The Plant Cell, Vol. 14, 1133–1146, May 2002, www.plantcell.org © 2002 American Society of Plant Biologists
Physical and Functional Interaction of the Arabidopsis K
Channel AKT2 and Phosphatase AtPP2CA
and Jean-Baptiste Thibaud
Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, Unité Mixte de Recherche 5004 Agro-M/Centre National
de la Recherche Scientifique/Institut National de la Recherche Agronomique/Université Montpellier II, Place Viala, 34060
Montpellier Cedex 1, France
Erwan Michard, Nadine Platet, Karine Mouline, Carine Alcon, Hervé Sentenac,
The AKT2 K
to date in Arabidopsis. The gene is expressed widely, mainly in the phloem but also at lower levels in leaf epiderm, me-
sophyll, and guard cells. The
AKT2 mRNA level is upregulated by abscisic acid. By screening a two-hybrid cDNA library,
we isolated a protein phosphatase 2C (AtPP2CA) involved in abscisic acid signaling as a putative partner of AKT2. We
further confirmed the interaction by in vitro binding studies. The expression of
gene) displayed a pattern largely overlapping that of
AtPP2CA with AKT2 in COS cells and
Xenopus laevis oocytes was found to induce both an inhibition of the AKT2 cur-
rent and an increase of the channel inward rectification. Site-directed mutagenesis and pharmacological analysis re-
vealed that this functional interaction involves AtPP2CA phosphatase activity. Regulation of AKT2 activity by AtPP2CA
in planta could allow the control of K
transport and membrane polarization during stress situations.
channel is endowed with unique functional properties, being the only weak inward rectifier characterized
and was upregulated by abscisic acid. Coexpression of
Potassium is the most abundant cation in the cytoplasm of
the living cell, where it is involved in the regulation of ionic
strength, osmotic potential, and membrane polarization. K
channels of the so-called Shaker family (nine genes in Arabi-
dopsis) have been shown to play a role in K
root periphery (AKT1: Lagarde et al., 1996; Hirsch et al.,
1998), K secretion into the root xylem sap (SKOR:
Gaymard et al., 1998), K transport in the phloem tissues
(AKT2: Marten et al., 1999; Lacombe et al., 2000), or K
ward (KAT1: Ichida et al., 1997; Szyroki et al., 2001; KAT2:
Pilot et al., 2001) and outward (GORK: Ache et al., 2000)
fluxes in guard cells, leading to stomatal opening/closing.
To adapt to fluctuating K
availability in the environment
and to cope with other stresses, plants need to tightly regu-
transport at both the whole plant and the cell level
(Kochian and Lucas, 1988; Schroeder et al., 1994). Studies
aimed at revealing the molecular determinants of these reg-
ulations have highlighted mechanisms likely to target
channels at both the transcriptional and post-
translational levels. Expression studies have revealed that
transcript levels of Shaker channels are sensitive to hor-
uptake by the
mones (Gaymard et al., 1998; Philippar et al., 1999; Lacombe
et al., 2000), sugar synthesis and accumulation, and envi-
ronmental signals (Deeken et al., 2000). At the post-trans-
lational level, indications have been found for regulation
by ATP and cyclic GMP (Hoshi, 1995), phosphorylation
events (Li et al., 1994; Armstrong et al., 1995; Tang and
Hoshi, 1999), functional interactions with the cytoskeleton
(Hwang et al., 1997), 14-3-3 proteins (Saalbach et al., 1997;
Booij et al., 1999), sulfonylurea receptors (Leonhardt et al.,
1997), syntaxins (Leyman et al., 1999), and G proteins (Wu
and Assmann, 1994; Wang et al., 2001).
Searches for interacting proteins have been focused on
the KAT1 guard cell channel, which has been shown to be
phosphorylated by calcium-dependent and abscisic acid
(ABA)–regulated protein kinase activities (Li et al., 1998; Mori
et al., 2000). A homolog of animal Shaker channel
has been identified in the Arabidopsis genome and shown to
interact physically with KAT1 (Tang et al., 1996). This channel
partner protein, named KAB1, has been suggested to stabi-
lize KAT1 in the membrane (Zhang et al., 1999). No other
couple channel-interacting protein has been identified at the
molecular level to date. In the work reported here, our aim
was to identify regulating partners of the AKT2 channel,
which displays a very broad expression pattern (Lacombe et
al., 2000), suggesting tissue-specific regulation mechanisms,
and a gating mode, which has been suggested to be regu-
lated post-translationally (Dreyer et al., 2001).
ensam.inra.fr; fax 33-499-612-930.
Article, publication date, and citation information can be found at
To whom correspondence should be addressed. E-mail cherel@
1134The Plant Cell
gene was isolated initially (Cao et al., 1995) as a
. In situ hybridization allowed the localiza-
expression in phloem tissues of aerial parts
(Marten et al., 1999). Use of the more sensitive technique of
promoter-driven expression of the
) reporter gene revealed
tivity mainly in the phloem of all plant organs but also, at a
lower level, in other cell types of the leaf blade, such as me-
sophyll, epidermis, and guard cells (Lacombe et al., 2000).
The presence of low levels of
been confirmed by reverse transcriptase–mediated poly-
merase chain reaction (RT-PCR) experiments (Szyroki et al.,
2001). Recently, electrophysiological characterization of
knockout mutant plants has provided definitive evi-
dence that AKT2 is expressed and functional in the meso-
phyll plasma membrane (Dennison et al., 2001).
AKT2 displays unique functional features among Shaker
channels, being the only member of this family able to medi-
ate both K
influx and efflux. Characterized in heterologous
systems, it behaves as a weakly inwardly rectifying channel,
displaying both time-dependent hyperpolarization-activated
and instantaneous leak-like current components (Marten
et al., 1999; Lacombe et al., 2000). These two kinetic com-
ponents of the total AKT2 current correspond to two sub-
populations of AKT2 channels that display distinct gating
modes (Dreyer et al., 2001). It has been demonstrated that a
given channel can switch from one gating mode to the
other. The mechanisms that trigger these transitions have
not been identified, but phosphorylation/dephosphorylation
events have been suggested (Dreyer et al., 2001). In this
context, it is interesting that AKT2 was isolated recently as a
putative partner of a plant protein phosphatase involved
in ABA signaling, AtPP2CA, on the basis of two-hybrid sys-
tem screening with this phosphatase as the bait (Vranova
et al., 2001). No additional experiments were reported to
confirm the interaction or to assess its functional signifi-
cance in plants.
Using AKT2 as the bait for two-hybrid system screening,
we found a positive cDNA encoding a large C-terminal frag-
ment of AtPP2CA. Here, we provide biochemical and func-
tional evidence that AtPP2CA interacts with AKT2, at least in
heterologous systems, and that AKT2 and AtPP2CA have
largely overlapping expression patterns. Unique functional
effects on the channel activity are presented, and their pos-
sible physiological meanings are discussed.
mRNA in guard cells has
Two-Hybrid Interactions in Yeast
With a large AKT2 C-terminal region as the bait (from Ser-
334 to the last amino acid of the channel, Ile-802) (Figure
1A), screening of the Arabidopsis cDNA library (see Meth-
ods) resulted in the isolation of numerous positive cDNA
clones. Cross-hybridization studies and sequence analyses
allowed us to identify nine groups of overlapping cDNA frag-
ments, each forming a pool of truncated copies of the same
mRNA. Among the cDNA groups for which high signals
were obtained in two-hybrid tests, one corresponded to
KAT1, a Shaker channel already shown to interact with
AKT2 in functional tests (Baizabal-Aguirre et al., 1999),
Figure 1. Two-Hybrid Interaction of AtPP2CA with Potassium
Channels of the Shaker Family.
(A) Scheme of the structural domains of AtPP2CA protein phos-
phatase and the AKT2 K? channel. The positions of the first amino
acids of AKT2 regions used as baits (Ser-334 for the construct used
for two-hybrid screening and Met-324 [beginning of the intracyto-
plasmic C terminus] for the construct used in subsequent two-
hybrid experiments) and of the first amino acid of the AtPP2CA
C-terminal region obtained after two-hybrid screening (Asn-74) are
indicated. In AtPP2CA, the hatched area represents the N-terminal
domain, which is not conserved within the PP2C family. S1 to S6 in-
dicate the six transmembrane segments of the channel hydrophobic
core, with a pore-forming domain (P) present between S5 and S6.
anky, ankyrin domain; CNBD, putative cyclic nucleotide binding do-
main; KHA, domain rich in acidic and hydrophobic amino acids,
characteristic of plant channels of the Shaker family.
(B) Two-hybrid interaction tests between AtPP2CA and other chan-
nels. The AtPP2CA catalytic domain (from Asn-74, cDNA fragment
in pGAD10 vector) was tested against the intracytoplasmic C-ter-
minal domains (downstream of the S6 last transmembrane segment,
cDNA inserts in pGBT9 vector) of Shaker potassium channels.
?-Galactosidase activities, measured for 1-mL culture pellets with
o-nitrophenyl ?-D-galactopyranoside as a substrate, are expressed
as OD420 per min ? 1000/OD600 of the culture medium. The left bar
shows AKT2 (from Met-324) tested with the free Gal4 activator do-
main (using the empty pGAD10 vector).
Interaction between the AKT2 Channel and a PP2C1135
and another corresponded to AtPP2CA (Kuromori and
Yamamoto, 1994), which is a member of the protein phos-
phatase 2C (PP2C) family (Rodriguez, 1998). Plant PP2Cs
display two structural domains: an N-terminal variable do-
main, and a conserved catalytic domain (Rodriguez, 1998).
Only the sequence (from amino acid 74) (Figure 1A) encod-
ing the catalytic domain was present in the
identified during the two-hybrid screening.
We further checked the specificity of the AKT2–AtPP2CA
interaction by testing the C-terminal domains of five differ-
ent Arabidopsis Shaker channels (including AKT2) with the
AtPP2CA construct. Hybrid constructs encoding the C-ter-
minal region (the whole cytoplasmic domain downstream of
the S6 segment) of AKT2 (from Met-324; see Methods),
AKT1 (from His-294; Daram et al., 1997), KAT1 (from Thr-
303; Urbach et al., 2000), KAT2 (from Arg-316; Pilot et al.,
2001), or AtKC1 (from His-330) were used. With the
AtPP2CA catalytic domain fused to the Gal4 activator do-
main, none of the DNA binding domain constructs except
the one corresponding to AKT2 gave a positive signal (Fig-
ure 1B). The reciprocal tests with AtPP2CA as the bait
(fused to the Gal4 DNA binding domain) were performed
and revealed much higher background levels, compared
with the previous two-hybrid configuration, as a result of
artifactual activation of the reporter gene by the AtPP2CA
hybrid polypeptide. However, a signal of higher than back-
ground level was observed with the AKT2 C-terminal region
(data not shown). It should be noted that all of the two-
hybrid constructs used in these experiments had been vali-
dated previously by the observation of positive results in
interaction tests with channel C-terminal regions, either
identical to the bait or different (Daram et al., 1997; Urbach
et al., 2000; Pilot et al., 2001; I. Chérel, unpublished results),
plant potassium channels being multimeric proteins.
AKT2 Is Retained on AtPP2CA-Coated Gel Beads
Experiments were performed with the AtPP2CA C-terminal
polypeptide (from Asn-74) tagged with a hexahistidine pep-
tide (His tag) at its N terminus. Small affinity columns (each
L of agarose beads) were prepared with
nickel–nitrilotriacetic acid agarose (Ni-NTA) matrix. In a first
step, the binding of His-tagged AtPP2CA produced in
to the Ni-NTA matrix was checked by incubating the matrix
extract, washing, and directly eluting with
250 mM imidazole to release proteins bound to nickel. The
most abundant polypeptide eluted from the gel had an
apparent molecular mass of
identified as the His-tagged polypeptide (predicted molecu-
lar mass of 40 kD) by protein gel blot analysis with an anti-
body raised against tetra-histidine peptide (Figure 2A). This
result indicates that the AtPP2CA His-tagged protein repre-
sented the major polypeptide retained on the matrix, con-
taminants being present as traces only.
In a second step, another column coated with His-tagged
AtPP2CA (AtPP2CA column) and washed as described
42 to 43 kD. This band was
above but without the elution step was used to test the
binding of the AKT2 C-terminal polypeptide (from Met-324,
hereafter referred to as AKT2-CT). Because nontagged pro-
teins can interact with the Ni-NTA matrix, especially when
the purification is performed under native conditions, a con-
L column, for which the AtPP2CA binding step
was omitted, was treated exactly like the AtPP2CA column.
Each column (with or without bound AtPP2CA) was loaded
with an extract from
9 cells (3.5 mg of protein) expressing
AKT2-CT, washed under mild conditions, and treated with
250 mM imidazole. Fractions collected at different steps of
the purification procedure were analyzed by SDS-PAGE.
AKT2-CT was not detectable after Coomassie blue staining
Figure 2. AKT2-CT Binding on Nickel-Affinity Agarose Beads
Loaded with His-Tagged AtPP2CA Fusion Polypeptide.
(A) His-tagged AtPP2CA binding on the metal affinity column. The
Ni-NTA matrix was mixed with an extract from bacteria expressing
the His-tagged AtPP2CA polypeptide, washed, and treated with 250
mM imidazole to elute the nickel from the matrix with proteins asso-
ciated to it. The eluate was analyzed by SDS-PAGE. At left is a Coo-
massie blue–stained gel, and at right is a protein gel blot revealed
with a monoclonal antibody directed against the tetrahistidine pep-
(B) Biochemical evidence for interaction between AtPP2CA and
AKT2. The Sf9 cell extract containing AKT2-CT (first lane) was
loaded either onto the AtPP2CA column (harboring AtPP2CA bound
by its hexahistidine tag) or onto an empty control Ni-NTA column.
Columns were washed twice (fractions W 1 and W 2), and proteins
were eluted with 250 mM imidazole (three successive elutions: frac-
tions Elu 1, Elu 2, and Elu 3). Every well was loaded with a 30-?L
1136 The Plant Cell
of the acrylamide gel, and silver staining did not allow its
identification with certainty. However, protein gel blot analy-
sis performed with anti-AKT2 serum revealed a polypeptide
(Figure 2B) with an apparent molecular mass (58 kD) higher
than the theoretical molecular mass of the AKT2-CT
polypeptide (54 kD) but corresponding exactly to that of the
band that appeared specifically in extracts from AKT2-CT–
9 cells (E. Michard, unpublished results).
The 58-kD band was detected at a relatively high level in
the first wash from both columns (Figure 2B, lanes W 1) but
was barely detectable in the second wash (Figure 2B, lanes
W 2). After elution with imidazole, a significant amount of
AKT2-CT was recovered from the AtPP2CA column in the
first two elution fractions (Figure 2B, AtPP2CA column,
lanes Elu 1 and Elu 2). On the other hand, the protein gel
blot corresponding to the control column revealed only a
faint band, probably resulting from nonspecific binding to
free sites of the matrix, and present solely in the lane corre-
sponding to the first elution fraction (Figure 2B, control col-
umn, lane Elu l). Similar results were obtained in a subse-
quent experiment comparing the AtPP2CA column with
another control column that was loaded previously with an
extract from bacteria that were able to synthesize only
the His tag (transformed with the empty vector; data not
shown). Thus, all of the data indicated that AtPP2CA and
Upregulated by ABA in Shoots and Roots
Gene Expression Overlaps That of AKT2 and Is
Four-week-old plants harboring the
3-kb region upstream from the initiator ATG) fused to the
reporter gene and grown in a greenhouse were tested
for GUS activity. Six independent transformants were exam-
ined. Blue staining (revealing GUS activity) was detected in
several organs of the plant in both roots and aerial parts
(Figure 3). In the root system, the staining was faint and
present mainly in the stele (Figure 3A). In aerial parts, stain-
ing was much more intense and was found in photosyn-
thetic and reproductive organs. It was concentrated in the
veins of cotyledons and leaves (Figures 3B and 3C), essen-
tially at the periphery and at the tip in the case of mature
leaves of the rosette and caulinary leaves. Staining also was
visible in mesophyll cells between stained veins and in hy-
dathodes, which are structures involved in the process of
guttation (Figure 3C). GUS activity also was detected in
guard cells (Figures 3F and 3G). In reproductive organs, in-
tense staining was present in the pollen (Figure 3D) and in
the receptacle below the green siliques (Figure 3E). Cross-
sections of roots and leaves revealed that in the vascular
system, GUS activity was detected only in phloem tissues
(Figures 3H and 3I).
The possibility of coexpression of the
genes in the same cell type was tested further by RT-PCR.
Mesophyll cells are easy to isolate, and AKT2 channels ac-
Figure 3. Expression Pattern of the AtPP2CA Gene Overlaps That
of the AKT2 Gene.
(A) to (I) Localization of AtPP2CA promoter activity in Arabidopsis
transgenic plants grown on compost in the greenhouse.
(A) Young lateral root.
(B) Cotyledon leaf.
(C) Mature leaf.
(E) Green silique.
(F) Mature leaf (enlargement showing guard cells).
(G) Cross-section of a stomatal chamber.
(H) Vascular area of a petiole.
(I) Root cross-section.
gc, guard cells; h, hydathode; ph, phloem; xyl, xylem.
(J) RT-PCR experiments performed on total RNA isolated from
whole leaves (L) or mesophyll protoplasts (M). Thin arrows indicate
the expected size of the genomic DNA fragments amplified with the
pairs of primers used in the test, and thick arrows indicate that of the
cDNA fragments. These sizes are in kilobases (cDNA/genomic): 600/
685 for AtPP2CA, 823/993 for AKT2, 654/987 for KAT1, 896/1301
for KAT2, and 546/639 for EFI?. g indicates an amplification of ge-
nomic DNA with the AtPP2CA primers.
Interaction between the AKT2 Channel and a PP2C1137
count for half of their K
RT-PCR experiments were performed with total RNA iso-
lated from mesophyll cells and whole leaves from the same
plants (Figure 3J). As negative controls, we used two other
Shaker channel genes:
, which is expressed in guard
cells (Nakamura et al., 1995), and
in guard cells and minor veins (Pilot et al., 2001). The tran-
scripts of these genes were detected in whole leaves but
not in mesophyll cells, for which only genomic DNA present
as a contaminant in our RNA preparation could be amplified.
On the contrary,
tected in mesophyll cells (Figure 3J). Other primer pairs (two
and one for
(data not shown).
Twelve independent experiments with transgenic plants
transformed with the
reporter gene construct and
grown in the greenhouse revealed reproducible expression
patterns but with a high variability in GUS staining intensity.
The entire set of observations led us to the hypothesis that
promoter activity was affected by unidentified
changes in environmental conditions. In this context, we
studied the effects of ABA on the expression of the
construct. Plants grown in the greenhouse but carefully wa-
tered to avoid any drought stress were transferred either
onto a control CaSO
solution or onto the same solution
supplemented with 100
M ABA. Whereas no GUS activity
was detected in the former plants, strong GUS staining was
observed in the latter plants after a 3-hr ABA treatment. The
induction by ABA was especially visible in leaf veins and in
the root stele (Figure 4A).
After longer ABA treatments (6 to 24 hr), roots and leaves
were stained uniformly (data not shown). Consistent with
these observations, RNA gel blot analyses revealed a strong
gene expression upon ABA treatment
(Figure 4B) in shoots (in agreement with the RNA gel blot
data presented by Tähtiharju and Palva ) but also in
roots (two independent experiments). The transcript distri-
bution between shoots and roots was largely in favor of
roots in this case, whereas the opposite situation was ob-
served with untreated plants grown in the greenhouse. This
kind of inversion is similar to that reported for other ABA-
induced genes when a physiological situation (drought
stress) is compared with an ABA treatment applied to the
root system (Gosti et al., 1995).
permeability (Dennison et al., 2001).
, which is expressed
transcripts were de-
) led to similar results
AtPP2CA Inhibits the AKT2 Current in COS Cells
The functional effects of the AtPP2CA–AKT2 interaction
were sought by patch clamping transfected COS cells (see
Methods). The currents recorded in cells expressing AKT2
alone were approximately three times larger than those in
cells coexpressing AKT2 and AtPP2CA (Figure 5A, middle
and bottom, respectively). To ascertain the specificity of the
inhibitory effect of AtPP2CA on AKT2 current, a control ex-
periment was performed by coexpressing AtPP2CA with an-
other Arabidopsis Shaker channel, KAT1. In accordance
with the fact that this plant potassium channel did not inter-
act with AtPP2CA in the two-hybrid system, KAT1 currents
were not affected by the presence of AtPP2CA in COS cells
As described previously (Lacombe et al., 2000), the AKT2
macroscopic current showed an instantaneous nonrectifying
(leak-like) component, which superimposed on a time-depen-
dent hyperpolarization-activated component (Figure 5A).
The inhibitory effects of AtPP2CA on these two components
were quantified, revealing a larger decrease of the instan-
taneous leak-like current. For instance, in the experiment
Figure 4. Induction of AtPP2CA Expression by ABA.
(A) AtPP2CA promoter activity (GUS reporter gene expression) in
3-week-old plantlets from the same transgenic line grown in the
greenhouse and transferred for 3 hr to 250 ?m CaSO4 (left) or 250
?m CaSO4 ? 100 ?m ABA (right). Top, whole plantlets; bottom,
(B) RNA gel blot analysis performed on 3-week-old plantlets grown
hydroponically in magenta boxes and transferred to control medium
(?) or treated for 3 hr with 100 ?m ABA (?).
1138The Plant Cell
reported in Figure 5C, the inhibition of AKT2 current (2.8-
fold) was attributable to a 4.5-fold decrease in the instanta-
neous leak-like component and a 2-fold decrease in the hy-
perpolarization-activated component (Figures 5D and 5E,
respectively; inhibition computed at
the AKT2 current was not only decreased in magnitude but
also changed qualitatively. A so-called “leak index” was de-
fined for the AKT2 current to appreciate the qualitative
In the experiments described above (Figure 5), where the
equilibrium potential for K
selective leak conductance (devoid of rectification) should re-
sult in a linear current-voltage (I-E) curve crossing the origin
of the graph. With such a conductance, the amount of cur-
rent recorded at
160 mV (|I
that recorded at
40 mV (4
a perfect K?-selective hyperpolarization-activated channel
should display a flat I-E curve for voltages above the activa-
tion potential with no outward current (4·I?40/|I?160| ? 0). The
(4·I?40/|I?160|) ratio was defined as the AKT2 leak index. Data
shown in Figure 5C yielded leak indexes of 0.39 ? 0.06 (n ?
6) for AKT2 expressed alone and 0.20 ? 0.04 (n ? 8) for
AKT2 coexpressed with AtPP2CA, highlighting the fact that
the functional interaction between AtPP2CA and AKT2 re-
sulted in a significant decrease in the leak-like current.
The requirement of protein phosphatase activity for the
observation of the AtPP2CA effect on AKT2 was checked by
introducing a null mutation in the catalytic site of the en-
zyme. The G139D mutation is known to result in the complete
loss of PP2C activity (Sheen, 1998). When coexpressed with
AKT2 in COS cells, the null G139D mutant protein poorly
modified the AKT2 currents (Figure 5C, closed circles).
160 mV). As a result,
) was 0 mV, an “ideal” K
|) would be four times
Coexpression in Xenopus laevis Oocytes Further
Confirms the Functional Interaction between
AtPP2CA and AKT2
In a first set of experiments, comparison of AKT2 currents in
oocytes expressing AKT2 alone or coexpressing AKT2 and
AtPP2CA provided further evidence that AtPP2CA inhibits
AKT2 activity (current reduced to 40% of the control value;
n ? 6; data not shown). Detailed analyses revealed that the
functional interaction also resulted in qualitative changes in
AKT2 current, as in COS cells, the leak-like component be-
ing more affected than the hyperpolarization-activated com-
ponent. Indeed, when calculated as described above, the
leak index was found to be 0.40 ? 0.02 (n ? 6) for AKT2 co-
expressed with AtPP2CA and 0.52 ? 0.07 (n ? 6) for AKT2
expressed alone (Figures 6B and 6D, closed symbols).
In a second set of experiments, the effects of AtPP2CA on
AKT2 current were investigated in the presence or absence
of vanadate, a permeant nonspecific phosphatase inhibitor
classically used for this kind of approach in Xenopus laevis
oocytes (McNicholas et al., 1994; Becq et al., 1997; Tsai et
al., 1999). It was verified that vanadate bath solution per-
Figure 5. Modulation of AKT2 Currents by Coexpression with
AtPP2CA in COS Cells.
(A) Typical current traces evoked by the voltage protocol indicated
at top in COS cells transfected with pIRES alone (control) or
cotransfected with pIRES and pCI-AKT2 (AKT2) or pIRES-AtPP2CA
and pCI-AKT2 (AKT2 ? AtPP2CA) at 3 days after transfection.
(B) Current-voltage relationship for KAT1 expressed alone (dia-
monds) or together with AtPP2CA (triangles).
(C) Current-voltage curves recorded in a batch of COS cells
cotransfected with pCI-AKT2 and pIRES (open squares), pIRES-
AtPP2CA (closed squares), or pIRES-AtPP2CA* (null mutation;
closed circles [note that closed circles are hidden by the open
squares at positive potential values]). Cs? controls (data not shown)
were used to ensure that currents were generated by potassium-
selective channels. Data are means ?SE, with n ? 6 in each case.
(D) and (E) Instantaneous (D) and time-dependent (E) currents taken
from data presented in Figure 3C for COS cells cotransfected with
pIRES-AtPP2CA and pCI-AKT2 (squares) or pIRES and pCI-AKT2
Interaction between the AKT2 Channel and a PP2C1139
fused for 1.5 hr before the current recordings had no effect
on the membrane conductance of water-injected oocytes
(Figure 6A). In oocytes expressing AKT2 alone, the vanadate
treatment induced a slight increase (?20%) in the AKT2 cur-
rent (Figure 6B). A much larger increase was observed in
oocytes coexpressing AKT2 and AtPP2CA (Figures 6C and
6D). Figure 6C shows current recordings obtained in an oo-
cyte expressing both the K? channel and the protein phos-
phatase before (top) and after (bottom) vanadate treatment.
The corresponding I-E curve shown in Figure 6D indicates
that the treatment resulted in a strong increase (?2.5 times)
of the AKT2 current.
The leak index was shifted from 0.40 ? 0.02 before vana-
date treatment to 0.60 ? 0.08 (n ? 5) after the treatment, in-
dicating that inhibition of phosphatase activity by vanadate
favored the leak-like component. Cs? controls (see Meth-
ods) were used to ascertain that neither an endogenous cur-
rent nor a nonspecific leak had appeared during the 1.5-hr
vanadate treatment (data not shown). Thus, the combined
data indicate both that the endogenous oocyte phos-
phatases affected AKT2 activity much less than AtPP2CA
and that the AKT2 inhibition by AtPP2CA was counteracted
by the use of a phosphatase inhibitor. In conclusion, mod-
ulations of AKT2 current intensity and rectification by
AtPP2CA are observed regardless of the heterologous sys-
tem, Xenopus oocytes or mammalian cells, and are caused
by the phosphatase activity of AtPP2CA.
Physical and Functional Interactions of AtPP2CA
Using the yeast two-hybrid system, we isolated AtPP2CA as
a putative AKT2-interacting protein. The two-hybrid system
allows a rapid screen for putative partners of a given pro-
tein. However, the isolation of a positive clone with this
technique cannot be taken as proof of interaction because
of the abundance of artifactual responses. The reciprocal
test, with the putative partner fused to the Gal4 DNA binding
domain and the target protein fused to the Gal4 activator
domain, eliminates many false-positive results. A positive
response in both test configurations is taken as a strong in-
dication of true physical association (Allen et al., 1995).
In the case of the AKT2–AtPP2CA interaction, in spite of a
high background signal, we confirmed the two-hybrid inter-
action in the reciprocal test, in agreement with the recent
isolation of AKT2 by screening of a two-hybrid cDNA library
using AtPP2CA as the bait (Vranova et al., 2001). In the
same report, it was indicated that a deletion of the last 180
amino acids of the AtPP2CA catalytic domain abolished the
two-hybrid interaction, revealing that this region either con-
tains the binding site for AKT2 or is necessary for the stabil-
ity and/or proper folding of the AtPP2CA polypeptide. The
AtPP2CA clone that was isolated during our two-hybrid
screen essentially encodes the AtPP2CA C-terminal (cata-
lytic) domain, providing evidence that this domain (Figure 1)
is involved directly in the interaction with AKT2.
Direct physical interaction between AKT2 and AtPP2CA
was further confirmed by the demonstration of in vitro bind-
ing of AKT2-CT onto a Ni-NTA agarose column coated with
AtPP2CA. The binding affinity between a protein phosphatase
and its substrate is expected to be low. Indeed, a large pro-
portion of AKT2-CT flowed through the AtPP2CA-coated
column. However, compared with the control columns,
which retained only trace amounts of AKT2-CT (nonspecific
Figure 6. Coexpression of AKT2 and AtPP2CA in Xenopus Oo-
(A) Control experiment performed with water-injected oocytes be-
fore and after a 1.5-hr treatment with 3 mM vanadate.
(B) Current-voltage (I-E) curves for oocytes injected with pCI-AKT2
alone before (closed symbols) and after (open symbols) vanadate
treatment. Mean values and standard errors for at least five indepen-
dent measurements (oocytes) are shown (n ? 5). Data were normal-
ized to the current value at ?150 mV in the presence of vanadate
(100%). The average of this reference value was ?1.78 ?A. Cs?
control recordings performed before and after vanadate treatment
(data not shown) indicated that the currents were generated by po-
(C) Typical currents recorded in oocytes coinjected with pCI-
AtPP2CA and pCI-AKT2 (ratio of 2:1) before and after vanadate
(D) Current-voltage (I-E) curves for oocytes coinjected with pCI-
AtPP2CA and pCI-AKT2 before (closed symbols) and after (open
symbols) vanadate treatment. Values shown and data normalization
were as in (B). The average reference value (100%) was ?2.18 ?A.
1140 The Plant Cell
binding), the AtPP2CA column bound a significant amount
of channel polypeptide (Figure 2). Thus, the hypothesis that
AtPP2CA can interact physically with AKT2 is supported by
results from two independent approaches, two-hybrid tests
and in vitro binding assays. Such an interaction involving a
PP2C and a channel has been observed between the hu-
man PP2C? and the cystic fibrosis transmembrane regula-
tor anion channel (Travis et al., 1997).
Coexpression of AKT2 and AtPP2CA in COS cells and Xe-
nopus oocytes revealed a modulation of AKT2 channel ac-
tivity that likely results from the physical association of the
two proteins. Dephosphorylation events mediate this func-
tional interaction, because the changes in channel activity
are sensitive to vanadate and suppressed by a point muta-
tion in the catalytic site of the AtPP2CA protein, resulting in
the loss of its phosphatase activity. Thus, all of the data
support the hypothesis that AtPP2CA can bind to and de-
phosphorylate AKT2, leading to changes in K? current fea-
tures. It should be noted that these changes are quantitative
and qualitative, with a decrease in both current amplitude
and leaky behavior. Conversion of a potassium leak channel
into a voltage-dependent channel induced by changes in
phosphorylation status was described in animal cells re-
cently, but in an opposite way, with phosphorylation events
leading to increased rectification: the addition of ATP and
commercially available protein kinase A was found to de-
crease the activity of the hippocampal K? channel KCNK2
and to convert this protein from a voltage-insensitive open
pore into a voltage-dependent outward rectifier (Bockenhauer
et al., 2001). AKT2 is a channel displaying such an intercon-
version for which a specific regulatory partner responsible
for this behavior has been identified.
Possible Links to Other ABA-Induced
AtPP2CA is one of the closest relatives of ABI1 and ABI2,
two PP2Cs known to be involved in ABA hormone signaling
(Leung and Giraudat, 1998; Rodriguez, 1998). Under
drought stress conditions, ABA mediates stomatal closure,
allowing limitation of water loss (reviewed by Blatt, 2000).
ABI1 and ABI2 genes are involved in most aspects of ABA
response (Leung et al., 1997). Genetic studies have estab-
lished that ABI1 and ABI2 are negative regulators of ABA
transduction signals (Gosti et al., 1999; Merlot et al., 2001).
The concomitant increases of ABI1 and ABI2 mRNA levels
and corresponding protein phosphatase activities induced
by ABA have led to a model (Merlot et al., 2001) in which
ABI1 and ABI2 take part in a negative feedback regulatory
loop that continuously resets the ABA signaling cascade to
adjust the response to the ABA level.
AtPP2CA overexpression results in the inactivation of an
ABA-responsive promoter (Sheen, 1998), and the encoded
protein plays a role during cold acclimation by counteract-
ing ABA effects (Tähtiharju and Palva, 2001). The inhibitory
effect of AtPP2CA on the AKT2 channel also may counter-
act the ABA-induced increase in AKT2 expression
(Lacombe et al., 2000). Like that of ABI1 and ABI2, the
AtPP2CA mRNA is induced by ABA. Together, these results
suggest that the general function of AtPP2CA could be sim-
ilar to that of ABI1 and ABI2, namely, to mediate a negative
control of ABA signaling in response to an increase in the
hormone level. Because of the pleiotropic effects of these
plant PP2Cs, it is likely that several substrates may be rec-
ognized as targets. Ion channels have been proposed to be
regulated by direct or indirect interactions with PP2Cs. For
example, overexpression of a mutant ABI1 protein in to-
bacco results in the deregulation of anion channels (Pei et
al., 1997) and of voltage-gated inward and outward K? recti-
fiers in guard cells (Armstrong et al., 1995). No substrate
protein of either ABI1 or ABI2, however, has been identified
Because the interaction between AKT2 and AtPP2CA in-
volves the well-conserved catalytic C-terminal domain of
AtPP2CA, the possibility exists for an interaction between
AKT2 and other closely related PP2Cs. These are ABI1
(48% identity with AtPP2CA within the catalytic domain),
ABI2 (46%), AtPP2C-HA (44%) (Rodriguez, 1998), and oth-
ers that are most similar to AtPP2CA (60 to 66% identity).
Although ABI1 was not found to react with AKT2 in a two-
hybrid assay (Vranova et al., 2001), this question is worth in-
Physiological Meaning of AKT2 Regulation by AtPP2CA
Under physiological conditions, the expression of both
AKT2 and AtPP2CA was observed mainly in shoots. De-
tailed comparison of the GUS data reveals that the expres-
sion pattern of AtPP2CA (Figure 3) clearly overlaps that of
AKT2 (Marten et al., 1999; Lacombe et al., 2000), especially
in the phloem vasculature and the receptacle below the si-
liques, tissues that constitute the main zones of expression
of the two genes. AtPP2CA also is expressed in leaf meso-
phyll and guard cells (Figure 3), where low levels of AKT2
promoter activity (Lacombe et al., 2000) and of AKT2 tran-
scripts (Szyroki et al., 2001) (Figure 3J) have been detected.
Thus, the AtPP2CA phosphatase can be expected to regu-
late AKT2 current amplitude and rectification in several cell
types. Also supporting this hypothesis, the electrophysio-
logical properties of AKT2 are similar to those of a K? chan-
nel detected in mesophyll cells, PKC1 (Spalding and
Goldsmith, 1993), which seems to be activated by phos-
In the phloem vasculature, where AKT2 and AtPP2CA dis-
play their highest expression levels, AKT2 could allow both
K? loading or unloading, as proposed by Lacombe et al.
(2000). High ABA levels have been found in phloem tissues,
and the hormone accumulation is enhanced during water
stress (Sotta et al., 1985). ABA has been proposed to pro-
Interaction between the AKT2 Channel and a PP2C1141
mote phloem unloading of Suc, possibly by inactivating the
proton pump (reviewed by Delrot and Bonnemain, 1985).
Under such conditions, AKT2 might contribute to potassium
release. Upregulation of AtPP2CA expression by ABA (Fig-
ure 4), resulting in the inhibition of AKT2 channels and the
switch to the rectifying gating mode, would control this re-
lease and thereby play a role in the control of membrane po-
larization (see below) and Suc transport. Also, AKT2 activity
and its regulation by AtPP2CA might modify the K? gradient
in the phloem vasculature between sources and sinks,
which has been hypothesized to play a major role in
steepening the gradient of hydrostatic pressure driving
the phloem sap toward the sinks (Vreugdenhil, 1985).
With respect to this hypothesis, it is worth noting that
concomitant sharp decreases in phloem sap flow rate and
phloem K? content can be evoked by stresses such as re-
peated cold shocks (Fromm and Bauer, 1994). Finally, it was
reported recently that the AKT2 gene and an AKT2-like gene
of Vicia faba, VFK1, are regulated by light in the phloem.
Both AKT2 and VFK1 transcripts are induced by light and
products of CO2 assimilation (Deeken et al., 2000; Ache et
al., 2001), and the depolarization evoked by Fru through Vicia
phloem cells is K? dependent (Ache et al., 2001). Thus, study-
ing the possible response of AtPP2CA activity to light and
photosynthesis should result in a more global understanding
of the role of the AKT2–AtPP2CA interaction in planta.
At the cell level, the AKT2–AtPP2CA interaction would al-
low the regulation of membrane potential, as discussed be-
low. In plant cells, two kinds of K? channels, activated either
by membrane hyperpolarization or membrane depolarization,
dominate membrane conductance (Blatt, 1992; Maathuis
and Sanders, 1995). As a result, a voltage range of low con-
ductance is found between the activation threshold of hy-
perpolarization-activated inwardly rectifying channels and
that of depolarization-activated outwardly rectifying chan-
nels. Compared with the other K? channels identified to date
at the molecular level, AKT2 is unique in its ability to be open
in the entire physiological voltage range (Figures 6B and
6D). Thus, in the low-conductance voltage range, changes in
AKT2 activity, involving functional interaction with AtPP2CA,
could markedly affect the membrane permeability to K? and
thereby play a role in the control of cell membrane potential.
For instance, in combination with a decrease in the activ-
ity of the electrogenic H?-ATPase, AKT2 activity could en-
able controlled membrane depolarization in a voltage range
within which strictly voltage-gated K? channels are inactive.
The AtPP2CA phosphatase then could control the ABA-
induced membrane depolarization that has been observed
in some cell types (guard cells: Thiel et al., 1992; mesophyll
cells: Felle et al., 2000) by inhibiting AKT2 current. AKT2
and the AtPP2CA phosphatase could thereby play a role
similar to the one fulfilled in animal cells by “background”
leak K? channels (Lesage and Lazdunski, 2000) and
their phosphatase/kinase partners (not yet identified;
Bockenhauer et al., 2001) in the control of cell membrane
potential. Poorly rectifying instantaneous currents often are
neglected in whole cell recording and simply subtracted,
being considered nonselective or even artifactual currents.
Our data clearly indicate that a leak current, controlled
by phosphorylation/dephosphorylation events and likely to
have major physiological importance, does exist in plant
In conclusion, AtPP2CA can interact physically and func-
tionally with AKT2, regulating the activity and unique gating
features of this channel. The expression patterns of the two
genes are very similar, not only with respect to tissue speci-
ficity but also regarding regulation by ABA. Therefore, the
functional interaction of AKT2 and AtPP2CA, under ABA
control, could play a role in the regulation of membrane con-
ductance to K? and membrane potential as well as in re-
lated functions at the whole plant level such as phloem
An AKT2 cDNA encoding a long C-terminal fragment (from amino
acid 334 to the end of the polypeptide sequence) was obtained from
a previous library screening with KAT1 as the bait. This cDNA frag-
ment was fused in frame with the sequence encoding the Gal4 DNA
binding domain in plasmid pGBT9 (Bartel and Fields, 1995). The re-
sulting plasmid was used to screen the library. For subsequent tests,
a larger fragment (encoding the sequence from amino acid 324) was
obtained via NcoI partial digestion and ligated into pGBT9, leading to
the creation of the pGBT9-AKT2 plasmid. pGBT9 constructs harbor-
ing AKT1, KAT1, and KAT2 sequences encoding channel C-terminal
regions were obtained previously (Daram et al., 1997; Urbach et al.,
2000; Pilot et al., 2001, respectively). The AtKC1 fragment (polypep-
tide sequence from His-330) was obtained by NcoI digestion.
Two-Hybrid cDNA Library Screening and Interaction Tests
Yeast strain Y190 Saccharomyces cerevisiae (Bartel and Fields,
1995) was cotransformed with pGBT9 plasmid harboring AKT2 se-
quence (75 ?g) and 15 ?g of DNA from a cDNA library designed for
two-hybrid screening (Clontech FL4000AB [Palo Alto, CA]) (prepared
from 3-week-old green vegetative tissue; 3 ? 106 independent trans-
formants), according to Gietz et al. (1992). Transformants were
plated on selective medium lacking His, Trp, and Leu (plating me-
dium described by Daram et al.  without His) supplemented
with 50 mM 3-aminotriazole. Yeast colonies were replicated on pa-
per sheets (80 g/m2) and tested for ?-galactosidase activity (colony
filter assay; Bartel and Fields, 1995). Positive colonies were amplified
at 30?C in 2 mL of liquid medium lacking Leu (plating medium [Daram
et al., 1997] without agar and supplemented with His and Trp).
After centrifugation, cell pellets were resuspended in 250 ?L of 10
mM Tris, 1 mM EDTA, and 200 mM NaCl, pH 8. Two hundred micro-
liters of 0.45-mm glass beads and 250 ?L of phenol:chloro-
form:isoamyl alcohol (25:24:1) were added to each cell suspension,
and tubes were vortexed (2 ? 1 min at maximum speed). After a
3-min centrifugation at 15,000g, the upper phases were mixed with
1142The Plant Cell
500 ?L of phenol:chloroform:isoamyl alcohol, vortexed (2 ? 1 min),
and centrifuged. Aqueous phases were precipitated with 3 volumes
of ethanol, and pellets were washed with 70% ethanol and resus-
pended in 20 ?L of water. These DNA preparations were used for
plasmid isolation after transformation of Escherichia coli and to de-
termine groups of cDNA clones originating from the same mRNA.
For the latter purpose, all cDNA inserts were amplified by polymer-
ase chain reaction (PCR) using oligonucleotides Matchmaker 5?AD
and 3?AD (Clontech). Amplification products were mixed with 20 ?
SSC (one-third volume) (1 ? SSC is 0.15 M NaCl and 0.015 M so-
dium citrate), heat denatured, and spotted onto a nylon membrane
(Hybond?). This membrane was hybridized with a 32P-radiolabeled
copy of one of the selected cDNAs, which were isolated previously
by SalI digestion of the library plasmid and purified twice on an aga-
rose gel. This allowed the identification of clones that hybridized with
each cDNA. The membrane then was dehybridized, and the experi-
ment was repeated with another cDNA insert.
For quantitative two-hybrid tests, cells from a 1-mL culture were
assayed for reporter gene expression using o-nitrophenyl ?-D-galac-
topyranoside as a substrate (Bartel and Fields, 1995).
Generation of a Full-Length AtPP2CA cDNA
An internal EcoRI site present in the AtPP2CA cDNA sequence was
used to generate the full-length cDNA. The original insert from the
cDNA library (3? fragment in pGAD10 vector) was digested with SalI
and cloned into pBluescript KS?. A clone displaying the proper ori-
entation (vector EcoRI site at the 5? side of the insert) was digested
with EcoRI for insertion of the 5? cDNA fragment. This 5? fragment
was obtained by PCR amplification from the two-hybrid cDNA library
with oligonucleotides 5?AD and 5?-CTTCTACCACAAACCGACGTC-
GTACCG-3? and cloned into vector pCR 2.1 (Invitrogen, Carlsbad,
CA). After sequencing, the 5? insert was reisolated by digestion with
EcoRI and subsequently ligated to the 3? end of AtPP2CA cDNA in
pBluescript KS? vector.
Production of the AtPP2CA Interaction Domain in E. coli
The AtPP2CA insert obtained from the cDNA library (in vector
pGAD10) was isolated by SalI digestion and then cloned into vector
pET28c(?) (Novagen, Madison, WI) using the same restriction site. E.
coli BL21(DE3) transformed with this construct (250 mL) was grown
until the exponential phase was reached (OD ? 0.6), and isopro-
pylthio-?-galactoside (300 ?M) was added to the culture. After 3 hr,
cells were pelleted and frozen in liquid nitrogen. The pellet was
treated as described in the Qiaexpressionist protocol (Qiagen, Valen-
cia, CA). Extraction was performed under native conditions after re-
suspension in 2.5 mL of lysis buffer (50 mM Na phosphate buffer, pH
8, containing 300 mM NaCl and 10 mM imidazole).
Production of AKT2-CT in Sf9 Cells
The AKT2 C-terminal domain (from amino acid 324 to the end) was
cloned into vector pGmAc34T (Davrinche et al., 1993). Sf9 cell ex-
tracts were obtained as described by Daram et al. (1997). They were
dialyzed for 2 hr against Sf9 buffer (50 mM Na phosphate buffer, pH
8, 150 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride,
and 7 mM ?-mercaptoethanol) containing 5 mM imidazole.
In Vitro Binding of AKT2-CT to AtPP2CA
A modified version of the Qiaexpressionist protocol (Qiagen) was
used. A nickel–nitrilotriacetic acid agarose gel (0.3 mL of a 50% so-
lution, washed with lysis buffer) was incubated for 1 hr with 1.25 mL
of bacterial lysate containing His-tagged AtPP2CA. A column was
obtained by pouring this mixture into a 1-mL pipette tip blocked with
glass wool. A control column was prepared with the same amount of
nickel–nitrilotriacetic acid agarose, which either had not been incu-
bated with the bacterial lysate or had been incubated with bacterial
extract from E. coli BL21 transformed with empty pET28c(?) vector.
Both columns were treated in parallel.
They were washed with 2 ? 1 mL of wash buffer and 1 mL of 50
mM Na phosphate buffer, pH 8. The dialyzed Sf9 cell extract, pre-
pared from AKT2-CT–expressing cells as described above (0.5 mL
per column, 3.5 mg of protein), then was loaded, and the flow-
through was loaded a second time. Columns were washed twice
with 1 mL of Sf9 buffer supplemented with 20 mM imidazole. Pro-
teins were eluted with 250 mM imidazole in Sf9 buffer (3 ? 170 ?L).
After SDS-PAGE (10% acrylamide), protein gel blot analysis was per-
formed using the Aurora kit (ICN, Costa Mesa, CA) and a polyclonal
antiserum raised against AKT2-CT. This antiserum was prepared us-
ing AKT2-CT expressed in E. coli as inclusion bodies and was puri-
fied by successive washes of the pellet and SDS-PAGE. The
absence of cross-reaction with proteins from Sf9 cells infected with
wild-type baculovirus was verified.
Localization of Promoter Expression Using ?-Glucuronidase
as a Reporter Gene
The AtPP2CA promoter region (3 kb) was amplified by PCR from an
Arabidopsis thaliana (cv Wassilewskija) genomic DNA preparation
with pfu DNA polymerase using oligonucleotides 5?-GGAGAAGCG-
CCGAATTCGAGTCATGG-3? and 5?-CCCAGCCATGGGATCTCT-
AACAAAACTTC-3?. This fragment was digested with NcoI (partial)
and EcoRI and inserted into pBI320.X (provided by R. Derose, Insti-
tute of Plant Molecular Biology, Strasbourg, France), leading to a fu-
sion between the AtPP2CA promoter and the ?-glucuronidase (GUS)
coding sequence. The resulting plasmid then was digested with
EcoRI and SacI, and the isolated fragment containing the AtPP2CA
promoter–GUS fusion was ligated into binary vector pMOG402 (pro-
vided by A. Hoekema, MOGEN International, Leiden, The Nether-
lands). All subsequent steps (plant transformation, GUS assay, and
cross-sections of plant tissues) were performed as described previ-
ously (Pilot et al., 2001). The insertion of the promoter-GUS fusion
into the plant genome was checked by PCR analysis.
RNA Gel Blot Analysis
RNAs were extracted and hybridized as described previously (Pilot et
al., 2001). The 32P-labeled probe was obtained by PCR amplification
using oligonucleotides 5?-CCGGTTGATTCAACTTCTCGAGC-3? and
5?-CTGAGGAGACTGCAATTCACATC-3?, which delineate an AtPP2CA-
specific region (nucleotides 49 to 648) at the beginning of the open
Plants were grown for 4 weeks in a growth chamber (8 hr of light at
22?C, 16 hr of darkness at 20?C, RH of 70%) for an optimal develop-
Interaction between the AKT2 Channel and a PP2C1143
ment of rosette leaves, then they were transferred for 24 hr in the
Mesophyll protoplasts were prepared as follows (protocol from E.
Hosy, personal communication). Abaxial epidermal strips from large
leaves were peeled, and epidermis-free areas were cut and digested
in a solution containing 5 mM Mes-KOH, pH 5.5, 1 mM CaCl2, 500
mM sorbitol, 2 mM ascorbic acid, 1% (w/v) cellulase, and 0.1% (w/v)
pectolyase for 15 min at 27?C. The digestion mixture was filtered on
nylon (0.05-mm mesh). The filtrate was recovered and diluted in en-
zyme-free digestion solution supplemented with 10 mM KCl. The
mesophyll protoplast preparation was checked with a microscope
for the absence of contaminating cells. The protoplast suspension
was allowed to decant in a tube at 0?C, and the resulting pellet was
frozen in liquid nitrogen until RNA extraction. Intact leaves of the
same plants were harvested and frozen in liquid nitrogen.
RNAs from mesophyll protoplasts and whole leaves were ex-
tracted as described previously (Pilot et al., 2001). First-strand DNA
synthesis was performed using Moloney murine leukemia virus re-
verse transcriptase (Promega) with 6 ?g of total RNA and 10 pmole
of oligo(dT)20 (final volume of 20 ?L). The reaction medium was di-
luted 10-fold in water, and 5 ?L was used for each PCR cycle (50 ?L).
Each couple of primers was chosen so as to surround at least one in-
tron. The elongation factor 1? gene (EF1?) (Axelos et al., 1989) was
used as a control. Primers were as follows: 5?-CCGGTTGAT-
TCAACTTCTCGAGC-3? and 5?-CTGAGGAGACTGCAATTCACATC-3?
for AtPP2CA; 5?-GCATTGAAGCAGCGTCAAACTTTGTTAACAG-3?
and 5?-CTTATGTGGATGTTGCAACCGTGC-3? for AKT2; 5?-GAC-
GCGATTTTCATCATCGACAAC-3? and 5?-GGTTCGGCTAGTCCA-
ATGAACGACGAGGTTGG-3? for KAT1; 5?-GCCTTCATCACCTAC-
AAGAAAG-3? and 5?-CCGTGAAATAGGTAGACGTTCTGAACGATT-
GGG-3? for KAT2; and 5?-CCACCACTGGTGGTTTTGAGGCTG-
GTATC-3? and 5?-CATTGAACCCAACGTTGTCACCTGGAAG-3? for
EF1?. The reaction was stopped after 36 cycles, and an aliquot was
loaded onto a 2% agarose gel.
The Altered Sites II In Vitro Mutagenesis System kit (Promega) was
used. The 0.6-kb EcoRI-XbaI internal fragment of AtPP2CA cDNA
was cloned into vector pAlter. Second-strand synthesis was
performed with mutagenic oligonucleotide 5?-CATCATTTCTAC-
GACGTCTTTGACGGC-3?, which was designed to create a G139D
mutation and an AatII site by replacement of the GGTGTC sequence
(amino acids Gly and Val) with GACGTC (amino acids Asp and Val). A
clone was selected on the basis of the AatII restriction pattern. The
native AatII internal site of AtPP2CA cDNA (not created by mutagen-
esis) was cut by partial digestion, and then complete NcoI digestion
was performed to generate a 108-bp fragment. This fragment was li-
gated into AtPP2CA cDNA digested with AatII and NcoI. The entire
region encompassing the 108-bp inserted fragment was verified by
Expression in COS Cells
Full-length cDNAs were cloned in pCI (Promega) or pIRES (Reyes et
al., 1998), which is a pCI-derived vector that allows coexpression of
the cDNA insert with a sequence encoding the membrane protein
CD8 (providing a transfection marker). COS-7 cells were cultured in
2 mL of Dulbecco’s modified Eagle’s medium (Gibco BRL) supple-
mented with 10% fetal bovine serum (Gibco BRL) at 37?C in 5% CO2.
One day before transfection, cells were detached with trypsin-EDTA
(Gibco BRL) and transferred to 35-mm dishes (20,000 cells per dish).
For one transfection, 80 ?L of HBS 2x (280 mM NaCl, 50 mM Hepes,
and 15 mM Na2HPO4, pH 7.15) were mixed gently with 80 ?L of TE-
CaCl2 (10 mM Tris, pH 8, 1 mM EDTA, and 250 mM CaCl2) containing
0.75 ?g of pCI-AKT2 plus 1.5 ?g of cotransfection plasmid: pIRES-
AtPP2CA or pIRES-AtPP2CA* (null G139D mutation), or empty
pIRES. The DNA precipitate that was formed was spread onto cells
in the culture medium.
After 8 hr, cells were rinsed twice with Dulbecco’s modified Eagle’s
medium. Electrophysiological analyses were performed 3 days later.
Transfected cells were detected with the anti-CD8 coated beads
method (Jurman et al., 1994). The bath solution contained 150 mM
KCl, 1 mM CaCl2, 1.5 mM MgCl2, and 10 mM Hepes/NaOH, pH 7.4.
The pipettes (Kimax 51; Kimble Glass, Inc., Owens, IL) were filled
with 150 mM KCl, 1.5 mM MgCl2, 3 mM EGTA, 2.5 mM MgATP, and
10 mM Hepes/NaOH, pH 7.2. In the whole-cell configuration (Hamill
et al., 1981), pulses of 1.6 sec were applied, from ?40 to ?160 mV
with a holding potential of ?40 mV (steps of 20 mV). Electrical signals
were amplified by Axopatch 200 A (CV-201 A, software Clampex-6;
Axon Instruments, Union City, CA). Control electrophysiological re-
cordings in media supplemented with 10 mM CsCl were performed
systematically (Cs? controls), as described previously (Lacombe et
al., 2000), to check that the recorded current essentially flowed
through K?-selective channels (AKT2) and not through a nonselec-
tive leak pathway.
Expression in Xenopus laevis oocytes
Xenopus laevis (Centre de Recherche de Biochimie Macromolécu-
laire, Centre National de la Recherche Scientifique, Montpellier,
France) oocytes were injected with 20 ng (0.02 ?L) of pCI-AKT2, ei-
ther alone or together with 40 ng of pCI-AtPP2CA. Control oocytes
were injected with 0.02 ?L of deionized water. Two-electrode volt-
age-clamp experiments were performed as described previously
(Véry et al., 1995; Lacombe and Thibaud, 1998).
The standard external solution contained 20 mM KCl, 80 mM NaCl,
1.5 mM CaCl2, 3 mM MgCl2, and 10 mM Hepes/NaOH, pH 7.5. Van-
adate is used classically at millimolar concentrations in the external
solution to inhibit phosphatase activities in oocyte experiments
(McNicholas et al., 1994; Becq et al., 1997; Tsai et al., 1999). To obtain
the 3 mM (theoretical) vanadate external solution, sodium metavana-
date (3 mM NaVO3) was allowed to dissolve (strong magnetic stirring
at 30?C) in the standard external solution for a few hours before the
experiments. Oocytes were subjected to a first voltage clamp exper-
iment (protocol: 1.6-sec pulses from ?30 to ?150 mV, in 20-mV
steps, and a holding potential of ?30 mV). Thereafter, the oocytes
were treated with the vanadate external solution for 1.5 hr and sub-
jected to a second voltage-clamp experiment (same protocol).
Cs? control recordings (see above for COS cells) were performed
systematically before and after vanadate treatment to ensure that
currents were generated by a potassium-selective channel.
The accession numbers for the sequences mentioned in this article
are Z83202 (AtKC1), GI8778547 (ABI1), GI9759243 (ABI2), and
1144The Plant Cell
We are grateful to Nicole Grignon for assistance with microscopy
techniques, to Eric Hosy for the protocol of mesophyll protoplast
preparation, and to Isabel Lefevre for critical reading of the
Received December 7, 2001; accepted February 18, 2002.
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