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1.
Summary Bilayer membranes were formed from decane, cholesterol, and three lipids isolated fromStaphylococcus aureus: positively charged lysyl phosphatidylglycerol (LysPG), negatively charged phosphatidylglycerol (PG), and neutral diglucosyldiglyceride (DiGluDiGly). The uncouplers of oxidative phosphorylation, 2,4-dinitrophenol (DNP) and 3-t-butyl,5-chloro,2-chloro,4-nitrosalicylanilide (S 13), increased the electrical conductance of all three differently charged bilayers. S 13 was found to be the most effective reagent of the known uncouplers in increasing conductance of the bilayers. The conductance induced by uncouplers was investigated as a function of pH and uncoupler concentration. The pH of maximum conductance for each uncoupling agent was dependent on both the uncoupler and the lipid; it was lower for each uncoupler in LysPG and higher in PG compared to DiGluDiGly bilayers. At a pH below the optimum for LysPG, the conductance of the positively charged membrane was 500 times and of the neutral one 10 times higher than that of the negatively charged bilayer at equal uncoupler concentration and pH. Above the pH optimum for DiGluDiGly, the conductance was approximately equal for the positive and neutral membranes, but was lower in PG bilayers. Conductance depended linearly on uncoupler concentration. The bilayer conductance induced by S 13 was entirely due to increased proton permeability in all three lipids. The findings are consistent with the role of uncouplers as carriers for protons across the hydrocarbon interior of lipid membranes. The differences in conductance of differently charged lipid bilayers at equal uncoupler concentration, as well as the change of pH optimum of conductance with lipid charge, can be explained in terms of an electrostatic energy contribution of the fixed lipid charges to the distribution of the uncoupler anion between the aqueous and the membrane phases. 相似文献
2.
John Gutknecht 《Journal of bioenergetics and biomembranes》1987,19(5):427-442
The proton/hydroxide (H+/OH–) permeability of phospholipid bilayer membranes at neutral pH is at least five orders of magnitude higher than the alkali or halide ion permeability, but the mechanism(s) of H+/OH– transport are unknown. This review describes the characteristics of H+/OH– permeability and conductance through several types of planar phospholipid bilayer membranes. At pH7, the H+/OH– conductances (G
H/OH) range from 2–6 nS cm–2, corresponding to net H+/OH– permeabilities of (0.4–1.7)×10–5 cm sec–1. Inhibitors ofG
H/OH include serum albumin, phloretin, glycerol, and low pH. Enhancers ofG
H/OH include chlorodecane, fatty acids, gramicidin, and voltages >80 mV. Water permeability andG
H/OH are not correlated. The characteristics ofG
H/OH in fatty acid (weak acid) containing membranes are qualitatively similar to the controls in at least eight different respects. The characteristics ofG
H/OH in gramicidin (water wire) containing membranes are qualitatively different from the controls in at least four different respects. Thus, the simplest explanation for the data is thatG
H/OH in unmodified bilayers is due primarily to weakly acidic contaminants which act as proton carriers at physiological pH. However, at low pH or in the presence of inhibitors, a residualG
H/OH remains which may be due to water wires, hydrated defects, or other mechanisms. 相似文献
3.
Jaromír Plášek David Babuka Milan Hoefer 《Journal of bioenergetics and biomembranes》2017,49(5):391-397
According to the common view, weak acid uncouplers increase proton conductance of biological (and phospholipid bilayer) membranes, thus effecting H+ fluxes driven by their electrochemical gradients. Under certain conditions, however, uncouplers can induce unexpected effects opposite to the dissipation of H+ gradients. Results are presented here demonstrating CCCP-induced proton influx into Saccharomyces cerevisiae cytosol driven by the electrochemical potentials of CCCP and its CCCP? anions, independent of electrochemical H+-gradient. Another view of week acid uncouplers’ action is proposed that is logically consistent with these observations. 相似文献
4.
Summary We report here a kinetic study of the mechanism by which the weak acid TTFB (4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole) transports protons across phospholipid bilayer membranes. A previous kinetic study of the homologous dichloro compound, DTFB, revealed that the rate limiting step for proton translocation was the back diffusion of the neutral, HA, form of the weak acid; we conclude here that this is also the rate limiting step for proton translocation with TTFB. At high concentrations of either DTFB or TTFB the charged permeant species is an HA
2
–
complex. The kinetic analysis and independent measurements reveal that the permeability of the membrane to HA and adsorption coefficients of A– and HA are an order of magnitude higher for TTFB than for DTFB. When either DTFB or TTFB was present in a solution where the pH was less than the pK of the weak acid, an unusual relaxation in the current was noted on application of a voltage step. The amplitude of the relaxation decreased as the voltage was increased. This relaxation is possibly due to a reorientation of the benzimidazole molecules at the membrane-solution interface. We also report experiments performed with DTFB on mitochondria. It was possible to reconcile these results with the bilayer data and, therefore, with the chemiosmotic hypothesis by postulating that the dielectric constant of the mitochondrial membrane is greater than that of a bilayer formed with decane as a solvent. To demonstrate the effect of dielectric constant on permeability, we replaced decane by 1-chlorodecane. This increased the capacitance of the artificial bilayer by a factor of two and the permeability of the bilayer to the A– form of DTFB by two orders of magnitude. 相似文献
5.
Summary We have examined the effects of two weak acid uncouplers of oxidative phosphorylation, 2,4-dinitrophenol and 5,6-dichloro-2-trifluoromethyl-benzimidazole, on the electrical properties of phospholipid bilayer membranes. All the effects they produce are consistent with the charged permeant species being a HA
2
–
complex formed between the neutral acid HA and its anion A– and the current in the aqueous phases being carried by buffered hydrogen ions. When both uncouplers are present simultaneously, all the evidence we have obtained is consistent with the charged permeant species being a HAB– complex formed between the neutral acid HA of one uncoupler and the anion B– of the other. It was necessary, however, to take into account interfacial processes and the unstirred layers adjacent to the membrane, the adsorption of anions to the bilayer and the buffer level in the aqueous phases to explain the results in terms of this model. The degree to which these factors will complicate a comparison of results obtained on artificial systems and mitochondria is also discussed. 相似文献
6.
Butacaine and certain other local anesthetics markedly stimulate the rate, extent, and efficiency of respiration-dependent contraction of heart mitochondria in nitrate salts at alkaline pH. The local anesthetics also induce respiratory control associated with contraction (i.e., the elevated rate of respiration during contraction declines to a State 4-like controlled rate when contraction is complete) so that the reaction at alkaline pH closely resembles the rapid and highly efficient process seen at neutral pH. Respiration-dependent contraction appears to be an osmotic response to cation extrusion on an endogenous cation/H+ exchanger (G. P. Brierley, M. Jurkowitz, E. Chavez, and D. W. Jung, 1977, J. Biol. Chem.252, 7932–7939). At alkaline pH, net ion extrusion is slow and inefficient due to the elevated permeability of the membrane to monovalent cations through a putative uniport pathway. Butacaine and other local anesthetics seem to decrease influx-efflux cycling of cations at alkaline pH by restricting cation influx through this uniport. Passive swelling at pH 8.3 in nitrate salts indicates that the uniport reaction is sensitive to Ca2+ and has a cation-selectivity of Na+ > K+ > Li+. Butacaine does not inhibit passive swelling under these conditions but produces effects identical to those of classical uncouplers and consistent with increased H+ conductance and accelerated influx of cations by cation/H+ exchange in nonrespiring mitochondria. However, since contraction in respiring mitochondria is inhibited by uncouplers but stimulated by butacaine, it is apparent that butacaine is not an effective proton conductor in energized mitochondria. 相似文献
7.
Roland Benz Angela Schmid Melitta Dihanich 《Journal of bioenergetics and biomembranes》1989,21(4):439-450
Reconstitution experiments were performed on lipid bilayer membranes in the presence of purified mitochondrial porin from yeast and of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant. The addition of the porin resulted in a strong increase of the membrane conductance, which was caused by the formation of ion-permeable channels in the membranes. Yeast porin has a single-channel conductance of 4.2 nS in 1 M KCl. In the open state it behaves as a general diffusion pore with an effective diameter of 1.7 nm and possesses properties similar to other mitochondrial porins. Surprisingly, the membrane conductance also increased in the presence of detergent extracts of the mitochondrial outer membrane of the mutant. Single-channel recordings of lipid bilayer membranes in the presence of small concentration of the mutant membranes suggested that this membrane also contained a pore. The reconstituted pores had a single-channel conductance of 2.0 nS in 1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. This means that the pores present in the mitochondrial outer membranes of the yeast mutant have a much smaller effective diameter than normal mitochondrial porins. Zero-current membrane potential measurements suggested that the second mitochondrial porin is slightly cation-selective, while yeast porin is slightly anion-selective in the open state but highly cation-selective in the closed state. The possible role of these pores in the metabolism of mitochondria is discussed. 相似文献
8.
Gaston Ducet 《Planta》1979,147(2):122-126
Pulsed acid base titrations, according to the procedure of Mitchell and Moyle, have been carried out on potato mitochondria in the presence and absence of Bovine Serum Albumine (BSA). The rate of the pH decay is slower when BSA is present. The buffering capacities of the outer and inner phases, the t1/2 of the pH decay after an acid pulse and the proton conductance of the inner membrane have been measured. The results show that plant mitochondria are relatively impermeable to H+ and OH–, but leakier than animal mitochondria. This may be related to the lower respiratory control ratios generally found with plant mitochondria.Abbreviations EGTA
ethylene glycol bis (aminoethyl ether) NN tetraacetic acid
- MERCAP
sodium mercaptobenzothiazole
- TRIS
tris (hydroxymethyl) aminomethane
- MOPS
morpholinopropane sulfonic acid
- BSA
bovine serum albumine
- RC
respiratory control ratio 相似文献
9.
Anton Misak Marian Grman Lubica Malekova Marta Novotova Jana Markova Olga Krizanova Karol Ondrias Zuzana Tomaskova 《European biophysics journal : EBJ》2013,42(9):709-720
Physiological and pathological functions of mitochondria are highly dependent on the properties and regulation of mitochondrial ion channels. There is still no clear understanding of the molecular identity, regulation, and properties of anion mitochondrial channels. The inner membrane anion channel (IMAC) was assumed to be equivalent to mitochondrial centum picosiemens (mCS). However, the different properties of IMAC and mCS channels challenges this opinion. In our study, we characterized the single-channel anion selectivity and pH regulation of chloride channels from purified cardiac mitochondria. We observed that channel conductance decreased in the order: Cl? > Br? > I? > chlorate ≈ formate > acetate, and that gluconate did not permeate under control conditions. The selectivity sequence was Br? ≥ chlorate ≥ I? ≥ Cl? ≥ formate ≈ acetate. Measurement of the concentration dependence of chloride conductance revealed altered channel gating kinetics, which was demonstrated by prolonged mean open time value with increasing chloride concentration. The observed mitochondrial chloride channels were in many respects similar to those of mCS, but not those of IMAC. Surprisingly, we observed that acidic pH increased channel conductance and that an increase of pH from 7.4 to 8.5 reduced it. The gluconate current appeared and gradually increased when pH decreased from pH 7.0 to 5.6. Our results indicate that pH regulates the channel pore diameter in such a way that dilation increases with more acidic pH. We assume this newly observed pH-dependent anion channel property may be involved in pH regulation of anion distribution in different mitochondrial compartments. 相似文献
10.
Summary
42K+ tracer flux and steady-state conductance measurements were carried out with bilayer lipid membranes containing grisorixin, a carboxylic polyether antibiotic. When the membranes are placed between two bulk aqueous solutions of identical composition, the exchange or self-diffusion transmembrane flux of potassium is measured by a method which allows the characterization of the bilayer K+ permeability at the equilibrium state. The K+ self-diffusion flux increases with the pH in the range pH 6 to pH 9 and reaches a constant value for values above 9. This can be directly related to the increase of the surface concentration of the 11 complex formed by K+ and the deprotonated polyether at both bilayer membrane interfaces. The transport model initially proposed by Pressman and coworkers (Proc. Natl. Acad. Sci. USA
58:1949–1956, 1967) is again taken into consideration in the quantitative analysis of the flux data. The transmembrane transport of K+ results from the translocation of its neutral complex with grisorixin and the association-dissociation of the antibiotic with either potassium or conditions by a translocation process of the acidic grisorixin. Using the data of some previous studies for mixed ionophorelipid monolayers at the air/water interface and the present results for the self-diffusion flux measurements, it was possible to propose an evaluation of the more important parameters characterizing the transport; namely, the total surface concentration of grisorixin, the interfacial pK and the translocation rate constant of its potassium neutral complex. The method proposed could be extended easily to other carboxylic polyethers, which would lead to an interesting comparison of their ionophoric properties using model membrane systems. 相似文献
11.
Karl E. O. Åkerman 《Journal of bioenergetics and biomembranes》1981,13(3-4):133-139
Respiration-driven Mg2+ efflux from rat heart mitochondria has been studied in different conditions. Almost total release of Mg2+ from the mitochondria occurs upon addition of a proton/bivalent cation exchanger, A23187. The content of Mg2+ remaining in mitochondria after A23187 treatment is the same if part of the mitochondrial Mg2+ has already been extruded through the energy-linked mechanism. Some inhibition of Mg2+ efflux is observed in the presence of high concentrations of La3+ (100 µM). A proton/monovalent cation exchanger, nigericin, completely prevents Mg2+ efflux, whereas a cation conductor, valinomycin, considerably stimulates it. The results indicate that the main part of mitochondrial Mg2+ is present in a membrane-bounded compartment, probably in the matrix space. The driving force of the Mg2+ efflux appears to be the proton gradient (pH) created by mitochondrial respiration. 相似文献
12.
13.
Summary Southern Corn Leaf Blight is caused by a toxin produced by a virulent form ofHelminthosporium maydis (Race T). The toxin has been shown to uncouple oxidative phosphorylation and dissipate Ca2+ gradients in mitochondria isolated from susceptible, but not resistant, corn. The possibility that the toxin acted by increasing the permeability of membranes to ions was tested using a planar bilayer membrane system. Addition of the toxin to the bilayer system, under voltage-clamp conditions, resulted in stepwise increases in current across the phospholipid bilayer, a response characteristic for channel formers. Single-channel conductance in 1m KCl is 27 pS which corresponds to 1.7×107 ions sec–1 channel–1 at 100 mV applied potential. The toxin channels are: (i) fairly uniform in conductance, (ii) ideally selective for K+ over Cl–, and (iii) most conductive to H+. The channel showed the following selectivity for alkali metal cations: Rb+>K+>Cs+>Na+>Li+ (169731) based on the most frequently observed conductance in 1m chloride salts. The toxin showed no voltage dependence over the range of –100 to +100 mV. Channel formation was also a property of a synthetic analog (Cmpd IV) of the toxin. The ability of the native toxin to form channels may be a mode of toxin action on mitochondrial membranes from susceptible corn. 相似文献
14.
Kathleen W. Kinnally Yuri N. Antonenko Dmitry B. Zorov 《Journal of bioenergetics and biomembranes》1992,24(1):99-110
Three classes of inner mitochondrial membrane (IMM) channel activities have been defined by direct measurement of conductance levels in membranes with patch clamp techniques in 150 mM K Cl. The 107 pS activity is slightly anion selective and voltage dependent (open with matrix positive potentials). Multiple conductance channel (MCC) activity includes several levels from about 40 to over 1000 pS and can be activated by voltage or Ca2+. MCC may be responsible for the Ca2+-induced permeability transition observed with mitochondrial suspensions. A low conductance channel (LCC) is activated by alkaline pH and inhibited by Mg2+. LCC has a unit conductance of about 15 pS and may correspond to the inner membrane anion channel, IMAC, which was proposed from results obtained from suspension studies. All of the IMM channels defined thus far appear to be highly regulated and have a low open probability under physiological conditions. A summary of what is known about IMM channel regulation and pharmacology is presented and possible physiological roles of these channels are discussed. 相似文献
15.
M. Tieffenberg Tosteson 《The Journal of membrane biology》1978,38(4):291-309
Summary Bilayer membranes formed from lipids dissolved in decane were exposed to glycophorin, a sialoglycoprotein which had been extracted from human red cell membranes. The interaction with the bilayer produced an increase in the steady state electrical conductance of the membrane proportional to the amount added. Fluctuations in membrane current when the electrical potential difference was constant were observed concommitantly with this increase in membrane conductance. The minimum size of the fluctuations corresponds to a conductance of 10–10 mho. The increase in conductance as well as the current fluctuations persisted after extensive washout of the chamber containing the protein (cisside). Subsequent addition of lectins (wheat germ agglutinin and phytohemoagglutinin) to the cis-side produced rupture of the membranes, whilst these hemoagglutinins added to the trans-side failed to produce an effect. Measurements of changes in surface potential using K+ nonactin as a probe indicated that glycophorin induces a negative surface charge. At high protein concentrations, the magnitude of the induced surface potential became independent of glycophorin concentration. The maximum number of charges introduced onto the membrane under these conditions was 1.4×105/m2. Cis (but not trans)-side addition of neuraminidase abolished these charges, indicating that they can be ascribed to the sialic acid residues that the protein bears. These results suggest that glycophorin incorporates into bilayer membranes with its N-terminal end (where the sialic acid and carbohydrates are located) facing the cis-side. Spectrin reversibly lowered the glycophorin-induced membrane conductance when added to the trans-side. Cis-side additions failed to produce an effect. Trypsin present on the trans-side irreversibly lowered the membrane conductance. These results indicate that parts of the glycophorin molecule, probably the C-terminal end, are accessible to reagents in the solution bathing the trans-side of the membrane. Thus glycophorin spans the planar bilayer in much the same way as it spans the red cell membrane. 相似文献
16.
A. G. Shugaev I. M. Andreev E. I. Vyskrebentseva 《Russian Journal of Plant Physiology》2005,52(2):182-188
Mitochondria isolated from sugar beet (Beta vulgaris L.) taproot were shown to swell spontaneously after the transfer from a sucrose-containing isolation medium to isoosmotic potassium chloride solutions. The kinetics of this process was strongly retarded after the replacement of potassium with sodium in the incubation medium and was substantially stimulated by the electron-transport chain activity and valinomycin. At neutral pH of the incubation medium, the rate of K+-dependent swelling of mitochondria decreased by 30–50% after adding 1 mM ATP but was insensitive to other nucleotides (GTP, UTP, and CTP). In the medium acidified to pH 6.0, the addition of ATP caused shrinkage of mitochondria that had been swollen in the KCl medium. In the absence of this nucleotide, the kinetics of K+-dependent swelling of mitochondria was considerably decelerated upon the acidification of the incubation medium. The effects of ATP were independent of the presence or absence of oligomycin and atractyloside. However, the ATP-dependent shrinkage of mitochondria was inhibited in the presence of quinine, and this agent also inhibited K+-dependent swelling of organelles in potassium acetate solutions. The presence of K+ ions in the incubation medium caused a rapid dissipation of the mitochondrial membrane potential () that was generated during succinate oxidation. The addition of ATP to the reaction medium resulted in the oligomycin-insensitive restoration of . The results are regarded as evidence that the membrane of taproot mitochondria is endowed with functionally active ATP-sensitive K+ uniporter. This system is likely to represent a K+ channel that catalyzes the electrogenic transfer of potassium ions to the mitochondrial matrix. It is supposed that the membrane of taproot mitochondria also contains a quinine-sensitive K+/H+ antiporter that catalyzes the efflux of potassium from the matrix or, on the contrary, the accumulation of K+ in the presence of potassium acetate.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 209–215.Original Russian Text Copyright © 2005 by Shugaev, Andreev, Vyskrebentseva.This revised version was published online in April 2005 with a corrected cover date. 相似文献
17.
《Comparative biochemistry and physiology. B, Comparative biochemistry》1993,104(2):423-426
- 1.1. It is shown that Ca2+-dependent activation of respiration of liver mitochondria from hibernating ground squirrels is accompanied by mitochondrial swelling.
- 2.2. The swelling of mitochondria from hibernating ground squirrels, as well as the activation of mitochondrial respiration, is precluded by cyclosporin A, p-bromphenacylbromide and oligomycin. Carboxyatractiloside, on the contrary, under these conditions favors the swelling and the acceleration of respiration.
- 3.3. It was concluded that Ca2+-dependent activation of hibernating ground squirrel liver mitochondrial respiration resulted from the appearance of a non-specific permeability pathway and from swelling of mitochondria.
18.
We identified three electrophoretic pathways by spectrophotometrically following the swelling of isolated yeast mitochondria:
Similarities and differences between these three electrophoretic pathways and the electrophoretic systems described in mammalian and plant mitochondria are discussed. 相似文献
| --An anion uniport whose activity could only be detected after depletion of divalent cations from the matrix by treatment with 1,10-phenanthroline. This uniport was inhibited by Mg2+ and dicyclohexylcarbodiimide. |
| --A K+(Na+) uniport which was detected only when mitochondria were suspended at low pH and low temperature. This uniport was sensitive to ruthenium red and oleic acid. |
| --A K+ selective uniport which was activated by alkaline pH and ATP depletion. This pathway was sensitive to glibenclamide and to various amphiphilic cations. |
19.
Summary A simple carrier model describes adequately the transport of protons across lipid bilayer membranes by the weak acid S-13. We determined the adsorption coefficients of the anionic, A–, and neutral, HA, forms of the weak acid and the rate constants for the movement of A– and HA across the membrane by equilibrium dialysis, electrophoretic mobility, membrane potential, membrane conductance, and spectrophotometric measurements. These measurements agree with the results of voltage clamp and charge pulse kinetic experiments. We considered three mechanisms by which protons can cross the membranesolution interface. An anion adsorbed to the interface can be protonated by (i) a H+ ion in the aqueous phase (protolysis), (ii) a buffer molecule in the aqueous phase or (iii) water molecules (hydrolysis). We demonstrated that the first reaction cannot provide the required flux of protons: the rate at which H+ must combine with the adsorbed anions is greater than the rate at which diffusion-limited reactions occur in the bulk aqueous phase. We also ruled out the possibility that the buffer is the main source of protons: the rate at which buffers must combine with the adsorbed anions is greater than the diffusion-limited rate when we reduced the concentration of polyanionic buffer adjacent to the membrane-solution interface by using membranes with a negative surface charge. A simple analysis demonstrates that a hydrolysis reaction can account for the kinetic data. Experiments at acid pH demonstrate that the transfer of H+ from the membrane to the aqueous phase is limited by the rate at which OH combines with adsorbed HA and that the diffusion coefficient of OH– in the water adjacent to the bilayer has a value characteristic of bulk water. Our experimental results demonstrate that protons are capable of moving rapidly across the membrane-solution interface, which argues against some mechanisms of local chemiosmosis. 相似文献
20.
1. The predictions of a general kinetic model for the chemiosmotic uptake of auxin and other weak acids are compared with experimental results for the auxin indoleacetic acid. The proposed mechanism involves diffusional flux of undissociated acid, a saturable, voltage-sensitive flux of anion (A-), and a carrier-mediated symport of H+ and A-, all operating in parallel. During much of uptake, the electrochemical gradients are such that the net symport and the net anion flux are in opposition: the symport contributes more to influx; the anion path, to efflux. The voltage-sensitive flux of A- therefore constitutes a leak. 2. The presence of a symport, whose carrier can distribute across the membrane in response to the internal and external concentrations of auxin, can speed the rate of uptake, but does not by itself alter the accumulation of auxin at equilibrium. 3. The accumulation ratio at equilibrium is less at low concentrations of auxin than at higher concentrations, indicating the presence of a saturable anion path. The concentration dependence of the transition depends on several factors, and is not a reliable indicator of the A--carrier binding constant. 4. Observed uptake near neutral pH appears larger than is consistent with a voltage-sensitive anion flux being the only carrier-mediated path across the membrane. This observation provides indirect evidence for the presence of an auxin-proton symport in addition to a saturable A- carrier. 5. The change in kinetics of uptake of [3H]indole-3-acetic acid (IAA), observed as the total concentration of IAA is raised from 0.1 to 100 M, is consistent with either (i) a symport that saturates at low concentrations, or (ii) activation of an A- efflux by intermediate concentrations of auxin. 6. The data on the concentration dependence of uptake of auxin are not consistent with a multi-proton symport.Abbreviations A-
auxin anion
- HA
weak acid, particularly IAA
- HXA
carrier in electroneutral complex with a proton and the auxin anion
- H2XA
carrier in electroneutral complex with two protons and the auxin anion
- IAA
indole-3-acetic acid
- X
auxin carrier
- XA
carrier-auxin anion complex 相似文献