Cation/proton antiport systems in Escherichia coli. Solubilization and reconstitution of delta pH-driven sodium/proton and calcium/proton antiporters

Uptake of 22Na+ and 45Ca2+ into everted membrane vesicles from Escherichia coli was measured with imposed transmembrane pH gradients, acid interior, as driving force. Vesicles loaded with 0.5 M KCl were diluted into 0.5 M choline chloride to create a potassium gradient. Addition of nigericin to produce K+/H+ exchange resulted in formation of a pH gradient. This imposed gradient was capable of driving 45Ca2+ accumulation. In another method vesicles loaded with 0.5 M NH4Cl were diluted into 0.5 M choline chloride, creating an ammonium diffusion potential. A gradient of H+ was produced by passive efflux of NH3. With an ammonium gradient as driving force, everted vesicles accumulated both 45Ca2+ and 22Na+. The data suggest that 22Na+ uptake was via the sodium/proton antiporter and 45Ca2+ via the calcium/proton antiporter. Uptake of both cations required alkaline pHout. A minimum pH gradient of 0.9 unit was needed for transport of either ion, suggesting gating of the antiporters. Octyl glucoside extracts of inner membrane were reconstituted with E. coli phospholipids in 0.5 M NH4Cl. NH4+-loaded proteoliposomes accumulated both 22Na+ and 45Ca2+, demonstrating that the sodium/proton and calcium/proton antiporters could be solubilized and reconstituted in a functional form.     

Cation/proton antiport systems in escherichia coli: properties of the sodium/proton antiporter.

The sodium/proton antiport system of Escherichia coli has been characterized by the effect of Na⁺ on the pH gradient established by respiration in everted membrane vesicles. The system has equal affinity for Na⁺ and Li⁺. Between pH 7 and 9 dissipation of Δψ, membrane potential, has no effect on the affinity for Na⁺ but decreases the V of the antiport reaction. Uptake of ²²Na⁺ by everted membrane vesicles was observed using flow dialysis.     

Sodium/proton antiporter in Streptococcus faecalis.

Streptococcus faecalis, like other bacteria, accumulates potassium ions and expels sodium ions. This paper is concerned with the pathway of sodium extrusion. Earlier studies (D.L. Heefner and F.M. Harold, Proc. Natl. Acad. Sci. USA 79:2798-2802, 1982) showed that sodium extrusion is effected by a primary, ATP-linked sodium pump. I report here that cells grown under conditions in which sodium ATPase is not induced can still expel sodium ions. This finding suggested the existence of an alternate pathway. Sodium extrusion by the alternate pathway requires the cells to generate a proton motive force. This conclusion rests on the following observations. (i) Sodium extrusion required glucose. (ii) Sodium extrusion was observed at neutral pH, which allows the cells to generate a proton motive force, but not at alkaline pH, which reduces the proton motive force to zero. (iii) Sodium extrusion was inhibited by the addition of dicyclohexylcarbodiimide and of proton-conducting ionophores. (iv) In response to an artificial pH gradient (with the exterior acid), energy-depleted cells exhibited a transient sodium extrusion which was unaffected by treatments that dissipated the membrane potential and which was blocked by proton conductors. I propose that streptococci have two independent systems for sodium extrusion: an inducible sodium ATPase and a constitutive sodium/proton antiporter.     

Cation/proton antiport systems in Escherichia coli.

Three distinct systems which function as proton/cation antiports have been identified in $\text{E.}\text{coli}$ by the ability of the ions to dissipate the ΔpH component of the protonmotive force in everted vesicles. System I exchanges H⁺ for K⁺, Rb⁺ or Na⁺; System II has Na⁺ and Li⁺ as substrates; and System III catalyzes proton exchange for Ca²⁺, Mn²⁺ or Sr²⁺.     

Stoichiometry of catecholamine/proton exchange across the chromaffin-granule membrane.

Catecholamines are accumulated by bovine chromaffin-granule "ghosts" in the presence of MgATP at 25 degrees C. With low concentrations of catecholamine, ratios of internal to external amine concentration of up to 20 000 were obtained. These values fit well with a transport model in which amine accumulation is both electrogenic and dependent on a pH gradient across the membrane.     

Observation of the phosphatidyl ethanolamine amino proton magnetic resonance in phospholipid vesicles: inside/outside ratios and proton transport.

The amino proton resonance of phosphatidyl ethanolamine in sonicated mixed phospholipid vesicles is observed 3.3 ppm downfield from H₂O. Above pH ～ 5 it is broadened beyond detectability as a result of exchange with H₂O protons. In low salt, resonances of amino protons inside the vesicles appear to persist as the pH is raised, while those on the outside disappear. Solvent catalized proton conduction along the surface is proposed, with an effective -NH₂ to -NH₃ transfer rate of about 8 × 10⁵ sec^?1 at 25°C.     

Influenza virus proton channels.

The M2 ion channel proteins of influenza A and B viruses are essential to viral replication. The two ion channels share a common motif, HXXXW, that is responsible for proton selectivity and activation. The ion channel for the influenza A virus, but not influenza B virus, is inhibited by the antiviral drug amantadine and amantadine-resistant escape mutants form in treated influenza A patients. The studies reviewed suggest that an antiviral compound directed against the conserved motif would be more useful than amantadine in inhibiting viral replication.     

Potassium/proton antiport system of growing Enterococcus hirae at high pH.

The cytoplasmic pH (pHin) of Enterococcus hirae growing at pH 9.2 was maintained at about 8.1. Membrane-permeating amines such as ammonia alkalinized the pHin from 8.1 to 9.0 at a high concentration and induced K+ extrusion. The pHin alkalinization was transient; the pHin fell from 9.0 to the original value of pH 8.1, at which point K+ extrusion ceased, and remained constant. Cells accumulated ammonium ion to an extent stoichiometrically equivalent to the K+ loss. This bacterium continued to grow well under this condition. These results suggest that the pHin-responsive primary K+/H+ antiport system (Y. Kakinuma, and K. Igarashi, J. Biol. Chem. 263:14166-14170, 1988) works for the pHin regulation of this organism growing at a high pH.     

Properties of Escherichia coli mutants altered in calcium/proton antiport activity.

Mutants sensitive to growth inhibition by CaCl2 were found to have alterations in calcium uptake in everted membrane vesicles. These mutations map at different loci on the Escherichia coli chromosomes. A mutation at the calA locus results in vesicles which have two- to threefold higher levels of uptake activity than vesicles from wild-type cells. The calA mutation is phenotypically expressed as increased sensitivity to CaCl2 in a strain also harboring a mutation in the corA locus, which is involved in Mg2+ transport. The calA locus maps very close to purA and cycA at about min 97. The calB mutation results both in sensitivity to CaCl2 at pH 5.6 and in vesicles with diminished calcium transport capability. The CalB phenotype is also expressed only in a corA genetic background; the calB locus appears to map very near, yet separately from, the calA locus. When the cor+ allele is present, calA and calB mutations still result in a defect in calcium transport in vesicles. In addition, both calC and calD mutations result in vesicles with impaired calcium transport activity. calC is cotransducible with kdp and nagA, whereas calD is cotransducible with proC.     

The proton ladder,a static mechanism for ion/proton coports and counterports

An ion/proton counterport is formed simply by locating a chain of ionizable residues connected by a proton conducting path near a passive ion pore which spans the membrane. The electric coupling between the ion in transit through the pore and the residues can ensure that for each ion passing through the pore in one direction a proton is driven along the chain of ionizable residues (the proton ladder) in the same or in the opposite direction. The mechanism is symmetrical in that a trans-membrane ion gradient may drive protons against their electrochemical potential gradient or a proton gradient may drive ions against theirs. The mechanism is applicable to cation or anion channels and to coports or counterports. No mechanical motion is required other than the motion of the ions and the protons. Monte Carlo computer simulations are performed on the model and its predicted properties are listed. The new type of counterport model is compared with currently used models. Offprint requests to: D. T Edmonds     

Crystallization of proton channel peptides.

Crystals have been grown of two similar peptides that form ion-conducting channels in diphytanoyl phosphatidylcholine bilayers. These crystals were grown by slow evaporation of the organic solvent, 2,2,2-trifluoroethanol. Crystals of one of the peptides have been characterized by X-ray diffraction, and X-ray data have been measured to 2.3 A resolution. Earlier it was proposed that the ion-conducting channels formed by these peptides consist of four peptides associated as a parallel alpha-helical tetramer. On the basis of the space group and unit cell dimensions of the crystals, a packing scheme for the peptide is proposed that is consistent with a tetrameric channel.     

Progress with proton pump inhibition.

The proton pump, a H+/K(+)-ATPase located on the secretory canalicular membrane of the parietal cell, forms the final pathway for gastric acid secretion. Omeprazole is concentrated in the secretory canaliculus, where it is converted to its active form, which binds covalently with the H+/K(+)-ATPase, thus inhibiting acid secretion arising from any stimulus. Meta-analysis has defined the primary determinants for peptic ulcer healing as the degree of acid suppression, the duration of suppression over 24 hours, and the length of treatment. The longer duration of acid suppression with omeprazole, particularly during the day, when food is ingested and H2-receptor antagonists are less effective, is reflected in the clinical superiority for symptom relief and ulcer healing and especially for the treatment of erosive esophagitis. Extensive clinical experience has proved omeprazole to be safe, and concerns over hypergastrinemia, ECL-cell hyperplasia, and carcinoid formation have not been substantiated in humans. Recent evidence has shown that omeprazole suppresses Helicobacter pylori and, in combination with antibiotics, can eradicate this organism in a substantial proportion of patients. This effect may result from enhancement of antibiotic bioavailability and optimizing host defense mechanisms.     

The sodium/proton antiport system in a newly isolated alkalophilic Bacillus sp.

The pH homeostasis and the sodium/proton antiport system have been studied in the newly isolated alkalophilic Bacillus sp. strain N-6, which could grow on media in a pH range from 7 to 10, and in its nonalkalophilic mutant. After a quick shift in external pH from 8 to 10 by the addition of Na2CO3, the delta pH (inside acid) in the cells of strain N-6 was immediately established, and the pH homeostatic state was maintained for more than 20 min in an alkaline environment. However, under the same conditions, the pH homeostasis was not observed in the cells of nonalkalophilic mutant, and the cytoplasmic pH immediately rose to pH 10. On the other hand, the results of the rapid acidification from pH 9 to 7 showed that the internal pH was maintained as more basic than the external pH in a neutral medium in both strains. The Na+/H+ antiport system has been characterized by either the effect of Na+ on delta pH formation or 22Na+ efflux in Na+-loaded right-side-out membrane vesicles of strain N-6. Na+- or Li+-loaded vesicles exhibited a reversed delta pH (inside acid) after the addition of electron donors (ascorbate plus tetramethyl-p-phenylenediamine) at both pH 7 and 9, whereas choline-loaded vesicles generated delta pHs of the conventional orientation (inside alkaline). 22Na+ was actively extruded from 22Na+-loaded vesicles whose potential was negative at pH 7 and 9. The inclusion of carbonyl cyanide m-chlorophenylhydrazone inhibited 22Na+ efflux in the presence of electron donors. These results indicate that the Na+/H+ antiport system in this strain operates electrogenically over a range of external pHs from 7 to 10 and plays a role in pH homeostasis at the alkaline pH range. The pH homeostasis at neutral ph was studied in more detail. K+ -depleted cells showed no delta pH (acid out) in the neutral conditions in the absence of K+, whereas these cells generated a delta pH if K+ was present in the medium. This increase of internal pH was accompanied by K+ uptake from the medium. These results suggest that electrogenic K+ entry allows extrusion of H+ from cells by the primary proton pump at neutral pH.     

Purified proton conductor in proton translocating adenosine triphosphatase of a thermophilic bacterium.

1. The membrane-integrated portion (TF0) of the proton translocating ATPase complex (TF0-F1) of the thermophilic bacterium PS3 was highly purified. Its proton-conducting activity was investigated in vesicles reconstituted from TF0 and phospholipids (TF0 vesicles). 2. The rate of proton conduction through TF0 was proportional to the membrane potential imposed (6H+ uptake/s/TF0 molecule with 103 mV at pH 8.0). The pH profile of the rate revealed that a proton, not a hydroxy ion, was the true substrate conducted and that there was a monoprotic proton binding site in TF0 (pKa = 6.8). The temperature coefficient of proton conductance of TF0 showed a considerable variation depending on the phospholipids of the vesicles with respective transition temperatures. 3. Passive proton conduction through TF0 was inhibited stoichiometrically by addition of either the soluble ATPase portion (TF1) of TF0-F1, or an energy transfer inhibitor dicyclohexylcarbodiimide or an antibody against TF0. 4. The proton conductance of TF0 was concluded to represent its intrinsic activity in the original TF0-F1 complex.     

Light-driven proton translocations in Halobacterium halobium

The purple membrane of Halobacterium halobium acts as a light-driven proton pump, ejecting protons from the cell interior into the medium and generating an electrochemical proton gradient across the cell membrane. However, the typical response of cells to light as measured with a pH electrode in the medium consists of an initial net inflow of protons which subsides and is then replaced by a net outflow which exponentially approaches a new lower steady state pH level. When the light is turned off a small transient acidification occurs before the pH returns to the original dark level. We present experiments suggesting that the initial inflow of protons is triggered by the beginning ejection of protons through the purple membrane and that the initial inflow rate is larger than the continuing light-driven outflow. When the initial inflow has decreased exponentially to a small value, the outflow dominates and causes the net acidification of the medium.The initial inflow is apparently driven by a pre-existing electrochemical gradient across the membrane, which the cells can maintain for extended times in the absence of light and oxygen. Treatments which collapse this gradient such as addition of small concentrations of uncouplers abolish the initial inflow.The triggered inflow occurs through the ATPase and is accompanied by ATP synthesis. Inhibitors of the ATPase such as N,N′-dicyclohexylcarbodiimide (DCCD) inhibit ATP synthesis and abolish the inflow. They also abolish the transient light-off acidification, which is apparently caused by a short burst of ATP hydrolysis before the enzyme is blocked by its endogenous inhibitor.Similar transient inflows and outflows of protons are also observed when anaerobic cells are exposed to short oxygen pulses.