Mutational analysis of potential pore-lining amino acids in TM IV of the Na/H exchanger

The Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. In this study we examined the effect of site-specific mutagenesis on the pore-lining amino acid Phe161 and effects of mutagenesis on the charged amino acids Asp159 and Asp172. There was no absolute requirement for a carboxyl side chain at amino acid Asp159 or Asp172. Mutation of Asp159 to Asn or Gln maintained or increased the activity of the protein. Similarly, for Asp172, substitution with a Gln residue maintained activity of the protein, even though substitution with an Asn residue was inhibitory. The Asp172Glu mutant possessed normal activity after correction for its aberrant expression and surface targeting. Replacement of Phe161 with a Leu demonstrated that it was not irreplaceable in NHE1 function. However, the mutation Phe161lys inhibited NHE1 function, while the Phe161Ala mutation caused altered NHE1 targeting and expression levels. Our results show that these three amino acids, while being important in NHE1 function, are not irreplaceable. This study demonstrates that multiple substitutions at a single amino acid residue may be necessary to get a clearer picture membrane protein function.     

Functional analysis of amino acids of the Na+/H+ exchanger that are important for proton translocation

The Na⁺/H⁺ exchanger is an integral membrane protein found in the plasma membrane of eukaryotic and prokaryotic cells. In eukaryotes it functions to exchange one proton for a sodium ion. In mammals it removes intracellular protons while in plants and fungal cells the plasma membrane form removes intracellular sodium in exchange for extracellular protons. In this study we used the Na⁺/H⁺ exchanger of Schizosaccharomyces pombe (Sod2) as a model system to study amino acids critical for activity of the protein. Twelve mutant forms of the Na⁺/H⁺ exchanger were examined for their ability to translocate protons as assessed by a cytosensor microphysiometer. Mutation of the amino acid Histidine 367 resulted in defective proton translocation. The acidic residues Asp145, Asp178, Asp266 and Asp267 were important in the proton translocation activity of the Na⁺/H⁺ exchanger. Mutation of amino acids His98, His233 and Asp241 did not significantly impair proton translocation by the Na⁺/H⁺ exchanger. These results confirm that polar amino acids are important in proton flux activity of Na⁺/H⁺ exchangers.     

Structure and analysis of the mouse Na+/H+ exchanger (NHE1) gene: Homology and conservation of splice sites

The Na⁺/H⁺ exchanger is a widely distributed integral membrane protein that is responsible for pH regulation in mammalian tissues. We have cloned and analyzed the NHE1 isoform of the mouse genomic Na⁺/H⁺exchanger. A clone from a mouse genomic library contained the NHE1 promoter region and the 5-untranslated region. It also contained the first 121 amino acids of the coding region of the Na⁺/H⁺ exchanger. A splice site occurred after amino acid 121, at the same region as in the human NHE1 gene. The deduced amino terminal coding sequence was 76 and 88% identical to the human and rat NHE1 sequences respectively. The 5-untranslated region was highly homologous to that of other species and two minicistrons contained in the human Na⁺/H⁺ exchanger were present in the mouse sequence. The results show that the deduced protein sequence of the mouse NHE1 gene has a high level of homology with other species and that the splice site of the first intron is conserved. These results suggest that the first large intron may play an important role in the NHE1 gene expression.     

Structural and functional analysis of extracellular loop 4 of the Nhe1 isoform of the Na+/H+ exchanger

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein. It regulates intracellular pH by removing a single intracellular H⁺ in exchange for one extracellular Na⁺. The membrane domain of NHE1 comprises the 500 N-terminal amino acids and is made of 12 transmembrane segments. The extracellular loops of the transmembrane segments are thought to be involved in cation coordination and inhibitor sensitivity. We have characterized the structure and function of amino acids 278–291 representing extracellular loop 4. When mutated to Cys, residues F277, F280, N282 and E284 of EL4 were sensitive to mutation and reaction with MTSET inhibiting NHE1 activity. In addition they were found to be accessible to extracellular applied MTSET. A peptide of the amino acids of EL4 was mostly unstructured suggesting that it does not provide a rigid structured link between TM VII and TM VIII. Our results suggest that EL4 makes an extension upward from TM VII to make up part of the mouth of the NHE1 protein and is involved in cation selectivity or coordination. EL4 provides a flexible link to TM VIII which may either allow movement of TM VII or allow TM VIII to not be adjacent to TM VII.     

Structural and Functional Analysis of Transmembrane Segment IV of the Salt Tolerance Protein Sod2

Sod2 is the plasma membrane Na⁺/H⁺ exchanger of the fission yeast Schizosaccharomyces pombe. It provides salt tolerance by removing excess intracellular sodium (or lithium) in exchange for protons. We examined the role of amino acid residues of transmembrane segment IV (TM IV) (¹²⁶FPQINFLGSLLIAGCITSTDPVLSALI¹⁵²) in activity by using alanine scanning mutagenesis and examining salt tolerance in sod2-deficient S. pombe. Two amino acids were critical for function. Mutations T144A and V147A resulted in defective proteins that did not confer salt tolerance when reintroduced into S. pombe. Sod2 protein with other alanine mutations in TM IV had little or no effect. T144D and T144K mutant proteins were inactive; however, a T144S protein was functional and provided lithium, but not sodium, tolerance and transport. Analysis of sensitivity to trypsin indicated that the mutations caused a conformational change in the Sod2 protein. We expressed and purified TM IV (amino acids 125–154). NMR analysis yielded a model with two helical regions (amino acids 128–142 and 147–154) separated by an unwound region (amino acids 143–146). Molecular modeling of the entire Sod2 protein suggested that TM IV has a structure similar to that deduced by NMR analysis and an overall structure similar to that of Escherichia coli NhaA. TM IV of Sod2 has similarities to TM V of the Zygosaccharomyces rouxii Na⁺/H⁺ exchanger and TM VI of isoform 1 of mammalian Na⁺/H⁺ exchanger. TM IV of Sod2 is critical to transport and may be involved in cation binding or conformational changes of the protein.     

Characterization of the ion transport activity of the budding yeast Na/H antiporter, Nha1p, using isolated secretory vesicles

The Saccharomyces cerevisiae Nha1p, a plasma membrane protein belonging to the monovalent cation/proton antiporter family, plays a key role in the salt tolerance and pH regulation of cells. We examined the molecular function of Nha1p by using secretory vesicles isolated from a temperature sensitive secretory mutant, sec4-2, in vitro. The isolated secretory vesicles contained newly synthesized Nha1p en route to the plasma membrane and showed antiporter activity exchanging H⁺ for monovalent alkali metal cations. An amino acid substitution in Nha1p (D266N, Asp-266 to Asn) almost completely abolished the Na⁺/H⁺ but not K⁺/H⁺ antiport activity, confirming the validity of this assay system as well as the functional importance of Asp-266, especially for selectivity of substrate cations. Nha1p catalyzes transport of Na⁺ and K⁺ with similar affinity (12.7 mM and 12.4 mM), and with lower affinity for Rb⁺ and Li⁺. Nha1p activity is associated with a net charge movement across the membrane, transporting more protons per single sodium ion (i.e., electrogenic). This feature is similar to the bacterial Na⁺/H⁺ antiporters, whereas other known eukaryotic Na⁺/H⁺ antiporters are electroneutral. The ion selectivity and the stoichiometry suggest a unique physiological role of Nha1p which is distinct from that of other known Na⁺/H⁺ antiporters.     

Characterization of a Histidine Rich Cluster of Amino Acids in the Cytoplasmic Domain of the Na+/H+ Exchanger

We examined the function of a highly conserved Histidine rich sequence ofamino acids found in the carboxyl-terminal of the Na⁺/H⁺exchanger (NHE1). A fusion protein containing the sequenceHYGHHH (540–545) and the balance of the carboxyl terminalof the protein did not bind calcium but bound to an immobilizedmetal affinity column and could be used to partially purify theexchanger protein. Mutation of the sequence to either HYGAAA orHYGRRR did not affect activity of the intact protein. Mutationto HHHHHH did not affect proton activation of the Na+/H+exchanger or localization but caused a decreased maximal velocitysuggesting that this conserved sequence is important in maximalactivity of the Na⁺/H⁺ exchanger.     

Overexpression of the NHE1 isoform of the Na+/H+ exchanger causes elevated apoptosis in isolated cardiomyocytes after hypoxia/reoxygenation challenge

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH in the myocardium and other tissues. NHE1 is an important mediator of myocardial damage that occurs after ischemia–reperfusion injury. It has also been implicated in apoptotic damage in many tissues and its expression and activity are elevated in disease states in the myocardium. In this study, we examined the effect of additional exogenous NHE1 expression on isolated cardiomyocytes susceptibility to ischemia/reperfusion damage. Exogenous NHE1 elevated Na⁺/H⁺ exchanger expression and activity when introduced into isolated cardiomyocytes through an adenoviral system. Isolated cardiomyocytes were subjected to simulated ischemia and reperfusion after infection with either control or NHE1-containing adenovirus. Cells were placed into an anaerobic chamber and effects of NHE1 expression after hypoxia/reoxygenation were examined. Hypoxia/reoxygenation increased caspase-3-like activity in controls, and the effect was greatly magnified in cells expressing NHE1 protein. It also elevated the percentage of apoptotic cardiomyocytes, which was also aggravated by expression of NHE1 protein. Hypoxia/reoxygenation also increased phospho-ERK levels. Elevated NHE1 expression was coincidental with increased expression of the ER stress protein, protein disulfide isomerase (PDI) and calreticulin (CRT). Our results demonstrate that increased NHE1 protein expression makes cells more susceptible to damage induced by hypoxia/reoxygenation in isolated cardiomyocytes. They suggest that elevated NHE1 in cardiovascular disease could predispose the human myocardium to enhanced apoptotic damage.     

Cloning of a novel human NHEDC1 (Na+/H+ exchanger like domain containing 1) gene expressed specifically in testis

The Na⁺/H⁺ exchangers (NHEs) catalyze the transport of Na⁺ in exchange for H⁺ across membranes in organisms and are required for numerous physiological processes. Here we report the cloning and characterization of a novel human NHEDC1 (Na⁺/H⁺ exchanger like domain containing 1) gene, which was mapped to human chromosome 4p24. This cDNA is 1859 bp in length, encoding a putative protein of 515 amino acids. The NHEDC1 proteins are highly conserved in mammals including human, mouse, rat, and Macaca fascicularis. One remarkable characteristic of human NHEDC1 gene is that it is exclusively expressed in the testis by RT-PCR analysis. Western blot analysis showed that the molecular weight of NHEDC1 is about 56 KDa. Guangming Ye and Cong Chen contributed equally to this work.     

Expression and characterization of the Na+/H+ exchanger in the mammalian myocardium

We examined two expression systems for studying the Na⁺/H⁺ exchanger in the mammalian myocardium. Mammalian NHE1 with a hemagglutinin (HA) tag and was cloned behind the alpha myosin heavy chain promoter. Transgenic mice were made with wild type NHE1 protein or with a hyperactive NHE1 protein mutated at the calmodulin-binding domain. Three lines of transgenic mice were made of each cDNA with expression levels of each type varying from high to low. Higher levels and activity of the Na⁺/H⁺ exchanger were associated with decreased long-term survival of mice, and with dilated or hypertrophic cardiomyopathy. The exogenous NHE1 protein was present in freshly made cardiomyocytes from transgenic mice, however, expression from the alpha myosin heavy chain promoter declined rapidly and little exogenous NHE1 was apparent on the fourth day after cardiomyocyte isolation. To express NHE1 protein in isolated cardiomyocytes, we transferred a mutated form of the protein into an adenoviral expression system. Infection of neonatal rat cardiomyocytes resulted in robust expression of the exogenous NHE1 protein. The mutant form of the NHE1 protein could be distinguished from the endogenous Na⁺/H⁺ exchanger by its resistance to inhibition by amiloride analogs. Our results suggest that for in vivo studies on intact hearts and animals, expression in transgenic mice is an appropriate system, however for long-term studies on cardiomyocytes, this model is inappropriate due to waning expression from the alpha myosin heavy chain promoter. Therefore, infection by adenovirus is a superior system for long-term studies on cardiomyocytes in culture.     

Characterization of modeled inhibitory binding sites on isoform one of the Na+/H+ exchanger

Mammalian Na⁺/H⁺ exchanger isoform one (NHE1) is a plasma membrane protein responsible for pH regulation in mammalian cells. Excess activity of the protein promotes heart disease and is a trigger of metastasis in cancer. Inhibitors of the protein exist but problems in specificity have delayed their clinical application. Here we examined amino acids involved in two modeled inhibitor binding sites (A, B) in human NHE1. Twelve mutations (Asp159, Phe348, Ser351, Tyr381, Phe413, Leu465, Gly466, Tyr467, Leu468, His473, Met476, Leu481) were made and characterized. Mutants S351A, F413A, Y467A, L468A, M476A and L481A had 40–70% of wild type expression levels, while G466A and H473A expressed 22% ~ 30% of the wild type levels. Most mutants, were targeted to the cell surface at levels similar to wild type NHE1, approximately 50–70%, except for F413A and G466A, which had very low surface targeting. Most of the mutants had measurable activity except for D159A, F413A and G466A. Resistance to inhibition by EMD87580 was elevated in mutants F438A, L465A and L468A and reduced in mutants S351A, Y381A, H473A, M476A and L481A. All mutants with large alterations in inhibitory properties showed reduced Na⁺ affinity. The greatest changes in activity and inhibitor sensitivity were in mutants present in binding site B which is more closely associated with TM4 and C terminal of extracellular loop 5, and is situated between the putative scaffolding domain and transport domain. The results help define the inhibitor binding domain of the NHE1 protein and identify new amino acids involved in inhibitor binding.     

Diverse residues of intracellular loop 5 of the Na+/H+ exchanger modulate proton sensing,expression, activity and targeting

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pH_i) by removing a single intracellular proton in exchange for one extracellular sodium ion. It is involved in cardiac hypertrophy and ischemia reperfusion damage to the heart and elevation of its activity is a trigger for breast cancer metastasis. NHE1 has an extensive 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments connected by intracellular and extracellular loops. Intracellular loops are hypothesized to modulate the sensitivity to pH_i. In this study, we characterized the structure and function of intracellular loop 5 (IL5), specifically amino acids 431–443. Mutation of eleven residues to alanine caused partial or nearly complete inhibition of transport; notably, mutation of residues L432, T433, I436, N437, R440 and K443 demonstrated these residues had critical roles in NHE1 function independent of effects on targeting or expression. The nuclear magnetic resonance (NMR) solution spectra of the IL5 peptide in a membrane mimetic sodium dodecyl sulfate solution revealed that IL5 has a stable three-dimensional structure with substantial alpha helical character. NMR chemical shifts indicated that K438 was in close proximity with W434. Overall, our results show that IL5 is a critical, intracellular loop with a propensity to form an alpha helix, and many residues of this intracellular loop are critical to proton sensing and ion transport.     

Structural and Functional Analysis of Transmembrane Segment VI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na⁺/H⁺ exchanger isoform 1 is a ubiquitously expressed integral membrane protein. It resides on the plasma membrane of cells and regulates intracellular pH in mammals by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. We characterized structural and functional aspects of the transmembrane segment (TM) VI (residues 227–249) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM VI was mutated to cysteine in the background of the cysteineless NHE1 protein, and the sensitivity to water-soluble sulfhydryl-reactive compounds (2-(trimethylammonium)ethyl)methanethiosulfonate (MTSET) and (2-sulfonatoethyl)methanethiosulfonate (MTSES) was determined for those residues with significant activity remaining. Three residues were essentially inactive when mutated to Cys: Asp²³⁸, Pro²³⁹, and Glu²⁴⁷. Of the remaining residues, proteins with the mutations N227C, I233C, and L243C were strongly inhibited by MTSET, whereas amino acids Phe²³⁰, Gly²³¹, Ala²³⁶, Val²³⁷, Ala²⁴⁴, Val²⁴⁵, and Glu²⁴⁸ were partially inhibited by MTSET. MTSES did not affect the activity of the mutant NHE1 proteins. The structure of a peptide representing TM VI was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. TM VI contains two helical regions oriented at an approximate right angle to each other (residues 229–236 and 239–250) surrounding a central unwound region. This structure bears a resemblance to TM IV of the Escherichia coli protein NhaA. The results demonstrate that TM VI of NHE1 is a discontinuous pore-lining helix with residues Asn²²⁷, Ile²³³, and Leu²⁴³ lining the translocation pore.     

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H⁺ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H⁺-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H⁺ in the course of cellular amino acid uptake by PAT1.     

Proline 146 is critical to the structure, function and targeting of sod2, the Na/H exchanger of Schizosaccharomyces pombe

Sod2 is the Na⁺/H⁺ exchanger of the fission yeast Schizosaccharomyces pombe that is principally responsible for salt tolerance. We examined the role of nine polar, membrane associated amino acids in the ability of the protein to confer salt tolerance in S. pombe. Wild type sod2 protein with a C-terminal GFP tag effectively rescued salt tolerance in S. pombe with deleted endogenous sod2. Sod2 protein with the mutations P163A, P183A, D298N, D389N, E390Q, E392Q and E397Q also conveyed salt tolerance as effectively as the wild type sod2 protein. In contrast, the mutation P146A resulted in a protein that did not convey salt tolerance nearly as effectively as the wild type and did not extrude Na⁺ as well as the wild type. Mutation of Pro¹⁴⁶ to Ser, Asp or Lys had an intermediate effect. Mutation of Thr¹⁴² to Ser resulted in a slightly defective protein. Western blot analysis showed that all mutant proteins were expressed at similar levels as wild type sod2 protein. Examination of the localization of the proteins showed that wild type and most sod2 mutants were present in the plasma membrane while the P146A mutant had an intracellular localization. Limited tryptic digestion suggested that the P146A sod2 protein had a change in conformation in comparison to the wild type protein. The results suggest that Pro¹⁴⁶ is an amino acid critical to sod2 structure, function and localization.     

The Na+/H+ Exchanger Present in Trout Erythrocyte Membranes is Not Responsible for the Amiloride-insensitive Na+/Li+ Exchange Activity

The protein responsible for the Na⁺/Li⁺ exchange activity across the erythrocyte membrane has not been cloned or isolated. It has been suggested that a Na⁺/H⁺ exchanger could be responsible for the Na⁺/Li⁺ exchange activity across the erythrocyte membrane. Previously, we reported that in the trout erythrocyte, the Li⁺/H⁺ exchange activity (mediated by the Na⁺/H⁺ exchanger βNHE) and the Na⁺/Li⁺ exchange activity respond differently to cAMP, DMA (dimethyl-amiloride) and O₂. We concluded that the DMA insensitive Na⁺/Li⁺ exchange activity originates from a different protein. To further examine these findings, we measured Li⁺ efflux in fibroblasts expressing the βNHE as the only Na⁺/H⁺ exchanger. Moreover, the internal pH of these cells was monitored with a fluorescent probe. Our findings indicate that acidification of fibroblasts expressing the Na⁺/H⁺ exchanger βNHE, induces a Na⁺ stimulated Li⁺ efflux activity in trout erythrocytes. This exchange activity, however, is DMA sensitive and therefore differs from the DMA insensitive Na⁺/Li⁺ exchange activity. In these fibroblasts no significant DMA insensitive Na⁺/Li⁺ exchange activity was found. These results support the hypothesis that the trout erythrocyte Na⁺/Li⁺ exchange activity is not mediated by the Na⁺/H⁺ exchanger (βNHE) present in these membranes. Received: 6 December 1996/Revised: 11 August 1997     

Sustained Na+/H+ Exchanger Activation Promotes Gliotransmitter Release from Reactive Hippocampal Astrocytes following Oxygen-Glucose Deprivation

Hypoxia ischemia (HI)-related brain injury is the major cause of long-term morbidity in neonates. One characteristic hallmark of neonatal HI is the development of reactive astrogliosis in the hippocampus. However, the impact of reactive astrogliosis in hippocampal damage after neonatal HI is not fully understood. In the current study, we investigated the role of Na⁺/H⁺ exchanger isoform 1 (NHE1) protein in mouse reactive hippocampal astrocyte function in an in vitro ischemia model (oxygen/glucose deprivation and reoxygenation, OGD/REOX). 2 h OGD significantly increased NHE1 protein expression and NHE1-mediated H⁺ efflux in hippocampal astrocytes. NHE1 activity remained stimulated during 1–5 h REOX and returned to the basal level at 24 h REOX. NHE1 activation in hippocampal astrocytes resulted in intracellular Na⁺ and Ca²⁺ overload. The latter was mediated by reversal of Na⁺/Ca²⁺ exchange. Hippocampal astrocytes also exhibited a robust release of gliotransmitters (glutamate and pro-inflammatory cytokines IL-6 and TNFα) during 1–24 h REOX. Interestingly, inhibition of NHE1 activity with its potent inhibitor HOE 642 not only reduced Na⁺ overload but also gliotransmitter release from hippocampal astrocytes. The noncompetitive excitatory amino acid transporter inhibitor TBOA showed a similar effect on blocking the glutamate release. Taken together, we concluded that NHE1 plays an essential role in maintaining H⁺ homeostasis in hippocampal astrocytes. Over-stimulation of NHE1 activity following in vitro ischemia disrupts Na⁺ and Ca²⁺ homeostasis, which reduces Na⁺-dependent glutamate uptake and promotes release of glutamate and cytokines from reactive astrocytes. Therefore, blocking sustained NHE1 activation in reactive astrocytes may provide neuroprotection following HI.     

Metagenome Cloning and Functional Analysis of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Genes from Keke Salt Lake in China

Na⁺/H⁺ antiporters are ubiquitous membrane proteins and play a central role in cell homeostasis including pH regulation, osmoregulation, and Na⁺/Li⁺ tolerance in bacteria. The microbial communities in extremely hypersaline soil are an important resource for isolating Na⁺/H⁺ antiporter genes. A metagenomic library containing 35,700 clones was constructed by using genomic DNA obtained from the hypersaline soil samples of Keke Salt Lake in Northwest of China. Two Na⁺/H⁺ antiporters, K1-NhaD, and K2-NhaD belonging to NhaD family, were screened and cloned from this metagenome by complementing the triple mutant Escherichia coli strain KNabc (nhaA ⁻ , nhaB ⁻ , chaA ⁻) in medium containing 0.2 M NaCl. K1-NhaD and K2-NhaD have 75.5% identity at the predicted amino acid sequence. K1-NhaD has 78% identity with Na⁺/H⁺ antiporter NhaD from Halomonas elongate at the predicted amino acid sequence. The predicted K1-NhaD is a 53.5 kDa protein (487 amino acids) with 13 transmembrane helices. K2-NhaD has 73% identity with Alkalimonas amylolytica NhaD. The predicted K2-NhaD is a 55 kDa protein (495 amino acids) with 12 transmembrane helices. Both K1-NhaD and K2-NhaD could make the triple mutant E. coli KNabc (nhaA ⁻ , nhaB ⁻ , chaA⁻) grow in the LBK medium containing 0.2–0.6 M Na⁺ or with 0.05–0.4 M Li⁺. Everted membrane vesicles prepared from E. coli KNabc cells carrying K1-NhaD or K2-NhaD exhibited Na⁺/H⁺ and Li⁺/H⁺ antiporter activities which were pH-dependent with the highest activity at pH 9.5. Little K⁺/H⁺ antiporter activity was also detected in vesicles form E. coli KNabc carrying K1-NhaD or K2-NhaD.