(Received for publication, February 15, 1995; and in revised form, May 17, 1995)
From the
The Arabidopsis hyperpolarization-activated
(inward-rectifying) K Voltage-dependent K K To investigate the
molecular structures that determine the direction of rectification, we
engineered a series of chimeric constructs between a plant
inward-rectifying K The Xsha2 cDNA clone (16) was kindly provided by Dr.
Nick Spitzer (University of California at San Diego). The KAT1 cDNA
clone (3) was reisolated from a Oocytes were
isolated as described (19) and injected with 5-20 ng of
mRNA in 50 nl of water. Ionic currents after leakage subtraction were
recorded 1-5 days after injection using the two-electrode
voltage-clamp technique as described previously(14) . Membrane
currents were normally measured in a K
Figure 3:
Effect of BAPTA injection on reversal
potentials and macroscopic currents recorded from KXH5 mRNA-injected
oocytes. Currents were recorded from a KXH5-expressing oocyte before (A and B) and after (C and D) BAPTA
injection and from a control oocyte after BAPTA injection (E). A and C, inward current and relaxation tail currents
were elicited by a step to -120 mV (A) or -140 mV (C), followed by depolarizing pulses to values indicated to
the left of tail current traces. B, D, and E, macroscopic currents were elicited by voltage pulse
protocols described in the legend to Fig. 2. F,
reversal potentials are plotted as a function of external
Cl
Figure 4:
Current-voltage relationship of chimeric
channels. Macroscopic currents were recorded from KXH5-injected (A) and KXS5-injected (B) oocytes with extracellular
K
Figure 2:
Macroscopic currents recorded from
uninjected or mRNA-injected oocytes. A, KAT1; B,
Xsha2 (16) ; C, uninjected oocyte showing measurable
endogenous currents(17, 22) ; D, KXH5; E, KXS5. The membrane potential of oocytes was held at
-40 to -60 mV and stepped for 1.5 s to potentials ranging
from -160 to +60 mV in
Sequence comparison between plant K
Figure 1:
Sequence alignment of the hydrophobic
portions of the KAT1 and Xsha2 channels and the design of chimeric
constructs. A, KAT1 and Xsha2 sequences were first aligned
with two Arabidopsis K
Four reciprocal pairs of chimeric constructs were
made to fuse fragments of the KAT1 and Xsha2 channels at conserved
residues at Asp To determine the ionic
selectivity of the chimeric channels, the reversal potential was
measured by relaxation (``tail'') current analysis.
Surprisingly, both KXH5 and KXS5 tail currents reversed direction at
-18.4 ± 2.1 mV (n = 9) in a 115 mM KCl solution (Fig. 3, A and F; data not
shown). This reversal potential was closer to the Cl Since oocytes
contain endogenous Ca To determine whether
the remaining currents after BAPTA injection were carried by cations
rather than anions, we measured inward-rectifying current-voltage
relationships in bath solutions with varying cation concentrations. As
illustrated in Fig. 4, removal of external K The
chimeric channels were also permeable to other cations. As shown in Table 1, replacing K
These data show that functional chimeric channels can be
formed between a plant inward-rectifying K Unlike the wild-type KAT1
inward-rectifying K Both the
KXH5 and KXS5 chimeric channels behave similarly, indicating that the
S5 segment and part of the S5-H5 linker are not critical for
determining the hyperpolarization-induced activation, in spite of a
significantly longer S5-H5 linker in plant K
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
channel KAT1 is structurally
more similar to animal depolarization-activated (outward-rectifying)
K
channels than to animal hyperpolarization-activated
K
channels. To gain insight into the structural basis
for the opposite voltage dependences of plant inward-rectifying and
animal outward-rectifying K
channels, we constructed
recombinant chimeric channels between the hyperpolarization-activated
K
channel KAT1 and a Xenopus depolarization-activated K
channel. We report
here that two of the chimeric constructs, which contain the first third
of the KAT1 sequence, including the first four membrane-spanning
segments (S1-S4) and the linker sequence between the fourth and
fifth membrane-spanning segments, express functional channels that
retain activation by hyperpolarization, but not depolarization. These
two chimeric channels are no longer selective for K
.
The chimeras are selective for cations over anions and are permeable to
Ca
. Therefore, unlike animal
hyperpolarizationactivated K
channels, in which the
carboxyl terminus is important for inward rectification induced by
Mg
and polyamine block, the plant KAT1 channel has
its major determinants for inward rectification in the amino-terminal
region, which ends at the end of the S4-S5 linker.
channels can be divided
into at least two major classes with respect to the membrane potential
ranges that produce activation. One class belongs to the
outward-rectifying K
(K
)
(
)channels, which are activated by membrane
depolarization. This class comprises the most diverse group of
K
channels, showing many distinct biophysical and
pharmacological properties(1, 2) . Potassium channels
of this class belong to a superfamily of genes that display common
structural motifs characterized by six membrane-spanning segments
(S1-S6), a putative voltage sensor (S4 segment), and an S5-S6
linker (pore or H5 region) involved in K
permeability
and ion selectivity (reviewed in (1) and (2) ).
channels that belong to a different class are the
inward-rectifying K
(K
)
channels, which are activated by membrane hyperpolarization. Genes
coding for this class of K
channels have recently been
cloned from both plants (3, 4) and
animals(5, 6, 7) . However, the general
structures of these K
channels differ
significantly between animals and plants. Animal inward-rectifying
K
channels possess only two predicted
membrane-spanning segments (M1 and M2), which surround a consensus
region corresponding to the H5 or pore region of the
K
channels. Activation of animal
inward-rectifying K
channels is mainly mediated by the
alleviation of cytosolic Mg
and polyamine block at
hyperpolarized membrane
potentials(8, 9, 10, 11) . Guard
cells and other plant cells express time- and voltage-dependent
inward-rectifying K
channels ((12) ; for
review, see (13) ). The Arabidopsis KAT1 cDNA (3) has been shown to confer the voltage and time dependence,
the cation selectivity, and TEA
and Ba
block properties of typical plant inward-rectifying K
channels (14) . However, plant K
channels are structurally more similar to
K
channels in
animals(3, 4) . Plant K
channels have at least six putative membrane-spanning segments, a
typical voltage sensor segment (S4), and a K
-selective
pore (H5 or P) domain. Furthermore, activation of inward-rectifying
K
channels in guard cells is independent of cytosolic
Mg
(15) . It is possible that activation of
plant K
channels is mediated by intrinsic
voltage-dependent gating, similar to that of animal
K
channels.
channel (KAT1) (3) and an
animal outward-rectifying K
channel (Xsha2) (16) and expressed corresponding mRNAs in Xenopus oocytes. Our results demonstrate that the region between the N
terminus and the end of the S4-S5 linker of the KAT1 channel is
sufficient for hyperpolarization-induced activation.
YES cDNA library of Arabidopsis(17) . Chimeric channels were produced by
a recombinant polymerase chain reaction method described by
Higuchi(18) . The chimeric constructs were subcloned into the
pBluescript KS vector (Stratagene, La Jolla, CA) or into a modified
pBluescript KS vector containing a poly(A)
segment (50
A residues) downstream of the cloning sites (kindly provided by Dr. C.
Labarca, California Institute of Technology) to increase the level of
expression. All polymerase chain reaction-generated DNA fragments were
verified by DNA sequencing. Capped mRNA was transcribed from linearized
plasmids in vitro using a T7 RNA polymerase kit (Ambion Inc.,
Austin, TX). DNA sequence alignment was performed with PCGENE computer
software (IntelliGenetics Inc.) initially and then slightly modified to
maximize the alignment of identical amino acids.
solution
containing 115 mM KCl, 1 mM CaCl
, 2
mM MgCl
, 10 mM Hepes (pH 7.2), or in a
Na
solution, which was the same as the K
solution except that 115 mM KCl was replaced with 115
mM NaCl. For experiments with varying Cl
concentrations (see Fig. 3F), KCl in the
K
solution was partially or completely replaced with
K
glutamate to obtain the desired Cl
concentrations. For experiments with varying K
or Ca
concentrations (see Fig. 4), KCl
in the 115 mM K
solution was removed, and
different amounts of K
glutamate or Ca
glutamate were added. The osmotic strength of these solutions was
adjusted to 240-260 mosm/kg with mannitol. To determine the
conductance ratios of the chimeric channels to monovalent cations,
oocytes were bathed in solutions containing 1 mM
MgCl
, 90 mM mannitol, 10 mM Hepes, and a
15 mM concentration of one of the following salts: KCl, NaCl,
CsCl, LiCl, and RbCl. pH was adjusted to 7.2 with 1 N Tris-Cl.
Bath electrode potentials were kept stable by using 3 M KCl-agar electrode bridges or by maintaining the Cl
concentration constant for individual oocytes. BAPTA was prepared
as a 50 mM stock solution (pH 7.2 with KOH) and was injected
into oocytes (50 nl/oocyte) 15-60 min before recording. The final
concentration of BAPTA in oocytes was estimated to be
2.5 mM assuming a spherical oocyte volume of 1 µl.
concentration. Reversal potentials were measured
from tail current experiments as shown in A and C. Error bars indicate S.D. (n = 4 for all
points). G, normalized macroscopic currents such as those
shown in B and D were plotted as a function of the
membrane potential. Current amplitudes were measured at the end of 1.5
s of stimulation after leakage subtraction from the same oocytes before
and after BAPTA injection and normalized with respect to current levels
recorded at -140 mV before BAPTA injection. Errorbars indicate S.D. (n = five
KXH5-injected oocytes). All recordings were performed in 115 mM KCl solution with the exception of F (see
``Materials and Methods'').
concentrations in the range of 0-115 mM K
(as indicated by the symbols). Oocytes
were injected with
2.5 mM BAPTA before recording.
Currents were elicited by voltage protocols as described in the legend
to Fig. 2and measured at the end of 1.5 s of stimulation after
linear leakage subtraction.
20-mV increments. The values of
some of the voltage pulses are indicated to the right of the
corresponding current traces. Currents were recorded in either a 115
mM KCl solution (A and C-E) or a 115
mM NaCl solution (B).
channels and animal K
channels has
revealed that the two types of channels share some similarities in the
hydrophobic core
region(3, 4, 13, 17) . As
illustrated in the alignment between KAT1 and Xsha2 (Fig. 1A), these similarities are not limited to the
voltage sensor segment (S4), the K
-selective pore
region (H5), and the overall hydrophobicity profile, but are extended
to some amino acids in other membrane-spanning segments as well. For
instance, the two negatively charged residues in S2 (Asp
,
Asp
), the negatively charged aspartic acid in S3
(Asp
), the leucine residue in S5 (Leu
), and
the alanine residue in S6 (Ala
), which are all highly
conserved among animal K
channels(20) , are also conserved in plant
K
channels (Fig. 1A).
These amino acids have been hypothesized to be important in forming
ionic interactions with the positively charged residues in S4 and to
stabilize the channel conformation in the open or closed
state(21) . The conservation of these residues in both animal
K
channels and plant
K
channels suggests that the overall
membrane topology and the possible arrangements of the six
membrane-spanning segments are similar between the two types of
channels and that chimeric constructs made by joining segments of the
two types of channels at conserved residues may retain the gross
membrane topology.
channels (AKT1 and
AKT2) (4, 17) and members of four separate Drosophila K
channel families
(Shaker, Shal, Shab, and Shaw)(30, 31, 32) ,
respectively. The identical residues among all three Arabidopsis K
channels or all five animal
K
channels are shown in boldface
upper-case letters and are underlined. The conservatively
substituted residues among all three Arabidopsis K
channels or at least four animal K
channels are shown in lightface upper-case letters. The
nonconserved residues are shown in lower-case letters. Amino
acids that are identical or conservatively substituted between KAT1 and
Xsha2 are marked by verticallines and asterisks, respectively. Members of the following groups of
amino acids were considered to be conserved: (Met, Ile, Leu, Val);
(Ala, Gly); (Ser, Thr); (Gln, Asn); (Asp, Glu); and (Lys, Arg), (Phe,
Tyr, Trp). Gaps were introduced for optimal alignment and are shown as dashes. The junction points for the construction of the
recombinant chimeric channels are marked by arrows. B, schematic representation of KAT1, Xsha2, and eight of the
chimeric cDNA constructs between KAT1 and Xsha2. The openbox in the C-terminal half of KAT1 represents a potential
cyclic nucleotide-binding site(3) .
(S2 segment), Ile
(S4
segment), Leu
(S5 segment), and Trp
(H5
region) (Fig. 1, A and B). Messenger RNAs were
synthesized in vitro from these chimeric constructs. 1-5
days after injection into Xenopus oocytes, currents elicited
by both depolarizing and hyperpolarizing pulses were analyzed. Control
KAT1 mRNA induced an inward-rectifying K
current (Fig. 2A)(14) , and Xsha2 mRNA induced an
outward-rectifying K
current in Xenopus oocytes (Fig. 2B)(16, 19) . These
currents were significantly larger than endogenous currents recorded in
some batches of uninjected or water-injected oocytes (Fig. 2C) (see (19) and (22) ).
However, not all chimeric constructs induced exogenous currents.
Careful analysis of these nonfunctional chimeras (XKS2, KXS2, XKS4,
KXS4, XKS5, and XKH5) showed that they did not give rise to
voltage-dependent currents, and they also did not increase linear
leakage currents in oocytes as judged by oocyte background resistance
measurements between -120 and +20 mV (n > 100
oocytes). Five additional chimeras were constructed: two in which the
KAT1 regions from Asp
(S2) to Leu
(S5) or to
Trp
(H5) were inserted into XSha2, two in which the
equivalent regions from XSha2 were inserted into KAT1, and one in which
only the XSha2 S4/55 linker was inserted into KAT1.
(
)These chimeras were also nonfunctional. Only the
chimeric KXH5 (n = 186 oocytes) and KXS5 (n = 69 oocytes) mRNAs (Fig. 1B) consistently
induced large inward currents upon membrane hyperpolarization (Fig. 2, D and E). Upon membrane
depolarization, no outward currents that were significantly larger than
endogenous currents were detected (Fig. 2, D and E). Thus, both KXH5 and KXS5 produced inward-rectifying
currents in oocytes. Similarly, the equivalent chimera to KXH5 between
KAT1 and the Drosophila K
channel EAG showed inward-rectifying currents (data not shown).
Further analysis focused on KXS5 and KXH5 (Fig. 1B).
The current induced by KXH5 was generally larger than that induced by
KXS5, but otherwise similar to the current induced by KXS5. Both
currents were activated at membrane potentials negative to
approximately -70 mV, which is similar to the activation
potential for KAT1(14, 17) . However, the activation
and deactivation time courses for both chimeras were much slower than
those for the KAT1 channel (Fig. 2).
equilibrium potential than to the K
equilibrium
potential. Substituting K
with Na
had
a negligible effect on the reversal potential (data not shown), whereas
varying the external Cl
concentration shifted the
reversal potential for both KXH5 and KXS5 channels with a slope of
42 mV/10-fold change in external Cl
concentration (Fig. 3F, opencircles), which is indicative of channels selective for
Cl
over K
. In addition, tail
currents of both chimeric channels showed an unusually large outward
conductance and a very slow relaxation time course (Fig. 3A), which are characteristic of endogenous
Cl
channel currents observed infrequently in some
uninjected control oocyte batches (see (22) for details).
Thus, initial results indicated that the currents induced by KXH5 and
KXS5 were mostly carried by Cl
ions.
-activated Cl
channels(23) , it is likely that the observed
Cl
current may be activated by Ca
if the chimeric channels are permeable to Ca
such that activation of chimeric channels induces endogenous
Ca
-activated Cl
currents. To test
this possibility, we injected oocytes with BAPTA before recording to
chelate intracellular Ca
and thus to inhibit
Ca
-activated Cl
channels in
oocytes. As predicted, injection of BAPTA greatly reduced the current
amplitude and changed the time dependence of KXH5-injected (n = 26) and KXS5-injected (n = 9) oocytes (Fig. 3, B, D, and G). BAPTA
injections had no effect in control oocytes, which showed no endogenous
currents (n = 5) (Fig. 3E).
Furthermore, BAPTA injections completely abolished
hyperpolarization-induced Cl
currents in the
subpopulation of oocyte batches in which uninjected oocytes showed
large hyperpolarization-activated Cl
currents (n = 4) (see (22) ). The KXH5 and KXS5 currents after
BAPTA injection continued to show inward rectification, a slow
activation time course, and no activation by depolarization (Fig. 3, D and G). In addition, the typical
large and slowly relaxing Cl
tail currents (Fig. 3A) were also completely abolished by BAPTA
injection (Fig. 3C). The reversal potential after BAPTA
injection was +2.9 ± 3.5 mV for KXH5 (n =
5) (Fig. 3, C and F) and +4.2 ±
2.8 mV for KXS5 (n = 4) in 115 mM KCl
solution. These reversal potentials were not dependent on the external
Cl
concentration (Fig. 3F, closedcircles), indicating that the remaining currents were no
longer carried by Cl
ions.
abolished the remaining inward currents, while readdition of
K
(as glutamate salt to keep the Cl
concentration unchanged) recovered the inward current. In control
uninjected oocytes, no K
-dependent inward currents
were observed (n = 3). Therefore, the chimeras KXH5 and
KXS5 produced a hyperpolarization-activated conductance that was
dependent on the external K
concentration.
in the bath solution with
equimolar concentrations of Rb
, Na
,
Cs
, or Li
did not change the
amplitude of inward currents significantly in both KXH5- and
KXS5-injected oocytes. Furthermore, replacing K
in the
115 mM K
solution with Ca
also induced inward currents, which depended on the extracellular
Ca
level (KXH5, n = 3; and KXS5, n = 3). Control oocytes did not show significant inward
currents in a bath solution with 90 mM Ca
after BAPTA injection (n = 3). These results
showed that both chimeric channels encode nonselective cation channels,
which are activated by hyperpolarization, but not depolarization.
channel and
an animal outward-rectifying K
channel. The two
functional chimeric channels retain hyperpolarization-induced
activation while losing depolarization-induced activation. Based on the
sequence composition of the two functional chimeras, the region that is
responsible for inward rectification is located in the first third of
the plant KAT1 channel, which includes the first four putative
membrane-spanning segments and the S4-S5 linker. This result is in
marked contrast to results obtained by chimeric analysis and
site-directed mutagenesis in animal inward-rectifying K
channels, in which the C terminus and a single charged amino acid
in the last putative membrane-spanning segment (M2) play a major role
in determining Mg
and polyamine block-dependent
inward
rectification(9, 10, 24, 25) . This
regional difference indicates different mechanisms for the generation
of inward rectification between plant and animal inward-rectifying
K
channels. Plant inward-rectifying K
channels usually display a steep voltage-dependent activation
(for review, see (13) ). This voltage dependence is not induced
by Mg
block(15) . The sequence and the
predicted structural similarities between plant
K
channels and animal
K
channels and the presence of a typical
S4 segment in plant K
channels (3, 4) (Fig. 1A) further suggest that
the activation of plant K
channels may be
induced by intrinsic voltagedependent gating, a process similar to the
gating of animal K
channels(26) . The S4 segment of plant
K
channels may respond to membrane
hyperpolarization by ``moving'' inwardly across the membrane
electrical field and in turn open the channel. This hypothesis would be
consistent with our results.
channel, the two chimeric channels
both display a much slower activation time course and lose K
selectivity. These results are not surprising because several
regions of voltage-dependent ion channels affect pore function and
activation kinetics. Furthermore, removal of the N- and C-terminal
regions of a delayed-rectifying K
channel (drk) have
been shown to produce a large increase in channel activation times and
inactivation times, similar to those found here, while retaining
depolarization-induced activation(27) . It is possible that
functional regions may not be completely compatible with each other if
they are derived from different sources as in our plant-animal chimeric
channels, which likely leads to nonfunctional chimeras, indicating a
limitation. Furthermore, the two functional chimeric channels may have
an aberrant structure, which results in changes in activation kinetics
and loss of K
selectivity while retaining activation
by hyperpolarization and cation over anion selectivity.
channels when compared with animal K
channels (Fig. 1A). In addition, the lack of
outward rectification in both chimeric channels suggests that the
structural component that links S4 movement to channel opening is
located in the region from the N terminus to the beginning of the S5
domain of the KAT1 channel. Durell and Guy (21) have proposed,
based on experimental and computational data, that the small S4-S5
linker region in animal K
channels may
function as a channel gate. Site-directed mutagenesis studies in animal
K
channels have also shown that the
S4-S5 linker contributes to formation of the inner mouth of
K
channels(28, 29) . It
is worth noting that the S4-S5 linker sequences of plant
K
channels are very different from those
of animal K
channels (Fig. 1A). Plant S4-S5 linkers are shorter and, most
noticeably, have two conserved negatively charged residues at the
beginning (Glu
and Asp
) and a positively
charged residue (Arg
) at the end, which are absent in
animal S4-S5 linkers (Fig. 1A). These charge
differences would imply structurally distinct gates that may be
relevant to the opposite voltage-dependent activation of plant
K
channels and animal
K
channels. More specific domain-swap
and site-directed mutagenesis analyses may allow further definition of
regions or amino acids that are important for the difference between
hyperpolarization- and depolarizationinduced activation.
, outward-rectifying;
K
, inward-rectifying; BAPTA,
1,2-bis(2-aminophenoxy)ethane-N, N, N`, N`-tetraacetic acid.
We thank Dr. Nick Spitzer for the Xsha2 cDNA clone,
Dr. C. Labarca for the pBS-KSAMV-pA50 vector, and
Audrey Ichida for comments on the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.