The Na ؉ -K ؉ co-transporter HKT1, first isolated from wheat, mediates high-affinity K ؉ uptake. The function of HKT1 in plants, however, remains to be elucidated, and the isolation of HKT1 homologs from Arabidopsis would further studies of the roles of HKT1 genes in plants. We report here the isolation of a cDNA homologous to HKT1 from Arabidopsis (AtHKT1) and the characterization of its mode of ion transport in heterologous systems. The deduced amino acid sequence of AtHKT1 is 41% identical to that of HKT1, and the hydropathy profiles are very similar. AtHKT1 is expressed in roots and, to a lesser extent, in other tissues. Interestingly, we found that the ion transport properties of AtHKT1 are significantly different from the wheat counterpart. As detected by electrophysiological measurements, AtHKT1 functioned as a selective Na ؉ uptake transporter in Xenopus laevis oocytes, and the presence of external K ؉ did not affect the AtHKT1-mediated ion conductance (unlike that of HKT1). When expressed in Saccharomyces cerevisiae, AtHKT1 inhibited growth of the yeast in a medium containing high levels of Na ؉ , which correlates to the large inward Na ؉ currents found in the oocytes. Furthermore, in contrast to HKT1, AtHKT1 did not complement the growth of yeast cells deficient in K ؉ uptake when cultured in K ؉ -limiting medium. However, expression of AtHKT1 did rescue Escherichia coli mutants carrying deletions in K ؉ transporters. The rescue was associated with a less than 2-fold stimulation of K ؉ uptake into K ؉ -depleted cells. These data demonstrate that AtHKT1 differs in its transport properties from the wheat HKT1, and that AtHKT1 can mediate Na ؉ and, to a small degree, K ؉ transport in heterologous expression systems.

The sequence of events following the addition of 0.5 M NaCl to cells of Escherichia coli growing in a minimal mineral medium was investigated. Immediately after upshock the cells took up a large amount of K+ and synthesized approximately half the equivalent amount of glutamate concomitantly. After 30 min the cells started to synthesize trehalose, and after 2 h they had replaced most of their initial osmoprotectants by the carbohydrate. Cell trehalose was rapidly replaced by proline, taken up from the medium when added to the osmoadapting cells. The initial rate of this proline uptake was extremely rapid, and with rates observed of up to 0.6 mmol x min-1 x g-1 of cell protein it was approximately ten times faster than that reported in the literature for non-growing cells. These results indicate that for osmoadaptation of growing cells of E. coli the uptake of proline has priority over the synthesis of trehalose, which in its turn is preferred above K+ and glutamate as osmoprotectants. We observed that two mutants with unknown lesions, but which are known to be impaired in osmoadaptation, were inhibited in replacing K+ and glutamate by trehalose, indicating that this is the basis for their defect in osmoadaptation. Further experiments revealed that neither internal pH nor the membrane potential nor the transmembrane protonmotive force are likely to be involved in osmoadaptation in E. coli. However, during osmoadaptation a high internal potassium concentration appeared to stimulate the derepression of proline-uptake systems (mainly system ProP).

Plant HKT proteins comprise a family of cation transporters together with prokaryotic KtrB, TrkH, and KdpA transporter subunits and fungal Trk proteins. These transporters contain four loop domains in one polypeptide with a proposed distant homology to K ؉ channel selectivity filters. Functional expression in yeast and Xenopus oocytes revealed that wheat HKT1 mediates Na ؉ -coupled K ؉ transport. Arabidopsis AtHKT1, however, transports only Na ؉ in eukaryotic expression systems. To understand the molecular basis of this difference we constructed a series of AtHKT1͞HKT1 chimeras and introduced point mutations to AtHKT1 and wheat HKT1 at positions predicted to be critical for K ؉ selectivity. A single-point mutation, Ser-68 to glycine, was sufficient to restore K ؉ permeability to AtHKT1. The reverse mutation in HKT1, Gly-91 to serine, abrogated K ؉ permeability. This glycine in P-loop A of AtHKT1 and HKT1 can be modeled as the first glycine of the K ؉ channel selectivity filter GYG motif. The importance of such filter glycines for K ؉ selectivity was confirmed by interconversion of Ser-88 and Gly-88 in the rice paralogues OsHKT1 and OsHKT2. Surprisingly, all HKT homologues known from dicots have a serine at the filter position in P-loop A, suggesting that these proteins function mainly as Na ؉ transporters in plants and that Na ؉ ͞K ؉ symport in HKT proteins is associated with a glycine in the filter residue. These data provide experimental evidence that the glycine residues in selectivity filters of HKT proteins are structurally related to those of K ؉ channels.

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Recently, a new type of K ؉ transporter, Ktr, has been identified in the bacterium Vibrio alginolyticus (T. Nakamura, R. Yuda, T. Unemoto, and E. P. Bakker, J. Bacteriol. 180:3491-3494, 1998). The Ktr transport system consists of KtrB, an integral membrane subunit, and KtrA, a subunit peripherally bound to the cytoplasmic membrane. The genome sequence of Bacillus subtilis contains two genes for each of these subunits: yuaA (ktrA) and ykqB (ktrC) encode homologues to the V. alginolyticus KtrA protein, and yubG (ktrB) and ykrM (ktrD) encode homologues to the V. alginolyticus KtrB protein. We constructed gene disruption mutations in each of the four B. subtilis ktr genes and used this isogenic set of mutants for K ؉ uptake experiments. Preliminary K ؉ transport assays revealed that the KtrAB system has a moderate affinity with a K m value of approximately 1 mM for K ؉ , while KtrCD has a low affinity with a K m value of approximately 10 mM for this ion. A strain defective in both KtrAB and KtrCD exhibited only a residual K ؉ uptake activity, demonstrating that KtrAB and KtrCD systems are the major K ؉ transporters of B. subtilis. Northern blot analyses revealed that ktrA and ktrB are cotranscribed as an operon, whereas ktrC and ktrD, which occupy different locations on the B. subtilis chromosome, are expressed as single transcriptional units. The amount of K ؉ in the environment or the salinity of the growth medium did not influence the amounts of the various ktr transcripts. A strain with a defect in KtrAB is unable to cope with a sudden osmotic upshock, and it exhibits a growth defect at elevated osmolalities which is particularly pronounced when KtrCD is also defective. In the ktrAB strain, the osmotically mediated growth defect was associated with a rapid loss of K ؉ ions from the cells. Under these conditions, the cells stopped synthesizing proteins but the transcription of the osmotically induced proHJ, opuA, and gsiB genes was not impaired, demonstrating that a high cytoplasmic K ؉ concentration is not essential for the transcriptional activation of these genes at high osmolarity. Taken together, our data suggest that K ؉ uptake via KtrAB and KtrCD is an important facet in the cellular defense of B. subtilis against both suddenly imposed and prolonged osmotic stress.

The hypothesis is presented that at least four families of putative K(+) symporter proteins, Trk and KtrAB from prokaryotes, Trk1,2 from fungi, and HKT1 from wheat, evolved from bacterial K(+) channel proteins. Details of this hypothesis are organized around the recently determined crystal structure of a bacterial K(+) channel: i. e., KcsA from Streptomyces lividans. Each of the four identical subunits of this channel has two fully transmembrane helices (designated M1 and M2), plus an intervening hairpin segment that determines the ion selectivity (designated P). The symporter sequences appear to contain four sequential M1-P-M2 motifs (MPM), which are likely to have arisen from gene duplication and fusion of the single MPM motif of a bacterial K(+) channel subunit. The homology of MPM motifs is supported by a statistical comparison of the numerical profiles derived from multiple sequence alignments formed for each protein family. Furthermore, these quantitative results indicate that the KtrAB family of symporters has remained closest to the single-MPM ancestor protein. Strong sequence evidence is also found for homology between the cytoplasmic C-terminus of numerous bacterial K(+) channels and the cytoplasm-resident TrkA and KtrA subunits of the Trk and KtrAB symporters, which in turn are homologous to known dinucleotide-binding domains of other proteins. The case for homology between bacterial K(+) channels and the four families of K(+) symporters is further supported by the accompanying manuscript, in which the patterns of residue conservation are demonstrated to be similar to each other and consistent with the known 3D structure of the KcsA K(+) channel.

The effect of protamine on growing cells of Escherichia coli K-12 strains containing different K+-uptake systems was investigated. Immediately after the addition of the toxic peptide, growth ceased and all strains lost most of their K+. In addition, these cells released a significant amount of their ATP into the medium, and the cytoplasmic volume of these cells decreased by 70%. Whereas cells without rapid K+-uptake systems did not recover, cells containing either the Trk systems or the overproduced Kup system slowly reversed the effects of protamine, and growth resumed after the cells had reached their original volume. Experiments with a set of strains carrying mutations in the K+-uptake gene trkA showed a reasonably satisfactory correlation between inhibition of net K+ uptake and the lag time for resumption of growth after addition of protamine. Cells carrying mutations in three extracytoplasmic proteases were hypersusceptible to protamine, suggesting that the toxic peptide is degraded by these proteases. Data on the effect of a second addition of protamine suggest that protamine degradation activity is inducible. These data are interpreted to mean that reaccumulation of K+ by protamine-treated cells triggers recovery of the cells, thereby allowing induction of extracytoplasmic proteases. These, in turn, degrade protamine, leading to complete recovery of the cells and resumption of growth. Cells that cannot take up K+ rapidly remain metabolically compromised to such an extent that extracytoplasmic protease activity is not induced, leading to a prolonged susceptibility of the cells to the toxic peptide.

The influence of K+ ions on the components of the transmembrane proton motive force (delta mu H+) in intact bacteria was investigated. In K+-depleted cells of the glycolytic bacterium STreptococcus faecalis the addition of K+ ions caused a depolarization of the membrane by about 60 mV. However, since the depolarization was compensated for by an increase in the transmembrane pH gradient (delta pH), the total proton motive force remained almost constant at about 120 mV. Half-maximal changes in the potential were observed at K+ concentrations at which the cells accumulated K+ ions extensively. In EDTA-treated, K+-depleted cells of Escherichia coli K-12, the addition of K+ ions to the medium caused similar, although smaller changes in the components of delta mu H+. Experiments with various E. coli K-12 K+ transport mutants showed that for the observed potential changes the cells required either a functional TrkA or Kdp K+ transport system. These data are interpreted to mean that the inward movement of K+ ions via each of these bacterial transport systems is electrogenic. Consequently, it leads to a depolarization of the membrane, which in its turn allows the cell to pump more protons into the medium.