Halotolerant cyanobacterium Aphanothece halophytica contains an Na(+)/H(+) antiporter, homologous to eukaryotic ones, with novel ion specificity affected by C-terminal tail

Recently, a cyanobacterium Synechocystis sp. PCC 6803 has been shown to contain an Na(+)/H(+) antiporter gene homologous to plants (SOS1 and AtNHX1 from Arabidopsis) and mammalians (NHEs from human) but not to Escherichia coli (nhaA and nhaB). Here, we examined whether a halotolerant cyanobacterium Aphanothece halophytica has homologous genes. It turned out that A. halophytica contains an Na(+)/H(+) antiporter homologous to plants, mammalians, and some bacteria (nhaP from Pseudomonas and synnhaP from Synechocystis) but with novel ion specificity. Its gene product, ApNhaP (Na(+)/H(+) antiporter from Aphanothece halophytica), exhibited the Na(+)/H(+) antiporter activity over a wide pH range between 5 and 9 and complemented the Na(+)-sensitive phenotype of the antiporter-deficient E. coli mutant. The ApNhaP had virtually no activity for the Li(+)/H(+) antiporter but showed high Ca(2+)/H(+) antiporter activity at alkaline pH. The ApNhaP complemented the Ca(2+)-sensitive phenotype of the E. coli mutant but not the Li(+)-sensitive phenotype. The replacement of a long C-terminal tail of ApNhaP with that of Synechocystis altered the ion specificity of the antiporter. These results suggest that the ion specificity of an Na(+)/H(+) antiporter is partly determined by the structural properties of the C-terminal tail, which was well exemplified in the case of A. halophytica.     

Influence of ischemic preconditioning on intracellular sodium,pH, and cellular energy status in isolated perfused heart

The possible relationships between intracellular Na(+) (Na(i)(+)), bioenergetic status and intracellular pH (pH(i)) in the mechanism for ischemic preconditioning were studied using (23)Na and (31)P magnetic resonance spectroscopy in isolated Langendorff perfused rat heart. The ischemic preconditioning (three 5-min ischemic episodes followed by two 5-min and one 10-min period of reperfusion) prior to prolonged ischemia (20 min stop-flow) resulted in a decrease in ischemic acidosis and faster and complete recovery of cardiac function (ventricular developed pressure and heart rate) after 30 min of reperfusion. The response of Na(i) during ischemia in the preconditioned hearts was characterized by an increase in Na(i)(+) at the end of preconditioning and an accelerated decrease during the first few minutes of reperfusion. During post-ischemic reperfusion, bioenergetic parameters (PCr/P(i) and betaATP/P(i) ratios) were partly recovered without any significant difference between control and preconditioned hearts. The reduced acidosis during prolonged ischemia and the accelerated decrease in Na(i)(+) during reperfusion in the preconditioned hearts suggest activation of Na(+)/H(+) exchanger and other ion transport systems during preconditioning, which may protect the heart from intracellular acidosis during prolonged ischemia, and result in better recovery of mechanical function (LVDP and heart rate) during post-ischemic reperfusion.     

Mutation E252C increases drastically the Km value for Na+ and causes an alkaline shift of the pH dependence of NhaA Na+/H+ antiporter of Escherichia coli

A single Cys replacement of Glu at position 252 (E252C) in loop VIII-IX of NhaA increases drastically the Km for Na(+) (50-fold) of the Na(+)/H(+) antiporter activity of NhaA and shifts the pH dependence of NhaA activity, by one pH unit, to the alkaline range. In parallel, E252C causes a similar alkaline pH shift to the pH-induced conformational change of loop VIII-IX. Thus, although both the Na(+)/H(+) antiporter activity of wild type NhaA and its accessibility to trypsin at position Lys(249) in loop VIII-IX increase with pH between pH 6.5 and 7.5, the response of E252C occurs above pH 8. Furthermore, probing accessibility of pure E252C protein in dodecyl maltoside solution to 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid revealed that E252C itself undergoes a pH-dependent conformational change, similar to position Lys(249), and the rate of the pH-induced conformational change is increased specifically by the presence of Na(+) or Li(+), the specific ligands of the antiporter. Chemical modification of E252C by N-ethylmaleimide, 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid; [2-(trimethylammonium)ethyl]methane thiosulfonate, or (2-sulfonatoethyl)methanethiosulfonate reversed, to a great extent, the pH shift conferred by E252C but had no effect on the K(m) of the mutant antiporter.     

Granulosa cells regulate oocyte intracellular pH against acidosis in preantral follicles by multiple mechanisms

Mammalian oocytes grow within ovarian follicles in which the oocyte is coupled to surrounding granulosa cells by gap junctions. We report here that growing oocytes isolated from mouse preantral follicles are incapable of recovering from an experimentally induced acidosis, and that oocytes acquire the ability to manage acid loads by activating Na(+)/H(+) exchange during growth. By contrast, granulosa cells from similar preantral follicles possess substantial Na(+)/H(+) exchange capacity, which is attributable to the simultaneous action of two Na(+)/H(+) exchanger isoforms: NHE1 and NHE3. Granulosa cells were also found to possess a V-type H(+)-ATPase that drives partial acidosis recovery when Na(+)/H(+) exchange is inactivated. By monitoring intracellular pH (pH(i)) in small follicle-enclosed oocytes, we found that the oocyte has access to each of these acidosis-correcting activities, such that small follicle-enclosed oocytes readily recover from acidosis in a manner resembling granulosa cells. However, follicle-enclosed oocytes are unable to access these activities if gap-junction communication within the follicle is inhibited. Together, these experiments identify the NHE isoforms involved in regulating oocyte pH(i), indicate that gap junctions allow granulosa cells to exogenously regulate oocyte pH(i) against acidosis until the oocyte has acquired endogenous pH(i) regulation, and reveal that granulosa cells possess multiple mechanisms for carrying out this function.     

Cloning and characterization of a Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene of the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus aidingensis</Emphasis> AD-6<Superscript>T</Superscript>

A gene encoding a Na(+)/H(+) antiporter was obtained from the genome of Halobacillus aidingensis AD-6(T), which was sequenced and designated as nhaH. The deduced amino acid sequence of the gene was 91% identical to the NhaH of H. dabanensis, and shared 54% identity with the NhaG of Bacillus subtilis. The cloned gene enable the Escherichia coli KNabc cell, which lack all of the major Na(+)/H(+) antiporters, to grow in medium containing 0.2 M NaCl or 10 mM LiCl. The nhaH gene was predicted to encode a 43.5 kDa protein (403 amino acid residues) with 11 putative transmembrane regions. Everted membrane vesicles prepared from E. coli KNabc cells carrying NhaH exhibited Na(+)/H(+) as well as Li(+)/H(+) antiporter activity, which was pH-dependent with the highest activity at pH 8.0, and no K(+)/H(+) antiporter activity was detected. The deletion of hydrophilic C-terminal amino acid residues showed that the short C-terminal tail was vital for Na(+)/H(+) antiporter activity.     

The Vibrio cholerae Mrp system: cation/proton antiport properties and enhancement of bile salt resistance in a heterologous host

The mrp operon from Vibrio cholerae encoding a putative multisubunit Na(+)/H(+) antiporter was cloned and functionally expressed in the antiporter-deficient strain of Escherichia coli EP432. Cells of EP432 expressing Vc-Mrp exhibited resistance to Na(+) and Li(+) as well as to natural bile salts such as sodium cholate and taurocholate. When assayed in everted membrane vesicles of the E. coli EP432 host, Vc-Mrp had sufficiently high antiport activity to facilitate the first extensive analysis of Mrp system from a Gram-negative bacterium encoded by a group 2 mrp operon. Vc-Mrp was found to exchange protons for Li(+), Na(+), and K(+) ions in pH-dependent manner with maximal activity at pH 9.0-9.5. Exchange was electrogenic (more than one H(+) translocated per cation moved in opposite direction). The apparent K(m) at pH 9.0 was 1.08, 1.30, and 68.5 mM for Li(+), Na(+), and K(+), respectively. Kinetic analyses suggested that Vc-Mrp operates in a binding exchange mode with all cations and protons competing for binding to the antiporter. The robust ion antiport activity of Vc-Mrp in sub-bacterial vesicles and its effect on bile resistance of the heterologous host make Vc-Mrp an attractive experimental model for the further studies of biochemistry and physiology of Mrp systems.     

Potassium/proton antiport system of Escherichia coli

The intracellular level of potassium (K(+)) in Escherichia coli is regulated through multiple K(+) transport systems. Recent data indicate that not all K(+) extrusion system(s) have been identified (15). Here we report that the E. coli Na(+) (Ca(2+))/H(+) antiporter ChaA functions as a K(+) extrusion system. Cells expressing ChaA mediated K(+) efflux against a K(+) concentration gradient. E. coli strains lacking the chaA gene were unable to extrude K(+) under conditions in which wild-type cells extruded K(+). The K(+)/H(+) antiporter activity of ChaA was detected by using inverted membrane vesicles produced using a French press. Physiological growth studies indicated that E. coli uses ChaA to discard excessive K(+), which is toxic for these cells. These results suggest that ChaA K(+)/H(+) antiporter activity enables E. coli to adapt to K(+) salinity stress and to maintain K(+) homeostasis.     

NhaA of Escherichia coli, as a model of a pH-regulated Na+/H+antiporter

Na(+)/H(+) antiporters are ubiquitous membrane proteins that are involved in homeostasis of H(+) and Na(+) throughout the biological kingdom. Corroborating their role in pH homeostasis, many of the Na(+)/H(+) antiporter proteins are regulated directly by pH. The pH regulation of NhaA, the Escherichia coli Na(+)/H(+) antiporter (EcNhaA), as of other, both eukaryotic and prokaryotic Na(+)/H(+) antiporters, involves a pH sensor and conformational changes in different parts of the protein that transduce the pH signal into a change in activity. Thus, residues that affect the pH response, the translocation or both activities cluster in separate domains along the antiporter molecules. Importantly, in the NhaA family, these domains are conserved. Helix-packing model of EcNhaA based on cross-linking data suggests, that in the three dimensional structure of NhaA, residues that affect the pH response may be in close proximity, forming a single pH sensitive domain. Therefore, it is suggested that, despite considerable differences in the primary structure of the antiporters from the bacterial NhaA to the mammalian NHEs, their three-dimensional architectures are conserved. Test of this possibility awaits the atomic resolution of the 3D structure of the antiporters.     

Na+/H+ antiporter from Synechocystis species PCC 6803, homologous to SOS1, contains an aspartic residue and long C-terminal tail important for the carrier activity

A putative Na(+)/H(+) antiporter gene whose deduced amino acid sequence was highly homologous to the NhaP antiporter from Pseudomonas aeruginosa and SOS1 antiporter from Arabidopsis was isolated from Synechocystis sp. PCC 6803. The Synechocystis NhaP antiporter (SynNhaP) was expressed in Escherichia coli mutant cells, which were deficient in Na(+)/H(+) antiporters. It was found that the SynNhaP complemented the salt-sensitive phenotype of the E. coli mutant. Membrane vesicles prepared from the E. coli mutant transformed with the SynNhaP exhibited the Na(+)/H(+) and Li(+)/H(+) antiporter activities, and their activities were insensitive to amiloride. Moreover, its activity was very high between pH 5 and 9. The replacement of aspartate-138 in SynNhaP with glutamate or tyrosine inactivated the SynNhaP antiporter activity. The deletion of a part of the long C-terminal hydrophilic tail significantly inhibited the antiporter activity. A topological model suggests that aspartate-138 in SynNhaP is conserved in NhaP, SOS1, and AtNHX1 and is involved in the exchange activity. Thus, it appeared that the SynNhaP would provide a model system for the study of structural and functional properties of eucaryotic Na(+)/H(+) antiporters.     

A Na+/H+ antiporter gene of the moderately halophilic bacterium Halobacillus dabanensis D-8T: cloning and molecular characterization

A gene encoding a Na(+)/H(+) antiporter was cloned from a chromosomal DNA of Halobacillus dabanensis strain D-8(T) by functional complementation. Its presence enabled the antiporter-deficient Escherichia coli strain KNabc to survive in the presence of 0.2 M NaCl or 5 mM LiCl. The gene was sequenced and designated as nhaH. The deduced amino-acid sequence of NhaH consists of 403 residues with a calculated molecular mass of 43,481 Da, which was 54% identical and 76% similar to the NhaG Na(+)/H(+) antiporter of Bacillus subtilis. The hydropathy profile was characteristic of a membrane protein with 12 putative transmembrane domains. Everted membrane vesicles prepared from E. coli cells carrying nhaH exhibited Na(+)/H(+) as well as Li(+)/H(+) antiporter activity, which was pH-dependent with highest activities at pH 8.5-9.0 and at pH 8.5, respectively. Moreover, nhaH confers upon E. coli KNabc cells the ability to grow under alkaline conditions.     

Functional characterization of a NapA Na(+)/H(+) antiporter from Thermus thermophilus

Na(+)/H(+) antiporters are ubiquitous membrane proteins and play an important role in cell homeostasis. We amplified a gene encoding a member of the monovalent cation:proton antiporter-2 (CPA2) family (TC 2.A.37) from the Thermus thermophilus genome and expressed it in Escherichia coli. The gene product was identified as a member of the NapA subfamily and was found to be an active Na(+)(Li(+))/H(+) antiporter as it conferred resistance to the Na(+) and Li(+) sensitive strain E. coli EP432 (DeltanhaA, DeltanhaB) upon exposure to high concentration of these salts in the growth medium. Fluorescence measurements using the pH sensitive dye 9-amino-6-chloro-2-methoxyacridine in everted membrane vesicles of complemented E. coli EP432 showed high Li(+)/H(+) exchange activity at pH 6, but marginal Na(+)/H(+) antiport activity. Towards more alkaline conditions, Na(+)/H(+) exchange activity increased to a relative maximum at pH 8, where by contrast the Li(+)/H(+) exchange activity reached its relative minimum. Substitution of conserved residues D156 and D157 (located in the putative transmembrane helix 6) with Ala resulted in the complete loss of Na(+)/H(+) activity. Mutation of K305 (putative transmembrane helix 10) to Ala resulted in a compromised phenotype characterized by an increase in apparent K(m) for Na(+) (36 vs. 7.6 mM for the wildtype) and Li(+) (17 vs. 0.22 mM), In summary, the Na(+)/H(+) antiport activity profile of the NapA type transporter of T. thermophilus resembles that of NhaA from E. coli, whereas in contrast to NhaA the T. thermophilus NapA antiporter is characterized by high Li(+)/H(+) antiport activity at acidic pH.     

Screening of Environmental DNA Libraries for the Presence of Genes Conferring Na+(Li+)/H+ Antiporter Activity on Escherichia coli: Characterization of the Recovered Genes and the Corresponding Gene Products

Environmental DNA libraries prepared from three different soils were screened for genes conferring Na(+)(Li(+))/H(+) antiporter activity on the antiporter-deficient Escherichia coli strain KNabc. The presence of those genes was verified on selective LK agar containing 7.5 mM LiCl. Two positive E. coli clones were obtained during the initial screening of 1,480,000 recombinant E. coli strains. Both clones harbored a plasmid (pAM1 and pAM3) that conferred a stable Li(+)-resistant phenotype. The insert of pAM2 (1,886 bp) derived from pAM1 contained a gene (1,185 bp) which encodes a novel Na(+)/H(+) antiporter belonging to the NhaA family. The insert of pAM3 harbored the DNA region of E. coli K-12 containing nhaA, nhaR, and gef. This region is flanked by highly conserved insertion elements. The sequence identity with E. coli decreased significantly outside of the insertion sequence elements, indicating that the unknown organism from which the insert of pAM3 was cloned is different from E. coli. The products of the antiporter genes located on pAM2 and pAM3 revealed functional homology to NhaA of E. coli and enabled the antiporter-deficient E. coli mutant to grow on solid media in the presence of up to 450 mM NaCl or 250 mM LiCl at pH 8.0. The Na(+)/H(+) antiporter activity in everted membrane vesicles that were derived from the E. coli strains KNabc/pAM2 and KNabc/pAM3 showed a substantial increase between pHs 7 and 8.5. The maximal activity was observed at pHs 8.3 and 8.6, respectively. The K(m) values of both antiporters for Na(+) were approximately 10-fold higher than the values for Li(+).     

Yeast plasma membrane Ena1p ATPase alters alkali-cation homeostasis and confers increased salt tolerance in tobacco cultured cells

In plants, the plasma membrane Na(+)/H(+) antiporter is the only key enzyme that extrudes cytosolic Na(+) and contributes to salt tolerance. But in fungi, the plasma membrane Na(+)/H(+) antiporter and Na(+)-ATPase are known to be key enzymes for salt tolerance. Saccharomyces cerevisiae Ena1p ATPase encoded by the ENA1/PMR2A gene is primarily responsible for Na(+) and Li(+) efflux across the plasma membrane during salt stress and for K(+) efflux at high pH and high K(+). To test if the yeast ATPase would improve salt tolerance in plants, we expressed a triple hemagglutinin (HA)-tagged Ena1p (Ena1p-3HA) in cultured tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY2) cells. The Ena1p-3HA proteins were correctly localized to the plasma membrane of transgenic BY2 cells and conferred increased NaCl and LiCl tolerance to the cells. Under moderate salt stress conditions, the Ena1p-3HA-expressing BY2 clones accumulated lower levels of Na(+) and Li(+) than nonexpressing BY2 clones. Moreover, the Ena1p-3HA expressing BY2 clones accumulated lower levels of K(+) than nonexpressing cells under no-stress conditions. These results suggest that the yeast Ena1p can also function as an alkali-cation (Na(+), Li(+), and K(+)) ATPase and alter alkali-cation homeostasis in plant cells. We conclude that, even with K(+)-ATPase activity, Na(+)-ATPase activity of the yeast Ena1p confers increased salt tolerance to plant cells during salt stress.     

GerN, an endospore germination protein of Bacillus cereus, is an Na(+)/H(+)-K(+) antiporter

GerN, a Bacillus cereus spore germination protein, exhibits homology to a widely distributed group of putative cation transporters or channel proteins. GerN complemented the Na(+)-sensitive phenotype of an Escherichia coli mutant that is deficient in Na(+)/H(+) antiport activity (strain KNabc). GerN also reduced the concentration of K(+) required to support growth of an E. coli mutant deficient in K(+) uptake (strain TK2420). In a fluorescence-based assay of everted E. coli KNabc membrane vesicles, GerN exhibited robust Na(+)/H(+) antiport activity, with a K(m) for Na(+) estimated at 1.5 mM at pH 8.0 and 25 mM at pH 7.0. Li(+), but not K(+), served as a substrate. GerN-mediated Na(+)/H(+) antiport was further demonstrated in everted vesicles as energy-dependent accumulation of (22)Na(+). GerN also used K(+) as a coupling ion without completely replacing H(+), as indicated by partial inhibition by K(+) of H(+) uptake into right-side-out vesicles loaded with Na(+). K(+) translocation as part of the antiport was supported by the stimulatory effect of intravesicular K(+) on (22)Na(+) uptake by everted vesicles and the dependence of GerN-mediated (86)Rb(+) efflux on the presence of Na(+) in trans. The inhibitory patterns of protonophore and thiocyanate were most consistent with an electrogenic Na(+)/H(+)-K(+) antiport. GerN-mediated Na(+)/H(+)-K(+) antiport was much more rapid than GerN-mediated Na(+)/H(+) antiport.     

The activity profile of the NhaD-type Na+(Li+)/H+ antiporter from the soda Lake Haloalkaliphile Alkalimonas amylolytica is adaptive for the extreme environment

In extreme alkaliphiles, Na(+)/H(+) antiporters play a central role in the Na(+) cycle that supports pH homeostasis, Na(+) resistance, solute uptake, and motility. Properties of individual antiporters have only been examined in extremely alkaliphilic soil Bacillus spp., whereas the most alkaline natural habitats usually couple high pH with high salinity. Here, studies were conducted on a Na(+)(Li(+))/H(+) antiporter, NhaD, from the soda lake haloalkaliphile Alkalimonas amylolytica. The activity profile of A. amylolytica NhaD at different pH values and Na(+) concentrations reflects its unique natural habitat. In membrane vesicles from antiporter-deficient Escherichia coli EP432 (DeltanhaA DeltanhaB), the pH optimum for NhaD-dependent Na(+)(Li(+))/H(+) antiport was at least 9.5, the highest pH that could be tested; no activity was observed at pH < or =8.5. NhaD supported low Na(+)/H(+) antiport activity at pH 9.5 that was detectable over a range of Na(+) concentrations from 10 mM to at least 800 mM, with a 600 mM optimum. Although A. amylolytica nhaD was isolated by complementing the Li(+) sensitivity of the triple mutant E. coli strain KNabc (DeltanhaA DeltanhaB DeltachaA), sustained propagation of nhaD-bearing plasmids in this strain resulted in a glycine (Gly(327))-->serine mutation in a putative cytoplasmic loop of the mutant transporter. The altered activity profile of NhaD-G327S appears to be adaptive to the E. coli setting: a much higher activity than wild-type NhaD at Na(+) concentrations up to 200 mM but lower activity at 400 to 600 mM Na(+), with a pH optimum and minimal pH for activity lower than those of wild-type NhaD.     

A major Li(+) extrusion system NhaB of Pseudomonas aeruginosa : comparison with the major Na(+) extrusion system NhaP

A gene encoding a Li(+) extrusion system was cloned from the chromosomal DNA of Pseudomonas aeruginosa and expressed in Escherichia coli cells. The gene enabled growth of E. coli KNabc cells, which were unable to grow in the presence of 10 mM LiCl or 0.1 M NaCl because of the lack of major Na(+) (Li(+))/H(+) antiporters. We detected Li(+)/H(+) and Na(+)/H(+) antiport activities in membrane vesicles prepared from E. coli KNabc cells that harbored a plasmid carrying the cloned gene. Activity of this antiporter was pH-dependent with an optimal pH activity between pH 7.5 and 8.5. These properties indicate that this antiporter is different from NhaP, an Na(+)/H(+) antiporter from P. aeruginosa that we reported previously, and that is rather specific to Na(+) but it cannot extrude Li(+) effectively. The gene was sequenced and an open reading frame (ORF) was identified. The amino acid sequence deduced from the ORF showed homology (about 60% identity and 90% similarity) with that of the NhaB Na(+)/H(+) antiporters of E. coli and Vibrio parahaemolyticus. Thus, we designated the antiporter as NhaB of P. aeruginosa. E. coli KNabc carrying the nhaB gene from P. aeruginosa was able to grow in the presence of 10 to 50 mM LiCl, although KNabc carrying nhaP was unable to grow in these conditions. The antiport activity of NhaB from P. aeruginosa was produced in E. coli and showed apparent Km values for Li(+) and Na(+) of 2.0 mM and 1.3 mM, respectively. The antiport activity was inhibited by amiloride with a Ki value for Li(+) and Na(+) of 0.03 mM and 0.04 mM, respectively.     

Two types of Bacillus subtilis tetA(L) deletion strains reveal the physiological importance of TetA(L) in K(+) acquisition as well as in Na(+), alkali, and tetracycline resistance

The chromosomally encoded TetA(L) protein of Bacillus subtilis is a multifunctional tetracycline-metal/H(+) antiporter that also exhibits monovalent cation/H(+) antiport activity and a net K(+) uptake mode. In this study, B. subtilis mutant strains JC112 and JC112C were found to be representative of two phenotypic types of tetA(L) deletion strains that are generated in the same selection. Both strains exhibited increased sensitivity to low tetracycline concentrations as expected. The mutants also had significantly reduced ability to grow in media containing low concentrations of K(+), indicating that the net K(+) uptake mode is of physiological consequence; the deficit in JC112 was greater than in JC112C. JC112 also exhibited (i) greater impairment of Na(+)- or K(+)-dependent growth at pH 8.3 than JC112C and (ii) a greater degree of Co(+2) as well as Na(+) sensitivity. Studies were initiated to explore the possibility of two different patterns of compensatory changes in other ion-translocating transporters in these mutants. Increased expression of two loci has thus far been shown. Increased expression of czcD-trkA, a locus with a proposed involvement in K(+) uptake, occurred in both mutants. The increase was highest in the presence of Co(2+) and was higher in JC112 than in JC112C. Deletion of czcD-trkA resulted in diminished growth of the wild-type and both mutant strains at low [K(+)], supporting a significant role for this locus in K(+) uptake. Expression of yheL, which is a homologue of the Na(+)/H(+) antiporter-encoding nhaC gene from Bacillus firmus OF4, was also increased in both tetA(L) deletion strains, again with higher up-regulation in JC112. The phenotypes resulting from deletion of yheL were consistent with a modest role for YheL in Na(+)-dependent pH homeostasis in the wild type. No major role for YheL was indicated in the mutants in spite of the overexpression. The studies underscore the multiple physiological functions of TetA(L), including tetracycline, Na(+), and alkali resistance and K(+) acquisition. The studies also reveal and begin to detail the complexity of the response to mutational loss of these functions.     

pH regulation in the intracellular malaria parasite, Plasmodium falciparum. H(+) extrusion via a V-type H(+)-ATPase

The mechanism by which the intra-erythrocytic form of the human malaria parasite, Plasmodium falciparum, extrudes H(+) ions and thereby regulates its cytosolic pH (pH(i)), was investigated using saponin-permeabilized parasitized erythrocytes. The parasite was able both to maintain its resting pH(i) and to recover from an imposed intracellular acidification in the absence of extracellular Na(+), thus ruling out the involvement of a Na(+)/H(+) exchanger in both processes. Both phenomena were ATP-dependent. Amiloride and the related compound ethylisopropylamiloride caused a substantial reduction in the resting pH(i) of the parasite, whereas EMD 96785, a potent and allegedly selective inhibitor of Na(+)/H(+) exchange, had relatively little effect. The resting pH(i) of the parasite was also reduced by the sulfhydryl reagent N-ethylmaleimide, by the carboxyl group blocker N,N'-dicyclohexylcarbodiimide, and by bafilomycin A(1), a potent inhibitor of V-type H(+)-ATPases. Bafilomycin A(1) blocked pH(i) recovery in parasites subjected to an intracellular acidification and reduced the rate of acidification of a weakly buffered solution by parasites under resting conditions. The data are consistent with the hypothesis that the malaria parasite, like other parasitic protozoa, has in its plasma membrane a V-type H(+)-ATPase, which serves as the major route for the efflux of H(+) ions.