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PubMed1980

Measurements of membrane potentials in Escherichia coli K-12 inner membrane vesicles with the safranine method.

The use of safranine, a positively-charged dye, as a probe for the determination of membrane potentials in Escherichia coli vesicles has been studied. 1. Shifts in the spectrum of safranine were observed during induction of potassium ion diffusion potentials with valinomycin or during oxidation of formate by vesicles prepared from cells of E. coli K-12 or ML 308-225 subjected to anaerobic growth with nitrate. The extent of the valinomycin-dependent spectral change correlated linearly with the magnitude of the K+ equilibrium potential, as calculated from the Nernst equation, from 50 to 160 mV (interior negative). The formate-induced changes could also be calibrated by increasing the concentration of potassium in the presence of valinomycin, after the formation of formate-dependent responses. In this case, results identical to those obtained with the first method were obtained. 2. O2 or nitrate-dependent oxidation of formate resulted in a membrane potential of the order of 170 mV. The oxidation of ascorbate-reduced N-methylphenazonium methosulphate resulted in a potential of similar magnitude, but anaerobically with nitrate only a small but definite potential was formed. 3. The water-soluble quinones, duroquinone and menadione, could produce membrane potentials when used in their oxidized or reduced forms in the presence of formate or nitrate (or oxygen). 2-Hydroxy-1,4-naphthoquinone was not only ineffective but was found to be inhibitory. 4. N,N'-dicyclohexylcarbodiimide at suitable concentrations increased the rate of formation and the extent of membrane potentials induced by respiration or by artificial means.

Biological Transport↗
PubMed1982

Ion pathways in renal brush border membranes.

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na+/H+ or Li+/H+ exchange was demonstrated by diluting Na2SO4 or Li2SO4 loaded vesicles into Na+-or Li+-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K+ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na+ conductance smaller than electroneutral Na+/H+ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na+ conductance greater than the H+ conductance was suggested. K+ gradients also induced changes of pH, at about 10% of the Na+ or Li+ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K+ was a low affinity substrate for the Na+/H+ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K+ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles has a low K+ conductance. Inward Cl- gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl- conductance greater than the H+ conductance and a Cl-/OH- exchange were present. The rate of Na+/H+ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent Km for Na+ or Li+ of about 10 mM and an EA of 14.3 kcal per mol. A 61-fold Na2SO4 gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower delta pH even in the presence of gramicidin showing that the H+ and Cl- pathways could alter the effect of the Na+/H+ exchange.

Acridine Orange↗
PubMed1992

Trans-potassium effects on the chloride/proton symporter activity of guinea-pig ileal brush-border membrane vesicles.

To investigate the inhibitory effect of trans potassium on the Cl-/H+ symporter activity of brush-border membrane vesicles from guinea pig ileum, we measured both 36Cl uptake and, by the pyranine fluorescence method, proton fluxes, in the presence of appropriate H+ and K+ gradients. In the absence of valinomycin, a time-dependent inhibitory effect of chloride uptake by trans K+ was demonstrated. This inhibition was independent of the presence or absence of any K+ gradient. Electrical effects cannot be involved to explain these inhibitions because the intrinsic permeability of these vesicles to Cl- and K+ is negligibly small. Rather, our results show that, in the absence of valinomycin, the inhibitory effect of intravesicular K+ involves an acceleration of the rate of dissipation of the proton gradient through an electroneutral exchange of trans K+ for cis H+, catalyzed by the K+/H+ antiporter also present in these membranes. Valinomycin can further accelerate the rate of pH gradient dissipation by facilitating an electrically-coupled exchange between K+ and H+. To evaluate the apparent rate of pH-dissipating, downhill proton influx, we measured chloride uptake by vesicles preincubated in the presence of alkaline-inside pH gradients (pHout/pHin = 5.0/7.5), charged or not with K+. In the absence of intravesicular K+, proton influx exhibited monoexponential kinetics with a time constant k = 11 s-1. Presence of 100 mM K+ within the vesicles significantly increased the rate of pH gradient dissipation which, furthermore, became bi-exponential and revealed the appearance of an additional, faster proton influx component with k = 71 s-1. This new component we interpret as representing the sum of the electroneutral and the electrically-coupled exchange of trans K+ for cis H+, mentioned above. Finally, by using the pH-sensitive fluorophore, pyranine, we demonstrate that, independent of the absence or presence of a pH gradient, either vesicle acidification or alkalinisation can be generated by adding, respectively, Cl- or K+ to the extravesicular medium. Such results confirm the independent existence of both Cl-/H+ symporter and K+/H+ antiporter activities in our vesicle preparations, the relative activity of the former being larger under the conditions of the present experiments. The possible interplay of these two proton-transfer mechanisms in the regulation of the intracellular pH is discussed.

Animals↗
PubMed1992

Black lipid membranes of tetraether lipids from Thermoplasma acidophilum.

Black lipid membranes were formed of tetraether lipids from Thermoplasma acidophilum and compared to the bilayer forming lipids diphytanoylphosphatidylcholine and diphythanylglucosylglycerol. Bilayer-forming lipids varied in thickness of black lipid membranes due to the organic solvent used. Measurements of the specific membrane capacitance (Cm = 0.744 microF/cm2) showed that the membrane-spanning tetraether lipids from Thermoplasma acidophilum form a monolayer of a constant thickness of 2.5-3.0 nm no matter from which solvent. This finding corresponds to the results of Gliozzi et al. for the lipids of another archaebacterium, Sulfolobus solfataricus. Black lipid membranes were formed at room temperature with a torus from bilayer-forming lipids, however, the torus could also be formed by the tetraether-lipid itself at room temperature and at defined concentration. In these stable black lipid membranes, conductance was measured in the presence of valinomycin, nonactin, and gramicidin. At 10(-7) M concentration, valinomycin mediated higher conductance in membranes from tetraether lipids (200-1200 microS/cm2) than from bilayer-forming lipids (125-480 microS/cm2). Nonactin, at 10(-6) M concentration, mediated a 6-fold higher conductance in a tetraether lipid membrane than in a bilayer, whereas conductance, in the presence of 5 x 10(-11) M gramicidin could reach higher values in bilayers than in tetraether lipid monolayers of comparable thickness. Monensin did not increase the conductance of black lipid membranes from tetraether lipids under all conditions applied in our experiments. Poly(L-lysine) destroyed black lipid membranes. Lipopolysaccharides from Thermoplasma acidophilum were not able to form stable black lipid membranes by themselves. The lipopolysaccharide complexes from Thermoplasma acidophilum and from Escherichia coli decreased the valinomycin-mediated conductance of monolayer and bilayer membranes. This influence was stronger than that of the polysaccharide dextran.

Electric Conductivity↗
PubMed1990

GABA agonist induced changes in ultrastructure and GABA receptor expression in cerebellar granule cells is linked to hyperpolarization of the neurons.

GABA has been shown to exert a neurotrophic like activity by enhancing the morphological and functional maturation of neurons. Mechanisms involved in this effect of GABA are largely unknown but since GABA has been shown to mediate a hyperpolarizing action on neurons it can be assumed that this action might be important. In order to investigate this possibility, the ability to mimic the trophic actions of GABA of different agents known to influence the membrane potential or the GABA gated chloride channels was studied. Hence, GABA receptor expression as well as the ultrastructure of cerebellar granule cells were monitored after exposure of the cells in culture to either bromide, valinomycin or picrotoxin. It was found that cells which at early developmental stages (4 days in culture) were exposed to bromide or valinomycin expressed low affinity GABA receptors similar to cells treated with the GABA agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol(THIP). This was in contrast to untreated cultures or cultures treated with both THIP and picrotoxin which expressed only high affinity GABA receptors and no low affinity receptors. In cultures at more mature stages (14 days in culture) bromide treatment did not lead to formation of low affinity GABA receptors. Studies of the ultrastructure of the cells (4-day-old cultures) showed that exposure to bromide or valinomycin mimicked the ability of THIP to enhance the cytoplasmic density of rough endoplasmic reticulum, Golgi apparatus, vesicles and coated vesicles. Again, in 14-day-old cultures treatment with bromide had no effect on the ultrastructure.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

Effect of phloretin on ionophore mediated electroneutral transmembrane translocations of H(+), K(+) and Na(+) in phospholipid vesicles.

Rates of M(+)/H(+) exchange (M(+)=K(+), Na(+)) across phospholipid membranes by ionophore mediated electroneutral translocations and transports through channels could either increase or decrease or change negligibly on adding the polar molecule phloretin to the membrane. The changes depend on pH, the concentration and choice of M(+) and choice of ionophore/channel. Such diverse behaviours have been inferred from studies on the decay of the pH difference across soybean phospholipid vesicular membrane (=Delta pH). The transporters used in this study are (a) the exchange ionophores: nigericin, monensin; (b) combinations of alkali metal ion carriers, valinomycin or nonactin with weak acids carbonyl cyanide m-chlorophenylhydrazone or 2,4-dinitrophenol and (c) channels formed by gramicidin A. All the diverse results can be rationally explained if we take note of the following. (i) The rate limiting steps are associated with the transmembrane translocations involving the rate limiting species identified in the literature. (ii) Phloretin in the membrane decreases the apparent M(+) dissociation constant, K(M), of the M(+) bound ionophores/channels which has the effect of increasing the concentration of these species. (iii) The concentrations of H(+) bound ionophores/channels decrease on adding phloretin. (iv) Phloretin inhibits ternary complex formation (involving valinomycin or nonactin, M(+) and an anion) by forming 1:2 complexes with valinomycin-M(+) or nonactin-M(+). (v) On adding 6-ketocholestanol to the membrane (instead of phloretin) K(M) increases. The decreases/increases in K(M) mentioned above are consistent with the consequences of a hypothesis in which phloretin decreases and 6-ketocholestanol increases the positive internal membrane dipole potential.

Hydrogen-Ion Concentration↗
PubMed1998

Effects of protein kinase and phosphatase inhibitors on slow shortening of guinea pig cochlear outer hair cells.

The intracellular mechanisms of slow shortening in isolated guinea pig cochlear outer hair cells were investigated using inhibitors and/or an activator of protein kinases and protein phosphatases. The slow shortening was induced by tetanic electrical field stimulation, and changes in the cell length, volume and intracellular Cl- concentration were microscopically monitored using a chloride-sensitive fluorescent dye. The slow shortening was inhibited by a calmodulin inhibitor, W-7, and a calcium calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN-62. The inhibition by W-7 or KN-62, was abolished by the supplemented conductance of K+ with valinomycin. Among the protein phosphatase inhibitors tested, a type 1 and 2A protein phosphatase inhibitor, calyculin A, inhibited the slow shortening. The inhibition by calyculin A was abolished by the increased Cl- permeability, but neither by the increased K+ conductance with valinomycin nor by the increased Ca2+ conductance with A23187. A protein serine/threonine phosphatase activator, N-acetylsphingosine, inhibited the shortening, which was abolished by either valinomycin or a type 2A protein phosphatase inhibitor, okadaic acid, but not by calyculin A. These findings suggest the following signaling mechanisms in the slow shortening of outer hair cells; the K+ channel opening is facilitated through protein phosphorylation by CaMKII and suppressed via okadaic acid-sensitive dephosphorylation, and the Cl- channel opening depends on calyculin A-sensitive protein phosphatase activity.

Animals↗
PubMed2000

The symbiotically essential cbb(3)-type oxidase of Bradyrhizobium japonicum is a proton pump.

Purified cbb(3)-type oxidase of Bradyrhizobium japonicum was reconstituted into phospholipid vesicles. Tight vesicles were obtained as shown by the disturbance of deltapH with CCCP and the membrane potential with valinomycin, which led to a six-fold increase in cytochrome c oxidase activity. The vesicles were thus suitable for proton translocation experiments. In the presence of valinomycin, a pulse with reduced cytochrome c caused an acidification with a subsequent alkalinization, whereas the same pulse caused only an alkalinization in the presence of valinomycin plus CCCP. We conclude that the cbb(3)-type oxidase of B. japonicum is a proton pump.

Bacterial Proteins↗
PubMed2001

Modulation of photosystem II chlorophyll fluorescence by electrogenic events generated by photosystem I.

In an attempt to uncover electric field interactions between PS I and PS II during their functioning, fluorescence induction curves were measured on hydroxylamine-treated thylakoids of Chenopodium album under conditions ensuring low and high levels of photogenerated membrane potentials. In parallel experiments with Peperomia metallica chloroplasts, the photocurrents were measured with patch-clamp electrodes and served as indicator of electrogenic activity of thylakoid membranes in continuous light. Inhibition of linear electron flow at PS II donor side by hydroxylamine (0.1 mM) eliminated a slow rise of chlorophyll fluorescence to a peak level and suppressed photoelectrogenesis. Activation of PS I-dependent electron transport using cofactors of either cyclic (phenazine methosulfate) or noncyclic electron transport (reduced TMPD or DCPIP in combination with methyl viologen) restored photoelectrogenesis in hydroxylamine-treated chloroplasts and led to reappearance of slow components in the fluorescence induction curve. Exposure of thylakoids to valinomycin reduced the peak fluorescence in the presence of KCl but not in the absence of KCl. Combined application of valinomycin and nigericin in the presence of KCl exerted stronger suppression of fluorescence than valinomycin alone but was ineffective in the absence of KCl. In samples treated with hydroxylamine and PS I cofactors (DCPIP/ascorbate and methyl viologen), preillumination with a single-turnover flash or a multiturnover pulse shifted the induction curves of both membrane potential and chlorophyll fluorescence to shorter times, which confirms the supposed influence of PS I-generated electrical field on PS II fluorescence. A model is presented that describes modulating effect of the membrane potential on chlorophyll fluorescence and roughly simulates the fluorescence induction curves measured at low and high membrane potentials.

Chenopodium album↗
PubMed1981

Photosensitized electron transport across lipid vesicle walls: Enhancement of quantum yield by ionophores and transmembrane potentials.

The photosensitized reduction of heptylviologen in the bulk aqueous phase of phosphatidylcholine vesicles containing EDTA inside and a membrane-bound tris(2,2'-bipyridine)ruthenium(2+) derivative is enhanced by a factor of 6.5 by the addition of valinomycin in the presence of K(+). A 3-fold stimulation by gramicidin and carbonyl cyanide m-chlorophenylhydrazone is observed. The results suggest that, under these conditions, the rate of photoinduced electron transfer across vesicle walls in the absence of ion carriers is limited by cotransport of cations. The rate of electron transfer across vesicle walls could be influenced further by generating transmembrane potentials with K(+) gradients in the presence of valinomycin. When vesicles are made with transmembrane potentials, interior more negative, the quantum yield of heptylviologen reduction is doubled, and, conversely, when vesicles are made with transmembrane potentials, interior more positive, the quantum yield is decreased and approaches the value found in the absence of valinomycin.

Journal Article↗
PubMed2003

Ion fluxes in giant excised cardiac membrane patches detected and quantified with ion-selective microelectrodes.

We have used ion-selective electrodes (ISEs) to quantify ion fluxes across giant membrane patches by measuring and simulating ion gradients on both membrane sides. Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE. For detection on the extracellular (pipette) side, ISEs are fabricated from flexible quartz capillary tubing (tip diameters, 2-3 microns), and an ISE is positioned carefully within the patch pipette with the tip at a controlled distance from the mouth of the patch pipette. Transport activity is then manipulated by solution changes on the cytoplasmic side. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. The sensitivity and utility of the methods are demonstrated with cardiac membrane patches by measuring (a) potassium fluxes via ion channels, valinomycin, and Na/K pumps; (b) calcium fluxes mediated by Na/Ca exchangers; (c) sodium fluxes mediated by gramicidin and Na/K pumps; and (d) proton fluxes mediated by an unknown electrogenic mechanism. The potassium flux-to-current ratio for the Na/K pump is approximately twice that determined for potassium channels and valinomycin, as expected for a 3Na/2K pump stoichiometery (i.e., 2K/charge moved). For valinomycin-mediated potassium currents and gramicidin-mediated sodium currents, the ion fluxes calculated from diffusion models are typically 10-15% smaller than expected from the membrane currents. As presently implemented, the ISE methods allow reliable detection of calcium and proton fluxes equivalent to monovalent cation currents <1 pA in magnitude, and they allow detection of sodium and potassium fluxes equivalent to <5 pA currents. The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion-coupled transporters in giant patches.

Cell Culture Techniques↗
PubMed1988

Oxaloacetate and malate transport by plant mitochondria.

The permeability of mitochondria from pea (Pisum sativum L. var Kleine Rheinländerin) leaves, etiolated pea shoots, and potato (Solanum tuberosum) tuber for malate, oxaloacetate, and other dicarboxylates was investigated by measurement of mitochondrial swelling in isoosmolar solutions of the above mentioned metabolites. For the sake of comparison, parallel experiments were also performed with rat liver mitochondria. Unlike the mammalian mitochondria, the plant mitochondria showed only little swelling in ammonium malate plus phosphate media but a dramatic increase of swelling on the addition of valinomycin. Similar results were obtained with oxaloacetate, maleate, fumarate, succinate, and malonate. n-Butylmalonate and phenylsuccinate, impermeant inhibitors of malate transport in mammalian mitochondria, had no marked inhibitory effect on valinomycin-dependent malate and oxaloacetate uptake of the plant mitochondria. The swelling of plant mitochondria in malate plus valinomycin was strongly inhibited by oxaloacetate, at a concentration ratio of oxaloacetate/malate of 10(-3). From these findings it is concluded: (a) In a malate-oxaloacetate shuttle transferring redox equivalents from the mitochondrial matrix to the cytosol, malate and oxaloacetate are each transported by electrogenic uniport, probably linked to each other for the sake of charge compensation. (b) The transport of malate between the mitochondrial matrix and the cytosol is controlled by the oxaloacetate level in such a way that a redox gradient can be maintained between the NADH/NAD systems in the matrix and the cytosol. (c) The malate-oxaloacetate shuttle functions mainly in the export of malate from the mitochondria, whereas the import of malate as a respiratory substrate may proceed by the classical malate-phosphate antiport.

Journal Article↗
PubMed1992

Proton Transport Activity of the Purified Vacuolar H-ATPase from Oats : Direct Stimulation by Cl.

To determine whether the detergent-solubilized and purified vacuolar H(+)-ATPase from plants was active in H(+) transport, we reconstituted the purified vacuolar ATPase from oat roots (Avena sativa var Lang). Triton-solubilized ATPase activity was purified by gel filtration and ion exchange chromatography. Incorporation of the vacuolar ATPase into liposomes formed from Escherichia coli phospholipids was accomplished by removing Triton X-100 with SM-2 Bio-beads. ATP hydrolysis activity of the reconstituted ATPase was stimulated twofold by gramicidin, suggesting that the enzyme was incorporated into sealed proteoliposomes. Acidification of K(+)-loaded proteoliposomes, monitored by the quenching of acridine orange fluorescence, was stimulated by valinomycin. Because the presence of K(+) and valinomycin dissipates a transmembrane electrical potential, the results indicate that ATP-dependent H(+) pumping was electrogenic. Both H(+) pumping and ATP hydrolysis activity of reconstituted preparations were completely inhibited by <50 nanomolar bafilomycin A(1), a specific vacuolar type ATPase inhibitor. The reconstituted H(+) pump was also inhibited by N,N'-dicyclohexylcarbodiimide or NO(3) (-) but not by azide or vanadate. Chloride stimulated both ATP hydrolysis by the purified ATPase and H(+) pumping by the reconstituted ATPase in the presence of K(+) and valinomycin. Hence, our results support the idea that the vacuolar H(+)-pumping ATPase from oat, unlike some animal vacuolar ATPases, could be regulated directly by cytoplasmic Cl(-) concentration. The purified and reconstituted H(+)-ATPase was composed of 10 polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. These results demonstrate conclusively that the purified vacuolar ATPase is a functional electrogenic H(+) pump and that a set of 10 polypeptides is sufficient for coupled ATP hydrolysis and H(+) translocation.

Journal Article↗
PubMed1997

Properties of chloride-conductive pathways in rat kidney cortical and outer-medulla brush-border membranes--inhibition by anti-(cystic fibrosis transmembrane regulator) mAbs.

The activity of the Cl(-)-conductive pathways, their regulation by protein kinase A (PKA) and their relationship to the cystic fibrosis transmembrane regulator (CFTR) protein were assessed in rat kidney cortical brush-border-membrane vesicles (cBBMV) and outer medullary vesicles (OMV) by measuring the rate of valinomycin-induced microsomal swelling by light scattering in the presence of an inward Cl- gradient. Valinomycin increased the rate of swelling of cBBMV and OMV, which is consistent with the presence of a Cl(-)-conductive pathway. PKA further increased these rates. This effect was blocked by the inhibitor of protein kinase A, suggesting that phosphorylation by PKA activates these pathways. Four anion-transport inhibitors were tested ¿N-phenylanthranilic acid (PhNHPhCOOH), 5-nitro-2-(3-phenylpropylamino)benzoic acid [N(PhPrNH2)BzOH], glybenclamide and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid¿. Ph2COOH and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid inhibited the basal Cl(-)-conductive pathways, while PKA-treated microsomes were sensitive also to N(PhPrNH2)BzOH and glybenclamide, suggesting that additional Cl- pathways were activated by phosphorylation. The pharmacological properties of these pathways were similar to those of the CFTR Cl- channel. Two anti-CFTR mAbs inhibited PKA-activated valinomycin-induced swelling in cBBMV and OMV, while immunoblot analysis of the corresponding proteins with the same antibodies indicated the presence of a 170-kDa protein. The results thus indicate the presence of a PKA-activated Cl(-)-conductive pathway in cBBMV and OMV, and suggest that CFTR protein is involved in PKA-activated Cl- fluxes in these vesicles.

Animals↗
PubMed1981

Effect of metabolic inhibitors on entry of exogenous deoxyribonucleic acid into Ca2+-treated Escherichia coli cells.

The effect of various metabolic inhibitors (carbonylcyanid-m-chlorophenylhydrazone, nigericin, valinomycin, dicyclocarbodiimide, arsenate, NaF, etc.) and lipid-soluble synthetic ions (tetraphenylphosphonium bromide and tetraphenylboron sodium) on deoxyribonucleic acid (DNA) entry during transformation of Ca2+-treated Escherichia coli cells with plasmid DNA and on cell viability was investigated. In contrast to intact cells, Ca2+-treated E. coli cells were permeable to nigericin, valinomycin, and the other drugs tested. The inhibitors differentially affected [14C]proline active transport, and whereas some drugs inhibited transformation, the effects did not correlate with the effects on transport. The most potent inhibitors of transformation were nigericin, dicyclocarbodiimide, and tetraphenylboron sodium. Carbonylcyanid-m-chlorophenylhydrazone, tetraphenylphosphonium bromide, and valinomycin were relatively inactive. Tetraphenylboron sodium- and nigericin-treated cells bound were plasmid [14C]DNA in the deoxyribonuclease-resistant form than the control and other sample cells. Nevertheless, te penetration of exogenous plasmid DNA into the cell was greatly reduced, at least in case of nigericin. Unlike the other drugs, nigericin and dicyclocarbodiimide drastically affected the cell viability, the former within very short times of interaction. It is concluded that proton motive force does not play any significant role in DNA entry into Ca2+-treated E. coli cells. The results also suggest that adenosine 5'-triphosphate is not required for DNA entry either. The inhibitory effect of certain drugs is discussed in terms of structural perturbations induced by the drugs in cell envelope membranes.

Adenosine Triphosphate↗
PubMed1984

Stimulation of oxyntic cell triggers K+ and Cl- conductances in apical H+-K+-ATPase membrane.

Vesicles isolated from the apical membrane of stimulated oxyntic cells [stimulation-associated (SA) vesicles] are highly permeable to KCl. The KCl flux is coupled to an electroneutral ATP-driven H+-K+ exchange (the H+-K+-ATPase) to produce net intravesicular HCl accumulation. In the past, we observed that rates of KCl transport were not accelerated by valinomycin and that dissipation of preformed H+ gradients in the presence of a protonophore (carbonyl cyanide, m-chlorophenylhydrazone, 10 microM) required the simultaneous presence of valinomycin. Consequently the fast KCl transport was attributed to an electroneutral cotransport system. Now we have been able to elicit fast H+ gradient dissipation in the absence of valinomycin by using the protonophore tetrachlorosalicylanilide. Experiments carried out in the absence of Cl- demonstrated the existence of a specific high-conductance pathway for K+. Experiments in K+-free medium demonstrated the existence of a high Cl- conductance. Parallel experiments in the equivalent H+-K+-ATPase-rich vesicles from nonsecreting oxyntic cells showed very little K+ and Cl- conductivity, suggesting that the appearance of large ionic conductance in the membrane is associated with the stimulation of the cell.

Adenosine Triphosphatases↗
PubMed1991

Passive chloride permeability charge coupled to H(+)-ATPase of avian osteoclast ruffled membrane.

We prepared proton-transporting membrane vesicles from the avian osteoclast's ruffled membrane, a specialized region of the cell surface that acidifies the bone resorption space. We demonstrated a unique conductive Cl- permeability that is charge coupled to the vesicle H(+)-ATPase and is required for acidification. Ion replacement indicated an anion selectivity of Br- approximately Cl- greater than SO4(2-) greater than NO3- approximately SCN- in supporting acidification. The anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (10 microM) was a competitive inhibitor of acidification and raised the Michaelis constant for ATP of the proton pump approximately 11-fold in 120 mM KCl. Inhibition was reversed by valinomycin, which provides an alternate path for charge neutralization. The Cl- dependence of acidification was nonlinear and yielded a Hill coefficient of 3-4, showing that it is distinct from a linear Cl- dependence reported for acidification of renal cortical endosomes. The K+ ionophore valinomycin augmented H+ transport in K2SO4, and not in KCl. Dependence of Cl- transport on membrane potential was confirmed by direct measurement of 36Cl- transport. We uncoupled charge transport from proton transport with a large excess of ammonia, which had no effect on 36Cl- accumulation in vesicles, and by measuring 36Cl- accumulation in response to a membrane diffusion potential, produced with a [K+] gradient and valinomycin in the absence of ATP. These experiments demonstrate that the electrogenic proton pump of the osteoclast ruffled membrane is charge coupled to a passive Cl- permeability in the same membrane.

Acridine Orange↗
PubMed1994

Na(+)-dependent and Na(+)-independent systems of choline transport by plasma membrane vesicles of A549 cell line.

Membrane vesicles of A549 lung cells accumulate choline by two pathways: the Na(+)-independent uphill uptake of choline [Michaelis-Menten constant (Km) approximately 44 microM; steady-state gradient approximately 45 at 5 microM external choline] is dependent on a transmembrane H+ gradient, is relatively insensitive to hemicholinium-3, is amiloride sensitive, and is abolished by valinomycin plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The Na(+)-dependent active choline uptake (Km approximately 4 microM, inhibitor constant for hemicholinium-3 approximately 0.1 microM), is specific for Na+, is amiloride and FCCP sensitive, and is electrogenic: the overshoot using K(+)-loaded vesicles and NaCl gradient was increased by valinomycin. The time of the overshoot peak, T was approximately 90 s in a NaSCN medium (or in presence of other lipid-soluble anions), a value close to that for alpha-aminoisobutyrate as substrate (T = approximately 1.5 min). T was lengthened in NaCl medium to approximately 10 min, and the overshoot was abolished by impermeant anions. External Cl- is not required for the choline uptake: valinomycin produced an overshoot in the presence of only impermeant anions, with T approximately 90 s. Most of the above properties are shared by the high-affinity Na(+)-dependent choline transport in synaptosomes. The characteristics of the Na(+)-dependent choline uptake by membrane vesicles of A549 cells are consistent with an electrogenic choline(+)-Na+ cotransport, with the rate-limiting anion (e.g., Cl-) influx balancing the positive charges transferred into the vesicles. The data are also consistent with an involvement of an amiloride-sensitive choline+/H+ antiport (or choline(+)-OH- symport) in the low- and high-affinity choline uptake pathways.

Anions↗