Báo cáo khoa học: Modulation of the Arabidopsis KAT1 channel by an activator of protein kinase C in Xenopus laevis oocytes potx

Modulation of the Arabidopsis KAT1 channel by an
activator of protein kinase C in Xenopus laevis oocytes
Aiko Sato
1
, Franco Gambale
2
, Ingo Dreyer
3
and Nobuyuki Uozumi
1
1 Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan
2 Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Genova, Italy
3 Heisenberg Group of Biophysics and Molecular Plant Biology, Institute for Biochemistry and Biology, University of Potsdam, Potsdam-
Golm, Germany
Introduction
Plants possess guard cells in leaves to control gas
exchange and water loss. Guard cells control stomatal
aperture by osmotic swelling and shrinking in response
to, for example, carbon dioxide concentration, humidity
and light irradiation. The volume change in guard
cells is regulated by fluxes of K
+
,Cl
)
and organic
compounds via diverse transport systems. The hyper-
polarization-activated (inward-rectifying) K
+
channel
KAT1 expressed in guard cells is of great interest as it
has been suggested to play a key role in controlling

Keywords
K
+
channel; KAT1; kinase; phosphorylation;
protein kinase C
Correspondence
N. Uozumi, Department of Biomolecular
Engineering, Graduate School of
Engineering, Tohoku University, Aobayama
6-6-07, Sendai 980-8579, Japan
Fax: +81 22 795 7293
Tel: +81 22 795 7280
E-mail:
(Received 19 November 2009, revised
17 February 2010, accepted 10 March
2010)
doi:10.1111/j.1742-4658.2010.07647.x
The Arabidopsis thaliana K
+
channel KAT1 has been suggested to play a
key role in the regulation of the aperture of stomatal pores on the surface
of plant leaves. Calcium-dependent and calcium-independent signaling
pathways are involved in abscisic acid-mediated regulation of guard cell
turgidity. Although the activity of the KAT1 channel is thought to be regu-
lated by calcium-dependent protein kinases, the effect of phosphorylation
on KAT1 and the phosphorylated target sites remain elusive. Because it
has been proposed that the phosphorylation recognition sequence of plant
calcium-dependent protein kinases resembles that of animal protein
kinases C, in this study, we used the Xenopus laevis oocyte protein kinase C
to identify the target sites of calcium-dependent protein kinases. KAT1

expressed in Xenopus oocytes was inhibited by the protein kinase C activa-
tor phorbol 12-myristate 13-acetate. On the basis of an in silico search, we
selected S ⁄ T-X-K ⁄ R motifs facing the cytosol, as it has been reported that
protein kinase C and calcium-dependent protein kinase share a common
consensus sequence. Mutagenesis analyses revealed that six Ser ⁄ Thr
residues were responsible for the reduction in activity after phorbol
12-myristate 13-acetate application. Simultaneous mutation of the five
residues located in the carboxyl-terminus region of KAT1 led to a
K
+
channel mutant that was insensitive to protein kinase C. These results
indicate that, in plant cells, a kinase analogous to protein kinase C might
exist that may modulate KAT1 channel activity through calcium-dependent
phosphorylation at some of the pinpointed residues in the cytosolic region
of KAT1.
Abbreviations
AAPK ⁄ ABR kinase, ABA-activated protein kinase ⁄ ABA-responsive kinase; ABA, abscisic acid; CDPK, calcium-dependent protein kinase;
DAG, diacylglycerol; InsP
3,
inositol 1,4,5-trisphosphate; Kv, voltage-activated K
+
channel; PAs, phosphatidic acids; PI, phosphatidylinositol;
PI-PLC, PI-specific phospholipase C; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SnRK, SNF1-related protein kinase;
WT, wild-type.
2318 FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS
the volume of guard cells in Arabidopsis thaliana leaves
[1–3]. KAT1 has been proposed to be involved in the
mediation of K
+
uptake during stomatal opening. The

plasma membrane H
+
-ATPase establishes a negative
membrane voltage which, in turn, results in the open-
ing of inward-rectifying K
+
channels, allowing the
influx of K
+
ions [4]. For stomatal closure, increased
levels of cytosolic Ca
2+
inhibit plasma membrane pro-
ton pumps [5], leading to a depolarization of the mem-
brane. This activates anion efflux channels and inhibits
inward K
+
uptake channels [6] to reduce the turgor
pressure of the cells. The involvement of KAT1 in
these regulatory processes has been suggested in sev-
eral reports. For example, the co-injection of KAT1
cRNA into oocytes with transcripts extracted from
Vicia faba guard cells decreases KAT1 channel
activity, unlike that with transcripts from mesophyll
cells [7]. In the same heterologous system, KAT1 cur-
rent amplitudes decrease in the presence of a soybean
calcium-dependent protein kinase (CDPK) [8]. Consis-
tent with this, CDPKs in guard cells are involved in
Ca
2+

and anion channel activation and stomatal
closure [9,10]. In line with these data is the finding
that Ca
2+
channels are activated by abscisic acid
(ABA) [11] and, as a consequence of cytosolic Ca
2+
elevation, inward K
+
channel activity is reduced,
resulting in stomatal closure [12]. In addition to
these Ca
2+
-dependent reactions, a calcium-indepen-
dent pathway also contributes to the control of guard
cell volume. The calcium-independent, ABA-activated
protein kinase ⁄ ABA-responsive kinase (AAPK ⁄ ABR
kinase) from Vicia faba has been found to be present
in guard cells and to control stomatal response to
ABA [13–15]. In an in vitro phosphorylation assay, the
Vicia AAPK ⁄ ABR kinase has been shown to phos-
phorylate the C-terminal region of KAT1 [16]. One of
the 10 members of the SNF1-related protein kinase 2
in A. thaliana, SnRK2.6, is an ortholog of AAPK and
shares 79% amino acid identity [15]. SnRK2.6 has
been identified as an essential element of the ABA
signaling pathway that mediates stomatal regulation
[17–20]. Recently, it has been shown that SnRK2.6,
after heterologous expression and purification from
Escherichia coli, can phosphorylate the residues T306

and T308 in KAT1. Modification of T306 abolished
KAT1 activity in oocyte recordings, whereas modifica-
tion of T308 did not cause a loss of function [21].
In animal cells, one type of Ser ⁄ Thr protein kinase,
protein kinase C (PKC), is involved in signal transduc-
tion pathways that govern a wide range of physiologi-
cal processes, such as proliferation, apoptosis, cell
survival and migration [22,23]. The animal Shaker
superfamily comprises the so-called voltage-activated
K
+
channel (Kv), Kv long QT, small-conductance cal-
cium-activated K
+
channel, large-conductance Ca
2+
-
and voltage-regulated K
+
channel, hyperpolarization-
activated cyclic nucleotide gated channel, ether-a-go-go
and cyclic nucleotide-gated channel members. It is well
known that some of these are modulated by PKC [24–
27]. In addition, G-protein-coupled inward rectifier
K
+
channels are inhibited by PKC phosphorylation
[28].
Diacylglycerol (DAG), a natural degradation prod-
uct of phosphatidylinositol (PI), allosterically activates

PKC and regulates the activity of other proteins
involved in carcinogenesis and metastasis, as well as in
cell growth, development, survival and apoptosis [29–
33]. DAG, generated from PI in the PI-specific phos-
pholipase C (PI-PLC) pathway, and elevated Ca
2+
induce the activation of conventional animal PKCs.
Although canonical PKC-encoding genes have not
been found in plants, a large family of CDPKs, some
of them being activated by phospholipids (e.g. CPK1
in A. thaliana), has been documented [34–36]. PKC
can be classified as conventional PKCs (cPKC; a, bI,
bII and c), which contain a putative Ca
2+
-binding
site, novel PKCs (nPKC; d, h, g and e), which lack
Ca
2+
-binding sites, and atypical PKCs (aPKC; f, k ⁄ i
and l), which are Ca
2+
-insensitive and are not acti-
vated by phorbol esters [37]. Both cPKCs and nPKCs
are activated by phorbol esters, such as phorbol 12-
myristate 13-acetate (PMA). In Xenopus oocytes, the
presence of all 11 PKC isozymes in mammals (a, bI,
bII, c, d, f, e, h, g, k ⁄ i and l) has been reported [38].
In Arabidopsis, the existence of a PI-PLC pathway
has been reported [39,40]. DAG has been considered
to be rapidly converted to phosphatidic acids (PAs) by

DAG kinases in plant cells [41]. Therefore, it may be
possible that the other downstream events uncovered
in animal cells may also have an equivalent in plant
cells. Ca
2+
plays an important role as an intracellular
signal in both plants and animals, including its
involvement in the regulation of CDPK activity [42].
Despite the absence of PKC in plant cells, PKC-like
enzymes have been reported to be present in protein
extracts from various plant species. For example, an
enzyme (ZmcPKC70) has been extensively purified and
characterized in leaf protein extracts from the C4 plant
maize [43], which belongs to the cPKC family, because
it is activated by both PMA, a well-known agonist of
animal PKC, and Ca
2+
. In addition, a PKC homolog,
which can be detected with the PKC antibody in
Brassica juncea, is activated by PMA and inhibited by
the general kinase inhibitor H-7 and the PKC-specific
inhibitor staurosporine [44]. Moreover, a large family
of CDPKs, including some showing cPKC-like charac-
A. Sato et al. Phosphorylation of KAT1 channel
FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS 2319
teristics, is present in plant genomes [34–36]. Maize
CDPK-1 phosphorylates in vitro sequence motifs simi-
lar to those recognized by animal PKCs [45].
On the basis of these facts, we examined the effect
of PKC activation on KAT1 channel activity by the

PKC activator PMA in Xenopus oocytes. We also pin-
pointed the phosphorylation target sites which regulate
KAT1 channel activity. We uncovered a complex pat-
tern of sites that are involved in channel regulation,
indicating that phospho-regulation of plant K
+
chan-
nels should not be considered as a ‘single switch’, but
rather as the result of a multistage process.
Results
Reduction of KAT1 currents by PKC activation
Earlier studies have reported the phosphorylation of
the KAT1 channel expressed in guard cells by CDPK.
On co-expression of KAT1 with a CDPK from soy-
bean in oocytes, a decrease in the current amplitude
was monitored [8,46]. To further evaluate whether
KAT1 channel activity is regulated by phosphoryla-
tion, we expressed KAT1 in Xenopus laevis oocytes
and applied PMA, which is known to activate endoge-
nous PKC. The recognition sequence of PKC for
phosphorylation resembles that of plant CDPKs
[45,47,48]. In KAT1-expressing oocytes, we measured
inward-rectifying K
+
currents as reported previously
[2,49]. After the addition of 1 lm PMA to the bath
solution [50], the current amplitude apparently began
to decrease. At 30 min after PMA application, currents
were inhibited by about 45.0 ± 5.6% (Fig. 1A). The
normalized current–voltage characteristics were almost

identical before and after PMA application (Fig. 1B).
Likewise, the normalized cord conductance was
not altered as a result of PMA treatment (Fig. 1C).
These results suggest that KAT1 is inhibited by PKC
in oocytes without affecting its voltage-dependent
properties.
To confirm the regulation of KAT1 channel activity
by PKC, we applied a different voltage pulse protocol
to record changes in KAT1 currents over time. For
this purpose, we applied a voltage pulse to –150 mV
every 30 s. The current amplitude decreased, and the
inhibition appeared to be saturated at 30 min after
PMA application (Fig. 2A). In addition, we measured
the current–voltage characteristics of KAT1 every
5.5 min with or without pre-incubation of oocytes
in 2 lm calphostin C, a PKC-specific inhibitor, for
12–24 h (Fig 2B, C). KAT1 currents measured in
calphostin-pre-incubated oocytes were less susceptible
to PMA than those in nontreated oocytes. Taken
together, these results demonstrate that KAT1 is
inhibited by PMA-stimulated activation of the oocyte
intrinsic PKC.
Identification of Ser
⁄
Thr PKC phosphorylation
sites influencing KAT1 channel activity
Several groups have reported different sequence
motifs that are recognized by PKC [51,52]. We used
2 µA
200 ms

0 min
15 min
30 min
–0.5
0
–200 –150 –100 –50 0
V (mV)
0 min
–2
–1.5
–1
Normalized current
15.5 min
32 min
A
B
C
PMA
No addition
Fig. 1. Effects of PMA on KAT1 WT chan-
nel activity. (A) Representative current
profile of KAT1 expressed in Xenopus
oocytes before (0 min) and 15 and 30 min
after the addition of 1 l
M PMA (a PKC
activator in oocytes). (B) Current–voltage
relationship of the current at 0, 15.5 and
32 min after PMA application. Currents
were normalized with respect to the current
at )150 mV. (C) Normalized KAT1 conduc-

tance G
Nor
before (full line; squares) and
after (broken line; circles) PMA application.
Phosphorylation of KAT1 channel A. Sato et al.
2320 FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS
the program prosite ( to
predict residues of KAT1 that might be phosphory-
lated by PKC. All of the resultant sequences con-
tained the classical S ⁄ T-X-K ⁄ R motif, which can be
recognized by PKCs in Xenopus laevis oocytes [38,53].
Moreover, ‘S ⁄ T-X-K ⁄ R’ matches with the consensus
sequences recognized by plant CDPKs [54,55]. In
addition, we employed the prediction program Netph-
osK 1.0 ( />using the Phospho.ELM database (http://phospho.
elm.eu.org/) containing experimentally verified phos-
phorylation patterns of Ser ⁄ Thr ⁄ Tyr residues in
eukaryotic proteins. The analysis for 11 Ser ⁄ Thr resi-
dues in the cytosolic C-terminus and for Thr at posi-
tion 45 resulted in a relatively high score (more than
0.7). Consequently, we selected these 12 Ser ⁄ Thr resi-
dues as possible phosphorylation sites for PKC within
the N- and C-terminal cytosolic regions of KAT1
(Fig. 3A). We systematically screened all of them by
replacing the Ser ⁄ Thr residues by an Ala residue
which mimics the dephosphorylated form. All vari-
ants, except KAT1 T303A, showed detectable
inwardly rectifying currents. We also tested the K
+
transport activities of KAT1 T303D. However, as for

the mutant T303A, we could not obtain K
+
currents
in oocytes (data not shown). The remaining mutants
could be subdivided into three datasets: (a) the muta-
tions T45A, T308A, S312A, S589A, S590A and
S641A displayed a decrease in PMA-induced channel
inhibition (27.1 ± 2.9%, 20.6 ± 3.2%, 17.0 ± 6.5%,
28.4 ± 6.0%, 23.9 ± 5.0% and 24.9 ± 6.6%, respec-
tively; Fig. 3B, top panel); (b) the mutants T22A,
S44A, S125A and S529A behaved similarly to the
wild-type (WT) (43.2 ± 7.2%, 36.9 ± 3.3%, 34.3 ±
5.6% and 44.5 ± 0.7%, respectively; Fig. 3B, middle
panel); (c) T458A showed slightly greater inhibition
by PMA-induced PKC activation compared with WT
(52.5 ± 2.3%; Fig. 3B, bottom panel). The data sug-
gested that six Ser ⁄ Thr residues – one in the cytosolic
N terminus (T45) and five in the cytosolic C-terminus
(T308, S312, S589, S590 and S641) – were candidates
for PKC phosphorylation target sites altering KAT1
channel activity.
Quintuple mutation renders KAT1 PKC-insensitive
The data in Fig. 3B show that the single mutations do
not completely abolish the inhibitory effect of PMA
application. This may indicate that PKC stimulates
simultaneously multiple phosphorylation events on
KAT1. To confirm this, we constructed the quintuple
mutant KAT1-T308A-S312A-S589A-S590A-S641A
eliminating all putative PKC target sites in the cyto-
solic C-terminus. After expression in oocytes, the quin-

tuple mutant was no longer sensitive to PMA-induced
PKC activation. Even after PMA application, the
–3
–4
–2
–1
0
PMA
A
B
C
–6
–5
–4.5 0 10 20 30 37.5
Time (min)
Current (µA)
0.8
0.6
1
1.2
0
0.2
0.4
0102030
Time (min)
No addition
PMA
PMA + calphostin C
60
37.5

10
20
30
40
50
0
No addition
PMA
PMA +
calphostin C
Current inhibition by PMA (%) I/I
control
Fig. 2. Inhibition of WT KAT1 activity by PKC activation. (A) Time
course of a representative WT KAT1 current amplitude at –150 mV
after the addition of 1 l
M PMA to the bath solution. A black bar
indicates the addition and removal of PMA. (B) Changes in the cur-
rent at –150 mV in response to 1 l
M PMA. PMA was added imme-
diately after the recording of currents at t = 0 min. The current
amplitude was normalized to the value measured before PMA
application (mean ± SEM, n = 3–4). Calphostin C indicates that
KAT1-expressing oocytes were pre-incubated for 12–18 h with
2 l
M calphostin C, a specific PKC inhibitor. (C) Percentage of cur-
rent inhibition 32 min after the addition of PMA.
A. Sato et al. Phosphorylation of KAT1 channel
FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS 2321
current amplitude behaved similarly to that of
WT KAT1 in the absence of PMA (Fig. 4A, B and

Table 1). These results suggest that PMA-induced
phosphorylation at some or all of the five residues
determines an inhibition of the K
+
currents.
Discussion
Phosphorylation and dephosphorylation events are
critical for the modulation of the activity of the guard
cell-expressed K
+
uptake channel KAT1. Nevertheless,
information on the kinase-mediated phosphorylation
of KAT1 and the target sites involved in the regulation
of channel activity is scarce. In this study, we investi-
gated the effect of the PKC-mediated phosphorylation
of KAT1 expressed in Xenopus oocytes. On stimulation
of the oocyte endogenous PKC by the application of
Putative cyclic
A
B
nucleotide
binding domain
(CNBD)
22
458
529
45
303
308
312

589
590
641
S
S
S
T
T
S
44
125
T
S
T
T
S
S
T45A
T308A
S312A
S589A
S590A
S641A
T22A
S44A
S125A
S529A
1
1.2
WT (no addition)

0
0.2
0.4
0.6
0.8
1
I/I
control
1
1.2
0
0.2
0.4
0.6
0.8
1
I/I
control
1.2
0
0.2
0.4
0.6
0.8
1
I/I
control
T458A
WT (PMA)
10 20 30 37.50

Time (min)
10 20 30 37.50
Time (min)
10 20 30 37.50
Time (min)
Fig. 3. Effects of mutations on possible phosphorylation sites on
KAT1 currents. (A) Schematic representation of the consensus
PKC phosphorylation sites at the cytosolic face of KAT1. (B)
Changes in the current amplitudes of the different KAT1 mutants
after PMA application. The characteristics of WT KAT1 in the
presence and absence of PMA are displayed as broken lines.
The mutants are divided into three groups: top panel, smaller
degree of inhibition compared with WT; middle panel, WT-like
behavior; bottom panel, larger degree of inhibition compared with
WT.
0.2
0.4
0.6
0.8
1
1.2
I/I
control
Quintuple mutant
WT (no addition)
WT (PMA)
0
0102030
Time (min)
40

50
60
37.5
N.D.
0
10
20
30
T22A
S44A
T45A
S125A
T303A
T308A
S312A
T458A
S529A
S589A
S590A
S641A
WT
Current inhibition
by PMA (%)
B
A
Quintuple mutant
No addition
Fig. 4. Quintuple mutations confer insensitivity to PKC activation.
(A) Change in the current amplitudes of the quintuple KAT1 mutant
KAT1-T308A-S312A-S589A-S590A-S641A after PMA application.

The characteristics of WT KAT1 in the presence and absence of
PMA are displayed as broken lines. (B) Inhibition of individual
mutants by PMA application at 32 min (mean ± SEM, n = 3–5).
*Student’s t-test, P < 0.05. N.D., no detectable currents.
Phosphorylation of KAT1 channel A. Sato et al.
2322 FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS
PMA to the bath medium, the KAT1 current ampli-
tude decreased more strongly than under control con-
ditions. This indicates that phosphorylation by PKC
has a downregulatory effect on KAT1. Subsequently,
we pinpointed by in silico analyses 12 putative target
sites for PKC in KAT1 and evaluated their role on
channel regulation. Among the 12 Ser ⁄ Thr sites in
KAT1, we identified experimentally six residues that
were involved in the regulation by PKC (Figs 3 and
5). The behavior of the different channel mutants on
PMA application could be divided into four groups:
(a) loss of function; (b) increase in the inhibitory
effect; (c) decrease in the inhibitory effect; (d) inhibi-
tion comparable with WT. In the first case, the
replacements T303A and T303D abolished the KAT1
current. It is possible that the mutation of Thr at
position 303 immediately after the S6 segment may
interfere with KAT1 channel gating, as illustrated
for residues a few positions upstream [56]. In addi-
tion, for other K
+
channels, it has been shown that
the region immediately after the last transmembrane
segment is strongly involved in channel gating [57–

61].
The inhibition of the KAT1-mediated K
+
current
on PMA application may depend on a decline in the
number of active channels or on a lower single-channel
conductance, as the voltage-dependent characteristics
were not affected by PMA application in oocytes
injected with the WT KAT1 channel (as shown in
Fig. 1B, C); notably, almost identical I–V characteris-
tics were also observed in oocytes injected with
mutants before and after the addition of PMA (data
not shown).
As the A. thaliana genome does not comprise a gene
encoding a protein that is homologous to animal
PKC, there is no evidence that the reduction in
KAT1 by phosphorylation suggested in this study
actually occurs in vivo in plant cells. Instead of PKC,
in the genome sequence of A. thaliana, 34 different
genes encoding CDPKs are present, which is currently
recognized as a major group of Ca
2+
-stimulated pro-
tein kinases [35]. A calcium-dependent kinase from
Vicia faba was found to phosphorylate the KAT1
protein translated in vitro [46], and a CDPK from
soybean decreased KAT1-mediated K
+
current ampli-
tudes in Xenopus oocytes [8]. To date, several CDPK

phosphorylation target sequences have been reported
[62–64]. The two most classical motifs are S-X-R ⁄ K
and R ⁄ K-X-X-S ⁄ T [54,55]. The S-X-R ⁄ K sequence is
included in the optimal oligopeptide which may be
phosphorylated by PKCs [53]. On the other hand,
PKC may also recognize Ser ⁄ Thr in the sequence, as
PKC recognizes preferentially substrates with a basic
residue at position )3 [53]. KAT1 comprises 10 R ⁄ K-
X-X-S ⁄ T motifs in the cytosolic N- and C-terminal
regions. Among them, T308 and S641 are matching
both motifs S ⁄ T-X-R ⁄ K as well as R-X-X-S ⁄ T (Figs 3
and 4).
Interestingly, in a recent study, residue T308 was
also identified as a target site for the Ca
2+
-indepen-
dent ABA-activated SnRK2.6 kinase [21]. In addition,
the SnRK2.6 kinase could phosphorylate T306 in an
in vitro kinase assay. This evidence suggests that multi-
ple protein kinases may participate in the regulation
of KAT1 channel activity to respond to various
Table 1. Percentage of current inhibition by PKC activation. Current
inhibition of KAT1 and its mutants by PKC activation at 32 min after
PMA (mean ± SEM, n = 3–6). Replacement of T303 by Ala or Asp
led to a loss of KAT1 activity.
Mutant Inhibition (%) n
T22A 43.2 ± 7.2 4
S44A 36.9 ± 3.3 4
T45A 27.1 ± 2.9 4
a

S125A 34.3 ± 5.6 3
T308A 20.6 ± 3.2 5
a
S312A 17.0 ± 6.5 3
a
T458A 52.5 ± 2.3 4
S529A 44.5 ± 0.7 3
S589A 28.4 ± 6.0 3
a
S590A 23.9 ± 5.0 3
a
S641A 24.9 ± 6.6 3
a
T308A ⁄ S312A ⁄ S589A ⁄ S590A ⁄ S641A 14.9 ± 5.2 4
a
WT 45.0 ± 5.6 3
Control 10.6 ± 4.5 3
WT + calphostin C 19.5 ± 4.8 4
a
P < 0.05.
K
+
KAT1 inhibition
45
308
589
590
641
S
S

S
T
T
S
Phosphorylation
306
303
T
T
312
Fig. 5. Possible regulation of KAT1 channel activity by phosphoryla-
tion via Ca
2+
-dependent ⁄ independent pathways. The target sites of
PKC (CDPK) and ABA-activated SnRK2.6 identified in heterologous
expression systems are indicated. T306 and T308 were possible
target sites for the Ca
2+
-independent, ABA-activated SnRK2.6
kinase [21]. T45, T308, S312, S589, S590 and S641 were possible
targets for PKC in Xenopus oocytes performed in this study.
Replacement of T303 by Ala or Asp led to a loss of KAT1 activity.
A. Sato et al. Phosphorylation of KAT1 channel
FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS 2323
physiological signals (Fig. 5). Indeed, the conversion of
T306 to Ala or Asp resulted in a loss of KAT1 activity
in Xenopus oocyte and yeast expression systems [21].
In addition, after the replacement of T303 by Asp, no
K
+

transport activity could be measured in oocytes.
The C-terminal region after the last transmembrane
region of S6 in plant K
+
channels is involved in chan-
nel gating [56,65,66]. If the residue at position 303 can
be recognized as a phosphorylation target site, the
modification of T303 may lead to a loss of K
+
trans-
port activity.
Although, in this study, we took advantage of a sig-
naling pathway in Xenopus oocytes to stimulate (by
PMA treatment), an animal-specific PKC, the results
obtained may have implications on signaling in plants.
The application of PMA to plant tissues has been
shown to alter the expression level of some genes
[67–70]. This fact may indicate that, in plants, similar
signaling pathways exist which connect the application
of phorbol esters to the activation of certain kinases
analogous to PKC. This is in line with other observa-
tions. Inositol 1,4,5-trisphosphate (InsP
3
) and Ca
2+
induce stomatal closure [71]. InsP
3
does not affect out-
wardly rectifying K
+

channels [72,73], but inhibits
only inwardly rectifying K
+
channels [72]. DAG is
rapidly converted to PAs by DAG kinases in plants
[41], and carrot CDPK, DcCPK1, and maize CDPK,
ZmCK11, are activated by PAs and Ca
2+
[48,74].
The phosphoinositide-dependent protein kinase-1 spe-
cifically binds PA [75]. In guard cell protoplasts, PA
inhibits the activity of inwardly rectifying K
+
channels and also induces stomatal closure and inhib-
its stomatal opening [76]. Through the serial trans-
duction pathway, the end result is the alteration of
the activity of inwardly rectifying K
+
channels by
phosphorylation.
It has been reported that exogenously supple-
mented animal protein kinase A greatly retards the
rundown rate of KAT1 [77,78]. In the same studies,
it was also shown that PKC application was not
effective in preventing rundown. This result is in
line with our study demonstrating that PKC appli-
cation has the inverse effect, and may be different
from the phosphorylation mechanism involved in
rundown.
Taken together, our results suggest the existence of

several PKC phosphorylation sites in the cytosolic
region of KAT1. K
+
channel modulation, e.g. K
+
-
uptake channel inhibition during stomatal closure,
may occur via protein kinases which have PKC-
like characteristics, such as CDPKs. Phospholipid
signaling may be involved in the preceding signaling
cascades.
Materials and methods
Channel expression in oocytes and electrical
recordings
The cDNAs encoding full-length KAT1 WT or its variants
were amplified by a two-step PCR using HindIII site-con-
taining sense primer and BamHI site-containing antisense
primer (Table S1, see Supporting information) [49]. The
HindIII-BamHI DNA fragments were ligated into the same
sites of a modified pYES2 vector (Invitrogen, Carlsbad,
CA, USA) for expression in oocytes and yeast [79]. Capped
cRNAs were synthesized in vitro from NotI-linearized plas-
mids using an in vitro transcription kit (Ambion, Austin,
TX, USA). Xenopus oocytes were defolliculated using colla-
genase and microinjected with either 1 or 2 ng of cRNAs
after a 1–3 day incubation in Barth’s buffer containing
88 mm NaCl, 1 mm KCl, 0.41 mm CaCl
2
, 0.33 mm
Ca(NO

3
)
2
,1mm MgSO
4
, 2.4 mm NaHCO
3
,5mm Hepes
and 50 lgÆmL
)1
gentamicin sulfate (pH 7.3) at 18 °C. The
two-electrode voltage clamp experiments were performed
using a voltage clamp amplifier (AxoClamp 2B, Axon
Instruments, Foster City, CA, USA) at room temperature
in Xenopus laevis oocytes [49]. Microelectrodes contained
3 m KCl with a resistance of 0.3–1.0 MX. The bath solu-
tion was 120 mm KCl, 1 mm MgCl
2
,1mm CaCl
2
and
10 mm Hepes (pH 7.3). Time-dependent changes in current
were recorded at –150 mV in single-step pulses every 30 s
and in step voltage pulses ()30 to )170 mV with a 20 mV
decrement) every 5.5 min. Step voltage pulses were applied
from a holding potential of –40 mV, the duration of
each pulse being 500 ms. Data acquisition and analysis
were performed using pclamp 9.2 (Molecular Devices,
Sunnyvale, CA, USA) and origin 5.0 software (Axon
Instruments).

Drug treatment and application
PMA (Alexis Biochemicals, Lausen, Switzerland) and
calphostin C (Alexis Biochemicals) were dissolved in dim-
ethylsulfoxide as stocks and mixed with the recording solu-
tion, reaching the final concentrations indicated in the
figures [50]. PMA was applied to oocytes after initial
measurements in its absence had been carried out.
Acknowledgements
This work was supported in part by Grants-in-Aid for
Scientific Research (17078005, 20246044 and 20-08103
to N.U.) from MEXT Japan Ministry of Education,
Culture, Sports, Science & Technology and JSPS
(Japan Society for the Promotion of Science) as well as
by the JSPS-CNR (National Research Council of
Italy) Bilateral Program to N.U. and F.G.
Phosphorylation of KAT1 channel A. Sato et al.
2324 FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS
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Supporting information
The following supplementary material is available:
Table S1. Primer sequences for plasmid construction.
This supplementary material can be found in the
online version of this article.
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2328 FEBS Journal 277 (2010) 2318–2328 ª 2010 The Authors Journal compilation ª 2010 FEBS