Neuronal cell surface ATP synthase mediates synthesis of extracellular ATP and regulation of intracellular pH

The ATP synthase is known to play important roles in ATP generation and proton translocation within mitochondria. Here, we now provide evidence showing the presence of functional ecto‐ATP synthase on the neuronal surface. Immunoblotting revealed that the α, β subunits of ATP synthase F₁ portion are present in isolated fractions of plasma membrane and biotin‐labelled surface protein from primary cultured neurons; the surface distribution of α, β subunits was also confirmed by immunofluorescence staining. Moreover, α and β subunits were also found in fractions of plasma membrane and lipid rafts isolated from rat brain, and flow cytometry analysis showed α subunits on the surface of acutely isolated brain cells. Activity assays showed that the extracellular ATP generation of cultured neurons could be compromised by α, β subunit antibodies and ATP synthase inhibitors. pH_i (intracellular pH) analysis demonstrated that at low extracellular pH, α or β subunit antibodies decreased pH_i of primary cultured neurons. Therefore, ATP synthase on the surface of neurons may be involved in the machineries of extracellular ATP generation and pH_i homoeostasis.     

The rotary mechanism of the ATP synthase

The F₀F₁ ATP synthase is a large complex of at least 22 subunits, more than half of which are in the membranous F₀ sector. This nearly ubiquitous transporter is responsible for the majority of ATP synthesis in oxidative and photo-phosphorylation, and its overall structure and mechanism have remained conserved throughout evolution. Most examples utilize the proton motive force to drive ATP synthesis except for a few bacteria, which use a sodium motive force. A remarkable feature of the complex is the rotary movement of an assembly of subunits that plays essential roles in both transport and catalytic mechanisms. This review addresses the role of rotation in catalysis of ATP synthesis/hydrolysis and the transport of protons or sodium.     

The rotary mechanism of ATP synthase

Since the chemiosmotic theory was proposed by Peter Mitchell in the 1960s, a major objective has been to elucidate the mechanism of coupling of the transmembrane proton motive force, created by respiration or photosynthesis, to the synthesis of ATP from ADP and inorganic phosphate. Recently, significant progress has been made towards establishing the complete structure of ATP synthase and revealing its mechanism. The X-ray structure of the F(1) catalytic domain has been completed and an electron density map of the F(1)-c(10) subcomplex has provided a glimpse of the motor in the membrane domain. Direct microscopic observation of rotation has been extended to F(1)-ATPase and F(1)F(o)-ATPase complexes.     

The mechanism of action of mitochondrial ATPase (ATP synthase)

The F₁ part of the ATP synthase contains 6 nucleotide binding sites, four of which can be occupied and covalently labeled with 8-azido-adenine nucleotides. The other two sites contain tightly bound nucleotides that cannot be replaced by 8-azido-adenine nucleotides. Of the four exchangeable sites two are directly ivolved in catalysis and these are located on -subunits, while the other two are located at - interfaces and have probably a regulatory role by influencing the affinity of the catalytic sites for substrate and product. When only one catalytic site contains substrate the affinity is very high, the rate of hydrolysis is slow, and the dissociation of products is even slower (single-site catalysis). When the second site also becomes occupied, the affinity decreases enormously, and the rate of hydrolysis and dissociation of products increases several orders of magnitude. When, however, the second site is occupied by substrate in such a way that turnover is not possible at this site (e.g., covalent linkage of nitreno-ATP), the first site is no longer active, apart from the very slow single-site catalysis. The two nonexchangeable, tightly bound nucleotides that cannot be replaced by 8-azido-nucleotides, can be replaced by 2-azido-nucleotides, due to their anticonfiguration. This anticonfiguration of the substrate is also required for binding with high affinity to a catalytic site. A picture emerges in which one of the three - pairs of F₁ contains tightly bound, nonexchangeable nucleotides, while the other two contain both one catalytic site (on ) and one regulatory site (at the - interface). Cooperativity exists both between the two catalytic sites and between the catalytic and the regulatory sites.     

Kinetic studies of ATP synthase: The case for the positional change mechanism

The mitochondrial ATP synthases shares many structural and kinetic properties with bacterial and chloroplast ATP synthases. These enzymes transduce the energy contained in the membrane's electrochemical proton gradients into the energy required for synthesis of high-energy phosphate bonds. The unusual three-fold symmetry of the hydrophilic domain, F₁, of all these synthases is striking. Each F₁ has three identical subunits and three identical subunits as well as three additional subunits present as single copies. The catalytic site for synthesis is undoubtedly contained in the subunit or an , interface, and thus each enzyme appears to contain three identical catalytic sites. This review summarizes recent isotopic and kinetic evidence in favour of the concept, originally proposed by Boyer and coworkers, that energy from the proton gradient is exerted not directly for the reaction at the catalytic site, but rather to release product from a single catalytic site. A modification of this binding change hypotheses is favored by recent data which suggest that the binding change is due to a positional change in all three subunits relative to the remaining subunits of F₁ and F₀ and that the vector of rotation is influenced by energy. The positional change, or rotation, appears to be the slow step in the process of catalysis and it is accelerated in all F₁F₀ ATPases studied by substrate binding and by the proton gradient. However, in the mammalian mitochondrial enzyme, other types of allosteric rate regulation not yet fully elucidated seem important as well.     

High affinity interaction between histidine-rich glycoprotein and the cell surface type ATP synthase on T-cells

Histidine-rich glycoprotein (HRG) is a plasma protein implicated in the innate immune system. In recent studies, we showed that either HRG, or the Arg23-Lys66 glycopeptide derived from HRG, in concert with concanavalin A (Con A), promotes a morphological change and adhesion of the human leukemic T-cell line MOLT-4 to culture dishes, and that cell surface glycosaminoglycan or Fcγ receptors do not participate in this cellular event. In the present study, we identified the α-subunit of ATP synthase as one of the HRG-binding proteins on the surface of T-cells by HRG-derived glycopeptide affinity chromatography and by a peptide mass finger printing method. HRG specifically interacted with mitochondrial ATP synthase with a dissociation constant of 66 nM. The presence of α- and β-subunits of ATP synthase on the plasma membrane of MOLT-4 cell was demonstrated by immunofluorescent staining and FACS analysis. The HRG/Con A-induced morphological changes of MOLT-4 cells were specifically inhibited by a monoclonal antibody against the β-subunit of ATP synthase. These results strongly suggest that the cell surface ATP synthase functions as a binding protein for HRG on MOLT-4 cells, which is required for the morphological changes observed in MOLT-4 cells following treatment with HRG/Con A.     

Molecular evolution of the modulator of chloroplast ATP synthase: origin of the conformational change dependent regulation

Chloroplast ATP synthase synthesizes ATP by utilizing a proton gradient as an energy supply, which is generated by photosynthetic electron transport. The activity of the chloroplast ATP synthase is regulated in several specific ways to avoid futile hydrolysis of ATP under various physiological conditions. Several regulatory signals such as Delta mu H(+), tight binding of ADP and its release, thiol modulation, and inhibition by the intrinsic inhibitory subunit epsilon are sensed by this complex. In this review, we describe the function of two regulatory subunits, gamma and epsilon, of ATP synthase based on their possible conformational changes and discuss the evolutionary origin of these regulation systems.     

Influence of two cell harvesting methods on intracellular ATP and amino acid concentrations in human fibroblast cultures

The intracellular ATP and amino acid concentrations were determined in human fibroblast cultures reaching confluence. The values obtained were very different, depending on the cell harvesting method: trypsinization or scraping. Trypsinization appeared to be the better method for measuring the ATP concentrations (21.25 +/- 0.96 nmol per mg cell protein), this level being much lower with scraping. On the contrary, scraping was the most appropriate method for amino acid measurement. This work underlines the importance of harvesting methods for metabolic studies in human cell cultures.     

Effect of ATP inhibitors on the translocation of luminal membrane between cytoplasm and cell surface of transitional epithelial cells during the expansion-contraction cycle of the rat urinary bladder

Summary Movement of asymmetric membrane plaques between the cytoplasm and surface of luminal urothelial cells was investigated during artificially induced contraction and expansion of untreated and ATP-depleted urinary bladders of the rat. Estimations of surface area, volume, and number of discoidal vesicles per unit volume of cytoplasm were determined by morphometric examination of electron micrographs. These values were compared in luminal cells from bladders incubated in control media or in media containing 0.15 mM 2,4-dinitrophenol and 0.02 mM sodium arsenate. The ATP inhibitors had no apparent effect upon the contraction of apical cells that had been incubated in an expanded state. In contrast, after distension of poisoned, contracted bladders, the orientation of intermediate filaments and the densities of discoidal vesicles were similar to the condition characterized by contracted cells. The results indicated that the normal reorientation of filaments, coincident with cell distension, had been suppressed by ATP inhibitors. This, in effect, impeded the filament-mediated translocation of membrane plaques to the surface. The reduction of surface area along the luminal border forced many cells to compensate by separating at their lateral margins.This work was supported by NIH Grant AM 32937     

Evidence for regulation in vivo of the ATP synthase in intact cells of the photosynthetic bacterium Rhodopseudomonas capsulata

(1) The cytoplasmic membrane potential (Δψ) of intact cells of Rhodopseudomonas capsulata, measured either from the uptake of butyltriphenylphosphonium cation or from the electrochromic carotenoid band shift, increased upon illumination (negative on the cytoplasmic side) and then, within the next 20 s, partly declined while the light was still on. In the presence of the F₀ inhibitor venturicidin the light-induced Δψ was increased by 30% and the partial decline was abolished. (2) From the ionic current/Δψ curves for the bacterial membranes it was concluded that the slow, partial decline of Δψ after the onset of illumination was the result of an increase in membrane conductance. The conductance increase seen in the ionic current/Δψ curves was blocked by venturicidin suggesting that it was caused by increased proton flux through the ATP synthase. (3) Analysis of the light-induced changes in adenine nucleotide levels in intact bacterial cells showed that the apparent increase in ATP synthase activity was not the result of a decrease in phosphorylation potential. The data were consistent with either an increase in the catalytic activity of the ATP synthase or with an increase in H⁺ flux through the enzyme without a proportionate increase in the rate of phosphorylation (increased ‘slip’). (4) This slow change in the properties of the ATP synthase, as judged by the venturicidin-sensitive partial decline of Δψ, required a minimum initial value of Δψ. When Δψ was reduced, either by decreasing the actinic light intensity or by adding carbonylcyanide trifluoromethoxyphenylhydrazone the partial decline in Δψ was abolished. (5) The slow change in ATP synthase properties reversed upon darkening the bacterial cell suspension. A second illumination period shortly after the first elicited a smaller initial Δψ and a smaller Δψ decline. The relaxation of the ATP synthase in the dark was measured from the dependence of the initial increase in Δψ after the second illumination period upon the dark-time between the two illumination periods.     

Use of photoaffinity nucleotide analogs to determine the mechanism of ATP regulation of a membrane-bound,cAMP-activated protein kinase

Using a radioactively tagged, photoaffinity analog of cAMP, 8-azidoadenosine-3′,5′-cyclic monophosphate (8-N₃ cAMP), and [γ³²P] ATP, the membranebinding properties of both the regulatory and catalytic subunits of the cAMP-activated protein kinase of human erythrocyte membranes were investigated. [³²P] 8-N₃ cAMP was used to locate and quantify regulatory subunits. Increased phosphorylation of specific membrane proteins by [γ³²P] ATP was used to determine the presence of the catalytic subunit. The data support a mechanism which operates through a tight membrane-bound regulatory subunit and a catalytic subunit that is released from the membrane when cAMP is present and the Mg · ATP concentration is below approximately 10 μM. The catalytic subunit is not required for the Mg · ATP inhibition of 8-N₃ cAMP binding. Experiments with a photoaffinity analog of ATP, 8-azidoadenosine triphosphate (8-N₃ATP), support the hypothesis that ATP hydrolysis and phosphorylation are not involved in the regulation. The data indicate that the regulatory subunit contains an ATP regulatory site which inhibits 8-N₃ cAMP binding and the release of the catalytic subunit. These results indicate that the membrane-bound type I enzyme (type IM) differs significantly from the soluble (type IS) enzyme studied in other tissues. These enzymes are compartmentalized by being in different cellular locations and are regulated differently by Mg · ATP.     

The rigid connecting loop stabilizes hairpin folding of the two helices of the ATP synthase subunit c

We have tested the role of the polar loop of subunit c of the Escherichia coli ATP synthase in stabilizing the hairpin structure of this protein. The structure of the c(32-52) peptide corresponding to the cytoplasmic region of subunit c bound to the dodecylphosphocholine micelles was solved by high-resolution NMR. The region comprising residues 41-47 forms a well-ordered structure rather similar to the conformation of the polar loop region in the solution structure of the full-length subunit c and is flanked by short alpha-helical segments. This result suggests that the rigidity of the polar loop significantly contributes to the stability of the hairpin formed by the two helices of subunit c. This experimental system may be useful for NMR studies of interactions between subunit c and subunits gamma and epsilon, which together form the rotor of the ATP synthase.     

The molecular mechanism of ATP synthesis by F1F0-ATP synthase

ATP synthesis by oxidative phosphorylation and photophosphorylation, catalyzed by F₁F₀-ATP synthase, is the fundamental means of cell energy production. Earlier mutagenesis studies had gone some way to describing the mechanism. More recently, several X-ray structures at atomic resolution have pictured the catalytic sites, and real-time video recordings of subunit rotation have left no doubt of the nature of energy coupling between the transmembrane proton gradient and the catalytic sites in this extraordinary molecular motor. Nonetheless, the molecular events that are required to accomplish the chemical synthesis of ATP remain undefined. In this review we summarize current state of knowledge and present a hypothesis for the molecular mechanism of ATP synthesis.     

Valsartan reverses post-translational modifications of the delta-subunit of ATP synthase during in vivo canine reperfused myocardial infarction

To determine whether reperfused myocardial infarction (RMI) induces PTM of the delta-subunit of the mitochondrial metabolic enzyme ATP synthase (ATP/delta) in the ischemic zone (IZ) and whether this can be reversed by the angiotensin II type 1 receptor (AT(1)R) blocker valsartan, we applied a pharmaco-proteomics approach in canine RMI hearts with or without valsartan pretreatment. Using the 2-DE technique, we identified differential regional expression of ATP/delta in the IZ compared to the non-ischemic zone (NIZ), with an approximately 2-fold increase in the IZ that was normalized by valsartan. Furthermore in the IZ, RMI triggered S-nitrosylation of cysteine-100, nitration of the two tyrosines 88 and 225, and hydroxylation of lysine-182 in ATP/delta followed by its myristoylation. Importantly, valsartan abolished these modifications of ATP/delta in the IZ, triggered phosphorylation of serine-76 in both the IZ and NIZ, and decreased necrosis, apoptosis, left ventricular dysfunction and remodeling. Thus, AT(1)R-blocker-induced cardioprotection during RMI is associated with phosphorylation of ATP/delta and inhibition of nitric oxide-related chemical modifications such as S-nitrosylation, nitration and hydroxylation. Targeting specific PTMs during RMI, such as those of ATP/delta with AT(1)R blockade, might be a potentially powerful novel therapeutic approach. However, the identification of S-nitrosylation was putative and requires MS/MS verification.     

The role of transmembrane span 2 in the structure and function of subunit a of the ATP synthase from Escherichia coli

The importance of the second transmembrane span of subunit a of the ATP synthase from Escherichia coli has been established by two approaches. First, biochemical analysis of five cysteine-substitution mutants, four of which were previously constructed for labeling experiments, revealed that only D119C, found within the second transmembrane span, was deleterious to ATP synthase function. This mutant had a greatly reduced growth yield, indicating inefficient ATP synthesis, but it retained a significant level of ATP-driven proton translocation and sensitivity to N,N(')-dicyclohexyl-carbodiimide, indicating more robust function in the direction of ATP hydrolysis. Second, the entire second transmembrane span was probed by alanine-insertion mutagenesis at six different positions, from residues 98 to 122. Insertions at the central four positions from residues 107 to 117 resulted in the inability to grow on succinate minimal medium, although normal levels of membrane-bound ATPase activity and significant levels of subunit a were detected. Double mutants were constructed with a mutation that permits cross-linking to the b subunit. Cross-linked products in the mutant K74C/114iA were seen, indicating no major disruption of the a-b interface due to the insertion at 114. Analysis of the K74C/110iA double mutant indicated that K74C is a partial suppressor of 110iA. In summary, the results support a model in which the amino-terminal, cytoplasmic end of the second transmembrane span has close contact with subunit b, while the carboxy-terminal, periplasmic end is important for proton translocation.     

Estradiol inhibits the effects of extracellular ATP in human sperm by a non genomic mechanism of action

Steroid hormones, beside their classical genomic mechanism of action, exert rapid, non genomic effects in different cell types. These effects are mediated by still poorly characterized plasma membrane receptors that appear to be distinct from the classic intracellular receptors. In the present study we evaluated the non genomic effects of estradiol (17βE₂) in human sperm and its effects on sperm stimulation by extracellular ATP, a potent activator of sperm acrosome reaction. In human sperm 17βE₂ induced a rapid increase of intracellular calcium (Ca²⁺) concentrations dependent on an influx of Ca²⁺ from the extracellular medium. The monitoring of the plasma membrane potential variations induced by 17βE₂ showed that this steroid induces a rapid plasma membrane hyperpolarization that was dependent on the presence of Ca²⁺ in the extracellular medium since it was absent in Ca²⁺ free-medium. When sperm were pre-incubated in the presence of the K⁺ channel inhibitor tetra-ethylammonium, the 17βE₂ induced plasma membrane hyperpolarization was blunted suggesting the involvement of K⁺ channels in the hyperpolarizing effects of 17βE₂. Extracellular ATP induced a rapid plasma membrane depolarization followed by acrosome reaction. Sperm pre-incubation with 17βE₂ inhibited the effects of extracellular ATP on sperm plasma membrane potential variations and acrosome reaction. The effects of 17βE₂ were specific since its inactive steroisomer 17αE₂ was inactive. Furthermore the effects of 17βE₂ were not inhibited by tamoxifen, an antagonist of the classic 17βE₂ intracellular receptor.     

The INA complex facilitates assembly of the peripheral stalk of the mitochondrial F1Fo‐ATP synthase

Mitochondrial F₁F_o‐ATP synthase generates the bulk of cellular ATP. This molecular machine assembles from nuclear‐ and mitochondria‐encoded subunits. Whereas chaperones for formation of the matrix‐exposed hexameric F₁‐ATPase core domain have been identified, insight into how the nuclear‐encoded F₁‐domain assembles with the membrane‐embedded F_o‐region is lacking. Here we identified the INA complex (INAC) in the inner membrane of mitochondria as an assembly factor involved in this process. Ina22 and Ina17 are INAC constituents that physically associate with the F₁‐module and peripheral stalk, but not with the assembled F₁F_o‐ATP synthase. Our analyses show that loss of Ina22 and Ina17 specifically impairs formation of the peripheral stalk that connects the catalytic F₁‐module to the membrane embedded F_o‐domain. We conclude that INAC represents a matrix‐exposed inner membrane protein complex that facilitates peripheral stalk assembly and thus promotes a key step in the biogenesis of mitochondrial F₁F_o‐ATP synthase.     

The effects of experimentally altered gill chloride cell surface area on acid-base regulation in rainbow trout during metabolic alkalosis

To evaluate the role of the gill chloride cells in regulating metabolic alkalosis in rainbow trout (Oncorhynchus mykiss), the surface area of branchial chloride cells was altered experimentally using combined cortisol/ovine growth hormone injections. Long-term (10-day) treatment of fish with cortisol/ovine growth hormone caused an increase in the two-dimensional chloride cell fractional surface area when compared to uninjected fish (from 8.4 to 29.7%). This was the combined result of an increase in the size of individual cells (from 34.6 to 59.2 m²) and increased numbers of cells (from 2368 to 5006 cells · mm^-2). Metabolic alkalosis was induced by intra-arterial infusion of 140 mmol · l^-1 NaHCO₃; control fish were infused with 140 mmol · l^-1 NaCl. Blood pH and plasma [HCO₃ ^-] increased in both the untreated and the cortisol/ovine growth hormone-treated fish. However, the increases in pH (from 8.05 to 8.53) and [HCO₃ ^-] (from 5.9 to 22.2 mmol · l^-1) in the untreated fish were significantly greater than in the cortisol/ovine growth hormone-treated fish (pH increased from 7.78 to 8.11; [HCO₃ ^-] increased from 5.5 to 13.9 mmol · l^-1). In all fish, NaHCO₃ infusion elicited an increase in the rate of branchial basic equivalent excretion (acidic equivalent uptake) which, in turn, was caused by decreases and increases in branchial Na⁺ uptake and Cl^- uptake, respectively. In the untreated fish, there was a pronounced increase (75%) in chloride cell surface area during NaHCO₃ infusion. The attenuation of the metabolic alkalosis during HCO₃ ^- infusion in the cortical/ovine growth hormone-treated fish was caused, at least in part, by an enhancement of branchial basic equivalent excretion. In these fish that already displayed a proliferation of chloride cells, there was no further increase in chloride cell surface area. The changes in Na⁺ influx and Cl^- influx were quantitatively similar during NaHCO₃ infusion in both groups. This suggests that the greater rate of base excretion in the cortisol/ovine growth hormone-treated fish was caused by a greater percentage of Cl^- uptake being coupled to HCO₃ ^- excretion and less to Cl^- excretion (Cl^- exchange diffusion).Abbreviations Amm total ammonia - bw body weight - CC chloride cell - CCFA chloride cell fractional area - cort/oGH cortisol/ovine growth hormone - dpm disintegrations per minute - J ^Amm net flux of total ammonia - J _in unidirectional influx - J _inCl^- chloride ion uptake - J _inNa⁺ sodium ion uptake - J _netH⁺ net acidic equivalent flux - J ^TA net flux of titrable alkalinity - MS 222 ethyl-m-aminobenzoate - oGH ovine growth hormone - PVC pavement cell - SEM scanning electron microscope - TA titrable alkalinity