From the Julius-von-Sachs-Institut für Biowissenschaften, Lehrstuhl Botanik I, Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany
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ABSTRACT |
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Acid-induced potassium uptake through
K+ channels is a prerequisite for stomatal opening.
Our previous studies identified a pore histidine as a major component
of the acid activation mechanism of the potato guard cell
K+ channel KST1 (1). Although this histidine is highly
conserved among all plant K+ uptake channels cloned so far,
the pH-dependent gating of the Arabidopsis
thaliana guard cell K+ channel KAT1 was not affected
by mutations of this histidine. In both channels, KST1 and KAT1,
aspartate mutants in the K+ channel consensus sequence GYGD
adjacent to the histidine (KST1-D269N and KAT1-D265N) were inhibited by
a rise in the extracellular proton concentration. pH changes affected
the half-maximal activation voltage V1/2 of the
KST1 mutant, whereas in the mutant channel KAT1-D265N an acid-induced
decrease in the maximum conductance gmax
indicated the presence of a proton block. In contrast to the wild type
KST1, the S4-mutant channel KST1-R181Q exhibited an activation upon
alcalization of the extracellular solution. From our
electrophysiological studies on channel mutants with respect to the
pore histidine as well as the aspartate, we conclude that the common
proton-supported shift in the voltage dependence of KST1 and KAT1 is
based on distinct molecular elements.
In the stomatal complex, acid-activated K+ uptake into
guard cells through inward rectifying K+ channels
(Kin+ channels) is fundamental for the
turgor-driven volume changes in guard cells (2-5). Extracellular
protons shift the voltage dependence of the hyperpolarization-activated
Kin+ channels in guard cells to more
positive voltages and thereby facilitate K+ uptake. When
expressed in Xenopus oocytes, the guard cell K+
channel All plant Kin+ channel With regard to the proposed structure for the six-transmembrane
K+ channels (23) KST1 contains only two extracellular
histidines. Both residues are key amino acids of the pH-sensing
structure (1). While the double mutant KST1-H160A/H271A completely
lacks pH-dependent gating, a single mutation to arginine at
position 271 even resulted in an inverted pH dependence. Because this
histidine is highly conserved in plant K+ channels only, it
was predicted that acid activation based on the pore histidine
represents a plant-specific feature. To test this hypothesis we studied
another guard cell Kin+ channel, KAT1,
with respect to the structural basis for its acid activation.
Histidine, aspartate, and glutamate mutants were generated in the plant
K+ channel consensus sequence GYGDXH and
electrophysiologically characterized in comparison to the wild type
after expression in Xenopus oocytes.
Generation of Channel Mutants--
KST1 and KAT1 single
histidine and glutamate mutants were generated as described previously
(1, 24). For the generation of aspartate mutants, site-directed
mutagenesis (QuikChangeTM Site-directed Mutagenesis
kit, Stratagene, Heidelberg, Germany) with primers
5'-ACCGGTTATGGAAACTTGCATGCTGAG-3' (KST1-D269N) and 5'-CCACGGGATATGGAAATTTTCATGCTGAGAACCC-3' (KAT1-D265N) was
performed on plasmids pKST1#8 and pKAT1 in the pGEMHE vector (25). All modifications were verified by DNA sequence analysis (ABI
PrismTM dRhodamine Terminator Cycle Sequencing Ready
Reaction kit, Perkin-Elmer).
Electrophysiology--
cRNAs of wild type and mutant channels
were generated by in vitro transcription (T7-Megascript kit,
Ambion Inc.) and injected in oocytes of Xenopus laevis
(Nasco, Fort Atkinson, WI) using a General Valve Picospritzer II
microinjector (Fairfield, NJ). Two to six days after injection
voltage-clamp recordings were performed with a Turbotec-01C amplifier
(npi Instruments, Tamm, Germany). The electrodes were filled with 3 M KCl and had typical input resistances of 2-8 M Biophysical Analysis--
To determine the half-maximal
activation voltage V1/2 in dependence of the
extracellular pH, relative open probabilities were deduced from double
voltage-step experiments. Following activation pulses of 1.5-5 s in
duration in the range of +20 mV to
The TEA+ block was described by the following equation:
The Role of Histidines for pH Sensing in KAT1--
To prove the
proposed plant-specific mechanism for acid activation in K+
uptake channels (1), the pH dependence of KAT1 wild type and channel
mutants with respect to the conserved pore histidine was studied after
expression in Xenopus oocytes. In double-electrode voltage-clamp experiments the hyperpolarization-induced K+
currents through KAT1 reversibly increased upon a pH drop from 7.4 to
5.6 of the extracellular solution (Fig.
1A and Ref. 6). As shown for
KST1 this acid activation was accompanied by a shift of the
half-maximal activation voltage V1/2 to more
positive voltages (Fig. 1B and Ref. 1). In contrast to KST1,
however, in the KAT1 channel mutants KAT1-H267A and KAT1-H267R the pH
dependence of V1/2 remained unaffected (not shown
and Fig. 1C). This might indicate a distinct geometry of the
two channel proteins with respect to the histidine residue. To verify
the predicted position of this conserved histidine in the pore region
of both channels (16), its requirement for the channel block by
TEA+ was analyzed. Whereas the wild type channels KST1 and
KAT1 were blocked by extracellular TEA+ in a
concentration-dependent manner (Km of 4 mM and 2 mM, respectively, Fig.
2, A and C), the
replacement of the histidine in the mutant channels KST1-H271A and
KAT1-H267A caused a loss of TEA+ sensitivity (Fig. 2,
B and C). Thus these residues are very likely located in the outer pore. With respect to the putative structure of
the six-transmembrane K+ channels (23) the second histidine
between the transmembrane helices S3 and S4, which was shown to play a
role in pH sensing of KST1 (1), is not found in KAT1. Because
additional histidines were not located on the putative extracellular
face of the membrane in KAT1, the tested pH range was extended to pH
4.5 to screen for more acidic residues like aspartate and glutamate,
which could account for acid activation in KAT1.
Mutations in the Pore Aspartate Affect the pH
Dependence--
While in KST1 a change of the external pH from 5.6 to
4.5 mediated only a slight shift in the half-maximal activation voltage V1/2 ( A Molecular Link between pH and Voltage Sensor in
KST1--
So far all mutant channels characterized by a striking pH
phenotype affected the voltage dependence of the potato inward
rectifier KST1 (Fig. 3 and Ref. 1). Thus, an interaction between the pH-sensing structure and the putative voltage sensor S4 was tested. To
identify interaction sites in S4, the KST1 channel mutant R181Q, which
was shifted in the half-activation 90 mV more positive than the wild
type (21), was studied regarding its pH sensitivity. When the
extracellular pH dropped from 6.5 to 5.6, the steady-state current
decreased (Fig. 4A). In the pH
range between pH 5.0 and pH 8.0, the half-maximal activation voltages
V1/2 were determined at five different pH values.
The inverted pH phenotype of mutant channel KST1-R181Q compared with
KST1 wild type derived from a shift of V1/2 to more
negative voltages upon protonation (Fig. 4B). Thus, this
arginine residue might represent an element within the link between
voltage sensor and pH sensor.
The use of site-directed mutagenesis and
electrophysiological characterization of the generated channel mutants
enabled the identification of structural elements essential for the
K+ selectivity, susceptibility toward blockers, voltage
dependence, and pH regulation of plant K+ channels (1, 20,
21, 24, 26, 27). In this study, we examined the molecular basis for
acid activation of the two guard cell K+ uptake channel
The K+ channel blocker TEA+ has been used
successfully to localize amino acid residues of the outward rectifying
Shaker K+ channel. Thereby MacKinnon and Yellen
(32) identified a threonine residue at position 449 in
Shaker mediating TEA+ blockade. This position
corresponds to the position of the pore histidine in KAT1 and KST1. In
blocking studies with extracellular TEA+, we could show
that substitution of the histidine by alanine resulted in an almost
TEA+-insensitive phenotype (Fig. 2). In homology to the
Shaker-type channels, the requirement of the histidine for
the TEA+ block of KAT1 and KST1 indicates its localization
in the outer pore of both guard cell channel proteins. The same
conclusion has been drawn from Cs+ and TEA+
inhibition experiments on the KAT1 double-mutant KAT1-H267T/E269V (33).
Our results indicate that the elimination of the histidine residue
might be sufficient to explain the reduced TEA+ sensitivity
of this double mutant. Furthermore the identical selectivity of KST1
wild type and the channel mutant
KST1-H271A4 suggests that the
constrained geometry of the selectivity filter (16) is not affected by
an adjacent single mutation.
The KAT1 and KST1 In conclusion, we could show that the proton-triggered shift in the
voltage dependence of KAT1 and KST1 is based on distinct amino acids.
Whereas in KST1 the histidine as well as the aspartate play a crucial
role in pH sensing, both residues do not contribute to the
pH-dependent gating in KAT1. In future experiments, chimera between plant K+ channels with altered pH phenotype will
help to identify in detail the distinct pH-sensitive domains and key
residues therein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunits KST1 and KAT1 maintained a pH dependence in the
absence of the biochemical machinery of the plant motor cell (1, 6-9).
Thus, the activation of these K+ uptake channels, upon a
rise in the apoplastic proton concentration, requires a
protein-intrinsic pH sensor.
-subunits
cloned so far are structurally related to the Shaker gene
family of outward rectifying K+ channels and to
Shaker-like hyperpolarization-activated
K+-permeable channels (7, 10-15). A common structure of
the pore region and the ion-conducting pathway for tetrameric
K+ channels is predicted from x-ray studies on the
Streptomyces lividans K+ channel KcsA (16). This
structure includes the two membrane-spanning helices of each subunit
adjacent to the pore loop (S5-S6 in Shaker-type channels).
In contrast to KcsA four additional transmembrane segments (S1-S4) for
the Shaker-type and the plant
Kin+ channels are proposed from
hydrophobicity analyses. Mutations in the positively charged
transmembrane helix S4 revealed that this segment represents part of
the voltage sensor of the six-transmembrane channels (17-22).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
.
Solutions were composed of 30 mM KCl, 2 mM
MgCl2, 1 mM CaCl2, 10 mM Mes/Tris1 or
Tris/Mes for pH values between 5.2 and 6.5 or between 6.8 and 8.4, respectively. pH 4.5 was adjusted with 10 mM citrate/Tris. pH-dependent currents of the aspartate mutants were
recorded in the absence of Ca2+. TEA+ blocking
experiments were performed at pH 5.6, and TEA+ was added to
a final concentration of 0.1, 0.5, 1, 5, 10, and 20 mM,
respectively. The ionic strength was balanced with choline chloride.
All solutions were adjusted to a final osmolality of 215-235 mosmol/kg
with D-sorbitol.
150 mV, inward currents relaxed
during the second pulse to
70 mV. Through extrapolation of the
relaxation time course to t = 0 the
I0-V relationship was obtained.
I0(V) is proportional to the open
probability of the channel at the end of the activation pulse. The data
were fitted by a single Boltzman distribution to obtain the
half-maximal activation voltages V1/2 and the
maximal conductances gmax. To determine the
relationship of V1/2 and pH we used the following
equation, which has previously been described in detail (1):
Here VS denotes the slope factor, which
is correlated to the gating charge, and
V
(Eq. 1)
1/2 corresponds to the half-maximal activation voltage of the completely deprotonated channel.
Kc and Ko denote the
reaction constants of the protonation reaction of the closed and open
channel, respectively.
Here [B] denotes the blocker concentration, and
Km indicates the half-maximal blocking concentration
(f = 0.5).
(Eq. 2)
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
pH dependence of KAT1 wild type and histidine
mutants. A, in Xenopus oocytes expressing
kat1 a 3-s voltage pulse to 150 mV from a holding voltage
of
20 mV elicited large K+ inward currents in 30 mM external K+ at the indicated pH values.
B, pH-dependent shift of the half-maximal
activation voltage V1/2 in both guard cell
K+ uptake channels KST1 (closed circles) and
KAT1 (open circles). Note the pronounced shift of
V1/2 between pH 5.6 and 4.5 in KAT1. C,
the mutant channel KAT1-H267R (open squares) closely
resembled the pH sensitivity of V1/2 of the wild
type KAT1 (closed squares). In B and C
the data points represent the means ± S.E. of at least three
measurements (for determination of V1/2 values see
"Experimental Procedures"). The solid lines are best
fits according to Equation 1.
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Fig. 2.
TEA+ blockade of the wild type
channels KST1 as well as KAT1 depended on the pore histidine.
A, upon hyperpolarization of the oocyte membrane to 150 mV
both guard cell K+ channels mediated pronounced inward
currents. In both channels KST1 (left traces, 5-s pulses)
and KAT1 (right traces, 3-s pulses) 5 mM
extracellular TEA+ significantly blocked K+
uptake. B, reduced inhibition of the mutant channels
KST1-H271A (left traces) and KAT1-H267A (right
traces) by external TEA+ compared with the wild type,
respectively. C, for various external TEA+
concentrations (0.1, 0.5, 1, 5, 10, and 20 mM) the blocking
percentages in relation to the steady-state current in zero
TEA+ are shown. The data points (KST1, n = 4; KST1-H271A, n = 4; KAT1, n = 3;
KAT1-H267A, n = 5) were fitted according Equation 2.
Whereas the half-maximal TEA+ blocking concentration
derived to 4 and 2 mM for KST1 (left side,
closed circles) and KAT1 (right side,
closed squares), respectively, 20 mM
TEA+ blocked the mutant channels KST1-H271A (left
side, open circles) and KAT1-H267A (right
side, open squares) less than 50%.
8 mV), the increased proton concentration
resulted in a positive shift of about 17 mV in KAT1 wild type (c.f.
Fig. 1B). To determine whether acidic amino acids in the
vicinity of the pore histidine create the strong pH sensitivity of KAT1
compared with KST1 in this pH range, we mutated the aspartate adjacent
to the histidine. In both channel mutants, KST1-D269N and KAT1-D265N,
the K+ uptake decreased upon extracellular acidification
(Fig. 3A). The K+
currents through KST1-D269N were already diminished at pH 8.0 and
almost totally suppressed at pH 7.4. In line with the histidine mutant
KST1-H271R (1), this inverted pH dependence compared with KST1 wild
type was due to a shift of V1/2 to more negative
voltages (Fig. 3B), leaving the maximum conductance gmax unchanged. In KAT1, however, channel
activity of the mutant KAT1-D265N was decreased at pH values
7.0 only. When the external pH changed from pH 7.4 to 7.0 the
steady-state K+ currents at
150 mV were diminished in
amplitude by 60.0 ± 0.5% (n = 3). Because the
current decline was accompanied by a change in
gmax
(
gmax(pH7.4
pH7.0) =
0.278 ± 0.035, n = 3) rather than a significant shift in the
half-maximal activation voltage V1/2 (Fig.
3B, unpaired t test (pH 7.0 and 7.4),
p value = 0.148), these results indicated a
proton block of the KAT1 mutant channel KAT1-D265N. The
carboxylate mutants KAT1-E273A and KAT1-E273R did not express
functional K+ channels in Xenopus oocytes (not
shown).
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Fig. 3.
pH sensitivity of aspartate mutants in KST1
and KAT1. A, in double-electrode voltage-clamp
experiments both mutants, KST1-D269N (left traces) and
KAT1-D265N (right traces), revealed
hyperpolarization-activated K+ inward currents with an
inverted pH dependence compared with the wild type channels. From the
holding voltage of 20 mV the membrane voltage was changed from 10 mV
to
140 mV (KST1-D269N) or
150 mV (KAT1-D265N), respectively, in
steps of 10 mV at the indicated pH values. B, steep increase
of the half-maximal activation voltage V1/2 with
decreasing proton concentration in the mutant channel KST1-D269N
(closed circles, n = 4), and pH
insensitivity of V1/2 for KAT1-D265N (open
circles, n = 3).
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Fig. 4.
Interaction of pH and voltage sensor.
A, upon acidification of the external solution from pH 6.5 to 5.6 K+ uptake at a membrane voltage of 150 mV was
reduced in the mutant channel KST1-R181Q. B, in contrast to
the KST1 wild type channel (closed circles,
n = 5), KST1-R181Q (closed triangles,
n = 4) revealed an inverted pH dependence of the
half-maximal activation voltage V1/2. Data points
were plotted as the means ± S.E., and solid lines
represent best fits according to Equation 1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunits KAT1 and KST1. The steady-state currents of both channels
increase upon acidification of the extracellular solution. A comparable
pH dependence was observed in another potato Kin+ channel, SKT1, and in the maize
channel ZMK1, which both represent members of the AKT1 family
(15).2 Acid activation of
KST1 and KAT1 is mediated by a shift of the half-maximal activation
voltage to more positive voltages with decreasing extracellular pH
(Fig. 1B and Refs. 1, 6, and 9). This
pH-dependent shift has been shown to depend on the only two
extracellular histidine residues in KST1 (1). In view of the
conservation of this pore histidine in all cloned
Kin+ channels, we supposed a
plant-specific pH-sensing structure. Because in KAT1 mutations of the
corresponding histidine at position 267, H267A and H267R, did not
affect the pH-dependent gating properties (Fig.
1C), the acid-induced voltage shift seems not just based on
this histidine. Evidence for the existence of additional sites is
further supported by our recent studies on the Arabidopsis thaliana K+ channel AKT3, which contains this pore
histidine, too, but is blocked by
protons.3 In contrast to all
plant K+ uptake channels but in line with the animal
Kin+ channels (30, 31) in AKT3
extracellular protons reduce the single channel conductance rather than
the voltage dependence.
-subunits are expressed in guard cells and share
the basic features of the in vivo characterized
GCKC1in channels (guard cell
K+ channel 1,
inward rectifying) from A. thaliana and
Solanum tuberosum, respectively (4, 7, 8). In contrast to
the potato channels, the GCKC1 from A. thaliana have been
shown to operate at more acidic pH values. This difference was also
observed between KAT1 and KST1 when expressed in Xenopus
oocytes (Fig. 1B) and might point to the participation of
acidic amino acids like aspartate or glutamate within the pH sensor of
KAT1. The consequently generated aspartate mutant KAT1-D265N, however,
revealed a proton block rather than an acid-induced shift in the
voltage dependence (Fig. 3B). Probably the same blocking
mechanism underlies the inverted pH dependence of the KAT1 mutant Y263R
in the GYGD consensus sequence (27). Mutations in the two pore
glutamates of KAT1 either did not affect the pH sensitivity
(KAT1-H267T/E269V; Ref. 33) or did not produce functional
K+ channels (KAT1-E273A or KAT1-E273R; data not shown).
Thus, the importance of the aspartate at position 265 for pH sensing is unique with respect to carboxylate residues within the KAT1 pore. In
KST1, however, in addition to the histidine we identified the pore
aspartate as a key amino acid of the pH sensor. As described for the
histidine mutant KST1-H271R (1), the mutant channel KST1-D269N showed
an inverted pH dependence with respect to the half-maximal activation
voltage V1/2 (Fig. 3B). Because all
described histidine and the aspartate mutants changed the gating
properties of KST1 in a pH-dependent manner, a molecular
link between the pH and the voltage sensor was anticipated. The
arginine residue at position 181 in the S4 segment represents a
candidate for this interaction, because the
voltage-dependent mutant channel KST1-R181Q shifted by
about 90 mV more positive and reversed its pH dependence. Studies on
the topology of the KAT1 channel following expression in
Escherichia coli show an interaction of amino acids in the
transmembrane segments S3 and S4 like in animal channels (28, 29, 34,
35). Because the histidine at position 160 in the S3-S4 linker of KST1
is part of the pH sensor, an interaction between His160 and
Arg181 might relate the voltage and pH sensor to each
other. Because it is impossible, however, to judge from this one
mutation in S4 whether this putative connection is of minor or major
importance in coupling, further mutations are required to elucidate the
complete structural link to the pH sensor.
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ACKNOWLEDGEMENTS |
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We thank K. Neuwinger for technical assistance and D. Becker and P. Dietrich for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant 1640/11 (to R. H.).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.
To whom correspondence should be addressed. Fax: 49-931-8886158;
E-mail: hedrich{at}botanik.uni-wuerzburg.de.
2 K. Philippar, I. Fuchs, H. Lüthen, S. Hoth, C. Bauer, K. Haga, G. Thiel, G. Ljung, G. Sandberg, M. Böttger, D. Becker, and R. Hedrich, submitted for publication.
3 I. Marten, S. Hoth, R. Deeken, P. Ache, K. Ketchum, R. Hedrich, and T. Hoshi, submitted for publication.
4 S. Hoth and R. Hedrich, unpublished results.
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ABBREVIATIONS |
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The abbreviation used is: Mes, 2-N-morpholineethanesulfonic acid.
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