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.     

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     

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.     

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.     

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.     

The proton pump/leak mechanism of unconsciousness

There are few explanations which account for the manner in which the catastrophic physiological consequences of anaesthesia, cold narcosis or, for that matter, a short, sharp upper-cut, come about. Most studies terminate with the presentation of ever-better correlations between an end-point in a model system (dough consistency, rubber elasticity, bacterial, protozoal or animal mobility, liposome permeability, luciferase activity, etc.) and oil/water partition co-efficients or with some arbitrary biological end-point. From what is currently known about (a) the permeating pathways of non-electrolytes, ions and protons across membranes e.g. liposomes, (b) the effect of anaesthetics on such pathways and (c) the effect of temperature and pressure on both liposomes and whole animals, it is possible to develop a testable hypothesis. It is called the ‘proton pump-leak’ hypothesis and involves a number of linked biophysical and biochemical processes. (d) It assumes that a living animal or plant is in a steady-state regarding all concentration gradients; passive leaks across membranes are balanced by temperature, pressure, and energy dependent ion/ion and/or proton/ion pumps (enzyme), working within an aqueous phase. (e) Consciousness is dependent upon inter-neuronal communication via release of transmitter substances. (f) Transmitter substances, characteristically either weak bases or weak acids e.g. catecholamines, accumulate passively in vesicles rich in acid-buffer, held to a low pH by the activity of H⁺/K⁺ energy-driven pumps. Interference with this finely-balanced system either by changing the chemical potential of the hydrophobic (membrane) phase at NTP (with anaesthetics), or by changing the chemical potential of both hydrophobic and aqueous (pump) phases by hyperbaric, hyphotermic, or anoxic conditions imposed (inevitably) on the whole animal, would result in the resetting of the steady-state parameters.     

The functions of plant cation/proton antiporters

The cation/H⁺ exchange is a basic process in transmembrane transport. The acquisition of genome sequences has now established that plants possess genes encoding a large number of cation/proton antiporter 1 (CPA1) proteins, few of which have been characterized with respect to their contribution to ion homeostasis. The CPA1s comprise plasma membrane, vacuolar, and endosomal forms, and they have been identified as important for a salinity tolerance. They are, however, also involved in both the control of cellular pH and K⁺ homeostasis, and regulate processes over a wide range of physiological events, from vesicle trafficking to development.     

Evolutionary origins of eukaryotic sodium/proton exchangers

More than 200 genes annotated as Na+/H+ hydrogen exchangers (NHEs) currently reside in bioinformation databases such as GenBank and Pfam. We performed detailed phylogenetic analyses of these NHEs in an effort to better understand their specific functions and physiological roles. This analysis initially required examining the entire monovalent cation proton antiporter (CPA) superfamily that includes the CPA1, CPA2, and NaT-DC families of transporters, each of which has a unique set of bacterial ancestors. We have concluded that there are nine human NHE (or SLC9A) paralogs as well as two previously unknown human CPA2 genes, which we have named HsNHA1 and HsNHA2. The eukaryotic NHE family is composed of five phylogenetically distinct clades that differ in subcellular location, drug sensitivity, cation selectivity, and sequence length. The major subgroups are plasma membrane (recycling and resident) and intracellular (endosomal/TGN, NHE8-like, and plant vacuolar). HsNHE1, the first cloned eukaryotic NHE gene, belongs to the resident plasma membrane clade. The latter is the most recent to emerge, being found exclusively in vertebrates. In contrast, the intracellular clades are ubiquitously distributed and are likely precursors to the plasma membrane NHE. Yeast endosomal ScNHX1 was the first intracellular NHE to be described and is closely related to HsNHE6, HsNHE7, and HsNHE9 in humans. Our results link the appearance of NHE on the plasma membrane of animal cells to the use of the Na+/K(+)-ATPase to generate the membrane potential. These novel observations have allowed us to use comparative biology to predict physiological roles for the nine human NHE paralogs and to propose appropriate model organisms in which to study the unique properties of each NHE subclass.     

The electrochemical proton potential and the proton/electron ratio of the electron transport with fumarate in Wolinella succinogenes

The electrochemical proton potential across the cytoplasmic membrane ( ) as well as the H⁺ / e ratio, which were brought about by the electron transport of Wolinella succinogenes, was measured with the aim of understanding the mechanism of electron-transport-coupled phosphorylation in this anaerobic bacterium. (1) Inverted vesicles derived from the bacterial membrane were found to take up protons from the external medium on initiation of fumarate reduction by H₂. Proton uptake was dependent on the presence of K⁺ within the vesicles, was enhanced by the presence of valinomycin and DCCD and high internal buffer concentration, and was abolished by protonophores. The maximum H⁺ / e ratio slightly exceeded 1. (2) The vesicles accumulated thiocyanate in the steady state of fumarate reduction by H₂. The concentration ratio (internal / external) was close to 1000 at an external thiocyanate concentration below 10 μM. Under the same conditions the uptake of methylamine was negligible. Thiocyanate uptake was abolished by the presence of a protonophore. (3) Cells of W. succinogenes accumulated tetraphenylphosphonium cation (TPP) in the steady state of fumarate reduction with H₂ or formate. Under the same conditions the uptake of benzoic acid was negligible. From the amount of TPP taken up by the bacteria, the free internal concentration of TPP was evaluated according to the procedure of Zaritsky et al. (Zaritsky, A., Kihara, M. and MacNab, R.M. (1981) J. Membrane Biol. 63, 215–231). The concentration ratio (internal / external) was 700 in the absence and close to 1 in the presence of a protonophore or in the absence of external Na⁺. (4) The experimental results are consistent with the view that the energy transduction from electron transport to phosphorylation is done by means of the across the bacterial membrane.     

A cation/proton antiport activity in Acholeplasma laidlawii

Sealed membrane vesicles of Acholeplasma laidlawii were obtained by controlled lysis of carotenoid-rich intact cells. An imposed delta pH was created by loading membrane vesicles or intact Acholeplasma laidlawii cells with 0.25 M NH4Cl and diluting them into 0.25 M choline chloride. The passive efflux of NH3 from the membrane vesicles or cells resulted in the creation of a delta pH (inside acid) that could be visualized by the quenching of the fluorescence of the weak base acridine orange. Whereas with isolated membrane vesicles, the fluorescence was dequenched by the addition of Na+, with intact cells, K+ in addition to Na+ was required. These results strongly suggest a Na+/H+ exchange activity that in intact Acholeplasma laidlawii cells is K+-dependent. The possible role of the Na+/H+ exchange activity in pH homeostasis at the more alkaline pH range, as well as in the extrusion of excess Na+ from the cells is discussed.     

Transmembrane proton translocation by cytochrome c oxidase

Respiratory heme-copper oxidases are integral membrane proteins that catalyze the reduction of molecular oxygen to water using electrons donated by either quinol (quinol oxidases) or cytochrome c (cytochrome c oxidases, CcOs). Even though the X-ray crystal structures of several heme-copper oxidases and results from functional studies have provided significant insights into the mechanisms of O₂-reduction and, electron and proton transfer, the design of the proton-pumping machinery is not known. Here, we summarize the current knowledge on the identity of the structural elements involved in proton transfer in CcO. Furthermore, we discuss the order and timing of electron-transfer reactions in CcO during O₂ reduction and how these reactions might be energetically coupled to proton pumping across the membrane.     

Elimination of proton donor strongly affects directionality and efficiency of proton transport in ESR,a light-driven proton pump from Exiguobacterium sibiricum

ESR from Exiguobacterium sibiricum is a retinal protein which functions as a proton pump. Unusual feature of ESR is that a lysine residue is present at a site for the internal proton donor, which in other proton pumps is a carboxylic residue. Replacement of Lys96 with alanine slows reprotonation of the Schiff base by two orders of magnitude, indicating that Lys96 and interacting water molecules function as internal proton donor to the Schiff base. In this work we examined time resolved generation of light-induced electric potential ΔΨ by the K96A mutant reconstituted into proteoliposomes. We found that the ΔΨ component, which accompanied reprotonation of the Schiff base in wild type ESR, was not only slowed but also decreased greatly in the mutant, and negative phase appeared at high pH. This indicates a higher probability of back reactions in ESR than in bacteriorhodopsin since no negative components have been observed in homologous mutants of BR, D96N and D96A. The higher rate of back reactions in ESR is probably caused by different arrangement of the proton acceptor site compared to that in BR and different sequence of proton release and uptake. Addition of sodium azide, which substitutes for the internal proton donor, restores both the rate and amplitude of the ΔΨ components related to the Schiff base reprotonation in the K96A mutant. This indicates that overall proton transport results from competition of forward and reverse reactions, and emphasizes the importance of internal donor for high efficiency and directionality of H⁺ transfer.