Hydroxytryptamine transport by the bovine chromaffin-granule membrane.

5-Hydroxytryptamine is accumulated by resealed chromaffin-granule 'ghosts' if a pH gradient (acid inside) is imposed across their membranes by preincubating them at low pH. This uptake, like that driven by MgATP, is reserpine- and uncoupler-sensitive. This strongly suggests that catecholamines are taken up by intact granules in response to a pH gradient. In line with this, it is shown that 5-hydroxytryptamine decreases the pH gradient generated in the presence of MgATP, an effect that is inhibited by reserpine; nigericin, which discharges the pH gradient in the presence of K+, inhibits uptake. Permeant anions, however, also inhibit uptake. It is suggested that this may be because they permit equilibration of amine cations directly across the membrane, down concentration gradients.     

Passive ion permeability of the chromaffin-granule membrane.

'Ghosts' of bovine chromaffin granules, in which the complex mixture of proteins and solutes normally found in the granule matrix is replaced by buffered sucrose are osmotically sensitive. They shrink when the osmotic pressure of the suspension medium is increased, and swell if solute entry is facilitated by the addition of ionophores. Swelling in the presence of ionophores has been used to investigate the passive ion permeability of these membranes. They have a very low permeability to K+ ions (of the order of 10(-10) cm/s); their permeability to protons, Na+ and choline ions is too low to be detected by these methods. Their passive permeability to anions decreases in the order: CNS- greater than I- greater than CCl3CO2- greater than Br- greater than Cl- greater than SO4(2)- greater than CH3CO2-, HCO3-, F-, PO4(3)- the permeability to hiocyanate being of the order of 10(-7) cm/s. Coupled proton and anion entry is extremely slow, except for weak acids. Fluoride, unexpectedly, also appears to enter rapidly when proton/K+ exchange is facilitated by nigericin. In the presence of K+ salts, nigericin, like valinomycin, induces lysis of intact granules, an effect that is not dependent on the presence of a permeant anion, but is dependent on the pH gradient across the membrane.     

Role of anions in low pH-induced translocation of diphtheria toxin

Previous work has shown that when Vero cells with surface-bound diphtheria toxin are exposed to low pH, toxin entry across the plasma membrane is induced and that this entry involves two steps, insertion of the B-fragment of the toxin into the membrane and translocation of the enzymatically active A-fragment to the cytosol. Here we have studied the role of permeant anions in this process. It was found that when the B-fragment was inserted into the membrane, part of it, a 25-kDa polypeptide, was shielded from externally added Pronase. This insertion did not require permeant anions. The translocation of the A-fragment was monitored by measuring either its ability to inhibit protein synthesis in the cells or the appearance of radioactively labeled 21-kDa fragment after treatment of the cells with externally applied Pronase. The translocation of the A-fragment was dependent on the presence of permeant anions in the medium. However, when the cells were depleted of Cl- by incubation in Cl- free buffer at high pH, translocation of the A-fragment did not require permeant anions in the medium. The possibility that translocation of the A-fragment is inhibited by an outward directed chloride gradient rather than by the absence of chloride is discussed.     

Interactions between diphtheria toxin entry and anion transport in Vero cells. IV. Evidence that entry of diphtheria toxin is dependent on efficient anion transport

Entry of prebound diphtheria toxin at low pH occurred rapidly in the presence of isotonic NaCl, NaBr, NaSCN, NaI, and NaNO3, but not in the presence of Na2SO4, 2-(N-morpholino)ethanesulfonic acid neutralized with Tris, or in buffer osmotically balanced with mannitol. SCN- was the most efficient anion to facilitate entry. Uptake studies with radioactively labeled anions showed that SCN- was transported into cells 3 times faster than Cl-, while the entry of SO2-4 occurred much more slowly. The anion transport inhibitors 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and piretanide inhibited entry at low pH even in the presence of permeant anions. When cells with bound toxin were exposed to low pH in the absence of permeant anions, then briefly exposed to neutral pH and subsequently exposed to pH 4.5 in the presence of isotonic NaCl, toxin entry was induced. The data indicate that efficient anion transport at the time of exposure to low pH is required for entry of surface-bound diphtheria toxin into the cytosol. Since insertion of diphtheria toxin into the membrane occurs even in the absence of permeant anions, the results indicate that low pH is required not only for insertion of fragment B into the membrane, but also for the subsequent entry of fragment A into the cytosol.     

Activation of hepatocyte protein kinase C by redox-cycling quinones.

Chromaffin granules, the secretory vesicles of the adrenal medulla, have a Na+/H+ exchange activity in their membranes which brings their proton gradient into equilibrium with a Na+ gradient. This explains why Na+ is mildly inhibitory to amine transport (which is driven by the H+ gradient) The activity can be demonstrated by using accumulation of 22Na+ in response to a pH gradient that is either imposed by diluting membrane 'ghosts' into alkaline media, or generated by ATP hydrolysis. It can also be monitored indirectly by fluorescence measurements in which the pH inside 'ghost' is monitored by quenching of a fluorescent weak base. This method has been used to monitor Na+ entry into acid-loaded 'ghosts' of H+ entry into methylamine accumulation. The exchanger appears to be reversible and non-electrogenic, with a stoichiometry of 1:1. Using an indirect assay we measured an apparent Km for Na+ of 4.7 mM, and a Ki for amiloride, a competitive inhibitor, of 0.26 mM. Direct assays using 22Na+ suggested a higher Km. Ethylisopropylamiloride was not inhibitory.     

THE PENETRATION OF THE MEMBRANE OF BRAIN MITOCHONDRIA BY ANIONS

The permeability of the membrane of rat brain non-synaptosomal mitochondria, towards inorganic and substrate anions, was assessed by measuring the rate of swelling that occurred when mitochondria were suspended in an iso-osmotic solution of a permeant anion, in the presence of a permeant cation such as NH⁺₄ or K⁺ in the presence or absence of valinomycin. In NH⁺₄-phosphate swelling was higher than it was in KCI or K⁺-phosphate, which showed the prevalence of the mechanism of phosphate transport previously demonstrated in liver mitochondria. The entry of succinate and L-malate seemed to require the presence in the inner mitochondrial membrane of specific carriers. as previously postulated for liver mitochondria, but the rate of swelling of brain mitochondria was lower than that of liver organelles. In K⁺-succinate, in the presence of antimycin, added ATP induced swelling and this was attributable to the simultaneous permeation both of the anion and the cation. Fumarate did not penetrate into brain mitochondria. Practically no swelling was recorded in NH⁺₄ or K⁺-citrate, which indicated that this anion penetrated poorly into the isolated brain mitochondria even in the presence of malate. Swelling occurred in NH⁺₄-L-glutamate in the presence of rotenone, and the entry of this anion seemed to follow a gradient of concentration although the presence of a specific translocator in the inner mitochondrial membrane might be concerned. The entry of glutamate was independent of that of phosphate and N-ethylmaleimide appeared to be a specific inhibitor of this entry. Swelling in K⁺-L-glutamate, in the presence of rotenone, was enhanced by the addition of valinomycin or ATP but in the latter case when osmotic equilibrium was reached swelling was not reversed by oligomycin. In conclusion, the lesser extent of swelling of isolated brain mitochondria compared with liver mitochondria could be attributed to the heterogeneity of the populations of these organelles, each population possessing its own characteristics of membrane permeability. Observations of electron micrographs of brain mitochondria incubated in iso-osmotic substrate anions confirmed the heterogeneous rate of swelling of these particles.     

Adenosine triphosphate in the bovine chromaffin granule.

1. pH and potential gradients are generated across the membranes of chromaffin granule 'ghost' by incubating them with MgATP: the inside of the 'ghosts' is positive and acid with respect to the incubation medium. 2. The pH gradient is partially dissipated by inclusion of a substrate for the catecholamine pump, or a mitochondrial uncoupling agent, but is enhanced by reserpine. 3. An imposed pH gradient leads to amine uptake by the 'ghosts': a potential gradient leads to ATP uptake. Studies with inhibitors confirm that amine accumulation by chromaffin granules is dependent on the former, and that ATP uptake results from ATPase-induced potential difference generation. 4. ATP has two known roles in chromaffin granule structure: the first is as a substrate for a membrane-bound proton-translocating ATPase; the second is as a component of the intragranular catecholamine storage complex.     

Mechanism of respiration-driven proton translocation in the inner mitochondrial membrane. Kinetics of proton translocation and role of cations

The kinetics and mechanism of passive and active proton translocation in submitochondrial vesicles, obtained by sonication of beef heart mitochondria, have been studied.Analysis of the anaerobic release of the protons taken up by submitochondrial particles in the respiring steady state shows that proton diffusion consists of two parallel, apparent first-order processes: a fast reaction which, on the basis of its kinetic properties and response to cations and various effectors, is considered to consist of a proton/monovalent cation exchange; and a slow process which, on analogous grounds, is considered as a single electrogenic flux.The study of the various parameters of the respiration-linked active proton translocation and of the accompanying migration of permeant anions and K⁺ led to the following conclusions: (i) The oxidoreduction-linked proton translocation is electrogenic. (ii) Cation counterflow is not a necessary factor in the respiration-driven proton translocation. (iii) The membrane potential developed by active proton translocation exerts a coupling with respect to permeant cations and anions. (iv) The respiration-driven proton translocation is secondarily coupled, through the Δμ_H component of the electrochemical proton gradient and at the level of a proton-cation exchange system of the membrane, to the flow of K⁺ and Na⁺.     

Monensin-induced swelling of Golgi apparatus cisternae mediated by a proton gradient

Monensin, a monovalent ionophore, caused swelling of mature cisternae of plant Golgi apparatus. The appearance of swollen cisternae was time-dependent and linear over a period of 1 h with an estimated maximum rate of production of one swollen cisterna every 3 to 4 min. Implicit in these observations was a need for the uptake of osmotically active monovalent cations to have occurred accompanied by a concomitant efflux of H+ and the entry of water. Furthermore, to sustain the H+ efflux, a source of H+ influx also would be required. To test for the latter, cisternal swelling, as visualized by electron microscopy, was monitored by treatment of wild carrot cells in suspension culture with drugs and inhibitors known to interfere with proton gradients. Swelling was inhibited by the protonophore, FCCP, by the inhibitor of lysosomal acidification, quercetin, and by the lysosomotropic amines, chloroquine and ammonia. While antimycin A, an inhibitor of mitochondrial oxidative phosphorylation, was ineffective, cyanide dramatically decreased swelling. The numbers of swollen cisternae produced could be reduced by prolonged treatment with arsenate, such that an ATP requirement is indicated, at least, for cisternal formation. Swelling was promoted by citrate, representative of a permeant organic anion. Reductions in numbers of monensin-induced swollen cisternae in the presence of quercetin, vanadate, and chloroquine could be compensated for by the addition of citrate. We conclude that the monensin-induced swelling of Golgi apparatus cisternae may involve a mechanism generating a proton gradient at or near the mature Golgi apparatus face.     

Anion regulation of active transport of protons across the membrane of the synaptic vesicles of the brain

The dependence of active transport of H+ on the presence of anions in synaptic vesicle membranes from rat brain was studied. The H+ transport was measured by monitoring the acidification of the vesicles with a permeant weak base-acridine orange. The fluorescence changes in the latter were proportional to the magnitude of artificially imposed pH gradients (delta pH). The ATP-dependent generation of delta pH was completely dependent on the presence of a permeant anion, was maximal at 150 mM Cl- and was inhibited, when the medium osmolarity was further increased by sucrose or KCl. At 150 mM only Br-, similar to Cl-, behaved as permeant anions, whereas I- was effective only at low (5-20 mM) concentrations. The anions--SCN-, ClO4-, HSO3- and I-(10-20 mM) as well as 4-acetamido-4'-isothiocyanatostilbene-2.2'-disulfonate (K0.5 = 14 microM) blocked the ATP-dependent generation of delta pH observed in the presence of Cl-, while other anions tested (F-, phosphate, bicarbonate, some organic anions) were virtually without effect and did not support the H+ transport. The dependence of the rate and extent of H+ accumulation on Cl- concentration was sigmoidal with a Hill coefficient of 2.8 and a Km value of 85-90 mM. The effects of anions point to the presence in the membrane of synaptic vesicles of an anion (chloride) channel whose conductance can regulate the H+ transport by switching it from an electrogenic to an electroneutral (coupled entry of H+ and Cl-) mode of operation.     

Delta pH, H+ diffusion potentials, and Mg2+ ATPase in neurosecretory vesicles isolated from bovine neurohypophyses

The proton gradient (delta pH) and electrical potential (delta psi) across the neurosecretory vesicles were measured using the optical probes 9-aminoacridine and Oxanol VI, respectively. The addition of neurosecretory vesicles to 9-aminoacridine resulted in a rapid quenching of the dye fluorescence which was reversed when the delta pH was collapsed with ammonium chloride or K+ in the presence of nigericin. From fluorescence quenching data and the intravesicular volume, delta pH across the membrane was calculated. Mg2+ ATP caused a marked carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive change in the membrane potential measured using Oxanol VI (plus 100 mV inside positive), presumably due to H+ translocation across the neurosecretory vesicle membrane. Imposition of this membrane potential was responsible for the lysis of vesicles in the presence of permeant anions. The effectiveness of these anions to support lysis reflected the relative permeability of the anion which followed the order acetate greater than I- greater than Cl greater than F- greater than SO4- = isethionate = methyl sulfate. These data showed that the neurosecretory vesicles possess a membrane H+-translocating system and prompted the study of Mg2+-dependent ATPase activities in the vesicle fractions. In intact vesicles a Mg2+ ATPase appeared to be coupled to electrogenic proton translocation, since the enzyme activity was enhanced by uncoupling the electrical potential, using proton ionophores. Inhibition of this enzyme with dicyclohexylcarbodiimide also inhibited the carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive delta psi across the vesicle membrane caused by H+ translocation. A second Mg2+ ATPase was also found on the vesicle membranes which is sensitive to vanadate. Complete inhibition of this enzyme with vanadate had little effect on the proton ionophore-uncoupled ATPase activity or on the Mg2+ ATP-induced membrane potential change.     

Anion-cation interactions in the pore of neuronal background chloride channels

Background Cl channels in neurons and skeletal muscle are significantly permeable for alkali cations when tested with asymmetrical concentrations of the same salt. Both anion and cation permeation were proposed to require binding of an alkali cation with the pore (Franciolini, F., and W. Nonner. 1987. Journal of General Physiology. 90:453-478). We tested this hypothesis by bilaterally substituting large alkali cations for Na and found no significant changes of unitary conductance at 300 mM symmetrical concentrations. In addition, all organic cations examined were permeant in a salt gradient test (1,000 mM internal@300 mM external), including triethanolamine, benzyltrimethylamine, and bis-tris-propane (BTP, which is divalent at the tested pH of 6.2). Inward currents were detected following substitution of internal NaCl by the Na salts of the divalent anions of phosphoric, fumaric, and malic acid. Zero-current potentials in gradients of the Na and BTP salts of varied anions (propionate, F, Br, nitrate) that have different permeabilities under bi-ionic conditions, were approximately constant, as if the permeation of either cation were coupled to the permeation of the anion. These results rule out our earlier hypothesis of anion permeation dependent on a bound alkali cation, but they are consistent with the idea that the tested anions and cations form mixed complexes while traversing the Cl channel.     

ATP-dependent proton transport by isolated brain clathrin-coated vesicles. Role of clathrin and other determinants of acidification

We have systematically investigated certain characteristics of the ATP-dependent proton transport mechanism of bovine brain clathrin-coated vesicles. H+ transport specific activity was shown by column chromatograpy to co-purify with coated vesicles, however, the clathrin coat is not required for vesicle acidification as H+ transport was not altered by prior removal of the clathrin coat. Acidification of the vesicle interior, measured by fluorescence quenching of acridine orange, displayed considerable anion selectively (Cl- greater than Br- much greater than NO3- much greater than gluconate, SO2-(4), HPO2-(4), mannitol; Km for Cl- congruent to 15 mM), but was relatively insensitive to cation replacement as long as Cl- was present. Acidification was unaffected by ouabain or vanadate but was inhibited by N-ethylmaleimide (IC50 less than 10 microM), dicyclohexylcarbodiimide (DCCD) (IC50 congruent to 10 microM), chlorpromazine (IC50 congruent to 15 microM), and oligomycin (IC50 congruent to 3 microM). In contrast to N-ethylmaleimide, chlorpromazine rapidly dissipated preformed pH gradients. Valinomycin stimulated H+ transport in the presence of potassium salts (gluconate much greater than NO3- greater than Cl-), and the membrane-potential-sensitive dye Oxonol V demonstrated an ATP-dependent interior-positive vesicle membrane potential which was greater in the absence of permeant anions (mannitol greater than potassium gluconate greater than KCl) and was abolished by N-ethylmaleimide, protonophores or detergent. Total vesicle-associated ouabain-insensitive ATPase activity was inhibited 64% by 1 mM N-ethylmaleimide, and correlated poorly with H+ transport, however N-ethylmaleimide-sensitive ATPase activity correlated well with proton transport (r = 0.95) in the presence of various Cl- salts and KNO3. Finally, vesicles prepared from bovine brain synaptic membranes exhibited H+ transport activity similar to that of the coated vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of anion-cation selectivity of amphotericin B channels

Summary Zero current potential and conductance of ionic channels formed by polyene antibiotic amphotericin B in a lipid bilayer were studied in various electrolyte solutions. Nonpermeant magnesium and sulphate ions were used to independently vary the concentration of monovalent anions and cations as well as to maintain the high ionic strength of the two solutions separated by the membrane. Under certain conditions the channels select very strongly for anions over cations. They are permeable to small inorganic anions. However, in the absence of these anions the channels are practically impermeable to any cation. In the presence of a permeant anion the contribution of monovalent cations to channel conductance grows with an increase in the anion concentration. The ratio of cation-to-anion permeability coefficients is independent of the membrane potential and cation concentration, but it does depend linearly on the sum of concentrations of a permeant anion in the two solutions. These results are accounted for on the assumption that a cation can enter only an anion-occupied channel to form an ionic pair at the center of the channel. The cation is also assumed to slip past the anion and then to leave the channel for the opposite solution. This model with only few parameters can quantitatively describe the concentration dependences of conductance and zero current potential under various conditions.     

On the inhibition of the mitochondrial inner membrane anion uniporter by cationic amphiphiles and other drugs

Depleting the mitochondrial matrix of divalent cations with the ionophore A23187 activates a pH-sensitive, anion uniport pathway which can transport many anions normally regarded as impermeant (Beavis, A. D., and Garlid, K. D. (1987) J. Biol. Chem. 262, 15085-15093). Addition of valinomycin to respiring mitochondria can also induce the uptake of a wide variety of anions; however, the mechanism of anion transport during this "respiration-induced" swelling is less certain. In this paper, I demonstrate that both of these processes are inhibited by a variety of cationic amphiphiles including propranolol, quinine, amiodarone, imipramine and amitriptyline, and the benzodiazepine R05-4864. Although the IC50 values for the two processes are not equal, the ratio of IC50 values for the two processes appears to be the same for all drugs. Measurements of net transmembrane proton fluxes that occur during the assays reveal that respiration-induced swelling is associated with extensive proton ejection, the peak of which coincides with the maximum rate of anion transport. Moreover, from measurements of matrix buffering power, it is estimated that the matrix pH is 3 units more alkaline during respiration-induced swelling than during A23187-induced swelling. It is also shown that the IC50 for A23187-induced transport is pH-dependent in a manner consistent with modulation of drug binding by protonation of two sites. These findings allow the difference in IC50 values for the two types of assay to be explained by the pH dependence of the binding constant for the drug. Furthermore, the pH gradient generated during respiration-induced swelling is so large that the electrical component of the proton-motive force will be negligible. Thus, despite the fact that the mitochondria are "energized," rapid electrophoretic anion influx is possible. These data provide evidence that the transport of anions in these two types of assay occurs via the same pathway.     

Evidence for a Cl-Stimulated MgATPase Proton Pump in Oat Root Membranes

The possibility that plant membrane-bound MgATPases may act as electrogenic proton pumps has been investigated. Using an oat (Avena sativa L. cv. Victory) root membrane preparation which is partially enriched in tightly sealed vesicles, we have shown that MgATP stimulates the uptake of the membrane-permeable anion [(14)C]SCN(-) by the vesicles; this indicates that an electrical potential (interior positive) is generated across the membrane. Both Cl(-) ions and the proton ionophore trifluoromethoxy(carbonyl-cyanide)phenylhydrazone inhibit the MgATP-driven [(14)C]SCN(-) uptake, presumably by collapsing the MgATP-generated membrane potential. The uptake of the pH gradient probe [(14)C]imidazole into the vesicles is also greatly stimulated by MgATP, indicating the presence of a transmembrane proton gradient (interior acid). MgATP-driven [(14)C]imidazole uptake is temperature sensitive, Cl(-)-stimulated, substrate specific for MgATP, sensitive to the MgATPase inhibitors vanadate and N,N'-dicyclohexylcarbodiimide, and completely eliminated by trifluoromethoxy(carboxyl-cyanide)phenylhydrazone. The mitochondrial ATPase inhibitor oligomycin has little effect on the MgATPase activity and on the MgATP-dependent [(14)C]SCN(-) and [(14)C]imidazole uptake. These data indicate that a class of oat root membrane-bound MgATPases, stimulated primarily by Cl ions, is capable of using the free energy of ATP-hydrolysis to generate an apparent electrochemical proton gradient in vitro.     

Energetics of the Na+-dependent transport of D-glucose in renal brush border membrane vesicles.

The energetics of the Na+-dependent transport of D-glucose into osmotically active membrane vesicles, derived from the brush borders of the rabbit renal proximal tubule, was studied by determining how alterations in the electrochemical potential of the membrane induced by anions, ionophores, and a proton conductor affect the uptake of the sugar. The imposition of a large NaCl gradient (medium is greater than vesicle) resulted in the transient uptake of D-glucose into brush border membranes against its concentration gradient. In the presence of Na+ salts of isethionate or sulfate, both relatively impermeable anions, there was no accumulation of D-glucose above the equilibrium value. With Na+ salts of two highly permeable lipophilic anions, NO3- and SCN-, the transient overshoot was enhanced relative to that with Cl-. With Na+ salts whose mode of membrane translocation is electroneutral, i.e. acetate, bicarbonate, and phosphate, no overshoot was found. These findings suggest that only anions which penetrate the brush border membrane and generate an electrochemical potential, negative on the inside, permit the uphill Na+-dependent transport of D-glucose.     

Anion competition for a volume-regulated current.

We have examined whether the anionic amino acids, glutamate and aspartate, permeate through the same volume-regulated conductance permeant to Cl- ions. Cell swelling was initiated in response to establishing a whole-cell configuration in the presence of a hyposmotic gradient. Volume-regulated anion currents carried by Cl-, glutamate, or aspartate developed with similar time courses and showed similar voltage-dependent inactivation. Permeability ratios (Paa/PCl) calculated from measured reversal potentials were dependent on the mole fraction ratio (MFR) of the permeant anions ([aa]/([aa] + [Cl-])). MFR was varied from 0.00 to 0.97. As the fraction of amino acid increased, Paa/PCl decreased. Current amplitude was similarly dependent on MFR. These results show that the permeation of anionic amino acids and that of Cl- ions are not independent of each other, indicating that the ion channel underlying the volume-regulated conductance can be occupied by more than one ion at a time. Application of Eyring rate theory indicated that the major barrier to Cl- ion permeation is at the intracellular side of the membrane, and that the major barrier to amino acid permeation is at the extracellular side of the membrane. The interactions between these permeant ions may have a physiological modulatory role in volume regulation through a volume-regulated anion conductance.     

Postillumination adenosine triphosphate synthesis in Rhodospirillum rubrum chromatophores. II. Stimulation by a K+ diffusion potential.

Addition of valinomycin, nonactin, or monactin plus KCl in the dark to preilluminated chromatophores induced the synthesis of a large amount of ATP. This stimulation of postillumination ATP synthesis by a dark-imposed K+ diffusion potential was different from the stimulation caused by addition of permeant anions or cations in the light, since it increases when the pH of the light stage decreased from 8.0 to 6.0. It was thus most pronounced when the chromatophores were preloaded with protons but the light-induced proton concentration gradient (deltapH) was low. Imposition of a Kplus diffusion potential resulted however in stimulation of ATP synthesis even when the light-induced deltapH was already above the threshold value required to initiate postillumination ATP synthesis. This situation was realized when valinomycin plus KCl were added in the dark to chromatophores preilluminated above pH 6.7 with thiocyanate as the permeant anion, and the amount of ATP formed was the sum of the yields obtained with each of these affectors by itself. On the other hand addition of thiocyanate together with valinomycin plus KCl in the dark led to inhibition of ATP synthesis. In this case the permeant anion could not affect the light-induced deltapH but it did eliminate the diffusion potential by decreasing the difference between the permeabilities of Kplus and the anion present in the reaction mixture.     

Molecular mechanism of uncoupling in brown adipose tissue mitochondria. The non-identity of proton and chloride conducting pathways

Specific permeability properties of the inner membrane of brown adipose tissue mitochondria were analysed with the aid of simultaneous pH measurements outside mitochondria and of mitochondria swelling. It was shown that valinomycin-induced potassium diffusion potential drives a parallel passive uptake of chlorides and extrusion of proton. Electrogenic H+ -extrusion was independent on anion transport, no competition was found between the two processes and the former process exerted a lower sensitivity to the inhibitory effect of GDP. The existence of two distinct, independent pathways for translocation of protons and halide anions across the membrane is suggested.