Non-selective voltage-activated cation channel in the human red blood cell membrane. (1/66)

Using the patch-clamp technique, a non-selective voltage-activated Na+ and K+ channel in the human red blood cell membrane was found. The channel operates only at positive membrane potentials from about +30 mV (inside positive) onwards. For sodium and potassium ions, similar conductances of about 21 pS were determined. Together with the recently described K+(Na+)/H+ exchanger, this channel is responsible for the increase of residual K+ and Na+ fluxes across the human red blood cell membrane when the cells are suspended in low ionic strength medium.  (+info)

Different foci for the regulation of the activity of the KefB and KefC glutathione-gated K+ efflux systems. (2/66)

KefB and KefC are glutathione-gated K+ efflux systems in Escherichia coli, and the proteins exhibit strong similarity at the level of both primary sequence and domain organization. The proteins are maintained closed by glutathione and are activated by binding of adducts formed between glutathione and electrophiles. By construction of equivalent mutations in each protein, this study has analyzed the control over inactive state of the proteins. A UV-induced mutation in KefB, L75S, causes rapid spontaneous K+ efflux but has only a minor effect on K+ efflux via KefC. Similarly amino acid substitutions that cause increased spontaneous activity in KefC have only small effects in KefB. Exchange of an eight amino acid region from KefC (HALESDIE) with the equivalent sequence from KefB (HELETAID) has identified a role for a group of acidic residues in controlling KefC activity. The mutations HELETAID and L74S in KefC act synergistically, and the activity of the resultant protein resembles that of KefB. We conclude that, despite the high degree of sequence similarity, KefB and KefC exhibit different sensitivities to the same site-specific mutations.  (+info)

Potassium ion efflux induced by cationic compounds in yeast. (3/66)

Potassium efflux in yeast induced by several cationic compounds showed different characteristics. All of the observed efflux required glucose as substrate at the concentrations used. For most of them, the phenomenon required binding of the cationic compound to the cell surface and increased with the negative cell surface charge, and for all the compounds tested, it depended on a metabolizable substrate. Efflux induced with terbium chloride appeared more likely due to the function of a K+/H+ antiporter. With DEAE-dextran and dihydrostreptomycin, potassium efflux was dependent on the cell potassium content and was also sensitive to osmotic changes of the medium. DEAE-dextran-provoked efflux was not due to cell disruption. Dihydrostreptomycin seemed to activate a potassium efflux system which could not be studied in isolation, but its inhibition of potassium uptake may also be involved. Except for cells treated with ethidium bromide, no appreciable cell disruption was observed. The potassium efflux observed appears to be a membrane phenomenon reversible after washing with magnesium chloride.  (+info)

Potassium/proton antiport system is dispensable for growth of Enterococcus hirae at low pH. (4/66)

An energy-dependent K+/H+ antiport system is found in Enterococcus hirae ATCC 9790 cultured in a standard complex medium (Y. Kakinuma, and K. Igarashi, J. Biol. Chem. 263:14166-14170, 1988). We have now found that the activity of this antiport system was totally missing in cells cultured in a defined medium. In this defined medium, E. hirae did not grow well at pH near 9, but grew normally at pH below 7.5. This antiport system is important at high pH but dispensable at lower pH for ion homeostasis of this bacterium.  (+info)

Isolation and properties of Enterococcus hirae mutants defective in the potassium/proton antiport system. (5/66)

A K+/H+ antiporter regulates cytoplasmic pH in Enterococcus hirae growing at alkaline pH. Mutants defective in this antiport activity were alkaline pH sensitive. One mutant, Pop1, lacked both K+/methylamine exchange at pH 9.5 and concomitant acidification of cytoplasmic pH. Pop1 grew well at pHs below 8 but did not at pHs above 9, conditions under which cytoplasmic pH was not fully acidified.  (+info)

Matrix free Mg(2+) and the regulation of mitochondrial volume. (6/66)

Mitochondria must maintain volume homeostasis in order to carry out oxidative phosphorylation. It has been postulated that the concentration of free Mg(2+) ([Mg(2+)]) serves as the sensor of matrix volume and regulates a K(+)-extruding K(+)/H(+) antiport (K. D. Garlid. J. Biol. Chem. 255: 11273-11279, 1980). To test this hypothesis, the fluorescent probe furaptra was used to monitor [Mg(2+)] and free Ca(2+) concentration ([Ca(2+)]) in the matrix of isolated beef heart mitochondria, and K(+)/H(+) antiport activity was measured by passive swelling in potassium acetate. Concentrations that result in 50% inhibition of maximum activity of 92 microM matrix [Mg(2+)] and 2.2 microM [Ca(2+)] were determined for the K(+)/H(+) antiport. Untreated mitochondria average 670 microM matrix [Mg(2+)], a value that would permit <1% of maximum K(+)/H(+) antiport activity. Hypotonic swelling results in large decreases in matrix [Mg(2+)], but swelling due to accumulation of acetate salts does not alter [Mg(2+)]. Swelling in phosphate salts decreases matrix [Mg(2+)], but not to levels that permit appreciable antiport activity. We conclude that 1) it is unlikely that matrix [Mg(2+)] serves as the mitochondrial volume sensor, 2) if K(+)/H(+) antiport functions as a volume control transporter, it is probably regulated by factors other than [Mg(2+)], and 3) alternative mechanisms for mitochondrial volume control should be considered.  (+info)

Leishmania plasma membrane Mg2+-ATPase is a H+/K+-antiporter involved in glucose symport. Studies with sealed ghosts and vesicles of opposite polarity. (7/66)

Experiments from other laboratories conducted with Leishmania donovani promastigote cells had earlier indicated that the plasma membrane Mg2+-ATPase of the parasite is an extrusion pump for H+. Taking advantage of the pellicular microtubular structure of the plasma membrane of the organism, we report procedures for obtaining sealed ghost and sealed everted vesicle of defined polarity. Rapid influx of H+ into everted vesicles was found to be dependent on the simultaneous presence of ATP (1 mm) and Mg2+ (1 mm). Excellent correspondence between rate of H+ entry and the enzyme activity clearly demonstrated the Mg2+-ATPase to be a true H+ pump. H+ entry into everted vesicle was strongly inhibited by SCH28080 (IC50 = approximately 40 microm) and by omeprazole (IC50 = approximately 50 microm), both of which are characteristic inhibitors of mammalian gastric H+,K+-ATPase. H+ influx was completely insensitive to ouabain (250 microm), the typical inhibitor of Na+,K+-ATPase. Mg2+-ATPase activity could be partially stimulated with K+ (20 mm) that was inhibitable (>85%) with SCH28080 (50 microm). ATP-dependent rapid efflux of 86Rb+ from preloaded vesicles was completely inhibited by preincubation with omeprazole (150 microm) and by 5,5'-dithiobis-(2-nitrobenzoic acid) (1 mm), an inhibitor of the enzyme. Assuming Rb+ to be a true surrogate for K+, an ATP-dependent, electroneutral stoichiometric exchange of H+ and K+(1:1) was established. Rapid and 10-fold active accumulation of [U-(14)C]2-deoxyglucose in sealed ghosts could be observed when an artificial pH gradient (interior alkaline) was imposed. Rapid efflux of [U-(14)C]d-glucose from preloaded everted vesicles could also be initiated by activating the enzyme, with ATP. Taken together, the plasma membrane Mg2+-ATPase has been identified as an electroneutral H+/K+ antiporter with some properties reminiscent of the gastric H+,K+-ATPase. This enzyme is possibly involved in active accumulation of glucose via a H+-glucose symport system and in K+ accumulation.  (+info)

Insect midgut K(+) secretion: concerted run-down of apical/basolateral transporters with extra-/intracellular acidity. (8/66)

In lepidopteran larvae, three transport mechanisms are involved in the active and electrogenic K(+) secretion that occurs in the epithelial goblet cells of the midgut. These consist of (i) basolateral K(+) channels, allowing K(+) entry from the haemolymph into the cytosol, (ii) apical electrogenic K(+)/2H(+) antiporters, which are responsible for secondary active extrusion of K(+) from the cell into the gut lumen via the goblet cavity and (iii) apical V-ATPase-type proton pumps. The latter energize apical K(+) exit by building up a large, cavity-positive electrical potential that drives the antiporters. Net K(+) secretion (I(K)) can be measured as short-circuit current (I(sc)) across the in vitro midgut mounted in an Ussing chamber. We investigated the influence of protons on the transepithelial I(K) and the partial reactions of the basolateral K(+) permeability (P(K)) and the apical, lumped 'K(+) pump' current (I(P)) at various extra- and intracellular pH values. In particular, we wanted to know whether increased cellular acidity could counteract the reversible dissociation of the V-ATPase into its V(1) and V(o) parts, as occurs in yeast after glucose deprivation and in the midgut of Manduca sexta during starvation or moulting, thus possibly enhancing K(+) transport. When intact epithelia were perfused with high-[K(+)] (32 mmol l(-1)) salines with different pH values, I(K) was reversibly reduced when pH values fell below 6 on either side of the epithelium. Attempts to modify the intracellular pH by pulsing with NH(4)(+) or propionate showed that intracellular acidification caused a reduction in I(K) similar to that obtained in response to application of external protons. Treatment with azide, a well-known inhibitor of the mitochondrial ATP synthase, had the same effect as pulsing with ammonium or propionate with, however, much faster kinetics and higher reversibility. Breakdown of the basolateral or apical barrier using the antibiotic nystatin allowed the intracellular pH to be clamped to that of the saline facing the nystatin-treated epithelial border. Cell acidification achieved by this manipulation led to a reduction in both apical I(P) and basolateral P(K). The transepithelial I(K) showed an approximately half-maximal reduction at external pH values close to 5 in intact tissues, and a similar reduction in I(P) and P(K) values was seen at an intracellular pH of 5 in nystatin-permeabilised epithelia. Thus, the hypothesized V(1)V(o) stabilization by cell acidity is not reflected in the pH-sensitivity of I(P). Moreover, all components that transport K(+) are synchronously inhibited below pH 6. The significance of our findings for the midgut in vivo is discussed.  (+info)