©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Strong Modifications in Cation Selectivity in an Arabidopsis Inward Rectifying Potassium Channel by Mutant Selection in Yeast (*)

(Received for publication, March 13, 1995; and in revised form, July 7, 1995)

Nobuyuki Uozumi (§) Walter Gassmann Yongwei Cao Julian I. Schroeder (¶)

From the Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0116

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(4)-selective channels with Rb and NH(4) currents that were approximately 10-13-fold greater in amplitude than K currents, whereas the NH(4) 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.


INTRODUCTION

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. (^1)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) .^1 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) . (^2)

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.


EXPERIMENTAL PROCEDURES

Plasmid Manipulations and Polymerase Chain Reaction

DNA manipulations were performed essentially as described by Sambrook et al.(24) . The KAT1 cDNA was reisolated by screening an Arabidopsis YES cDNA library.^1 The KAT1 expression plasmid was modified to generate unique silent EcoRI and XbaI sites on opposite sides of the pore coding region (H5) of KAT1. KAT1 was subcloned into a modified pYES2 expression vector (Invitrogen Inc., San Diego, CA) in which the EcoRI and XbaI sites in the polylinker were removed by ligation into HindIII and NotI sites in the vector, resulting in KAT1 expression under control of the GAL1 promoter and T7 promoter and a unique EcoRI site proximal to the H5 region at amino acid position 166. To generate a silent XbaI site in KAT1, the nucleotide A at nucleotide position 963 was substituted by a T. A mutagenesis procedure based on polymerase chain reaction (PCR) (^3)was used as described elsewhere(25) . Two PCR reactions for the first cycle were performed. The first reaction was performed with a T3 primer and the oligonucleotide 5`-CAGAGTTTGCTTCTAGAAATCAACT-3` at nucleotide positions 950-974, and the other reaction was performed with a T7 primer and 5`-GTTGATTTCTAGAAGCAAACTCTG-3` at complementary nucleotide positions 973-950. The products of each reaction were subjected to agarose gel electrophoresis and extracted from the gel. The mixture of the extracted PCR products and T3 and T7 primers were used for the second cycle reaction. The full-length PCR product was subsequently digested with SphI and cloned into two flanking SphI sites in KAT1. The DNA sequence between the SphI sites on the plasmid was confirmed by DNA sequencing. To introduce random mutations at amino acid position 256, we applied the same procedure as mentioned above by using two sets of oligonucleotide primers containing 3 inosines at the nucleotides that encode amino acid 256 (5`-GAAGTTACGAGAAACTTG-3` at 1155-1135, 5`-CTTTACTGGTCCATTIIIACATTAACGACCACG-3` at 751-783, (5`-ACCATTTCAGCCACTAAGCCTC-3` at 438-459, and 5`-CGTGGTCGTTAATGTIIIAATGGACCAGTAAAG-3` at 783-751).

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(2), 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.

Yeast Complementation

The Saccharomyces cerevisiae strain 9.3 (MATa, trk1(d), trk2(d), ena1(d)::HIS3::ena4(d), leu2, ura3, trp1, ade2), a kind gift of Dr. A. Rodríguez-Navarro, (Universidad Polytecnica, Madrid, Spain) was transformed as described previously(26) . Transformants were first selected on YNB medium (0.6% yeast nitrogen base) supplemented with 100 mM KCl, 2% glucose, and all nutrients with the exception of uracil. For selection of functional mutants, arginine-based medium was prepared as described elsewhere with some modifications(27) : 10 mML-arginine base, 1 mM MgSO(4), 0.1 mM CaCl(2), 0.16 or 1 mM KCl, 20 mg/liter adenine, 40 mg/liter leucine, 20 mg/liter histidine, 30 mg/liter tryptophan, 30 mg/liter lysine, 0.1% trace mineral solution, 1% vitamin stock solution, 1.5% agarose, 2% sucrose, 2% galactose, and phosphoric acid to adjust the pH to 6.5. When required, NaCl or NH(4)Cl were added as indicated in the text.

Recordings in Xenopus Oocytes

Messenger RNAs were synthesized in vitro, and Xenopus laevis oocytes were isolated, injected, and incubated as described previously(13) . Recordings of K currents were performed in a solution that was composed of 115 mM KCl, 1.8 mM CaCl(2), 1.0 mM MgCl(2), and 10 mM Hepes with Tris to adjust the pH to 7.3, and mannitol was added to raise the osmolality to 250 mosmol/kg. For Na, Li, Rb, NH(4), and Cs solutions, KCl was replaced with NaCl, LiCl, RbCl, NH(4)Cl, or CsCl, respectively. When other solutions were used, adequate concentrations of KCl and/or NaCl were supplemented as indicated in the text. Two electrode voltage clamp recordings, on-line data acquisition, and analysis were performed as described previously (13) . From a holding potential of -40 mV, oocytes were hyperpolarized with a series of seven stepwise voltage pulses from -60 to -150 mV in - 15-mV increments. Linear leak and capacitive currents were subtracted from the wild type and mutant traces using a depolarizing P/6 subtraction protocol(13) . Total remaining current magnitudes instantaneous plus time-dependent were analyzed for selectivity determinations(13) , which allows a model-independent analysis while also producing an unbiased larger statistical variation.


RESULTS

Mutant Selection in Yeast

Threonine at position 256 has been proposed to interact with the GYG sequence in the K-selective pore region of K channels (23) (Fig. 1A). To understand the possible role of this threonine residue in determining ion selectivity, we replaced threonine with various amino acids by random site-directed mutagenesis (see ``Experimental Procedures''). Mutant K channel cDNAs were transformed into a K uptake-deficient mutant yeast line to test whether individual site-directed mutants conferred K uptake and growth in yeast. K uptake-deficient yeast mutants do not grow on media containing low K (e.g. 0.16 mM K) but can be propagated in media containing geq7 mM K(10) . Sequence analysis of a pool of randomly mutagenized cDNAs revealed eleven mutants that had a single amino acid substitution at this location. Six mutants, which had the amino acids alanine, aspartic acid, glutamic acid, glycine, glutamine, and serine at position 256, were found to confer significant K uptake and growth of the mutant yeast strain (Fig. 1B). The other five mutants, phenylalanine, leucine, proline, arginine, and tryptophan, did not complement the K uptake-deficient yeast mutant (Fig. 1B).

Rband NHConductivity of T256D and T256G KAT1 Mutants

Yeast lines were transformed with functional K channel mutants and grown in the presence of toxic cations to identify alterations in growth rates when compared with the wild type KAT1 channel. When the mutant channels T256D and T256G were expressed in yeast, the transformed cells became more sensitive to large external (50 mM) NH(4) concentrations than yeast expressing wild type KAT1 (Fig. 2) or than the T256A and T256S mutations (data not shown). These data indicate an increase in NH(4) uptake to toxic levels in yeast or block of K uptake by NH(4) (Fig. 2).


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(4) 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(4) of the mutant channels were analyzed in Xenopus oocytes. In uninjected control oocytes, NH(4) produced an increase in linear time-independent background currents (Fig. 3A). The NH(4)-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(4)-induced background currents in oocytes therefore did not affect the analysis of wild type and mutant K channel-mediated NH(4) currents(13) , contrary to a recent hypothesis(14) . The value for the current amplitude ratio for NH(4) 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(4)-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(4) (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(4). 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(4)-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(4) currents 39-40-fold relative to K currents ( Fig. 4and Table 1). These data are consistent with the increased NH(4) 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 approx NH(4) > K Na approx Li approx 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(4) in yeast. Furthermore, the T256Q mutant showed a time-dependent NH(4) 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(4) 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).



Altered NaSensitivity of KAT1 Mutants

Data from recordings in oocytes showed that functional channel mutants displayed only small differences in the selectivity for Na, Li, or Cs, all of which showed negligible or very small relative inward conductances (Table 1) that correspond statistically to the previously reported selectivity of the wild type channel KAT1 (13, 28) (Table 1). External Na (115 mM) did however lead to small outward currents in KAT1-expressing oocytes (not shown), which may be related to the effect of low external cations on inward rectifying K channel activation, that abolishes activation in K-free external solutions (29) (for similar effects of Li see (28) ). Interaction of Na with K uptake transporters in plants is of potential significance, because Na competition with K for uptake has been proposed to contribute to detrimental effects of Na on plant growth(30, 31, 32) . From initial current amplitude determinations (Table 1), it could not be determined whether the wild type or mutant K channels are impermeable to Na or whether Na blocks these channels. To determine whether Na may interact with the wild type or mutant K channels, yeast strains transformed with K channels were grown in arginine-based medium, to which 100 mM Na was added. Two other mutant K channels, T256E and T256Q, were identified that conferred increased Na sensitivity to yeast (Fig. 5). The T256D and T256G mutants ( Fig. 3and Fig. 4) also showed an increased sensitivity to Na but the mechanisms were not further characterized here.


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.


DISCUSSION

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) ^1 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.^1 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.

Mutations That Produce Strong Alteration of Ion Selectivity and Pore Blockade

The seven functional K channels (including wild type) were assayed for alteration of yeast growth patterns in the presence of the toxic cation NH(4). The K channel mutants T256G, T256D, T256Q, and T256E rendered transformed yeast lines sensitive to NH(4). Two mutants (T256G and T256D) showed a dramatic inversion in the conductance ratios of Rb and NH(4) over K, without abolishing the selectivity of the channel for K over Na, Li, and Cs. Yool and Schwarz (36) reported a 1.9-4.7-fold increase in relative Rb and NH(4) selectivity from four kinds of outward rectifying K channel mutants that had a single amino acid mutation at different positions in the H5 region, as compared with a 39-72-fold increase in the present study. De Biasi et al.(37) reported the transformation of a K-selective channel into a Cs-permeable channel by mutating the position corresponding to 256 from an isoleucine to a histidine, but the Cs conductance was lower than the K conductance. To date, such extreme and yet ion-specific inversions in selectivity relative to K by a single point mutation as reported here have not been identified for K channels to our knowledge. Transformation of a Na channel into a Ca-selective channel by mutations is an example in which an extreme change in the cation selectivity of a voltage-dependent cation channel, while maintaining specificity, has been found(38) .

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) .

Effects of KChannel Mutants on Yeast Growth

The results of growth studies of yeast transformed with the mutants T256D and T256G showed a correlation with the electrophysiological data obtained from oocyte experiments. Excess NH(4) uptake made yeast less viable when transformed with either mutant as compared with transformation with the wild type KAT1 channel (Fig. 2). On the other hand, Rb, which is not toxic in yeast or plants, did not show any detrimental effects on yeast growth in medium containing K and Rb. Although NH(4) is an important ion that acts as a major nitrogen source for yeast, cytosolic NH(4) is kept at very low concentrations by NH(4) fixation via glutamine synthase and glutamate dehydrogenase, which alleviate toxicities resulting from excess NH(4) accumulation and the resulting cytosolic acidification(22) . The finding that NH(4) toxicity in yeast was observed suggests that uptake of NH(4) through T256D or T256G was more rapid than NH(4) fixation leading to adverse effects on cell growth. Sensitivity of yeast to NH(4) provides an assay to detect extreme changes in K channel structure, which alter NH(4) selectivity as demonstrated here.

The reasons for the discrepancy in the NH(4) 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(4) currents in yeast (14) , the NH(4)-induced cytosolic acidification(22) , the reduction in KAT1 currents by cytosolic acidification(16) , or other unknown factors may contribute to reduced NH(4) 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(4) 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.


FOOTNOTES

*
This research was supported by Department of Energy Grant DE-FG03-94-ER20148 and in part by a National Science Foundation Presidential Young Investigator Award (to J. I. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biotechnology, Faculty of Engineering, Nagoya University, Chikusa-ku, Nagoya, 464-01, Japan.

To whom correspondence should be addressed. Tel.: 619-534-7759; Fax: 619-534-7108.

(^1)
Y. Cao, J. M. Ward, W. B. Kelly, A. M. Ichida, R. F. Gaber, N. Uozumi, J. I. Schroeder, and N. M. Crawford,(1995) Plant Physiol. (Bethesda), in press.

(^2)
R. L. Nakamura and R. F. Gaber, submitted for publication.

(^3)
The abbreviation used is: PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Alonso Rodríguez-Navarro for providing the 9.3 yeast line and Walter Kelly, Audrey Ichida, Martin Schwarz, and Francisco Rubio for discussions and comments on the manuscript.


REFERENCES

  1. Schroeder, J. I., Raschke, K., and Neher, E. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,4108-4112 [Abstract]
  2. Schroeder, J. I. (1988) J. Gen. Physiol. 92,667-683 [Abstract]
  3. Fairley-Grenot, F. A., and Assmann, S. M. (1993) Planta 189,410-419
  4. Bush, D. S., Hedrich, R., Schroeder, J. I., and Jones, R. L. (1988) Planta 176,368-377
  5. Colombo, R., and Cerana, R. (1991) Plant Physiol. (Bethesda) 97,1130-1135
  6. Kourie, J., and Goldsmith, M. H. M. (1992) Plant Physiol. 98,1087-1097
  7. VanDujn, B., Ypey, D., and Libbenga, K. (1993) Plant Physiol. 101,81-88 [Abstract/Free Full Text]
  8. Gassmann, W., and Schroeder, J. I. (1994) Plant Physiol. 105,1399-1408 [Abstract/Free Full Text]
  9. Schroeder, J. I., Ward, J. M., and Gassmann, W. (1994) Annu. Rev. Biophys. Biomol. Struct. 23,441-471 [CrossRef][Medline] [Order article via Infotrieve]
  10. Anderson, J. A., Huprikar, S. S., Kochian, L. V., Lucas, W. J., and Gaber, R. F. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,3736-3740 [Abstract]
  11. Sentenac, H., Bonneaud, N., Minet, M., Lacroute, F., Salmon, J., Gaymard, F., and Grignon, C. (1992) Science 256,663-665 [Medline] [Order article via Infotrieve]
  12. Pongs, O. (1993) J. Membr. Biol. 136,1-8 [Medline] [Order article via Infotrieve]
  13. Schachtman, D. P., Schroeder, J. I., Lucas, W. J., Anderson, J. A., and Gaber, R. F. (1992) Science 258,1654-1658 [Medline] [Order article via Infotrieve]
  14. Bertl, A., Anderson, J. A., Slayman, C. L., and Gaber, R. F. (1995) Proc. Natl. Acad. Sci. U. S. A. 92,2701-2705 [Abstract]
  15. Schroeder, J. I. (1995) FEBS Lett. 363,157-160 [CrossRef][Medline] [Order article via Infotrieve]
  16. Hoshi, T. (1995) J. Gen. Physiol. 105,309-328 [Abstract]
  17. Cao, Y., Crawford, N. M., and Schroeder, J. I. (1995) J. Biol. Chem. 270,17697-17701 [Abstract/Free Full Text]
  18. Jan, L. Y., and Jan, Y. N. (1992) Annu. Rev. Physiol. 54,537-555 [CrossRef][Medline] [Order article via Infotrieve]
  19. Heginbotham, L., Abramson, T., and MacKinnon, R. (1992) Science 258,1152-1155 [Medline] [Order article via Infotrieve]
  20. Pongs, O. (1992) Physiol. Rev. 72,69-88
  21. Anderson, J. A., Nakamura, R. L., and Gaber, R. F. (1995) Soc. Exp. Biol.Symp. 48,85-95
  22. Lee, R. B., and Ratcliffe, R. G. (1991) Planta 183,359-367
  23. Taglialatela, M., and Brown, A. M. (1994) News Physiol. Sci. 9,169-173 [Abstract/Free Full Text]
  24. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  25. Higuchi, R. (1990) in PCR Protocols: A Guide to Methods and Applications (Innis, M. S., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds) pp. 177-183, Academic Press, Inc., Orlando, FL
  26. Schachtman, D. P., and Schroeder, J. I. (1994) Nature 370,655-658 [CrossRef][Medline] [Order article via Infotrieve]
  27. Rodríguez-Navarro, A., and Ramos, J. (1984) J. Bacteriol. 159,940-945 [Medline] [Order article via Infotrieve]
  28. Very, A.-A., Gaymard, F., Bosseux, C., Sentenac, H., and Thibaud, J.-B. (1995) Plant J. 7,321-332 [CrossRef][Medline] [Order article via Infotrieve]
  29. Schroeder, J. I., and Fang, H. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,11583-11587 [Abstract]
  30. Rains, D. W., and Epstein, E. (1965) Science 148,1611
  31. Rains, D. W., and Epstein, E. (1967) Plant Physiol. (Bethesda) 42,319-323
  32. Rains, D. W., and Epstein, E. (1967) Plant Physiol. (Bethesda) 42,314-318
  33. Ho, K., Nichols, C., Lederer, W. J., Lytton, J., Vassilev, P. M., Kanazirska, M. V., and Hebert, S. C. (1993) Nature 362,31-38 [CrossRef][Medline] [Order article via Infotrieve]
  34. Kubo, Y., Baldwin, T. J., Jan, Y. N., and Jan, L. Y. (1993) Nature 362,127-133 [CrossRef][Medline] [Order article via Infotrieve]
  35. Kubo, Y., Reuveny, E., Slesinger, P., Jan, Y., and Jan, L. (1993) Nature 364,802-806 [CrossRef][Medline] [Order article via Infotrieve]
  36. Yool, A. J., and Schwarz, T. L. (1991) Nature 349,700-704 [CrossRef][Medline] [Order article via Infotrieve]
  37. De Biasi, M. D., Drewe, J. A., Kirsch, G. E., and Brown, A. M. (1993) Biophys. J. 65,1235-1242 [Abstract]
  38. Heinemann, S., Terlau, H., Stuhmer, W., Imoto, K., and Numa, S. (1992) Nature 356,441-443 [CrossRef][Medline] [Order article via Infotrieve]
  39. Hille, B. (1992) Ionic Channels of Excitable Membranes , Sinauer Associates, Inc., Sunderland, MA

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.