Chloroplast envelope K+/H+ antiporters are involved in cytosol pH regulation.

Variations in light intensity induce cytosol pH changes in photosynthetic tissues, providing a possible signal to adjust a variety of biochemical, physiological and developmental processes to the energy status of the cells. It was shown that these pH changes are partially due to the transport of protons in or out of the thylakoid lumen. However, the ion transporters in the chloroplast that transmit these pH changes to the cytosol are not known. KEA1 and KEA2 are K+/H+ antiporters in the chloroplast inner envelope that adjust stromal pH in light-to-dark transitions. We previously determined that stromal pH is higher in kea1kea2 mutant cells. In this study, we now show that KEA1 and KEA2 are required to attenuate cytosol pH variations upon sudden light intensity changes in leaf mesophyll cells, showing they are important components of the light-modulated pH signalling module. The kea1kea2 mutant mesophyll cells also have a considerably less negative membrane potential. Membrane potential is dependent on the activity of the plasma membrane proton ATPase and is regulated by secondary ion transporters, mainly potassium channels in the plasma membrane. We did not find significant differences in the activity of the plasma membrane proton pump but found a strongly increased membrane permeability to protons, especially potassium, of the double mutant plasma membranes. Our results indicate that chloroplast envelope K+/H+ antiporters not only affect chloroplast pH but also have a strong impact on cellular ion homeostasis and energization of the plasma membrane.

Structure and mechanism of the K+/H+ exchanger KefC.

Intracellular potassium (K+) homeostasis is fundamental to cell viability. In addition to channels, K+ levels are maintained by various ion transporters. One major family is the proton-driven K+ efflux transporters, which in gram-negative bacteria is important for detoxification and in plants is critical for efficient photosynthesis and growth. Despite their importance, the structure and molecular basis for K+-selectivity is poorly understood. Here, we report ~3.1 Å resolution cryo-EM structures of the Escherichia coli glutathione (GSH)-gated K+ efflux transporter KefC in complex with AMP, AMP/GSH and an ion-binding variant. KefC forms a homodimer similar to the inward-facing conformation of Na+/H+ antiporter NapA. By structural assignment of a coordinated K+ ion, MD simulations, and SSM-based electrophysiology, we demonstrate how ion-binding in KefC is adapted for binding a dehydrated K+ ion. KefC harbors C-terminal regulator of K+ conductance (RCK) domains, as present in some bacterial K+-ion channels. The domain-swapped helices in the RCK domains bind AMP and GSH and they inhibit transport by directly interacting with the ion-transporter module. Taken together, we propose that KefC is activated by detachment of the RCK domains and that ion selectivity exploits the biophysical properties likewise adapted by K+-ion-channels.

Ethylene-induced potassium transporter AcKUP2 gene is involved in kiwifruit postharvest ripening.

BACKGROUND: Potassium (K) is important in the regulation of plant growth and development. It is the most abundant mineral element in kiwifruit, and its content increases during fruit ripening. However, how K+ transporter works in kiwifruit postharvest maturation is not yet clear. RESULTS: Here, 12 K+ transporter KT/HAK/KUP genes, AcKUP1 ~ AcKUP12, were isolated from kiwifruit, and their phylogeny, genomic structure, chromosomal location, protein properties, conserved motifs and cis-acting elements were analysed. Transcription analysis revealed that AcKUP2 expression increased rapidly and was maintained at a high level during postharvest maturation, consistent with the trend of K content; AcKUP2 expression was induced by ethylene, suggesting that AcKUP2 might play a role in ripening. Fluorescence microscopy showed that AcKUP2 is localised in the plasma membrane. Cis-elements, including DER or ethylene response element (ERE) responsive to ethylene, were found in the AcKUP2 promoter sequence, and ethylene significantly enhanced the AcKUP2 promoter activity. Furthermore, we verified that AcERF15, an ethylene response factor, directly binds to the AcKUP2 promoter to promote its expression. Thus, AcKUP2 may be an important potassium transporter gene which involved in ethylene-regulated kiwifruit postharvest ripening. CONCLUSIONS: Therefore, our study establishes the first genome-wide analysis of the kiwifruit KT/HAK/KUP gene family and provides valuable information for understanding the function of the KT/HAK/KUP genes in kiwifruit postharvest ripening.

Exogenous sucrose influences KEA1 and KEA2 to regulate abscisic acid-mediated primary root growth in Arabidopsis.

Arabidopsis K+-efflux antiporter (KEA)1 and KEA2 are chloroplast inner envelope membrane K+/H+ antiporters that play an important role in plastid development and seedling growth. However, the function of KEA1 and KEA2 during early seedling development is poorly understood. In this work, we found that in Arabidopsis, KEA1 and KEA2 mediated primary root growth by regulating photosynthesis and the ABA signaling pathway. Phenotypic analyses revealed that in the absence of sucrose, the primary root length of the kea1kea2 mutant was significantly shorter than that of the wild-type Columbia-0 (Col-0) plant. However, this phenotype could be remedied by the external application of sucrose. Meanwhile, HPLC-MS/MS results showed that in sucrose-free medium, ABA accumulation in the kea1kea2 mutant was considerably lower than that in Col-0. Transcriptome analysis revealed that many key genes involved in ABA signals were repressed in the kea1kea2 mutant. We concluded that KEA1 and KEA2 deficiency not only affected photosynthesis but was also involved in primary root growth likely through an ABA-dependent manner. This study confirmed the new function of KEA1 and KEA2 in affecting primary root growth.

Loss of inner-envelope K+/H+ exchangers impairs plastid rRNA maturation and gene expression.

The inner-envelope K+ EFFLUX ANTIPORTERS (KEA) 1 and 2 are critical for chloroplast development, ion homeostasis, and photosynthesis. However, the mechanisms by which changes in ion flux across the envelope affect organelle biogenesis remained elusive. Chloroplast development requires intricate coordination between the nuclear genome and the plastome. Many mutants compromised in plastid gene expression (PGE) display a virescent phenotype, that is delayed greening. The phenotypic appearance of Arabidopsis thaliana kea1 kea2 double mutants fulfills this criterion, yet a link to PGE has not been explored. Here, we show that a simultaneous loss of KEA1 and KEA2 results in maturation defects of the plastid ribosomal RNAs. This may be caused by secondary structure changes of rRNA transcripts and concomitant reduced binding of RNA-processing proteins, which we documented in the presence of skewed ion homeostasis in kea1 kea2. Consequently, protein synthesis and steady-state levels of plastome-encoded proteins remain low in mutants. Disturbance in PGE and other signs of plastid malfunction activate GENOMES UNCOUPLED 1-dependent retrograde signaling in kea1 kea2, resulting in a dramatic downregulation of GOLDEN2-LIKE transcription factors to halt expression of photosynthesis-associated nuclear-encoded genes (PhANGs). PhANG suppression delays the development of fully photosynthesizing kea1 kea2 chloroplasts, probably to avoid progressing photo-oxidative damage. Overall, our results reveal that KEA1/KEA2 function impacts plastid development via effects on RNA-metabolism and PGE.

Mitochondrial K+ Transport: Modulation and Functional Consequences.

The existence of a K+ cycle in mitochondria has been predicted since the development of the chemiosmotic theory and has been shown to be crucial for several cellular phenomena, including regulation of mitochondrial volume and redox state. One of the pathways known to participate in K+ cycling is the ATP-sensitive K+ channel, MitoKATP. This channel was vastly studied for promoting protection against ischemia reperfusion when pharmacologically activated, although its molecular identity remained unknown for decades. The recent molecular characterization of MitoKATP has opened new possibilities for modulation of this channel as a mechanism to control cellular processes. Here, we discuss different strategies to control MitoKATP activity and consider how these could be used as tools to regulate metabolism and cellular events.

c-di-AMP, a likely master regulator of bacterial K+ homeostasis machinery, activates a K+ exporter.

bis-(3',5')-cyclic diadenosine monophosphate (c-di-AMP) is a second messenger with roles in virulence, cell wall and biofilm formation, and surveillance of DNA integrity in many bacterial species, including pathogens. Strikingly, it has also been proposed to coordinate the activity of the components of K+ homeostasis machinery, inhibiting K+ import, and activating K+ export. However, there is a lack of quantitative evidence supporting the direct functional impact of c-di-AMP on K+ transporters. To gain a detailed understanding of the role of c-di-AMP on the activity of a component of the K+ homeostasis machinery in B. subtilis, we have characterized the impact of c-di-AMP on the functional, biochemical, and physiological properties of KhtTU, a K+/H+ antiporter composed of the membrane protein KhtU and the cytosolic protein KhtT. We have confirmed c-di-AMP binding to KhtT and determined the crystal structure of this complex. We have characterized in vitro the functional properties of KhtTU and KhtU alone and quantified the impact of c-di-AMP and of pH on their activity, demonstrating that c-di-AMP activates KhtTU and that pH increases its sensitivity to this nucleotide. Based on our functional and structural data, we were able to propose a mechanism for the activation of KhtTU by c-di-AMP. In addition, we have analyzed the impact of KhtTU in its native bacterium, providing a physiological context for the regulatory function of c-di-AMP and pH. Overall, we provide unique information that supports the proposal that c-di-AMP is a master regulator of K+ homeostasis machinery.

Bacterial infection reinforces host metabolic flux from arginine to spermine for NLRP3 inflammasome evasion.

Hosts recognize cytosolic microbial infection via the nucleotide-binding domain-like receptor (NLR) protein family, triggering inflammasome complex assembly to provoke pyroptosis or cytokine-related caspase-1-dependent antimicrobial responses. Pathogens have evolved diverse strategies to antagonize inflammasome activation. Here, Edwardsiella piscicida gene-defined transposon library screening for lactate dehydrogenase (LDH) release in nlrc4-/- bone marrow-derived macrophages (BMDMs) demonstrates that genes clustered in the bacterial arginine metabolism pathway participate in NLRP3 inflammasome inhibition. Blocking arginine uptake or putrescine export significantly relieves NLRP3 inflammasome inhibition, indicating that this bacterium rewires its arginine metabolism network during infection. Moreover, intracellular E. piscicida recruits the host arginine importer (mCAT-1) and putrescine exporter (Oct-2) to bacterium-containing vacuoles, accompanied by reduced arginine and accumulated cytosolic spermine. Neutralizing E. piscicida-induced cytosolic spermine enhancement by spermine synthetase or extracellular spermine significantly alters NLRP3 inflammasome activation. Importantly, accumulated cytosolic spermine inhibits K+ efflux-dependent NLRP3 inflammasome activation. These data highlight the mechanism of bacterial gene-mediated arginine metabolism control for NLRP3 inflammasome evasion.

Functional characterization of proton antiport regulation in the thylakoid membrane.

During photosynthesis, energy is transiently stored as an electrochemical proton gradient across the thylakoid membrane. The resulting proton motive force (pmf) is composed of a membrane potential (ΔΨ) and a proton concentration gradient (ΔpH) and powers the synthesis of ATP. Light energy availability for photosynthesis can change very rapidly and frequently in nature. Thylakoid ion transport proteins buffer the effects that light fluctuations have on photosynthesis by adjusting pmf and its composition. Ion channel activities dissipate ΔΨ, thereby reducing charge recombinations within photosystem II. The dissipation of ΔΨ allows for increased accumulation of protons in the thylakoid lumen, generating the signal that activates feedback downregulation of photosynthesis. Proton export from the lumen via the thylakoid K+ exchange antiporter 3 (KEA3), instead, decreases the ΔpH fraction of the pmf and thereby reduces the regulatory feedback signal. Here, we reveal that the Arabidopsis (Arabidopsis thaliana) KEA3 protein homo-dimerizes via its C-terminal domain. This C-terminus has a regulatory function, which responds to light intensity transients. Plants carrying a C-terminus-less KEA3 variant show reduced feed-back downregulation of photosynthesis and suffer from increased photosystem damage under long-term high light stress. However, during photosynthetic induction in high light, KEA3 deregulation leads to an increase in carbon fixation rates. Together, the data reveal a trade-off between long-term photoprotection and a short-term boost in carbon fixation rates, which is under the control of the KEA3 C-terminus.

Insights into the mechanisms of transport and regulation of the arabidopsis high-affinity K+ transporter HAK51.

The high-affinity K+ transporter HAK5 from Arabidopsis (Arabidopsis thaliana) is essential for K+ acquisition and plant growth at low micromolar K+ concentrations. Despite its functional relevance in plant nutrition, information about functional domains of HAK5 is scarce. Its activity is enhanced by phosphorylation via the AtCIPK23/AtCBL1-9 complex. Based on the recently published three-dimensionalstructure of the bacterial ortholog KimA from Bacillus subtilis, we have modeled AtHAK5 and, by a mutational approach, identified residues G67, Y70, G71, D72, D201, and E312 as essential for transporter function. According to the structural model, residues D72, D201, and E312 may bind K+, whereas residues G67, Y70, and G71 may shape the selective filter for K+, which resembles that of K+shaker-like channels. In addition, we show that phosphorylation of residue S35 by AtCIPK23 is required for reaching maximal transport activity. Serial deletions of the AtHAK5 C-terminus disclosed the presence of an autoinhibitory domain located between residues 571 and 633 together with an AtCIPK23-dependent activation domain downstream of position 633. Presumably, autoinhibition of AtHAK5 is counteracted by phosphorylation of S35 by AtCIPK23. Our results provide a molecular model for K+ transport and describe CIPK-CBL-mediated regulation of plant HAK transporters.

Close Temporal Relationship between Oscillating Cytosolic K+ and Growth in Root Hairs of Arabidopsis.

Root hair elongation relies on polarized cell expansion at the growing tip. As a major osmotically active ion, potassium is expected to be continuously assimilated to maintain cell turgor during hair tip growth. However, due to the lack of practicable detection methods, the dynamics and physiological role of K+ in hair growth are still unclear. In this report, we apply the small-molecule fluorescent K+ sensor NK3 in Arabidopsis root hairs for the first time. By employing NK3, oscillating cytoplasmic K+ dynamics can be resolved at the tip of growing root hairs, similar to the growth oscillation pattern. Cross-correlation analysis indicates that K+ oscillation leads the growth oscillations by approximately 1.5 s. Artificially increasing cytoplasmic K+ level showed no significant influence on hair growth rate, but led to the formation of swelling structures at the tip, an increase of cytosolic Ca2+ level and microfilament depolymerization, implying the involvement of antagonistic regulatory factors (e.g., Ca2+ signaling) in the causality between cytoplasmic K+ and hair growth. These results suggest that, in each round of oscillating root hair elongation, the oscillatory cell expansion accelerates on the heels of cytosolic K+ increment, and decelerates with the activation of antagonistic regulators, thus forming a negative feedback loop which ensures the normal growth of root hairs.

A single nucleotide substitution in TaHKT1;5-D controls shoot Na+ accumulation in bread wheat.

Improving salinity tolerance in the most widely cultivated cereal, bread wheat (Triticum aestivum L.), is essential to increase grain yields on saline agricultural lands. A Portuguese landrace, Mocho de Espiga Branca accumulates up to sixfold greater leaf and sheath sodium (Na+ ) than two Australian cultivars, Gladius and Scout, under salt stress in hydroponics. Despite high leaf and sheath Na+ concentrations, Mocho de Espiga Branca maintained similar salinity tolerance compared to Gladius and Scout. A naturally occurring single nucleotide substitution was identified in the gene encoding a major Na+ transporter TaHKT1;5-D in Mocho de Espiga Branca, which resulted in a L190P amino acid residue variation. This variant prevents Mocho de Espiga Branca from retrieving Na+ from the root xylem leading to a high shoot Na+ concentration. The identification of the tissue-tolerant Mocho de Espiga Branca will accelerate the development of more elite salt-tolerant bread wheat cultivars.

Arabidopsis K+ transporter HAK5-mediated high-affinity root K+ uptake is regulated by protein kinases CIPK1 and CIPK9.

The high-affinity K+ transporter HAK5 is the major contributor to root K+ uptake from dilute solutions in K+-starved Arabidopsis plants. Its functionality is tightly regulated and its activity is enhanced under K+ starvation by the transcriptional induction of the AtHAK5 gene, and by the activation of the transporter via the AtCBL1-AtCIPK23 complex. In the present study, the 26 members of the Arabidopsis CIPK protein kinase family were screened in yeast for their capacity to activate HAK5-mediated K+ uptake. Among them, AtCIPK1 was the most efficient activator of AtHAK5. In addition, AtCIPK9, previously reported to participate in K+ homeostasis, also activated the transporter. In roots, the genes encoding AtCIPK1 and AtCIPK9 were induced by K+ deprivation and atcipk1 and atcipk9 Arabidopsis KO mutants showed a reduced AtHAK5-mediated Rb+ uptake. Activation of AtHAK5 by AtCIPK1 did not occur under hyperosmotic stress conditions, where AtCIPK1 function has been shown to be required to maintain plant growth. Taken together, our data contribute to the identification of the complex regulatory networks that control the high-affinity K+ transporter AtHAK5 and root K+ uptake.

Solution structure of the cytoplasmic domain of NhaP2 a K+/H+ antiporter from Vibrio cholera.

NhaP2 is a K+/H+ antiporter from Vibrio cholerae which consists of a transmembrane domain and a cytoplasmic domain of approximately 200 amino acids, both of which are required for cholera infectivity. Here we present the solution structure for a 165 amino acid minimal cytoplasmic domain (P2MIN) form of the protein. The structure reveals a compact N-terminal domain which resembles a Regulator of Conductance of K+ channels (RCK) domain connected to a more open C-terminal domain via a flexible 20 amino acid linker. NMR titration experiments showed that the protein binds ATP through its N-terminal domain, which was further supported by waterLOGSY and Saturation Transfer Difference NMR experiments. The two-domain organisation of the protein was confirmed by BIOSAXS, which also revealed that there are no detectable-ATP-induced conformational changes in the protein structure. Finally, in contrast to all known RCK domain structures solved to date, the current work shows that the protein is a monomer.

Engineering salinity tolerance in plants: progress and prospects.

MAIN CONCLUSION: There is a need to integrate conceptual framework based on the current understanding of salt stress responses with different approaches for manipulating and improving salt tolerance in crop plants. Soil salinity exerts significant constraints on global crop production, posing a serious challenge for plant breeders and biotechnologists. The classical transgenic approach for enhancing salinity tolerance in plants revolves by boosting endogenous defence mechanisms, often via a single-gene approach, and usually involves the enhanced synthesis of compatible osmolytes, antioxidants, polyamines, maintenance of hormone homeostasis, modification of transporters and/or regulatory proteins, including transcription factors and alternative splicing events. Occasionally, genetic manipulation of regulatory proteins or phytohormone levels confers salinity tolerance, but all these may cause undesired reduction in plant growth and/or yields. In this review, we present and evaluate novel and cutting-edge approaches for engineering salt tolerance in crop plants. First, we cover recent findings regarding the importance of regulatory proteins and transporters, and how they can be used to enhance salt tolerance in crop plants. We also evaluate the importance of halobiomes as a reservoir of genes that can be used for engineering salt tolerance in glycophytic crops. Additionally, the role of microRNAs as critical post-transcriptional regulators in plant adaptive responses to salt stress is reviewed and their use for engineering salt-tolerant crop plants is critically assessed. The potentials of alternative splicing mechanisms and targeted gene-editing technologies in understanding plant salt stress responses and developing salt-tolerant crop plants are also discussed.

H+ Transport by K+ EXCHANGE ANTIPORTER3 Promotes Photosynthesis and Growth in Chloroplast ATP Synthase Mutants.

The composition of the thylakoid proton motive force (pmf) is regulated by thylakoid ion transport. Passive ion channels in the thylakoid membrane dissipate the membrane potential (Δψ) component to allow for a higher fraction of pmf stored as a proton concentration gradient (ΔpH). K+/H+ antiport across the thylakoid membrane via K+ EXCHANGE ANTIPORTER3 (KEA3) instead reduces the ΔpH fraction of the pmf. Thereby, KEA3 decreases nonphotochemical quenching (NPQ), thus allowing for higher light use efficiency, which is particularly important during transitions from high to low light. Here, we show that in the background of the Arabidopsis (Arabidopsis thaliana) chloroplast (cp)ATP synthase assembly mutant cgl160, with decreased cpATP synthase activity and increased pmf amplitude, KEA3 plays an important role for photosynthesis and plant growth under steady-state conditions. By comparing cgl160 single with cgl160 kea3 double mutants, we demonstrate that in the cgl160 background loss of KEA3 causes a strong growth penalty. This is due to a reduced photosynthetic capacity of cgl160 kea3 mutants, as these plants have a lower lumenal pH than cgl160 mutants, and thus show substantially increased pH-dependent NPQ and decreased electron transport through the cytochrome b 6 f complex. Overexpression of KEA3 in the cgl160 background reduces pH-dependent NPQ and increases photosystem II efficiency. Taken together, our data provide evidence that under conditions where cpATP synthase activity is low, a KEA3-dependent reduction of ΔpH benefits photosynthesis and growth.

The topology of plastid inner envelope potassium cation efflux antiporter KEA1 provides new insights into its regulatory features.

The plastid potassium cation efflux antiporters (KEAs) are important for chloroplast function, development, and photosynthesis. To understand their regulation at the protein level is therefore of fundamental importance. Prior studies have focused on the regulatory K+ transport and NAD-binding (KTN) domain in the C-terminus of the thylakoid carrier KEA3 but the localization of this domain remains unclear. While all three plastid KEA members are highly conserved in their transmembrane region and the C-terminal KTN domain, only the inner envelope KEA family members KEA1 and KEA2 carry a long soluble N-terminus. Interestingly, this region is acetylated at lysine 168 by the stromal acetyltransferase enzyme NSI. If an odd number of transmembrane domains existed for inner envelope KEAs, as it was suggested for all three plastid KEA carriers, regulatory domains and consequently protein regulation would occur on opposing sides of the inner envelope. In this study we therefore set out to investigate the topology of inner envelope KEA proteins. Using a newly designed antibody specific to the envelope KEA1 N-terminus and transgenic Arabidopsis plants expressing a C-terminal KEA1-YFP fusion protein, we show that both, the N-terminal and C-terminal, regulatory domains of KEA1 reside in the chloroplast stroma and not in the intermembrane space. Considering the high homology between KEA1 and KEA2, we therefore reason that envelope KEAs must consist of an even number of transmembrane domains.

Roles for intracellular cation transporters in respiratory growth of yeast.

Potassium is involved in copper and iron metabolism in eukaryotic Golgi apparatus, but it is not clear yet whether potassium distributions in other vesicles also affect copper and iron metabolism. Here we show that respiratory growth and iron acquisition by the yeast Saccharomyces cerevisiae relies on potassium (K+) compartmentalization to the mitochondria, as well as the vacuole and late endosome via K+/H+ exchangers Mdm38p, Vnx1p and Nhx1p, respectively. The data indicate that NHX1 and VNX1 knock-out cells grow better than wild type cells on non-fermentable YPEG media, while MDM38 knock-out cells display a growth defect on YPEG media. The over expression of the KHA1 gene located on the Golgi apparatus partially compensates for the growth defect of the MDM38 knock-out strain. The results suggest that the vacuole and late endosome are important potassium storage vesicles and Mdm38p affects the mitochondrial function by regulating copper and iron metabolism. Our study reveals potassium compartmentalization to the subcellular vesicles is relevant for respiratory growth by improving copper utilization and promoting iron absorption.

Modification of Activity of the Thylakoid H+/K+ Antiporter KEA3 Disturbs ∆pH-Dependent Regulation of Photosynthesis.

The thylakoid K+ efflux antiporter 3 (KEA3) is required for regulating components of the proton motive force (pmf), proton concentration gradient (ΔpH), and membrane potential (Δψ). The Arabidopsis (Arabidopsis thaliana) disturbed proton gradient regulation mutant (dpgr) is a dominant allele of KEA3, conferring disturbed transport activity. Here, we show that overexpressing the DPGR-type KEA3 (DPGRox) retarded plant growth, whereas overexpressing the wild-type KEA3 (KEA3ox) did not. In KEA3ox lines, the contribution of Δψ to pmf was enhanced, but in DPGRox lines, the size of pmf was reduced. In DPGRox plants, proton conductivity of the thylakoid membrane (g H +) was elevated under high light, implying disturbed stoichiometry of H+/K+ antiport through DPGR-type KEA3 rather than simply enhanced activity. The ΔpH-dependent regulation consisting of thermal dissipation of excessively absorbed light energy and downregulation of cytochrome b 6 f complex activity was severely and mildly affected in DPGRox and KEA3ox plants, respectively. Consequently, photosystem I was sensitive to fluctuating light in both transgenic plants. Both photosystems were sensitive to constant high light and were slightly photodamaged even at standard growth light intensity in DPGRox plants. KEA3 regulates the components of pmf and optimizes the operation of ∆pH-dependent regulation of electron transport.

Evidence for potassium transport activity of Arabidopsis KEA1-KEA6.

Arabidopsis thaliana contains the putative K+ efflux transporters KEA1-KEA6, similar to KefB and KefC of Escherichia coli. KEA1-KEA3 are involved in the regulation of photosynthetic electron transport and chloroplast development. KEA4-KEA6 mediate pH regulation of the endomembrane network during salinity stress. However, the ion transport activities of KEA1-KEA6 have not been directly characterized. In this study, we used an E. coli expression system to examine KEA activity. KEA1-KEA3 and KEA5 showed bi-directional K+ transport activity, whereas KEA4 and KEA6 functioned as a K+ uptake system. The thylakoid membrane-localized Na+/H+ antiporter NhaS3 from the model cyanobacterium Synechocystis is the closest homolog of KEA3. Changing the putative Na+/H+ selective site of KEA3 (Gln-Asp) to that of NhaS3 (Asp-Asp) did not alter the ion selectivity without loss of K+ transport activity. The first residue in the conserved motif was not a determinant for K+ or Na+ selectivity. Deletion of the possible nucleotide-binding KTN domain from KEA3 lowered K+ transport activity, indicating that the KTN domain was important for this function. The KEA3-G422R mutation discovered in the Arabidopsis dpgr mutant increased K+ transport activity, consistent with the mutant phenotype. These results indicate that Arabidopsis KEA1-KEA6 act as K+ transport systems, and support the interpretation that KEA3 promotes dissipation of ΔpH in the thylakoid membrane.