrane potentials (Johannes et d.,1992a). Analysis of the voltage dependence of the response leads to the conclusion that the binding sites for Ca" gating are located 30% through the electric field of the membrane from the luminal side (Johannes and Sanders, 199Sa). Activation by luminal Ca2+

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is curious, because the large size of the vacuolar Ca2+ pool implies that rapid changes in channel gating compatible with fine control of activity cannot be attained through changes in luminal Ca2+.Thus it might be that the activation of the channel by luminal Ca2+ serves a more general purpose, such as restriction on the accumulation of excess Ca2+ in the vacuole. Luminal pH also plays a significant role in the control of channel activity (Johannes et al., 1992b; Allen and Sanders, 1994a). At non-physiological pH around neutrality, channel activity is high - to such an extent that the retention of such activity in vivo would outstrip the ability of vacuolar Ca2+ sequestration mechanisms to sustain a Ca2+ gradient across the vacuolar membrane (Bush, 1993). However, activity progressively decreases with luminal pH, until at physiological values around p H 5.5, channel openings are very infrequent indeed (Allen and Sanders, 1994a). The physiological implications are significant. Modest alkalinization of the vacuole has been observed during stomata1 opening (Bowling and Edwards, 1984), and this might play a role in the short-term control of channel activity in stimulusresponse coupling. Thus, luminal H+ might serve not only to prevent uncontrolled leakage of Ca2+ through VVCa channels in normal conditions but also, unlike luminal Ca2+, to regulate Ca2+ release in short-term responses to environmental stimuli.

2. Permeation and selectivity With 5-20 mM Ca2+ as a charge carrier on the luminal side, single-channel studies have revealed unitary conductances of 6 and 12 pS (beet: Gelli and Blumwald, 1993; Johannes et ad., 1992a) and 14 and 27 pS (guard cells: Allen and Sanders, 1994a). Intriguingly, in beet, spontaneous and reversible transitions between the 12 pS conductance state and one at 4 pS have been reported, and this is accompanied be a marked decrease in open probability (Johannes and Sanders, 1995b). Ionic selectivity for Ca2+ over K + , determined by measurement of reversal potentials in bi-ionic conditions, has been reported as 20:l (beet: Johannes et al., 1992a; Gelli and Blumwald, 1993) and 6:l (guard cells: Allen and Sanders, 1994a). Other alkali earth cations are also transported well by these channels, especially Ba2+, which, as with many animal Ca2+ channels, yields greater currents than those produced by Ca2+ (Gelli and Blumwald, 1993; Allen and Sanders, 1994a; Johannes and Sanders, 1995a). However, detailed permeation studies (Johannes and Sanders, 1995a) have revealed that, like SV channels, VVCa channels behave as multi-ion pores. A bona fide quantitative assessment of permeability ratios is therefore not possible (for the same reasons as given for SV channels). Nevertheless, VVCa channels bear many of the hallmark qualities of Ca2+ channels, including larger currents carried by those ions (K+, Mg2+) which exhibit low apparent permeability ratios. The explanation for this apparent paradox is that ion-binding site(s) within the channel which are required to endow the

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channel with ionic selectivity are also instrumental in slowing the passage of the ion (Hille, 1992). Those ions binding most strongly are therefore those for which the channel exhibits selectivity in complex solutions, but in simple solutions with only one permeant ion present, the unitary currents appear smaller than for those ions binding less strongly. The corollary is that large unitary currents carried through VVCa channels at high luminal K + concentrations can be titrated with modest additions of Ca2+. The affinity and location of the putative ion-binding sites endowing selectivity can then be investigated by monitoring the Ca2+ concentration and voltage dependence of the inhibited current. Such studies have resulted in the conclusion that the half-maximal inhibition constant at 0 mV is 300 pM Ca2+ (Johannes and Sanders, 1995a). This inhibition constant is only rather weakly voltage dependent, and quantitative analysis suggests a membrane location 9% through the electrical field from the luminal side. Some circumstantial evidence has been obtained for a second ion-binding site nearer to the cytosolic side (Johannes and Sanders, 1995a).

3. Pharmacology VVCa channels are potently inhibited by lanthanides, in particular La'+ (Gelli and Blumwald, 1993) and Gd3+ (Johannes et ul., 1992a; Allen and Sanders, 1994a). Blockade is associated with a drop in open state probability rather than in unitary current. In addition, verapamil and nifedipine - which are normally thought of as plasma membrane Ca2+ channel antagonists are effective in decreasing activity (Gelli and Blumwald, 1993; Allen and Sanders, 1994a). 4. Function VVCa channels are ideally poised to play a role in intracellular Ca2+ mobilization, and hence in intracellular signalling. They catalyse a relatively specific influx of Ca2+ into the cytosol, and respond over a range of membrane potentials believed to pertain in vivo. There are hints of involvement in signal transduction networks involving elevation of vacuolar pH, since this is a critical determinant of activity. The switch from the low-activity, low-conductance state to the high-activity, high-conductance state (Johannes and Sanders, 199%) might also confer essential physiological properties, although until it is known what triggers these state transitions, implications for function must remain speculative. 5 . Deyolarization-activated Ca2+ channels Calcium-selective channels activating on depolarization have been described in vacuoles from beet (Pantoja et a f . , 1992b), Arubidopsis (Ping et al., 1992a) and tobacco (Ping et al., 1992b). These channels are outwardly rectifying and hence could in principle carry Ca2+ from cytosol to vacuole. One proposed

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function for these channels is that they clear cytosolic Ca2+ loads, for example after vacuolar release of Ca2+ in cell signalling (Pantoja et a f . , 1992b). However, this function is not possible, since the release channels would not be capable of generating an outward Ca2+ gradient from the cytosol to the lumen. Furthermore, as shown in Table I, the electrochemical potential for Ca2+ is strongly directed into the cytosol. It seems very likely that these depolarization-activated Ca2+ channels are actually manifestations of the SV channel, as suggested by Ward and Schroeder (1994). G.

INOSITOL 1,4,5-TRISPHOSPHATE-GATED Ca2+ CHANNELS

Additional pathways exist for mobilization of vacuolar Ca2+. These are gated by ligands thought to have roles in intracellular signalling. The best characterized are those gated by inositol 1,4,S-trisphosphate (Imp3). An increasing body of evidence from microinjection and metabolic studies suggests that InsP3 has a role in controlling a number of processes in plant cells, including stomata1 closure (Gilroy et al., 1991), osmoregulation (Einspahr et af., 1988; Srivastava et al., 1989; Cho et af., 1993), modulation of turgor in the motor cells of leaf pulvini by red light (Kim et al., 1996), and pollen tube growth (Franklin-Tong et al., 1996). According to models developed from work on animal cells, InsP3 is produced by G proteinmediated hydrolysis of the plasma membrane lipid phosphatidylinositol 4,s-bisphosphate on perception of an appropriate agonist (Berridge, 1993). Binding of InsP3 to its receptor then results in Ca2+ mobilization from the endoplasmic reticulum lumen. Both the receptor and Ca2+ release properties are conferred by a homo-tetrameric array on the endoplasmic reticulum. Many of the details of the pathway for InsP3 production in plants remain to be determined, and it cannot be assumed that the animal cell model will apply in detail to plants, although evidence for stimulus-induced inositol lipid turnover is beginning to emerge in plants ( D r ~ b a k 1992). , The presence of an InsP3-gated channel on the vacuolar membrane of plants points to a role for this organelle as an InsP3-mobilizable store of Ca2+,at least in some cases. In this context, it should be noted that other intracellular locations for InsP3-gated channels in plants are not excluded. Indeed, although membrane fractionation studies on carrot suspension cultures failed to detect InsP3sensitive Ca2+ release at any membrane other than the vacuole (Canut et a f . , 1993), recent work on cauliflower florets has yielded a firm indication that InsP3 will also mobilize Ca2+ from a membrane fraction with much greater buoyant density than that of the vacuolar membrane - perhaps the plasma membrane (Muir, 1996).

I . ZnsI', binding and specificity Mobilization of Ca2+ from plant membrane vesicles was first demonstrated by Drobak and Ferguson (1985) using Ca2+-loaded zucchini hypocotyl

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microsomes. Subsequent studies using vacuole-enriched microsomes from the roots of oats or beet (Schumaker and Sze, 1987; Brosnan and Sanders, 1990) or intact vacuoles from Acer cell cultures (Ranjeva et al., 1988) established that the vacuole comprised an InsP3-mobilizable Ca2+ store, and that the dose dependence on InsP3 is similar to that for animal systems, with half-maximal effects at 200-600 nM. Binding sites specific for InsP3 have been solubilized from red beet microsomes, with binding co-purifying with vacuolar membranes (Brosnan and Sanders, 1993). The Kd for InsP3 binding is 120nM, which is in good agreement with the dose dependence found for Ca2+ release once allowance is made for the fact that ATP - which competes with InsP3 for binding - is also present in the Ca2+ release media. Other inositol phosphates (Imp2, InsP4) are ineffective in Ca2+release and in competing for InsP3 binding sites when applied at concentrations < 1 pM (Schumaker and Sze, 1987; Brosnan and Sanders, 1990, 1993). InsP3-binding sites have also been identified in Chenopodium, although specific binding is apparent only at elevated levels of Ca’+ in the millimolar concentration range (Scanlon et al., 1996). The physiological relevance of these sites might therefore be questioned, although it is possible that the membrane isolation protocol results in modification of binding properties. The Imp3-binding site density has been estimated from the equilibrium binding studies on beet to be < 1 pmol mg-’ (Brosnan and Sanders, 1993). Despite this low abundance, Biswas et ul. (1995) were able to use heparin affinity chromatography to purify to apparent homogeneity a protein of M, = 110 000 which could be reconstituted to yield InsP3-gated Ca2+ release activity. The disparity in M , between this protein and the mammalian receptor (250 000 for the monomer) is surprising, given that many of the Ca2+ release and InsP3-binding properties are conserved between animals and plants, and that the InsP3-binding and Ca” channel domains of the mammalian receptor are at the N and C termini, respectively (Taylor and Marshall, 1992). 2. Gating of InsP.3-dependent currents Whole vacuole currents are elicited by InsP3 with a half-saturation constant of 220nM (Alexandre et a / . , 1990), which is within the range observed in vesicle studies. Currents are inwardly rectifying over physiological negative membrane potentials, and this is reflected in the behaviour of Imp3-gated single channels for which open-state probability increases continuously over the range -20 to -90mV. Although several reports have commented on a failure to replicate the observation of InsP3-gated currents in vacuoles (Johannes et al., 1992a; Ping et a / . , 1992a; Gelli and Blumwald, 1993), it appears that, in beet at least, substantial hyperosmotic shock is required prior to vacuole isolation in order that measurable currents are obtained (Allen and Sanders, 1994b). Indeed, the magnitude of the InsP3-induced current

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increases as an exponential function of the osmotic pressure of the pretreatment solution. It is not known whether this sensitization of vacuoles to InsP3 relates to increased receptor expression, to post-translational events, or even, perhaps, to protection of InsP3 sensitivity during vacuole isolation. Currents elicited by InsP3 are readily reversible on InsP3 washout, as anticipated for a bonafide ligand-gated channel (Alexandre et al., 1990; Allen and Sanders, 1994b). There is no evidence that whole-vacuole currents are sensitive to the prevailing [Ca2+], (Allen and Sanders, 1994b), and, in this respect, gating of plant InsP3 channels appears to differ from that of many animal counterparts, where activation by [Ca”], comprises part of a positive-feedback mechanism for Ca2+-induced Ca2+ release (Taylor and Marshall, 1992). The effects of luminal Ca2+ remain to be investigated. 3. Permeation and selectivity Whether assayed at the single-channel or the whole-vacuole level, InsP3gated currents appear to be very highly selective for Ca2+ over K + , with reported values in excess of 100:l (Alexandre et al., 1990; Allen and Sanders, 1994b). Single-channel conductance has been reported as 30 pS with 5 mM Ca2+ on the luminal side (Alexandre et al., 1990). Subsequent investigations yielded ill-defined and variable estimates of single-channel conductance, largely as a result of extremely rapid gating kinetics (Allen and Sanders, 1994b). 4. Pharmacology Antagonists of InsP3-gated Ca2+ release from vacuoles of plants are similar to those identified in animals. The most potent inhibitor described to date is low molecular mass heparin (M,= 5000), which inhibits Ca2+ release by competition with InsP3 for binding with a Ki = 34 nM in beet (Brosnan and Sanders, 1990, 1993; Johannes et al., 1992b). Heparin with a higher M , is considerably less efficacious (Johannes et al., 1992b). Electrophysiological studies suggest that heparin might also be inhibitory to an unidentified Ca2+-sensitive current in the same membrane, thereby possibly limiting its use as a selective inhibitor of InsP3-gated Ca2+ currents (Alexandre and Lassalles, 1992). A second, less potent inhibitor is 8-(N,N-diethylamino)octyl 3,4,5trimethoxybenzoate (TMB-8), which inhibits in the micromolar range (Schumaker and Sze, 1987; Ranjeva et al., 1988; Johannes et al., 1992a). By contrast, ruthenium red and ryanodine, which interact with mammalian endomembrane Ca2+ channels, are ineffective (Muir et al., 1997). 5.

Function While the very high selectivity and the nature of the activating ligand leave little doubt that InsP3-gated channels function to release Ca2+ during signal

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transduction, the spectrum of physiological stimuli to which the vacuolar receptor responds has yet to be elucidated. In the pollen tube, for example, where InsP3 might play a crucial role in the control of growth, available evidence indicates a paucity of vacuolar material in the tip and subtip regions which are responsive to InsP3 (Franklin-Tong et al., 1996). Conversely, the prominence of the vacuole in beet storage root, coupled with the upregulation of vacuolar InsP3-gated channels during osmotic stress (Allen and Sanders, 1994b), points to a role for the vacuolar channels in turgor regulation (which is well established in beet: Cram, 1980). The electrophysiological studies on vacuolar InsP3-gated Ca2+ channels give rise to the interesting possibility that these channels could function in stimulus-response coupling without changes in InsP3 levels in one of two ways. First, a general increase in activity might be achieved (through increased expression or post-translational modification of the receptor), as for the response of beet to osmotic stress. This would place InsP3-gated Ca2+ release away from the early signalling events. Second, as originally pointed out by Alexandre et al. (1990), the strong inward rectification of the channel enables large changes in activity to be generated by membrane hyperpolarization alone. In either case, InsP3 would need to be present, but only at constant, basal concentration, to permit activation. H . RYANODINE RECEPTOR HOMOLOGUES

Ryanodine receptors were originally identified in the sarcoplasmic reticulum as the voltage-gated ion channels responsible fbr Ca2+ release during contraction of skeletal muscle. However, in many other animal cell types, coordinate action of ryanodine receptors with InsP3 receptors is thought to give rise to complex patterns of Ca2+ signalling (Berridge, 1993). In such instances, ryanodine receptors are thought to reside in the endoplasmic reticulum. At least one ryanodine receptor isoform (RYR2) is activated - either directly or indirectly -by the NAD metabolite cyclic ADP-ribose (cADPR), and cell types in which cADPR is thought to play a role in Ca2+signalling include non-skeletal muscle, brain and sea urchin eggs (Galione, 1994). The possibility of a similar role for cADPR in plants has yet to be examined critically. Nevertheless, plant vacuoles possess ryanodine receptor-like channels which are activated by cADPR. +

1. Ligand and voltage gating

Release of Ca2+ by cADPR can be demonstrated from vacuolar-enriched membrane vesicles of beet using a radiometric approach, or from intact vacuoles using whole-vacuole patch clamp (Allen et al., 1995). Using either methodology, the cADPR concentrations eliciting a half-maximal response are in the range 2 W O n M (Allen et al., 1995; Muir and Sanders, 1996). The non-cyclic isomer, ADPR, is ineffective at 100 nM.

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Although the dose-response relationship for cADPR is voltage insensitive, cADPR-elicited whole-vacuole currents are markedly voltage sensitive (Allen et al., 1995). The response to voltage is similar to that of InsP3-gated channels in the same membrane: instantaneous, and with strong inward rectification over physiological membrane potentials. This behaviour is also reflected in the properties of single cADPR-gated channels. Rectification is induced by the presence of Ca2+ on the luminal side of the membrane (G. J. Allen, unpublished data). As in the case of InsP3-gated channels in animal cells, [Ca2+], is a key activator of animal ryanodine receptors, which also participate in Ca2+-induced Ca2+ release. However, there is no evidence for Ca2+ activation of cADPR-elicited currents in beet vacuoles, and at concentrations greater than 1 p M , the response in Vicia guard cell vacuoles is actually inhibited (G. J . Allen, unpublished data). 2. Permeation and selectivity Although electrical activity is induced by cADPR in outside-out membrane patches, channels appear very fast in their gating characteristics, and it is not possible to resolve bona fide single-channel events. The best estimates of selectivity have therefore come from reversal potential measurements of cADPR-elicited whole-vacuole currents, where the permeability ratio Pc,:PK- 15:l has been determined for beet (Allen et al., 1995). This implies that the channel is significantly less selective for Ca2+ than plant InsP3-gated channels. 3. Pharmacology It is with respect to agonists and antagonists that cADPR-gated Ca2+ release at the vacuole exhibits most functional similarity to the ryanodine receptors of animals. Ryanodine itself - a plant alkaloid - can have agonistic or antagonistic effects in animals, depending on concentration. Ryanodine alone, applied to vacuole-enriched microsomes, elicited release of Ca2+ with a half-saturation constant of 40 nM (Muir and Sanders, 1996). Significantly, pretreatment with ryanodine releases just that fraction of the intravesicular Ca2+ pool which is cADPR sensitive, such that subsequent treatment with cADPR is without effect on Ca2+ release. Caffeine, also an agonist of ryanodine receptors in animals, likewise induces Ca2+ release while rendering the vesicles insensitive to subsequent addition of cADPR. Conversely, cADPR-elicited Ca2+ release is blocked by the ryanodine receptor antagonists ruthenium red and procaine (Allen et al., 1995; Muir and Sanders, 1996). 4. Function Calcium channels activated by cADPR co-reside in the same vacuoles as InsP3-gated channels, and yet the two channel types can clearly be distinguished on the basis of pharmacological profile, ionic selectivity and

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the discrete identities of the activating ligand (Allen et al., 1995). While it is anticipated that cADPR-elicited release of Ca2+ from the vacuole will play a role in signal transduction, there is as yet no information on which pathways are involved.

111. ANION CHANNELS By contrast with cation channels, far less is known about the behaviour of anion channels. The principal anionic constituents of higher plant vacuoles are normally malate and/or C1-, and consequently most studies have addressed the issue of permeation by these ions. A . MALATE (VMAL) CHANNELS

A consistent picture is now beginning to emerge regarding the mechanism of channel-mediated export of malate from the cytosol into the vacuole. Some of these studies have been performed on the CAM plants Graptopetalum paraguayense (Iwasaki et al., 1992) and Kalanchoe daigremontiana (Cheffings et al., 1997), which comprise favourable material because the vacuole undergoes diurnal cycling of vacuolar malate filling.

1 . Gating Malate-permeable channels have been identified from whole-vacuole and single-channel patch clamp studies both in CAM plants (see above) and in sugar beet (Pantoja et al., 1992c) and Arabidopsis thaliana (Cerana et al., 1995). The currents are very strongly inward rectifying, corresponding to anion uptake into the vacuolar lumen over the physiological range of negative membrane potentials. Activation occurs only at potentials negative of the reversal potential for the divalent species (maI2-: Iwasaki et al., 1992; Cerana et al., 1995), suggesting that malate efflux though the channel is prevented even in the event of a favourable membrane potential. Time constants for activation of the current are remarkably slow: in Kalanchoe, for example, Cheffings et al. (1997) report two exponential components with time constants of 0.8 and 5 . 3 s , together with an instantaneous one. The mechanism by which rectification is achieved is unclear at present. One possibility is that luminal C1- blocks malate re-entry to the cytosol (i.e. outward current), and some evidence for this is provided by the observation that luminal C1- inhibits VMAL channels by decreasing the open state probability (Plant et al., 1994). The single-channel conductance is not affected by luminal CI-. Cytosolic Ca2+ and ATP are without effect on VMAL channels (Iwasaki et al., 1992; Cerana et al., 1995; Cheffings et al., 1997).

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2. Permeation and selectivity Thermodynamically, a membrane potential of -10 to -40 mV is competent in driving cytosolic export of malate into the vacuole (even to the high vacuolar levels occurring in CAM plants) providing the permeant species is maI2- and, in the case of CAM plants, protonation to the monoanionic form (Hmal-) occurs on the luminal side. Selectivity studies have confirmed that the reversal potentials of VMAL-mediated currents are in accord with permeation of maI2- (Pantoja el al., 1992c; Cerana et al., 1995). Singlechannel recordings with 10 mM malate on the cytosolic side yield a unitary conductance of 120 pS in Graptopetalum. Reversal potential measurements in Arabidopsis indicate that a number of other organic divalent anions also permeate with equal efficacy to malate, including succinate and fumarate, while oxaloacetate is somewhat less permeant (Cerana et al., 1995). In sugar beet, permeation of malate channels by NO3-, acetate and even H2P042- has been described (Plant et al., 1994). 3. Function It seems clear that VMAL channels are ideally suited for vacuolar uptake of malate by CAM and other plants, but not for malate mobilization into the cytosol. The identity of the latter pathway remains to be established. In addition, there is the distinct possibility that VMAL channels in some species are responsible for the vacuolar accumulation of a number of inorganic ions, although there is still substantial uncertainty on this point: Cerana et al. (1995) point out that activation by cytosolic malate of an inorganic anion conductance in Arabidopsis might arise either as a result of permeation though VMAL channels, or through separate inorganic ion channels. B. CHLORIDE (VCI) CHANNELS

There is some evidence that, based on their different inhibitor profiles, uptake of malate and of Cl- into vacuoles proceeds by different pathways (Martinoia et al., 1990). However, reports of Cl--permeable channels in higher plant vacuoles have been scant. Ping et al. (1992b) recorded the activity of single C1- channels in tobacco vacuoles. The single-channel conductance is 11OpS in 100mM C1-, and the channel opens at negative potentials, thereby putatively carrying C1- from the cytosol to the vacuole. Klughammer et al. (1992) have detected a number of single-channel conductances for C1-, NO3- and S042- in planar lipid bilayers into which membrane vesicles from barley have been fused. However, one problem with the bilayer approach is that even small amounts of contaminating membranes can contribute to the observations of single channels, so that unless copurification studies are performed with marker enzymes, localization cannot be assured. In addition, there is no possibility with vacuolar membranes to relate channel orientation in the bilayer to that in the native

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membrane, so the physiological meaning of any observed rectification cannot be ascertained.

IV. SUMMARY OF INDIVIDUAL CHANNEL CHARACTERISTICS The properties of vacuolar ion channels, as described in detail in the preceding account, are summarized in 'Table 11. This table comprises a generalized account, and is not intended to describe in detail those interesting variations in channel behaviour which relate to cell or tissue type and which are noted in the text. Rather, a general catalogue is intended, and this will doubtless require revision as new channel types are discovered and new functions ascertained.

V.

INTEGRATION OF VACUOLAR CHANNEL ACTIVITY

Characterization of the behaviour of individual channel types with respect to ion permeation and gating properties is a prerequisite to appreciation of their respective physiological roles. Nevertheless, a full understanding of channel function can only be attained when the activity of a given channel type is viewed against the background of other transport processes at the same membrane. These other transport processes might influence a number of factors which in the preceding discussion have been shown to impact on the activities of various channels, including membrane voltage, pH,, [Ca"], and, as a result of these potentially localized changes, the activities of phosphatases and kinases. Some intriguing interactions have been proposed in the context of vacuolar Ca2+ mobilization (Ward and Schroeder, 1994), and while these proposals remain largely hypothetical, they do form the basis for further experimental investigation. The proposals centre around the role of the SV channel as a vehicle for Ca2+-induced Ca2+ release (CICR). The insensitivity of the InsP3-gated channel and ryanodine receptor homologues to [Ca2'], in plants has already been noted as a principal point of divergence between ligandgated endomembrane Ca2+ release channels in plant and animal systems. Ward and Schroeder (1994) propose that in guard cells Ca2+ release through these or VVCa channels could trigger activation of Ca2+-permeable SV channels in two ways. First, direct activation of SV channels could arise through elevation of [Ca2+],. Second, activation of [ CaZf],-sensitive VK channels would depolarize the vacuolar membrane towards E K , with the result that SV channels might then enter their range of voltage activation. Essentially, amplification of the primary Ca'+ signal would be achieved even though the channels responsible for initial CaZf release are insensitive to [Ca2'lc. Localized changes in [Ca2'], could play a special role, not only in

[Ca2+lCactivated, instantaneous, voltage-independent Pressure activated, osmotically activated, voltage independent

K+

VK

K+ (Cl-)

Positive and negative voltages, instantaneous.

K+ (Cl-)

Fv

HOP

[Ca2+lCactivated, time-dependent, positive voltages

Ca2+, K +

Permeant ion(s)

sv

Channel

Gating kineticdvoltagedependence Other regulators

Inhibitors

-

PHC

70 pS (100 KCI) 20pS (200 KCI)

PHC

30 pS (200 KC1)

Volume regulation

K+ release -

-

K+ release, “shunt” for ATPase

Ca2+ K+ release flux,

Function

-

Calmodulin, Zn2+, DIDS, SITS, 50-250 pS (100 mM KCI) phosphorylation, TEA, PHC 9-aminoacridine, Decreases in quinacrine , increasing Ca2+ quinine, +turbocuraine, charybdotoxin

Unitary conductance

TABLE TI Summary of the properties of vacuolar ion channels

Ca2+

Ca2+

ma12-

c1-

IP3 gated

cADPR gated

VMAL

VCl

Negative voltages, instantaneous.

Voltages negative of Emal,time dependent

Negative voltages, instantaneous

Negative voltages, instantaneous

Negative voltages, instantaneous (single channel), time dependent (whole vacuole)

pH,, cytosolic pH; pH,, vacuolar pH.

Ca2+

WCa

110pS (100mM KCl)

120 p s (10 mM malate)

-

30 pS ( 5 mM Ca2+)

6-27 pS (5-20 mM Ca2+)

-

[Cl-lv

[Ca2+Ic (guard cells)

-

PHV [Ca”],

-

-

Ruthenium red, ryanodine, procaine

Low-M, heparin, TMB-8

La3+, Gd3+, verapamil, nifedipine

C1- into vacuole

Malate into vacuole

Ca’+ release

Ca2+ release

Ca2+ release

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the activation of SV channels, but also in the inactivation of FV channels (Alexandre and Lassalles, 1991). This latter aspect of control could be important, since FV channels tend to clamp the membrane potential to E K . Down-regulation of FV channel activity in conditions of elevated [Ca2'], could then enable the membrane potential to move from the restricted range imposed by the K + gradient. Positive feedback in Ca2+ signalling at the level of SV channels must inevitably be subject to negative-feedback regulation, since mobilization of anything other than a small proportion of the sizeable vacuolar Ca2' pool into the cytosol would be potentially lethal. The inhibition of SV channel activity by the Ca2+-dependent protein phosphatase calcineurin (Allen and Sanders, 1995) might provide one mechanism for control and downregulation of CICR. However, if this is indeed the case, then the action of phosphatase must be delayed with respect to initial SV channel activation by elevation of [Ca2'],. Such delay could occur if activation of phosphatase were to occur at higher [Ca2'], than the activation of CICR. The contrasting dependence of VK. and FV channels on both pH, and [Ca2'], has already been noted. These diverse responses might give clues to the ways in which Ca2+-dependent and -independent signalling events can converge on the same response (Allen and Sanders, 1996). Thus, stomatal closure could be achieved by vacuolar K+ release either through FV channels in a Ca2+-independent event controlled by an increase in pH, or through VK channels in a Ca2+-dependent event without an increase in pH,. These findings could go some way to reconciling seemingly disparate reports in the literature regarding the centrality or otherwise of cytosolic Ca2+ signalling in stomatal closure (MacRobbie, 1997).

VI. CONCLUSIONS This review has highlighted many areas of ignorance in our understanding of vacuolar channels, especially with respect to function, but even in relation to such basic properties as ion permeation (Gradmann, 1996). However, these uncertainties must be viewed against a background in which just over a decade ago, vacuolar ion channels were essentially uncharacterized in higher plants. Progress during that decade has been astonishing with respect to identification and characterization of the properties of vacuolar ion channels at the electrophysiological level. Despite this, and the progress in our molecular understanding of water channels at the same membrane (see Chrispeels et af., this volume), not one vacuolar ion channel has, to date, been cloned. The challenge for the next decade will be to place the pioneering electrophysiological work with vacuolar ion channels on a firm physiological footing with the range of molecular, optical and biochemical techniques which have yet to be applied to this experimental system.

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ACKNOWLEDGEMENTS We thank Susan Brudenell for valuable and accurate help with manuscript preparation and the Biotechnology and Biological Sciences Research Council for continuing support to this laboratory. This review is dedicated to the memory of LGDS - scholar and teacher.

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