The Effect of Valinomycin on Potassium and Sodium Permeability of HK and LK Sheep Red Cells

Tosteson, D. C.; Cook, Philip M.; Andreoli, Thomas E.; Tieffenberg, M.

doi:10.1085/jgp.50.11.2513

Cited by 109 publications

(37 citation statements)

References 15 publications

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“…Both TNC and valinomycin produced slight reductions of questionable significance in particle density on the B-fracture surfaces. Valinomycin at 10-6M increases K + permeability of sheep red cell membranes by more than two orders of magnitude (Tosteson et al, 1967), while 10-2M TNC reduces C1-permeability by about four orders of Table 2. Closed circles: ouabainsensitive K-influx.…”

Section: Discussionmentioning

confidence: 93%

Cation transport and membrane morphology

Kirk

Tosteson

1973

J. Membrain Biol.

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Summary. Freeze-fracture electron-microscopy has been used to study membrane ultrastructure in (1) red cells from five species of mammals which have naturally occurring differences in cation transport and (2) red cells which have been treated with various drugs known to affect transport. A reproducible method for estimation of the intramembrane particle density is described. Considerable differences in the densities of intramembrane particles on the A-fracture faces were noted in five species of red cells studied. Such differences were not noted on the B-fracture faces. The differences correlated with species differences in active potassium transport and membrane phospholipid composition. Ouabain and trinitrocresolate-treated red cell membranes were found to have small but reproducible reductions in intra-membrane particle densities on the B-fracture faces, but such differences were not seen in valinomycin and amphotericin B-treated red cells. It was found that dimethylsulfoxide (DMSO) and glycerol drastically reduced intra-membrane particle densities. However, over the range of glycerol and DMSO concentrations in which membrane morphology was altered, no effects on either passive or active potassium transport were observed. It appears that the particles which are altered by DMSO and glycerol are not involved in cation transport. This paper reports experiments designed to explore the relation between ion transport and membrane ultrastructure as revealed by freeze-fracture electron-microscopy. We have directed particular attention to the intra~ membrane particles revealed by this method (Branton, 1966). We have used three approaches to this problem. First, we have examined the ultrastructure of the membranes of red cells from two genotypes of sheep and four other species of mammals known to have different red cell cation transport systems

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Section: Discussionmentioning

confidence: 93%

Cation transport and membrane morphology

Kirk

Tosteson

1973

J. Membrain Biol.

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“…To address the role of acidification and Δψ in yeast vacuole fusion, we used a pair of well-studied ionophores, valinomycin and nigericin, in combination with bafilomycin. Valinomycin is a neutral K + -specific ionophore, which transports K + across the membrane down the concentration gradient (35,36), and nigericin is an electroneutral K + /H + antiporter (37). In the presence of V-ATPase activity, valinomycin and nigericin together equilibrate H + and K + ions to collapse both the electrochemical and pH gradients of the vacuole (Fig.…”

Section: Significancementioning

confidence: 99%

Vibrio effector protein VopQ inhibits fusion of V-ATPase–containing membranes

Sreelatha

Bennett²,

Carpinone³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Vesicle fusion governs many important biological processes, and imbalances in the regulation of membrane fusion can lead to a variety of diseases such as diabetes and neurological disorders. Here we show that the Vibrio parahaemolyticus effector protein VopQ is a potent inhibitor of membrane fusion based on an in vitro yeast vacuole fusion model. Previously, we demonstrated that VopQ binds to the V o domain of the conserved V-type H + -ATPase (V-ATPase) found on acidic compartments such as the yeast vacuole. VopQ forms a nonspecific, voltage-gated membrane channel of 18 Å resulting in neutralization of these compartments. We now present data showing that VopQ inhibits yeast vacuole fusion. Furthermore, we identified a unique mutation in VopQ that delineates its two functions, deacidification and inhibition of membrane fusion. The use of VopQ as a membrane fusion inhibitor in this manner now provides convincing evidence that vacuole fusion occurs independently of luminal acidification in vitro.V esicle fusion governs many important physiological processes including neurotransmitter release and exocytosis. As such, many studies have focused on understanding this process and the proteins involved in fusion using various models such as yeast vacuoles and Drosophila synaptic vesicles (1, 2). Yeast vacuoles are an established and elegant model to study eukaryotic membrane fusion because of the ease of their isolation and the conserved nature of the fusion machinery required for their homotypic fusion (3). Although the core SNARE and Rab GTPase fusion machinery alone can drive the physiologically relevant fusion of liposomes in vitro (2), genetic and biochemical experiments have identified a number of additional regulators of vacuole fusion, including the membrane sector of the highly conserved V-type H + -ATPase (V-ATPase) (4, 5). The eukaryotic V-ATPase is the main electrogenic proton pump involved in the acidification of many intracellular organelles such as endosomes, lysosomes, and the yeast vacuole (6). The V-ATPase consists of two conserved, multisubunit domains: the cytoplasmic V 1 domain and the membrane bound V o domain. The V 1 domain hydrolyzes ATP, providing the energy for proton translocation through the membrane-bound V o proteolipid proton channel, thus acidifying the lumen of the vesicle. The loss of V-ATPase subunits is lethal in higher eukaryotes, highlighting the importance of this vital protein complex for normal eukaryotic physiology. However, yeast that lack subunits of the V-ATPase exhibit conditional lethality that is rescued by growth on acidic media, thus providing a unique and powerful system for the study of V-ATPase functions in vivo. In addition to its acidification function, the V-ATPase has been implicated in a broad range of biological processes, including the proper trafficking of secreted and endocytosed cargos (7), viral fusion (8), exocytosis (1, 9, 10), and the SNARE-dependent membrane fusion of yeast vacuoles (4,5,11,12). Even though the role of V-ATPase in fusion has been demons...

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“…The valinomycin could slowly dissolve into the aqueous phase and, from there, into the erythrocyte lipids. The potassium outflux would then be increased markedly as has been shown by Tosteson, Cook, Andreoli and Tieffenberg [24]. To verify that this effect is either too small or too slow to affect the measurements significantly, an experiment was performed where 0.5 ml of the valinomycin solution was added directly to the cell suspension.…”

Section: Electrodesmentioning

confidence: 88%

A study of passive potassium efflux from human red blood cells using ion-specific electrodes

Morel

1973

J. Membrain Biol.

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Summary. Using ion-specific electrodes, the potassium leakage induced by ouabain in human erythrocytes can be measured continuously and precisely near physiological conditions. Upon small additions of isotonic sucrose solution to a suspension of red cells in physiological saline the passive potassium efflux increases proportionally to the chloride ratio. The same result is obtained upon addition of hypertonic sucrose solution, suggesting that neither osmolarity nor intracellular concentrations have any influence on the passive potassium efflux. The independence of the potassium efflux and osmolarity can be verified by addition of a penetrating substance like glucose to the cell suspension. Adding water or hypertonic sodium chloride solution shows that the potassium efflux increases slightly in more concentrated salt solutions. Inasmuch as it can be interpreted as a pure ionic strength effect, this result supports the hypothesis of independence of potassium efflux and intracellular concentrations. The results of this investigation together with other studies show that the passive permeability of the human red blood cell to potassium depends uniquely on the membrane potential near physiological conditions, while it depends on parameters such as pH or concentrations for large membrane potentials. This suggests that two different mechanisms of transport might be involved: one would control the permeability under normal conditions; the other would represent a leak through the route normally used by anions and become important only under extreme conditions.Because it is readily available in great quantities and its structure is simpler than that of other cells, the erythrocyte is an object of choice for the study of cell membrane properties. The active pumping of sodium and potassium across the red cell membrane has been demonstrated for some time [13] and it is probably now the best studied of all active transport processes.A necessary lemma to the complete elucidation of active transport mechanisms in the red cell membrane is the explanation of the cell's passive transport properties. How does the red cell discriminate between ions and, above all, between anions and cations? What parameters determine the

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The Effect of Valinomycin on Potassium and Sodium Permeability of HK and LK Sheep Red Cells

Cited by 109 publications

References 15 publications

Cation transport and membrane morphology

Cation transport and membrane morphology

Vibrio effector protein VopQ inhibits fusion of V-ATPase–containing membranes

A study of passive potassium efflux from human red blood cells using ion-specific electrodes

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