Two Trk/Ktr/HKT-type potassium transporters, TrkG and TrkH, perform distinct functions in Escherichia coli K-12.

Escherichia coli K-12 possesses two versions of Trk/Ktr/HKT-type potassium ion (K+) transporters, TrkG and TrkH. The current paradigm is that TrkG and TrkH have largely identical characteristics, and little information is available regarding their functional differences. Here, we show using cation uptake experiments with K+ transporter knockout mutants that TrkG and TrkH have distinct ion transport activities and physiological roles. K+-transport by TrkG required Na+, whereas TrkH-mediated K+ uptake was not affected by Na+. An aspartic acid located five residues away from a critical glycine in the third pore-forming region might be involved in regulation of Na+-dependent activation of TrkG. In addition, we found that TrkG but not TrkH had Na+ uptake activity. Our analysis of K+ transport mutants revealed that TrkH supported cell growth more than TrkG; however, TrkG was able to complement loss of TrkH-mediated K+ uptake in E. coli. Furthermore, we determined that transcription of trkG in E. coli was downregulated but not completely silenced by the xenogeneic silencing factor H-NS (histone-like nucleoid structuring protein or heat-stable nucleoid-structuring protein). Taken together, the transport function of TrkG is clearly distinct from that of TrkH, and TrkG seems to have been accepted by E. coli during evolution as a K+ uptake system that coexists with TrkH.

Analysis of Arabidopsis TPK2 and KCO3 reveals structural properties required for K+ channel function.

Arabidopsis thaliana contains five tandem-pore domain potassium channels, TPK1-TPK5 and the related one-pore domain potassium channel, KCO3. Although KCO3 is unlikely to be an active channel, it still has a physiological role in plant cells. TPK2 is most similar to KCO3 and both are localized to the tonoplast. However, their function remains poorly understood. Here, taking advantage of the similarities between TPK2 and KCO3, we evaluated Ca2+ binding to the EF hands in TPK2, and the elements of KCO3 required for K+ channel activity. Presence of both EF-hand motifs in TPK2 resulted in Ca2+ binding, but EF1 or EF2 alone failed to interact with Ca2+. The EF hands were not required for K+ transport activity. EF1 contains two cysteines separated by two amino acids. Replacement of both cysteines with serines in TPK2 increased Ca2+ binding. We generated a two-pore domain chimeric K+ channel by replacing the missing pore region in KCO3 with a pore domain of TPK2. Alternatively, we generated two versions of simple one-pore domain K+ channels by removal of an extra region from KCO3. The chimera and one of the simple one-pore variants were functional channels. This strongly suggests that KCO3 is not a pseudogene and KCO3 retains components required for the formation of a functional K+ channel and oligomerization. Our results contribute to our understanding of the structural properties required for K+ channel activity.

Functional characterization of multiple PAS domain-containing diguanylate cyclases in Synechocystis sp. PCC 6803.

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger known to control a variety of bacterial processes. The model cyanobacterium, Synechocystis sp. PCC 6803, has a score of genes encoding putative enzymes for c-di-GMP synthesis and degradation. However, most of them have not been functionally characterized. Here, we chose four genes in Synechocystis (dgcA-dgcD), which encode proteins with a GGDEF, diguanylate cyclase (DGC) catalytic domain and multiple Per-ARNT-Sim (PAS) conserved regulatory motifs, for detailed analysis. Purified DgcA, DgcB and DgcC were able to catalyze synthesis of c-di-GMP from two GTPs in vitro. DgcA had the highest activity, compared with DgcB and DgcC. DgcD did not show detectable activity. DgcA activity was specific for GTP and stimulated by the divalent cations, magnesium or manganese. Full activity of DgcA required the presence of the multiple PAS domains, probably because of their role in protein dimerization or stability. Synechocystis mutants carrying single deletions of dgcA-dgcD were not affected in their growth rate or biofilm production during salt stress, suggesting that there was functional redundancy in vivo. In contrast, overexpression of dgcA resulted in increased biofilm formation in the absence of salt stress. In this study, we characterize the enzymatic and physiological function of DgcA-DgcD, and propose that the PAS domains in DgcA function in maintaining the enzyme in its active form.

Identification of regions responsible for the function of the plant K+ channels KAT1 and AKT2 in Saccharomyces cerevisiae and Xenopus laevis oocytes.

The Arabidopsis K+ channel KAT1 complements in K+-limited medium the growth of the K+ uptake defective Saccharomyces cerevisiae mutant strain CY162, while another K+ channel, AKT2, does not. To gain insight into the structural basis for this difference, we constructed 12 recombinant chimeric channels from these two genes. When expressed in CY162, only three of these chimeras fully rescued the growth of CY162 under K+-limited conditions. We conclude that the transmembrane core region of KAT1 is important for its activity in S. cerevisiae. This involves not only the pore region but also parts of its voltage-sensor domain.

Kup-mediated Cs+ uptake and Kdp-driven K+ uptake coordinate to promote cell growth during excess Cs+ conditions in Escherichia coli.

The physiological effects of caesium (Cs) on living cells are poorly understood. Here, we examined the physiological role of Cs+ on the activity of the potassium transporters in E. coli. In the absence of potassium (K+), Kup-mediated Cs+ uptake partially supported cell growth, however, at a much lower rate than with sufficient K+. In K+-limited medium (0.1 mM), the presence of Cs+ (up to 25 mM) in the medium enhanced growth as much as control medium containing 1 mM K+. This effect depended on the maintenance of basal levels of intracellular K+ by other K+ uptake transporters. Higher amounts of K+ (1 mM) in the medium eliminated the positive effect of Cs+ on growth, and revealed the inhibitory effect of high Cs+ on the growth of wild-type E. coli. Cells lacking Kdp, TrkG and TrkH but expressing Kup grew less well when Cs+ was increased in the medium. A kdp mutant contained an increased ratio of Cs+/K+ in the presence of high Cs+ in the medium and consequently was strongly inhibited in growth. Taken together, under excess Cs+ conditions Kup-mediated Cs+ influx sustains cell growth, which is supported by intracellular K+ supplied by Kdp.

The topogenic function of S4 promotes membrane insertion of the voltage-sensor domain in the KvAP channel.

Voltage-dependent K+ (KV) channels control K+ permeability in response to shifts in the membrane potential. Voltage sensing in KV channels is mediated by the positively charged transmembrane domain S4. The best-characterized KV channel, KvAP, lacks the distinct hydrophilic region corresponding to the S3-S4 extracellular loop that is found in other K+ channels. In the present study, we evaluated the topogenic properties of the transmembrane regions within the voltage-sensing domain in KvAP. S3 had low membrane insertion activity, whereas S4 possessed a unique type-I signal anchor (SA-I) function, which enabled it to insert into the membrane by itself. S4 was also found to function as a stop-transfer signal for retention in the membrane. The length and structural nature of the extracellular S3-S4 loop affected the membrane insertion of S3 and S4, suggesting that S3 membrane insertion was dependent on S4. Replacement of charged residues within the transmembrane regions with residues of opposite charge revealed that Asp72 in S2 and Glu93 in S3 contributed to membrane insertion of S3 and S4, and increased the stability of S4 in the membrane. These results indicate that the SA-I function of S4, unique among K+ channels studied to date, promotes the insertion of S3 into the membrane, and that the charged residues essential for voltage sensing contribute to the membrane-insertion of the voltage sensor domain in KvAP.