(Received for publication, September 4, 1996)
From the Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208
Among the highly conserved sites in K+ channel pores, the tyrosine-glycine sequence is believed to play an important role in selectivity. Here we describe a novel approach in which comprehensive mutagenesis of the YG sites of the voltage-gated K+ channel, Kat1, is combined with phenotypic screening in Saccharomyces cerevisiae and electrophysiological analysis in Xenopus oocytes to determine the roles of these sites in K+ selectivity. We show that structural constraints necessitate a tyrosine or phenylalanine at the first position to confer full K+ selectivity. Substitution to arginine creates a channel titratable by external pH, suggesting that the side group at this position may line the channel pore. Permeation is abolished by any increase in bulk at the adjacent glycine position unless accompanied by a compensatory mutation at the tyrosine site. These results suggest a model in which the selectivity filter of the K+ channel requires an aromatic residue paired with glycine within the pore loop in order to maintain maximal K+ selectivity.
Potassium uptake in Saccharomyces cerevisiae is mediated by two putative potassium transporters encoded by the TRK1 and TRK2 genes (1, 2). Cells in which both genes are deleted exhibit a 1000-fold increase in the requirement of potassium in the medium to sustain normal growth compared with wild-type cells (2). The phenotype of trk mutants facilitated cloning of Arabidopsis K+ channel cDNAs, KAT1 and AKT1, by their ability to mediate K+ uptake and thus restore yeast cell growth on potassium-limiting media (3, 4).
Kat1 and Akt1 are founding members of a class of voltage-gated inward
rectifying K+ channels that harbor the six-transmembrane
architecture of the Shaker superfamily of channels (5). Particularly
noteworthy are the amphipathic helix (S4), thought to constitute the
voltage sensor (6), and, between the S5 and S6, the conserved pore region containing the tyrosine-glycine signature sequence found in most K+-selective channels (Fig.
1).
Although structurally closer to Shaker than to Irk1 or RomK1, Kat1 was shown to be an inward rectifying K+ channel by its ability to elicit slowly activating, hyperpolarization-dependent, K+-selective currents when expressed in Xenopus oocytes (14). Similar results were obtained from recordings of KAT1 expressed in S. cerevisiae (15). The electrophysiological properties of Kat1 are characteristic of the inward rectifying K+ channels described in the guard cells of higher plants (16, 17), and KAT1 promoter-driven expression of a glucoronidase reporter gene in transgenic Arabidopsis was shown to occur only within guard cells of young plants (18). Thus, KAT1 likely encodes a guard cell-specific inward rectifying K+ channel associated with stomatal opening and closing.
The ion selectivity of Kat1 is typical of a K+-selective
channel with monovalent cation permeability following the order
K+ > NH4+ > Rb+ Na+ ~ Li+ ~ Cs+ (14, 19). Like most K+ channels, Kat1
contains within its pore loop the highly conserved glycine-tyrosine-glycine
(Gly262-Tyr263-Gly264) triplet,
which has been shown to be intimately involved with ion selectivity
(20, 21, 22). Recent investigations of K+ channel structure
using cysteine substitution and modification, toxin binding, and mutant
cycle analysis (23, 24, 25, 26, 27) support a model in which an hour glass-shaped
pore is formed from wide external and internal vestibules, which
converge rapidly to a short, narrow pore. Pascual et al.
(24) have proposed that the
Gly375-Tyr376-Gly377 triplet of the
Kv2.1 channel forms the narrowest region, because cysteine mutants at
the adjacent residues Val374 and Asp378 are
accessible to probes from the inside and outside of the membrane, respectively. Ranganathan et al. (26) further specified that the highly conserved Tyr445 of the Shaker channel
(analogous to Tyr263 of Kat1) is centrally located within
the pore and likely interacts directly with potassium ions.
We previously described how the expression of an inward rectifying K+ channel in S. cerevisiae can be used as a model system to identify and analyze mutations that alter the ability of the channel to discriminate between physiological ions (28). This system offers the ability to screen large numbers of mutant channels for changes in ion selectivity observed as an increase in sensitivity of the yeast cells to potentially toxic ions such as sodium. Uozumi et al. (29) have recently used this strategy to identify several mutations within the pore region of Kat1 that alter ion selectivity of the channel. Here, we show that each of the 400 possible amino acid sequences at the highly conserved tyrosine-glycine sequence of the K+ channel pore can be generated and tested in a combinatorial fashion to assess the role of these sites in ion selectivity.
The results of this comprehensive mutagenesis screen revealed
extremely stringent structural requirements for the YG region of the
K+ channel pore. Single mutations at Gly264
never produced functional channels in S. cerevisiae.
Furthermore, although many amino acid substitutions at
Tyr263 retain channel function, only a single mutation, Tyr
Phe, retained wild-type levels of selectivity for K+
over other physiological ions.
The KAT1 coding
sequence was cloned into the multicopy yeast expression vector pYES2
(Invitrogen) under the control of the galactose-inducible promoter
GAL1. The resulting plasmid was modified to include a
poly(A) tail at the 3-end of the gene. To facilitate subcloning of
mutant pore region fragments, unique HindIII and BglII sites that did not alter the amino acid coding
sequence of Kat1 were introduced at nucleotide positions 726 and 1239, respectively. A degenerate primer was designed such that the codons corresponding to amino acids Tyr263 and Gly264
were changed to NN(G/T). This combination of 32 codons is sufficient to
encode all 20 amino acids. Polymerase chain reaction was used to create
the degenerate fragments, which were cloned into the HindIII-BglII sites. 14,500 individual
Escherichia coli transformants were obtained in generating
the YG library. This was sufficient to ensure a 14-fold redundancy for
representing each of the possible 1024 combinations of the YG codons in
the library.
Plasmid DNAs from 46 bacterial transformants were isolated at random and sequenced to verify the quality of the library. Mutations to all amino acid codons were found at both positions 263 and 264. Each of these clones contained guanine or thymine at the third position of the amino acid codons, corresponding to Tyr263 and Gly264, consistent with the degenerate primer used to generate the library. The frequencies of the expected and observed ratios of each amino acid codon are shown in Table I.
|
Additional site-directed mutations were generated using a recombinant polymerase chain reaction approach (30). Mutant polymerase chain reaction fragments were cloned into the unique HindIII-BglII sites.
Media and StrainsThe transport characteristics of the
wild-type (strain R757) and K+ uptake-defective (strain
CY162, trk1 trk2
) strains of S. cerevisiae used in this study have been previously described (1, 2). Yeast
transformations were performed by electroporation (31), and the
Ura+ transformants were plated on yeast nitrogen-based
media without uracil, supplemented with the required amino acids, 100 mM KCl, and 1 M sorbitol. After several days of
growth at 30 °C, colonies that developed were replica-plated to
various test media. These media all contained 2% galactose and 2%
sucrose for induction of the GAL1 promoter. Yeast
nitrogen-based medium supplemented with the required amino acids but
lacking uracil, pH 5.9, contained 7 mM KCl. Low salt medium
was made as described previously to 0.2 mM KCl (1, 32).
Some media were supplemented with NaCl to 400 or 700 mM.
Low salt media were made essentially free of ammonium. Plasmids were
isolated from yeast (33) and transformed into E. coli.
Plasmid DNA was prepared and sequenced by dideoxy methods (34) (U.S.
Biochemical Corp.). Plasmids were retransformed into S. cerevisiae to confirm phenotypes.
Media used to analyze the pH sensitivity of the RG mutant (see Fig. 5)
were low salt, containing 1 mM potassium. These media were
buffered with 20 mM Tris base and 20 mM
MES,1 which maintained the pH of the media
over several days of growth when tested with pH paper.
Electrophysiology
RNA from KAT1 and mutant KAT1 cDNAs was synthesized using mMessage mMachine capped RNA transcription kit (Ambion Inc.). Oocytes were isolated from Xenopus laevis (Nasco), defolliculated, injected with RNA, and incubated at 18 °C in modified Barth's solution containing 96 mM KCl (35). Between 2.5 and 25 ng of RNA was injected, depending on the expression or activity level of the channel. Recordings were made 1-4 days after RNA injection using an oocyte clamp OC-725B amplifier (Warner Instruments), filtered at 1 kHz (Frequency Devices 902). Data were recorded and analyzed using Axon Instruments software AxoData and AxoGraph on a Macintosh PowerPC 7100/80. Leakage currents were subtracted using a P/6 subtraction method (36).
Bath solutions contained 100 mM XCl (X = K+, Rb+, NH4+, or Na+), 1.8 mM CaCl2, 1.0 mM KHC03, 1.0 mM MgCl2, 10 mM HEPES (pH 7.4 with Tris base; solutions at low pH were prepared by the addition of HCl). At least 10 ml of bath solution was perfused during solution changes.
Conductance Ratio and Permeability Ratio CalculationsThe
amplitude of current was determined at the end of a 1-s pulse to 150
mV in the bath solutions containing potassium, rubidium, ammonium, and
sodium. Conductance ratios were determined by dividing the current
measurement of the test ions by the potassium current. All values are
means ± S.E. Reversal potentials were measured by first
activating channels with a 1-s hyperpolarizing pulse to
150 mV and
then stepping to different test potentials. Conductance measurements
for the low pH experiments were taken at the end of a 3-s pulse to
130 mV. The permeability ratio
PX/PK was determined using the
equation PX/PK = (eVrevF/RT)[K]/[X] based on the
Nernst-Planck electrodiffusion equation (37).
A yeast genetic screen was utilized to study the effects of amino
acid substitutions at the highly conserved tyrosine-glycine amino acid
pair in the pore of the Arabidopsis potassium channel Kat1.
A potassium uptake-deficient strain of S. cerevisiae (CY162, trk1 trk2
) can grow normally on media containing 100 mM K+ but exhibits slow growth on media with 7 mM K+ and virtually no growth on media
containing 0.2 mM K+ (Fig.
2A) (2). Expression of the wild-type
KAT1 allele from the galactose-inducible promoter
GAL1 confers strong growth of trk1
trk2
cells on media containing as little as 50 µM
K+ (3). In contrast, on glucose-containing medium, the
GAL1 promoter is repressed and the KAT1 cDNA
cannot suppress the trk1
trk2
phenotype.
A yeast expression library was constructed to contain all possible amino acid combinations at the Tyr263-Gly264 positions of Kat1. Mutant Kat1 channels from this YG library were first screened for those that retained function. Approximately 10,000 individual transformants of strain CY162 were allowed to develop into colonies on glucose medium containing a permissive concentration of potassium (100 mM KCl). This number of colonies was sufficient to ensure approximately a 10-fold coverage of the library combinations based on 32 nucleotide combinations at each codon. These colonies were replica-plated to galactose media containing either 7 mM or 0.2 mM K+.
Of the 10,000 potential mutants, only 230 exhibited growth on 7 mM K+, 64 of which could also grow on media
containing 0.2 mM K+. This observation
suggested that the majority of substitutions at the YG positions
produced channels incapable of suppressing the potassium
uptake-defective phenotype of trk1 trk2
cells. Plasmids from colonies capable of growing on 7 mM
K+ were isolated, prepared for DNA sequencing, and
retransformed into yeast to confirm the mutant phenotypes. Mutant Kat1
channels deemed functional by this test conferred a wide range of
growth phenotypes ranging from weak growth on 7 mM
K+ to strong growth on 0.2 mM K+.
Fig. 2A shows representative phenotypes of mutant Kat1
channels. A complete listing of mutant phenotypes is shown in Table
II. If other mutants retained function, the rate of
K+ uptake was too low to even partially suppress the
trk1
trk2
phenotype. The 7 mM cut-off
point was chosen because higher K+ concentrations result in
semipermissive growth of the trk1
trk2
recipient cells
and would thus prevent identification of weak mutants.
|
DNA sequence analysis of plasmid DNA from transformants that grew on medium containing 0.2 mM KCl identified only three amino acid combinations capable of allowing strong growth at this concentration of potassium: wild type (YG), FG, and PG (Fig. 2A). The conservative tyrosine to phenylalanine substitution conferred growth as strong as the wild-type channel on media containing 7 mM or 0.2 mM K+. The PG mutant, too, conferred strong growth on 0.2 mM K+, although growth was not quite as robust as that conferred by the wild-type or FG channel. Two other substitutions, IG and TG, sustained weaker growth on the media containing 0.2 mM K+.
The remaining mutant channels exhibited partial suppression of the
trk1 trk2
phenotype by conferring growth on medium
containing 7 mM KCl, but not on 0.2 mM KCl. DNA
sequence analysis of these mutants revealed that most amino acid
substitutions at Tyr263 did not abolish channel function.
When glycine was retained at position 264, all amino acid substitutions
at position 263 with the exception of histidine, lysine, arginine, and
tryptophan resulted in channels capable of taking up sufficient
K+ to allow growth on 7 mM K+. The
HG, KG, RG, and WG substitutions were identified during random
sequencing of the mutant library plasmids isolated from E. coli. In trk1
trk2
cells, they either did not
suppress or only very weakly suppressed the K+ uptake
deficiency under the conditions tested (data not shown; see
"pH-sensitive RG Channel").
In striking contrast to the results at Tyr263, no
mutant containing only a single amino acid substitution at
Gly264 was identified by this screen. Based on the number
of mutant channels examined and the confirmed complexity of the
library, these results strongly suggested that single amino acid
substitutions at this position cannot produce channels capable of
suppressing the potassium uptake-defective phenotype of trk1
trk2
cells. To test this, seven mutants containing amino acid
substitutions at position 264 (YC, YF, YK, YL, YP, YS, and YT) were
constructed by site-directed mutagenesis. None of these mutants were
able to suppress the low potassium phenotype of trk1
trk2
cells, providing further evidence that single point
mutations at Gly264 cannot form functional channels (data
not shown). 15 mutants isolated in the library screening contained
amino acid substitutions at both Tyr263 and
Gly264 (Table II). The mutations at Gly264 were
represented by substitutions to alanine, serine, threonine, and
asparagine. Thus, structural changes at Gly264 can be
tolerated and allow channel function, but evidently only in combination
with a compensatory mutation at Tyr263.
Selectivity of Mutant Kat1 Channels
The selectivity of each of the functional mutant Kat1 channels was
tested in two ways. First, the ability of mutant channels to suppress
the K+ requirement of trk1 trk2
cells was
tested in the presence of a competing ion, sodium. In the second test,
sodium permeation through mutant Kat1 channels was examined using a
strain of wild-type TRK1 TRK2 cells.
Although very high concentrations of extracellular sodium are toxic,
S. cerevisiae cells tolerate growth on media containing as
much as 1 M NaCl (38). Growth of trk1 trk2
cells expressing a wild-type KAT1 allele was essentially
unaffected on medium containing 7 mM KCl/400 mM
NaCl (Fig. 2A). From these observations we conclude that for
cells expressing the wild-type K+-selective channel (i)
K+ permeated the channel at levels sufficient to allow
strong growth of trk1
trk2
cells, (ii) Na+
could not permeate the channel at levels sufficient to cause toxicity
to the cell and, (iii) passage of K+ was not substantially
blocked by sodium.
The collection of mutants showed a wide range in the ability of
Na+ to inhibit rescue of the trk1 trk2
phenotype. At one extreme, the FG channel in the presence of high
sodium appeared indistinguishable from the wild type, suggesting that
it retained wild-type properties of selectivity (Fig. 2A).
Rescue by the IG and TG mutants was also quite resistant to the
presence of high sodium (Fig. 2A). These mutants are
considered to be selective, although not as selective as the wild-type
or FG channel. This class distinction became more obvious when higher
amounts of sodium were added to the media, and growth was more
inhibited compared to the wild-type channel (data not shown).
Rescue of the trk1 trk2
phenotype by the remaining
mutants was severely inhibited by the presence of sodium, suggesting a
decrease in channel selectivity. For example, mutants such as CG and
DS, which conferred strong growth on 7 mM KCl, exhibited no
growth when the same medium was supplemented with 400 mM
NaCl (Fig. 2A). Even the PG mutant, which conferred growth
nearly as strong as the wild-type channel on 0.2 mM KCl,
was unable to rescue the K+ uptake defect of trk1
trk2
cells on the sodium media. Thus, the ability of a mutant
channel to suppress the K+ requirement of trk1
trk2
cells can be independent of channel selectivity. Whether
the Na+ sensitivity of these channels was due to actual
permeation by Na+ or merely blockage or inactivation of the
pore by Na+ could not be determined by this test but was
revealed by an independent assay (see below).
Phenotypic Test for Na+ Permeation in S. cerevisiae
The inability of most mutant Kat1 channels to confer growth to
trk1 trk2
cells on media containing high levels of
sodium could be attributed to several possibilities. For example,
inhibition may be caused by blockage of potassium uptake, altered
channel gating, toxicity due to the uptake of sodium ions, or a
combination of factors that could result in inactivation of the
channel. The ability of Na+ to permeate wild-type and
mutant channel pores was assessed by expressing the channels in
S. cerevisiae cells that also expressed the TRK1
and TRK2 genes. These cells are not dependent upon
KAT1 for growth on K+-limiting media due to the
activity of the endogenous K+ transporters. Thus, cells
expressing either wild-type KAT1 or the empty vector
resulted in equally strong growth on low potassium media (Fig.
2B). Furthermore, cation uptake by
TRK1-expressing cells is highly selective for K+
over Na+; TRK1 cells expressing either wild-type
KAT1 or vector alone grew equally strongly on 2 mM KCl medium or 2 mM KCl supplemented with 700 mM NaCl (Fig. 2B). However, mutant Kat1 channels
with increased sodium permeation conferred sodium sensitivity to these cells.
Most of the mutant channels identified as nonselective based on the
ability of Na+ to inhibit their rescue of the trk1
trk2
phenotype conferred various degrees of Na+
sensitivity to wild-type cells (Table II). The most striking example
was the PG mutant, which, when expressed in TRK1 TRK2 cells
on medium supplemented with 700 mM Na+,
severely inhibited growth. Other examples of nonselective mutants inferred to permeate sodium by this test included the DS and CG channels (Fig. 2B). Thus, a major component of the sodium
sensitivity conferred to S. cerevisiae by the mutant Kat1
channels was likely through increased sodium permeability.
The FG mutant, which appeared to be as selective as the wild-type
channel when expressed in trk1 trk2
cells, did not
confer sodium sensitivity to the TRK1 TRK2 cells, suggesting
again that very little sodium permeates this channel. Of note, the TG
and IG substitutions, identified as modestly selective because of their
Na+-resistant rescue of trk1
trk2
cells,
conferred sodium sensitivity to the wild-type cells. Thus, mutations of
Tyr263 to Thr or Ile seem to confer increased permeation to
Na+ and therefore decreased selectivity of the pore.
Several channels that were identified as nonselective due to their
Na+-sensitive rescue of the trk1 trk2
phenotype seemed to have little or no effect on the growth of wild-type
cells on the high sodium medium. In these cases it is possible that the
channels were primarily blocked by, but not permeable to, sodium. A
fairly strong suppressor of the trk1
trk2
phenotype
such as the VT channel may be a likely candidate for this hypothesis.
Alternatively, if total conduction of ions through these channels is
low, even mutant channels with increased sodium-potassium permeability
might not substantially affect growth of wild-type yeast in the
presence of potentially toxic concentrations of sodium. Weaker
suppressors of the trk1
trk2
phenotype such as LG and
LA are more consistent with this explanation.
Electrophysiology of Wild-type Kat1 and Mutant Kat1 Channels
The selectivity of wild-type and mutant Kat1 channels identified from the genetic screen was also assessed by two-electrode voltage clamp analysis in Xenopus oocytes. Many of the mutant channels expressed weakly or not at all in this system. However, functional representatives of the different classes of mutants identified in the yeast screen were obtained and found to exhibit similar phenotypes when expressed in oocytes.
Channel selectivity was assessed under bi-ionic conditions when the
bath solution was substituted with the test ions potassium, sodium,
rubidium, and ammonium. Conductance ratios for the test ions were
determined relative to the potassium conductance at the end of a 1-s
pulse at a membrane potential of 150 mV. When possible, reversal
potentials were used to determine permeability ratios as predicted by
the Nernst-Planck electrodiffusion equation (37).
The potassium currents
elicited by the wild-type Kat1 channel upon hyperpolarization were
essentially identical to those previously described for this channel
(Fig. 3A). Significant
KAT1-dependent currents were detected when the
bath was switched to rubidium or ammonium (Fig. 3, A and
D). Conductance of these ions relative to potassium when the
cell was hyperpolarized to a potential of 150 mV was 18 ± 3%
and 44 ± 17% (n = 3), respectively (conductance ratios are summarized in Table III). In addition,
reversal potentials of tail currents were measured in the potassium,
rubidium, and ammonium baths and determined to be
5 ± 2 mV,
29 ± 1 mV, and
75 ± 5 mV, respectively, representing
permeability ratios of PRb+/PK+ = 39% and
PNH4+/PK+ = 6%. When the bath solution was exchanged for sodium, very little inward current was detected upon hyperpolarization (Fig.
3A). Tail current analysis in this bath solution was not
performed, since the reversal potential fell below
160 mV. Potassium
currents could be restored upon exchange back to the potassium bath
(data not shown).
|
Consistent with findings in the yeast system, only one mutant channel,
the YG to FG substitution, duplicated the selectivity properties of the
wild-type channel (Fig. 3 B). Currents measured in response
to hyperpolarization with potassium in the bath solution were larger
than those of ammonium and rubidium (Fig. 3, B and D; Table III). Sodium was highly impermeant, since no
current was detected in the sodium bath. Reversal potentials measured
in the potassium, rubidium, and ammonium baths were 2 ± 3 mV,
28 ± 3 mV, and
62 ± 10 mV, representing permeability
ratios of PRb+/PK+ = 36%
and
PNH4+/PK+ = 9%.
Based on the phenotypes
exhibited in S. cerevisiae, the TG mutant was inferred to be
highly selective for K+ over Na+ but not as
selective as the wild type or FG mutant. Expressed in oocytes, the TG
mutant elicited slowly activating potassium currents under
hyperpolarization, similar to the wild-type channel (Fig.
3C). However, currents conducted by ammonium and rubidium ions were equal or greater in magnitude to the potassium currents (Fig.
3, C and D. Conductance ratios measured at 150
mV for Rb+ and NH4+ were
130 ± 32% and 123 ± 15% (n = 4),
respectively. The reversal potential measurements taken in potassium,
rubidium, and ammonium baths were
1 ± 2 mV,
15 ± 5 mV,
and
60 ± 2 mV, respectively, representing permeability ratios
of PRb+/PK+ = 57% and PNH4+/PK+ = 10%. Despite the alteration in channel selectivity, currents were
not detected in the sodium bath, consistent with this channel being
selective for potassium over sodium. Thus, a single amino acid
substitution at this highly conserved site (Y263T) can increase the
magnitude of currents carried by Rb+ or
NH4+ relative to K+ without
a major decrease in selectivity for K+ over
Na+.
The majority of mutant Kat1
channels identified in the yeast screen were inferred to be
nonselective by inhibition of growth on media containing high sodium.
Two examples of this class of mutant channels, DS and CG, conferred
detectable currents when expressed in oocytes. The DS substitution lost
all wild-type properties of selectivity. Potassium currents conducted
by this channel were clearly smaller than either rubidium or ammonium
currents (Fig. 4, A and C).
Furthermore, sodium currents equal in magnitude to the potassium
currents were conducted by this channel (Fig. 4A). The
conductance ratios for Rb+,
NH4+, and Na+ relative to
K+ at a membrane potential of 150 mV were determined to
be 296 ± 40, 293 ± 72, and 78 ± 20%
(n = 4), respectively. The CG mutant lost potassium
selectivity properties in a similar fashion: rubidium and ammonium
conductance ratios were increased, and significant sodium currents were
detected (Fig. 4, B and C). The conductance ratios relative to potassium for the CG channel for the test ions Rb+, NH4+, and
Na+ were 80 ± 11, 76 ± 30, and 47 ± 14%
(n = 4), respectively.
Tail current analysis for the nonselective channels revealed complex
inactivation kinetics possibly due to activity of endogenous channels.
These currents may be attributed to Ca2+-activated
Cl channels similar to those observed in recordings of
chimeric channels of Kat1 and the Shaker potassium channel (39).
Another possibility is that this mutant channel can be blocked in a
voltage-dependent manner by a divalent cation, resulting in
the "hump" in the tail currents. However, we are confident that the
inward currents measured in response to hyperpolarization in the
various test baths are mostly carried by the bath cation, because the
kinetics of activation and saturation are similar to those of the
wild-type Kat1 channel.
pH-sensitive RG Channel
Mutants in which a basic amino acid occupied the
Tyr263 site did not form channels capable of rescuing
growth of trk1 trk2
cells under the conditions of the
screen (data not shown). Given that most other substitutions formed
functional channels, we suspected that potassium conduction might be
blocked by protonated side groups.
Although the growth medium used in the screen was prepared to pH 5.9, after several days of growth it became acidic (data not shown). Medium
buffered with MES and Tris-base maintained its pH after several days.
On low potassium medium buffered to pH 7.5, the RG channel conferred
strong growth of trk1 trk2
cells, suggesting that this
channel can be titrated by a shift in pH of the medium (Fig.
5A). A similar effect was seen for the RG
channel expressed in oocytes. The current amplitudes approached zero as
the pH of the bath was lowered from 7.4 to 4.7 (Fig. 5, B
and C). This result is striking because the current
amplitudes of the wild-type channel following the same pH shift
actually increase 10-fold (Fig. 5, B and C) (19,
40). We estimate that the pKa of the residue in the
RG channel responsible for this event is approximately 5, given the
50% reduction in K+ current at this pH.
We have presented the results of a combinatorial saturation
mutagenesis of two highly conserved sites,
Tyr263-Gly264, in the pore of Kat1. At these
positions, almost any substitution alters channel selectivity. Of the
400 possible amino acid combinations at these positions, only the
conservative tyrosine to phenylalanine mutation at position 263 resulted in a channel with completely wild-type permeation properties
assayed in the S. cerevisiae and Xenopus systems.
All other mutants isolated in the screen were phenotypically distinct
from cells expressing the wild-type Kat1 channel. Growth of
trk1 trk2
cells expressing these mutants on low
K+ was always weaker than growth conferred by the wild-type
channel. More importantly, on media containing high concentrations of
sodium, all of the mutant channels except FG exhibited phenotypes
strongly suggestive of a decrease in ion selectivity.
The phenotypes of Kat1 channels expressed in both the yeast and oocyte systems were similar. The YG to FG substitution, which retained all selective properties of the wild-type channel in two separate tests in yeast, also maintained wild-type selectivity when expressed in oocytes. Two channels that appeared nonselective in the yeast system, the CG and DS mutants, were shown in oocytes to have completely lost wild-type properties of ion selectivity. Both mutants were highly permeable to sodium and exhibited alterations in permeability with other test ions.
The TG substitution, which was inferred to be highly selective for
K+ over Na+ in S. cerevisiae was
impermeable to sodium when expressed in oocytes. Alterations in the
selectivity of the semipermeant ions rubidium and ammonium suggested
that the mutation caused only a minor change in the ability of the pore
to discriminate between monovalent cations. The Na+
sensitivity of wild-type TRK1 TRK2 cells expressing the TG
channel suggested that sodium, too, permeated this channel in S. cerevisiae. We believe that for this channel the apparent
difference in sodium phenotypes observed in Xenopus oocytes
and S. cerevisiae is due to a fundamental difference between
the two systems of analysis. The most extreme hyperpolarization episode
of oocytes lasted 1 s at a membrane potential of 160 mV, whereas
yeast cells are likely to maintain potentials at least this large over
periods of time several orders of magnitude longer. Thus, the S. cerevisiae system seems to be more sensitive with regard to the
ability to detect increases in uptake of Na+. A change in
channel selectivity that results in a very low velocity of
Na+ uptake may nevertheless be sufficient to confer
toxicity when uptake of the toxic ion is accumulated over the time
scale of cell division.
A recent model of the pore of the Shaker channel has positioned the
side chain at the tyrosine position away from the center of the pore
(41). However, our results indicate a strict requirement for either
tyrosine or phenylalanine, providing strong support for hypotheses that
aromatic rings form a key component of the K+ channel
selectivity filter through cation- interactions (20, 42, 43). The
solvent accessibility of the titratable group in the RG mutant further
supports this model. Although we can only speculate about the mechanism
of the pH sensitivity for this mutant, a compelling model positions the
side chain of the arginine residue into the lining of the pore
regulating potassium flux directly by the presence or absence of a
proton.
Our results also support predictions that the tyrosine residue is positioned at the narrowest region of the pore loop (24, 25, 26, 41, 42, 44). Substitutions to smaller side chains, which would be predicted to directly increase the diameter of the pore, decreased selectivity, again with the notable exception of phenylalanine. Conversely, the tyrosine substitution to the larger tryptophan did not form a functional channel, possibly due to a decreased diameter of the pore.
The ability of the genetic screen to test the effect of simultaneous mutations at both Tyr263 and Gly264 revealed a highly interdependent relationship between these sites. Single amino acid substitutions at Gly264 apparently do not result in functional channels. However, substitutions at this site can be accommodated if a second, compensatory mutation, occurs at Tyr263. Although we cannot rule out the possibility that single mutations at this site prevent expression or reduce stability of the channel, the fact that double mutations at YG sometimes result in functional channels indicates that a glycine at position 263 is not an absolute requirement for expression or stability.
We suggest two possibilities to explain why channels containing single mutations at Gly264 are nonfunctional. The increase in size of the amino acid side chain at this position could either occlude the pore directly or could cause the adjacent tyrosine residue to move further into the pore and thereby block current. Either model is consistent with the observation that, for the most part, the amino acids that can be accommodated at Gly264 (when accompanied by a change at Tyr263), are those that contain the smallest side groups, i.e. alanine, serine, and threonine. Notably, one larger side group, that of asparagine, can also be accommodated at Gly264 in combination with a proline at Tyr263. The potential of prolines to confer turns along the peptide may be necessary to compensate for the occlusion that would otherwise result from the large side group of asparagine. Interestingly, in Shaker, mutations are tolerated at the analogous glycine position (20). Nevertheless, the observation that these channels are highly nonselective is consistent with the requirement of an aromatic amino acid paired with glycine in formation of the optimally selective pore.
In apparent contradiction to our conclusions, the Y445V mutation in Shaker was deemed selective for K+ over Na+, based on a low Na+:K+ permeability ratio (<0.20) (20). However, as shown for the TG mutant, the sensitivity of the yeast system allows the identification of changes in ion selectivity that are exceedingly small or even undetectable by conventional electrophysiological analyses. The Y263V mutation of Kat1 resulted in decreased selectivity but failed to express currents in Xenopus oocytes.
Clearly, channels that are selective for potassium over sodium can be formed with nonaromatic substitutions at the tyrosine position. However, given the comprehensive analysis of this position described here and the sensitivity of the yeast growth assays, maximal potassium selectivity in Kat1 was achieved only by the tyrosine/phenylalanine-glycine pair.
We thank N. Spruston and L. Pinto for critical reading of the manuscript. We also thank R. MacKinnon and L. Heginbotham for extensive help and guidance and J. Schroeder, W. Kelly, A. Ichida, and W. Gassmann for providing invaluable training and advice with electrophysiology.