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Annals of Botany 86: 449±469, 2000doi: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,
PolandReceived: 12 April 2000 Returned for revision: 7 May 2000 Accepted: 12 June 2000 Published electronically: 21 July 2000
that these
0305-7364/0
AbbreviatioA-9-C, anthralight; cADPRprotein kinasequilibrium pinositol triphoacid; OA, okadependent onand phosphoTMB-8, 8 (N
* For corre
A precise control of ion channel opening is essential for the physiological functioning of plant cells. This process istermed gating. Ion channel gating can be e�ected by ligand-binding, ¯uctuations in membrane potential, membranestretch and light quality. Modern electrophysiological and molecular-biological techniques have enabled thecharacterization and classi®cation of many ion channels according to their gating phenomena. Indications are thatgating mechanisms are complex and that individual ion channels can be regulated by a number of factors. In thispaper, gating mechanisms are reviewed following a standard classi®cation of ion channels based on permeability. Thegating of K�, Ca2� and anion channels in the plasma membrane, tonoplast and endomembranes of plant cells isdescribed. # 2000 Annals of Botany Company
Key words: Review, ion channel, ligand-gating, voltage-gating, stretch-gating, light-gating, plasmalemma, tonoplast.
INTRODUCTION
Ion channels are integral components of all membranes andthey can be viewed as dynamic ion transport systemscoupled via membrane electrical activities (White et al.,1999). Not only do they in¯uence membrane voltagethrough the ionic currents they mediate, but their activitiescan also be regulated by membrane voltage. Ion channelscan be divided into four `historically-based' groups accord-ing to gating mechanism: ligand-gated, voltage-gated,stretch-activated and light-activated. Ligand-gated ionchannels bind intracellular second messengers which pro-vide the essential links between external stimuli and speci®cintracellular responses (Leckie et al., 1998). Moreover,additional modulations by ATP or protons make thechannels capable of sensing changes in energy status oracid metabolism, respectively (Schulz-Lessdorf et al., 1996).Voltage-dependent channels appear optimally suited forelectrical signal transmission via membrane depolarization(e.g. through action potentials) and/or for signal trans-duction in response to changes in membrane potential(e.g. models investigating the coupling between membranepotential and voltage-dependent Ca2�-channels suggest
are engaged in intracellular signalling). They
0/090449+21 $35.00/00
ns: ABA, Abscisic acid; ABC, ATP binding cassette;cene-9-carboxylic acid; AP, action potential; BL, blue, cyclic ADP-ribose; CDPK, calmodulin-like domaine; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea; E,otential; I, current intensity; IAA, indol-3-acetic acid; IP3,sphate; NPPB, (5-nitro-2-3-phenylpropylamino) benzoicdaic acid; PLC, phospholipase C; PKA, protein kinasecyclic AMP; PKC, protein kinase dependent on [Ca2�]cytlipids; PKG, protein kinase dependent on cyclic GMP;,N diethylamino) octyl-3,4,5-trimethoxybenzoate.
spondence. E-mail trebacz@biotop.umcs.lublin.pl
are also involved in membrane voltage stabilization, whichis critical for maintaining ionic gradients and nutritionalion ¯uxes. Stretch-activated ion channels serve as addi-tional speci®c transmembrane `receptors' co-existing withother cellular volume-sensing mechanisms. Light-activatedchannels are in fact ligand-gated, although a preciseindication of the ligands is not yet possible because theprocess of light signal transduction remains unclear. Thesechannels are distinguished particularly because of a specialimportance of light stimuli in plant signalling processes.
Modern biomolecular techniques reveal how complicatedthe processes controlling channel behaviour are. It becomesincreasingly apparent that the activity of a channel maydepend on the developmental and metabolic stage of thecell. Moreover, regulation of ion channels relies not only onthe channel proteins themselves, but also to a great extenton regulatory polypeptides, such as auxiliary b-subunits,cytoskeletal components, 14-3-3 proteins, phosphates,kinases, and G-proteins (Czempinski et al., 1999).
Jan and Jan (1997) recently reviewed receptor-regulatedion channels in excitable and nonexcitable animal tissues(G-protein-gated and cGMP-gated K� channels; voltage-gated K�-, Na�-, Clÿ-, Ca2� channels; voltage-insensitiveCa2� channels; Ca2�-activated K� channels; ligand-gatedCa2� channels). The activities of these channels are sensi-tive to external and internal signals that are mediated byreceptors for hormones and transmitters. There are alsoplant-derived elicitor-speci®c receptors, which are closelycoupled with plasma membrane ion channels important forsignal transduction in plant cells (Ward et al., 1995;Blumwald et al., 1998). Studies on receptor-regulated ionchannels suggest that they too are gated via G-proteins,either by direct protein-protein interaction or indirectly
by kinase (PKA, PKG, PKC)/phosphatase cascades or# 2000 Annals of Botany Company
Czempinski et al., 1999).
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 membranepotential changes
Trebacz et al., 1994; Elzenga et al., 1995, 1997; Blom-Zandstra et al., 1997; SchoÈ nknecht et al.,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
450 Krol and TrebaczÐIon Channel Gating in Plant Cells
second messenger binding (Ca2�, IP3 , cGMP, cAMP). Agrowing body of evidence indicates that G-proteins, secondmessengers and phosphorylation/dephosphorylation pro-cesses mediate various plant responses through ion channeland other transport system regulation (Table 1).
Moreover, plant transmembrane receptors resemblingreceptor kinases of animal cells are involved in mediating avariety of cellular processes and responses to diverseextracellular signals (Braun and Walker, 1996; Trewavasand Malho, 1997). PCR, advanced homology-based clon-ing and function-complementation techniques have alreadyled to identi®cation of more than 70 plant protein kinasegenes (Stone and Walker, 1995). However, the precisefunction of speci®c protein kinases and phosphatasesduring plant growth and development has been elucidated
in only a few cases (Stone and Walker, 1995).sensitivity of voltage-gated K� channels.
POTASSIUM CHANNELS
Ion transport across all biological membranes is highlyselective and thus electrochemical potentials can begenerated. The electrochemical potentials largely dependon the potassium ion gradient, so most of the potassiumchannels must remain active for long periods of time. Suchgradients are indispensable for long-term cell functionssuch as nutrition, elongation, turgor and water regulation
or osmotically driven movements (Schroeder et al., 1984;Schroeder, 1989; Roberts and Tester, 1995; Hedrich andDietrich, 1996; Logan et al., 1997; Maathuis et al., 1997;
Ligand-gated potassium channels
Ligand binding causes conformational changes inchannel proteins. It is a process of great importance,especially during signal transduction cascades when secondmessengers synchronize the metabolism of the cell withenvironmental conditions and enhance the input stimuli.There are many K� channels a�ected by calcium ionbinding (namely: plasmalemma K�out channels, KORC,NORC, VK, FV, SVÐfor more information see below) inplant cells (Katsuhara and Tazawa, 1992; Allen andSanders, 1996; Czempinski et al., 1997, 1999; Maathuiset al., 1997; Muir et al., 1997; Allen et al., 1998a). BesidesCa2�, H� ions, nucleotides, proteins and plant hormonescan serve as potassium channel ligands (see below). Theirattachment corresponds accordingly to changes in voltage
Voltage-gated potassium channels in the plasmalemma
Voltage-gated plasmalemma K� channels are generallydivided into inward �K�� and outward (K� ) recti®ers. K�
in out inchannels are activated by hyperpolarizing potentials while
a
K�out are activated by membrane depolarization. Both K�inand K�out channels serve as membrane safeguards prevent-ing membrane voltage from becoming too negative orpositive, respectively. Such a role of voltage-gated K�channels in stabilizing membrane voltages is universalamong all eukaryotes (Maathuis et al., 1997). Voltage-dependent plasma membrane-bound outward potassiumrecti®ers responsible for K� e�ux are involved in turgorregulation (Liu and Luan, 1998), stomatal closure(MacRobbie, 1997; Grabov and Blatt, 1998a), organmovements (Iijima and Hagiwara, 1987; Stoeckel andTakeda, 1993), cation release into xylem (Roberts andTester, 1995), light-induced potential changes of theplasmalemma (Blom-Zandstra et al., 1997) or repolariza-tion during action potentials (APs), and prevention ofexcessive depolarization (Stoeckel and Takeda, 1993;Trebacz et al., 1994; Maathuis et al., 1997). These rolesare summarized in Table 2. K�in channels are involved in:potassium uptake into a cell during cell expansion, growthprocesses, organ movements and stomatal openings; low-a�nity uptake pathway in root hair cells; xylem unloadingby conducting cations from xylem to symplast of growingshoots; membrane voltage prevention against excessivehyperpolarization (reviewed by Maathuis et al., 1997)
Krol and TrebaczÐIon Ch
(summarized in Table 2).
Regulation of plasmalemma voltage-gated potassiumchannels
In addition to membrane potential, e�ectors like H�,Ca2�, nucleotides and K� ions can either interact directly(ligand binding) with both inward and outward plasma-lemma K� channels or act indirectly via membrane-bound,attached or soluble regulators (Hedrich and Dietrich, 1996;Kurosaki, 1997; Blatt, 1999; Czempinski et al., 1999).Inwardly and outwardly rectifying K� channels are con-trolled by cytosolic calcium, ATP and pH in very di�erentways (Grabov and Blatt, 1997). The action of pHcyt is mostpronounced on the depolarization-activated outward-rectifying K� channels which are virtually insensitive toincreased [Ca2�]cyt (Grabov and Blatt, 1997). They do notshow such pronounced sensitivity towards external pH butrequire slightly alkaline cytosolic pH for activation (Blattand Grabov, 1997a). Alkaline pHcyt activates IKout in avoltage-dependent manner through a co-operative bindingof two protons (Grabov and Blatt, 1997). Moreover,their activation by depolarization depends critically onphosphorylation (e.g. by a kinase tightly associated with thechannel protein in Samanea saman motor cellsÐMoran,1996) or dephosphorylation events associated with [Ca2�]cytincrease (e.g. by calcium-dependent phosphatase in Arabi-dopsis thaliana guard cellsÐMacRobbie, 1997). In meso-phyll and guard cells of Vicia faba there are outward-rectifying K� channels regulated by calcium and G-proteininteraction as well (Li and Assmann, 1993). On the otherhand, there are potential Ca2�-binding sites (EF-handmotifs) found at the C-terminus of a-subunits from putativeoutward potassium recti®ers. These ion channels are verylikely to be directly regulated by Ca2� (Czempinski et al.,
1997, 1999). This also applies to KORC and NORCchannels which become active at depolarized membranepotentials, but their respective activation depends on thecytoplasmic Ca2� level (De Boer and Wegner, 1997).KORC, NORC and SKOR are di�erent channels fromplasmalemma of root xylem parenchyma cells. They areresponsible for xylem loading (Roberts and Tester, 1995;De Boer and Wegner, 1997; Maathuis et al., 1997; Gaymardet al., 1998). KORC channels also show a considerableconductance for Na� but very low permeability for Li� andCs�. This indicates that KORC channels may also act as a`®lter' protecting the shoot from harmful Cs� or Li� ions(Maathuis et al., 1997). NORC channels discriminate onlyslightly between cations and their role in solute release intoxylem is limited. However, they do provide a function inresetting the membrane potential after excessive depolariza-tion (Maathuis et al., 1997). Kout currents conducted bySKOR are e�ectively inhibited by both cytosolic andexternal acidi®cation (Lacombe et al., 2000). SKORchannels have no Ca2�-binding sites, but they containankyrin and cyclic nucleotide-binding domains (Gaymardet al., 1998). Direct binding of nucleotides, calcium ions(De Boer and Wegner, 1997; Czempinski et al., 1997, 1999)or protons (Blatt and Grabov, 1997a) to the channelproteins illustrates that voltage-gated outward-rectifyingplasmalemma potassium channels may be regarded asligand-gated in certain experimental conditions.
Recently Ca2�-gated outward rectifying potassiumchannels have been described in the plasmalemma of thealga Eremosphaera viridis (SchoÈ nknecht et al., 1998). Thesechannels show very steep Ca2�-dependence and they can beCa2�-stimulated both directly and indirectly by interactionwith calmodulin (SchoÈ nknecht et al., 1998). They areinvolved in hyperpolarizing currents during darkening-induced transient hyperpolarizations of the plasmamembrane (Table 2).
The gating of K�out current is e�ected by [K�]ext , so thatits voltage dependence shifts in parallel with EK (Blatt,1999). K� ions bind in a co-operative fashion to a set ofsites exposed on the extracellular face of the membrane toinactivate K�out channels and they may be substituted withRb� or Cs� (Blatt, 1999). This inactivating binding ofmonovalent ions to the channel protein is facilitated byinside negative membrane voltage. Recent studies haveshown that IKout activation is also dependent on thecooperative interaction of two K� ions with the channel,but at sites di�erent from the channel pore (Grabov andBlatt, 1998a).
Voltage-dependent plant plasmalemma K�-uptake-channels represent various types (KAT, AKT) of di�erentspatial expression patterns (Bei and Luan, 1998), di�erentfunctions (Bei and Luan, 1998; Tang et al., 1998) anddi�erent sensitivities to voltage, Cs�, Ca2� and H� (Dreyeret al., 1997; Bei and Luan, 1998). This diversity partlyresults from nonselective heteromerization of di�erenta-subunits (Dreyer et al., 1997) as well as from the abilityof b-subunits to associate with more than one type ofa-subunit in vivo (Tang et al., 1996, 1998). All voltage-dependent plant plasmalemma K�-uptake-channels con-tain a conserved GYGD motif within a pore region, which
nnel Gating in Plant Cells 451
is responsible for K� conductivity (Czempinski et al.,
TABLE2.Plasm
alemmaionchannels
Channel
Permeability
Gatingmechanism
Physiologicalrole
References
Potassium
channels
K� outfrom
Vicia
fabaguard
cell
K�
Depolarization-dependentopening
Up-regulatedbypH
inincrease
(strong
voltage-dependentstim
ulation)
Co-operativebindingoftw
oprotons
K�gradientsensitive
Inhibited
byexternalK� -binding
RegulatedbyG-protein-inducedCa2� -increase
Stomatalclosure
Preventionfrom
re¯uxofK�into
theguard
cell
LiandAssmann,1993;Blattand
Grabov,1997a;Maathuiset
al.,1997;
MacRobbie,1997;Grabovand
Blatt,1998a;Leckie
etal.,1998;
Pei
etal.,1998;Blatt,1999
K� outfrom
Arabidopsisthaliana
guard
cells
K�
Depolarization-dependentopeningstim
ulated
byCa-dependentphosphatase
Up-regulatedbypH
inincrease
Stomatalclosure
MacRobbie,1997
K� outfrom
Samanea
saman
motorcells
K�Rb�Na�
Cs�
Li�
Depolarization-inducedactivation
Phosphorylationbyakinase
tightlyassociated
withK� o
utchannel
Leafmovements
Invo
lvem
entin
circadianclock
Moran,1996;Maathuiset
al.,1997
K� outfrom
Mim
osa
pudica
motorcells
K�
Activationbydepolarization
Rapid
movements
inMim
osa
RepolarizationduringAP
Stoeckel
andTakeda,1993
K� outfrom
Dionaea
muscipula
trap-lobecells
K�
Voltage-dependence
(depolarizationactivated)
Outw
ard
recti®cationstrongly
dependsonthe
concentrationofintracellularK�
Closure
oftrap-lobes
IijimaandHagiwara,1987
K� outfrom
Conocephalum
conicum
K�
Voltage-dependence
(depolarizationactivated)
RepolarizationduringAP
Trebaczet
al.,1994
KORC
(K�outw
ard
rectifying
conductance)
K�Na�
Activated
atmem
branevo
ltages
more
positive
thanÿ5
0mV
Ca2� -dependentactivation
Xylem
loading
Shootprotectionfrom
harm
ful
Cs�
andLi�
ions
Roberts
andTester,1995;DeBoer
and
Wegner,1997;Maathiuset
al.,1997
SKOR
(Shaker-typeK�outw
ard
rectifyingchannel)
K�
Voltage-dependent
Changes
inboth
pH
cytandpH
extregulate
the
number
ofchannelsavailable
foractivation
Xylem
loading
Gaymard
etal.,1998;
Lacombeet
al.,2000
NORC
(non-selective
outw
ard
rectifyingconductance)
Non-selective
amongcations
Activeat
mem
branevo
ltages
more
positive
than�3
0mV
Ca2� -dependentactivation
Protectionagainst
high
depolarization
Xylem
loading
Roberts
andTester,1995;DeBoer
and
Wegner,1997;Maathiuset
al.,1997;
White,
1998
Maxicationchannel
from
ryeroots
Non-selective
amongcations
Activeat
mem
branevo
ltages
more
positive
thanEK
Mem
branevo
ltagestabilization
White,
1998
K� outfrom
Nitellopsisobtusa
K�Na�
Ligand-binding:ATP-and[Ca2� ]
ext-dependent
regulation(inhibition)
Saltstress
tolerance
Katsuhara
etal.,1990;Katsuhara
and
Tazawa,1992
K� outfrom
Eremosphaeraviridis
K�
Ca-dependentandstim
ulatedboth
bydirect
Ca2� -bindingandindirectlybysome
calm
odulininteractions
Dark-inducedhyperpolarization
ofV
mandtherebydivalent
cationuptake
Scho Ènknechtet
al.,1998
K� outfrom
Nicotianatabacum
L.mesophyllcells
K�
Light-activation
Voltage-dependence
Mem
branedepolarizationupon
lighttransition
Blom-Zandstra
etal.,1997
K� outfrom
guard
cellsof
Vicia
fabaL.
K�
Stretch-activated
Volumeandturgorregulationand
therebycontrolofleaf
gasexchange
Cosgrove
andHedrich,1991
452 Krol and TrebaczÐIon Channel Gating in Plant Cells
K� in(K
AT1)from
Vicia
faba
guard
cell
K�
Hyperpolarization-dependentopening
LoweringpH
extpromotesK�currentin
voltage-
dependentmanner
CDPK
dependentphosphorylationofKAT1
protein
inaCa2�dependentmanner
Inhibited
byIP
3-induced[Ca2� ]
inelevation
Inhibited
bypolymerized
actin®laments
Modulatedbyauxin
Controlled
byactin®laments
RequireexternalK�ionsforactivation
ModulatedbycA
MP-dependentsignallingsystem
and/ordirectcyclic
nucleotidebinding
Stomataopening
Regulationofstomatalaperture
Osm
oticvo
lumereadjustment
Blatt
etal.,1990;Fairley-G
renot
andAssmann,1992;Blatt
andThiel,
1994;WuandAssmann,1995;
Ilanet
al.,1996;BlattandGrabov,
1997a;Claussen
etal.,1997;Grabovand
Blatt,1997,1998a;Hwanget
al.,1997;
Maathuiset
al.,1997;MacRobbie,1997;
McA
insh
etal.,1997;Leckieet
al.,1998;
Liet
al.,1998;Liu
andLuan,1998;
Pei
etal.,1998;Blatt,1999;
Czempinskiet
al.,1999;Jinand
Wu,1999
KAT1from
Arabidopsisthaliana
andKST1from
guard
cellsand
¯owersofSolanum
tuberosum
K� ,
NH� 4,Rb� ,
Na� ,
Li�
Voltagedependent(hyperpolarizationactivated)
ATPandcG
MPactivation
Ionpermeationmay
feed
backongating
Competitivelyinhibited
byCa2�andCs�
ions
pH
regulated(pH
extacidi®cationshifts
voltage-dependence
toward
less
negativevo
ltages)
Regulationbycytoskeletalproteins
Modulatedbycyclic
nucleotidebinding
Stomatalopening
K�uptakeduringother
osm
otic
movements
Arm
stronget
al.,1995;Mu Èller-R
o Èber
etal.,1995;Becker
etal.,1996;Hedrich
andDietrich,1996;Hoth
etal.,1997;
Maathuiset
al.,1997;
Czempinskiet
al.,1999
AKT1from
Arabidopsisthaliana,
SKT1Ð
Solanum
tuberosum
root
cellsandchannel
analogue
from
corn
roots
K� ,
Rb� ,
Na� ,
Cs�,Li�
Hyperpolarization-dependentopening
Inward
K�gradientsensitive
Regulationofmem
branevo
ltage
Low-a�nityK�uptake
Hedrich
andDietrich,1996;
Bertlet
al.,
1997;Maathuiset
al.,1997;
Czempinskiet
al.,1999
KIR
C(K�inward
rectifying
conductance)
K� ,
Rb� ,
Na� ,
Cs�,Li�
Activeat
mem
branevo
ltages
more
negative
thanÿ1
10mV
Xylem
unloading
Maathiuset
al.,1997
VIC
(voltage-insensitive
cation
channel)
NH� 4,Rb� ,
K� ,
Cs�,Na� ,
Li�,
TEA�
Open
60±80%
ofthetimeat
voltages
more
positive
thanÿ1
20mV
Inhibited
bydivalentcations
Low-a�nityNH� 4-uptake
Osm
oticadjustmentindependentof
themem
branepotentials
Compensatory
cation¯uxes
White,
1997,1999
K� infrom
Zea
mayscoleoptile
K� ,
Rb�
Hyperpolarization-dependentopening
LoweringpH
ext
Inhibited
byCa2�
Modulatedbyauxin
Cellelongation
Hedrich
andDietrich,1996;
Thielet
al.,1996;Claussen
etal.,1997
K� infrom
Avenasativa
mesophyll
cells
K�
Voltage-dependent
Plasm
alemmaV
mstabilization
Stabilizationofionic
andosm
otic
conditionsduringcellexpansion
Kourie,
1996
K� infrom
Samanea
samanmotor
cells
K�
ActivationbyH�pump-inducedhyperpolarization
InhibitionbyPLC-m
ediatedIP
3-induced
Ca2�increase
Directresponse
tolight
Leafmovements
Kim
etal.,1992,1996;
Maathuiset
al.,1997
K� infrom
culturedcarrotcells
K�
Controlled
bycytoplasm
icconcentrationofcA
MP
Mem
branechanges
andthusactivation
ofvo
ltage-gated
channels
Kurosaki,1997
Stretch
activated
K� infrom
Vicia
fabaguard
cells
K�
Osm
oticum
gradient-sensitive
Voltage-dependence
Regulatedbyactin®laments
Osm
oregulation
Ramahaleoet
al.,1996;Liu
and
Luan,1998
Table
2continued
onnextpage
Krol and TrebaczÐIon Channel Gating in Plant Cells 453
TABLE2.Continued
Channel
Permeability
Gatingmechanism
Physiologicalrole
References
Calcium
channels
VDCCÐ
voltage-dependent
Ca-channel
from
guard
cells
Ca2�
Depolarizationactivated
Earlyevents
ofplanthorm
one-induced
responses
McA
insh
etal.,1995;Grabovand
Blatt,1998b
VDCC
from
ryeroots
VDCC
from
wheatroots
(rca
channel)
Ba2� ,
Sr2� ,
Ca2� ,
Mg2� ,
Mn2� ,
K� ,
Na� ,
Rb� ,
Li�
Depolarizationactivated
Strongvo
ltage-dependence
(depolarization
activated)
CytosolicATPshifts
activationto
more
negative
potentials
Divalentcationuptakeinto
roots
Signallingmechanismsandpriming
thecellforresponse
White,
1998;PinerosandTester,1997;
White,
1998
VDCC
from
Arabidopsisroots
andDaucuscarota
suspension
protoplasts
Ba2� ,
Sr2� ,
Ca2� ,
Mg2� ,
K�
Depolarizationactivated
Activeunder
conditionofmicrotubule
disorganization
Slow
inactivationat
negativevo
ltages
Cationuptake
Maintainingappropriateelectrochem
ical
gradients
importantforthetransport
ofother
ionsandcellvo
lume
regulation
Signallingmechanismsandpriming
thecellforresponse
Thionet
al.,1996;Whiteet
al.,1998
VDCC
from
characeancells
Ca2�
Depolarizationactivated
APinduction
Earlyevents
ofturgorregulationand
salttolerance
Katsuhara
andTazawa,1992;
Shim
men,1997
VDCC
from
Chara
corallina
Ca2�
Depolarizationactivated
DuringCa-starvationchannelsmight
open
toscavengeavailable
Ca2�
Reidet
al.,1997
Voltage-dependentCa-channels
from
liverwort
Conocephalum
conicum
andmoss
Physcomitrellapatens
Ca2�
Depolarizationactivated
Light-inducedmem
branedepolarization
Trebaczet
al.,1994;Erm
olayevaet
al.,
1996,1997
Ca-channelsfrom
mosses
Ca2�
Cytokinin-induceddepolarizationactivated
Earlyevents
ofcytokinin-induced
responses
Schumaker
andGizinski,1993
VDCC
from
Mim
osa
pudica
motorcells
Ca2�
Hyperpolarization-activated
Activationofchannelsinvo
lved
inleaf
movements
Stoeckel
andTakeda,1995
VDCC
from
pollen
tubes
Ca2�
Voltage-dependent
Stretch-activated
Growth
processes
Holdaw
ay-C
larkeet
al.,1997
VDCC
from
Fucusrhizoids
Ca2�
Voltage-dependent
Stretch-activated
Growth
processes
Tayloret
al.,1996
SAC
from
Fucuszygotes
Non-selective
Stretch-activated
Mechanosensitive
Ca-channels
from
rootcells
Non-selective
Stretch-activated
Regulatedbycytoskeletalproteins
Regulationofturgor
Determinationoftheallometry
ofcell
expansionandmorphogenesis
Thionet
al.,1996;Whiteet
al.,1998
Mechanosensitive
Ca-channels
from
guard
cells
Ca2�
Stretch-activated
Regulatedbycytoskeletalproteins
Transm
issionofCa-signalsinto
thecytoplasm
Guard
cellvo
lumeandturgorregulation
andtherebycontrolofleaf
gas
exchange
Controlofother
ionchannelswith
Ca-dependentactivities
Cosgrove
andHedrich,1991;
MacRobbie,1997;McA
insh
etal.,1997
454 Krol and TrebaczÐIon Channel Gating in Plant Cells
Mechanosensitive
Ca-channels
from
Fucusrhizoids
Ca2�
Stretch-activated
Regulatedbycytoskeletonproteins
Transm
issionofCa-signalsinto
thecytoplasm
Cellvo
lumeregulation
Tayloret
al.,1996;McA
insh
etal.,1997
Receptor-regulatedCa-channels
from
parsleyprotoplastsand
rootcells
Non-selective
Elicitor-activated
Earlyevents
ofpathogen
defence
system
activation
Zim
mermannet
al.,1997;
Whiteet
al.,1998
Receptor-regulatedCa-channels
Ca2�
Elicitor-activated
Voltage-gated
Earlyevents
ofpathogen
defence
system
activation
Blumwald
etal.,1998
Receptor-regulatedCa-from
tomatoprotoplasts
Ca2� ,
K�
Elicitor-activated
Hyperpolarization-activated
Ca-in¯uxasanearlyresponse
tovarious
signalsincludingfungalelicitors
GelliandBlumwald,1997
Anionchannels
GCAC1from
Vicia
fabaand
Commelinacommunis
Clÿ,malate
S-typeshowsweakvo
ltagedependence
S-typerequires
hydrolysable
ATPandactivationof
protein
kinase
OA-sensitive
phosphatasesare
invo
lved
indown-regulationofS-typechannel
S-typemay
beABCprotein
oritistightlycontrolled
bysuch
protein
R-typeisactivated
byparallel
voltagemem
brane
depolarization,pH
cytacidi®cation,[Ca2� ]
cyt
increase
andnucleotidebinding
Directauxin
bindingshifts
activationpotential
towardsrestingpotentialsto
favo
urchannel
opening
S-typeserves
asmajorpathway
for
anione�
uxduringstomatalclosure
andasnegativefeedbackduring
stomatalopening
R-typeresponsible
forsignal
transductionvia
mem
brane
depolarization
GCAC
channelsare
capable
ofsensing
changes
intheenergystatus,acid
metabolism
andprotonpumpactivity
inguard
cells,because
oftime-
and
voltagedependentactivitystrongly
modulatedbyATPandH�
Kelleret
al.,1989;Marten
etal.,1991;
Hedrich
andJeromin,1992;
Linder
andRaschke,
1992;
Schroeder
andKeller,1992;
Schroeder
etal.,1993;Dietrichand
Hedrich,1994;
Schmidtet
al.,1995;
Ward
etal.,1995;LiandAssmann,1996;
Esser
etal.,1997;MoriandMuto,1997;
Pei
etal.,1997,1998;Grabovand
Blatt,1998a;Schwarz
and
Schroeder,1998;Leonhardtet
al.,1999
GCAC1from
Nicotiana
benthamianaandArabidopsis
thaliana
S-typeshowsweakvo
ltagedependence,requires
protein
phosphatase
activitiesandisdown-
regulatedbyprotein
kinase
S-typeisin¯uencedbypH
gradient
R-typeisactivated
byparallel
voltagemem
brane
depolarization,pH
cytacidi®cation,[Ca2� ]
cyt
increase
andnucleotidebinding
R-typeismodulatedbyphosphorylation/
dephosphorylationprocesses
Arm
stronget
al.,1995;Ward
etal.,1995;
Schulz-Lessdorfet
al.,1996;Elzengaand
VanVolkenburgh,1997b;
Lew
iset
al.,1997;Grabovand
Blatt,1998a;Pei
etal.,1997,1998
TSACÐ
tobacco
suspension-cell
anionchannel
Clÿ
ATP-controlled
voltage-dependence
(depolarization
activated)
Modulatedbyauxin
Anionrelease
duringinhibitionof
cellelongation
Zim
mermannet
al.,1994
Anionchannelsfrom
mesophyll
cellsofPisum
sativum
Clÿ
Voltage-dependent(hyperpolarizationactivated)
Twomodekineticsdi�erentlycontrolled
byATP
(R-andS-type,
S-typeoccurs
inthepresence
ofATP)
Ca-dependentactivation
Light-inducedtransientdepolarization
ElzengaandVanVolkenburgh,1997a,b
Anionchannelsfrom
suspension-
culturedcarrotcells
Clÿ
Voltage-dependent(hyperpolarizationactivated)
Voltage-dependentinactivationunder
large
hyperpolarization
Controlofmem
branepotential
Regulationofosm
oticbalance
Barbara
etal.,1994
Table
2continued
onnextpage
Krol and TrebaczÐIon Channel Gating in Plant Cells 455
TABLE2.Continued
Channel
Permeability
Gatingmechanism
Physiologicalrole
References
Anionchannelsfrom
Eremosphaeraviridis
Clÿ
Hyperpolarizationactivated
Lim
itingtheamplitudeofdark-induced
transienthyperpolarizationcausedby
K� -release
Scho Ènknechtet
al.,1998
Anionchannelsfrom
Charophyta
cells
Clÿ
Ca-dependentactivation
DepolarizingcurrentduringAP
Okihara
etal.,1991;Katsuhara
and
Tazawa,1992;Thielet
al.,1993;
Shim
men,1997
Anionchannelsfrom
liverw
ort
C.conicum
Trebaczet
al.,1994
Anionchannelsfrom
Aldrovanda
vesiculosa
IijimaandSibaoka,1985
Anionchannelsfrom
Physcomitrellapatens
Clÿ
Ca-dependentactivation
Phytochrome-mediatedsignalling
pathway
Erm
olayevaet
al.,1996,1997
Anionchannelsfrom
Charophyta
cells
Clÿ
H� -
andCa2� -dependentactivation(directbinding)
Phosphorylation/dephosphorylationprocesses
Facilitationofenhancedprotone�
ux
under
intracellularacidosis
Johannes
etal.,1998
Anionchannelsfrom
epidermal
cellsofArabidopsishypocotyls
Clÿ
Strongandweakvo
ltage-dependence
ofR-and
S-typeunitary
conductances,respectively
(activationbyV
mdepolarization)
R-andS-types
havethesameconductance
but
di�erentopen
probabilities
Thesw
itch
betweenR-andS-typeiscontrolled
by
ATP(R
-typeoccurs
inthepresence
ofATP)
Modulatedbyphosphorylation/dephosphorylation
processes
R-typemay
beinvo
lved
inthe
transductionofexternalsignalsand
transm
issionofAP
S-typemay
beinvo
lved
inturgor
regulationandhypocotylmovements
Thomineet
al.,1995,1997;Choand
Spalding,1996;ElzengaandVan
Volkenburgh,1997b;Lew
iset
al.,1997;
Parkset
al.,1998
Anionchannelsfrom
epidermal
cellsofArabidopsishypocotyls
Clÿ
BL-activation(increase
inopen
probability)
Light-inducedinhibitionofcell
elongation
ChoandSpalding,1996
Anionchannelsfrom
mesophyll
cellsofPisum
sativum
Clÿ
Light-inducedactivation(increase
inopen
probability)
Ca-dependentactivation
Light-inducedtransientmem
brane
potentialdepolarization
Chargebalance
forlight-inducedH�
pumpactivation,thuscontrolof
pH
ext,mem
branevo
ltageandosm
otic
potential
Elzengaet
al.,1995,1997;Elzengaand
VanVolkenburgh,1997a,b
SAC
from
guard
cellsof
Vicia
fabaL.
Non-selective
Stretch-activated
Reductionofcellturgor
Activationofvo
ltage-dependention
channelsthroughmem
brane
depolarization
Cosgrove
andHedrich,1991
SAC
from
Arabidopsisthaliana
guard
cells
Controlofleaf
gasexchange
Teodoro
etal.,1998
456 Krol and TrebaczÐIon Channel Gating in Plant Cells
1994) (Table 2).
a
1999). Kourie (1996) demonstrated that the relative numberof opened voltage-activated inward rectifying potassiumchannels increased sigmoidally as a function of hyper-polarized membrane potential. The kinetics of inwardrectifying K� currents in Avena sativa mesophyll cellsreported by Kourie was independent of [K�]ext and it lackedtime-dependent inactivation. Neither low [K�]ext nor[Na�]ext caused inactivation of the above-mentionedcurrents, while Cs�-induced block was reversible andstrongly voltage-dependent. A role of preventing largemembrane hyperpolarization resulting from electrogenicproton pumping was proposed for these K�in channels byKourie (1996). On the other hand, there are reportsconcerning K�in channels (AKT1) `sensing' external potas-sium concentration (Bertl et al., 1997). AKT1 channels arepresent in root cells. Extracellular K� binds to a modulatorsite thereby enhancing the rate of opening of AKT1 protein.Blatt (1999) also noticed that IKin current in guard cellsrequires external millimolar K� concentrations for itsactivity. In submillimolar [K�]ext , K
�in channels appear to
enter a long-lived inactive state (Blatt, 1999).Control of plasmalemma K�in channels is modulated by
increasing [Ca2�]cyt (inactivation) and increasing externalproton concentration (voltage-dependent activation) ordecreasing pHcyt (voltage-independent activation; increasein the pool of active channels through allosteric interaction)(Ilan et al., 1996; Grabov and Blatt, 1997, 1998a; Hothet al., 1997; MacRobbie, 1997). Ca2�-dependent inactiva-tion can proceed even when pH is bu�ered. Equally,changes in pH and channel gating may occur withoutmeasurable changes in calcium concentration (Allan et al.,1994; Armstrong et al., 1995). Thus, the e�ects of cellularpH and calcium are separable, although these two ionicmessengers do interact. In other words, pHcyt may act inparallel with, but independently of, [Ca2�]cyt in controllingK�in channels (Grabov and Blatt, 1997). Kim et al. (1996)reported that phosphoinositide turnover, phospholipase C(PLC) activation or the presence of inositol triphosphate(IP3) is correlated with K�in channel closure. Earlier, Blattet al. (1990) demonstrated the possibility of controlling K�inchannel activity by IP3-mediated Ca2� release. Both theabove-mentioned results indicate that increase in [Ca2�]cyt isresponsible for K�in channel inactivation and they support agrowing body of evidence that G-proteins function inregulating IKin (reviewed by Blatt and Grabov, 1997a,b).Recently, Li et al. (1998) identi®ed a Ca2�-dependentprotein kinase, with a calmodulin-like domain (CDPK),which phosphorylates K�in channels of Vicia faba guard cellprotoplasts. Moreover, the cAMP-dependent signallingsystem `cross-talks' with Ca2�-dependent inhibition of K�inchannels from Vicia faba guard cells by reversing inhibitorycalcium e�ects (Jin and Wu, 1999). In contrast to K�inchannels from guard cells, K�in channels in the plasma-lemma of rye root cells are insensitive to [Ca2�]cyt (White,1997).
K�in channels (KAT1ÐArabidopsis thaliana, KST1ÐSolanum tuberosum) can also be inhibited by Ca2� and Cs�via competition in binding to the pore forming regionexposed to the aqueous lumen of the channel (Becker et al.,
Krol and TrebaczÐIon Ch
1996). Thiel et al. (1996) showed that Ca2�-binding to the
K� channel protein is responsible for fast and reversibleinactivation of inward K� currents in maize coleoptileprotoplasts.
In addition to their Ca2� and pH dependence, voltage-gated plasmalemma K�in channels seem also to require ATP(Hoshi, 1995; MuÈ ller-RoÈ ber et al., 1995; Wu and Assmann,1995). Their structures contain ATP and cyclic nucleotide-binding cassettes in the C-terminal domains. The rundownof K�in recti®ers in the absence of ATP is explained in termsof a shift in the voltage-dependence (Hedrich and Dietrich,1996). Kurosaki (1997) surveyed some of the inward K�channels (located in the plasma membrane of culturedcarrot cells) whose gating was controlled by cytoplasmicconcentration of cAMP. Their activation resulted intransient membrane potential changes, which in turnactivated voltage-gated Ca2� channels. Because plasma-lemma voltage-gated inward K�-channels described byHedrich and Dietrich (1996) and Kurosaki (1997) areregulated via direct nucleotide binding to the channelprotein, they can be classi®ed as ligand-gated ones as well.
There is an obvious correlation between inward rectifyingK� channels and cytoskeletal proteins (Table 2). As aconserved structural feature, proteins of the AKT subfamilycontain so-called ankyrin repeats which are potentialdomains for interaction with the cytoskeleton (Czempinskiet al., 1999). Because proteins from the KAT subfamily lacksuch ankyrin sequences, but they are `sensitive' tocytoskeletal drugs, there must be other channel domainsparticipating in the regulation by cytoskeletal compounds(Hwang et al., 1997; Czempinski et al., 1999). Pharmaco-logical studies on guard cells have shown that actin®laments contribute to regulation of K�in channels as wellas of stomatal aperture (Hwang et al., 1997). CytochalasinD, which induces depolymerization of actin ®laments,activates inward potassium currents, while phalloidinÐastabilizer of ®lamentous actinÐinhibits them (Hwang et al.,1997). These authors demonstrated that polymerized actinstabilizes K�in channels in the closed state and thus makesthem unresponsive to membrane hyperpolarization. Asactin ®laments depolymerize, the closed state of K�inchannels becomes less stable and more channels becomeready to respond to the hyperpolarized membrane poten-tial. Liu and Luan (1998) also correlated regulation of IKinwith the pattern of organization of actin ®laments. Theystated that actin structure may be a critical component inthe osmosensing pathway conducted by K�in channels inplants.
There are also reports of auxin-induced modulation ofK�-inward recti®ers at the plasma membrane in coleoptilecells (Claussen et al., 1997) and guard cells (Blatt and Thiel,
nnel Gating in Plant Cells 457
Voltage-gated potassium channels in the tonoplast
In the tonoplast of higher plants there are three distinctkinds of voltage-sensitive potassium channels (FV, fastactivating; SV, slow activating; and VK, strongly K� select-ive) (Table 3). FV channels are instantaneously activated atthe resting levels of [Ca2�] and pH by changes in
cyt cyttonoplast voltage (Allen et al., 1998). They open at cytosolTABLE3.Ionchannelsin
plantendomem
branes
Channel
Permeability
Gatingmechanism
Physiologicalrole
References
Potassium
channels
Tonoplast
FV
(fast-activating)
cationchannels
NH� 4,K� ,
Rb� ,
Cs�,
Na� ,
Li�
Voltage-dependentopen
probability
Preferred
outw
ard
recti®cationat
positive
potentials
(relativeto
thecytoplasm
)Activeat
therestinglevelsof[Ca2� ]
cytandpH
cyt
Inhibited
byvacuolarandcytosolicCa-increases
FV
currents
are
reducedat
acidic
pH
cyt
ATPregulated
Blocked
byMg2�andpolyamines
Controlofthetonoplast
electrical
potentialdi�erence
aroundEK
Ashuntconductance
forthe
vacuolarH�pumps
Invo
lvem
entin
potassium
release
duringstomatalclosure
Invo
lvem
entin
increase
incellular
osm
olarity
Smallmonovalentcationuptake
LinzandKo Èhler,1994;Allen
andSanders,
1996,1997;Maathuiset
al.,1997;
Tikhonovaet
al.,1997;Allen
etal.,1998a;
GrabovandBlatt,1998a;B
ruÈggem
annetal.,
1999a,b;Dobrovinskayaet
al.,1999
Tonoplast
SV
(slow-activating)
cationchannels
K� ,
Na� ,
Rb� ,
Li�,
NH� 4,Ca2� ,
Mg2� ,
polyamines
Tim
e-dependentactivationat
cytosol-positive
potentials
Outw
ard-rectifying
Strongvo
ltage-dependence
modulatedbyCa2� ,
Mg2�
andH�ions(C
a-andMg-activationanddown-
regulationofSV
channel
activitybyprotons)
Ca2�inducesloweringofthevo
ltagethreshold
for
activation
RequirealkalinepH
atboth
sides
oftonoplast
Regulatedbyprotein
phosphorylationandcalm
odulin
interaction
Single
channel
conductance
dependenton[K� ]
cyt
Modulatedbyredoxagents
(increasedopen
probability
inthepresence
ofantioxidants)
Blocked
bypolyamines
inavo
ltage-dependentmanner
Vacuolarreceptorsite
forcalcium
duringstomatalclosure
Possible
participationin
CIC
R(C
a-inducedCa-release)
Turgorregulation
Vacuolariontransport
Ward
andSchroeder,1994;Allen
and
Sanders,1995,1996,1997;Schulz-Lessdorf
andHedrich,1995;Ward
etal.,1995;
Gambale
etal.,1996;Bethkeand
Jones,1997;Maathuiset
al.,1997;
MacRobbie,1997;McA
insh
etal.,1997;
Allen
etal.,1998b;GrabovandBlatt,
1998a;Carpanetoet
al.,1999;Ceranaet
al.,
1999;Dobrovinskayaet
al.,1999
Tonoplast
VK
(vacuolarK� )
channels
K� ,
Rb� ,
NH� 4
Activated
bymicromolar[Ca2� ]
cytandacidic
pH
cyt
Voltage-independent,non-rectifyingchannel
Ca-dependentpotassium
uptakeand
release
duringstomatal
movements
(e.g.ABA-induced
stomatalclosure)
Activationofvo
ltage-gated
tonoplast
channels
Ward
etal.,1995;Allen
andSanders,1996,
1997;Maathuiset
al.,1997;
MacRobbie,1997;McA
insh
etal.,1997;
Allen
etal.,1998a;GrabovandBlatt,1998a
Cation-selective
channel
from
tonoplast
ofalgae
Lamprothamnium,Chara
buckellii,Chara
australisand
Nitellopsisobtusa
K� ,
Na�
Activated
bymicromolar[Ca2� ]
cyt
Voltagedependence
StrongpH-dependence
(inhibitionbyacidic
pH)
Osm
oticvo
lumeandturgor
regulation
Katsuhara
andTazawa,1992;
Lu Èhring,1999
Cation-selective
channel
from
thylakoidsofSpinacea
oleracea
andPisum
sativum
cotyledons
K� ,
Ca2� ,
Mg2�
Voltage-gated
(activated
bypositive
mem
branepotentials
ofstromarelative
tolumen)
Compensationoflight-induced
proton¯uxes
PottosinandScho Ènknecht,1996;
HinnahandWagner,1998
Cation-selective
channel
from
chloroplast
envelope
Multi-cation
Voltage-dependent
Metabolite
di�usion
Heiber
etal.,1995
Cation-selective
channel
from
chloroplast
envelope
K�
Voltage-dependent
ATP-regulated
ModulatedbyCs�,Mg2�
Compensationoflight-driven
protonmovements
Heiber
etal.,1995
Cation-selective
channel
from
nuclearenvelopefrom
redbeet
Multi-cation
Ca-regulatedvo
ltage-dependence
Ca-regulatedpathwaysfornuclear
processes
GrygorczykandGrygorczyk,1998
458 Krol and TrebaczÐIon Channel Gating in Plant Cells
Calcium
channels
Ligand-gated
Ca-channel
from
vacuole
andER
ofcauli¯ower
andvacuolesofguard
cells,
zucchinihypocotyls,oat
roots,
carrotandredbeetroots,mung
beanhypocotyls,maizecells
Ca2�
ActivationbyIP
3-binding
Ca-currentrecti®cationoverphysiologicaltonoplast
potentials(cytosolnegativewithreference
tolumen)
Ca2� -release
duringsignal
transduction
MuirandSanders,1996,1997;Allen
and
Sanders,1997;Muiret
al.,1997;Leckie
etal.,1998;MacRobbie,1997;McA
insh
etal.,1997
Ligand-gated
Ca-channel
invacuolesfrom
redbeets
and
cauli¯ower
¯orets
Ca2� ,
K�
ActivationbycA
DPR-binding
Ca2� -release
duringsignal
transduction
Allen
etal.,1995;MuirandSanders,1996;
Allen
andSanders,1997;McA
insh
etal.,
1997;Muiret
al.,1997;Leckie
etal.,1998
Ligand-gated
Ca-channel
from
algaEremosphaeraviridis
Ca2�
ActivationbycA
DPR-binding
Ca2� -release
duringsignal
transduction
Bauer
etal.,1998
VVCa(voltage-gated
Ca-
channelsfrom
vacuolesofVicia
fabaguard
cellsandBeta
vulgarisroots)
Ca2� ,
K�
Voltage-dependent(hyperpolarizationactivated)
pH-sensitive
Requiretw
oCa2�ionsbindingto
open
LuminalCa2�shiftsthethreshold
forvo
ltageactivationto
less
negativepotentials
Inhibited
by[Ca2� ]
cytincreases
IntracellularCa2� -release
Allen
andSanders,1994,1997;Johannes
andSanders,1995;McA
insh
etal.,1997;
PinerosandTester,1997
Ca-channel
from
ER
ofBryonia
diodicatendrils
Ca2�
Voltage-dependent
Ca2� -gradientsensitive
IntracellularCa2� -release
during
responsesto
mechanicalstim
uli
KluÈsener
etal.,1995
Anionchannels
Vacuolarmalate
channelÐ
VMal
from
Arabidopsisthaliana
vacuoles
Malate
fumarate,
acetateNOÿ 3;
H2POÿ 4
Activationbypotentialsmore
negativethanEMal
Stronginward
recti®cationbecause
ofluminalClÿ
blockadeofmalate
re-entry
Vacuolarmalate
uptake
Ceranaet
al.,1995;Allen
andSanders,
1997;Che�
ngset
al.,1997
VacuolarchloridechannelÐ
VCl
Clÿ,NOÿ 3;
SO
2ÿ
4
Activationbytonoplast
hyperpolarization(negative
potentialsrelative
tothecytoplasm
)Ðinward-recti®cation
Vacuolaranionuptake
Allen
andSanders,1997
VacuolarVClfrom
Vicia
faba
guard
cells
Clÿ
Activationbytonoplast
hyperpolarization
Channel
activationdependsonprotein
phosphorylation
Inward-recti®cation
Anionuptakeduringstomatal
opening
Pei
etal.,1996;GrabovandBlatt,1998a
Vacuolaranionchannel
from
Characeancells
Clÿ,NOÿ 3
Outw
ard
rectifyingCa-dependentregulation
Turgorregulation
Katsuhara
andTazawa,1992
VDAC
(voltage-dependentanion
channelsin
outermem
braneof
mitochondria)
Non-selective
Voltage-dependent
pH-dependent
Secondmessenger-binding
Controlofmitochondrialmem
brane
potential
ControlofATPdi�usion
Controlofsignaltransduction
Elkeles
etal.,1997;Mannella
etal.,1997,
1998;RostovtsevaandColombini,1997;
Green
andReed,1998;Songet
al.,1998;
Shim
izuet
al.,1999
Inner
mem
braneanionchannel
from
mitochondria
Non-selective
pH-regulated(activated
bylow
matrixpH)
Anionuniport
BeavisandVercesi,1992
Anionchannel
from
outer
envelopeofchloroplasts
Non-selective
Voltage-dependent
Metabolite
di�usion
Heiber
etal.,1995;Pohlm
eyer
etal.,1998
Anionchannel
from
inner
envelopeofchloroplasts
Clÿ
Voltage-dependent
Compensationoflight-driven
protonmovements
Heiber
etal.,1995;FuksandHomble,1999
Anionchannel
from
thylakoids
Clÿ
Voltage-dependent
Compensationoflight-driven
protonmovements
Heiber
etal.,1995;Pottosinand
Scho Ènknecht,1995
Krol and TrebaczÐIon Channel Gating in Plant Cells 45
9vacuolar membrane face (LuÈ hring, 1999).
a
positive vacuolar membrane potential for longer times thanat negative potentials and hence they mainly allow K� andNH�4 e�ux from the cytoplasm into the vacuole (preferredoutward recti®cation) (Tikhonova et al., 1997; BruÈ ggemannet al., 1999b). Their function is to control the electricalpotential di�erence across the tonoplast (Tikhonova et al.,1997). Vacuolar Ca2� suppresses FV channels in a voltage-dependent manner while cytosolic Ca2� blocks them in avoltage-independent manner (Allen and Sanders, 1996;Tikhonova et al., 1997; Allen et al., 1998a). One of the mostpronounced features of FV channels is their blockade byMg2�. Increasing cytosolic free Mg2� decreases the openprobability of FV channels without a�ecting single currentamplitudes (BruÈ ggemann et al., 1999a). FV currents werealso shown to be reduced at acidic pHcyt (Linz and KoÈ hler,1994) or by cytosolic polyamines (Dobrovinskaya et al.,1999). Recent studies on FV currents in red beet vacuolesindicate that FV channels may be ATP regulated (Allenet al., 1998a).
SV channels are strictly outward rectifying, cationselective and they show characteristics typical of a multi-ion pore, i.e. more than one ion can occupy the channelpore at the same time (Allen and Sanders, 1996). Theydisplay time-dependent activation at cytosol-positive poten-tials and when [Ca2�]cyt is higher than approx. 0.5 mM(Schulz-Lessdorf and Hedrich, 1995; Allen and Sanders,1996). Calcium and protons modulate the voltage-dependence of SV channels (Schulz-Lessdorf and Hedrich,1995). These two cations interact strongly with the voltagesensor without changing the unitary conductance. Theopen probability of the SV-type channel is a function of[Ca2�]cyt (Gambale et al., 1996). Schulz-Lessdorf andHedrich (1995) showed that there is a regulatory Ca2�-binding site on the cytoplasmic face of the SV channel andthat calmodulin may be involved in the modulation of theactivation threshold of the SV-type channel. This is inagreement with recent results of Bethke and Jones (1997)who examined SV currents stimulated by both calmodulin-like domain protein kinase (CDPK) and okadaic acid-sensitive phosphatases. On the other hand, Ca2�-dependentprotein phosphatase can induce the inhibition of SVchannels (Allen and Sanders, 1995). Bethke and Jones(1997) proposed a model in which SV channel activity isregulated by protein phosphorylation at two sites. In theabsence of calcium ions, Mg2� can activate SV currents(Allen and Sanders, 1996; Cerana et al., 1999). Moreover,the single channel conductance increases as a function ofthe potassium concentration (Gambale et al., 1996). Thisbehaviour can be explained by a multi-ion occupancymechanism. However, at negative transtonoplast voltages,the closure of SV channels is una�ected by either Ca2� orMg2�, indicating that the channel belongs to the voltage-gated superfamily (Cerana et al., 1999). SV channels arealso reversibly activated by a variety of sulphydryl reducingagents at the cytoplasmic side of the vacuole (Carpanetoet al., 1999). Increase in the open probability in the presenceof antioxidants may correlate ion transport with othercrucial mechanisms that in plants control turgor regulation,response to oxidative stresses, detoxi®cation and resistance
460 Krol and TrebaczÐIon Ch
to heavy metals (Carpaneto et al., 1999).
Cytosolic polyamines are strong inhibitors of SVchannels, but in contrast to the inhibition of FV channels,the blockage of SV channels displays a pronounced voltage-dependence (Dobrovinskaya et al., 1999). Hence,polyamine-blockage is relieved at a large depolarization(because of the permeation of polyamines through thechannel pore) and in the presence of high concentrations ofpolyamines the slow vacuolar channels are converted intoinward recti®ers (Dobrovinskaya et al., 1999).
VK channels are non-rectifying and are activated atmicromolar [Ca2�]cyt and acidic pHcyt by tonoplast poten-tials ranging from ÿ100 to �60 mV (Allen and Sanders,1996; Allen et al., 1998a). Therefore, they can be involved invacuolar potassium uptake and loss. So far their presencehas been proved only in guard cells.
Di�erent sensitivities of FV-, SV- and VK-channels to[Ca2�]cyt and pH may provide a mechanism whereby stimuliactivating various signalling pathways can generatevacuolar ion uptake or loss. Muir et al. (1997) concludedthat this di�erential regulation of vacuolar channels byCa2� represents a downstream event in signal transductioncascades induced by Ca2�-release. SV channels are thoughtto participate in signalling processes because of their abilityto release Ca2� after Ca2�-dependent activation (CICRÐCa2�-induced Ca2�-release) (Allen et al., 1998b). However,Pottosin et al. (1997) demonstrated that the SV channel isnot suited for CICR from vacuoles, at least in the case ofbarley mesophyll cells. Thus, the physiological role of SVchannels remains a matter for discussion.
The most frequently observed voltage-gated K�in -channelin the tonoplast of Chara was examined by LuÈ hring (1999)(Table 3). Acidi®cation on both sides of the membranedecreases open probability of the channel and changes itsvoltage-dependence, most probably through protonation ofnegatively charged residues in neighbouring voltage-sensingtransmembrane domains (LuÈ hring, 1999). The channelbehaves like animal maxi-K channels and its gating kineticsresponds to cytosolic Ca2�. Under natural conditions,pH changes contribute mainly to channel regulation at the
nnel Gating in Plant Cells
Voltage-gated potassium channels in other intracellularmembranes
Heiber et al. (1995) showed that the chloroplast envelopecontains voltage-dependent cation channels (Table 3) withcomplex gating behaviour and subconductace states, as wellas cation-selective pores with high conductances. Voltage-dependent cation channels favour potassium uptake andtheir gating is a�ected by monovalent cations (Cs�),divalent cations (Mg2�) and millimolar concentrations ofATP. Hinnah and Wagner (1998) observed potassiumselective pore-like channels in osmotically swollen thyla-koids from pea protoplasts derived from cotyledons ofyoung Pisum sativum plants (Table 3). There is also anonselective (PK 4 PMg 4 PCa) cation channel in nativespinach thylakoid membranes (Table 3) found by Pottosinand SchoÈ nknecht (1996). This cation channel displaysbursting behaviour and its open probability increases at
positive membrane potentials (Pottosin and SchoÈ nknecht,a
1996). It has only a moderate voltage-dependence com-pared to classical voltage-dependent recti®ers. It is postu-lated that its function is to compensate the light-drivenproton uptake into thylakoids (Pottosin and SchoÈ nknecht,1996).
A Ca2�- and voltage-dependent non-speci®c channel wasfound in the nuclear envelope of red beet (Grygorczyk andGrygorczyk, 1998) (Table 3). Micromolar [Ca2�] on thenucleoplasmic side of the envelope activates this cationchannel. The channel voltage-dependent activity changeswith the nucleoplasmic calcium concentration. Such achannel may provide a Ca2�-regulated pathway for Ca2�-
Krol and TrebaczÐIon Ch
dependent nuclear processes (e.g. gene transcription).
skeletal ®bres (Sievers et al., 1996).
Plasmalemma voltage-insensitive cation channels (VIC)The VIC channels are responsible for an in¯ux of a rangeof monovalent cations into cereal root cells (Table 2). It hasbeen postulated that they could contribute to low-a�nityNH�4 uptake and rapid osmotic adjustment independent ofmembrane potential. They may also compensate electro-genic cation ¯uxes (White, 1999). Under saline conditionsVIC channels along with K�in channels play a major role inthe toxic Na� in¯ux across the plasma membrane (White,1999). Inward currents through the VIC channels are
inhibited by Ca2� and Ba2�.(discussed by Szarek and Trebacz, 1999).
( facilitating Ca2� in¯ux to the cytosol).
Stretch-activated potassium channels
Changes in turgor pressure induced by hyper- or hypo-osmotic stress induce an early change in activities of stretch-sensitive channels. Stretch-activated channels (SACs) alsorespond when mechanical forces are exerted on the cell(Ramahaleo et al., 1996). For the translation of membranestretch into channel gating it is generally argued thatattachment of membrane proteins to tension-transmittingcomponents is necessary, by linkage to cell wall proteins, orcytoskeletal proteins, or both (MacRobbie, 1997). Anionic,cationic, as well as non-selective SACs, have been reportedto occur in plasma membranes (Table 2). There is agrowing body of evidence for involvement of stretch-activated ion channels in regulation of the response ofguard cells to ABA through interactions with the cyto-skeleton (MacRobbie, 1997; McAinsh et al., 1997). Liu andLuan (1998) identi®ed two kinds of stretch-activatedpotassium channels in Vicia faba guard cells: voltage-gated and insensitive to membrane potential. This was the®rst evidence that plants contain osmosensitive, voltage-dependent channels, those previously described by Rama-haleo et al. (1996) being voltage-independent. Negativepressure activates voltage-insensitive currents with conduct-ance very di�erent from that of voltage-dependent K�-channels. Voltage-dependent currents (IKin and IKout) are inturn sensitive to osmotic gradient rather than changes inpressure, although actin ®laments are involved in IKinregulation (Liu and Luan, 1998). Hypotonic conditionsactivate IKin and inactivate IKout , while hypertonic con-ditions act in the opposite way. An alternation in channelopening frequency is responsible for regulating I and
KinIKout under di�erent osmotic conditions. Hypertonicinhibition of IKin can be prevented by disruption of actin®laments. Actin ®lament disruption occurs in hypotonicconditions providing a link between hypotonic stress andhypotonic activation of the inward K� channels. Alsocytochalasin D (a cytoskeleton disrupting drug) modulatesIKin in a similar way to hypotonic conditions (Liu andLuan, 1998), which is consistent with the report of Hwanget al. (1997). It seems reasonable that stretch-activatedchannels in the plant plasma membrane, which is undercontinuous compression resulting from turgor pressure andthe presence of the cell wall, interact with cytoskeletalstructures providing local stretch of the membrane. It ispostulated that during perception of gravitational stimuli,statoliths exert local stretch on the membrane via cyto-
nnel Gating in Plant Cells 461
Light-activated potassium channels
Blom-Zandstra et al. (1997) examined light e�ects onvoltage-gated K�out channels in mesophyll protoplasts ofNicotiana tabacum (Table 2). Single channel data frompatch-clamp studies indicate that the activity of the channelincreases upon dark-light transition. The e�ect of light wasnot observed in root cells or chlorophyll-de®cient cells,suggesting that such a response requires photosyntheticactivity. These results are consistent with those of Kim et al.(1992) who showed that K� channels display responses tolight. The light activated ion channels and electrogenicproton pump are regarded as important factors in the notyet fully understood light stimulus transduction cascade
CALCIUM CHANNELS
Calcium ions are universal second messengers in plant andanimal cells. They mediate in various signalling pathways(reviewed by Brownlee et al., 1999; Sanders et al., 1999)from signal perception to gene expression, through theactivation of ion channels and enzyme cascades. Stimulus-induced increases in [Ca2�]cyt encode information as speci®cspatial and temporal changes in frequency of [Ca2�]cytoscillationsÐthe `calcium signature' (McAinsh et al.,1997; Leckie et al., 1998). After signal transition, excessCa2� must be sequestered into external and internal storesto keep [Ca2�]cyt at a low level ranging from tens tohundreds nM. Thus, all Ca2� channels located in Ca2�-sequestering membranes are strongly inward rectifying
Ligand-gated calcium channels in plasma membrane
Recently, Zimmermann et al. (1997) reported a novelCa2�-permeable, La3�-sensitive plasma membrane ionchannel of large conductance (Table 2). The channel isactivated by elicitors and is essential in pathogen defence.Receptor-mediated stimulation of these channels appears tobe involved in the signalling cascade triggering a pathogendefence system. The activation of plasma membrane Ca2�-channels by speci®c and non-speci®c elicitors provides a
direct demonstration of a pathway by which [Ca2�]cyt(Muir et al., 1997).
a
increases to levels that can initiate the production of activeoxygen species, callose and phytoalexins via Ca2�±
462 Krol and TrebaczÐIon Ch
dependent gene expression (Blumwald et al., 1998).
aperture changes.
Ligand-gated calcium channels in inner membranes
Ligand-gated Ca2� channels in plant cells reported todate represent two classes: IP3 (inositol triphosphate)- orcADPR (cyclic ADP-ribose)-gated (Table 3). Recently anew signalling moleculeÐNAADP (nicotinic acid adeninedinucleotide phosphate)Ðhas been found in animal cells(Lee, 2000). Ligand-gated Ca2� channels are present only inintracellular compartments, and thus their existence pro-vides a convenient mechanism for linking perception ofstimuli (e.g. light, IAA, ABA, osmotic shock, pollination,Nod-factors, cold shock) to intracellular calcium mobiliza-tion (Knight et al., 1996; McAinsh et al., 1997; Muir et al.,1997; Trewavas and Malho, 1997). The IP3-induced Ca2�-release originates mainly from vacuolar stores, although incauli¯ower, Muir and Sanders (1997) found at least twodistinct membrane populations sensitive to IP3. IP3-inducedCa2�-currents are inwardly rectifying and highly selectivefor calcium (Allen and Sanders, 1997). A speci®c IP3-binding 400-kDa protein, which is competent to releaseCa2� when incorporated into proteoliposomes (Biswaset al., 1995), was puri®ed from mung bean, though nosubsequent reports on this protein have appeared. There issome indirect evidence for the presence of IP3-gated Ca2�channels in the tonoplast of the algae Chara and Nitella(Katsuhara and Tazawa, 1992).
As well as IP3-gated channels, cADPR-gated Ca2�channels act as instantaneous strong inward recti®ers overphysiological membrane potentials and they are activatedby ligand binding only in the presence of calcium on theluminal side of the membrane. Pharmacological studiessuggest that cADPR has the capacity to act as a Ca2�-mobilizing intracellular messenger and an endogenousmodulator of Ca2�-induced Ca2� release (CICR) (Willmottet al., 1996). Ryanodine and ca�eine (agonists of ryanodinereceptors in animal cells) are able to cause activation ofcADPR-gated channels in a dose-dependent manner (Allenet al., 1995), while ruthenium red and procaine (antagonistsof ryanodine receptors in animal cells) block Ca2� release(Allen et al., 1995; Muir and Sanders, 1996; Bauer et al.,1998) in plant cells. Heparin of low molecular mass andTMB-8, well known competitive inhibitors of IP3-receptorsin plant and animal cells, are without e�ect on cADPR-gated Ca2�-channels (Muir and Sanders, 1996). Allen et al.(1995) demonstrated that there is a relatively low density ofcADPR-gated channels in beet microsomes. cADPR-gatedchannels could participate in calcium release only up to25% in comparison to the dominating IP3-induced Ca2�-release. Similar results were obtained from cauli¯owermicrosomes (Muir et al., 1997) and the unicellular greenalga Eremosphaera viridis (Bauer et al., 1998). Preliminaryexperiments on sea urchin egg homogenates indicate thatcADPR may bind to an accessory 100±140 kDa protein(Galione and Summerhill, 1996).
The lack of modulation of plant ligand-gated Ca2�-
channels by cytosolic Ca2� is the most notable di�erencerecognized to date between these and animal channels
nnel Gating in Plant Cells
Voltage-gated calcium channels in the plasmalemma
Many voltage-gated Ca2� channels have been describedin a variety of plant tissues and species (reviewed by Pinerosand Tester, 1997) (Table 2). Most of these are activatedthrough membrane depolarization and stimuli causingmembrane depolarization such as increased [K�]ext(Thuleau et al., 1994), Ca2� starvation (Reid et al., 1997),cytokinins (Schumaker and Gizinski, 1993), light orelectrical pulses (Trebacz et al., 1994; Ermolayeva et al.,1996, 1997) mechanical stimulation (Shimmen, 1997), ABA(McAinsh et al., 1995; Grabov and Blatt, 1998b) andmicrotubule inhibitors (Thion et al., 1996). White (1998),focusing on Ca2� channels in the plasma membrane of rootcells, distinguished between them based on their di�erentsensitivities to La3�, Gd3� and verapamil. He discussedtheir roles in mineral nutrition, intracellular signalling andpolarized growth.
Kiegle et al. (2000), Gelli and Blumwald (1997) andStoeckel and Takeda (1995) described the hyperpolariza-tion-activated in¯uxes of Ca2� through the plasmalemma.The hyperpolarization-activated calcium current is postu-lated to allow nutritive Ca2� uptake. Hyperpolarization-activated Ca2� channels described in the plasma membraneof Vicia faba guard cells by Fairley-Grenot and Assmann(1992) are in fact the inwardly rectifying K� channelsmediating Ca2� in¯ux prior to their closure and they maybe involved in the regulatory mechanism of stomatal
Voltage-gated calcium channels in inner membranes
Voltage-gated Ca2�-channels are also present in othercell compartments such as the vacuole, thylakoids or ER(Pineros and Tester, 1997) (Table 3). Vacuolar voltage-gated Ca2� channels (VVCa), characterized by Allen andSanders (1994), behave as multi-ion pores inwardly rectify-ing over the voltage range between ÿ20 and ÿ50 mV(hyperpolarization). Their activity is inhibited by lantha-nides, verapamil, nifedipine and by [Ca2�]cyt above 1 mM.Luminal Ca2� shifts the threshold for VVCa activation to aless negative potential, and therefore restricts the accumu-lation of calcium excess in the vacuole. Luminal pH ofabout 5.5 prevents uncontrolled leakage of Ca2�, because atthis physiological pH value the channel openings are veryinfrequent (the highest activation is around pH 7).Johannes and Sanders (1995) showed that a binding oftwo calcium ions is required to open the VVCa channel.Voltage-gated vacuolar Ca2� channels, previouslydescribed in tonoplasts of beet, Arabidopsis and tobacco,are in fact manifestations of SV K� channels (Ward andSchroeder, 1994).
KluÈ sener et al. (1995, 1997) have shown the voltage-gatedCa2� channels derived from endoplasmic reticulum mem-branes of Bryonia dioica touch-sensitive tendrils. The rangeof membrane potentials activating these channels was
a�ected by the Ca2� gradient across the membrane. Singlea
channel currents were modulated by divalent cations,protons and H2O2 . H2O2 is a strong inhibitor of thesechannels. The channel conductance increases with cytosolacidi®cation. These channels play an important role in themodulation of [Ca2�] in response to changes in [H O ]
Krol and TrebaczÐIon Ch
cyt 2 2 cytor pH .
in turgor/volume regulation and signal transduction.
[Ca2�] is assumed to occur during turgor regulation.
cyt
Stretch-activated calcium channels
Taylor et al. (1996) examined both stretch-activated andvoltage-gated mechanosensitive Ca2�-permeable cationchannels in subprotoplasts prepared from di�erent regionsof rhizoid and thallus cells of Fucus zygotes (Table 2). Theirresults suggest that intercellular signal transduction ispatterned by interactions of the cell wall, plasma membraneand intracellular Ca2� stores.
Thion et al. (1996) observed activation of voltage-gatedCa2� channels by microtubule disruption. Their results areconsistent with a previous report of Davies (1993), whopostulated that variation potentials can be transduced viamechano-sensitive Ca2� channels into gene expressionthrough Ca2�-dependent cytoskeleton-associated phos-phorylation/dephosphorylation processes. In addition,Ca2� in¯ux through `volume sensing' voltage-gated Ca2�channels is essential for an apical Ca2� gradient to bemaintained in a growing cell (Taylor et al., 1996; Holdaway-
Clarke et al., 1997).ANION CHANNELS
Plant anion channels regulate anion e�ux from a cellthrough plasmalemma (Table 2) and/or tonoplast(Table 3). Anion e�ux from the cytoplasm into theextracellular space is driven by the anion gradient and thenegative membrane potential causing plasma membranedepolarization, which in turn activates outward rectifyingvoltage-gated K� channels. Anion-induced depolarizationplays a crucial role in such processes as xylem loading,generation and propagation of action potentials or light-induced transient voltage changes of membrane potential.In addition, anion and potassium losses promote osmo-regulation, stomatal closure, tissue and organ movements.Since plant cells experience low extracellular anion concen-trations, anion uptake must be energetically coupled with
proton pumps.Ligand-gated anion channels in the plasmalemma
There are many anion channels activated by cytoplasmiccalcium widespread in plant cells (Katsuhara and Tazawa,1992). Ca2�-dependent anion channels are responsible forthe main depolarizing current during action potential inCharophyta (Okihara et al., 1991; Katsuhara and Tazawa,1992; Thiel et al., 1993; Shimmen, 1997), the liverwortConocephalum conicum (Trebacz et al., 1994), Aldrovandavesiculosa (Iijima and Sibaoka, 1985) and during phyto-chrome-mediated transient depolarization in the mossPhyscomitrella patens (Ermolayeva et al., 1996, 1997).
Johannes et al. (1998) showed a direct e�ect of cyto-
plasmic protons on Clÿ e�ux in Chara corallina duringintracellular acidosis. H�-activated anion channels respon-sible for Clÿ currents act to facilitate an enhanced protone�ux under conditions of low pHcyt . Activity of thesechannels is also indirectly pH- and Ca2�-dependentthrough phosphorylation/dephosphorylation processes.The above-mentioned ®ndings imply that plasmamembrane anion channels play a central role in pHcytregulation in plants, in addition to their established roles
nnel Gating in Plant Cells 463
Ligand-gated anion channels in the tonoplast
Katsuhara and Tazawa (1992) summarized calcium-regulated channels and their bearing on physiologicalactivities in characean cells. They presented some evidencefor the presence of Ca2�-regulated anion channels in thetonoplast of Chara, Nitellopsis and Lamprothamnium giantinternodal cells (Table 3). Activation of these channels by
cyt
Voltage gated anion channels in the plasma membrane
In the plasma membrane, voltage-gated anion channelsare activated by depolarization and under an excess ofcytoplasmic Ca2�. They deactivate under hyperpolarizingpotentials (Keller et al., 1989; Hedrich et al., 1990; Hedrichand Jeromin, 1992; Linder and Raschke, 1992; Schroederand Keller, 1992; Dietrich and Hedrich, 1994; Thomineet al., 1995; Schultz-Lessdorf et al., 1996; Lewis et al., 1997;Pei et al., 1998). Inverse voltage dependence (activation byhyperpolarization) has been reported infrequently to date.Barbara et al. (1994) reported hyperpolarization-activatedchloride currents contributing both to the control ofmembrane potential and to osmotic balance regulation incarrot cells. Neither calcium ions nor MgATP werenecessary for fast activation of these channels. Underlarge hyperpolarization, Barbara et al. (1994) observedrapid and voltage-dependent channel inactivation.Recently, hyperpolarization-activated anion channels havealso been found in the plasmalemma of the unicellulargreen alga Eremosphaera viridis (SchoÈ nknecht et al., 1998).They conduct an anion e�ux and hence they are respons-ible for limiting the amplitude of dark-induced transienthyperpolarization caused by K�-release. The well-knownanion 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 areCa2�-dependent anion currents activated by hyperpolar-izing pulses. These anion channels display ATP-dependentbi-modular ( fast and slow) kinetics. R-mode ( fast acti-vation and deactivation of the channel) occurs in theabsence of ATP. However when 3 mM MgATP is added tothe pipette solution facing the cytoplasmic side of themembrane, the current shows slow but clear time-inactivation (S-mode).
Dietrich and Hedrich (1994) showed the bimodularkinetics of the guard cell anion channel (GCAC1) in Viciafaba protoplasts. Previously these two modes of one guardcell anion channel were considered as two anion channels
contributing to di�erent depolarization-associated processesextracellular face of the channel, eliciting stomatal opening.
kinase dependent anion uptake (Pei et al., 1996).
a
during regulation of stomatal movements (Schroeder andKeller, 1992). Dietrich and Hedrich (1994) noted that themode of action of GCAC1 is under the control ofcytoplasmic factors. Later Thomine et al. (1995) alsoidenti®ed a voltage-dependent anion channel in epidermalcells of Arabidopsis hypocotyls which showed two-modefunction: rapid and slow mode in the presence or absence ofintracellular ATP, respectively. R-type and S-type channelsare voltage-regulated in a quite di�erent way and theydisplay di�erent kinetics. Only R-type anion channelsdisplay strong voltage-dependence, while weak voltage-dependence of S-type channels leaves them partially activeeven when the membrane is strongly hyperpolarized. Suchbehaviour of S-type channels makes them responsible forsustained e�ux of anions (Keller et al., 1989; Linder andRaschke, 1992; Schroeder and Keller, 1992; Schroeder et al.,1993; Thomine et al., 1995), which serves as a negativeregulator during stomatal opening (Schroeder et al., 1993;Pei et al., 1998) or hypocotyl movements (Cho andSpalding, 1996). Transition between R- and S-mode of ananion channel may correspond to ATP binding (Schulz-Lessdorf et al., 1996; Thomine et al., 1997) or alternativelyto ATP-dependent phosphorylation/dephosphorylationprocesses (Schmidt et al., 1995; Thomine et al., 1995).R-type guard cell anion channels (GCAC1) in Arabidopsiswere shown not to be directly regulated by phosphorylationevents (Schulz-Lessdorf et al., 1996). They require cyto-plasmic ATP to undergo voltage- and Ca2�-dependentactivation, involving strongly cooperative binding of fourATP molecules (Schulz-Lessdorf et al., 1996). On the otherhand, S-type GCAC1 channels are strongly activated byphosphorylation (in Vicia faba and Commelina communisguard cells) or dephosphorylation (in Arabidopsis andNicotiana cells) (Armstrong et al., 1995; Schmidt et al.,1995; Cho and Spalding, 1996; Li and Assmann, 1996;Schulz-Lessdorf et al., 1996; Esser et al., 1997; Mori andMuto, 1997; Pei et al., 1997, 1998; Schwarz and Schroeder,1998). Therefore, guard cell anion channels characterized inArabidopsis (GCAC1) can also be classi®ed as ligand-gatedchannels, since Schulz-Lessdorf et al. (1996) showed directbinding of ATP to the channel protein. Leonhardt et al.(1999) in turn, suggest that the slow anion channel in guardcells may belong to the class of ATP binding cassette (ABC)proteins. The same situation applies in the case of voltage-gated and nucleotide-regulated anion channels of Arabi-dopsis hypocotyls described by Thomine et al. (1997). Theycon®rmed that nucleotide binding (ATP 4ADP�AMP)regulates channel activity (alters the kinetics and voltage-dependence, causing a shift toward depolarized potentialsand thus leading to a strong reduction of anion currentamplitude). This regulation may couple the electricalproperties of the membrane with the metabolic status ofthe whole cell.
Rapid- and slow-modes of the Arabidopsis guard cellanion channel (GCAC1) are also variously in¯uenced bypHcyt (Schulz-Lessdorf et al., 1996). The kinetics of S-modeis in¯uenced by the pH gradient across the plasmalemma(the inactivation gate responds to pH gradient, which maybe converted into a change of a channel structure). Such
464 Krol and TrebaczÐIon Ch
pH gradient-dependence of slow inactivation resembles a
carrier-mode action. In the case of R-mode, the protongradient does not seem to a�ect channel activationfollowing ATP-binding. The single channel activity ofR-type GCAC1 increases as a function of [H�]cyt (proto-nation of the cytoplasmic site of the channel), while singlechannel conductance is una�ected either by pHcyt or pHext .Similar pH sensitivity was determined for anion-permeablevacuolar channels (Schulz-Lessdorf and Hedrich, 1995).Since the time- and voltage-dependent activity of guard cellanion channels (GCAC1) was shown to be stronglymodulated by ATP and H� (Schulz-Lessdorf et al., 1996),these channels have been thought to be capable of sensingchanges in the energy status, the proton pump activity andacid metabolism of the cell.
Patch-clamp studies revealed that growth hormones candirectly a�ect voltage-dependent activity of inwardlyrectifying anion channels in a dose-dependent manner(Hedrich and Jeromin, 1992). Auxin binding is side- andchannel-speci®c, and results in a shift of the activationpotentials towards the resting potential favouring transientchannel opening (Marten et al., 1991). These authorsdemonstrated that auxin can interact directly with the
nnel Gating in Plant Cells
Voltage-gated anion channels in the tonoplast
In the tonoplast (Table 3) there are two types of cytosol-negative-potential-activated (hyperpolarization-activated)anion channels: VCl and VMal (Allen and Sanders,1997). The ®rst is responsible for carrying Clÿ to thevacuole (inward rectifying), while the second is mainlypermeable for malate, but also for succinate, fumarate,acetate, oxaloacetate, NOÿ3 and H2PO
ÿ4 : VMal is very
strongly inward rectifying over the physiological range ofnegative potentials, but more negative than Emal (Ceranaet al., 1995). Cytosolic Ca2� and ATP do not a�ect VMalchannels (Cerana et al., 1995; Che�ngs et al., 1997). On theother hand, Pei et al. (1996) reported that calmodulin-likedomain protein kinase (CDPK) activates vacuolar malateand chloride conductances (VCl) in guard cell vacuoles ofVicia faba. Activation of both currents depends on Ca2�and ATP, enabling anion uptake into the vacuole even atphysiological potentials. CDPK-activated VCl currentswere also observed in red beet vacuoles, suggesting thatthese channels may provide a more general mechanism for
Voltage-dependent anion channels in other endomembranes
Voltage-dependent anion channels (VDACs or mitochon-drial porins) in the outer membrane of mitochondriaregulate the mitochondrial membrane potential, amongother things, during transduction of an apoptotic signal intothe cell (Green and Reed, 1998; Shimizu et al., 1999) ormetabolite di�usion (Elkeles et al., 1997; Rostovtseva andColombini, 1997; Mannella, 1998) (Table 3). According toRostovtseva and Colombini (1997) these channels areideally suited to controlling the ¯ow of ATP between thecytosol and the mitochondrial spaces. VDAC pore is formed
by a single �30-kDa protein (Song et al., 1998) whichling cellular pH, transmembrane and osmotic potential.
a
undergoes a major conformational change at pH5 5(Mannella, 1997, 1998). However, functional VDAC is aheterodimer including one pore protein and other modulat-ing subunits (Elkeles et al., 1997). Apart from transmem-brane voltage and pH, VDACs can be regulated by directbinding of signalling proteins (Shimizu et al., 1999).
Anion uniport in plant mitochondria is mediated by apH-regulated inner membrane anion channel that is acti-vated by matrix H� (Beavis and Vercesi, 1992). Voltage-dependent inner mitochondrial anion channels (IMACs),which serve as a safeguard mechanism for recharging themitochondrial membrane potential, have been found inanimal tissues (Ballarin and Sorgato, 1996; Borecky et al.,1997).
Voltage-dependent anion channels were characterized bya patch-clamp study in osmotically swollen thylakoids fromPeperomia metallica (SchoÈ nknecht et al., 1988) and the algaNitellopsis obtusa (Pottosin and SchoÈ nknecht, 1995).Voltage-gated anion channels found in thylakoids are mostprobably responsible for the compensation of light-drivenH� movements (SchoÈ nknecht et al., 1988; Heiber et al.,1995).
Recently, Pohlmeyer et al. (1998) discovered a new typeof voltage-dependent solute channel of high conductance inthe outer envelope of chloroplasts, etioplasts and non-greenroot plastids (Table 3). The channels are permeable fortriosephosphate, ATP, Pi , dicarboxylic acids, amino acids,and sugars. Their open probability is highest at 0 mV (whichis consistent with the absence of transmembrane potentialacross the plastidic outer membranes). Previously, Heiberet al. (1995) reported a voltage-dependent anion channel oflow conductance in the chloroplast envelope. There are alsoanion channels found in the inner envelope membrane of
Krol and TrebaczÐIon Ch
isolated intact chloroplasts (Fuks and Homble, 1999).
growth and development.
Stretch-activated anion channels
Falke et al. (1988) ®rst reported large conductance,stretch-activated, anion-selective channels in protoplasts oftobacco. Cosgrove and Hedrich (1991) then showed theexistence of stretch-activated Clÿ, Ca2� and K� channels inthe plasma membrane of guard cells. Teodoro et al. (1998)suggested that the changes in turgor pressure induced byhyper-/hypo-osmotic stress may cause an early inactivation/
activation of stretch-sensitive anion channels, respectively.from the State Committee for Scienti®c Research.
vacuoles by cytosolic and luminal calcium. The Plant Journal 10:
Light-activated anion channels
By patch clamping hypocotyl cells isolated from dark-grown Arabidopsis thaliana seedlings, Cho and Spalding(1996) revealed the existence of blue-light activated anionchannels responsible for light induced membrane depolar-ization (Table 2). Their results are consistent with previousreports of Elzenga et al. (1995). Further studies on blue-light activated anion channels in Arabidopsis hypocotylconducted by Lewis et al. (1997) showed that the openprobability of the channel depends on [Ca2�]cyt and thatwithin the calcium concentration range of 1±10 mM theprobability of channel activation increases. Their results
indicate that cytoplasmic calcium does not a�ect the anionchannel directly, but that it does so through intermediates(e.g. Ca2�-dependent kinases or phosphatases). Activationof blue light-induced anion channels plays a central role intransducing light signals into hypocotyl growth inhibition(Cho and Spalding, 1996; Parks et al., 1998).
Light-activated anion channels, resembling those above,were also reported by Elzenga and Van Volkenburgh(1997a) (Table 2). They examined light-induced transientdepolarization in Pisum sativum mesophyll cells due toincreased conductance for anions and concluded that:(1) under illumination the anion current increases three-fold because of an increase in the open probability of a 32-pS anion channel; (2) this change in channel activity is notdue to light-induced changes in membrane potential; (3) theanion current depends on light intensity and can be totallyblocked by the photosynthetic inhibitor DCMU; (4) theanion current is strongly Ca2�-dependent; and (5) light-induced anion e�ux may balance light-induced protonextrusion and therefore participate in a mechanism control-
nnel Gating in Plant Cells 465
CONCLUSIONS
From year to year the number of characterized ion channelsincreases, which bene®ts our understanding of their roles innumerous physiological processes. Modern electrophysio-logical and molecular biological techniques have enabledthe characterization and classi®cation of novel channeltypes. On the other hand, some channels previouslydescribed as di�erent types are in fact `synonyms'. Thesemainly originate from multiple gating mechanisms that cansense the energy status of the cell and thus make the cellresponsive to various stimuli in a very e�cient way. The ®netuning of channel activities depends on e�ectors availablein a certain cell type, i.e. it is plant and tissue speci®c(Barbier-Brygoo et al., 1999). Further research concerningregulation and gating of the ion channels described here willhelp to unravel the intermediate signalling mechanisms usedby plants in dynamic responses to the environment during
ACKNOWLEDGEMENTS
We thank Professor M. A. Venis and the reviewers forhelpful comments and critical reading of the manuscript.The investigation was supported by the grant 6P04 C 04218
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