Cation Proton Antiporter Research Articles

Genome-Wide Identification of the Cation/Proton Antiporter (CPA) Gene Family and Functional Analysis of AtrNHX8 under Salt Stress.

Amaranthus tricolor is an important vegetable, and its quality is affected by salt stress. Cation/proton antiporters (CPA) contribute to plant development and tolerance to salt stress. In this study, 35 CPA genes were identified from a genome database for A. tricolor, including 9 NHX, 5 KEA, and 21 CPA2 genes. Furthermore, in A. tricolor, the expression levels of most AtrNHX genes were higher at a low salinity level (50 or 100 mM NaCl) than in the control or 200 mM NaCl treatment. Levels of most AtrNHX genes were elevated in the stem. Moreover, AtrNHX8 was homologous to AtNHX4, which is involved in the regulation of sodium homeostasis and salt stress response. After AtrNHX8 overexpression in Arabidopsis thaliana, seed germination was better, and the flowering time was earlier than that of wild-type plants. Additionally, the overexpression of AtrNHX8 in A. thaliana improved salt tolerance. These results reveal the roles of AtrNHX genes under salt stress and provide valuable information on this gene family in amaranth.

Site-directed mutagenesis at the Glu78 in Ec-NhaA transporter impacting ion exchange: a biophysical study.

Na+/H+ antiporters facilitate the exchange of Na+ for H+ across the cytoplasmic membrane in prokaryotic and eukaryotic cells. These transporters are crucial to maintain the homeostasis of sodium ions, consequently pH, and volume of the cells. Therefore, sodium/proton antiporters are considered promising therapeutic targets in humans. The Na+/H+ antiporter in Escherichia coli (Ec-NhaA), a prototype of cation-proton antiporter (CPA) family, transports two protons and one sodium (or Li+) in opposite direction. Previous mutagenesis experiments on Ec-NhaA have proposed Asp164, Asp163, and Asp133 amino acids with the significant implication in functional and structural integrity and create site for ion-binding. However, the mechanism and the sites for the binding of the two protons remain unknown and controversial which could be critical for pH regulation. In this study, we have explored the role of Glu78 in the regulation of pH by Ec-NhaA. Although we have created various mutants, E78C has shown a considerable effect on the stoichiometry of NhaA and presented comparable phenotypes. The ITC experiment has shown the binding of ~ 5 protons in response to the transport of one lithium ion. The phenotype analysis on selective medium showed a significant expression compared to WT Ec-NhaA. This represents the importance of Glu78 in transporting the H+ across the membrane where a single mutation with Cys amino acid alters the number of H+ significantly maintaining the activity of the protein.

Genome-wide identification of the monovalent cation/proton antiporter (CPA) superfamily and expression analysis in tomato

The cation/proton antiporter (CPA) superfamily, crucial for regulating Na+, K+, and pH equilibrium in plant cells, plays an essential role in plant growth and stress adaptation. However, limited research has been conducted on the CPA gene superfamily in tomatoes. In this study, a total of 31 SlCPA members were identified in the tomato genome, comprising 18 cation/H+ exchangers (SlCHXs), 7 Na+/H+ exchangers (SlNHXs), and 6 K+ efflux antiporters (SlKEAs). A comprehensive analysis encompassing their chromosomal locations, gene structures, phylogenetic relationships, collinearity, tissue-specific expression profiles, and responses to abiotic stress was undertaken. Collinearity and phylogenetic analyses unveiled two groups, represented by Solyc08g081810, Solyc01g008190, Solyc12g056160, and Solyc06g060630, Solyc08g078880, suggesting potential redundancy resulting from duplication events. Tissue-specific expression analysis revealed SlNHXs and SlKEAs expression at multiple developmental stages, while SlCHXs were predominantly expressed during early flower and fruit development, indicating their likely involvement in these processes. Salt treatment notably induced the expression of SlSOS1 (Solyc01g005020) and Solyc04g056600, implicating their role in responding to salt stress. Under drought stress, the expression of Solyc01g005020 and Solyc08g081820 was also significantly induced, supported by promoter element analyses indicating the presence of drought response-related cis-elements like ABRE and MYB binding sites. This study establishes a foundation for further elucidating the gene functions of individual members within the tomato SlCPA family.

Genome-wide characterization of the CPA gene family in potato and a preliminary functional analysis of its role in NaCl tolerance.

The cation/proton antiporter (CPA) superfamily plays a crucial role in regulating ion homeostasis and pH in plant cells, contributing to stress resistance. However, in potato (Solanum tuberosum L.), systematic identification and analysis of CPA genes are lacking. A total of 33 StCPA members were identified and classified into StNHX (n = 7), StKEA (n = 6), and StCHX (n = 20) subfamilies. StCHX owned the highest number of conserved motifs, followed by StKEA and StNHX. The StNHX and StKEA subfamilies owned more exons than StCHX. NaCl stress induced the differentially expression of 19 genes in roots or leaves, among which StCHX14 and StCHX16 were specifically induced in leaves, while StCHX2 and StCHX19 were specifically expressed in the roots. A total of 11 strongly responded genes were further verified by qPCR. Six CPA family members, StNHX1, StNHX2, StNHX3, StNHX5, StNHX6 and StCHX19, were proved to transport Na+ through yeast complementation experiments. This study provides comprehensive insights into StCPAs and their response to NaCl stress, facilitating further functional characterization.

Genome-wide identification of the cation/proton antiporter (CPA) gene family and functional characterization of the key member BnaA05.NHX2 in allotetraploid rapeseed

Rapeseed (Brassica napus L.) is susceptible to nutrient stresses during growth and development; however, the CPA (cation proton antiporter) family genes have not been identified in B. napus and their biological functions remain unclear. This study was aimed to identify the molecular characteristics of rapeseed CPAs and their transcriptional responses to multiple nutrient stresses. Through bioinformatics analysis, 117 BnaCPAs, consisting of three subfamilies: Na+/H+ antiporter (NHX), K+ efflux antiporter (KEA), and cation/H+ antiporter (CHX), were identified in the rapeseed genome. Transcriptomic profiling showed that BnaCPAs, particularly BnaNHXs, were transcriptionally responsive to diverse nutrient stresses, including Cd toxicity, K starvation, salt stress, NH4+ toxicity, and low Pi. We found that the salt tolerance of the transgenic rapeseed lines overexpressing BnaA05.NHX2 was significantly higher than that of wild type. Subcellular localization showed that BnaA05.NHX2 was localized on the tonoplast, and TEM combined with X-ray energy spectrum analysis revealed that the vacuolar Na+ concentrations of the BnaA05.NHX2-overexpressing rapeseed plants were significantly higher than those of wild type. The findings of this study will provide insights into the complexity of the BnaCPA family and a valuable resource to explore the in-depth functions of CPAs in B. napus.

Genome-Wide Analysis of Cation/Proton Antiporter Family in Soybean (Glycine max) and Functional Analysis of GmCHX20a on Salt Response

Monovalent cation proton antiporters (CPAs) play crucial roles in ion and pH homeostasis, which is essential for plant development and environmental adaptation, including salt tolerance. Here, 68 CPA genes were identified in soybean, phylogenetically dividing into 11 Na+/H+ exchangers (NHXs), 12 K+ efflux antiporters (KEAs), and 45 cation/H+ exchangers (CHXs). The GmCPA genes are unevenly distributed across the 20 chromosomes and might expand largely due to segmental duplication in soybean. The GmCPA family underwent purifying selection rather than neutral or positive selections. The cis-element analysis and the publicly available transcriptome data indicated that GmCPAs are involved in development and various environmental adaptations, especially for salt tolerance. Based on the RNA-seq data, twelve of the chosen GmCPA genes were confirmed for their differentially expression under salt or osmotic stresses using qRT-PCR. Among them, GmCHX20a was selected due to its high induction under salt stress for the exploration of its biological function on salt responses by ectopic expressing in Arabidopsis. The results suggest that the overexpression of GmCHX20a increases the sensitivity to salt stress by altering the redox system. Overall, this study provides comprehensive insights into the CPA family in soybean and has the potential to supply new candidate genes to develop salt-tolerant soybean varieties.

TMCO3, a Putative K+ :Proton Antiporter at the Golgi Apparatus, Is Important for Longitudinal Growth in Mice and Humans.

Isolated short stature, defined as short stature without any other abnormalities, is a common heterogeneous condition in children. Exome sequencing identified the homozygous nonsense variant c.1832G>A/p.(Trp611*) in TMCO3 in two sisters with isolated short stature. Radiological studies, biochemical measurements, assessment of the skeletal status, and three-dimensional bone microarchitecture revealed no relevant skeletal and bone abnormalities in both sisters. The homozygous TMCO3 variant segregated with short stature in the family. TMCO3 transcript levels were reduced by ~50% in leukocyte-derived RNA of both sisters compared with controls, likely due to nonsense-mediated mRNA decay. In primary urinary cells of heterozygous family members, we detected significantly reduced TMCO3 protein levels. TMCO3 is functionally uncharacterized. We ectopically expressed wild-type TMCO3 in HeLa and ATDC5 chondrogenic cells and detected TMCO3 predominantly at the Golgi apparatus, whereas the TMCO3W611* mutant did not reach the Golgi. Coordinated co-expression of TMCO3W611* -HA and EGFP in HeLa cells confirmed intrinsic instability and/or degradation of the mutant. Tmco3 is expressed in all relevant mouse skeletal cell types. Highest abundance of Tmco3 was found in chondrocytes of the prehypertrophic zone in mouse and minipig growth plates where it co-localizes with a Golgi marker. Knockdown of Tmco3 in differentiated ATDC5 cells caused reduced and increased expression of Pthlh and Ihh, respectively. Measurement of long bones in Tmco3tm1b(KOMP)Wtsi knockout mice revealed significant shortening of forelimbs and hindlimbs. TMCO3 is a potential member of the monovalent cation:proton antiporter 2 (CPA2) family. By in silico tools and homology modeling, TMCO3 is predicted to have an N-terminal secretory signal peptide, forms a dimer localized to the membrane, and is organized in a dimerization and a core domain. The core domain contains the CPA2 motif essential for K+ binding and selectivity. Collectively, our data demonstrate that loss of TMCO3 causes growth defects in both humans and mice. © 2023 American Society for Bone and Mineral Research (ASBMR).

Structure-Guided Identification of Critical Residues in the Vacuolar Cation/Proton Antiporter NHX1 from Arabidopsis thaliana.

Cation/Proton Antiporters (CPA) acting in all biological membranes regulate the volume and pH of cells and of intracellular organelles. A key issue with these proteins is their structure-function relationships since they present intrinsic regulatory features that rely on structural determinants, including pH sensitivity and the stoichiometry of ion exchange. Crystal structures are only available for prokaryotic CPA, whereas the eukaryotic ones have been modeled using the former as templates. Here, we present an updated and improved structural model of the tonoplast-localized K+, Na+/H+ antiporter NHX1 of Arabidopsis as a representative of the vacuolar NHX family that is essential for the accumulation of K+ into plant vacuoles. Conserved residues that were judged as functionally important were mutated, and the resulting protein variants were tested for activity in the yeast Saccharomyces cerevisiae. The results indicate that residue N184 in the ND-motif characteristic of CPA1 could be replaced by the DD-motif of CPA2 family members with minimal consequences for their activity. Attempts to alter the electroneutrality of AtNHX1 by different combinations of amino acid replacements at N184, R353 and R390 residues resulted in inactive or partly active proteins with a differential ability to control the vacuolar pH of the yeast.

Cation/proton antiporters: novel structure-driven pharmaceutical opportunities

Cation/proton antiporters (CPAs) regulate cells’ salt concentration and pH. Their malfunction is associated with a range of human pathologies, yet only a handful of CPA-targeting therapeutics are presently in clinical development. Here, we discuss how recently published mammalian protein structures and emerging computational technologies may help to bridge this gap.

Prokaryotic Na+/H+ Exchangers-Transport Mechanism and Essential Residues.

Na+/H+ exchangers are essential for Na+ and pH homeostasis in all organisms. Human Na+/H+ exchangers are of high medical interest, and insights into their structure and function are aided by the investigation of prokaryotic homologues. Most prokaryotic Na+/H+ exchangers belong to either the Cation/Proton Antiporter (CPA) superfamily, the Ion Transport (IT) superfamily, or the Na+-translocating Mrp transporter superfamily. Several structures have been solved so far for CPA and Mrp members, but none for the IT members. NhaA from E. coli has served as the prototype of Na+/H+ exchangers due to the high amount of structural and functional data available. Recent structures from other CPA exchangers, together with diverse functional information, have allowed elucidation of some common working principles shared by Na+/H+ exchangers from different families, such as the type of residues involved in the substrate binding and even a simple mechanism sufficient to explain the pH regulation in the CPA and IT superfamilies. Here, we review several aspects of prokaryotic Na+/H+ exchanger structure and function, discussing the similarities and differences between different transporters, with a focus on the CPA and IT exchangers. We also discuss the proposed transport mechanisms for Na+/H+ exchangers that explain their highly pH-regulated activity profile.

Cation/Proton Antiporter Genes in Tomato: Genomic Characterization, Expression Profiling, and Co-Localization with Salt Stress-Related QTLs

The cation/proton antiporter (CPA) family represents a class of transmembrane transporter proteins that play a crucial role in plants during high salinity stress by maintaining the cell’s ionic balance and pH homeostasis. So far, the CPA genes have not been systematically characterized in tomato (Solanum lycopersicum). In this study, we identified and analyzed 33 putative CPA genes in tomato. Phylogenetic analysis showed that tomato CPAs could be classified into three subgroups, i.e., CHX (18 genes), KEA (8 genes), and NHX (7 genes). CPA genes within each subgroup shared similar motifs, conserved catalytic domains and gene structure. Further analysis revealed that the CPA genes were unevenly distributed on the chromosomes and segmental duplication events played a major role in the expansion of the CPA gene family in tomato. Gene expression analysis exhibited that CPA genes were differentially expressed in different tissues, various stages of fruit development, and differentially regulated in response to abiotic stresses, especially salt stress. Further, co-localization of tomato CPA genes with quantitative trait loci (QTL) of salt stress-related phenotypes revealed their broader functions in salt stress tolerance. Finally, predicted protein–protein interactions of tomato CPAs, gene ontology analysis, and the presence of putative cis-elements in the promoter further support the diverse role of tomato CPAs in plant development and plant stress tolerance. In brief, this study highlights the potential role of tomato CPAs in plant development and abiotic stress tolerance, especially in salt stress, and provides comprehensive information to explore new candidate genes for salt tolerance in tomato.

Slipknotted and unknotted monovalent cation-proton antiporters evolved from a common ancestor.

While the slipknot topology in proteins has been known for over a decade, its evolutionary origin is still a mystery. We have identified a previously overlooked slipknot motif in a family of two-domain membrane transporters. Moreover, we found that these proteins are homologous to several families of unknotted membrane proteins. This allows us to directly investigate the evolution of the slipknot motif. Based on our comprehensive analysis of 17 distantly related protein families, we have found that slipknotted and unknotted proteins share a common structural motif. Furthermore, this motif is conserved on the sequential level as well. Our results suggest that, regardless of topology, the proteins we studied evolved from a common unknotted ancestor single domain protein. Our phylogenetic analysis suggests the presence of at least seven parallel evolutionary scenarios that led to the current diversity of proteins in question. The tools we have developed in the process can now be used to investigate the evolution of other repeated-domain proteins.

Pharmacophore-based screening and modification of amiloride analogs for targeting the NhaP-type cation-proton antiporter in Vibrio cholerae.

The genome of Vibrio cholerae contains three structural genes for the NhaP-type cation-proton antiporter paralogues, Vc-NhaP1, Vc-NhaP2, and Vc-NhaP3, mediating exchange of K+ and or Na+ for protons across the membrane. Based on phenotypic analysis of chromosomal Vc-NhaP1, Vc-NhaP2, and Vc-NhaP3 triple deletion mutants, we suggest that Vc-NhaP paralogues are primarily K+/H+ antiporters and might play a role in the acid tolerance response of V.cholerae as it passes through the gastric acid barrier of the stomach. Comparison of the biochemical properties of Vc-NhaP isoforms revealed that Vc-NhaP2 was the most active among all three paralogues. Therefore, the Vc-NhaP2 antiporter is a plausible therapeutic target for developing novel inhibitors targeting these ion exchangers. Our structural and mutational analysis of Vc-NhaP2 identified a putative cation-binding pocket formed by antiparallel extended regions of two transmembrane segments (TMSs V and XII) along with TMS VI. Molecular dynamics simulations suggested that the flexibility of TMSs V and XII is crucial for intramolecular conformational events in Vc-NhaP2. In this study, we developed putative Vc-NhaP2 inhibitors from amiloride analogs. Molecular docking of the modified amiloride analogs revealed promising binding properties. The four selected drugs potentially interacted with functionally important amino acid residues located on the cytoplasmic side of TMS VI, the extended chain region of TMSs V and XII, and the loop region between TMSs VIIII and IX. Molecular dynamics simulations revealed that binding of the selected drugs can potentially destabilize Vc-NhaP2 and alter the flexibility of functionally important TMS VI. This work presents the utility of in silico approaches for the rational identification of potential targets and drugs that could target NhaP2 cation proton antiporters to control V. cholerae. The goal was to identify potential drugs that could be validated in future experiments.

Molecular insights into the role of plant transporters in salt stress response.

Salt stress disturbs the cellular osmotic and ionic balance, which then creates a negative impact on plant growth and development. The Na+ and Cl- ions can enter into plant cells through various membrane transporters, including specific and non-specific Na+ , K+ , and Ca2+ transporters. Therefore, it is important to understand Na+ and K+ transport mechanisms in plants along with the isolation of genes, their characterization, the structural features, and their post-translation regulation under salt stress. This review summarizes the molecular insights of plant ion transporters, including non-selective cation transporters, cyclic nucleotide-gated cation transporters, glutamate-like receptors, membrane intrinsic proteins, cation proton antiporters, and sodium proton antiporter families. Further, we discussed the K+ transporter families such as high-affinity K+ transporters, HAK/KUP/KT transporters, shaker type K+ transporters, and K+ efflux antiporters. Besides the ion transport process, we have shed light on available literature on epigenetic regulation of transport processes under salt stress. Recent advancements of salt stress sensing mechanisms and various salt sensors within signaling transduction pathways are discussed. Further, we have compiled salt-stress signaling pathways, and their crosstalk with phytohormones.

Identification and characterisation of monovalent cation/proton antiporters (CPAs) in Phyllostachys edulis and the functional analysis of PheNHX2 in Arabidopsis thaliana

Plant monovalent cation/proton antiporters (CPAs), types of transmembrane transporters, play important roles in resistance to salt stress. In this study, 37 CPA genes from moso bamboo (Phyllostachys edulis) were identified and characterised. The expression profiles of 10 CPA1 genes (PheNHXs) of moso bamboo were detected by qRT-PCR, which showed that they were specifically expressed in six tissues. In addition, the expression of 10 PheNHXs in leaves and roots changed significantly under 150/200 mM NaCl and 100 μM ABA treatments. In particular, the expression of PheNHX2 in leaves and roots was significantly upregulated under NaCl treatment, thus, we cloned PheNHX2 and analysed its function. Subcellular localisation analysis showed that PheNHX2 was located on the vacuolar membrane. Overexpression of PheNHX2 reduced seed germination and root growth of Arabidopsis thaliana under salt stress, as well as severely affecting cellular Na+ and K+ content, which in turn reduced the salt tolerance of transgenic Arabidopsis. Measurements of physiological indicators, including chlorophyll content, malondialdehyde content, peroxidase and catalase enzyme activities and relative electrical conductivity, all supported this conclusion. Under salt stress, PheNHX2 also inhibited the expression of some stress-related and ion transport-related genes in transgenic Arabidopsis. Overall, these results indicate that overexpression of PheNHX2 reduces the salt tolerance of transgenic Arabidopsis. This investigation establishes a foundation for subsequent functional studies of moso bamboo CPA genes, and it provides a deeper understanding of PheNHX2 regulation in relation to the salt tolerance of moso bamboo.

Genome-wide identification, characterization, and expression analysis of the monovalent cation-proton antiporter superfamily in maize, and functional analysis of its role in salt tolerance

Na+, K+ and pH homeostasis are important for plant life and they are controlled by the monovalent cation proton antiporter (CPA) superfamily. The roles of ZmCPAs in salt tolerance are not fully elucidated. In this study, we identified 35 ZmCPAs comprising 13 Na+/H+ exchangers (ZmNHXs), 16 cation/H+ exchanger (ZmCHXs), and 6 K+ efflux antiporters (ZmKEAs). All ZmCPAs have transmembrane domains and most of them were localized to plasma membrane or tonoplast. ZmCHXs were specifically highly expressed in anthers, while ZmNHXs and ZmKEAs showed high expression in various tissues. ZmNHX5 and ZmKEA2 were up-regulated in maize seedlings under both NaCl and KCl stresses. Yeast complementation experiments revealed the roles of ZmNHX5, ZmKEA2 in NaCl tolerance. Analysis of the maize mutants further validated the salt tolerance functions of ZmNHX5 and ZmKEA2. Our study highlights comprehensive information of ZmCPAs and provides new gene targets for salt tolerance maize breeding.

An angular motion of a conserved four-helix bundle facilitates alternating access transport in the TtNapA and EcNhaA transporters

There is ongoing debate regarding the mechanism through which cation/proton antiporters (CPAs), like Thermus thermophilus NapA (TtNapA) and Escherichia coli NapA (EcNhaA), alternate between their outward- and inward-facing conformations in the membrane. CPAs comprise two domains, and it is unclear whether the transition is driven by their rocking-bundle or elevator motion with respect to each other. Here we address this question using metadynamics simulations of TtNapA, where we bias conformational sampling along two axes characterizing the two proposed mechanisms: angular and translational motions, respectively. By applying the bias potential for the two axes simultaneously, as well as to the angular, but not the translational, axis alone, we manage to reproduce each of the two known states of TtNapA when starting from the opposite state, in support of the rocking-bundle mechanism as the driver of conformational change. Next, starting from the inward-facing conformation of EcNhaA, we sample what could be its long-sought-after outward-facing conformation and verify it using cross-linking experiments.

Genome-Wide Identification and Functional Characterization of the Cation Proton Antiporter (CPA) Family Related to Salt Stress Response in Radish (Raphanus sativus L.).

The CPA (cation proton antiporter) family plays an essential role during plant stress tolerance by regulating ionic and pH homeostasis of the cell. Radish fleshy roots are susceptible to abiotic stress during growth and development, especially salt stress. To date, CPA family genes have not yet been identified in radish and the biological functions remain unclear. In this study, 60 CPA candidate genes in radish were identified on the whole genome level, which were divided into three subfamilies including the Na+/H+ exchanger (NHX), K+ efflux antiporter (KEA), and cation/H+ exchanger (CHX) families. In total, 58 of the 60 RsCPA genes were localized to the nine chromosomes. RNA-seq. data showed that 60 RsCPA genes had various expression levels in the leaves, roots, cortex, cambium, and xylem at different development stages, as well as under different abiotic stresses. RT–qPCR analysis indicated that all nine RsNHXs genes showed up regulated trends after 250 mM NaCl exposure at 3, 6, 12, and 24h. The RsCPA31 (RsNHX1) gene, which might be the most important members of the RsNHX subfamily, exhibited obvious increased expression levels during 24h salt stress treatment. Heterologous over-and inhibited-expression of RsNHX1 in Arabidopsis showed that RsNHX1 had a positive function in salt tolerance. Furthermore, a turnip yellow mosaic virus (TYMV)-induced gene silence (VIGS) system was firstly used to functionally characterize the candidate gene in radish, which showed that plant with the silence of endogenous RsNHX1 was more susceptible to the salt stress. According to our results we provide insights into the complexity of the RsCPA gene family and a valuable resource to explore the potential functions of RsCPA genes in radish.

Phylogenetic Diversity and Physiological Roles of Plant Monovalent Cation/H+ Antiporters.

The processes of plant nutrition, stress tolerance, plant growth, and development are strongly dependent on transport of mineral nutrients across cellular membranes. Plant membrane transporters are key components of these processes. Among various membrane transport proteins, the monovalent cation proton antiporter (CPA) superfamily mediates a broad range of physiological and developmental processes such as ion and pH homeostasis, development of reproductive organs, chloroplast operation, and plant adaptation to drought and salt stresses. CPA family includes plasma membrane-bound Na+/H+ exchanger (NhaP) and intracellular Na+/H+ exchanger NHE (NHX), K+ efflux antiporter (KEA), and cation/H+ exchanger (CHX) family proteins. In this review, we have completed the phylogenetic inventory of CPA transporters and undertaken a comprehensive evolutionary analysis of their development. Compared with previous studies, we have significantly extended the range of plant species, including green and red algae and Acrogymnospermae into phylogenetic analysis. Our data suggest that the multiplication and complexation of CPA isoforms during evolution is related to land colonisation by higher plants and associated with an increase of different tissue types and development of reproductive organs. The new data extended the number of clades for all groups of CPAs, including those for NhaP/SOS, NHE/NHX, KEA, and CHX. We also critically evaluate the latest findings on the biological role, physiological functions and regulation of CPA transporters in relation to their structure and phylogenetic position. In addition, the role of CPA members in plant tolerance to various abiotic stresses is summarized, and the future priority directions for CPA studies in plants are discussed.

Acanthamoeba spp. monoclonal antibody against a CPA2 transporter: a promising molecular tool for acanthamoebiasis diagnosis and encystment study.

Free-living amoeba of the genus Acanthamoeba are ubiquitous protozoa involved in opportunistic and non-opportunistic infection in humans, such as granulomatous amoebic encephalitis and amoebic keratitis. Both infections have challenging characteristics such as the formation of the resistant cysts in infected tissues, hampering the treatment and most usual diagnosis depending on time-consuming and/or low sensitivity techniques. The use of monoclonal antibodies presents itself as an opportunity for the development of more effective alternative diagnostic methods, as well as an important and useful tool in the search for new therapeutic targets. This study investigated the possibility of using a previously produced monoclonal antibody (mAb3), as a diagnostic tool for the detection of Acanthamoeba trophozoites by direct and indirect flow cytometry and immunofluorescence. Immunoprecipitation assay and mass spectrometry allowed the isolation of the antibody's target and suggested it is a transporter part of the CPA (cation: proton antiporter) superfamily. In vitro tests indicate an important role of this target in Acanthamoeba's encystment physiology. Our results support the importance of studying the role of CPA2 transporters in the context of acanthamoebiasis, as this may be a way to identify new therapeutic candidates.