From the Biochimie et Physiologie Moléculaire des Plantes, UMR 5004 Agro-M/CNRS/INRA/UM2, Place Viala, F-34060 Montpellier Cedex 1, France
Received for publication, August 11, 2000
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ABSTRACT |
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Stomatal opening, which controls gas exchanges
between plants and the atmosphere, results from an increase in turgor
of the two guard cells that surround the pore of the stoma. KAT1
was the only inward K+ channel shown to be expressed
in Arabidopsis guard cells, where it was proposed to
mediate a K+ influx that enables stomatal opening. We
report that another Arabidopsis K+
channel, KAT2, is expressed in guard cells. More than KAT1, KAT2 displays functional features resembling those of native inward K+ channels in guard cells. Coexpression in
Xenopus oocytes and two-hybrid experiments indicated that
KAT1 and KAT2 can form heteromultimeric channels. The data indicate
that KAT2 plays a crucial role in the stomatal opening machinery.
The epidermis of the aerial organs of plants presents a waxy
cuticle that prevents water loss, but impedes direct access of the
photosynthesizing tissues to atmospheric CO2. Pores in the epidermis, called stomata, allow atmospheric CO2 to enter
the plant for photosynthesis. By providing an access to the outer atmosphere, they also allow transpiration, i.e. controlled
diffusion of H2O vapor from the plant into the atmosphere,
which is a driving force for the ascent of crude sap from the roots to
the shoots.
Regulation of the stomatal aperture allows the plant to tune and
optimize uptake of CO2 and transpiration under diverse
environmental conditions. Stomatal movements result from changes in
turgor of the two guard cells surrounding the pore, an increase in
turgor resulting in increased opening of the stomatal pore. The changes in turgor involve ion transport from and into the guard cells through
K+ and anion channels. These channels are the targets of
complex transduction pathways that allow the plant to regulate stomatal opening (1). In this context, the molecular identification of guard
cell ion channels was an early challenge when plant channels began to
be cloned. By chance, one of the first cloned plant K+
channels, KAT1 (2), was rapidly demonstrated to be endowed with
functional properties compatible with a role in mediating K+ influx (3) and to be expressed in guard cells (4),
opening the way to molecular approaches. By expressing a KAT1
Cs+-resistant mutant channel in transgenic
Arabidopsis, evidence has been obtained that this channel
functions as an inward channel (Kin
channel)1 in the guard cell
plasma membrane and plays a role in stomatal opening (5). Biochemical
approaches have revealed that Ca2+-dependent
protein kinases phosphorylate the KAT1 protein (6). A homolog of the
animal K+ channel regulatory On the other hand, two reports (5, 8) pointed out differences in
sensitivity to external pH and to the channel blocker Cs+
between the current mediated by KAT1 after heterologous expression and
the current in situ. Two nonexclusive hypotheses can be put forward to explain these differences. First, the differences could be
due to the fact that information obtained with heterologous systems
gives a distorted view of the functional properties in planta because of, for example, lack of interactions with
endogenous proteins and/or artifactual interactions with host cell
proteins (9-11). The second hypothesis is that another channel is
expressed in guard cells and contributes per se to the
inward current by forming homomeric channels and/or in interaction with
KAT1 by forming heteromeric channels.
We have now identified a second gene expressed in guard cells,
KAT2, for which a partial cDNA had been previously
cloned (12). Electrophysiological characterization of KAT2 current in
Xenopus oocytes revealed basic properties very similar to
those of the inward current in guard cells. Regarding the sensitivity
to external pH and Cs+, KAT2 is more reminiscent of the
guard cell Kin channels than KAT1. Like their animal
counterparts, plant Shaker channels are tetrameric proteins (13). Here
we also bring evidence that KAT1 and KAT2 polypeptides can assemble in
heterotetramers. The results provide evidence that KAT2 is a major
determinant of the inward K+ current through the guard cell
membrane, a fact that should stimulate research on stomatal opening
mechanisms and their regulation.
KAT2 cDNA Cloning--
The 5'-region of KAT2
cDNA (see Fig. 1C; GenBankTM/EBI
accession number AJ288900) was determined by RACE-PCR on
poly(A)+ mRNA isolated from 14-day-old plantlet leaves.
Amplified fragments corresponding to the 5'-end of
KAT2 cDNA were cloned and sequenced. The
full-length KAT2 open reading frame was then isolated by
reverse transcription-PCR on poly(A)+ mRNA using a
primer hybridizing at the ATG codon and containing a SpeI
site (5'-ACTAGTATGTTGAAGAGAAAGCACCTCAACAC-3') and a reverse primer
hybridizing at the stop codon and containing a NotI site (5'-GCGGCCGCTTAAGAGTTTTCATTGATGAGAATATACAAATG-3'). The amplified fragment (2.1 kilobases) was cloned at the EcoRV site
of pBluescript and sequenced.
Northern Blot and Reverse Transcription-PCR
Experiments--
Plants were grown in vitro in magenta
boxes as described previously (14). Total RNA extraction and Northern
blotting were performed as described previously (15). Specific probes
corresponding to KAT1 and KAT2 and used in Northern blot experiments
were generated by PCR (cDNA fragments encoding sequences 511-587
and 524-608 of KAT1 and KAT2, respectively). Reverse transcription and
PCR were performed with Superscript II (Life Technologies, Inc.) and Extra-Pol I (Eurobio), respectively, following the manufacturers' recommendations.
Transgenic Plants and GUS Assay--
The KAT2
promoter region was isolated from genomic DNA (ecotype Columbia) by PCR
walking (16) with a reverse primer introducing a unique NcoI
site just upstream from the ATG codon
(5'-CCATGGGGTTAGTTATAAATATAGTGATGAAACTTGTG-3'). A
1.8-kilobase fragment was isolated, cloned into pBluescript, and
sequenced. The construct was digested by BamHI and
NcoI and introduced into pBI320.X (from Dr. R. Derose; this
plasmid bears a unique NcoI site at the initiation codon of
the promoterless GUS 3'-nopaline synthase gene), leading to
a translational fusion between the KAT2 promoter region and
the GUS coding sequence. This construct was digested by
BamHI and SacI and introduced into the pMOG402
binary vector (from Dr. H. Hoekema). The resulting plasmid was
introduced into Agrobacterium tumefaciens MP90 (17). Arabidopsis thaliana (ecotype Columbia) was transformed with
agrobacteria using the floral dip method (18). Selection of T1
seedlings was performed in vitro on the half-strength medium
described by Murashige and Skoog (19) supplemented with 1% sucrose,
0.7% agar, and 50 µg·ml Expression in Xenopus Oocytes and
Electrophysiology--
KAT1 and KAT2 cDNAs
were introduced into the pCi plasmid (Promega) under the control of the
cytomegalovirus promoter. The resulting plasmids, pCi-KAT1 and
pCi-KAT2, were injected into Xenopus oocytes (purchased from
Centre de Recherches de Biochimie Macromoléculaire, CNRS,
Montpellier, France) using a 10-15-µm tip diameter micropipette and
a pneumatic injector (10 nl of 1 µg·µl
Whole-cell currents were recorded as described previously (21) using
the two-electrode voltage-clamp technique, 3-7 days after injection,
on oocytes continuously perfused with bath solution (see figure
legends). Quantitative analyses of macroscopic current that yielded the
gating parameters given in Table I were performed as described
previously (21).
Patch-clamp experiments were performed on devitellinized oocytes as
described previously (22). Voltage-pulse protocol application, data
acquisition, and data analyses were performed using pClamp (Axon
Instruments, Inc., Foster City, CA), Winascd (Dr. G. Droogmans, University of Leuven, Leuven, Belgium), and Sigmaplot (Jandel Scientific, Erkrath, Germany) software.
Two-hybrid Experiments in Yeast--
Plasmid vectors pGBT9 (23)
and pACTII (kindly provided by S. Elledge, Baylor College of Medicine,
Houston, TX) were used for the generation of fusion proteins with the
DNA-binding domain and the activator domain of Gal4,
respectively. In-frame fusions were made with the C-terminal
cytoplasmic regions from KAT1 and KAT2. For KAT1, a NcoI
site was created at the end of the S6 segment coding sequence by
site-directed mutagenesis (24), the GTCGTTCATTGGACT sequence from KAT1
being replaced by GTCGTCCATGGGACT. The KAT2 insert was obtained
by digestion with BsrI and blunt-ending with mung bean
nuclease, thus allowing the polypeptide sequence to be synthesized
from Arg316. Yeast cell transformations and the assay for
reporter gene expression (using
o-nitrophenyl- Cloning and Sequence Analysis--
A partial KAT2
cDNA sequence has been reported (GenBankTM/EBI
accession number U25694) (12). A complete KAT2 cDNA
(GenBankTM/EBI accession number AJ288900) was isolated by
5'-RACE, and the promoter region was isolated by PCR walking. The
sequence of a bacterial artificial chromosome containing the
KAT2 gene then became available (BAC T9A21;
GenBankTM/EBI accession number AL021713). Comparison of the
PCR-amplified fragments (KAT2 cDNA and promoter) with
that of this bacterial artificial chromosome indicated that the PCR
steps had introduced no mutation.
Sequence analysis of the deduced KAT2 polypeptide identified the three
domains exhibited by all plant Shaker-like channels (see
"Discussion") cloned up to now (Fig.
1A): from the N to C terminus,
(i) the hydrophobic core with the typical six transmembrane segments
(S1-S6) and the pore-forming domain (called the P domain, between S5
and S6), (ii) the putative cyclic nucleotide-binding domain, and (iii)
the so-called KHA domain, rich in hydrophobic and acidic
residues and thought to play a role in channel tetramerization (13)
and/or channel clustering in the membrane (26). Within the
Arabidopsis Shaker-like family, the strongest similarities are found to KAT1. The similarities of KAT2 to KAT1 are detected all
along the polypeptide (~85% identity from the first residue to the
end of the putative cyclic nucleotide-binding domain; ~65% identity
in the KHA domain), except in the region lying
between the putative cyclic nucleotide-binding domain and the
KHA domain (residues 509-610 in KAT1 and residues 520-639
in KAT2) (Fig. 1A). The P domain of KAT2 differs from that
of KAT1 by a single residue (Fig. 1B), and the S4 segments
(with a role in voltage sensing) are identical.
Comparison of the 5'-untranslated region sequence with the
sequence of the genomic clone (Fig. 1C and BAC T9A21,
respectively) revealed a typical TATA box ~40 base pairs upstream
from the first nucleotide of the KAT2 cDNA we have
cloned, supporting the hypothesis that this cDNA could be
full-length. The KAT2 5'-untranslated region is unusually
long (at least 455 base pairs) compared with other
Arabidopsis genes (27). Furthermore, it contains four ATG
codons defining four small upstream open reading frames (open reading
frames upstream from the main one). Such features strongly suggest a
role of the transcript leader in regulation of expression (28-30).
KAT2 Expression Pattern--
Northern blot experiments detected
KAT2 mRNA in aerial organs and not in roots (Fig.
2A). Reverse transcription-PCR
experiments failed to detect KAT2 transcripts in roots (data
not shown), indicating that expression of KAT2 is restricted
to aerial organs. Localization of expression was further investigated
using transgenic plants carrying the Escherichia coli
Functional Characterization in Xenopus Oocytes--
In
Xenopus oocytes injected with the pCi-KAT2 plasmid,
hyperpolarization of the membrane beyond
Under our experimental conditions (expression level, size of patch),
macroscopic KAT2 currents mimicking whole-oocyte currents could be
recorded in the cell-attached patch-clamp configuration (Fig.
3C, trace c.a.). Upon patch excision, however,
the KAT2 current decreased rapidly (inside-out configuration) (Fig.
3C, traces i.o.1 and i.o.2). In this
configuration, unitary currents could be resolved, which are shown in
Fig. 3D. KAT2 rundown could be overcome and the initial
current partly restored by cramming the patch back into the oocyte
(Fig. 3C, trace p.c.). This suggested that KAT2
required intracellular factors, available in the oocyte cytoplasm, to
open. From the single-channel recordings obtained in the inside-out
configuration at different potentials, we were able to determine the
single-channel slope conductance of KAT2: 6.7 picosiemens in
symmetrical 100 mM K+ solution (Fig.
3D), a value quite similar to that reported for KAT1 (31,
32).
As native guard cell inwardly rectifying K+ channels are
known to be stimulated upon external acidification (33, 34), we compared the sensitivity of KAT1 and KAT2 to external pH in parallel experiments carried out on oocytes from the same batch (Fig.
4). The activation potential of KAT2 was
shifted positively when the pH was decreased from 7.5 to 6.0 (Fig.
4B), leading to an increase in current amplitude at a given
potential. Analyses of the corresponding G/Gmax versus potential
curves (data not shown) showed that the Gmax
value was not changed and yielded the gating parameters (Table I).
Although the apparent gating charge (zg) was not changed, the half-activation potential
(Ea50) was indeed shifted by
approximately +15 mV when the pH was decreased from 7.5 to 6.0. By
contrast, and as previously reported (35), KAT1 gating parameters were
left unchanged by the pH drop (Fig. 4A), an increase in
macroscopic conductance (Gmax) being responsible for the increase in current (Table I).
Block by external Cs+ is a classical feature of plant and
animal K+ channels that is believed to involve the binding
of Cs+ to some site within the pore. Pore penetration by
Cs+ often results in some voltage dependence of the
K+ channel block (36). As previously reported (9, 31),
addition of 0.5 mM Cs+ to the external medium
resulted in a voltage-dependent block of the KAT1 current
(Table I). In parallel experiments on the same batch of oocytes,
addition of Cs+ resulted in a voltage-independent block of
KAT2 inward currents (Fig. 5,
A and B; and Table I). The inhibition constant of
this block, estimated from the data shown in Fig. 5B, is 2.5 mM. It is worth noting that the sequences of the pore
domains of KAT1 and KAT2 differ by a single amino acid,
Phe266 in KAT1 corresponding to Leu in KAT2 (Fig.
1B). In this context, a KAT1-F266L mutant channel (a gift
from R. Hedrich, University of Würzburg, Würzburg,
Germany), i.e. a KAT1 mutant with the pore sequence of
KAT2, was studied. The Cs+ sensitivity of the mutant
channel is clearly voltage-dependent and reminiscent of
that of KAT1 (Fig. 5C). Therefore, the pore domain sequence
is not the only determinant of KAT1 and KAT2 sensitivity to
Cs+. Further pharmacological characterization
revealed that KAT2 has a lower sensitivity to external Ba2+
than KAT1 and a sensitivity to external tetraethylammonium similar to
that of KAT1 (Table I).
Interactions with KAT1--
Single-point mutations in the KAT1 P
domain have been recently reported to yield dominant-negative mutants
(37). We introduced a mutation in KAT1 (W253G) that produced
electrically silent channels in Xenopus oocytes.
Coexpression of this mutant with the wild-type KAT1 channel in
Xenopus oocytes resulted in a lower inward current than did
the expression of the wild-type channel alone (Fig.
6A). Such an effect indicates
that the KAT1-W253G mutant has a dominant-negative capability,
i.e. that it can interact with the wild-type
polypeptide, leading to formation of channels that are not functional
or not targeted to the membrane. Interestingly, coexpression of the
KAT1-W253G mutant with KAT2 decreased the inward current as well (Fig.
6A), providing evidence that the polypeptides encoded by
KAT1 and KAT2 can interact and form
heterotetrameric channels.
Plant K+ channels have been shown to form tetramers through
interactions involving the cytoplasmic C-terminal domain (13). We
therefore investigated the possibility of interactions between the
C-terminal domains of KAT1 and KAT2 using the two-hybrid system in
yeast (38) and obtained positive results (Fig. 6B).
Coexpression of the wild-type KAT1 and KAT2 polypeptides in oocytes
resulted in an inward current that activated at a threshold potential
between those of KAT1 and KAT2. We failed to find any typical feature
of this current (i.e. a feature that would not be
reminiscent of KAT1 or KAT2 properties). Interestingly, however, the
current monitored in control oocytes injected with 10 ng of either KAT1
or KAT2 plasmid was significantly smaller than that monitored in
oocytes coinjected with 5 ng of each plasmid, suggesting some synergy.
The physiological significance of this effect cannot be assessed at the
present time. However, in the animal field, similar observations due to
interaction between channel subunits have been shown to play a role
in situ in the organism (39, 40).
KAT1 and KAT2 belong to the Shaker-like family of K+
channels (41), of which nine members have been identified in
Arabidopsis (the whole genome having been sequenced). These
channels share sequence similarities and structural homologies with
animal Shaker channels (42). Shaker channels consist of four subunits
arranged around a central pore (42). The core region of each subunit of
the tetramer is predicted to consist of six transmembrane segments (S1-S6). S4 is characterized by the presence of several basic amino
acids and forms the voltage sensor of the channel. The highest degree
of sequence identity among Shaker channels is found in the so-called P
domain sequence (~20 amino acids), located between S5 and
S6. In the functional protein, the four P domains are assembled around
the aqueous pore at the center of the structure (42).
Animal Shaker channel subunits can form heteromultimeric structures,
and this process generates diversity in potassium channel activity
(43-45). Similarly, coexpression of couples of plant K+
channel subunits in Xenopus oocytes has revealed formation
of heteromultimeric structures (10, 37), providing the first support
for the hypothesis that, in plants, too, functional diversity of
K+ channel activity could result from temporal and
developmental overlapping expression patterns. However, our present
knowledge of the expression pattern of the various K+
channels in the plant is still very poor and mainly concerns four genes
in Arabidopsis, namely KAT1 (4), AKT1
(20), AKT2 (46, 47), and SKOR (14), the first
three of which encode Kin channels and the fourth an
outward channel. The available data identified KAT1 as the only
Kin channel expressed in guard cells (4).
KAT2 is mainly expressed in the phloem of minor veins, together with
the AKT2 K+ channel (46, 47), and in guard cells, together
with KAT1 (4). The present data demonstrate that, in plants, too,
several Shaker K+ channel subunits can be expressed within
a single cell type. In minor veins, KAT2 could be involved in
K+ loading into the phloem sap, as suggested for AKT2 (46,
47). Although this transport plays major roles, taking part in both control of phloem sap flow rate and integration of K+
fluxes at the whole-plant level (48, 49), electrophysiological properties of the cell membrane in phloem tissues have never been analyzed yet because of methodological difficulties.
Electrophysiological investigations have been mainly focused on guard
cells for both biological (a crucial role in the control of gas
exchanges between the plant and the atmosphere) and methodological
(cells easily accessible to electrophysiological techniques) reasons.
The discussion below concerns only K+ channel activity and
KAT1-KAT2 interactions in guard cells.
The functional properties of KAT2 are very similar to those reported
for KAT1 (3, 31, 32, 35). Both polypeptides form voltage-gated inwardly
rectifying channels, highly selective for K+ and displaying
the same unitary conductance (~7 picosiemens) and small differences
in gating parameters (half-activation potential and gating charge). The
main differences between these channels detected so far concern the
sensitivity to external pH and Cs+. The sensitivity of KAT1
(expressed in Xenopus oocytes) to external pH has been
analyzed in studies, leading to partly conflicting results. Hoshi (32)
reported that acidification from pH 7.2 to 6.2 was without any effect
on the current. For a larger acidification, from pH 7.4 to 4.5, Hedrich
et al. (31, 50) reported a positive shift of the
half-activation potential, but no effect on the
Gmax parameter, whereas Véry et
al. (35) reported that acidification from pH 6.4 to 5.0 was
without any effect on the gating parameters (half-activation potential
and gating charge), but resulted in an increase in
Gmax. As experimental conditions in heterologous systems may vary somewhat (because of endogenous reactivity to pH), we
decided to check in parallel experiments, using the same batch of
oocytes, whether KAT1 and KAT2 were differently affected by a moderate
acidification from pH 7.5 to 6.0. The results indicate that the two
channels actually display different pH sensitivities, with KAT2 being
more sensitive to external acidification than KAT1. It has already been
pointed out that KAT1 exhibits a weaker pH sensitivity compared with
the native inward K+ channels characterized in
Arabidopsis guard cells (8). Thus, regarding the effect of
external pH, KAT2 is more reminiscent of the native guard cell
Kin channels (8, 34) than KAT1. Similarly, the
Cs+ sensitivity of KAT2 is clearly closer to that of the
native guard cell Kin channels (the block of steady-state
currents by Cs+ is not voltage-dependent) (5)
compared with KAT1 (voltage-dependent block) (9, 31,
51).
Guard cells respond to a number of environmental and physiological
factors, including light intensity and quality, CO2
availability, humidity, plant water status, and hormones (1), with
different sensitivity between abaxial and adaxial stomata (52). The
differences in basic properties revealed in the heterologous context
between KAT1 and KAT2 are weak and therefore unlikely to play,
per se, crucial roles in guard cell physiology. Furthermore,
the interactions that the present data reveal between KAT1 and KAT2 do
not result in new current features strongly differing from those of the
currents mediated by each channel expressed separately. Coexpression of these two channels is therefore not likely to provide guard cells with
a mechanism generating, per se, diversity in channel
function properties. We propose that the presence of the two genes
supports channel expression control and/or generates diversity in
regulatory mechanisms modulating channel function properties. In
relation to this hypothesis, the length of the KAT2 leader and the
presence of several upstream open reading frames strongly suggest
translational regulation (28, 29). Also, the region that lies between
the cyclic nucleotide monophosphate-binding domain and the
KHA domain in both channels displays the lowest level of
sequence similarities. It could therefore be involved in specific
interactions with specific regulatory proteins. Furthermore, this
region bears several specific putative phosphorylation sites, present
in one channel and absent from the other, which could allow specific
regulation. Such diversity, together with specific expression patterns
(e.g. leaf guard cells expressing both KAT1 and
KAT2 and stem guard cells expressing only KAT2,
as suggested by the present data), could allow the plant to tune,
independently in each organ, the guard cell membrane Kin
activity in relation to environmental conditions. In leaf guard cells,
heteromultimerization of KAT1 and KAT2 polypeptides could further
increase the diversity of the regulatory mechanisms. In conclusion, the
expression of two different guard cell Kin channel subunits
has to be taken into account in investigating stomatal opening
mechanisms and their regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunits has been
identified and shown to interact with KAT1 (7).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 kanamycin under
the following conditions: 21/18 °C day/night temperature, 16-h
photoperiod, and 150 microeinsteins·m
2·s
1.
For GUS assay, plants were either grown in vitro on the same medium and under the same conditions as described above or grown in a
greenhouse on attapulgite-peat compost (14). GUS histochemical staining was performed as described previously (20). Cross-sections of
GUS-stained material were prepared on hydroxyethyl methacrylate (Technovit 7100, Heraus-Kulzer GmBH, Wehrheim, Germany)-embedded tissues with an Amersham Pharmacia Biotech microtome and were counterstained in purple by periodic acid-Schiff staining.
1
plasmid solution/oocyte). Control oocytes were injected with 10 nl of
empty plasmid solution.
-D-galactopyranoside as
substrate) were performed as described previously (23, 25).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence analysis. A, dot
matrix comparison (DNA Strider program, polypeptide homology matrix,
stringency = 2, and window = 15 amino acids) of the deduced
KAT2 amino acid sequence (GenBankTM/EBI accession number
AJ288900) with that of KAT1 (GenBankTM/EBI accession number
M86990). Schematic representations of the predicted channel domains are
presented (bars lining the graph) on the same scale as the
dot plot. S1-S6 are the six transmembrane segments forming the channel
transmembrane domain. The P domain is the pore domain between S5 and
S6. cNBD, putative cyclic nucleotide-binding domain. The
KHA domain is the C-terminal domain thought to play a role
in channel tetramerization and/or clustering in the membrane.
B, alignment of the KAT1 and KAT2 pore sequences.
C, sequence of the 5'-region of the KAT2
cDNA. The 5'-untranslated sequence is in lowercase
letters, and the first 7 residues of the coding sequence are
in uppercase letters. The four small upstream open reading
frames present in the untranslated region are
underlined.
-glucuronidase gene (GUS) under the control of the
KAT2 promoter region (1.8 kilobases). Reporter gene activity
was analyzed on the F1 and F2 progeny of 10 independent transgenic
plants. GUS activity was never detected in roots. In developing leaves,
GUS staining was present in all cells (Fig. 2B). In mature
leaves, the activity was mainly detected in guard cells and in minor
veins (Fig. 2, C and D). Mature leaf
cross-sections revealed that GUS staining in minor veins was present in
phloem and not in xylem parenchyma (Fig. 2E). In hypocotyls,
stems (Fig. 2F), and petioles, GUS activity was present only
in guard cells. In parallel experiments on transgenic
Arabidopsis expressing GUS under the control of
the KAT1 promoter region (stock CS3763,
Arabidopsis Biological Resource Center), stem guard cells
never displayed GUS staining (data not shown), in accordance with the
lack of expression of the reporter gene in these cells (4). The data suggest that guard cells in the stem express KAT2, but not KAT1, whereas guard cells in petioles and leaves express both channels.
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Fig. 2.
KAT2 expression pattern.
A, Northern blot analysis (5-week-old plants). Ten
µg of total RNA extracted from roots (R), rosette leaves
(RL), caulinary leaves (CL), and stem
(S) were size-fractionated on an agarose gel, transferred to
a nylon membrane, and hybridized with a specific probe corresponding to
either KAT2 or KAT1. B-F, localization of
KAT2 promoter activity in transgenic Arabidopsis
(ecotype Columbia) using the GUS reporter gene. Shown are a
2-week-old seedling (B) and a rosette leaf (C), a
magnification of the rosette leaf epidermis (D), a 30-µm
cross-section of a rosette leaf showing staining in the phloem
(E), and the stem (F) of a 4-week-old plant.
ep, epidermis; ms, mesophyll; x, xylem
parenchyma; ph, phloem.
100 mV elicited an inward current (Fig. 3) that was not recorded in
control oocytes injected with empty pCi (data not shown). The exogenous
macroscopic current displayed slow activation and deactivation kinetics
(Fig. 3A) with voltage-dependent time constants
(Table I). No inactivation could be seen
even during hyperpolarizing pulses lasting 50 s (data not shown).
The steady-state current-voltage plots (Fig. 3B) show a
strong inward rectification with a threshold potential of about
100
mV irrespective of external K+ concentration. The reversal
potential of the KAT2 current was determined at different external
K+ concentrations. Following a change in the external
K+ concentration from 10 to 100 mM, the
reversal potential shifted by 57 ± 2 mV (n = 5),
remaining close to the K+ equilibrium potential (Fig.
3B, inset), thus indicating that the inward
current mediated by KAT2 was mainly carried by K+ ions.
Determination of reversal potential under pseudo bi-ionic conditions
(data not shown) allowed the determination of relative permeability
ratios. KAT2 displays the following permeability sequence (Eisenman's
series IV): K+ > Rb+ >
Na+
Li+ (Table I).
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Fig. 3.
Functional expression of KAT2 in
Xenopus oocytes. A and B,
two-electrode voltage clamp. The bath solution contained 1 mM CaCl2, 1.5 mM MgCl2,
5 mM HEPES-NaOH (pH 7.4), and 100 mM [K + Na]Cl (K+ concentration indicated below). From a holding
potential of 40 mV, the membrane potential was clamped (11 successive
pulses of 2 s each) at values ranging from +10 to
140 mV in
15-mV increments. Current traces recorded in 10 mM
K+ are shown in A. The steady-state current at
the end of the activation step was plotted against membrane potential
for three different external K+ concentrations: 100 mM (100K;
), 10 mM
(10K;
), and 2 mM (2K;
). The
resulting current-voltage plots in B indicate that the
potential threshold at which the current significantly increased
(activation potential) was approximately
100 mV. Inset,
plotting the reversal potential (Erev) for the KAT2
current (
) versus the external concentration of
K+ revealed that, in this example, the reversal potential
shifted by 56.5 mV for a 10-fold increase in external K+
concentration, as expected for a highly selective K+
channel. The solid line in the inset indicates
the K+ equilibrium potential (EK)
calculated with the Nernst equation. C and D,
patch clamp. Both bath and pipette solutions contained 100 mM KCl, 2 mM MgCl2, and 10 mM HEPES-NaOH (pH 7.4). In C, the
current-voltage plots were elicited by 10-s potential ramps from
180
to
40 mV. In the cell-attached patch clamp configuration, the
current-voltage curve (trace c.a.) is reminiscent of those
obtained by two-electrode voltage clamping (shown in B).
After patch excision (inside-out configuration), the current amplitude
decreased very quickly (traces i.o.1 and i.o.2;
obtained 10 and 20 s after patch excision, respectively). Patch
cramming into the oocyte made the current increase (trace
p.c.). In the inside-out configuration (D), when few
channels remained active in the patch (at least two in this example), a
pulse to
150 mV allowed the determination of the unitary current
(0.75 pA in symmetrical 100 mM KCl). The unitary current
amplitudes were obtained from gaussian fit to amplitude histograms. The
single-channel current-voltage curve over the voltage range from
130
to
160 mV (experimental points: mean ± S.E., n = 3) has been fit to a linear function, which yields a unitary
conductance of 6.7 picosiemens.
Comparison of functional properties of KAT1 and KAT2 channels
deactivation is the time for
half-deactivation (obtained by a single decaying exponential fit to
tail currents). Each value is mean ± S.E. (n = number of oocytes). TEA, tetraethylammonium.
View larger version (10K):
[in a new window]
Fig. 4.
Effect of external acidification on KAT1 and
KAT2 macroscopic currents. Membrane currents were recorded from
oocytes injected with 10 ng of either pCi-KAT2 or pCi-KAT1 using a
standard staircase protocol (see Fig. 3, A and
B). The external solution contained 1 mM
CaCl2, 1.5 mM MgCl2, 10 mM KCl, 90 mM NaCl, and 5 mM
HEPES-NaOH (pH 7.5) or 5 mM MES-NaOH (pH 6.0).
A, KAT1 current-voltage relationship at pH 7.5 ( ) and at pH 6.0 (
) (mean ± S.E., n = 4); B, KAT2 current-voltage relationship at pH 7.5 (
) and at pH 6.0 (
) (mean ± S.E., n = 6).
View larger version (9K):
[in a new window]
Fig. 5.
Effect of Cs+ on KAT2
currents. Membrane currents were recorded from oocytes injected
with 10 ng of pCi-KAT2 or pCi-KAT1-F266L. The external solution
contained 1 mM CaCl2, 1.5 mM
MgCl2, 5 mM HEPES-NaOH (pH 7.4), 10 mM KCl, and 90 mM [Na + Cs]Cl.
A and B, sensitivity of the KAT2 current to
Cs+. Increasing Cs+ concentrations in the
external medium (0 ( ), 0.5 (
), 1 (
), and 5 (
)
mM) induced a decrease in the KAT2 current as shown in the
current-voltage plots (A). This block was
voltage-independent (B). C, the KAT1 mutant F266L
(which corresponds to a KAT1 channel with the KAT2 pore) displays a
voltage-dependent Cs+ block.
View larger version (21K):
[in a new window]
Fig. 6.
Interactions between KAT1 and KAT2.
A, dominant-negative experiments in Xenopus
oocytes. KAT1 (left panel) and KAT2 (right panel)
were either expressed alone (open symbols) or coexpressed
with the KAT1-negative mutant KAT1-W253G (mutation W253G, not
functional) in a 1:4 molar ratio (closed symbols). From a
holding potential of 40 mV, the membrane potential was clamped (12 successive pulses of 2 s each) at values ranging from +15 to
150
mV in
15-mV increments. Coexpression of KAT1 with KAT1-W253G
(left panel) led to a decrease in the KAT1 current
consistent with a dominant-negative effect on KAT1 expression. The same
experiment with KAT2 instead of KAT1 (right panel) led to a
decrease in the KAT2 current similar to the one observed for KAT1.
B, interactions between the C-terminal domains of KAT1 and
KAT2 revealed using the two-hybrid system. The pGBT9 and pACTII
plasmids bearing the KAT1 or KAT2 C-terminal insert (encoding the
cytoplasmic region of the channel downstream from the last
transmembrane segment) or no insert (empty) were tested in
the different combinations indicated.
-Galactosidase activities were
measured following the hydrolysis of
o-nitrophenyl-
-D-galactopyranoside in liquid
medium. The data (mean ± S.E., n = 3) are given
in the following units: 1000 × ((A420 nm)/(reaction time in
min)/(A600 nm of the yeast culture)). Note the
logarithmic scale. C, coexpression of wild-type KAT1 and
KAT2 channels in Xenopus oocytes. Membrane currents were
recorded from oocytes injected with 10 ng of pCi-KAT1 (
), 10 ng of
pCi-KAT2 (
), or 5 ng of pCi-KAT1 + 5 ng of pCi-KAT2 (coexpression;
). The external solution contained 1 mM
CaCl2, 1.5 mM MgCl2, 5 mM HEPES-NaOH (pH 7.4), 90 mM NaCl, and 10 mM KCl. From a holding potential of
40 mV, the membrane
potential was clamped (17 successive pulses of 2 s each) at values
ranging from 0 to
160 mV in
10-mV increments. Left
panel, steady-state current-voltage relationship; right
panel, normalized (to
160 mV) current-voltage relationship
(mean ± S.E., n = 4).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Isabel Lefevre, Anne-Aliénor Véry, and Ingo Dreyer for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by European Communities BIOTECH Program Grant BIO4-CT96 and Rhône-Poulenc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ288900.
These authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 33-499-612-609; Fax: 33-467-525-737; E-mail: sentenac@ensam.inra.fr.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M007303200
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ABBREVIATIONS |
---|
The abbreviations used are:
Kin
channel, inwardly rectifying K+ channel;
RACE, rapid
amplification of cDNA ends;
PCR, polymerase chain reaction;
GUS, E. coli -glucuronidase gene;
P domain, pore-forming domain;
KHA domain, domain rich in hydrophobic
and acidic residues;
MES, 4-morpholineethanesulfonic acid.
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