Progress in identification of plant ion channels and
development of electrophysiological analyses in heterologous expression systems and in planta, in combination with reverse genetic approaches, are providing the possibility of
associating molecular entities with physiological functions. Recently, the first attempts to determine in vivo functions using knockout mutants demonstrated the roles of root ion channels. The search for proteins interacting with such channels leads to an even more complex view of the concerted action in protein networks.
Addresses
*Max-Planck-Institut für Molekulare Pflanzenphysiologie, D-14424 Potsdam, Germany; e-mail: [email protected] †Biochimie et Physiologie Moléculaire des Plantes, INRA/ENSA-M/CNRS URA 2133, Place Viala, F-34 060 Montpellier cedex 1, France; e-mail [email protected]
Current Opinion in Plant Biology1999, 2:477–482
1369-5266/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved.
Abbreviations
ABA abscisic acid
AKT1 ArabidopsisK+transporter 1
GFP green fluorescent protein IRK inward-rectifying K+
KAT1 K+Arabidopsis thaliana channel 1
KCO K+channel family related to the Ca2+-activated, outwardly rectifying K+channel
KST1 K+channel of Solanum tuberosum
P domain pore domain
SKOR Stellar outward-rectifying K+channel
TWIK tandem of P domains in a weak inward-rectifying K+
Introduction
Channels, when open, form selective pores through which transported ions can move without inducing a general conformational change of the protein. The max-imum velocity at which ions can move through channels (107ions per second) is therefore several orders of mag-nitude higher than the velocity at which ions can be transported by carriers. Ion channels are involved in the control of membrane potential and signal transduction in plants [1], as in animals. In plants, they are also involved in sustained transport, such as uptake of ions from the soil solution or secretion of ions into the xylem sap [2].
The first channels identified in plants were two K+
chan-nels from Arabidopsis, AKT1 (ArabidopsisK+ transporter 1)
[3] and KAT1 (K+Arabidopsis thaliana channel 1 ) [4], both
cloned in 1992 by functional complementation of yeast mutant strains defective for K+transport. Various
molecu-lar approaches and electronic cloning have thereafter revealed a large multigene family of channels related to
AKT1 and KAT1, sharing homologies with animal Shaker -type channels and grouped in the so-called plant Shaker family, two other families of K+ channels related to the
animal TWIK (tandem of P-domains in a weak inward-rectifying K+) and IRK (inward-rectifying K+) channels
[5•], and four other gene families likely to encode cation or
anion channels from their homologies with animal chan-nels. Given that ∼70% of the open reading frames in the Arabidopsis genome have now been fully or partially sequenced, it may be assumed that most plant ion channel families with counterparts in animals have been identified. This advancement has shifted research priorities from cloning to characterization of the gene products and iden-tification of their roles in planta. Although work in this field is in its infancy, molecular approaches, aimed at broaden-ing the view by identifybroaden-ing regulatory mechanisms and interacting networks, have commenced.
The first part of this review is focused on the work of the past two years on plant Shakerchannels, which has provid-ed both highly significant results at the biological level and a paradigm in this field of research by closely associating tools from molecular biology, reverse genetics and electro-physiology. The second and third part of the review present exciting progress in characterization of other ion channel families and analysis of channel regulation.
Plant K
+channels of the
Shaker
family
At present, all K+channels cloned, from prokaryotes to eukaryotes, are classified into four groups according to their structural features [6] (Figure 1). They all contain a characteristic domain, named H5 or the pore (P) domain, which forms part of the aqueous pore of the channel and controls permeation and ionic selectivity [7••]. They
dif-fer in the number of transmembrane segments (two, four, six or eight) and P domains (one or two) present per polypeptide. A milestone in elucidating the struc-ture–function relationship of K+ channels was the
determination of the three-dimensional structure of a bacterial K+ channel from the two transmembrane
seg-ments/one pore family [7••].
Structure–function relationship
Regions similar to the six transmembrane segments (called S1–S6) and the P domain of animal Shaker channels are present in the first channels identified in Arabidopsis, AKT1, KAT1 and in their relatives (Figure 1). The postu-lated topology derived from animal Shaker channels (Figure 2) has been confirmed for KAT1 [8]. Experimental evidence has been obtained indicating that the S4 segment and the P domain in the plant Shaker-like channels play the same roles as their animal counterparts [6]. Namely, the S4 domain senses the transmembrane voltage and
Plant ion channels: from molecular structures to
physiological functions
controls channel activation, while the P domain forms the aqueous pore and controls permeation.
In this field of research, KAT1 and its counterpart KST1 (K+channel of Solanum tuberosum) in potato [9] have been
used as models, at least in part because these channels can be easily characterized in Xenopus oocytes. Furthermore, within the Shakerfamily, KAT1 was the first channel known to be endowed with inward rectification (i.e. mediating K+influx), as all the animal channels cloned at that time
were characterized as outward rectifiers (i.e. mediating K+efflux). This discovery gave rise to a question debated
within the electrophysiologist community (plant and ani-mal), namely whether hyperpolarization-induced KAT1 inward currents were related to the channel activation [10]
or to recovery from inactivation [11]. The former hypothe-sis has recently received further support [12,13••].
Furthermore, recent identification of an outwardly rectify-ing K+ channel SKOR (Steller outward-rectifying
K+ channel) [14••] and of a weakly rectifying K+channel
AKT3 [15•] among the plant Shakerfamily clearly indicates
that similar structures can be endowed with different recti-fication mechanisms. The plant Shakerfamily is therefore providing an exciting model for electrophysiologists inter-ested in structure–function relationship analyses.
Site-directed mutagenesis of the KAT1S4 domain coupled to electrophysiological characterization of mutated channnels in Xenopusoocytes has recently identified posi-tively-charged amino acids as voltage sensing residues [12,13••]. A similar strategy has identified mutations in the
sequence encoding the P domain of KAT1 that alter chan-nel permeation properties and confer increased sensitivity to blocking by Ca2+ [16] or stimulation by H+ [17•].
Although the AKT1 channel cannot be characterized in Xenopusoocytes (for reasons that have not yet been eluci-dated), random mutagenesis coupled to characterization of mutated channels by functional expression in yeast has pro-vided evidence that the P domain controls permeation in Figure 1
Secondary structure of the four K+channel types. Channels belonging to the Shakerfamily share a typical hydrophobic structure consisting of six transmembrane segments (S1–S6), the P domain being present between the fifth and the sixth segments. The second family, named IRK in animal cells, includes channels displaying two transmembrane segments, the P domain being present between them. The third and fourth groups, named TWIK in animal cells and TOK (two-pore outwardly rectifying K+channel) in yeast, correspond to polypeptides with two P domains and either four or eight putative transmembrane segments. In the former case, the hydropathy profile suggests a union of two IRK-like structures. In the latter case, the hydropathy profile is reminiscent of a Shaker-like structure attached to an IRK-like structure. The first three families have members in both animal [6] and plant cells [5•]. Only in yeast has a channel of the fourth family been identified [5•].
Shaker-type
TWIK-type
P1 P2
IRK-type P
P + + + + +
C N
Cytosol S1 Voltage sensor S6
Membrane Exterior
TOK-type P1 P2
Current Opinion in Plant Biology
Figure 2
Structure of plant Shaker-like channels. The proposed structure is derived from sequence and structural homologies with animal Shaker channels, direct analyses of the transmembrane topology, mutagenesis of pore mutants and biochemical characterization (see Figure 1 and text). (a)The P domain between the fifth and sixth transmembrane region forms part of the aqueous pore [12,13••]. The voltage sensor in the fourth transmembrane span [16,18] is surrounded by the other transmembrane spans to isolate positive charges. Functional domains are present in the cytosolic carboxyl terminus: a putative cyclic nucleotide-binding domain (cNMP), an interaction domain (KHA), and in the AKT-like subfamily only, ankyrin repeats (Anky). (b)Tetramerization of four α-subunits results in the formation of functional channels.
S1 S4 S5
S6 S3
S2
Anky
KHA
Carboxyl-terminus S1
S2 S3
S4 S5
S6
cNMP P
Amino terminus
P
(a) (b)
Aqueous pore
this channel also [18]. In KST1, two histidine residues pre-sent at the external face of the channel, one within the P domain and the other within the S3–S4 linker, have been shown to act as pH sensing elements, upregulating the channel activity upon acidification of the medium [19]. A similar upregulation has been shown for KAT1, but involves distinct molecular mechanisms [17•]. Comparison of KAT1
currents at different K+/Rb+ratios has led to the conclusion
that KAT1 conduction requires several K+ions to be present
simultaneously within the pore [20]. This so-called multi-ion pore behavior is also supported by the demonstratmulti-ion that the impermeant ion methylammonium blocks K+and
NH4+currents through KAT1 differently [21].
In plant Shaker-like K+ channels, a putative cyclic
nucleotide binding site and a domain rich in hydrophobic and acidic residues, called KHA, are present downstream
from the hydrophobic core, in the cytoplasmic carboxyter-minal domain (Figure 1). In some channels (the AKT subfamily), several ankyrin repeats are present between these two domains and could play a role in protein–protein interactions [3]. Expression of AKT1 or KAT1 cDNAs in insect cells results in the appearance of membrane proteins with an apparent molecular weight that is four times that of the corresponding predicted polypeptide, indicating that AKT1 and KAT1 are tetrameric channels ([22]; S Urbach, I Cherel, H Sentenac, F Gaymard, unpublished data), like their animal counterparts. When expressed in insect cells, the cytoplasmic carboxyterminal region of AKT1 forms highly stable homotetrameric structures, suggesting a role for this region in channel tetramerization [22]. Expression of a fusion protein between KST1 and green fluorescent protein (GFP) has revealed that the KHA domain is not
essential for the expression of functional channels in insect cells but plays a role in channel clustering [23].
Knockout mutants highlight the role of Shakerchannels in mineral nutrition
Expression of the AKT2 gene (also called AKT3) in phloem tissues has been reported recently, suggesting a role for the encoded K+channel in long distance K+
trans-port via the phloem vasculature [15•]. Expression studies
and electrophysiological approaches support the hypothe-sis that KAT1 [24,25] and its counterpart KST1 in potato [9] mediate K+ influx in guard cells leading to stomatal
opening, probably in both the low (<100µM) and high (> 1 mM) K+ concentration range [26]. It should be noted,
however, that differences have been found between the functional properties of the major guard cell inward chan-nel and those of KAT1 expressed in Xenopusoocytes [27•].
Such differences could result from the fact that character-ization in heterologous systems might provide a distorted view, because of artefactual interactions and/or lack of control by plant proteins [28•].
Using knockout mutants, clear-cut evidence has been obtained for the roles of two ArabidopsisK+channels AKT1
and SKOR (Figure 3). AKT1 was characterized as an
inwardly rectifying K+ channel [29] preferentially
expressed in the root epiderm and cortex [30], suggesting a role for AKT1 in K+uptake from the soil solution. This has
been definitively confirmed by phenotype characterization of a knockout mutant [31••]. Growth of the mutant is
reduced under limiting K+concentrations (<100µM) in the
presence of NH4+, indicating that AKT1 is involved in K+
uptake in the low concentration range, probably together with other NH4+-sensitive transporters [31••,32•]. Until this discovery, K+channels were not thought to play a role in
high-affinity uptake. The Arabidopsis SKOR channel was identified by electronic cloning [14••]. The transcript
accu-mulation could be detected only in roots, and expression was localized in pericycle and xylem parenchyma cells. Functional characterization in Xenopusoocytes identified S OR as an outwardly rectifying channel. Characterization of a knockout mutant demonstrated that this channel is involved in K+ release into the xylem sap [14••]. SKOR
transcript abundance is dramatically decreased by abscisic acid (ABA) treatment, suggesting that control of K+
translo-cation towards the shoots is part of the plant response to water stress [14••]. K+fluxes through the plant are
integrat-ed at the organism level and are modifiintegrat-ed by changes in soil K+ availability and the K+status of the plant. With the
iden-tification of AKT1, SKOR and AKT2 (which could be involved in K+transport in phloem cell tubes [15•]),
mole-cular tools are now available for analyzing K+transport at
the whole plant level, and its regulation during develop-ment and upon hormonal or environdevelop-mental signals.
New molecularly identified plant ion channels
Plant counterparts of animal K+channels of the TWIKfam-ily, harboring two pore domains and four transmembrane Figure 3
K+transport within Arabidopsis thaliana. Ion channels have been identified by (a)employing insertion mutants to mediate K+uptake in the root (AKT1 [31••]) and (b)K+secretion into the xylem for long distance transport (SKOR [14••]). (c)AKT2/3 [15•] is assumed to contribute to K+distribution via the phloem. (d)KAT1 was shown to be responsible for K+uptake into guard cells during stomatal opening [25].
X
ylem Phloem
(a) (b)
Stem
(c) Leaf
Root
(d)
segments within one α-subunit (Figure 1), have been identified by searches in databases [5•,33]. They form the
so-called KCO (K+ channel family related to the Ca2+
-activated, outwardly rectifying K+ channel [KCO1])
family. The first member identified (in Arabidopsis), KCO1, is an outwardly rectifying K+channel activated by
increased Ca2+levels in the physiological concentration
range (100–500 nM) [33]. Interestingly, in addition to structural elements in animal two-pore channels, KCO1 and its relatives possess Ca2+-binding sites (EF-hand
motifs , See [33]) in their cytosolic carboxyl termini, and Ca2+ binding has been demonstrated in vitro by Ca2+
-dependent mobility shifts [5•]. The first plant ion
channel structurally related to animal IRK channels (Figure 1) has been identified by its sequence similarities to the two-pore channel KCO2 [5•]. It also harbors two
EF-hand motifs.
Homologs of animal anion channels of the ClC (chloride channel) family [34] have been identified in tobacco [35], Arabidopsis[36] and potato [5•] but their functional
prop-erties are presently unknown. A putative cation channel sharing sequence and structure homologies with animal cyclic nucleotide gated cation channels was identified in barley by use of a two-hybrid system with calmodulin as the bait protein [37•]. Search in databases have revealed
that this channel belongs to a multigene family, with at least six members in Arabidopsis[38•], all harboring a cyclic
nucleotide binding domain overlapping with a calmodulin binding site. Plant homologs of animal ionotropic gluta-mate-receptors have been recently identified [39•].
Pharmacological data [39•] and expression of antisense
constructs (JM Kwak et al., Abstract 6-4, 10thInternational
Conference on Arabidopsis Research, July 4-6 1999, Melbourne, Australia) have suggested that these proteins play a role in light-signal transduction. Finally, the Arabidopsis genome sequencing program has recently revealed genes likely to encode homologs of animal Ca2+
channels. This is likely to open the field of Ca2+signaling
to molecular approaches.
In conclusion, six multigene families sharing homologies with animal channels have been identified during the last two or three years by electronic cloning. Ion channel activ-ity has, however, been demonstrated for none of the encoded polypeptides except for KCO1 [33]. Furthermore, questions about subcellular membrane local-ization have been recently raised by the finding of ion channels that cannot be characterized in heterologous sys-tems or that are not related to ion channels previously described in planta[35].
A new frontier: towards identification of
interacting proteins involved in channel
activity control
Whether as one stage or the final target of signaling events, ion channel activity must be tightly controlled [1]. In animal cells, besides control by voltage or ligands, a
large array of regulation mechanisms involving protein–protein interactions have now been characterized. They include association of channel subunits encoded by genes belonging to distinct channel subfamilies, phospho-rylation/dephosphorylation, association with regulatory subunits, interaction with cytoskeletal elements, or con-trol of retrieval/insertion into the membrane.
Various approaches developed during the past two to three years have provided data indicating that control of channel activity in plants involves similar mechanisms but detailed knowledge is still lacking, as illustrated by the following examples. Although regulation by phosphorylation/dephos-phorylation is well documented [40,41•,42], presently no
specific gene encoding a kinase or phosphatase involved in channel phosphorylation/dephosphorylation has been cloned. Evidence that distinct K+ channel subunits can
assemble within heteromultimeric structures has been obtained by two-hybrid tests [23] and co-expression in Xenopusoocytes [43,44], but this phenomenon has not been demonstrated in planta. Systematic sequencing programs in plants have revealed polypeptides that share homologies with animal K+ channel β-subunits and seem to interact
with AKT1 and KAT1 channels in biochemical tests [45]. No functional evidence for such interaction has, however, been obtained by co-expression in heterologous systems. Effects of various drugs known to modulate K+ channel
activity in animal cells suggest that sulfonylurea-receptor-like proteins are tightly involved in regulation of channel activity during stomatal movements [46•], but neither guard
cell outward K+channels nor sulfonylurea-receptor-like
pro-teins have been cloned. Interactions with cytoskeleton proteins seem to play a role in the control of K+and Ca2+
channel activity [47,48•], but none of the interacting
pro-teins have been identified. The hypothesis that regulation of vesicle trafficking and secretion by syntaxins can control channel activity has received pharmacological support and a syntaxin has been cloned [49••], but syntaxin binding
part-ners are still black boxes. Finally, a function for protein farnesylation in control of channel activity in guard cells has been demonstrated by using farnesyltransferase inhibitors and characterizing an Arabidopsismutant with a disrupted farnesyltransferase gene [50••], but again the pathway and
targets are unknown.
Conclusions
Irruption of increasingly documented databases has allowed rapid progress in molecular identification of ion channels, triggering a revolution in this field of plant biol-ogy [51•], as in many other fields. The power of
electrophysiological tools is providing further strong sup-port for research on ion channels, allowing functional characterization of cloned channels in heterologous sys-tems and structure–function relationship analyses. Of course, the final aim is to determine the role in plantaof each identified channel. Recently, this has been achieved for two K+channels, by (electro)physiological
families of (putative) ion channels have been identified in Arabidopsisand the present knowledge mainly concerns a few channels of a single family (Shaker). Obviously, knowl-edge in this field is just emerging. However, it is clear that new research goals will now shift towards the identification of regulatory proteins and interacting networks.
Acknowledgements
We are grateful to John Vidmar and Siobhan Staunton for critical reading of the manuscript.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
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in the presence of ammonium. The whole set of data provides evidence for a role for AKT1 in K+ uptake from the soil in the low concentration range.
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rec-tifier AKT1 is analyzed in detail.
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