Annals of Botany 86: 449±469, 2000 doi:10.1006/anbo.2000.1226, available online at http://www.idealibrary.com on

REVIEW

Ways of Ion Channel Gating in Plant Cells

ELZBIETA KROL and KAZIMIERZ TREBACZ*

Department of Biophysics, Institute of Biology, Maria Curie-Skl/ odowska University, Akademicka 19, 20-033 Lublin, Poland

Received: 12 April 2000 Returned for revision: 7 May 2000 Accepted: 12 June 2000 Published electronically: 21 July 2000

A precise control of ion channel opening is essential for the physiological functioning of plant cells. This process is termed gating. Ion channel gating can be e€ected by ligand-binding, ¯uctuations in membrane potential, membrane stretch and light quality. Modern electrophysiological and molecular-biological techniques have enabled the characterization and classi®cation of many ion channels according to their gating phenomena. Indications are that gating mechanisms are complex and that individual ion channels can be regulated by a number of factors. In this paper, gating mechanisms are reviewed following a standard classi®cation of ion channels based on permeability. The gating of K‡,Ca2‡ and anion channels in the plasma membrane, tonoplast and endomembranes of plant cells is described. # 2000 Annals of Botany Company

Key words: Review, ion channel, ligand-gating, voltage-gating, stretch-gating, light-gating, plasmalemma, tonoplast.

INTRODUCTION are also involved in membrane voltage stabilization, which is critical for maintaining ionic gradients and nutritional Ion channels are integral components of all membranes and ion ¯uxes. Stretch-activated ion channels serve as addi- they can be viewed as dynamic ion transport systems tional speci®c transmembrane `receptors' co-existing with coupled via membrane electrical activities (White et al., other cellular volume-sensing mechanisms. Light-activated 1999). Not only do they in¯uence membrane voltage channels are in fact ligand-gated, although a precise through the ionic currents they mediate, but their activities indication of the ligands is not yet possible because the can also be regulated by membrane voltage. Ion channels process of light signal transduction remains unclear. These can be divided into four `historically-based' groups accord- channels are distinguished particularly because of a special ing to gating mechanism: ligand-gated, voltage-gated, importance of light stimuli in plant signalling processes. stretch-activated and light-activated. Ligand-gated ion Modern biomolecular techniques reveal how complicated channels bind intracellular second messengers which pro- the processes controlling channel behaviour are. It becomes vide the essential links between external stimuli and speci®c increasingly apparent that the activity of a channel may intracellular responses (Leckie et al., 1998). Moreover, depend on the developmental and metabolic stage of the additional modulations by ATP or protons make the cell. Moreover, regulation of ion channels relies not only on channels capable of sensing changes in energy status or the channel proteins themselves, but also to a great extent acid metabolism, respectively (Schulz-Lessdorf et al., 1996). on regulatory polypeptides, such as auxiliary b-subunits, Voltage-dependent channels appear optimally suited for cytoskeletal components, 14-3-3 proteins, phosphates, electrical signal transmission via membrane depolarization kinases, and G-proteins (Czempinski et al., 1999). (e.g. through action potentials) and/or for signal trans- Jan and Jan (1997) recently reviewed receptor-regulated duction in response to changes in membrane potential ion channels in excitable and nonexcitable animal tissues (e.g. models investigating the coupling between membrane (G-protein-gated and cGMP-gated K‡ channels; voltage- potential and voltage-dependent Ca2‡-channels suggest gated K‡-, Na‡-, Cl-, Ca2‡ channels; voltage-insensitive that these are engaged in intracellular signalling). They Ca2‡ channels; Ca2‡-activated K‡ channels; ligand-gated Ca2‡ channels). The activities of these channels are sensi- * For correspondence. E-mail [email protected] tive to external and internal signals that are mediated by Abbreviations: ABA, Abscisic acid; ABC, ATP binding cassette; receptors for hormones and transmitters. There are also A-9-C, anthracene-9-carboxylic acid; AP, action potential; BL, blue light; cADPR, cyclic ADP-ribose; CDPK, calmodulin-like domain plant-derived elicitor-speci®c receptors, which are closely protein kinase; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea; E, coupled with plasma membrane ion channels important for equilibrium potential; I, current intensity; IAA, indol-3-acetic acid; IP3, signal transduction in plant cells (Ward et al., 1995; inositol triphosphate; NPPB, (5-nitro-2-3-phenylpropylamino) benzoic Blumwald et al., 1998). Studies on receptor-regulated ion acid; OA, okadaic acid; PLC, phospholipase C; PKA, protein kinase 2‡ channels suggest that they too are gated via G-proteins, dependent on cyclic AMP; PKC, protein kinase dependent on [Ca ]cyt and phospholipids; PKG, protein kinase dependent on cyclic GMP; either by direct protein-protein interaction or indirectly TMB-8, 8 (N,N diethylamino) octyl-3,4,5-trimethoxybenzoate. by kinase (PKA, PKG, PKC)/phosphatase cascades or 0305-7364/00/090449+21 $35.00/00 # 2000 Annals of Botany Company 450 Krol and TrebaczÐIon Channel Gating in Plant Cells

TABLE 1. Plant responses controlled by ion channel regulation

Plant response Reference

Blue- and red-light induced phototropism Cho and Spalding, 1996; Ermolayeva et al., 1996, 1997; Elzenga and Van Volkenburgh, 1997a; Lewis et al., 1997; Parks et al., 1998; Suh et al., 1998 Leaf movement Kim et al., 1992, 1996; Stoeckel and Takeda, 1993, 1995; Moran, 1996; Mayer et al., 1997 Plant excitability Katsuhara and Tazawa, 1992; Thiel et al., 1993 Light-induced hypocotyl elongation Sidler et al., 1998 Light-induced transient membrane Trebacz et al., 1994; Elzenga et al., 1995, 1997; Blom-Zandstra et al., 1997; SchoÈ nknecht et al., potential changes 1998; Szarek and Trebacz, 1999 Light-induced stomatal opening Kinoshita and Shimazaki, 1997; Suh et al., 1998 ABA-induced stomatal closure Armstrong et al., 1995; McAinsh et al., 1995, 1997; Schmidt et al., 1995; Ward et al., 1995; Li and Assmann, 1996; Blatt and Grabov, 1997a,b; Esser et al., 1997; MacRobbie, 1997; Mori and Muto, 1997; Pei et al., 1997, 1998; Grabov and Blatt, 1998a; Leckie et al., 1998; Li et al., 1998; Schwarz and Schroeder, 1998; Barbier-Brygoo et al., 1999 Plant hormone-induced responses Marten et al., 1991; Hedrich and Jeromin, 1992; Schumaker and Gizinski, 1993; Blatt and Thiel, 1994; Zimmermann et al., 1994; Ward et al., 1995; Venis et al., 1996; Claussen et al., 1997; Barbier-Brygoo et al., 1999 Ethylene-mediated responses Berry et al., 1996 Cold-shock responses Knight et al., 1996; Lewis et al., 1997 Nod- and pathogen-induced responses Ward et al., 1995; Zimmermann et al., 1997; Blumwald et al., 1998 Pollination Holdaway-Clarke et al., 1997; Brownlee et al., 1999 Water and solute transport Johansson et al., 1996, 1998; Logan et al., 1997; Eckert et al., 1999 Salt tolerance and turgor regulation Katsuhara and Tazawa, 1992; Taylor et al., 1996; Liu and Luan, 1998; Teodoro et al., 1998; Brownlee et al., 1999 Cellular pH regulation Johannes et al., 1998 Proton pump regulation De Boer, 1997; Claussen et al., 1997; Logan et al., 1997

2‡ second messenger binding (Ca ,IP3 , cGMP, cAMP). A Schroeder, 1989; Roberts and Tester, 1995; Hedrich and growing body of evidence indicates that G-proteins, second Dietrich, 1996; Logan et al., 1997; Maathuis et al., 1997; messengers and phosphorylation/dephosphorylation pro- Czempinski et al., 1999). cesses mediate various plant responses through ion channel and other transport system regulation (Table 1). Moreover, plant transmembrane receptors resembling Ligand-gated potassium channels receptor kinases of animal cells are involved in mediating a Ligand binding causes conformational changes in variety of cellular processes and responses to diverse channel proteins. It is a process of great importance, extracellular signals (Braun and Walker, 1996; Trewavas especially during signal transduction cascades when second and Malho, 1997). PCR, advanced homology-based clon- messengers synchronize the metabolism of the cell with ing and function-complementation techniques have already environmental conditions and enhance the input stimuli. led to identi®cation of more than 70 plant protein kinase There are many K‡ channels a€ected by calcium ion genes (Stone and Walker, 1995). However, the precise ‡ binding (namely: plasmalemma Kout channels, KORC, function of speci®c protein kinases and phosphatases NORC, VK, FV, SVÐfor more information see below) in during plant growth and development has been elucidated plant cells (Katsuhara and Tazawa, 1992; Allen and in only a few cases (Stone and Walker, 1995). Sanders, 1996; Czempinski et al., 1997, 1999; Maathuis et al., 1997; Muir et al., 1997; Allen et al., 1998a). Besides Ca2‡,H‡ ions, nucleotides, proteins and plant hormones POTASSIUM CHANNELS can serve as potassium channel ligands (see below). Their Ion transport across all biological membranes is highly attachment corresponds accordingly to changes in voltage selective and thus electrochemical potentials can be sensitivity of voltage-gated K‡ channels. generated. The electrochemical potentials largely depend on the potassium ion gradient, so most of the potassium Voltage-gated potassium channels in the plasmalemma channels must remain active for long periods of time. Such gradients are indispensable for long-term cell functions Voltage-gated plasmalemma K‡ channels are generally ‡ ‡ ‡ such as nutrition, elongation, turgor and water regulation divided into inward Kin † and outward (Kout) recti®ers. Kin or osmotically driven movements (Schroeder et al., 1984; channels are activated by hyperpolarizing potentials while Krol and TrebaczÐIon Channel Gating in Plant Cells 451

‡ ‡ Kout are activated by membrane depolarization. Both Kin channels which become active at depolarized membrane ‡ and Kout channels serve as membrane safeguards prevent- potentials, but their respective activation depends on the ing membrane voltage from becoming too negative or cytoplasmic Ca2‡ level (De Boer and Wegner, 1997). positive, respectively. Such a role of voltage-gated K‡ KORC, NORC and SKOR are di€erent channels from channels in stabilizing membrane voltages is universal plasmalemma of root xylem parenchyma cells. They are among all eukaryotes (Maathuis et al., 1997). Voltage- responsible for xylem loading (Roberts and Tester, 1995; dependent plasma membrane-bound outward potassium De Boer and Wegner, 1997; Maathuis et al., 1997; Gaymard recti®ers responsible for K‡ e‚ux are involved in turgor et al., 1998). KORC channels also show a considerable regulation (Liu and Luan, 1998), stomatal closure conductance for Na‡ but very low permeability for Li‡ and (MacRobbie, 1997; Grabov and Blatt, 1998a), organ Cs‡. This indicates that KORC channels may also act as a movements (Iijima and Hagiwara, 1987; Stoeckel and `®lter' protecting the shoot from harmful Cs‡ or Li‡ ions Takeda, 1993), cation release into xylem (Roberts and (Maathuis et al., 1997). NORC channels discriminate only Tester, 1995), light-induced potential changes of the slightly between cations and their role in solute release into plasmalemma (Blom-Zandstra et al., 1997) or repolariza- xylem is limited. However, they do provide a function in tion during action potentials (APs), and prevention of resetting the membrane potential after excessive depolariza- excessive depolarization (Stoeckel and Takeda, 1993; tion (Maathuis et al., 1997). Kout currents conducted by Trebacz et al., 1994; Maathuis et al., 1997). These roles SKOR are e€ectively inhibited by both cytosolic and ‡ are summarized in Table 2.Kin channels are involved in: external acidi®cation (Lacombe et al., 2000). SKOR potassium uptake into a cell during cell expansion, growth channels have no Ca2‡-binding sites, but they contain processes, organ movements and stomatal openings; low- ankyrin and cyclic nucleotide-binding domains (Gaymard anity uptake pathway in root hair cells; xylem unloading et al., 1998). Direct binding of nucleotides, calcium ions by conducting cations from xylem to symplast of growing (De Boer and Wegner, 1997; Czempinski et al., 1997, 1999) shoots; membrane voltage prevention against excessive or protons (Blatt and Grabov, 1997a) to the channel hyperpolarization (reviewed by Maathuis et al., 1997) proteins illustrates that voltage-gated outward-rectifying (summarized in Table 2). plasmalemma potassium channels may be regarded as ligand-gated in certain experimental conditions. Recently Ca2‡-gated outward rectifying potassium Regulation of plasmalemma voltage-gated potassium channels have been described in the plasmalemma of the channels alga Eremosphaera viridis (SchoÈ nknecht et al., 1998). These In addition to membrane potential, e€ectors like H‡, channels show very steep Ca2‡-dependence and they can be Ca2‡, nucleotides and K‡ ions can either interact directly Ca2‡-stimulated both directly and indirectly by interaction (ligand binding) with both inward and outward plasma- with calmodulin (SchoÈ nknecht et al., 1998). They are lemma K‡ channels or act indirectly via membrane-bound, involved in hyperpolarizing currents during darkening- attached or soluble regulators (Hedrich and Dietrich, 1996; induced transient hyperpolarizations of the plasma Kurosaki, 1997; Blatt, 1999; Czempinski et al., 1999). membrane (Table 2). ‡ ‡ ‡ Inwardly and outwardly rectifying K channels are con- The gating of Kout current is e€ected by [K ]ext , so that trolled by cytosolic calcium, ATP and pH in very di€erent its voltage dependence shifts in parallel with EK (Blatt, ‡ ways (Grabov and Blatt, 1997). The action of pHcyt is most 1999). K ions bind in a co-operative fashion to a set of pronounced on the depolarization-activated outward- sites exposed on the extracellular face of the membrane to ‡ ‡ rectifying K channels which are virtually insensitive to inactivate Kout channels and they may be substituted with 2‡ ‡ ‡ increased [Ca ]cyt (Grabov and Blatt, 1997). They do not Rb or Cs (Blatt, 1999). This inactivating binding of show such pronounced sensitivity towards external pH but monovalent ions to the channel protein is facilitated by require slightly alkaline cytosolic pH for activation (Blatt inside negative membrane voltage. Recent studies have and Grabov, 1997a). Alkaline pHcyt activates IKout in a shown that IKout activation is also dependent on the voltage-dependent manner through a co-operative binding cooperative interaction of two K‡ ions with the channel, of two protons (Grabov and Blatt, 1997). Moreover, but at sites di€erent from the channel pore (Grabov and their activation by depolarization depends critically on Blatt, 1998a). phosphorylation (e.g. by a kinase tightly associated with the Voltage-dependent plant plasmalemma K‡-uptake- channel protein in Samanea saman motor cellsÐMoran, channels represent various types (KAT, AKT) of di€erent 2‡ 1996) or dephosphorylation events associated with [Ca ]cyt spatial expression patterns (Bei and Luan, 1998), di€erent increase (e.g. by calcium-dependent phosphatase in Arabi- functions (Bei and Luan, 1998; Tang et al., 1998) and dopsis thaliana guard cellsÐMacRobbie, 1997). In meso- di€erent sensitivities to voltage, Cs‡,Ca2‡ and H‡ (Dreyer phyll and guard cells of Vicia faba there are outward- et al., 1997; Bei and Luan, 1998). This diversity partly rectifying K‡ channels regulated by calcium and G-protein results from nonselective heteromerization of di€erent interaction as well (Li and Assmann, 1993). On the other a-subunits (Dreyer et al., 1997) as well as from the ability hand, there are potential Ca2‡-binding sites (EF-hand of b-subunits to associate with more than one type of motifs) found at the C-terminus of a-subunits from putative a-subunit in vivo (Tang et al., 1996, 1998). All voltage- outward potassium recti®ers. These ion channels are very dependent plant plasmalemma K‡-uptake-channels con- likely to be directly regulated by Ca2‡ (Czempinski et al., tain a conserved GYGD motif within a pore region, which 1997, 1999). This also applies to KORC and NORC is responsible for K‡ conductivity (Czempinski et al., 452

TABLE 2. Plasmalemma ion channels

Channel Permeability Gating mechanism Physiological role References

Potassium channels

‡ ‡ Kout from Vicia faba guard cell K Depolarization-dependent opening Stomatal closure Li and Assmann, 1993; Blatt and ‡ Up-regulated by pHin increase (strong Prevention from re¯ux of K into Grabov, 1997a; Maathuis et al., 1997; voltage-dependent stimulation) the guard cell MacRobbie, 1997; Grabov and Co-operative binding of two protons Blatt, 1998a; Leckie et al., 1998; K‡ gradient sensitive Pei et al., 1998; Blatt, 1999 ‡ Inhibited by external K -binding Cells Plant in Gating Channel TrebaczÐIon and Krol Regulated by G-protein-induced Ca2‡-increase ‡ ‡ Kout from Arabidopsis thaliana K Depolarization-dependent opening stimulated Stomatal closure MacRobbie, 1997 guard cells by Ca-dependent phosphatase

Up-regulated by pHin increase ‡ ‡ ‡ ‡ Kout from Samanea saman K Rb Na Depolarization-induced activation Leaf movements Moran, 1996; Maathuis et al., 1997 motor cells Cs‡ Li‡ Phosphorylation by a kinase tightly associated Involvement in circadian clock ‡ with K out channel ‡ ‡ Kout from Mimosa pudica K Activation by depolarization Rapid movements in Mimosa Stoeckel and Takeda, 1993 motor cells Repolarization during AP ‡ ‡ Kout from Dionaea muscipula K Voltage-dependence (depolarization activated) Closure of trap-lobes Iijima and Hagiwara, 1987 trap-lobe cells Outward recti®cation strongly depends on the concentration of intracellular K‡ ‡ ‡ Kout from Conocephalum conicum K Voltage-dependence (depolarization activated) Repolarization during AP Trebacz et al., 1994 KORC (K‡ outward rectifying K‡ Na‡ Activated at membrane voltages more positive Xylem loading Roberts and Tester, 1995; De Boer and conductance) than 50 mV Shoot protection from harmful Wegner, 1997; Maathius et al., 1997 Ca2‡-dependent activation Cs‡ and Li‡ ions SKOR (Shaker-type K‡ outward K‡ Voltage-dependent Xylem loading Gaymard et al., 1998;

rectifying channel) Changes in both pHcyt and pHext regulate the Lacombe et al., 2000 number of channels available for activation NORC (non-selective outward Non-selective Active at membrane voltages more positive Protection against high Roberts and Tester, 1995; De Boer and rectifying conductance) among cations than ‡30 mV depolarization Wegner, 1997; Maathius et al., 1997; Ca2‡-dependent activation Xylem loading White, 1998 Maxi cation channel from Non-selective Active at membrane voltages more positive Membrane voltage stabilization White, 1998

rye roots among cations than EK ‡ ‡ ‡ 2‡ Kout from Nitellopsis obtusa K Na Ligand-binding: ATP- and [Ca ]ext-dependent Salt stress tolerance Katsuhara et al., 1990; Katsuhara and regulation (inhibition) Tazawa, 1992 ‡ ‡ Kout from Eremosphaera viridis K Ca-dependent and stimulated both by direct Dark-induced hyperpolarization SchoÈ nknecht et al., 1998 2‡ Ca -binding and indirectly by some of Vm and thereby divalent calmodulin interactions cation uptake ‡ ‡ Kout from Nicotiana tabacum K Light-activation Membrane depolarization upon Blom-Zandstra et al., 1997 L. mesophyll cells Voltage-dependence light transition ‡ ‡ Kout from guard cells of K Stretch-activated Volume and turgor regulation and Cosgrove and Hedrich, 1991 Vicia faba L. thereby control of leaf gas exchange ‡ ‡ Kin (KAT1) from Vicia faba K Hyperpolarization-dependent opening Stomata opening Blatt et al., 1990; Fairley-Grenot ‡ guard cell Lowering pHext promotes K current in voltage- Regulation of stomatal aperture and Assmann, 1992; Blatt and Thiel, dependent manner Osmotic volume readjustment 1994; Wu and Assmann, 1995; CDPK dependent phosphorylation of KAT1 Ilan et al., 1996; Blatt and Grabov, protein in a Ca2‡ dependent manner 1997a; Claussen et al., 1997; Grabov and 2‡ Inhibited by IP3-induced [Ca ]in elevation Blatt, 1997, 1998a; Hwang et al., 1997; Inhibited by polymerized actin ®laments Maathuis et al., 1997; MacRobbie, 1997; Modulated by auxin McAinsh et al., 1997; Leckie et al., 1998; Controlled by actin ®laments Li et al., 1998; Liu and Luan, 1998; Require external K‡ ions for activation Pei et al., 1998; Blatt, 1999; Modulated by cAMP-dependent signalling system Czempinski et al., 1999; Jin and and/or direct cyclic nucleotide binding Wu, 1999

‡ ‡ ‡ KAT1 from Arabidopsis thaliana K ,NH4 ,Rb , Voltage dependent (hyperpolarization activated) Stomatal opening Armstrong et al., 1995; MuÈ ller-RoÈ ber Cells Plant in Gating Channel TrebaczÐIon and Krol and KST1 from guard cells and Na‡,Li‡ ATP and cGMP activation K‡ uptake during other osmotic et al., 1995; Becker et al., 1996; Hedrich ¯owers of Solanum tuberosum Ion permeation may feed back on gating movements and Dietrich, 1996; Hoth et al., 1997; Competitively inhibited by Ca2‡ and Cs‡ ions Maathuis et al., 1997;

pH regulated (pHext acidi®cation shifts Czempinski et al., 1999 voltage-dependence toward less negative voltages) Regulation by cytoskeletal proteins Modulated by cyclic nucleotide binding AKT1 from Arabidopsis thaliana, K‡,Rb‡,Na‡, Hyperpolarization-dependent opening Regulation of membrane voltage Hedrich and Dietrich, 1996; Bertl et al., SKT1ÐSolanum tuberosum root Cs‡,Li‡ Inward K‡ gradient sensitive Low-anity K‡ uptake 1997; Maathuis et al., 1997; cells and channel analogue Czempinski et al., 1999 from corn roots KIRC (K‡ inward rectifying K‡,Rb‡,Na‡, Active at membrane voltages more negative Xylem unloading Maathius et al., 1997 conductance) Cs‡,Li‡ than 110 mV

‡ ‡ ‡ ‡ VIC (voltage-insensitive cation NH4 ,Rb ,K , Open 60±80 % of the time at voltages more Low-anity NH4 -uptake White, 1997, 1999 channel) Cs‡,Na‡,Li‡, positive than 120 mV Osmotic adjustment independent of TEA‡ Inhibited by divalent cations the membrane potentials Compensatory cation ¯uxes

‡ ‡ ‡ Kin from Zea mays coleoptile K ,Rb Hyperpolarization-dependent opening Cell elongation Hedrich and Dietrich, 1996; Lowering pHext Thiel et al., 1996; Claussen et al., 1997 Inhibited by Ca2‡ Modulated by auxin

‡ ‡ Kin from Avena sativa mesophyll K Voltage-dependent Plasmalemma Vm stabilization Kourie, 1996 cells Stabilization of ionic and osmotic conditions during cell expansion

‡ ‡ ‡ Kin from Samanea saman motor K Activation by H pump-induced hyperpolarization Leaf movements Kim et al., 1992, 1996; cells Inhibition by PLC-mediated IP3-induced Maathuis et al., 1997 Ca2‡ increase Direct response to light

‡ ‡ Kin from cultured carrot cells K Controlled by cytoplasmic concentration of cAMP Membrane changes and thus activation Kurosaki, 1997 of voltage-gated channels

‡ ‡ Stretch activated Kin from K Osmoticum gradient-sensitive Osmoregulation Ramahaleo et al., 1996; Liu and Vicia faba guard cells Voltage-dependence Luan, 1998 Regulated by actin ®laments 453 Table 2 continued on next page 454 TABLE 2. Continued

Channel Permeability Gating mechanism Physiological role References

Calcium channels

VDCCÐvoltage-dependent Ca2‡ Depolarization activated Early events of plant hormone-induced McAinsh et al., 1995; Grabov and Ca-channel from guard cells responses Blatt, 1998b VDCC from rye roots Ba2‡,Sr2‡,Ca2‡, Depolarization activated Divalent cation uptake into roots White, 1998; Pineros and Tester, 1997; VDCC from wheat roots Mg2‡,Mn2‡,K‡, Strong voltage-dependence (depolarization Signalling mechanisms and priming White, 1998 (rca channel) Na‡,Rb‡,Li‡ activated) the cell for response Cytosolic ATP shifts activation to more negative rladTeazInCanlGtn nPatCells Plant in Gating Channel TrebaczÐIon and Krol potentials VDCC from Arabidopsis roots Ba2‡,Sr2‡,Ca2‡, Depolarization activated Cation uptake Thion et al., 1996; White et al., 1998 and Daucus carota suspension Mg2‡,K‡ Active under condition of microtubule Maintaining appropriate electrochemical protoplasts disorganization gradients important for the transport Slow inactivation at negative voltages of other ions and cell volume regulation Signalling mechanisms and priming the cell for response VDCC from characean cells Ca2‡ Depolarization activated AP induction Katsuhara and Tazawa, 1992; Early events of turgor regulation and Shimmen, 1997 salt tolerance VDCC from Chara corallina Ca2‡ Depolarization activated During Ca-starvation channels might Reid et al., 1997 open to scavenge available Ca2‡ Voltage-dependent Ca-channels Ca2‡ Depolarization activated Light-induced membrane depolarization Trebacz et al., 1994; Ermolayeva et al., from liver wort Conocephalum 1996, 1997 conicum and moss Physcomitrella patens Ca-channels from mosses Ca2‡ Cytokinin-induced depolarization activated Early events of cytokinin-induced Schumaker and Gizinski, 1993 responses VDCC from Mimosa pudica Ca2‡ Hyperpolarization-activated Activation of channels involved in Stoeckel and Takeda, 1995 motor cells leaf movements VDCC from pollen tubes Ca2‡ Voltage-dependent Growth processes Holdaway-Clarke et al., 1997 Stretch-activated VDCC from Fucus rhizoids Ca2‡ Voltage-dependent Growth processes Taylor et al., 1996 Stretch-activated SAC from Fucus zygotes Non-selective Stretch-activated Mechanosensitive Ca-channels Non-selective Stretch-activated Regulation of turgor Thion et al., 1996; White et al., 1998 from root cells Regulated by cytoskeletal proteins Determination of the allometry of cell expansion and morphogenesis Mechanosensitive Ca-channels Ca2‡ Stretch-activated Transmission of Ca-signals into Cosgrove and Hedrich, 1991; from guard cells Regulated by cytoskeletal proteins the cytoplasm MacRobbie, 1997; McAinsh et al., 1997 Guard cell volume and turgor regulation and thereby control of leaf gas exchange Control of other ion channels with Ca-dependent activities Mechanosensitive Ca-channels Ca2‡ Stretch-activated Transmission of Ca-signals into Taylor et al., 1996; McAinsh et al., 1997 from Fucus rhizoids Regulated by cytoskeleton proteins the cytoplasm Cell volume regulation Receptor-regulated Ca-channels Non-selective Elicitor-activated Early events of pathogen defence Zimmermann et al., 1997; from parsley protoplasts and system activation White et al., 1998 root cells Receptor-regulated Ca-channels Ca2‡ Elicitor-activated Early events of pathogen defence Blumwald et al., 1998 Voltage-gated system activation Receptor-regulated Ca- from Ca2‡,K‡ Elicitor-activated Ca-in¯ux as an early response to various Gelli and Blumwald, 1997 tomato protoplasts Hyperpolarization-activated signals including fungal elicitors Anion channels rladTeazInCanlGtn nPatCells Plant in Gating Channel TrebaczÐIon and Krol

GCAC1 from Vicia faba and Cl, malate S-type shows weak voltage dependence S-type serves as major pathway for Keller et al., 1989; Marten et al., 1991; Commelina communis S-type requires hydrolysable ATP and activation of anion e‚ux during stomatal closure Hedrich and Jeromin, 1992; protein kinase and as negative feedback during Linder and Raschke, 1992; OA-sensitive phosphatases are involved in stomatal opening Schroeder and Keller, 1992; down-regulation of S-type channel R-type responsible for signal Schroeder et al., 1993; Dietrich and S-type may be ABC protein or it is tightly controlled transduction via membrane Hedrich, 1994; Schmidt et al., 1995; by such protein depolarization Ward et al., 1995; Li and Assmann, 1996; R-type is activated by parallel voltage membrane GCAC channels are capable of sensing Esser et al., 1997; Mori and Muto, 1997; 2‡ depolarization, pHcyt acidi®cation, [Ca ]cyt changes in the energy status, acid Pei et al., 1997, 1998; Grabov and increase and nucleotide binding metabolism and proton pump activity Blatt, 1998a; Schwarz and Direct auxin binding shifts activation potential in guard cells, because of time- and Schroeder, 1998; Leonhardt et al., 1999 towards resting potentials to favour channel voltage dependent activity strongly opening modulated by ATP and H‡ GCAC1 from Nicotiana S-type shows weak voltage dependence, requires Armstrong et al., 1995; Ward et al., 1995; benthamiana and Arabidopsis protein phosphatase activities and is down- Schulz-Lessdorf et al., 1996; Elzenga and thaliana regulated by protein kinase Van Volkenburgh, 1997b; S-type is in¯uenced by pH gradient Lewis et al., 1997; Grabov and R-type is activated by parallel voltage membrane Blatt, 1998a; Pei et al., 1997, 1998 2‡ depolarization, pHcyt acidi®cation, [Ca ]cyt increase and nucleotide binding R-type is modulated by phosphorylation/ dephosphorylation processes TSACÐtobacco suspension-cell Cl ATP-controlled voltage-dependence (depolarization Anion release during inhibition of Zimmermann et al., 1994 anion channel activated) cell elongation Modulated by auxin Anion channels from mesophyll Cl Voltage-dependent (hyperpolarization activated) Light-induced transient depolarization Elzenga and Van Volkenburgh, 1997a,b cells of Pisum sativum Two mode kinetics di€erently controlled by ATP (R- and S-type, S-type occurs in the presence of ATP) Ca-dependent activation Anion channels from suspension- Cl Voltage-dependent (hyperpolarization activated) Control of membrane potential Barbara et al., 1994 cultured carrot cells Voltage-dependent inactivation under large Regulation of osmotic balance hyperpolarization 455 Table 2 continued on next page 456 TABLE 2. Continued

Channel Permeability Gating mechanism Physiological role References

Anion channels from Cl Hyperpolarization activated Limiting the amplitude of dark-induced SchoÈ nknecht et al., 1998 Eremosphaera viridis transient hyperpolarization caused by K‡-release Anion channels from Cl Ca-dependent activation Depolarizing current during AP Okihara et al., 1991; Katsuhara and Charophyta cells Tazawa, 1992; Thiel et al., 1993; Shimmen, 1997 Anion channels from liverwort Trebacz et al., 1994 C. conicum Cells Plant in Gating Channel TrebaczÐIon and Krol Anion channels from Aldrovanda Iijima and Sibaoka, 1985 vesiculosa Anion channels from Cl Ca-dependent activation Phytochrome-mediated signalling Ermolayeva et al., 1996, 1997 Physcomitrella patens pathway Anion channels from Cl H‡- and Ca2‡-dependent activation (direct binding) Facilitation of enhanced proton e‚ux Johannes et al., 1998 Charophyta cells Phosphorylation/dephosphorylation processes under intracellular acidosis Anion channels from epidermal Cl Strong and weak voltage-dependence of R- and R-type may be involved in the Thomine et al., 1995, 1997; Cho and cells of Arabidopsis hypocotyls S-type unitary conductances, respectively transduction of external signals and Spalding, 1996; Elzenga and Van

(activation by Vm depolarization) transmission of AP Volkenburgh, 1997b; Lewis et al., 1997; R- and S-types have the same conductance but S-type may be involved in turgor Parks et al., 1998 di€erent open probabilities regulation and hypocotyl movements The switch between R- and S-type is controlled by ATP (R-type occurs in the presence of ATP) Modulated by phosphorylation/dephosphorylation processes Anion channels from epidermal Cl BL-activation (increase in open probability) Light-induced inhibition of cell Cho and Spalding, 1996 cells of Arabidopsis hypocotyls elongation Anion channels from mesophyll Cl Light-induced activation (increase in Light-induced transient membrane Elzenga et al., 1995, 1997; Elzenga and cells of Pisum sativum open probability) potential depolarization Van Volkenburgh, 1997a,b Ca-dependent activation Charge balance for light-induced H‡ pump activation, thus control of

pHext , membrane voltage and osmotic potential SAC from guard cells of Non-selective Stretch-activated Reduction of cell turgor Cosgrove and Hedrich, 1991 Vicia faba L. Activation of voltage-dependent ion channels through membrane depolarization SAC from Arabidopsis thaliana Control of leaf gas exchange Teodoro et al., 1998 guard cells Krol and TrebaczÐIon Channel Gating in Plant Cells 457

1999). Kourie (1996) demonstrated that the relative number K‡ channel protein is responsible for fast and reversible of opened voltage-activated inward rectifying potassium inactivation of inward K‡ currents in maize coleoptile channels increased sigmoidally as a function of hyper- protoplasts. polarized membrane potential. The kinetics of inward In addition to their Ca2‡ and pH dependence, voltage- ‡ ‡ rectifying K currents in Avena sativa mesophyll cells gated plasmalemma Kin channels seem also to require ATP ‡ reported by Kourie was independent of [K ]ext and it lacked (Hoshi, 1995; MuÈ ller-RoÈ ber et al., 1995; Wu and Assmann, ‡ time-dependent inactivation. Neither low [K ]ext nor 1995). Their structures contain ATP and cyclic nucleotide- ‡ [Na ]ext caused inactivation of the above-mentioned binding cassettes in the C-terminal domains. The rundown ‡ ‡ currents, while Cs -induced block was reversible and of Kin recti®ers in the absence of ATP is explained in terms strongly voltage-dependent. A role of preventing large of a shift in the voltage-dependence (Hedrich and Dietrich, membrane hyperpolarization resulting from electrogenic 1996). Kurosaki (1997) surveyed some of the inward K‡ ‡ proton pumping was proposed for these Kin channels by channels (located in the plasma membrane of cultured Kourie (1996). On the other hand, there are reports carrot cells) whose gating was controlled by cytoplasmic ‡ concerning Kin channels (AKT1) `sensing' external potas- concentration of cAMP. Their activation resulted in sium concentration (Bertl et al., 1997). AKT1 channels are transient membrane potential changes, which in turn present in root cells. Extracellular K‡ binds to a modulator activated voltage-gated Ca2‡ channels. Because plasma- site thereby enhancing the rate of opening of AKT1 protein. lemma voltage-gated inward K‡-channels described by

Blatt (1999) also noticed that IKin current in guard cells Hedrich and Dietrich (1996) and Kurosaki (1997) are requires external millimolar K‡ concentrations for its regulated via direct nucleotide binding to the channel ‡ ‡ activity. In submillimolar [K ]ext ,Kin channels appear to protein, they can be classi®ed as ligand-gated ones as well. enter a long-lived inactive state (Blatt, 1999). There is an obvious correlation between inward rectifying ‡ ‡ Control of plasmalemma Kin channels is modulated by K channels and cytoskeletal proteins (Table 2). As a 2‡ increasing [Ca ]cyt (inactivation) and increasing external conserved structural feature, proteins of the AKT subfamily proton concentration (voltage-dependent activation) or contain so-called ankyrin repeats which are potential decreasing pHcyt (voltage-independent activation; increase domains for interaction with the cytoskeleton (Czempinski in the pool of active channels through allosteric interaction) et al., 1999). Because proteins from the KAT subfamily lack (Ilan et al., 1996; Grabov and Blatt, 1997, 1998a; Hoth such ankyrin sequences, but they are `sensitive' to et al., 1997; MacRobbie, 1997). Ca2‡-dependent inactiva- cytoskeletal drugs, there must be other channel domains tion can proceed even when pH is bu€ered. Equally, participating in the regulation by cytoskeletal compounds changes in pH and channel gating may occur without (Hwang et al., 1997; Czempinski et al., 1999). Pharmaco- measurable changes in calcium concentration (Allan et al., logical studies on guard cells have shown that actin ‡ 1994; Armstrong et al., 1995). Thus, the e€ects of cellular ®laments contribute to regulation of Kin channels as well pH and calcium are separable, although these two ionic as of stomatal aperture (Hwang et al., 1997). Cytochalasin messengers do interact. In other words, pHcyt may act in D, which induces depolymerization of actin ®laments, 2‡ parallel with, but independently of, [Ca ]cyt in controlling activates inward potassium currents, while phalloidinÐa ‡ Kin channels (Grabov and Blatt, 1997). Kim et al. (1996) stabilizer of ®lamentous actinÐinhibits them (Hwang et al., reported that phosphoinositide turnover, phospholipase C 1997). These authors demonstrated that polymerized actin ‡ (PLC) activation or the presence of inositol triphosphate stabilizes Kin channels in the closed state and thus makes ‡ (IP3) is correlated with Kin channel closure. Earlier, Blatt them unresponsive to membrane hyperpolarization. As ‡ ‡ et al. (1990) demonstrated the possibility of controlling Kin actin ®laments depolymerize, the closed state of Kin 2‡ channel activity by IP3-mediated Ca release. Both the channels becomes less stable and more channels become 2‡ above-mentioned results indicate that increase in [Ca ]cyt is ready to respond to the hyperpolarized membrane poten- ‡ responsible for Kin channel inactivation and they support a tial. Liu and Luan (1998) also correlated regulation of IKin growing body of evidence that G-proteins function in with the pattern of organization of actin ®laments. They regulating IKin (reviewed by Blatt and Grabov, 1997a,b). stated that actin structure may be a critical component in 2‡ ‡ Recently, Li et al. (1998) identi®ed a Ca -dependent the osmosensing pathway conducted by Kin channels in protein kinase, with a calmodulin-like domain (CDPK), plants. ‡ which phosphorylates Kin channels of Vicia faba guard cell There are also reports of auxin-induced modulation of protoplasts. Moreover, the cAMP-dependent signalling K‡-inward recti®ers at the plasma membrane in coleoptile 2‡ ‡ system `cross-talks' with Ca -dependent inhibition of Kin cells (Claussen et al., 1997) and guard cells (Blatt and Thiel, channels from Vicia faba guard cells by reversing inhibitory 1994)(Table 2). ‡ calcium e€ects (Jin and Wu, 1999). In contrast to Kin ‡ channels from guard cells, Kin channels in the plasma- 2‡ Voltage-gated potassium channels in the tonoplast lemma of rye root cells are insensitive to [Ca ]cyt (White, 1997). In the tonoplast of higher plants there are three distinct ‡ Kin channels (KAT1ÐArabidopsis thaliana, KST1Ð kinds of voltage-sensitive potassium channels (FV, fast Solanum tuberosum) can also be inhibited by Ca2‡ and Cs‡ activating; SV, slow activating; and VK, strongly K‡ select- via competition in binding to the pore forming region ive) (Table 3). FV channels are instantaneously activated at 2‡ exposed to the aqueous lumen of the channel (Becker et al., the resting levels of [Ca ]cyt and pHcyt by changes in 1996). Thiel et al. (1996) showed that Ca2‡-binding to the tonoplast voltage (Allen et al., 1998). They open at cytosol 458 TABLE 3. Ion channels in plant endomembranes

Channel Permeability Gating mechanism Physiological role References

Potassium channels

‡ ‡ Tonoplast FV (fast-activating) NH4 ,K , Voltage-dependent open probability Control of the tonoplast electrical Linz and KoÈ hler, 1994; Allen and Sanders, ‡ ‡ cation channels Rb ,Cs , Preferred outward recti®cation at positive potentials potential di€erence around EK 1996, 1997; Maathuis et al., 1997; Na‡,Li‡ (relative to the cytoplasm) A shunt conductance for the Tikhonova et al., 1997; Allen et al., 1998a; 2‡ ‡ Active at the resting levels of [Ca ]cyt and pHcyt vacuolar H pumps Grabov and Blatt, 1998a; BruÈ ggemann et al., Inhibited by vacuolar and cytosolic Ca-increases Involvement in potassium release 1999a,b; Dobrovinskaya et al., 1999

FV currents are reduced at acidic pHcyt during stomatal closure ATP regulated Involvement in increase in cellular Cells Plant in Gating Channel TrebaczÐIon and Krol Blocked by Mg2‡ and polyamines osmolarity Small monovalent cation uptake Tonoplast SV (slow-activating) K‡,Na‡, Time-dependent activation at cytosol-positive potentials Vacuolar receptor site for calcium Ward and Schroeder, 1994; Allen and cation channels Rb‡,Li‡, Outward-rectifying during stomatal closure Sanders, 1995, 1996, 1997; Schulz-Lessdorf ‡ 2‡ 2‡ 2‡ NH4 ,Ca , Strong voltage-dependence modulated by Ca ,Mg Possible participation in CICR and Hedrich, 1995; Ward et al., 1995; Mg2‡, and H‡ ions (Ca- and Mg-activation and down- (Ca-induced Ca-release) Gambale et al., 1996; Bethke and polyamines regulation of SV channel activity by protons) Turgor regulation Jones, 1997; Maathuis et al., 1997; Ca2‡ induces lowering of the voltage threshold for Vacuolar ion transport MacRobbie, 1997; McAinsh et al., 1997; activation Allen et al., 1998b; Grabov and Blatt, Require alkaline pH at both sides of tonoplast 1998a; Carpaneto et al., 1999; Cerana et al., Regulated by protein phosphorylation and calmodulin 1999; Dobrovinskaya et al., 1999 interaction ‡ Single channel conductance dependent on [K ]cyt Modulated by redox agents (increased open probability in the presence of antioxidants) Blocked by polyamines in a voltage-dependent manner ‡ ‡ ‡ 2‡ Tonoplast VK (vacuolar K ) K ,Rb , Activated by micromolar [Ca ]cyt and acidic pHcyt Ca-dependent potassium uptake and Ward et al., 1995; Allen and Sanders, 1996, ‡ channels NH4 Voltage-independent, non-rectifying channel release during stomatal 1997; Maathuis et al., 1997; movements (e.g. ABA-induced MacRobbie, 1997; McAinsh et al., 1997; stomatal closure) Allen et al., 1998a; Grabov and Blatt, 1998a Activation of voltage-gated tonoplast channels ‡ ‡ 2‡ Cation-selective channel from K ,Na Activated by micromolar [Ca ]cyt Osmotic volume and turgor Katsuhara and Tazawa, 1992; tonoplast of algae Voltage dependence regulation LuÈ hring, 1999 Lamprothamnium, Chara Strong pH-dependence (inhibition by acidic pH) buckellii, Chara australis and Nitellopsis obtusa Cation-selective channel from K‡,Ca2‡, Voltage-gated (activated by positive membrane potentials Compensation of light-induced Pottosin and SchoÈ nknecht, 1996; thylakoids of Spinacea oleracea Mg2‡ of stroma relative to lumen) proton ¯uxes Hinnah and Wagner, 1998 and Pisum sativum cotyledons Cation-selective channel from Multi-cation Voltage-dependent Metabolite di€usion Heiber et al., 1995 chloroplast envelope Cation-selective channel from K‡ Voltage-dependent Compensation of light-driven Heiber et al., 1995 chloroplast envelope ATP-regulated proton movements Modulated by Cs‡,Mg2‡ Cation-selective channel from Multi-cation Ca-regulated voltage-dependence Ca-regulated pathways for nuclear Grygorczyk and Grygorczyk, 1998 nuclear envelope from red beet processes Calcium channels

2‡ 2‡ Ligand-gated Ca-channel from Ca Activation by IP3-binding Ca -release during signal Muir and Sanders, 1996, 1997; Allen and vacuole and ER of cauli¯ower Ca-current recti®cation over physiological tonoplast transduction Sanders, 1997; Muir et al., 1997; Leckie and vacuoles of guard cells, potentials (cytosol negative with reference to lumen) et al., 1998; MacRobbie, 1997; McAinsh zucchini hypocotyls, oat roots, et al., 1997 carrot and red beet roots, mung bean hypocotyls, maize cells Ligand-gated Ca-channel in Ca2‡,K‡ Activation by cADPR-binding Ca2‡-release during signal Allen et al., 1995; Muir and Sanders, 1996; vacuoles from red beets and transduction Allen and Sanders, 1997; McAinsh et al., cauli¯ower ¯orets 1997; Muir et al., 1997; Leckie et al., 1998 Ligand-gated Ca-channel from Ca2‡ Activation by cADPR-binding Ca2‡-release during signal Bauer et al., 1998 alga Eremosphaera viridis transduction Cells Plant in Gating Channel TrebaczÐIon and Krol VVCa (voltage-gated Ca- Ca2‡,K‡ Voltage-dependent (hyperpolarization activated) Intracellular Ca2‡-release Allen and Sanders, 1994, 1997; Johannes channels from vacuoles of Vicia pH-sensitive and Sanders, 1995; McAinsh et al., 1997; faba guard cells and Beta Require two Ca2‡ ions binding to open Pineros and Tester, 1997 vulgaris roots) Luminal Ca2‡ shifts the threshold for voltage activation to less negative potentials 2‡ Inhibited by [Ca ]cyt increases Ca-channel from ER of Bryonia Ca2‡ Voltage-dependent Intracellular Ca2‡-release during KluÈ sener et al., 1995 diodica tendrils Ca2‡-gradient sensitive responses to mechanical stimuli Anion channels

Vacuolar malate channelÐVMal Malate Activation by potentials more negative than EMal Vacuolar malate uptake Cerana et al., 1995; Allen and Sanders, from Arabidopsis thaliana fumarate, Strong inward recti®cation because of luminal Cl 1997; Chengs et al., 1997 vacuoles acetate NO3 ; blockade of malate re-entry H2PO4 Vacuolar chloride channelÐVCl Cl ,NO3 ; Activation by tonoplast hyperpolarization (negative Vacuolar anion uptake Allen and Sanders, 1997 2 SO4 potentials relative to the cytoplasm)Ðinward-recti®cation Vacuolar VCl from Vicia faba Cl Activation by tonoplast hyperpolarization Anion uptake during stomatal Pei et al., 1996; Grabov and Blatt, 1998a guard cells Channel activation depends on protein phosphorylation opening Inward-recti®cation Vacuolar anion channel from Cl ,NO3 Outward rectifying Ca-dependent regulation Turgor regulation Katsuhara and Tazawa, 1992 Characean cells VDAC (voltage-dependent anion Non-selective Voltage-dependent Control of mitochondrial membrane Elkeles et al., 1997; Mannella et al., 1997, channels in outer membrane of pH-dependent potential 1998; Rostovtseva and Colombini, 1997; mitochondria) Second messenger-binding Control of ATP di€usion Green and Reed, 1998; Song et al., 1998; Control of signal transduction Shimizu et al., 1999 Inner membrane anion channel Non-selective pH-regulated (activated by low matrix pH) Anion uniport Beavis and Vercesi, 1992 from mitochondria Anion channel from outer Non-selective Voltage-dependent Metabolite di€usion Heiber et al., 1995; Pohlmeyer et al., 1998 envelope of chloroplasts Anion channel from inner Cl Voltage-dependent Compensation of light-driven Heiber et al., 1995; Fuks and Homble, 1999 envelope of chloroplasts proton movements Anion channel from thylakoids Cl Voltage-dependent Compensation of light-driven Heiber et al., 1995; Pottosin and proton movements SchoÈ nknecht, 1995 459 460 Krol and TrebaczÐIon Channel Gating in Plant Cells positive vacuolar membrane potential for longer times than Cytosolic polyamines are strong inhibitors of SV at negative potentials and hence they mainly allow K‡ and channels, but in contrast to the inhibition of FV channels, ‡ NH4 e‚ux from the cytoplasm into the vacuole (preferred the blockage of SV channels displays a pronounced voltage- outward recti®cation) (Tikhonova et al., 1997; BruÈ ggemann dependence (Dobrovinskaya et al., 1999). Hence, et al., 1999b). Their function is to control the electrical polyamine-blockage is relieved at a large depolarization potential di€erence across the tonoplast (Tikhonova et al., (because of the permeation of polyamines through the 1997). Vacuolar Ca2‡ suppresses FV channels in a voltage- channel pore) and in the presence of high concentrations of dependent manner while cytosolic Ca2‡ blocks them in a polyamines the slow vacuolar channels are converted into voltage-independent manner (Allen and Sanders, 1996; inward recti®ers (Dobrovinskaya et al., 1999). Tikhonova et al., 1997; Allen et al., 1998a). One of the most VK channels are non-rectifying and are activated at 2‡ pronounced features of FV channels is their blockade by micromolar [Ca ]cyt and acidic pHcyt by tonoplast poten- Mg2‡. Increasing cytosolic free Mg2‡ decreases the open tials ranging from 100 to ‡60 mV (Allen and Sanders, probability of FV channels without a€ecting single current 1996; Allen et al., 1998a). Therefore, they can be involved in amplitudes (BruÈ ggemann et al., 1999a). FV currents were vacuolar potassium uptake and loss. So far their presence also shown to be reduced at acidic pHcyt (Linz and KoÈ hler, has been proved only in guard cells. 1994) or by cytosolic polyamines (Dobrovinskaya et al., Di€erent sensitivities of FV-, SV- and VK-channels to 2‡ 1999). Recent studies on FV currents in red beet vacuoles [Ca ]cyt and pH may provide a mechanism whereby stimuli indicate that FV channels may be ATP regulated (Allen activating various signalling pathways can generate et al., 1998a). vacuolar ion uptake or loss. Muir et al. (1997) concluded SV channels are strictly outward rectifying, cation that this di€erential regulation of vacuolar channels by selective and they show characteristics typical of a multi- Ca2‡ represents a downstream event in signal transduction ion pore, i.e. more than one ion can occupy the channel cascades induced by Ca2‡-release. SV channels are thought pore at the same time (Allen and Sanders, 1996). They to participate in signalling processes because of their ability display time-dependent activation at cytosol-positive poten- to release Ca2‡ after Ca2‡-dependent activation (CICRÐ 2‡ . 2‡ 2‡ tials and when [Ca ]cyt is higher than approx. 0 5 mM Ca -induced Ca -release) (Allen et al., 1998b). However, (Schulz-Lessdorf and Hedrich, 1995; Allen and Sanders, Pottosin et al. (1997) demonstrated that the SV channel is 1996). Calcium and protons modulate the voltage- not suited for CICR from vacuoles, at least in the case of dependence of SV channels (Schulz-Lessdorf and Hedrich, barley mesophyll cells. Thus, the physiological role of SV 1995). These two cations interact strongly with the voltage channels remains a matter for discussion. ‡ sensor without changing the unitary conductance. The The most frequently observed voltage-gated Kin -channel open probability of the SV-type channel is a function of in the tonoplast of Chara was examined by LuÈ hring (1999) 2‡ [Ca ]cyt (Gambale et al., 1996). Schulz-Lessdorf and (Table 3). Acidi®cation on both sides of the membrane Hedrich (1995) showed that there is a regulatory Ca2‡- decreases open probability of the channel and changes its binding site on the cytoplasmic face of the SV channel and voltage-dependence, most probably through protonation of that calmodulin may be involved in the modulation of the negatively charged residues in neighbouring voltage-sensing activation threshold of the SV-type channel. This is in transmembrane domains (LuÈ hring, 1999). The channel agreement with recent results of Bethke and Jones (1997) behaves like animal maxi-K channels and its gating kinetics who examined SV currents stimulated by both calmodulin- responds to cytosolic Ca2‡. Under natural conditions, like domain protein kinase (CDPK) and okadaic acid- pH changes contribute mainly to channel regulation at the sensitive phosphatases. On the other hand, Ca2‡-dependent vacuolar membrane face (LuÈ hring, 1999). protein phosphatase can induce the inhibition of SV channels (Allen and Sanders, 1995). Bethke and Jones Voltage-gated potassium channels in other intracellular (1997) proposed a model in which SV channel activity is membranes regulated by protein phosphorylation at two sites. In the absence of calcium ions, Mg2‡ can activate SV currents Heiber et al. (1995) showed that the chloroplast envelope (Allen and Sanders, 1996; Cerana et al., 1999). Moreover, contains voltage-dependent cation channels (Table 3) with the single channel conductance increases as a function of complex gating behaviour and subconductace states, as well the potassium concentration (Gambale et al., 1996). This as cation-selective pores with high conductances. Voltage- behaviour can be explained by a multi-ion occupancy dependent cation channels favour potassium uptake and mechanism. However, at negative transtonoplast voltages, their gating is a€ected by monovalent cations (Cs‡), the closure of SV channels is una€ected by either Ca2‡ or divalent cations (Mg2‡) and millimolar concentrations of Mg2‡, indicating that the channel belongs to the voltage- ATP. Hinnah and Wagner (1998) observed potassium gated superfamily (Cerana et al., 1999). SV channels are selective pore-like channels in osmotically swollen thyla- also reversibly activated by a variety of sulphydryl reducing koids from pea protoplasts derived from cotyledons of agents at the cytoplasmic side of the vacuole (Carpaneto young Pisum sativum plants (Table 3). There is also a et al., 1999). Increase in the open probability in the presence nonselective (PK 4 PMg 4 PCa) cation channel in native of antioxidants may correlate ion transport with other spinach thylakoid membranes (Table 3) found by Pottosin crucial mechanisms that in plants control turgor regulation, and SchoÈ nknecht (1996). This cation channel displays response to oxidative stresses, detoxi®cation and resistance bursting behaviour and its open probability increases at to heavy metals (Carpaneto et al., 1999). positive membrane potentials (Pottosin and SchoÈ nknecht, Krol and TrebaczÐIon Channel Gating in Plant Cells 461

1996). It has only a moderate voltage-dependence com- inhibition of IKin can be prevented by disruption of actin pared to classical voltage-dependent recti®ers. It is postu- ®laments. Actin ®lament disruption occurs in hypotonic lated that its function is to compensate the light-driven conditions providing a link between hypotonic stress and proton uptake into thylakoids (Pottosin and SchoÈ nknecht, hypotonic activation of the inward K‡ channels. Also 1996). cytochalasin D (a cytoskeleton disrupting drug) modulates 2‡ ACa - and voltage-dependent non-speci®c channel was IKin in a similar way to hypotonic conditions (Liu and found in the nuclear envelope of red beet (Grygorczyk and Luan, 1998), which is consistent with the report of Hwang Grygorczyk, 1998)(Table 3). Micromolar [Ca2‡] on the et al. (1997). It seems reasonable that stretch-activated nucleoplasmic side of the envelope activates this cation channels in the plant plasma membrane, which is under channel. The channel voltage-dependent activity changes continuous compression resulting from turgor pressure and with the nucleoplasmic calcium concentration. Such a the presence of the cell wall, interact with cytoskeletal channel may provide a Ca2‡-regulated pathway for Ca2‡- structures providing local stretch of the membrane. It is dependent nuclear processes (e.g. gene transcription). postulated that during perception of gravitational stimuli, statoliths exert local stretch on the membrane via cyto- skeletal ®bres (Sievers et al., 1996). Plasmalemma voltage-insensitive cation channels (VIC) The VIC channels are responsible for an in¯ux of a range Light-activated potassium channels of monovalent cations into cereal root cells (Table 2). It has been postulated that they could contribute to low-anity Blom-Zandstra et al. (1997) examined light e€ects on ‡ ‡ NH4 uptake and rapid osmotic adjustment independent of voltage-gated Kout channels in mesophyll protoplasts of membrane potential. They may also compensate electro- Nicotiana tabacum (Table 2). Single channel data from genic cation ¯uxes (White, 1999). Under saline conditions patch-clamp studies indicate that the activity of the channel ‡ VIC channels along with Kin channels play a major role in increases upon dark-light transition. The e€ect of light was the toxic Na‡ in¯ux across the plasma membrane (White, not observed in root cells or chlorophyll-de®cient cells, 1999). Inward currents through the VIC channels are suggesting that such a response requires photosynthetic inhibited by Ca2‡ and Ba2‡. activity. These results are consistent with those of Kim et al. (1992) who showed that K‡ channels display responses to light. The light activated ion channels and electrogenic Stretch-activated potassium channels proton pump are regarded as important factors in the not Changes in turgor pressure induced by hyper- or hypo- yet fully understood light stimulus transduction cascade osmotic stress induce an early change in activities of stretch- (discussed by Szarek and Trebacz, 1999). sensitive channels. Stretch-activated channels (SACs) also respond when mechanical forces are exerted on the cell CALCIUM CHANNELS (Ramahaleo et al., 1996). For the translation of membrane stretch into channel gating it is generally argued that Calcium ions are universal second messengers in plant and attachment of membrane proteins to tension-transmitting animal cells. They mediate in various signalling pathways components is necessary, by linkage to cell wall proteins, or (reviewed by Brownlee et al., 1999; Sanders et al., 1999) cytoskeletal proteins, or both (MacRobbie, 1997). Anionic, from signal perception to gene expression, through the cationic, as well as non-selective SACs, have been reported activation of ion channels and enzyme cascades. Stimulus- 2‡ to occur in plasma membranes (Table 2). There is a induced increases in [Ca ]cyt encode information as speci®c 2‡ growing body of evidence for involvement of stretch- spatial and temporal changes in frequency of [Ca ]cyt activated ion channels in regulation of the response of oscillationsÐthe `calcium signature' (McAinsh et al., guard cells to ABA through interactions with the cyto- 1997; Leckie et al., 1998). After signal transition, excess skeleton (MacRobbie, 1997; McAinsh et al., 1997). Liu and Ca2‡ must be sequestered into external and internal stores 2‡ Luan (1998) identi®ed two kinds of stretch-activated to keep [Ca ]cyt at a low level ranging from tens to potassium channels in Vicia faba guard cells: voltage- hundreds nM. Thus, all Ca2‡ channels located in Ca2‡- gated and insensitive to membrane potential. This was the sequestering membranes are strongly inward rectifying ®rst evidence that plants contain osmosensitive, voltage- (facilitating Ca2‡ in¯ux to the cytosol). dependent channels, those previously described by Rama- haleo et al. (1996) being voltage-independent. Negative Ligand-gated calcium channels in plasma membrane pressure activates voltage-insensitive currents with conduct- ance very di€erent from that of voltage-dependent K‡- Recently, Zimmermann et al. (1997) reported a novel 2‡ 3‡ channels. Voltage-dependent currents (IKin and IKout)arein Ca -permeable, La -sensitive plasma membrane ion turn sensitive to osmotic gradient rather than changes in channel of large conductance (Table 2). The channel is pressure, although actin ®laments are involved in IKin activated by elicitors and is essential in pathogen defence. regulation (Liu and Luan, 1998). Hypotonic conditions Receptor-mediated stimulation of these channels appears to activate IKin and inactivate IKout , while hypertonic con- be involved in the signalling cascade triggering a pathogen ditions act in the opposite way. An alternation in channel defence system. The activation of plasma membrane Ca2‡- opening frequency is responsible for regulating IKin and channels by speci®c and non-speci®c elicitors provides a 2‡ IKout under di€erent osmotic conditions. Hypertonic direct demonstration of a pathway by which [Ca ]cyt 462 Krol and TrebaczÐIon Channel Gating in Plant Cells increases to levels that can initiate the production of active recognized to date between these and animal channels oxygen species, callose and phytoalexins via Ca2‡± (Muir et al., 1997). dependent gene expression (Blumwald et al., 1998). Voltage-gated calcium channels in the plasmalemma Ligand-gated calcium channels in inner membranes Many voltage-gated Ca2‡ channels have been described Ligand-gated Ca2‡ channels in plant cells reported to in a variety of plant tissues and species (reviewed by Pineros date represent two classes: IP3 (inositol triphosphate)- or and Tester, 1997)(Table 2). Most of these are activated cADPR (cyclic ADP-ribose)-gated (Table 3). Recently a through membrane depolarization and stimuli causing ‡ new signalling moleculeÐNAADP (nicotinic acid adenine membrane depolarization such as increased [K ]ext dinucleotide phosphate)Ðhas been found in animal cells (Thuleau et al., 1994), Ca2‡ starvation (Reid et al., 1997), (Lee, 2000). Ligand-gated Ca2‡ channels are present only in cytokinins (Schumaker and Gizinski, 1993), light or intracellular compartments, and thus their existence pro- electrical pulses (Trebacz et al., 1994; Ermolayeva et al., vides a convenient mechanism for linking perception of 1996, 1997) mechanical stimulation (Shimmen, 1997), ABA stimuli (e.g. light, IAA, ABA, osmotic shock, pollination, (McAinsh et al., 1995; Grabov and Blatt, 1998b) and Nod-factors, cold shock) to intracellular calcium mobiliza- microtubule inhibitors (Thion et al., 1996). White (1998), tion (Knight et al., 1996; McAinsh et al., 1997; Muir et al., focusing on Ca2‡ channels in the plasma membrane of root 2‡ 1997; Trewavas and Malho, 1997). The IP3-induced Ca - cells, distinguished between them based on their di€erent release originates mainly from vacuolar stores, although in sensitivities to La3‡,Gd3‡ and verapamil. He discussed cauli¯ower, Muir and Sanders (1997) found at least two their roles in mineral nutrition, intracellular signalling and distinct membrane populations sensitive to IP3.IP3-induced polarized growth. Ca2‡-currents are inwardly rectifying and highly selective Kiegle et al. (2000), Gelli and Blumwald (1997) and for calcium (Allen and Sanders, 1997). A speci®c IP3- Stoeckel and Takeda (1995) described the hyperpolariza- binding 400-kDa protein, which is competent to release tion-activated in¯uxes of Ca2‡ through the plasmalemma. Ca2‡ when incorporated into proteoliposomes (Biswas The hyperpolarization-activated calcium current is postu- et al., 1995), was puri®ed from mung bean, though no lated to allow nutritive Ca2‡ uptake. Hyperpolarization- subsequent reports on this protein have appeared. There is activated Ca2‡ channels described in the plasma membrane 2‡ some indirect evidence for the presence of IP3-gated Ca of Vicia faba guard cells by Fairley-Grenot and Assmann channels in the tonoplast of the algae Chara and Nitella (1992) are in fact the inwardly rectifying K‡ channels (Katsuhara and Tazawa, 1992). mediating Ca2‡ in¯ux prior to their closure and they may 2‡ As well as IP3-gated channels, cADPR-gated Ca be involved in the regulatory mechanism of stomatal channels act as instantaneous strong inward recti®ers over aperture changes. physiological membrane potentials and they are activated by ligand binding only in the presence of calcium on the Voltage-gated calcium channels in inner membranes luminal side of the membrane. Pharmacological studies suggest that cADPR has the capacity to act as a Ca2‡- Voltage-gated Ca2‡-channels are also present in other mobilizing intracellular messenger and an endogenous cell compartments such as the vacuole, thylakoids or ER modulator of Ca2‡-induced Ca2‡ release (CICR) (Willmott (Pineros and Tester, 1997)(Table 3). Vacuolar voltage- et al., 1996). Ryanodine and ca€eine (agonists of ryanodine gated Ca2‡ channels (VVCa), characterized by Allen and receptors in animal cells) are able to cause activation of Sanders (1994), behave as multi-ion pores inwardly rectify- cADPR-gated channels in a dose-dependent manner (Allen ing over the voltage range between 20 and 50 mV et al., 1995), while ruthenium red and procaine (antagonists (hyperpolarization). Their activity is inhibited by lantha- 2‡ 2‡ of ryanodine receptors in animal cells) block Ca release nides, verapamil, nifedipine and by [Ca ]cyt above 1 mM. (Allen et al., 1995; Muir and Sanders, 1996; Bauer et al., Luminal Ca2‡ shifts the threshold for VVCa activation to a 1998) in plant cells. Heparin of low molecular mass and less negative potential, and therefore restricts the accumu-

TMB-8, well known competitive inhibitors of IP3-receptors lation of calcium excess in the vacuole. Luminal pH of in plant and animal cells, are without e€ect on cADPR- about 5.5 prevents uncontrolled leakage of Ca2‡, because at gated Ca2‡-channels (Muir and Sanders, 1996). Allen et al. this physiological pH value the channel openings are very (1995) demonstrated that there is a relatively low density of infrequent (the highest activation is around pH 7). cADPR-gated channels in beet microsomes. cADPR-gated Johannes and Sanders (1995) showed that a binding of channels could participate in calcium release only up to two calcium ions is required to open the VVCa channel. 2‡ 2‡ 25 % in comparison to the dominating IP3-induced Ca - Voltage-gated vacuolar Ca channels, previously release. Similar results were obtained from cauli¯ower described in tonoplasts of beet, Arabidopsis and tobacco, microsomes (Muir et al., 1997) and the unicellular green are in fact manifestations of SV K‡ channels (Ward and alga Eremosphaera viridis (Bauer et al., 1998). Preliminary Schroeder, 1994). experiments on sea urchin egg homogenates indicate that KluÈ sener et al. (1995, 1997) have shown the voltage-gated cADPR may bind to an accessory 100±140 kDa protein Ca2‡ channels derived from endoplasmic reticulum mem- (Galione and Summerhill, 1996). branes of Bryonia dioica touch-sensitive tendrils. The range The lack of modulation of plant ligand-gated Ca2‡- of membrane potentials activating these channels was channels by cytosolic Ca2‡ is the most notable di€erence a€ected by the Ca2‡ gradient across the membrane. Single Krol and TrebaczÐIon Channel Gating in Plant Cells 463 channel currents were modulated by divalent cations, intracellular acidosis. H‡-activated anion channels respon- protons and H2O2 .H2O2 is a strong inhibitor of these sible for Cl currents act to facilitate an enhanced proton channels. The channel conductance increases with cytosol e‚ux under conditions of low pHcyt . Activity of these acidi®cation. These channels play an important role in the channels is also indirectly pH- and Ca2‡-dependent 2‡ modulation of [Ca ]cyt in response to changes in [H2O2]cyt through phosphorylation/dephosphorylation processes. or pHcyt . The above-mentioned ®ndings imply that plasma membrane anion channels play a central role in pHcyt regulation in plants, in addition to their established roles Stretch-activated calcium channels in turgor/volume regulation and signal transduction. Taylor et al. (1996) examined both stretch-activated and voltage-gated mechanosensitive Ca2‡-permeable cation Ligand-gated anion channels in the tonoplast channels in subprotoplasts prepared from di€erent regions of rhizoid and thallus cells of Fucus zygotes (Table 2). Their Katsuhara and Tazawa (1992) summarized calcium- results suggest that intercellular signal transduction is regulated channels and their bearing on physiological patterned by interactions of the cell wall, plasma membrane activities in characean cells. They presented some evidence and intracellular Ca2‡ stores. for the presence of Ca2‡-regulated anion channels in the Thion et al. (1996) observed activation of voltage-gated tonoplast of Chara, Nitellopsis and Lamprothamnium giant Ca2‡ channels by microtubule disruption. Their results are internodal cells (Table 3). Activation of these channels by 2‡ consistent with a previous report of Davies (1993), who [Ca ]cyt is assumed to occur during turgor regulation. postulated that variation potentials can be transduced via mechano-sensitive Ca2‡ channels into gene expression Voltage gated anion channels in the plasma membrane through Ca2‡-dependent cytoskeleton-associated phos- phorylation/dephosphorylation processes. In addition, In the plasma membrane, voltage-gated anion channels Ca2‡ in¯ux through `volume sensing' voltage-gated Ca2‡ are activated by depolarization and under an excess of channels is essential for an apical Ca2‡ gradient to be cytoplasmic Ca2‡. They deactivate under hyperpolarizing maintained in a growing cell (Taylor et al., 1996; Holdaway- potentials (Keller et al., 1989; Hedrich et al., 1990; Hedrich Clarke et al., 1997). and Jeromin, 1992; Linder and Raschke, 1992; Schroeder and Keller, 1992; Dietrich and Hedrich, 1994; Thomine et al., 1995; Schultz-Lessdorf et al., 1996; Lewis et al., 1997; ANION CHANNELS Pei et al., 1998). Inverse voltage dependence (activation by Plant anion channels regulate anion e‚ux from a cell hyperpolarization) has been reported infrequently to date. through plasmalemma (Table 2) and/or tonoplast Barbara et al. (1994) reported hyperpolarization-activated (Table 3). Anion e‚ux from the cytoplasm into the chloride currents contributing both to the control of extracellular space is driven by the anion gradient and the membrane potential and to osmotic balance regulation in negative membrane potential causing plasma membrane carrot cells. Neither calcium ions nor MgATP were depolarization, which in turn activates outward rectifying necessary for fast activation of these channels. Under voltage-gated K‡ channels. Anion-induced depolarization large hyperpolarization, Barbara et al. (1994) observed plays a crucial role in such processes as xylem loading, rapid and voltage-dependent channel inactivation. generation and propagation of action potentials or light- Recently, hyperpolarization-activated anion channels have induced transient voltage changes of membrane potential. also been found in the plasmalemma of the unicellular In addition, anion and potassium losses promote osmo- green alga Eremosphaera viridis (SchoÈ nknecht et al., 1998). regulation, stomatal closure, tissue and organ movements. They conduct an anion e‚ux and hence they are respons- Since plant cells experience low extracellular anion concen- ible for limiting the amplitude of dark-induced transient trations, anion uptake must be energetically coupled with hyperpolarization caused by K‡-release. The well-known proton pumps. anion channel inhibitors such as A-9-C, NPPB and Zn2‡ block these channels. Elzenga and Van Volkenburgh (1997b) reported that in pea mesophyll cells there are Ligand-gated anion channels in the plasmalemma Ca2‡-dependent anion currents activated by hyperpolar- There are many anion channels activated by cytoplasmic izing pulses. These anion channels display ATP-dependent calcium widespread in plant cells (Katsuhara and Tazawa, bi-modular (fast and slow) kinetics. R-mode (fast acti- 1992). Ca2‡-dependent anion channels are responsible for vation and deactivation of the channel) occurs in the the main depolarizing current during action potential in absence of ATP. However when 3 mM MgATP is added to Charophyta (Okihara et al., 1991; Katsuhara and Tazawa, the pipette solution facing the cytoplasmic side of the 1992; Thiel et al., 1993; Shimmen, 1997), the liverwort membrane, the current shows slow but clear time- Conocephalum conicum (Trebacz et al., 1994), Aldrovanda inactivation (S-mode). vesiculosa (Iijima and Sibaoka, 1985) and during phyto- Dietrich and Hedrich (1994) showed the bimodular chrome-mediated transient depolarization in the moss kinetics of the guard cell anion channel (GCAC1) in Vicia Physcomitrella patens (Ermolayeva et al., 1996, 1997). faba protoplasts. Previously these two modes of one guard Johannes et al. (1998) showed a direct e€ect of cyto- cell anion channel were considered as two anion channels plasmic protons on Cl e‚ux in Chara corallina during contributing to di€erent depolarization-associated processes 464 Krol and TrebaczÐIon Channel Gating in Plant Cells during regulation of stomatal movements (Schroeder and carrier-mode action. In the case of R-mode, the proton Keller, 1992). Dietrich and Hedrich (1994) noted that the gradient does not seem to a€ect channel activation mode of action of GCAC1 is under the control of following ATP-binding. The single channel activity of ‡ cytoplasmic factors. Later Thomine et al. (1995) also R-type GCAC1 increases as a function of [H ]cyt (proto- identi®ed a voltage-dependent anion channel in epidermal nation of the cytoplasmic site of the channel), while single cells of Arabidopsis hypocotyls which showed two-mode channel conductance is una€ected either by pHcyt or pHext . function: rapid and slow mode in the presence or absence of Similar pH sensitivity was determined for anion-permeable intracellular ATP, respectively. R-type and S-type channels vacuolar channels (Schulz-Lessdorf and Hedrich, 1995). are voltage-regulated in a quite di€erent way and they Since the time- and voltage-dependent activity of guard cell display di€erent kinetics. Only R-type anion channels anion channels (GCAC1) was shown to be strongly display strong voltage-dependence, while weak voltage- modulated by ATP and H‡ (Schulz-Lessdorf et al., 1996), dependence of S-type channels leaves them partially active these channels have been thought to be capable of sensing even when the membrane is strongly hyperpolarized. Such changes in the energy status, the proton pump activity and behaviour of S-type channels makes them responsible for acid metabolism of the cell. sustained e‚ux of anions (Keller et al., 1989; Linder and Patch-clamp studies revealed that growth hormones can Raschke, 1992; Schroeder and Keller, 1992; Schroeder et al., directly a€ect voltage-dependent activity of inwardly 1993; Thomine et al., 1995), which serves as a negative rectifying anion channels in a dose-dependent manner regulator during stomatal opening (Schroeder et al., 1993; (Hedrich and Jeromin, 1992). Auxin binding is side- and Pei et al., 1998) or hypocotyl movements (Cho and channel-speci®c, and results in a shift of the activation Spalding, 1996). Transition between R- and S-mode of an potentials towards the resting potential favouring transient anion channel may correspond to ATP binding (Schulz- channel opening (Marten et al., 1991). These authors Lessdorf et al., 1996; Thomine et al., 1997) or alternatively demonstrated that auxin can interact directly with the to ATP-dependent phosphorylation/dephosphorylation extracellular face of the channel, eliciting stomatal opening. processes (Schmidt et al., 1995; Thomine et al., 1995). R-type guard cell anion channels (GCAC1) in Arabidopsis Voltage-gated anion channels in the tonoplast were shown not to be directly regulated by phosphorylation events (Schulz-Lessdorf et al., 1996). They require cyto- In the tonoplast (Table 3) there are two types of cytosol- plasmic ATP to undergo voltage- and Ca2‡-dependent negative-potential-activated (hyperpolarization-activated) activation, involving strongly cooperative binding of four anion channels: VCl and VMal (Allen and Sanders, ATP molecules (Schulz-Lessdorf et al., 1996). On the other 1997). The ®rst is responsible for carrying Cl to the hand, S-type GCAC1 channels are strongly activated by vacuole (inward rectifying), while the second is mainly phosphorylation (in Vicia faba and Commelina communis permeable for malate, but also for succinate, fumarate, guard cells) or dephosphorylation (in Arabidopsis and acetate, oxaloacetate, NO3 and H2PO4 : VMal is very Nicotiana cells) (Armstrong et al., 1995; Schmidt et al., strongly inward rectifying over the physiological range of

1995; Cho and Spalding, 1996; Li and Assmann, 1996; negative potentials, but more negative than Emal (Cerana Schulz-Lessdorf et al., 1996; Esser et al., 1997; Mori and et al., 1995). Cytosolic Ca2‡ and ATP do not a€ect VMal Muto, 1997; Pei et al., 1997, 1998; Schwarz and Schroeder, channels (Cerana et al., 1995; Chengs et al., 1997). On the 1998). Therefore, guard cell anion channels characterized in other hand, Pei et al. (1996) reported that calmodulin-like Arabidopsis (GCAC1) can also be classi®ed as ligand-gated domain protein kinase (CDPK) activates vacuolar malate channels, since Schulz-Lessdorf et al. (1996) showed direct and chloride conductances (VCl) in guard cell vacuoles of binding of ATP to the channel protein. Leonhardt et al. Vicia faba. Activation of both currents depends on Ca2‡ (1999) in turn, suggest that the slow anion channel in guard and ATP, enabling anion uptake into the vacuole even at cells may belong to the class of ATP binding cassette (ABC) physiological potentials. CDPK-activated VCl currents proteins. The same situation applies in the case of voltage- were also observed in red beet vacuoles, suggesting that gated and nucleotide-regulated anion channels of Arabi- these channels may provide a more general mechanism for dopsis hypocotyls described by Thomine et al. (1997). They kinase dependent anion uptake (Pei et al., 1996). con®rmed that nucleotide binding (ATP 4 ADP  AMP) regulates channel activity (alters the kinetics and voltage- Voltage-dependent anion channels in other endomembranes dependence, causing a shift toward depolarized potentials and thus leading to a strong reduction of anion current Voltage-dependent anion channels (VDACs or mitochon- amplitude). This regulation may couple the electrical drial porins) in the outer membrane of mitochondria properties of the membrane with the metabolic status of regulate the mitochondrial membrane potential, among the whole cell. other things, during transduction of an apoptotic signal into Rapid- and slow-modes of the Arabidopsis guard cell the cell (Green and Reed, 1998; Shimizu et al., 1999)or anion channel (GCAC1) are also variously in¯uenced by metabolite di€usion (Elkeles et al., 1997; Rostovtseva and pHcyt (Schulz-Lessdorf et al., 1996). The kinetics of S-mode Colombini, 1997; Mannella, 1998)(Table 3). According to is in¯uenced by the pH gradient across the plasmalemma Rostovtseva and Colombini (1997) these channels are (the inactivation gate responds to pH gradient, which may ideally suited to controlling the ¯ow of ATP between the be converted into a change of a channel structure). Such cytosol and the mitochondrial spaces. VDAC pore is formed pH gradient-dependence of slow inactivation resembles a by a single 30-kDa protein (Song et al., 1998) which Krol and TrebaczÐIon Channel Gating in Plant Cells 465 undergoes a major conformational change at pH 5 5 channel directly, but that it does so through intermediates (Mannella, 1997, 1998). However, functional VDAC is a (e.g. Ca2‡-dependent kinases or phosphatases). Activation heterodimer including one pore protein and other modulat- of blue light-induced anion channels plays a central role in ing subunits (Elkeles et al., 1997). Apart from transmem- transducing light signals into hypocotyl growth inhibition brane voltage and pH, VDACs can be regulated by direct (Cho and Spalding, 1996; Parks et al., 1998). binding of signalling proteins (Shimizu et al., 1999). Light-activated anion channels, resembling those above, Anion uniport in plant mitochondria is mediated by a were also reported by Elzenga and Van Volkenburgh pH-regulated inner membrane anion channel that is acti- (1997a) (Table 2). They examined light-induced transient vated by matrix H‡ (Beavis and Vercesi, 1992). Voltage- depolarization in Pisum sativum mesophyll cells due to dependent inner mitochondrial anion channels (IMACs), increased conductance for anions and concluded that: which serve as a safeguard mechanism for recharging the (1) under illumination the anion current increases three- mitochondrial membrane potential, have been found in fold because of an increase in the open probability of a 32- animal tissues (Ballarin and Sorgato, 1996; Borecky et al., pS anion channel; (2) this change in channel activity is not 1997). due to light-induced changes in membrane potential; (3) the Voltage-dependent anion channels were characterized by anion current depends on light intensity and can be totally a patch-clamp study in osmotically swollen thylakoids from blocked by the photosynthetic inhibitor DCMU; (4) the Peperomia metallica (SchoÈ nknecht et al., 1988) and the alga anion current is strongly Ca2‡-dependent; and (5) light- Nitellopsis obtusa (Pottosin and SchoÈ nknecht, 1995). induced anion e‚ux may balance light-induced proton Voltage-gated anion channels found in thylakoids are most extrusion and therefore participate in a mechanism control- probably responsible for the compensation of light-driven ling cellular pH, transmembrane and osmotic potential. H‡ movements (SchoÈ nknecht et al., 1988; Heiber et al., 1995). CONCLUSIONS Recently, Pohlmeyer et al. (1998) discovered a new type of voltage-dependent solute channel of high conductance in From year to year the number of characterized ion channels the outer envelope of chloroplasts, etioplasts and non-green increases, which bene®ts our understanding of their roles in root plastids (Table 3). The channels are permeable for numerous physiological processes. Modern electrophysio- triosephosphate, ATP, Pi , dicarboxylic acids, amino acids, logical and molecular biological techniques have enabled and sugars. Their open probability is highest at 0 mV (which the characterization and classi®cation of novel channel is consistent with the absence of transmembrane potential types. On the other hand, some channels previously across the plastidic outer membranes). Previously, Heiber described as di€erent types are in fact `synonyms'. These et al. (1995) reported a voltage-dependent anion channel of mainly originate from multiple gating mechanisms that can low conductance in the chloroplast envelope. There are also sense the energy status of the cell and thus make the cell anion channels found in the inner envelope membrane of responsive to various stimuli in a very ecient way. The ®ne isolated intact chloroplasts (Fuks and Homble, 1999). tuning of channel activities depends on e€ectors available in a certain cell type, i.e. it is plant and tissue speci®c (Barbier-Brygoo et al., 1999). Further research concerning Stretch-activated anion channels regulation and gating of the ion channels described here will Falke et al. (1988) ®rst reported large conductance, help to unravel the intermediate signalling mechanisms used stretch-activated, anion-selective channels in protoplasts of by plants in dynamic responses to the environment during tobacco. Cosgrove and Hedrich (1991) then showed the growth and development. existence of stretch-activated Cl,Ca2‡ and K‡ channels in the plasma membrane of guard cells. Teodoro et al. (1998) suggested that the changes in turgor pressure induced by ACKNOWLEDGEMENTS hyper-/hypo-osmotic stress may cause an early inactivation/ We thank Professor M. A. Venis and the reviewers for activation of stretch-sensitive anion channels, respectively. helpful comments and critical reading of the manuscript. The investigation was supported by the grant 6P04 C 04218 Light-activated anion channels from the State Committee for Scienti®c Research. By patch clamping hypocotyl cells isolated from dark- grown Arabidopsis thaliana seedlings, Cho and Spalding LITERATURE CITED (1996) revealed the existence of blue-light activated anion Allan AC, Fricker MD, Ward JL, Beale MH, Trewavas AJ. 1994. 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