Genetic Selection of Mutations in the High Affinity K+ Transporter HKT1 That Define Functions of a Loop Site for Reduced Na+ Permeability and Increased Na+ Tolerance*

Francisco RubioDagger , Martin Schwarz§, Walter Gassmann, and Julian I. Schroederparallel

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

    ABSTRACT
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Abstract
Introduction
References

Potassium is an important macronutrient required for plant growth, whereas sodium (Na+) can be toxic at high concentrations. The wheat K+ uptake transporter HKT1 has been shown to function in yeast and oocytes as a high affinity K+-Na+ cotransporter, and as a low affinity Na+ transporter at high external Na+. A previous study showed that point mutations in HKT1, which confer enhancement of Na+ tolerance to yeast, can be isolated by genetic selection. Here we report on the isolation of mutations in new domains of HKT1 showing further large increases in Na+ tolerance. By selection in a Na+ ATPase deletion mutant of yeast that shows a high Na+ sensitivity, new HKT1 mutants at positions Gln-270 and Asn-365 were isolated. Several independent mutations were isolated at the Asn-365 site. N365S dramatically increased Na+ tolerance in yeast compared with all other HKT1 mutants. Cation uptake experiments in yeast and biophysical characterization in Xenopus oocytes showed that the mechanisms underlying the Na+ tolerance conferred by the N365S mutant were: reduced inhibition of high affinity Rb+ (K+) uptake at high Na+ concentrations, reduced low affinity Na+ uptake, and reduced Na+ to K+ content ratios in yeast. In addition, the N365S mutant could be clearly distinguished from less Na+-tolerant HKT1 mutants by a markedly decreased relative permeability for Na+ at high Na+ concentrations. The new mutations contribute to the identification of new functional domains and an amino acid in a loop domain that is involved in cation specificity of a plant high affinity K+ transporter and will be valuable for molecular analyses of Na+ transport mechanisms and stress in plants.

    INTRODUCTION
Top
Abstract
Introduction
References

Potassium is an important macronutrient required for plant growth. K+ uptake into plant root cells is generally thought to be mediated by transport systems with high and low affinities for K+ (1-5). A cDNA named HKT1 was isolated from wheat roots and was characterized as a member of high affinity K+ transport systems from plants (6). HKT1 is highly expressed in the root cortex and in cells surrounding the vasculature in leaves (6) and shows homology to the yeast plasma membrane high affinity K+ uptake transporters TRK1 and TRK2 (6, 7). HKT1 has been shown to mediate high affinity Na+-K+ symport (8), suggesting that additional high affinity K+ uptake pathways should exist in roots (9) (see "Discussion").

Detailed biophysical studies showed that HKT1 is highly selective for the alkali cations K+ and Na+ (9). When the extracellular concentrations of K+ and Na+ are similar, HKT1 functions as a Na+-coupled K+ uptake transporter. HKT1 is modelled to have a high affinity K+-coupling site with an apparent affinity for K+ of 3 µM, and a high affinity Na+-coupling site with an apparent affinity for Na+ of 175 µM (8, 9). At toxic, high millimolar, extracellular Na+ concentrations, however, Na+ competes with K+ at the high affinity K+-coupling site, leading to a block of high affinity K+ uptake; large rates of detrimental low affinity Na+ uptake are mediated via HKT1 in yeast and oocytes (8, 9). The apparent affinity of the high affinity K+-coupling site for Na+ is approximately 5 mM (8, 9). These findings suggest that HKT1 may be one of the pathways for Na+ transport across plant root and leaf membranes that is important for salt toxicity in plants.

Characterization of the molecular mechanisms underlying Na+ transport in plants and identifying structural components that allow Na+ uptake via these transporters are required for a fundamental understanding of Na+ homeostasis. It has been proposed that important Na+ entry pathways into plant cells represent K+ uptake transporters (10, 11), and multiple Na+ uptake pathways are likely to exist (12, 13). Salinization of irrigated lands in arid regions is affecting agriculture world-wide, reducing production because most crop plants are glycophytes and sensitive to high millimolar NaCl concentrations that occur in saline soils (14). Plant responses to salt stress are a complex phenomenon involving several processes such as osmoprotectant accumulation (14, 15), enzyme sensitivity to Na+ (16, 17), and ion transport across different plant membranes (14, 18-20).

Structural studies of the wheat transporter HKT1 led to the identification of point mutations located in the 6th hydrophobic domain that decrease low affinity Na+ uptake and confer Na+ tolerance to yeast (8). Here we report on the isolation of new Na+-tolerant HKT1 mutations in novel domains, by using yeast mutants with increased Na+ sensitivity. These new HKT1 mutants cause greatly enhanced Na+ tolerance in yeast compared with previous mutants. The mechanisms by which mutants cause Na+ tolerance were characterized, and mechanistic distinctions among Na+-tolerant HKT1 mutants are revealed. The new mutations contribute to the identification of a new site in a loop domain, N365, that is involved in cation specificity of HKT1 and in HKT1-mediated Na+ tolerance. These findings contribute to a molecular physiological understanding of HKT1 structure and function. Furthermore, highly tolerant HKT1 mutants can be used to determine contributions of HKT1 to plant Na+ transport in molecular physiological studies.

    EXPERIMENTAL PROCEDURES

Yeast Growth-- Saccharomyces cerevisiae strains CY162 (Mat a, ura3-52, his3Delta 200, his44-15, trk1Delta , trk2::pCK64) (21) and 9.3 (Mat a, ena1Delta :HIS3::ena4Delta , leu2, ura3-1, trp1-1, ade2-1, trk1Delta , trk2::pCK64) kind gifts of Dr. Richard Gaber (Northwestern University) and Dr. Alonso Rodríguez-Navarro (Universidad Politécnica de Madrid, Spain), respectively, were used for selection and physiological characterization of HKT1 mutants. Standard minimal medium (22) and arginine phosphate medium (AP)1 (23) supplemented with KCl and NaCl as indicated were used for yeast growth. Yeast transformation was carried out by the polyethylene glycol method as described previously (24). Standard procedures were used for Escherichia coli growth and transformation and DNA manipulations (25).

Random HKT1 Mutagenesis and Mutant Selection-- The HKT1 cDNA cloned in the pYES2 vector (Invitrogen, La Jolla, CA) was mutagenized randomly by error PCR as described elsewhere (26). Two different sets of DNA fragments generated by error PCR that covered the coding region of the HKT1 cDNA were employed: the first reaction produced DNA fragments from position -36 to 1152, and the second reaction from position 657 to 1570. The DNA fragments were cotransformed into yeast to allow homologous recombination (27) with a gapped pDR195-based plasmid containing the yeast PMA1 gene promoter (28) and the HKT1 cDNA lacking the BgII (387 position) to the BclI (939 position) fragment for the first set of PCR reactions or the BclI (939 position) to the XbaI (1478 position) fragment for the second set of PCR reactions. URA+ transformants were first selected on standard minimal medium lacking uracil and supplemented with 100 mM K+ and then replica-plated to AP medium supplemented with 0.1 mM K+ plus 400 mM Na+, where strain 9.3 containing the wild type HKT1 did not grow because of Na+ toxicity. Growing colonies were selected, and their plasmids were isolated and reintroduced into yeast to retest for Na+ tolerance. Clones conferring the highest Na+ tolerance were chosen for further characterization.

Yeast Uptake Experiments-- The K+ uptake-deficient yeast strain CY162 described above (21) and the same strain transformed with a plasmid containing the wild type HKT1 cDNA or the mutated HKT1 cDNA under the control of the yeast PMA1 gene promoter (8, 28) were used for uptake experiments. Cells were grown in arginine phosphate medium (23) supplemented with 30 mM K+. For Na+ uptake experiments in the low affinity range of concentrations (1-300 mM Na+), cells were used directly. For Rb+ and Na+ uptake experiments in the high affinity range of concentrations (1-300 µM Rb+ or Na+), cells were previously starved of K+ for 5-6 h (29). For the uptake experiments, cells were harvested and resuspended in uptake buffer (10 mM Mes, 0.1 mM MgCl2, 2% glucose brought to pH 6.0 with Ca(OH)2) and incubated at 30 °C. For high affinity Rb+ and Na+ uptake experiments, 86Rb+ or 22Na+ (NEN Life Science Products) were added at 0.05 µCi nmol-1 at time 0. Samples were filtered at different time points through a Millipore membrane (0.8 µm) and washed with a 10 mM RbCl or NaCl solution. The internal Rb+ or Na+ content was calculated from the external activity, and the counts accumulated in the cells. Samples were counted in a Beckman Ls-230 scintillation counter. For low affinity Na+ uptake experiments, NaCl was added at time 0. Samples were collected as described above, washed with a 20 mM MgCl2 solution, and extracted with acid. The internal Na+ content was determined by atomic emission spectrophotometry using a Perkin-Elmer 5000 spectrophotometer. Initial rates of Rb+ or Na+ uptake are reported and represent averages of at least three independent experiments. Error bars denote standard error of the mean.

Internal K+ and Na+ Content Determination in Yeast-- Yeast cells expressing HKT1 or the Na+-tolerant HKT1 mutants were incubated in AP medium supplemented with 0.1 mM K+ and 300 or 500 mM Na+. After 24 h, samples were collected on filters as described above and the internal K+ and Na+ contents determined by atomic emission spectrophotometry. Error bars denote standard error of the mean.

Statistical analyses for uptake experiments and ionic content determination in yeast were performed with the Instat3 software (GraphPad Software Inc., San Diego, CA) using an unpaired t test. The significance level was p < 0.05.

Xenopus Oocyte Expression and Electrophysiology-- HKT1 and HKT1 mutant mRNA synthesis and injection (20 ng) into oocytes, voltage clamp recordings with a Dagan Cornerstone TEV-200 voltage clamp amplifier (Dagan, Minneapolis, MN) one day after mRNA injection, and data acquisition and analysis with the program Axotape (Axon Instruments, Foster City, CA) were performed as described previously (6). Data were low pass filtered at 20 Hz. Oocytes were impaled with electrodes filled with 1 M KCl and were bathed in a solution containing (in mM): 6 MgCl2, 1.8 CaCl2, 10 Mes-Tris, pH 5.5, osmolality 240-260 mosmol kg-1 with D-sorbitol. K+ and Na+ were added as glutamate salts. Tris+ (as glutamate) was added in experiments where the total alkali cation concentration varied. Error bars denote standard error of the mean.

pK+/pNa+ Permeability Ratio Determinations-- To calculate the pK+/pNa+ permeability ratios, the Goldman equation (Eq. 1) for ion channels was employed.
E<SUB>rev</SUB>=<FR><NU>RT</NU><DE>zF</DE></FR> <UP>ln</UP><FR><NU>p<UP>K<SUP>+</SUP></UP>[<UP>K<SUP>+</SUP></UP>]<SUB>o</SUB>+p<UP>Na<SUP>+</SUP></UP>[<UP>Na<SUP>+</SUP></UP>]<SUB>o</SUB></NU><DE>p<UP>K<SUP>+</SUP></UP>[<UP>K<SUP>+</SUP></UP>]<SUB>i</SUB>+p<UP>Na<SUP>+</SUP></UP>[<UP>Na<SUP>+</SUP></UP>]<SUB>i</SUB></DE></FR> (Eq. 1)

In addition, and to more accurately reflect the pK+/pNa+ permeability ratios of just the K+-coupling site for the HKT1 model with two separate Na+ and K+-coupling sites (8), an expanded version of the Goldman equation was employed (Eq. 2).
E<SUB>rev</SUB>=<FR><NU>RT</NU><DE>(m+n)F</DE></FR><FENCE>m <UP>ln</UP><FR><NU>p<UP>K<SUP>+</SUP></UP>[<UP>K<SUP>+</SUP></UP>]<SUB>o</SUB>+p<UP>Na<SUP>+</SUP></UP>[<UP>Na<SUP>+</SUP></UP>]<SUB>o</SUB></NU><DE>p<UP>K<SUP>+</SUP></UP>[<UP>K<SUP>+</SUP></UP>]<SUB>i</SUB>+p<UP>Na<SUP>+</SUP></UP>[<UP>Na<SUP>+</SUP></UP>]<SUB>i</SUB></DE></FR>+n <UP>ln</UP><FR><NU>p′<UP>K<SUP>+</SUP></UP>[<UP>K<SUP>+</SUP></UP>]<SUB>o</SUB>+p′<UP>Na<SUP>+</SUP></UP>[<UP>Na<SUP>+</SUP></UP>]<SUB>o</SUB></NU><DE>p′<UP>K<SUP>+</SUP></UP>[<UP>K<SUP>+</SUP></UP>]<SUB>i</SUB>+p′<UP>Na<SUP>+</SUP></UP>[<UP>Na<SUP>+</SUP></UP>]<SUB>i</SUB></DE></FR></FENCE> (Eq. 2)

Eq. 2 consists of the addition of two terms that consider the permeability of HKT1 for the K+-coupling site (m term) and the permeability of HKT1 for the Na+ coupling (n term). To solve for pK+/pNa+ in the expanded Goldman equation (Eq. 2), the K+ permeability of the high affinity Na+-coupling site was approximated to be negligible (9), and the stoichiometries were set to m = 2 and n = 1 (8).
<FR><NU>p<UP>K<SUP>+</SUP></UP></NU><DE>p<UP>Na<SUP>+</SUP></UP></DE></FR>=<FR><NU>[<UP>Na<SUP>+</SUP></UP>]<SUB>o</SUB>−10<SUP>S</SUP>[<UP>Na<SUP>+</SUP></UP>]<SUB>i</SUB></NU><DE>10<SUP>S</SUP>[<UP>K<SUP>+</SUP></UP>]<SUB>i</SUB></DE></FR> (Eq. 3)
where
<UP>S</UP>=<FR><NU>(m+n)<UP>F</UP></NU><DE>2 RT</DE></FR>E<SUB>rev</SUB>−<UP>log</UP><FR><NU>1</NU><DE>2</DE></FR> <FR><NU>[<UP>Na<SUP>+</SUP></UP>]<SUB>o</SUB></NU><DE>[<UP>Na<SUP>+</SUP></UP>]<SUB>i</SUB></DE></FR> (Eq. 4)

For all equations, Erev is the reversal potential, R is the gas constant, T is the absolute temperature, z is the charge of the ion, F is the Faraday constant, pNa+ is the relative permeability of the K+-coupling site of HKT1 for Na+, pK+ is the relative permeability of the K+-coupling site of HKT1 for K+. p'Na+ and p'K+ refer to the Na+-coupling site. The ionic concentrations are given in brackets (o = external, i = internal). The internal K+ and Na+ concentrations were assumed to be 92.5 mM K+ and 6.2 mM Na+ as has been determined for Xenopus oocytes (30).

    RESULTS

Isolation of New Na+ Tolerant HKT1 Mutants-- A nonbiased random selection approach was used to identify new amino acid positions in HKT1 that affect Na+ transport. To isolate new HKT1 mutants that conferred even larger Na+ tolerance than those found previously (8), we used a yeast strain, 9.3, which is more Na+ sensitive than the CY162 strain. The 9.3 yeast strain has a deletion in the four ENA1 to ENA4 genes that encode plasma membrane Na+-extruding ATPases (31, 32). In addition, the yeast K+ uptake transporter genes TRK1 and TRK2 were deleted in the 9.3 strain. Mutants were isolated at 0.1 mM K+ where strain 9.3 does not grow because it lacks the endogenous high affinity K+ uptake system, thus including selective pressure for maintaining high affinity K+ uptake in HKT1 mutants, and 400 mM Na+ to select for Na+ tolerance.

The HKT1 cDNAs were randomly mutagenized by error-prone PCR amplification (see "Experimental Procedures"). By screening 5,000 clones, putative HKT1 mutants were isolated that showed increased growth in the presence of 0.1 mM K+ plus 400 mM Na+. Subsequent plasmid isolation and retransformation of yeast was performed to retest HKT1 mutants for Na+ tolerance. Sequencing of the strongest Na+ tolerance-conferring HKT1 mutants showed that five new HKT1 mutants had been isolated (Table I). Interestingly, four of five mutants were altered in the Asn-365 residue in independent PCR reactions, indicating that this residue is important for Na+ tolerance.

                              
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Table I
Mutations in HKT1 that confer Na+ tolerance

The single base Q270L and N365S HKT1 mutants were analyzed in detail here, because they conferred the highest degree of Na+ tolerance (Table I). Fig. 1A shows that at high Na+, the yeast strain 9.3 expressing HKT1 did not grow, whereas 9.3 expressing the HKT1 mutants Q270L, N365S, and the former mutant A240V grew. To quantify the Na+ tolerance conferred by the two new mutations, the growth of the 9.3 strain expressing HKT1 and the Na+-tolerant HKT1 mutants was followed in liquid culture at several Na+ concentrations. The HKT1 mutant A240V was included for comparison because it was the more Na+-tolerant of the two previously described HKT1 mutants (8). At 150 mM Na+, 9.3 cells expressing HKT1 did not grow and 9.3 cells expressing the HKT1 mutants A240V, Q270L, and N365S grew well (Fig. 1B). The control strain 9.3 did not show growth because the medium contained low K+ and high Na+ concentrations.


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Fig. 1.   The new HKT1 mutations N365S and Q270L confer Na+ tolerance to yeast. Panel A, growth of the yeast strain in which Na+-extruding ATPases were deleted (9.3 strain) was used to isolate and assay the Na+-tolerant HKT1 mutants N365S, Q270L, and A240V. Yeast cells were grown on an AP medium supplemented with 0.1 mM K+ plus 350 mM Na+. Panel B, liquid culture growth of the yeast strain 9.3 expressing wild type HKT1 (open circle ) and the mutants N365S (black-square), Q270L (), A240V (), and untransformed 9.3 strain (triangle ) in liquid AP medium supplemented with 0.1 mM K+ plus 150 mM Na+. The ability of Na+-resistant HKT1 mutant expressing lines to grow in high Na+ is evident. At time 0, media were inoculated with 106 cells/ml of each strain and the optical density (OD) at 600 nm of the culture was recorded. Panel C, increasing the Na+ concentration to 250 mM shows the strong Na+ resistance of the N365S mutant. Conditions were the same as in panel B but in the presence of 0.1 mM K+ plus 250 mM Na+. Panel D, according to the Kyte and Doolittle algorithm, HKT1 comprises approx 12 hydrophobic domains. Mutants A240V and L247F are located in the 6th hydrophobic domain, mutant Q270L in the 7th hydrophobic domain, and mutant N365S in the loop between the 8th and 9th hydrophobic domains.

When the Na+ concentration was increased to 250 mM Na+, only yeast cells expressing the N365S mutation showed large growth rates (Fig. 1C; N365S doubling time 10 ± 0.7 h). Thus, this mutant dramatically increased Na+ tolerance compared with the previously isolated Na+-tolerant HKT1 mutant A240V. According to hydrophobicity analysis of HKT1, the new N365S and Q270L mutations lie in the loop between the 8th and 9th hydrophobic domains (N365S) and in the 7th hydrophobic domain (Q270L) (Fig. 1D). These results provide evidence that HKT1 domains in addition to the 6th hydrophobic domain are involved in HKT1-mediated Na+ tolerance.

Kinetic Characterization of Rb+ and Na+ Transport in the Na+-tolerant HKT1 Mutants-- To investigate whether the two new HKT1 mutations Q270L and N365S affected high affinity transport characteristics under non-Na+ stress conditions, Rb+ and Na+ uptake experiments at micromolar Rb+ and Na+ concentrations were carried out in the yeast strain CY162 expressing the HKT1 mutants. Micromolar Na+ concentrations produced a similar activation of high affinity Rb+ uptake in HKT1 and in the N365S and Q270L mutants (data not shown). Rb+ uptake was measured in the presence of 1 mM Na+ for maximal activation of high affinity Rb+ uptake (8). Fig. 2A shows that CY162 expressing wild type HKT1 and the mutants Q270L and N365S displayed approximately similar rates of high affinity Rb+ uptake. Detailed analysis of uptake kinetics showed a slight 1.5-fold increase in the affinity for Rb+ in the Na+-tolerant N365S mutant. The Km (Rb+) values for Rb+ uptake in the presence of 1 mM Na+ were 70.2 ± 8 µM for HKT1, 49.8 ± 5 µM for Q270L, and 45.8 ± 9 µM for N365S, and the Vmax (nmol Rb+ mg-1 min-1) values 2.7 ± 0.1 for HKT1, 2.2 ± 0.1 for Q270L, and 2.0 ± 0.1 for N365S. Statistical analyses of the data showed that there were not significant differences among the Rb+ Km values (p values were 0.079 for the HKT1, N365S pair and 0.084 for the HKT1, Q270L pair). The untransformed control strain CY162 did not show Rb+ uptake in the range of Rb+ concentrations used in these experiments because of the deletion of the high affinity K+ uptake systems encoded by TRK1 and TRK2 genes.


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Fig. 2.   HKT1 and the Na+-tolerant HKT1 mutants Q270L and N365S display high affinity Rb+ and Na+ uptake. Panel A, initial rates of Rb+ uptake in the presence of 1 mM Na+ as a function of the external Rb+ concentration were analyzed in CY162 yeast cells expressing wild type HKT1 (open circle ), the HKT1 mutants Q270L () and N365S (black-square), and untransformed strain CY162 (triangle ). Panel B, initial rates of Na+ uptake in the presence of 100 µM K+ as a function of the external Na+ concentration. In panels A and B high affinity Rb+ and Na+ uptake were analyzed in the K+ uptake-deficient yeast strain CY162 (trk1-, trk2-) expressing the indicated HKT1 mutants.

High affinity Na+ uptake experiments were performed in the presence of 100 µM K+ for maximal activation of high affinity Na+ uptake (8). The two mutations showed small effects on high affinity Na+ uptake (Fig. 2B). The KmM Na+) values for Na+ uptake in the presence of 100 µM K+ were 65.3 ± 6.9 µM for HKT1, 36.5 ± 13.3 µM for Q270L, and 33.2 ± 9.8 µM for N365S, and the Vmax (nmol mg-1 min-1) values 6.8 ± 0.2 for HKT1, 5.5 ± 0.2 for Q270L, and 5.0 ± 0.2 for N365S. Statistical analyses of the data showed that the Na+ Km values were not significantly different (p = 0.054 for the HKT1, N365S pair and p = 0.11 for the HKT1, Q270L pair), whereas the Na+ Vmax values were significantly different (p = 0.005 for the HKT1, N365S pair and p = 0.009 for the HKT1, Q270L pair). The untransformed control strain CY162 did not show Na+ uptake in the range of Na+ concentrations used in these experiments. The Na+-tolerant mutants showed significantly lower Vmax values for high affinity Rb+ and Na+ uptake. This could be a consequence of a lower expression of the mutants in yeast as compared with wild type HKT1. However, a reduced expression level would decrease the rate at which yeast expressing the mutants grow in low K+ medium, which was not observed (data not shown). Although the lower Vmax for high affinity Na+ uptake showed by the mutants could contribute to Na+ tolerance, we hypothesized that other substantial changes in transport parameters would be required in the HKT1 mutants to achieve the observed dramatic increase in Na+ tolerance (Fig. 1C).

An earlier study showed that high Na+ concentrations inhibited high affinity Rb+ uptake through HKT1 (8). The effect of millimolar Na+ concentrations on high affinity Rb+ uptake was investigated for the new HKT1 mutants, Q270L and N365S. Fig. 3A shows the rates of Rb+ uptake from a 100 µM Rb+ solution in yeast cells expressing HKT1 mutants as a function of increasing millimolar Na+ concentrations in the range from 1 to 400 mM Na+. High Na+ concentrations inhibited high affinity K+ (Rb+) uptake to a lesser extent in cells expressing the Na+-resistant HKT1 mutants than in cells expressing wild type HKT1 (Fig. 3A). The three mutations N365S, Q270L, and A240V showed a similar reduction in the inhibition of high affinity Rb+ uptake by high Na+ concentrations. The Na+ concentrations (mM Na+) that produced 50% inhibition of Rb+ uptake were 40 ± 4.3 for HKT1, 90 ± 2.6 for N365S (p = 0.0044), 90 ± 1.7 for Q270L (p = 0.0054), and 112 ± 2.9 for A240V (p = 0.0016). These data show that the ability of Na+ to bind to a modelled high affinity K+-coupling site (9) is reduced in the HKT1 mutants. However, the 50% inhibition constants show that Na+ block of K+ uptake alone cannot account for the increased Na+ tolerance of the N365S mutant with respect to A240V (Figs. 3A and 1C).


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Fig. 3.   Toxic concentrations of Na+ inhibit high affinity Rb+ uptake to a lesser extent and promote a reduced low affinity Na+ uptake in the Na+-tolerant HKT1 mutants when compared with wild type HKT1. A, initial rates of Rb+ uptake at 100 µM Rb+ were determined in the presence of 1-400 mM Na+ in the K+ uptake-deficient yeast strain CY162 (trk1-, trk2-). A clear enhanced resistance to high Na+ was found in cells expressing the Na+ resistant mutants N365S (black-square), Q270L (), and A240V (), when compared with cells expressing wild type HKT1 (open circle ). The Rb+ uptake rates are expressed as a percentage of the Rb+ uptake rate at 1 mM Na+ (100%). B, high and low affinity HKT1-mediated Na+ uptake can be clearly distinguished by Eadie-Hofstee analysis. For Na+ uptake experiments in the micromolar range of concentrations, 0.1 mM K+ was added. C, Na+-tolerant HKT1 mutants show reduced toxic low affinity Na+ uptake. Initial rates of Na+ uptake at external Na+ concentrations in the range 0-100 mM Na+ were measured in the K+ uptake-deficient yeast strain CY162 (trk1-, trk2-) expressing wild type HKT1 (open circle ) and the HKT1 mutants N365S (black-square), A240V (), Q270L (), and the control strain CY162 (triangle ).

In addition to high affinity Na+-coupled K+ uptake, HKT1 also promotes low affinity Na+ uptake in yeast and oocytes, with an apparent Km of approximately 5 mM Na+ (8, 9). High and low affinity components of Na+ uptake mediated by HKT1 could be clearly separated by Eadie-Hofstee analyses (Fig. 3B). This is consistent with previous studies showing two distinct transport mechanisms for high and low affinity Na+ uptake by HKT1 (8, 9). Low affinity Na+ uptake was investigated and kinetic parameters were compared in the Na+-tolerant mutants. Fig. 3C shows that CY162 cells expressing the more Na+-tolerant mutants A240V and N365S showed a substantial reduction in low affinity Na+ uptake compared with the wild type HKT1. The A240V mutant showed a significant lower Vmax value and the N365S showed significant lower affinity and lower Vmax of low affinity Na+ uptake (Table II). The results described above illustrate that HKT1 mutants become more Na+ resistant by simultaneously reducing Na+ inhibition of Rb+ uptake (Fig. 3A) and by reducing low affinity Na+ uptake (Fig. 3C, Table II). This dual effect of the mutations cannot be explained by a reduction in the expression level of the mutated transporter in yeast. To further test this hypothesis, we measured the internal K+ and Na+ contents of yeast cells expressing HKT1 and the HKT1 mutants after incubation in media with high Na+ concentrations (Fig. 4). When the yeast cells were incubated for 24 h in medium with 0.1 mM K+ and 500 mM Na+, the internal Na+/K+ ratios were 6.8 ± 1.4 for HKT1 (n = 6), 2.7 ± 0.4 for A240V (n = 6, p = 0.013), 1.9 ± 0.2 for Q270L (n = 6, p = 0.0065), and 0.6 ± 0.1 for N365S (n = 6, p = 0.0025). The most Na+-tolerant mutant N365S showed the lowest internal Na+/K+ ratio at both 300 and 500 mM Na+ (Fig. 4).

                              
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Table II
Km and Vmax values for low affinity Na+ uptake in yeast cells expressing HKT1, Na+-tolerant HKT1 mutants and the control yeast strain CY162


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Fig. 4.   The internal Na+/K+ ratio of yeast cells expressing HKT1 is higher than in yeast cells expressing the Na+-tolerant HKT1 mutants. Yeast cells expressing HKT1 or the Na+-tolerant HKT1 mutants were incubated in AP media supplemented with 0.1 mM K+ and 300 mM Na+ (solid bars) or 500 mM Na+ (open bars). After 24 h of incubation, the internal Na+ and K+ content was determined. The more Na+-tolerant mutant N365S showed the lower internal Na+/K+ ratio. At 500 mM Na+, the internal K+ and Na+ concentrations (nmol mg-1) were 14 ± 1.7 K+ and 94 ± 2.9 Na+ for HKT1-expressing cells, 37.7 ± 4.6 K+ and 99.7 ± 4.6 Na+ for A240V-expressing cells, 193 ± 11 K+ and 107.7 ± 7 Na+ for N365S-expressing cells, and 58 ± 1.7 K+ and 113 ± 7.2 Na+ for Q270L-expressing cells.

Electrophysiological Characterization of the Na+-tolerant HKT1 Mutants-- Differences in kinetic parameters between the two most Na+-tolerant N365S and A240V mutations were observed as described above. However, a strong differentiation between the N365S and A240V mutations was not apparent, except for low affinity Km (Na+) values (Table II) and Na+ to K+ content ratios in yeast. The stronger Na+ resistance of the N365S mutant when compared with the A240V mutant was quantitatively characterized further by studying biophysical transport characteristics. Yeast uptake experiments suggest that Na+ toxicity of HKT1 is mediated by the large rate of low affinity Na+ uptake recorded at toxic millimolar Na+ concentrations (Fig. 3C) and by Na+ inhibition of K+ nutrition (Fig. 3A). To allow a more quantitative biophysical characterization of this Na+ uptake component at millimolar Na+ concentrations in N365S, two electrode voltage-clamp experiments were performed in Xenopus oocytes expressing wild type HKT1 and the Na+-tolerant HKT1 mutants.

We first investigated Na+-coupled K+ currents induced by increasing external K+ or Na+ concentrations in the range of 1 to 10 mM while maintaining the other cation at a concentration of 1 mM. Voltage ramps were applied to oocytes expressing HKT1 or the mutant N365S (Fig. 5). When the external Na+ concentration was maintained at a constant value of 1 mM Na+ and K+ was increased from 1 to 10 mM K+, no significant differences for the shifts in the current-voltage relationships were observed for HKT1 and N365S expressing oocytes (HKT1: n = 4; N365S: n = 5; p = 0.42) (Fig. 5, A and B). In complementary experiments (Fig. 5, C and D), a 10-fold increase in the external Na+ concentration from 1 to 10 mM Na+ at a constant K+ concentration of 1 mM produced reversal potential shifts of +23.1 ± 0.9 mV for HKT1 (n = 4) and +24.6 ± 2.6 mV for N365S (n = 4). This indicated that the N365S mutation did not greatly affect Na+-coupled K+ uptake mediated by HKT1, which was also deduced from uptake studies in yeast at micromolar K+ and Na+ concentrations.


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Fig. 5.   Wild type HKT1 and the mutant N365S show similar reversal potential shifts for Na+-K+ symport. Increasing external K+ concentrations from 1 to 10 mM in the presence of 1 mM Na+ resulted in similar shifts toward more positive potentials of the current-voltage relationships of HKT1-mediated currents (A) and N365S-mediated currents (B) in oocytes. Increasing external Na+ concentrations from 1 to 10 mM in the presence of 1 mM K+ produced similar shifts toward more positive potentials of the current-voltage relationships of HKT1 (C) and N365S (D) in oocytes. The above treatments did not produce measurable currents in uninjected oocytes (not shown). Voltage ramps of 40 s duration from -40 to -120 mV were applied. Illustrated recordings show raw current data after filtering and use of the indicated symbols for clarity.

Experiments were then carried out to study the currents induced by high millimolar Na+ concentrations in oocytes expressing HKT1 and N365S to examine the low affinity Na+ uptake mode of HKT1 (8, 9) (Fig. 6, A and B). In wild type HKT1 expressing oocytes (n = 10), the 100 mM Na+-induced inward currents were much larger than the 1 mM K+ + 1 mM Na+-induced currents (Fig. 6A). In N365S-expressing oocytes (n = 10), the size of the currents induced by 100 mM Na+ were smaller than those induced by 1 mM K+ + 1 mM Na+ (Fig. 6B), confirming suppression of low affinity Na+ uptake by N365S (Fig. 3C). Note that the magnitude of the 1 mM K+ + 1 mM Na+-induced currents is higher in N365S than in HKT1-expressing oocytes (-2.06 ± 0.4 µA for N365S and -1.13 ± 0.07 µA for HKT1, at a membrane potential of -120 mV), suggesting that a reduced expression of the N365S mutant in oocytes is not the cause of the reduction in the currents induced by 100 mM Na+.


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Fig. 6.   The Na+-tolerant HKT1 mutant N365S shows a dramatic reduction in Na+ uptake current induced by 100 mM Na+ when compared with wild type HKT1. The large Na+ uptake (negative) currents induced by 100 mM Na+ () in wild type HKT1-expressing Xenopus oocytes (panel A) are repressed by the N365S point mutation (panel B). Panels A and B, magnitude of HKT1-induced currents as a function of the imposed membrane potentials recorded from oocytes expressing wild type HKT1 (panel A) or the HKT1 mutant N365S (panel B) exposed to 1 mM K+ plus 1 mM Na+ (open circle ) or to 100 mM Na+ (). The above treatments did not elicit currents in uninjected control oocytes (panel B, inset).

We also investigated the currents induced by 1 mM K+ + 1 mM Na+ and compared these to currents mediated by 100 mM Na+ in oocytes expressing the A240V, Q270L, and L247F mutants to allow a comparison among these mutants. In A240V- and Q270L-expressing oocytes, the magnitude of the 100 mM Na+-induced currents were smaller than the 1 mM K+ + 1 mM Na+-induced currents, similar to N365S-expressing oocytes. We calculated the ratio of the current induced by 100 mM Na+ over the current induced by 1 mM K+ + 1 mM Na+ at a membrane potential of -120 mV. The ratio was 2.56 ± 0.12 for HKT1-expressing oocytes (n = 20), 1.55 ± 0.12 for L247F-expressing oocytes (n = 11), 0.82 ± 0.05 for A240V-expressing oocytes (n = 17), 0.78 ± 0.1 for N365S-expressing oocytes (n = 11), and 0.65 ± 0.16 for Q270L-expressing oocytes (n = 6). For the less Na+-resistant L247F HKT1 mutant (n = 4), 100 mM Na+-induced currents were larger than the 1 mM K+ + 1 mM Na+-induced currents, but were smaller than wild type HKT1 currents. This result correlates to the intermediate level of Na+ resistance conferred by L247F (8). These data illustrate the strong reduction in low affinity Na+ uptake in the more Na+-tolerant HKT1 mutants.

The results described above indicate that the HKT1 mutations had decreased the permeability for Na+ at high Na+ concentrations. Changes in permeabilities of cations would affect the reversal potential of the transporter (33). To directly analyze this hypothesis, and to quantitatively determine whether the low affinity Na+ transport mode of HKT1 mutants can be biophysically distinguished among mutants, we measured the reversal potentials of currents in the presence of 10 mM or 100 mM Na+ in oocytes expressing HKT1 and the Na+-tolerant mutants. Fig. 7 shows the shift in the reversal potential of HKT1-mediated currents in response to an increase in the external Na+ concentration from 10 to 100 mM (equivalent to an 8.5-fold Na+ activity shift after correction for ionic activities). Wild type HKT1 expression produced the previously described large shifts in the reversal potential of +48.2 ± 0.9 mV (n = 6) upon increasing the extracellular Na+ concentration (8). The more Na+-tolerant mutants, N365S and A240V, showed dramatic reductions in reversal potential shifts, whereas wild type HKT1 and the Na+-tolerant mutants, L247F (n = 6) and Q270L (n = 5), showed large shifts of approx 50 mV. The most Na+-tolerant N365S mutant showed the smallest shift in the reversal potential of +16.6 ± 4.2 mV (n = 7), which correlates with the strong Na+-tolerant phenotype of this mutant when compared with all other mutants. The A240V mutation, which shows less Na+ resistance than N365S, also showed an intermediate shift in the reversal potential of +26.1 ± 3.3 (n = 11), revealing a larger Na+ permeability than the N365S mutant (Fig. 7).


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Fig. 7.   The Na+-tolerant N365S mutant shows a reduction in reversal potential shifts induced by increasing external Na+ from 10 to 100 mM. Average shifts in reversal potentials (Delta Erev) of HKT1-mediated currents in response to an increase in the external Na+ concentration from 10 to 100 mM. Reversal potentials of steady-state currents of HKT1 and HKT1 mutants were recorded as in Fig. 6 (8, 9). Data from 5 to 11 cells (±S.E.) were averaged for each HKT1 construct (see text).

We analyzed the permeability ratio for K+ over Na+ using the Goldman equation for ion channels (see "Experimental Procedures"). Because in contrast to ion channels, HKT1 has been modelled to possess two independent ion-coupling sites, one of which is highly selective for Na+ and the other is selective for K+, we also calculated the K+ to Na+ permeability ratios using an expansion of the Goldman equation (see "Experimental Procedures") to more accurately reflect the pK+/pNa+ permeability ratios of just the K+-coupling site. Using the Goldman equation, the permeability ratios pK+/pNa+ for HKT1 and the Na+-tolerant mutants derived from absolute reversal potential measurements in the presence of 100 mM external Na+ were 5.3 ± 0.8 (n = 4) for HKT1, 12.1 ± 2.3 (n = 7) for A240V, and 15.5 ± 1.8 (n = 5) for N365S. Using the expanded model for two ion-coupling sites (see "Experimental Procedures"), pK+/pNa+ permeability ratio values for the K+ selective site were 51 for HKT1, 187 for A240V, and 257 for N365S. The high relative permeabilities of the K+-coupling site for K+ over Na+ derived from the two-site model are consistent with the predicted high selectivity of this site for K+ over other cations (8, 9). Permeability ratio analyses show a K+ to Na+ selectivity sequence of N365S > A240V > wild type HKT1, independent of the model used.

    DISCUSSION

Expression of the HKT1 protein in yeast was shown to confer typical characteristics of high affinity K+ uptake, including a micromolar apparent K+ affinity (Km approx  3 µM K+) and saturation of uptake at micromolar K+ and Rb+ concentrations (6, 8, 9). High millimolar Na+ concentrations have two important effects on cation transport by HKT1 leading to toxicity in yeast (8). First, high affinity K+ uptake via HKT1 is blocked by millimolar Na+ concentrations (Fig. 3A). Second, at these millimolar Na+ concentrations, HKT1 functions as a low affinity transporter mediating high rates of Na+ uptake (Fig. 3C) (8, 9).

Flux studies have shown both high and low affinity Na+ uptake pathways in plants and suggested that high affinity Na+ uptake is mediated by K+ transporters (10, 11). However, Na+ toxicity related to salt stress has been specifically attributed to the low affinity components of Na+ uptake (11, 14, 19). Therefore, the finding that HKT1 mediates low affinity Na+ uptake at millimolar Na+ concentrations indicates a putative contribution of HKT1 as one of several components mediating low affinity Na+ uptake during salt stress in plants (12, 13). RNA in situ hybridizations show HKT1 expression in root and leaf tissues that are relevant to Na+ transport and stress (6). Expression of HKT1 in leaf cells indicates a role for Na+ transport in leaves (6), where Na+ is particularly deleterious to plant metabolism and growth. Note that Na+ uptake studies in roots show several low affinity Na+ uptake components (12). Recently, several plant cDNAs encoding cation transporters have been isolated that may encode additional low affinity Na+ uptake components depending on the ionic conditions, including LCT1 from wheat (34) and HvHAK1 from barley (35). The Na+-coupled high affinity K+ uptake mechanism of HKT1 suggested that additional high affinity K+ uptake pathways should exist in plant cells (8, 9). Both the Arabidopsis, AtKT, or AtKUP cDNAs and the homologous barley HvHAK1 cDNA have been shown to mediate K+ uptake (35-38).

Na+-tolerant mutants in HKT1 were isolated here to identify and characterize important amino acid residues and mechanisms involved in Na+ transport and to isolate HKT1 mutants with reduced Na+ uptake for molecular physiological studies to determine contributions of HKT1 to Na+ transport. Wheat HKT1 is highly selective for the alkali cations K+ and Na+, and micromolar K+ concentrations strongly inhibit high affinity Rb+ uptake.2 At a concentration of approx 15 µM, K+ produces a approx 15-fold higher uptake rate than Rb+ (9). However, when K+ is present at high millimolar concentrations, K+ partially impairs high affinity Na+-coupled K+ uptake (9).

Identification of HKT1 Mutants with Reduced Na+ Permeability-- In the present study, the use of a yeast strain deficient in the ENA family of Na+-extruding ATPases allowed a stringent screen to isolate highly Na+-tolerant HKT1 mutations. The N365S mutation that showed the strongest increase in Na+ tolerance was analyzed in detail here. The N365S mutation is a relatively conservative substitution. Interestingly, the Asn-365 amino acid is conserved in the Arabidopsis HKT1 homologue.3 Four of the five newly identified mutants showed a mutation at position Asn-365, indicating that this site is of central importance for determining the interaction of HKT1 with Na+ (Table I).

Consistent with the above model, the Na+-tolerant HKT1 mutants characterized here show a reduced block of Rb+ uptake by Na+ (Fig. 3A). At the same time, the apparent Km of low affinity Na+ uptake was increased, and the Vmax for Na+ decreased (Fig. 3C, Table II). Note that the Km for low affinity Na+ uptake of the Na+-tolerant HKT1 mutants (Fig. 3C) does not coincide with the Na+ concentration that inhibits high affinity Rb+ uptake (Fig. 3A). The higher value obtained for Na+ Ki (Fig. 3A) compared with the low affinity Na+ Km (Fig. 3C) may be a consequence of the micromolar Rb+ concentration (100 µM) present in the Ki experiment (Fig. 3A), which will compete at the high affinity K+-coupling site, reducing the affinity of the transporter for millimolar Na+. However, the existence of additional low affinity Na+ binding sites in HKT1 involved in activation, inhibition, or transport of Na+ cannot be ruled out. Although N365S displayed the lowest apparent affinity for low affinity Na+ uptake (Km = 10.2 mM Na+) and also had a reduced maximal uptake rate (Fig. 3C, Table II), these parameters alone did not appear to resolve the strikingly higher Na+ resistance of N365S as compared with the A240V mutant.

An additional effect of the N365S mutation is a lower Na+ permeability of HKT1. The selectivity for Na+ of wild type HKT1 and the mutants was determined by measuring current reversal potentials in oocytes when external Na+ concentrations were changed from 10 mM Na+ to 100 mM Na+ (Fig. 7). Indeed, the N365S mutant showed a dramatic reduction in the reversal potential shift (16.6 mV versus 48.2 mV), reflecting a markedly reduced Na+ permeability in the N365S mutant. Calculations of relative K+ to Na+ permeability ratios with 100 mM Na+ using either a channel model or a two-site coupled transporter model confirmed that the N365S mutant has the highest selectivity for K+ over Na+ of all mutants isolated so far. Calculated K+ to Na+ selectivity values were lower for the channel model, because both K+ and Na+ transport sites are treated as one entity. The model for the two-site coupled transporter quantifies the previously predicted high K+ to Na+ selectivity of the K+-coupling site (8, 9), with K+ to Na+ permeability ratios ranging from 51 for HKT1 to 257 for N365S. A recent study has identified point mutations at other sites in HKT1 that affect the Na+-coupling site (39), which differs mechanistically from the K+-coupling site mutations described here. The reduction in relative Na+ permeability of the N365S mutant leads to a reduction in low affinity Na+ uptake as shown in oocyte (Fig. 6) and yeast (Fig. 3B) experiments and to increased K+ content (Fig. 4). Note that permeability ratio values of HKT1 will depend on conditions, because of competition of ionic interactions in HKT1 (9).

The N365S mutation affects several important parameters in HKT1 function: (i) reduction in low affinity Na+ uptake, (ii) reduction in Na+ to K+ permeability, and (iii) reduction in the inhibition of high affinity K+ uptake by millimolar Na+ concentrations. The combined effects of these changes, integrated over physiological time periods, contribute to maintain a low internal Na+/K+ ratio during long-term uptake studies (Fig. 4), resulting in Na+-resistant cell growth. The results presented here suggest a model to explain the increase in Na+ tolerance conferred by the N365S mutation. In this mutant, the selectivity between micromolar K+ and millimolar Na+ at the high affinity K+-coupling site is increased in comparison with wild type HKT1 and also with the A240V mutant. As a result, the internal Na+/K+ concentration ratio is kept below the threshold level that would inhibit yeast growth.

Interestingly, Asn-365 is not located in a hydrophobic domain but in a hydrophilic loop domain. In the case of K+ channels the selectivity filter has been ascribed largely to a short hydrophilic loop ("P-domain") rather than hydrophobic domains (40-43). In the bacterial high affinity K+ pump, Kdp, 33 independent mutations affecting 13 amino acid residues were identified that reduced K+ affinity (44). Of these 13 residues, 12 were located in hydrophilic loop domains (44). The Asn-365 site in a loop domain of HKT1 substantially affects Na+ sensitivity, Na+ transport, and Na+ permeability, which correlates well with findings in K+ channels and in the bacterial K+ pump Kdp on the importance of loop domains for cation selectivity.

Physiological Significance of K+ versus Na+ Uptake-- In yeast, high affinity Na+ uptake via the K+-Na+ symport activity of HKT1 (Fig. 2B), does not cause Na+ toxicity. This is most likely because of the low Na+ uptake rates under high affinity uptake conditions. At these low rates, transfer of Na+ from the cytosol into the vacuole would be sufficiently rapid to prevent toxic levels of Na+ in the cytosol. In addition, because the stoichiometry of high affinity uptake by HKT1 at micromolar cation concentrations was found to be approximately 1.7 K+ per Na+ ion (8), the low levels of Na+ uptake would be sufficiently balanced by K+ uptake to prevent displacement of K+ by Na+ in the cytosol. (Note that because of competition, the exact K+ to Na+ uptake stoichiometry of HKT1 was shown to depend on ionic conditions (9, 45).) It has recently been proposed that the HKT1 protein may be located in the vacuolar membrane rather than in the plasma membrane (46). Here we report on HKT1 mutants that reduce HKT1-mediated Na+ transport into the cytosol. These findings would be relevant even if HKT1 were targeted to an endomembrane in plants because Na+ transport by HKT1 would affect the cytoplasmic Na+/K+ content ratio.

Sodium-tolerant HKT1 mutants increased the ratio of K+ to Na+ content in yeast (8). In plants, analysis of the Na+-sensitive Arabidopsis mutant sos1 supports the model that the balance of cytosolic K+ versus Na+ concentrations is an important determinant of Na+ toxicity. The sos1 mutant shows strongly reduced high affinity K+ uptake and a reduction in the ratio of K+ to Na+ content (47, 48). Interestingly, sos1 at the same time also shows reduced low affinity Na+ uptake. The sos1-affected low affinity Na+ uptake component is induced in wild type Arabidopsis roots in response to K+ starvation, which also induces high affinity K+ uptake systems (5, 49). These data indicate a genetic relationship of high affinity K+ uptake and low affinity Na+ uptake in Arabidopsis roots (48). Note that the sos1 mutation appears to affect several different transporters and therefore probably has a regulatory function (47, 48).

Conclusions-- The identification and characterization of the strongly Na+-tolerant N365S HKT1 mutant described here provides a new tool to study contributions of HKT1 transporters to Na+ sensitivity, Na+ transport, regulation of Na+ transport, and K+ versus Na+ content ratios in transgenic plants. New HKT1 sites that reduce toxic effects of Na+ in yeast were identified and characterized by a combination of random mutagenesis, functional selection in yeast, and flux and biophysical transport studies. Mutants maintain the physiologically beneficial function of Na+-coupled high affinity K+ uptake because they were selected in K+ uptake-deficient yeast mutant backgrounds (21). A new critical site in HKT1, Asn-365, and new biophysical transport parameters were identified that can be altered to enhance Na+ resistance in yeast. In plants, toxic low affinity Na+ uptake is thought to be mediated by multiple Na+ uptake transporters (see above and Ref. 12). The use of an Na+-ATPase yeast deletion mutant line (9.3 ena-) in combination with uptake deficiency (e.g. trk-) to select for Na+-tolerant mutants, as presented for HKT1 here, can be applied to other plant Na+ uptake transporters and could provide a potent approach to identify strong Na+-tolerant mutants in individual Na+ transporters in the future. Isolation and analysis of additional HKT1 mutants should allow insights into further structural sites and mechanisms that affect Na+ transport. Mutations characterized here provide molecular tools to test physiological contributions of HKT1 to Na+ transport in complex multicellular systems such as plant tissues.

    ACKNOWLEDGEMENTS

We thank Judie Murray for assistance in preparing the manuscript, Eugene Kim for comments on the manuscript, Alonso Rodríguez-Navarro for providing the 9.3 yeast strain, and Richard F. Gaber for providing the CY162 strain.

    FOOTNOTES

* This research was supported by grants from the United States Department of Agriculture (to J. I. S.) and by a Ministerio de Educación y Ciencia (Spain) postdoctoral fellowship (to F. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, 28040 Madrid, Spain.

§ Present address: Zentrum f. Molekulare Neurobiologie, Martinistr. 52, 20246 Hamburg, Germany.

Present address: Dept. of Plant Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720-3102.

parallel To whom correspondence should be addressed. Tel.: 619-534-7759 or 619-534-6296; Fax: 619-534-7108; E-mail: Julian{at}biomail.ucsd.edu.

2 F. Rubio, D. P. Schachtman, W. Gassmann, and J. I. Schroeder, unpublished material.

3 N. Uozumi, E. J. Kim, F. Rubio, S. Muto, and J. I. Schroeder, unpublished material.

    ABBREVIATIONS

The abbreviations used are: AP, arginine phosphate; PCR, polymerase chain reaction; Mes, 4-morpholineethanesulfonic acid.

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Abstract
Introduction
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