(Received for publication, March 13, 1995; and in revised form, July 7, 1995)
From the
The Arabidopsis thaliana cDNA, KAT1, encodes a
hyperpolarization-activated K channel. In the present
study, we utilized a combination of random site-directed mutagenesis,
genetic screening in a potassium uptake-deficient yeast strain, and
electrophysiological analysis in Xenopus oocytes to identify
strong modifications in cation selectivity of the inward rectifying
K
channel KAT1. Threonine at position 256 was replaced
by 11 other amino acid residues. Six of these mutated KAT1 cDNAs complemented a K
uptake-deficient yeast
strain at low concentrations of potassium. Among these, two mutants
(T256D and T256G) showed a sensitivity of yeast growth toward high
ammonium concentrations and a dramatic increase in current amplitudes
of rubidium and ammonium ions relative to K
by
39-72-fold. These single site mutations gave rise to
Rb
- and NH
-selective channels with
Rb
and NH
currents that were approximately 10-13-fold greater in
amplitude than K
currents, whereas the NH
to K
current
amplitude ratio of wild type KAT1 was 0.28. This strong conversion in
cation specificity without loss of general selectivity exceeds those
reported for other mutations in the pore domain of voltage-dependent
K
channels. Yeast growth was greatly impaired by
sodium in two other mutants at this site (T256E and T256Q), which were
blocked by millimolar sodium (K = 1.1 mM for
T256E), although the wild type channel was not blocked by 110 mM sodium. Interestingly, the ability of yeast to grow in the
presence of toxic cations correlated to biophysical properties of KAT1 mutants, illustrating the potential for qualitative
K
channel mutant selection in yeast. These data
suggest that the size of the side chain of the amino acid at position
256 in KAT1 is important for enabling cation permeation and that this
site plays a crucial role in determining the cation selectivity of
hyperpolarization-activated potassium channels.
Hyperpolarization-activated potassium channels in plants provide
a mechanism for membrane potential control and low affinity potassium
uptake in specific cell types, such as guard
cells(1, 2, 3) , aleurone layer
cells(4) , mesophyll cells(5, 6) , suspension
cells(7) , and root hair cells (8) (for more complete
references see (9) ). The proton-extruding ATPase in plants
creates a sufficiently negative plasma membrane potential to drive
passive low affinity potassium uptake via these noninactivating
potassium-selective channels. Recently, complementation of K uptake-deficient yeast mutants and molecular cloning studies have
led to the isolation of three Arabidopsis potassium channel
clones: KAT1(10) , AKT1(11) , and AKT2. (
)By heterologous expression in Xenopus oocytes, KAT1 was characterized as a hyperpolarization-activated
(inward rectifying) potassium channel that displays the hallmark
macroscopic properties of inward rectifying K
channels
in plant cells(13) . Patch clamp studies in KAT1-expressing
yeast have recently shown that yeast can also be used for
electrophysiological analysis of KAT1(14) . Interestingly, KAT1, AKT1, and AKT2 show structural
similarities to the superfamily of depolarization-activated (outward
rectifying) potassium channels, including 6 putative membrane-spanning
segments, an amphipathic S4 domain, and a highly conserved pore domain
(called P or H5 domain) (see Fig. 1A). The lack of
cytosolic Mg
block(15, 16) , the
voltage- and time-dependence of KAT1(13, 16) , and
hyperpolarization-induced activation in chimeras of KAT1 and outward
rectifying K
channels (17) together strongly
suggest that KAT1 activation occurs via an intrinsic gating mechanism.
Figure 1:
A,
diagram of putative membrane-spanning domains of the KAT1 polypeptide.
Threonine (T) at position 256 was mutated. Dimensions of the
H5 domain and other regions were selected for illustrative purposes
only. NB indicates a consensus sequence for a nucleotide
binding domain. B, complementation of the K uptake-deficient yeast mutant (S. cerevisiae) strain 9.3
with KAT1 wild type (Wt) and mutants at amino acid
position 256 (A, D, E, F, G, L, P, Q, R, S,
and W). Yeast was grown in arginine-based medium supplemented
with 0.16 mM KCl, which prohibited growth of the
nontransformed control line(10) . The single letter code amino
acid mutations stand for the following mutations: A, T256A; D, T256D; E, T256E; F, T256F; G,
T256G; L, T256L; P, T256P; Q, T256Q; R, T256R; S, T256S; W,
T256W.
The K selectivity is believed to be determined by
the amino acid sequence of the pore region of the
channel(10, 11, 18, 19, 20) .
In the pore domain, a Gly-Tyr-Gly sequence and several
surrounding amino acids in KAT1 are highly conserved in inward and
outward rectifying potassium
channels(18, 19, 20) . The ability to select
functional mutants in the Arabidopsis inward rectifying
potassium channel, KAT1, using complementation of K
uptake-deficient yeast strains has been recently indicated to
provide a powerful approach for studying determinants of the
selectivity filter domain(21) . Functional mutations in the GYG
sequence that change sensitivity to Na
and lose
K
specificity could be identified using yeast
selection, indicating that the structural constraints on the GYG motif
for maintaining cation specificity are stringent and can be studied
using this approach(21) . (
)
In the present study,
we pursued random site-directed mutagenesis using completely degenerate
oligonucleotides at the nucleotides encoding the amino acid at position
256 (see Fig. 1A) to determine the role of this site
for cation selectivity. This amino acid has been proposed to interact
with the GYG motif (positions 262-264 in KAT1) (23) and
may therefore influence the selectivity of K channels.
Mutant selection in yeast combined with biophysical analysis of inward
rectifying potassium channels in oocytes, as pursued here, suggests an
important role for the length and nature of the amino acid side chain
at position 256 in KAT1 for cation specificity. Furthermore, altered
growth rates of yeast transformed with mutant channels in the presence
of toxic cations demonstrate the crucial importance of the selectivity
of these K
uptake channels for cell growth.
PCR was
performed in 100-µl volumes in a thermal cycler (Ericomp, San
Diego, CA). 50 ng of DNA template was used in reactions containing 50
mM KCl, 100 mM Tris-HCl, pH 8.3, 1.5 mM MgCl, 0.1% glycerol (w/v), 0.20 mM of each
deoxynucleotide triphosphate, 1 µM of each primer, and 2.5
units of Taq polymerase. Amplification was performed in 30
cycles as follows: 2 min at 94 °C, 2 min at 50 °C, and 2 min at
72 °C. Reaction products were analyzed on 1% agarose gels.
Figure 2:
Growth inhibition of the S. cerevisiae 9.3 line containing wild type KAT1 (Wt) and the
T256D (D) and T256G (G) mutants by addition of 50
mM NH to the arginine-based
medium supplemented with 0.16 mM KCl.
The
ion conduction properties and selectivities for Kversus Li
, Na
,
Rb
, Cs
, and NH
of the mutant channels
were analyzed in Xenopus oocytes. In uninjected control
oocytes, NH
produced an
increase in linear time-independent background currents (Fig. 3A). The NH
-induced current in
uninjected oocytes resembled an increased leakage current under the
imposed conditions (Fig. 3A) and was completely
eliminated when current recordings were performed with P/6 leak
subtraction (see ``Experimental Procedures'') (Fig. 3B), as had been previously found(13) . NH
-induced background
currents in oocytes therefore did not affect the analysis of wild type
and mutant K
channel-mediated NH
currents(13) ,
contrary to a recent hypothesis(14) . The value for the current
amplitude ratio for NH
to
K
of the wild type KAT1 channel reported here (0.28; Table 1) is consistent with previous results (0.30; (13) ).
Figure 3:
NH-induced
background currents in uninjected control Xenopus oocytes are
eliminated by P/6 leak subtraction (see ``Experimental
Procedures''). Currents were recorded by two-electrode voltage
clamp by stepping the membrane potential from a holding potential of
-40 mV to potentials ranging from +30 mV to -130 mV in
-20-mV increments. Data from one representative oocyte are shown;
it was bathed in 115 mM K
(A and B, top) and 115 mMNH
(A and B, middle). Currents were recorded without leak subtraction (A) and with P/6 leak subtraction (B). The bottom
plots in A and B show the average current-voltage
curves of currents recorded from 15 oocytes without (A) and
with (B) leak subtraction in K
and NH
. Error bars denote
S.D.
In oocytes injected with K channel
messenger RNA, the six mutants that confer K
uptake in
yeast (Fig. 1B) all showed typical
hyperpolarization-activated (inward rectifying) K
currents ( Fig. 4and Fig. 6, and data not shown).
All functional mutants were analyzed with respect to alkali metal
cation selectivity. Relative current amplitudes of each cation were
analyzed (Table 1) rather than permeability ratios because it is
likely that net cation transport or block determine growth phenotypes
in yeast (see ``Discussion''), which are not reflected by
zero current-derived permeability ratios(39) . Interestingly,
the two NH
-sensitive
mutants T256D and T256G (Fig. 2) showed a striking
55-72-fold increase in the Rb
current amplitude
relative to K
( Fig. 4and Table 1). These
mutants also showed increased NH
currents 39-40-fold relative to K
currents
( Fig. 4and Table 1). These data are consistent with the
increased NH
sensitivity
of these mutations (Fig. 2). The voltage and time dependence of
the T256D and T256G mutants showed variations. The rate of activation
in T256G was slower than that of wild type (Fig. 4C).
Importantly, the selectivity of the T256D and T256G mutants was not
significantly changed for K
over Na
,
Cs
, and Li
(Table 1). A
conductance sequence of Rb
NH
> K
Na
Li
Cs
for T256D and T256G was estimated from the data in Table 1.
Note that the T256Q and T256E mutants also showed an increased
sensitivity to 50 mMNH
in yeast. Furthermore, the T256Q mutant showed a time-dependent NH
block of K
currents, suggesting open channel block, which may contribute to
the yeast phenotype (data not shown).
Figure 4:
Cation
selectivity of hyperpolarization-induced currents in Xenopus oocytes injected with cRNAs for wild type KAT1 (A) and the mutants T256D (B) and T256G (C). Currents recorded from oocytes in 115 mM
K (top row), 115 mM Rb
in the absence of K
(second row from
top), and 115 mMNH
solution in the absence of K
(third row from
top). Current voltage curves obtained from the illustrated records
are shown at the bottom.
Figure 6:
Hyperpolarization-activated currents from Xenopus oocytes injected with cRNAs for wild type KAT1 (A) and the mutants T256E (B) and T256Q (C). Currents were obtained in solutions containing 30 mM K (top) and 30 mM K
and 85 mM Na
(bottom).
Figure 5: Sodium-induced growth inhibition of the S. cerevisiae 9.3 line expressing wild type KAT1 (Wt) and the mutants T256E (E) and T256Q (Q). Sodium (100 mM) was added to the arginine-based medium supplemented with 0.16 mM KCl.
Voltage clamp data in the absence of
K in the bath suggested that the T256E and T256Q
mutants were not significantly different in Na
conductivity when compared with the wild type KAT1 channel (Table 1). To determine whether Na
blocked any
of the mutant K
channels, voltage clamp recordings
were performed in the simultaneous presence of Na
and
K
. Initially, the solution bathing oocytes contained
30 mM K
(Fig. 6, top traces in A,
B, C). Oocytes were subsequently exposed to a bath solution
containing 30 mM K
and 85 mM
Na
(Fig. 6, bottom traces in A, B, and C). In wild type, T256A, T256D,
T256G, and T256S, Na
did not block K
permeation under these conditions (Fig. 6A and
data not shown). However, K
currents elicited by the
T256E and T256Q mutants, which conferred Na
sensitivity in yeast (Fig. 5), were inhibited by
Na
(Fig. 6B and 6C). In bath
solutions with 5 mM K
and 110 mM Na
, oocytes also showed increased Na
inhibition of K
currents for the T256E and T256Q
mutants (data not shown). The concentration for half-maximal block in
T256E was 1.1 mM Na
in the 5 mM K
bath solution (n = 5). These
data indicate that Na
blocks the T256E channels at low
millimolar Na
, whereas the wild type channels are not
significantly inhibited at 100-fold higher (110 mM)
Na
levels.
Complementation of K uptake-deficient yeast
mutants has led to the cloning of channels and transporters from higher
plants that confer K
uptake(10, 11, 26) . We have used this
approach to screen for functional mutants of inward rectifying
K
channels that confer K
uptake (Fig. 1B) or show altered cation sensitivities ( Fig. 2and 5). The amino acid sequence TXXTXGYG
(amino acid positions 257-264 in the KAT1 sequence), which is
thought to contribute to the ion selectivity filter, is conserved in
the center of the H5 region of voltage-dependent K
channels including all three Arabidopsis clones(10, 11)
and three animal
inward rectifying K
channels with only two predicted
membrane-spanning domains(33, 34, 35) . The
narrowest part of the pore in tetrameric channels has been suggested to
be formed by the tyrosine in all four subunits(20) . The
threonine at position 256 in KAT1 immediately preceding the conserved
consensus sequence TXXTXGYG has been suggested to be
juxtaposed to the GYG motif(23) . The amino acid Thr-256 is
conserved in the other Arabidopsis K
channels, AKT1 (11) and AKT2.
Sequence
alignments of amino acids in the H5 regions of outward rectifying
K
channels show that the amino acid position
equivalent to 256 in KAT1 is occupied by threonine, isoleucine, valine,
or glutamic acid(23) .
Among the 11 residues analyzed in
this study substituting Thr-256, 5 residues (Phe, Leu, Pro, Arg, and
Trp) inhibited growth of the potassium uptake-deficient yeast strain.
All of these 5 residues have bulky side chains. In addition, arginine
possesses a positive charge at neutral local pH. These side chains are
likely to obstruct the channel pore or otherwise change the tertiary
structure. Taglialatela and Brown (23) reported that the
replacement of isoleucine with leucine in the Shaker outward rectifying
potassium channel at the position corresponding to 256 in KAT1 resulted
in complete loss of function. Our mutant T256L in this present report
is in agreement with their results (Fig. 1B). Selection
of mutants by analyzing growth of yeast colonies allowed identification
of functional K channel mutants. The amino acids of
the 6 functional mutants (Ala, Asp, Glu, Gly, Gln, and Ser), which
conferred K
uptake in yeast, are smaller or slightly
larger in size when compared with threonine in wild type KAT1.
If the channel
consists of a symmetrical tetramer, a single residue substitution would
produce a 4-fold symmetrical change in the channel. A consistent
observation was that large bulky amino acid side chains prohibited
function, whereas smaller side chains allowed K permeation, indicating a possible limitation in side chain size
for K
channel function. Note however that a
substitution of threonine by serine, for instance, would enlarge the
pore diameter by up to the equivalent of two methyl groups, but the
T256S mutant did not show any strong changes in relative cation
selectivities (Table 1). Steric hindrance arguments therefore do
not suffice for explaining extreme differences in cationic
selectivities of the T256D and T256G mutants. These findings are
consistent with models that involve a combination of structural
constraints, hydration energies, and ionic charge densities
contributing to the mechanism for ionic selectivity (12, 20, 38, 39) .
The reasons for the
discrepancy in the NH current amplitude between KAT1 expressed in oocytes ( (13) and this report) and patch clamp studies in yeast (14) require further analysis; the endogenous voltage-dependent NH
currents in yeast (14) , the NH
-induced cytosolic
acidification(22) , the reduction in KAT1 currents by cytosolic
acidification(16) , or other unknown factors may contribute to
reduced NH
currents in
yeast(14) . Furthermore, apparent differences in
half-activation potentials of KAT1 in yeast and oocytes used oocyte
KAT1 currents for analysis that did not come near to
saturation(14) .
High sodium concentrations did not interact
significantly with the wild type KAT1 channel, indicating that the
structure of this particular K channel may not account
for effects of Na
on K
uptake in
plant cells(30, 31, 32) . Note, however, that
other inward rectifying plant K
channels exist that
may interact with Na
(6) . Na
impaired the growth of yeast expressing T256E, T256Q, T256D, and
T256G. In oocytes, Na
inhibition of K
currents was detected in the T256E and T256Q mutants when an
external solution containing 30 mM K
and 85
mM Na
(or 5 mM K
and 110 mM Na
) was applied to the
oocytes. This was not observed in the T256D and T256G mutants,
indicating that further biophysical parameters may lead to yeast
phenotypes. In the wild type also no significant Na
inhibition of K
currents was observed. These
results suggest that Na
blocks the pore of T256E and
T256Q mutants, thereby inhibiting K
uptake in yeast.
Although the side chains of glutamine and glutamate are slightly larger
than that of threonine, the lack of significant Na
block in wild type and the other mutants suggests a more specific
effect of T256E or T256Q mutation on K
channel
structure that allows Na
block. For example these
mutations might lead to partial but insufficient dehydration of
Na
in the pore, resulting in Na
block.
In summary, the present study and a recent study on the
GYG motif (21) demonstrate that the combination of yeast
complementation and electrophysiological recording in oocytes is a
powerful approach for identification and analysis of extreme structural
ion specificity mutants in voltage-dependent K channels. The results obtained from yeast and oocyte experiments
indicate that strong inversions in the current amplitude ratios of NH
and Rb
to K
can be obtained by single mutations at
position 256 without loss of specificity toward other alkali metal
ions. The results suggest that the residue at this position plays an
important role in determining the selectivity filter-forming structure
of voltage-dependent K
channels.