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1.
The Nicotiana plumbaginifolia pma2 (plasma membrane H+-ATPase) gene is capable of functionally replacing the H+-ATPase genes of the yeast Saccharomyces cerevisiae, provided that the external pH is kept above 5.0. Single point mutations within the pma2 gene were previously identified that improved H+-ATPase activity and allowed yeast growth at pH 4.0. The aim of the present study was to identify most of the PMA2 positions, the mutation of which would lead to improved growth and to determine whether all these mutations result in similar enzymatic and structural modifications. We selected additional mutants in total 42 distinct point mutations localized in 30 codons. They were distributed in 10 soluble and membrane regions of the enzyme. Most mutant PMA2 H+-ATPases were characterized by a higher specific activity, lower inhibition by ADP, and lower stimulation by lysophosphatidylcholine than wild-type PMA2. The mutants thus seem to be constitutively activated. Partial tryptic digestion and immunodetection showed that the PMA2 mutants had a conformational change making the C-terminal region more accessible. These data therefore support the hypothesis that point mutations in various H+-ATPase parts displace the inhibitory C-terminal region, resulting in enzyme activation. The high density of mutations within the first half of the C-terminal region suggests that this part is involved in the interaction between the inhibitory C-terminal region and the rest of the enzyme. 相似文献
2.
M Piotrowski P Morsomme M Boutry C Oecking 《Canadian Metallurgical Quarterly》1998,273(45):30018-30023
Accumulating evidence suggests that the H+-ATPase of the plant plasma membrane is activated by a direct, reversible interaction with 14-3-3 proteins involving the displacement of the C-terminal autoinhibitory domain of the enzyme. The fungal phytotoxin fusicoccin (FC) appears to stabilize this H+-ATPase.14-3-3 complex, thus leading to a persistent activation of the H+-ATPase in vivo. In this study we show that functional replacement of the Saccharomyces cerevisiae H+-ATPase genes by a Nicotiana plumbaginifolia H+-ATPase (pma2) results in the generation of a high affinity fusicoccin binding site that is exceptionally abundant. Acquisition of FC binding capacity is accompanied by a significant increase in the amount of plasma membrane-associated yeast 14-3-3 homologs. The existence of a (plant) PMA2.(yeast)14-3-3 complex was demonstrated using two-dimensional gel systems (native/denaturing). After expression of PMA2 lacking most of its C-terminal region, neither H+-ATPase.14-3-3 complex formation nor FC binding activity could be observed. Furthermore, we obtained direct biochemical evidence for a minimal FC binding complex consisting of the C-terminal PMA2 domain and yeast 14-3-3 homologs. Thus we demonstrated unambiguously the relevance of this regulatory ATPase domain for 14-3-3 interaction as well as its requirement for FC binding. 相似文献
3.
Novel localization of a Na+/H+ exchanger in a late endosomal compartment of yeast. Implications for vacuole biogenesis 总被引:1,自引:0,他引:1
Na+/H+ exchangers catalyze the electrically silent countertransport of Na+ and H+, controlling the transmembrane movement of salt, water, and acid-base equivalents, and are therefore critical for Na+ tolerance, cell volume control, and pH regulation. In contrast to numerous well studied plasma membrane isoforms (NHE1-4), much less is known about intracellular Na+/H+ exchangers, and thus far no vertebrate isoform has been shown to have an exclusively endosomal distribution. In this context, we show that the yeast NHE homologue, Nhx1 (Nass, R., Cunningham, K. W., and Rao, R. (1997) J. Biol. Chem. 272, 26145-26152), localizes uniquely to prevacuolar compartments, equivalent to late endosomes of animal cells. In living yeast, we show that these compartments closely abut the vacuolar membrane in a striking bipolar distribution, suggesting that vacuole biogenesis occurs at distinct sites. Nhx1 is the founding member of a newly emergent cluster of exchanger homologues, from yeasts, worms, and humans that may share a common intracellular localization. By compartmentalizing Na+, intracellular exchangers play an important role in halotolerance; furthermore, we hypothesize that salt and water movement into vesicles may regulate vesicle volume and pH and thus contribute to vacuole biogenesis. 相似文献
4.
Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear. 相似文献
5.
CD Holyoak M Stratford Z McMullin MB Cole K Crimmins AJ Brown PJ Coote 《Canadian Metallurgical Quarterly》1996,62(9):3158-3164
The weak acid sorbic acid transiently inhibited the growth of Saccharomyces cerevisiae in media at low pH. During a lag period, the length of which depended on the severity of this weak-acid stress, yeast cells appeared to adapt to this stress, eventually recovering and growing normally. This adaptation to weak-acid stress was not due to metabolism and removal of the sorbic acid. A pma1-205 mutant, with about half the normal membrane H+-ATPase activity, was shown to be more sensitive to sorbic acid than its parent. Sorbic acid appeared to stimulate plasma membrane H+-ATPase activity in both PMA1 and pma1-205. Consistent with this, cellular ATP levels showed drastic reductions, the extent of which depended on the severity of weak-acid stress. The weak acid did not appear to affect the synthesis of ATP because CO2 production and O2 consumption were not affected significantly in PMA1 and pma1-205 cells. However, a glycolytic mutant, with about one-third the normal pyruvate kinase and phosphofructokinase activity and hence a reduced capacity to generate ATP, was more sensitive to sorbic acid than its isogenic parent. These data are consistent with the idea that adaptation by yeast cells to sorbic acid is dependent on (i) the restoration of internal pH via the export of protons by the membrane H+-ATPase in an energy-demanding process and (ii) the generation of sufficient ATP to drive this process and still allow growth. 相似文献
6.
7.
S Wakabayashi B Bertrand T Ikeda J Pouysségur M Shigekawa 《Canadian Metallurgical Quarterly》1994,269(18):13710-13715
The ubiquitous plasma membrane Na+/H+ exchanger (NHE1) is rapidly activated in response to various extracellular signals. To understand how the intracellular Ca2+ is involved in this activation process, we investigated the effect of Ca2+ ionophore ionomycin on activity of the wild-type or mutant NHE1 expressed in the exchanger-deficient fibroblasts (PS120). In wild-type transfectants, a short (up to 1 min) incubation with ionomycin induced a significant alkaline shift (approximately 0.2 pH unit) in the intracellular pH (pHi) dependence of the rate of 5-(N-ethyl-N-isopropyl) amiloride-sensitive 22Na+ uptake, without changes in the cell volume and phosphorylation state of NHE1. Mutations that prevented calmodulin (CaM) binding to a high affinity binding region (region A, amino acids 636-656) rendered NHE1 constitutively active by inducing a similar alkaline shift in pHi dependence of Na+/H+ exchange. These same mutations abolished the ionomycin-induced NHE1 activation. These data suggest that CaM-binding region A functions as an "autoinhibitory domain" and that Ca2+/CaM activates NHE1 by binding to region A and thus abolishing its inhibitory effect. Furthermore, we found that a short stimulation with thrombin and ionomycin had apparently no additive effects on the alkaline shift in the pHi dependence of Na+/H+ exchange and that deletion of region A also abolished such an alkaline shift induced by a short thrombin stimulation. The results strongly suggest that the early thrombin response and the ionomycin response share the same activation mechanism. Based on these data and the results shown in the accompanying paper (Bertrand, B., Wakabayashi, S., Ikeda, T., Pouysségur, J., and Shigekawa, M. (1994) J. Biol. Chem. 269, 13703-13709), we propose that CaM is one of the major "signal transducers" that mediate distinct extracellular signals to the "pHi sensor" of NHE1. 相似文献
8.
Membrane segment 5 (M5) is thought to play a direct role in cation transport by the sarcoplasmic reticulum Ca2+-ATPase and the Na+, K+-ATPase of animal cells. In this study, we have examined M5 of the yeast plasma membrane H+-ATPase by alanine-scanning mutagenesis. Mutant enzymes were expressed behind an inducible heat-shock promoter in yeast secretory vesicles as described previously (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940-7949). Three substitutions (R695A, H701A, and L706A) led to misfolding of the H+-ATPase as evidenced by extreme sensitivity to trypsin; the altered proteins were arrested in biogenesis, and the mutations behaved genetically as dominant lethals. The remaining mutants reached the secretory vesicles in sufficient amounts to be characterized in detail. One of them (Y691A) had no detectable ATPase activity and appeared, based on trypsinolysis in the presence and absence of ligands, to be blocked in the E1-to-E2 step of the reaction cycle. Alanine substitution at an adjacent position (V692A) had substantial ATPase activity (54%), but was likewise affected in the E1-to-E2 step, as evidenced by shifts in its apparent affinity for ATP, H+, and orthovanadate. Among the mutants that were sufficiently active to be assayed for ATP-dependent H+ transport by acridine orange fluorescence quenching, none showed an appreciable defect in the coupling of transport to ATP hydrolysis. The only residue for which the data pointed to a possible role in cation liganding was Ser-699, where removal of the hydroxyl group (S699A and S699C) led to a modest acid shift in the pH dependence of the ATPase. This change was substantially smaller than the 13-30-fold decrease in K+ affinity seen in corresponding mutants of the Na+, K+-ATPase (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764-22771). Taken together, the results do not give firm evidence for a transport site in M5 of the yeast H+-ATPase, but indicate a critical role for this membrane segment in protein folding and in the conformational changes that accompany the reaction cycle. It is therefore worth noting that the mutationally sensitive residues lie along one face of a putative alpha-helix. 相似文献
9.
Ionic fluxes that contribute to changes in membrane potential and variations of pHi (intracellular pH) are not well known in mast cells, although they can be important in the stimulus-secretion coupling. Cellular volume regulation implies changes in the concentration of intracellular ions, such as sodium and potassium and volume changes can be imposed varying the tonicity of the medium. We studied the physiology of sodium and examined the effect of ouabain on [22Na] entry in mast cells in isotonic and hypertonic media. We also recorded changes in membrane potential and pHi using the fluorescent dyes bis-oxonol (Bis-(1,3-diethylthiobarbituric acid) trimethineoxonol) a n d BCECF (2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester) in hypertonic conditions. The results show that [22Na] influx increases four fold in hypertonic solutions and it is mediated mainly by an amiloride-sensitive Na+/H+ exchanger. This transporter is involved in the shrinkage-activated cellular alkalinization and the pHi recovery is accelerated by inhibition of the Na+/K+ ATPase with ouabain in the absence of extracellular calcium. Under hypertonic conditions 22Na influx is apparently not increased by ouabain, while the Na+/K+ ATPase inhibitor clearly increases [22Na] uptake and also induces membrane depolarization in isotonic conditions. All together, these findings suggest that Na+/K+ ATPase is partially inhibited in hypertonic conditions. 相似文献
10.
The H+-ATPase from the plasma membrane of Neurospora crassa is an integral membrane protein of relative molecular mass 100K, which belongs to the P-type ATPase family that includes the plasma membrane Na+/K+-ATPase and the sarcoplasmic reticulum Ca2+-ATPase. The H+-ATPase pumps protons across the cell's plasma membrane using ATP as an energy source, generating a membrane potential in excess of 200mV. Despite the importance of P-type ATPases in controlling membrane potential and intracellular ion concentrations, little is known about the molecular mechanism they use for ion transport. This is largely due to the difficulty in growing well ordered crystals and the resulting lack of detail in the three-dimensional structure of these large membrane proteins. We have now obtained a three-dimensional map of the H+-ATPase by electron crystallography of two-dimensional crystals grown directly on electron microscope grids. At an in-plane resolution of 8 A, this map reveals ten membrane-spanning alpha-helices in the membrane domain, and four major cytoplasmic domains in the open conformation of the enzyme without bound ligands. 相似文献
11.
MG Martín MP Lostao E Turk J Lam M Kreman EM Wright 《Canadian Metallurgical Quarterly》1997,112(4):1206-1212
BACKGROUND & AIMS: Defects in the Na+-dependent glucose transporter (SGLT1) are associated with the disorder glucose-galactose malabsorption, characterized by severe diarrhea. This study focused on a unique proband with glucose-galactose malabsorption who was investigated 30 years ago, and the aims of the study were to identify mutations in the SGLT1 gene and to determine the defect in sugar transport. METHODS: Mutations were identified by sequencing, and each mutant protein was then studied using a Xenopus oocyte heterologous expression system. Analysis included Western, freeze fracture, radiotracer uptake, and electrophysiological assays. RESULTS: Two heterozygous missense mutations (Cys355Ser and Leu147Arg) were identified that entirely eliminated Na+/sugar cotransport activity. Western blot analysis showed that the levels of both mutant proteins in the oocyte were comparable to wild-type SGLT1, but no complex glycosylation was detected. No SGLT1 charge movements were observed with the mutant proteins, and freeze fracture data showed that neither mutant protein reached the plasma membrane. CONCLUSIONS: The Cys355Ser and Leu147Arg mutations eliminate the Na+/sugar cotransport by blocking the transfer of SGLT1 protein from the endoplasmic reticulum to the plasma membrane. This is consistent with earlier studies on phlorizin binding to the brush border membrane of duodenal biopsy specimens from this patient. 相似文献
12.
M Ritter P Schratzberger H Rossmann E W?ll K Seiler U Seidler N Reinisch CM K?hler H Zwierzina HJ Lang F Lang M Paulmichl CJ Wiedermann 《Canadian Metallurgical Quarterly》1998,124(4):627-638
1. Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N-formyl-Met-Leu-Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pHi) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pHi and migration of fMLP stimulated PMNs. 2. Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling. 3. Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP-induced cell swelling. 4. Step omission of extracellular Na+ caused intracellular acidification, which was accelerated by subsequent addition of gastric H+/K+ ATPase inhibitor SCH 28080, or by omission of extracellular K+ ions. In addition Na+ removal caused cell swelling, which was further enhanced by fMLP. 5. H+/K+ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP-induced increase in CV. 6. Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na+/H+ exchanger. In fMLP-stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited. 7. The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole. 8. Western analysis with a monoclonal anti gastric H+/K+ ATPase beta-subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs. 9. The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na+/H+ exchanger and a K+-dependent H+-extruding mechanism, presumably an H+/K+ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration. 相似文献
13.
M Ito AA Guffanti J Zemsky DM Ivey TA Krulwich 《Canadian Metallurgical Quarterly》1997,179(12):3851-3857
Application of protoplast transformation and single- and double-crossover mutagenesis protocols to alkaliphilic Bacillus firmus OF4811M (an auxotrophic strain of B. firmus OF4) facilitated the extension of the sequence of the previously cloned nhaC gene, which encodes an Na+/H+ antiporter, and the surrounding region. The nhaC gene is part of a likely 2-gene operon encompassing nhaC and a small gene that was designated nhaS; the operon is preceded by novel direct repeats. The predicted alkaliphile NhaC, based on the extended sequence analysis, would be a membrane protein with 462 amino acid residues and 12 transmembrane segments that is highly homologous to the deduced products of homologous genes of unknown function from Bacillus subtilis and Haemophilus influenzae. The full-length version of nhaC complemented the Na+-sensitive phenotype of an antiporter-deficient mutant strain of Escherichia coli but not the alkali-sensitive growth phenotypes of Na+/H+-deficient mutants of either alkaliphilic B. firmus OF4811M or B. subtilis. Indeed, NhaC has no required role in alkaliphily, inasmuch as the nhaC deletion strain of B. firmus OF4811M, N13, grew well at pH 10.5 at Na+ concentrations equal to or greater than 10 mM. Even at lower Na+ concentrations, N13 exhibited only a modest growth defect at pH 10.5. This was accompanied by a reduced capacity to acidify the cytoplasm relative to the medium compared to the wild-type strain or to N13 complemented by cloned nhaC. The most notable deficiency observed in N13 was its poor growth at pH 7.5 and Na+ concentrations up to 25 mM. During growth at pH 7.5, NhaC is apparently a major component of the relatively high affinity Na+/H+ antiport activity available to extrude the Na+ and to confer some initial protection in the face of a sudden upshift in external pH, i.e., before full induction of additional antiporters. Consistent with the inference that NhaC is a relatively high affinity, electrogenic Na+/H+ antiporter, N13 exhibited a defect in diffusion potential-energized efflux of 22Na+ from right-side-out membrane vesicles from cells that were preloaded with 2 mM Na+ and energized at pH 7.5. When the experiment was conducted with vesicles loaded with 25 mM Na+, comparable efflux was observed in preparations from all the strains. 相似文献
14.
pH controls the activity of the NhaA Na+/H+ antiporter of Escherichia coli. In the present work we show that replacement of glycine 338 of NhaA with serine (G338S) alleviates the pH control of the antiporter. Monitoring Na+-dependent collapse of DeltapH, to assess antiporter activity in isolated membrane vesicles, shows that the mutant protein is practically independent of pH, between pH 7 and 9, and even at pH 6 is 70% active. Similarly the purified reconstituted mutant protein catalyzes pH-independent passive efflux of 22Na from proteoliposomes as well as DeltapH-driven influx. Whereas the native NhaA in isolated membrane vesicles is exposed to digestion by trypsin only above pH 7, the mutated protein is degraded already at pH 6.5. DeltanhaA DeltanhaB cells transformed with a plasmid encoding the pH-independent antiporter are sensitive to Na+ but not to K+ at alkaline pH, while growing in the presence of both ions at neutral pH. Several possibilities that could explain the Na+ sensitivity of the mutant at alkaline pH were excluded; Western analysis and measurement of Na+/H+ antiporter activity in membrane vesicles, isolated from cells shifted to the non-permissive growth conditions, showed neither reduced expression of G338S-NhaA nor defective activity. The finding that the mutated protein is electrogenic led to the retraction of the idea that the protein is active in vitro but not in vivo at alkaline pH, when only Deltapsi exists in the cells. The Na+ concentration needed for half-maximal activity of G338S in isolated everted membrane vesicles is similar to that of the wild type. Therefore an increase in intracellular Na+ due to a reduced antiporter affinity could not explain the results. It is suggested that the loss of growth at alkaline pH in the presence of Na+ is due to the loss of the pH control of the mutated NhaA. Indeed, in the four mutations suppressing G338S phenotype, growth at alkaline pH was restored together with the pH regulation of NhaA. Three of the four suppressor mutations cluster in helix IV, whereas the original mutation is in helix XI, suggesting that the two helixes interact. 相似文献
15.
E Estrada P Agostinis JR Vandenheede J Goris W Merlevede J Fran?ois A Goffeau M Ghislain 《Canadian Metallurgical Quarterly》1996,271(50):32064-32072
The plasma membrane H+-ATPase of Saccharomyces cerevisiae is subject to phosphorylation by a casein kinase I activity in vitro. We show this casein kinase I activity to result from the combined function of YCK1 and YCK2, two highly similar and plasma membrane-associated casein kinase I homologues. First, H+-ATPase phosphorylation is severely impaired in the plasma membrane of YCK-deficient yeast strains. Furthermore, the wild-type level of the phosphoprotein is restored by the addition of purified mammalian casein kinase I to the mutant membranes. We used the H+-ATPase as well as a synthetic peptide substrate that contains a phosphorylation site for casein kinase I to compare kinase activity in membranes prepared from yeast cells grown in the presence or absence of glucose. The addition of glucose results in increased H+-ATPase activity which is associated with a decline in the phosphorylation level of the enzyme. Mutations in both YCK1 and YCK2 affect this regulation, suggesting that H+-ATPase activity is modulated by glucose via a combination of a "down-regulating" casein kinase I activity and another, yet uncharacterized, "up-regulating" kinase activity. Biochemical mapping of phosphorylated H+-ATPase identifies a major phosphopeptide that contains a consensus phosphorylation site (Ser-507) for casein kinase I. Site-directed mutagenesis of this consensus sequence indicates that Glu-504 is important for glucose-induced decrease in the apparent Km for ATP. 相似文献
16.
T Hiramatsu K Kodama T Kuroda T Mizushima T Tsuchiya 《Canadian Metallurgical Quarterly》1998,180(24):6642-6648
We cloned several genes encoding an Na+/H+ antiporter of Staphylococcus aureus from chromosomal DNA by using an Escherichia coli mutant, lacking all of the major Na+/H+ antiporters, as the host. E. coli cells harboring plasmids for the cloned genes were able to grow in medium containing 0.2 M NaCl (or 10 mM LiCl). Host cells without the plasmids were unable to grow under the same conditions. Na+/H+ antiport activity was detected in membrane vesicles prepared from transformants. We determined the nucleotide sequence of the cloned 7-kbp region. We found that seven open reading frames (ORFs) were necessary for antiporter function. A promoter-like sequence was found in the upstream region from the first ORF. One inverted repeat followed by a T-cluster, which may function as a terminator, was found in the downstream region from the seventh ORF. Neither terminator-like nor promoter-like sequences were found between the ORFs. Thus, it seems that the seven ORFs comprise an operon and that the Na+/H+ antiporter consists of seven kinds of subunits, suggesting that this is a novel type of multisubunit Na+/H+ antiporter. Hydropathy analysis of the deduced amino acid sequences of the seven ORFs suggested that all of the proteins are hydrophobic. As a result of a homology search, we found that components of the respiratory chain showed sequence similarity with putative subunits of the Na+/H+ antiporter. We observed a large Na+ extrusion activity, driven by respiration in E. coli cells harboring the plasmid carrying the genes. The Na+ extrusion was sensitive to an H+ conductor, supporting the idea that the system is not a respiratory Na+ pump but an Na+/H+ antiporter. Introduction of the plasmid into E. coli mutant cells, which were unable to grow under alkaline conditions, enabled the cells to grow under such conditions. 相似文献
17.
M Gekle N Golenhofen H Oberleithner S Silbernagl 《Canadian Metallurgical Quarterly》1996,93(19):10500-10504
There is increasing evidence for an additional acute, nongenomic action of the mineralocorticoid hormone aldosterone on renal epithelial cells, leading to a two-step model of mineralocorticoid action on electrolyte excretion. We investigated the acute effect of aldosterone on intracellular free Ca2+ and on intracellular pH in an aldosterone-sensitive Madin-Darby canine kidney cell clone. Within seconds of application of aldosterone, but not of the glucocorticoid hydrocortisone, there was a 3-fold sustained increase of intracellular Ca2+ at a half-maximal concentration of 10(-10) mol/liter. Omission of extracellular Ca2+ prevented this hormone response. In the presence of extracellular Ca2+ aldosterone led to intracellular alkalinization. The Na+/H+ exchange inhibitor ethyl-isopropanol-amiloride (EIPA) prevented the aldosterone-induced alkalinization but not the aldosterone-induced increase of intracellular Ca2+. Omission of extracellular Ca2+ also prevented aldosterone-induced alkalinization. Instead, aldosterone led to a Zn(2+)-dependent intracellular acidification in the presence of EIPA, indicative of an increase of plasma membrane proton conductance. Under control conditions, Zn2+ prevented the aldosterone-induced alkalinization completely. We conclude that aldosterone stimulated net-entry of Ca2+ from the extracellular compartment and a plasma membrane H+ conductance as prerequisites for the stimulation of plasma membrane Na+/H+ exchange which in turn modulates K+ channel acitivity. It is probable that the aldosterone-sensitive H+ conductance maintains Na+/H+ exchange activity by providing an acidic environment in the vicinity of the exchanger. Thus, genomic action of aldosterone determines cellular transport equipment, whereas the nongenomic action regulates transporter activity that requires responses within seconds or minutes, which explains the rapid effects on electrolyte excretion. 相似文献
18.
Identification of a mitochondrial Na+/H+ exchanger 总被引:1,自引:0,他引:1
The electroneutral exchange of protons for Na+ and K+ across the mitochondrial inner membrane contributes to organellar volume and Ca2+ homeostasis. The molecular nature of these transporters remains unknown. In this report, we characterize a novel gene (YDR456w; renamed NHA2) in Saccharomyces cerevisiae whose deduced protein sequence is homologous to members of the mammalian Na+/H+ exchanger gene family. Fluorescence microscopy showed that a Nha2-green fluorescent protein chimera colocalizes with 4',6-diamidino-2-phenylindole staining of mitochondrial DNA. To assess the function of Nha2, we deleted the NHA2 gene by homologous disruption and found that benzamil-inhibitable, acid-activated 22Na+ uptake into mitochondria was abolished in the mutant strain. It also showed retarded growth on nonfermentable carbon sources and severely reduced survival during the stationary phase of the cell cycle compared with the parental strain, consistent with a defect in aerobic metabolism. Sequence comparisons revealed that Nha2 has highest identity to a putative Na+/H+ exchanger homologue (KIAA0267; renamed NHE6) in humans. Northern blot analysis demonstrated that NHE6 is ubiquitously expressed but is most abundant in mitochondrion-rich tissues such as brain, skeletal muscle, and heart. Fluorescence microscopy showed that a NHE6-green fluorescent protein chimera also accumulates in mitochondria of transfected HeLa cells. These data indicate that NHA2 and NHE6 encode homologous Na+/H+ exchangers and suggest they may be important for mitochondrial function in lower and higher eukaryotes, respectively. 相似文献
19.
Previously we have shown that the Na+-translocating Escherichia coli (F1-delta)/Propionigenium modestum (Fo+delta) hybrid ATPase acquires a Na+-independent phenotype by the c subunit double mutation F84L, L87V that is reflected by Na+-independent growth of the mutant strain MPC8487 on succinate [Kaim, G., and Dimroth, P. (1995) J. Mol. Biol. 253, 726-738]. Here we describe a new class of mutants that were obtained by random mutagenesis and screening for Na+-independent growth on succinate. All six mutants of the new class contained four mutations in the a subunit (S89P, K220R, V264E, I278N). Results from site-specific mutagenesis revealed that the substitutions K220R, V264E, and I278N were sufficient to create the new phenotype. The resulting E. coli mutant strain MPA762 could only grow in the absence but not in the presence of Na+ ions on succinate minimal medium. This effect of Na+ ions on growth correlated with a Na+-specific inhibition of the mutant ATPase. The Ki for NaCl was 1. 5 mM at pH 6.5, similar to the Km for NaCl in activating the parent hybrid ATPase at this pH. On the other hand, activation by Li+ ions was retained in the new mutant ATPase. In the absence of Na+ or Li+, the mutant enzyme had the same pH optimum at pH 6.5 and twice the specific activity as the parent hybrid ATPase. In accordance with the kinetic data, the reconstituted mutant ATPase catalyzed H+ or Li+ transport but no Na+ transport. These results show for the first time that the coupling ion selectivity of F1Fo ATPases is determined by structural elements not only of the c subunit but also of the a subunit. 相似文献
20.
We identified amino acid residues important for activity of sod2, the Na+/H+ antiporter of Schizosaccharomyces pombe. We mutated all eight His residues of sod2 into Arg. Only His367-->Arg affected function and resulted in complete inability of sod2 to allow growth of S. pombe in LiCl-containing medium. Mutant S. pombe (H367R) could not expel sodium in acidic (pH 4.0) medium and were defective in their ability to alkalinize external medium. When His367 was replaced by Asp, sodium export of S. pombe was suppressed at acidic pH while the sodium-dependent proton influx at pH 6.1 was increased compared to wild type. We also mutated three residues conserved in putative membrane regions of various eukaryotic and prokaryotic Na+/H+ exchangers. S. pombe containing Asp241-->Asn and Asp266, 267-->Asn mutations had greatly impaired growth in LiCl-containing medium. In addition, sodium-dependent proton influx at external pH 6. 1 was impaired. Sodium export from S. pombe cells at external pH 4.0 was also almost completely abolished by the D266,267N mutation; however, the D241N mutant protein retained almost normal Na+ export. The results demonstrate that His367, Asp241, and Asp266,267 are important in the function of the eukaryotic Na+/H+ exchanger sod2. 相似文献