Transient hyperpolarization of yeast by glucose and ethanol

At pH 7, addition of glucose under anaerobic conditions to a suspension of the yeast Saccharomyces cerevisiae causes both a transient hyperpolarization and a transient net efflux of K⁺ from the cells. Hyperpolarization shows a peak at about 3 min and a net K⁺ efflux at 4–5 min. An additional transient hyperpolarization and net K⁺ efflux are found after 60–80 and 100 min, respectively. Addition of 2-deoxyglucose instead of glucose does not lead to hyperpolarization of the cells or K⁺ efflux. At low pH, neither transient hyperpolarization nor a transient K⁺ efflux are found. With ethanol as substrate and applying aerobic conditions, both a transient hyperpolarization and a transient K⁺ efflux are found at pH 7. The fluorescent probe 2-(dimethylaminostyryl)-1-ethylpyridinium appears to be useful for probing changes in the membrane potential of S. cerevisiae. It is hypothesized that the hyperpolarization of the cells is due to opening of K⁺ channels in the plasma membrane. Accordingly, the hyperpolarization of the cells at pH 7 is almost completely abolished by 1.25 mM K⁺, whereas the same amount of Na⁺ does not reduce the hyperpolarization     

Active extrusion of potassium in the yeast Saccharomyces cerevisiae induced by low concentrations of trifluoperazine

Trifluoperazine (TFP), the antipsychotic drug, induces substantial K+ efflux, membrane hyperpolarization and inhibition of H+-ATPase in the yeast Saccharomyces cerevisiae. Investigations on the mechanism of these effects revealed two different processes observed at different incubation conditions. At an acidic pH of 4.5 and an alkaline pH of 7.5, K+ efflux was accompanied by substantial proton influx which led to intracellular acidification and dissipation of delta psi formed by cation efflux. The results indicated nonspecific changes in membrane permeability. Similar results were also observed when cells were incubated at pH 5.5-6.0 with higher concentrations of TFP (above 75 microM). On the other hand, low concentrations of TFP (30-50 microM) at pH 5.5-6.0 caused marked membrane hyperpolarization and K+ efflux unaccompanied by the efflux of other cations and by H+ influx. Our experiments indicate that under these conditions K+ efflux was an active process. (1) K+ efflux proceeded only in the presence of a metabolic substrate and was inhibited by metabolic inhibitors. (2) When 0.3-0.9 mM-KCl was present in the medium at pH 6.0, the concentration of K+ within the cells (measured at the end of the incubation with TFP) was much lower than the theoretical concentration of Kin+ if the distribution of K+ between medium and cell water was at equilibrium (at zero electrochemical gradient). (3) Valinomycin decreased the net K+ efflux and decreased the membrane hyperpolarization induced by TFP, probably by increasing the flux of K+ into the cells along its electrochemical gradient. (4) Conditions which led to active K+ efflux also led to a marked decrease in cellular ATP level. The results indicate that under a specific set of conditions TFP induces translocation of K+ against its electrochemical gradient.     

A putative K(+)-selective channel in the plasma membrane of yeast that is blocked by micromolar concentrations of external divalent cations and is insensitive to tetraethylammonium

At pH 7, addition of glucose to an anaerobic suspension of non-metabolizing yeast cells causes a transient net efflux of K+ from the cells and a concomitant transient hyperpolarization of the plasma membrane (Van de Mortel, J.B.J., et al. (1988) Biochem. Biophys. Acta 936, 421-428). Both phenomena are effectively suppressed in the presence of low concentrations of polyvalent cations. The concentrations of Mn2+, Ca2+, Ba2+, Mg2+, Sr2+ and La3+ required for half-maximal suppression of the transient hyperpolarization are 10, 17, 20, 38, 47 and 5 microM, respectively. Subsequent addition of EDTA 90 s after that of Ca2+ immediately restores both K+ efflux and cellular uptake of the fluorescent membrane potential probe 2-(dimethylaminostyryl)-1-ethylpyridinium (DMP). This suggests that an interaction of polyvalent cations with an external binding site blocks the putative K(+)-selective channel. Opening of this channel is not blocked by 20 mM tetraethylammonium nor by 100 microM 3,4-diaminopyridine. It is argued that this glucose-induced K(+)-conductive pathway is not identical to the voltage-gated K+ channels identified until now in patch-clamp studies of the yeast plasma membrane.     

Hyperpolarization and intracellular acidification in Trichoderma viride as a response to illumination.

Using indirect methods based on uptake of [3H]tetraphenylphosphonium cation and [14C]benzoic acid by cells of the fungus Trichoderma viride we found that the illumination-induced transient hyperpolarization of the plasma membrane is followed immediately by a rapid temporary decrease in intracellular pH. Hyperpolarization and intracellular acidification were completely suppressed by 150 mM-KCl and by the K(+)-ionophore valinomycin. The light-induced acidification of the cytoplasm was not observed in the presence of the cytochrome respiratory chain inhibitors antimycin A and mucidin. Based on these results, we hypothesize that the hyperpolarization of the cells is the consequence of an efflux of K+ through a light-activated K(+)-channel in the plasma membrane. The loss of positive charge in the cytoplasm caused by this efflux of cations is counterbalanced by H+ originating from the light-activated mitochondrial respiratory chain.     

Calcium-induced oscillations in K+ conductance and membrane potential of human erythrocytes mediated by the ionophore A23187

The time-dependence of ionophore A23187-induced changes in the conductance of the Ca2+-sensitive K+ channels of the human red cell has been monitored with ion-specific electrodes. The membrane potential was reflected in CCCP-mediated pH changes in a buffer-free extracellular medium, and changes in extracellular K+ activity and electrode potential of an extracellular Ca2+-electrode were recorded. Within a narrow range of ionophore-mediated Ca2+ influx, the above-mentioned parameters were found to oscillate when ionophore was added to a suspension of glucose-fed cells. The period of oscillation was about 2 min/cycle depending on ionophore concentration, and the amplitude of hyperpolarization was about 60 mV, corresponding to a maximal gK+ of the same magnitude as gCl-. Without CCCP present no oscillation in K+ conductance was observed. The Ca2+ affinity for the opening process was in the micromolar range. The closing of the K+ channels was a spontaneous process in that the depolarization was well under way before the Ca2+-ATPase-mediated Ca2+ net efflux started. Below the Ca2+ influx range for oscillations, no response was observed for up to 20 min after the addition of ionophore. Above the upper limit, a permanent hyperpolarization resulted with an extracellular K+ activity increasing monotonically as a function of time. In experiments with ATP-depleted cells, responses of the latter type ensued at all ionophore concentrations above the lower limit. Addition of surplus EGTA to suspensions of hyperpolarized cells restores the normal membrane potential in the case of glucose-fed cells, whereas the K+-channels in ATP-depleted cells remained open.     

The effects of cesium chloride on insulin release, ionic fluxes and membrane potential in pancreatic B-cells

Cs+ decreases K+ permeability in nerve and muscle cells. Its effects on the pancreatic B-cell function were studied with mouse islets. In the presence of 3 mM glucose, Cs+ substitution for K+ steadily inhibited 86Rb+ efflux and hyperpolarized the B-cell membrane. Addition of Cs+ to a K+-medium also inhibited 86Rb+ efflux, but depolarized the B-cell membrane. None of these changes altered insulin release. Substitution of Cs+ for K+ in a medium containing 10 mM glucose caused a Ca2+-dependent stimulation of insulin release and 45Ca2+ efflux, produced an initial fall and a secondary rise in 86Rb+ efflux and augmented the electrical activity in B-cells. Reintroduction of K+ to the medium was followed by a marked and transient inhibition of insulin release, that was blocked by ouabain and accompanied by an inhibition of 45Ca2+ and 86Rb+ efflux and by a hyperpolarization of the B-cell membrane. Addition of Cs+ to a K+ medium containing 10 mM glucose stimulated insulin release, 45Ca2+ efflux and 86Rb+ efflux. It also increased the electrical activity in B-cells. In the absence of Ca2+, however, Cs+ addition decreased the rate of 86Rb+ efflux. The effects of Cs+ on the B-cell function may be explained by its ability to decrease K+ permeability of the plasma membrane, by its inability to activate the sodium pump, and by a third unidentified effect likely brought about by the accumulation of intracellular Cs+.     

Activation of Ca2+ influx by metabolic substrates in Saccharomyces cerevisiae: role of membrane potential and cellular ATP levels

Influx of Ca2+ into cells of Saccharomyces cerevisiae was measured under non-steady-state conditions, which enable measurements of the initial rate of transport across plasma membranes without interference by the vacuolar Ca2+ transport system. Removal of glucose from the incubation medium led to inactivation of Ca2+ influx within 5 min. Readdition of glucose led to a transient increase in the rate of Ca2+ transport, reaching a peak after 3-5 min. A second increase was observed 60-80 min later. To examine whether the first transient activation of Ca2+ influx by glucose was mediated by membrane hyperpolarization, influx of 45Ca2+ was measured in the presence and absence of metabolic substrates (glucose, glycerol, and glucose plus antimycin A) in cells hyperpolarized to different values of membrane potential (delta psi). Logarithms of the rate of Ca2+ influx were plotted against values of delta psi. Two different slopes were obtained, depending upon whether the metabolic substrate was present or absent. Ca2+ influx in the presence of the metabolic substrates was always higher than expected by their effect on delta psi. Glycerol plus antimycin A did not affect Ca2+ influx. It was concluded that metabolized substrates activate Ca2+ influx not only by effects on delta psi but also by additional mechanism(s). Since no simple correlation between Ca2+ influx and intracellular ATP levels was observed, it was concluded that ATP levels do not affect the initial rates of Ca2+ transport across the plasma membrane of S. cerevisiae.     

Changes in 45Ca and 109Cd uptake, membrane potential and cell pH in Saccharomyces cerevisiae provoked by Cd2+

The effect of Cd2+ poisoning of Saccharomyces cerevisiae on 45Ca, 109Cd and [14C]tetraphenylphosphonium (TPP) uptake and cell pH was examined. At Cd2+ concentrations that produced substantial K+ efflux the rates of uptake of 45Ca, 109Cd and [14C]TPP increased progressively during incubation of the cells with Cd2+, and the cell pH was lowered concomitantly. The initial rates of uptake of the divalent cations and of TPP were increased in cells pre-loaded with Cd2+, which shows that stimulation of the ion fluxes was exerted by the Cd2+ that accumulated in the cells. The distribution ratio of TPP between cells and medium, however, was decreased by Cd2+. Although hyperpolarization of the cell membrane by Cd2+ cannot be excluded, it is argued that Cd2+ primarily stimulated divalent cation uptake by increasing the cation permeability of the cell membrane allowing the cations to enter the cells more easily.     

Effects of potassium ions on the electrical and pH gradients across the membrane of Streptococcus lactis cells.

Bacteria transduce and conserve energy at the plasma membrane in the form of an electrochemical gradient of hydrogen ions (deltap). Energized cells of Streptococcus lactis accumulate K+ ions presumably in exchange for H+. We reasoned that if the movement of H+ is limited, then an increase in H+ efflux, effected by potassium transport inward, should result in changes in the steady-state deltap. We determined the electrical gradient (deltapsi) from the fluorescence of a membrane potential-sensitive cyanine dye, and the chemical H+ gradient (deltapH) from the distribution of a weak acid. The deltap was also determined independently from the accumulation levels of the non-metabolizable sugar thiomethyl-beta-galactoside. KCl addition to cells fermenting glucose or arginine at pH 5 changed the deltap very little, but lowered the deltapsi, while increasing the deltapH. At pH 7, the deltapH only increased slightly; thus, the decrease in deltapsi, effected by addition of potassium ions, resulted in a lowered steady-state deltap. These effects were shown not to be due to swelling or shrinking of the cells. Thus, in these nongrowing cells, under conditions of energy utilization for the active transport of K+, the components of deltap can vary depending on the limitations on the net movement of protons.     

Transient increase in Ca2+ influx in Saccharomyces cerevisiae in response to glucose: effects of intracellular acidification and cAMP levels

Influx of 45Ca2+ into Saccharomyces cerevisiae was measured under experimental conditions which enabled measurements of initial rate of transport across the plasma membrane, without interference by the vacuolar Ca2+ transport system. Addition of glucose or glycerol to the cells, after pre-incubation in glucose-free medium for 5 min, caused a rapid, transient increase in 45Ca2+ influx, reaching a peak at 3-5 min after addition of substrate. Ethanol, or glycerol added with antimycin A, had no effect on 45Ca2+ influx. We have shown previously that this increase is not mediated by an effect of the substrates on intracellular ATP levels. Changes in membrane potential accounted for only a part of the glucose-stimulated 45Ca2+ influx. The roles of intracellular acidification and changes in cellular cAMP in mediating the effects of glucose on 45Ca2+ influx were examined. After a short preincubation in glucose-free medium addition of glucose caused a decrease in the intracellular pH, [pH]i, which reached a minimum value after 3 min. A transient increase in the cellular cAMP level was also observed. Addition of glycerol also caused intracellular acidification, but ethanol or glycerol added with antimycin A had no effect on [pH]i. Artificial intracellular acidification induced by exposure to isobutyric acid or to CCCP caused a transient rise in Ca2+ influx but the extent of the increase was smaller than that caused by glucose, and the time-course was different. We conclude that intracellular acidification may be responsible for part of the glucose stimulation of Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the cAMP level in the yeast Saccharomyces cerevisiae: the glucose-induced cAMP signal is not mediated by a transient drop in the intracellular pH

Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae is known to cause a rapid, transient increase in the cAMP level, which lasts for 1-2 min and induces a cAMP-dependent protein phosphorylation cascade. The glucose-induced cAMP signal cannot be explained solely on the basis of an increased ATP level. Transient membrane depolarization and transient intracellular acidification have been suggested as possible triggers for the cAMP peak. Addition of glucose to cells in which the plasma membrane had been depolarized still produced the increase in the cAMP level excluding membrane depolarization as the possible trigger. Using in vivo 31P NMR-spectroscopy we followed phosphate metabolism and the time course of the drop in the intracellular pH after addition of glucose with a time resolution of 15 s. Under aerobic conditions the initial pH and ATP level were high. On addition of glucose, they both showed a rapid, transient drop, which lasted for about 30 s. Under anaerobic conditions, the initial pH and ATP level were low and on addition of glucose they both increased relatively slowly compared to aerobic conditions. Several conditions were found in which the pH drop which occurs under aerobic conditions could be blocked completely without effect on the cAMP signal or without completely preventing it: addition of NH4Cl together with glucose at high extracellular pH and addition of a low concentration of glucose before a high concentration. Also, when glucose was added twice to the same cells no consistent relationship was observed between the pH drop and the cAMP peak. These results appear to exclude transient intracellular acidification as the trigger for the cAMP signal. Hence, we conclude that the effect of glucose cannot be explained on the basis of effects known to be caused by the membrane depolarizing compounds which cause increases in the cAMP level. A new, more specific kind of interaction appears to be involved.     

The influence of cellular amino acids and the Na+ : K+ pump on the membrane potential of the Ehrlich ascites tumor cell.

The membrane potential of the Ehrlich ascites tumor cell was shown to be influenced by its amino acid content and the activity of the Na+ :K+ pump. The membrane potential (monitored by the fluorescent dye, 3,3'-dipropylthiodicarbocyanine iodide) varied with the size of the endogenous amino acid pool and with the concentration of accumulated 2-aminoisobutyrate. When cellular amino acid content was high, the cells were hyperpolarized; as the pool declined in size, the cells were depolarized. The hyperpolarization seen with cellular amino acid required cellular Na+ but not cellular ATP. Na+ efflux was more rapid from cells containing 2-aminoisobutyrate than from cells low in internal amino acids. These observations indicate that the hyperpolarization recorded in cells with high cellular amino acid content resulted from the electrogenic co-efflux of Na+ and amino acids. Cellular ATP levels were found to decline rapidly in the presence of the dye and hence the influence of the pump was seen only if glucose was added to the cells. When the cells contained normal Na+ (approx. 30mM), the Na+ :K+ pump was shown to have little effect on the membrane potential (the addition of ouabain had little effect on the potential). When cellular Na+ was raised to 60mM, the activity of the pump changed the membrane potential from the range -25 to -30 mV to -44 to -63 mV. This hyperpolarization required external K+ and was inhibited by ouabain.     

Effects of monensin on metabolism of isolated rat islets of Langerhans.

Monensin, a univalent ionophore, is a carboxylic acid produced by Streptomyces cinnamonensis. It will complex various alkali-metal ions, but most readily binds Na+. Because of interest in the possible role of Na+ in the regulation of insulin secretion, we examined its effects on several aspects of the metabolism of isolated rat islets of Langerhans. The ionophore inhibited glucose-stimulated insulin release in a concentration-dependent manner, completely inhibiting secretion evoked by 20 mM-glucose at concentrations as low as 0.1 microM in static incubations. In perifusion experiments, both phases of insulin release were equally affected. Monensin (0.1 microM) had no significant effect on glucose oxidation as measured by the generation of 14CO2 from [14C]glucose. Monensin increased the rate of 22Na+ efflux from preloaded islets and net 22Na+ uptake over 30 min, in the absence of changes in islet volume or extracellular space. The ionophore increased the Rb+/K+ permeability of islet cells, as shown by its inhibition of 86Rb+ retention and stimulation of 86Rb+ efflux. At 0.1 microM, monensin abolished glucose-stimulated 45Ca2+ uptake by islets during 5 min incubations, and stimulated 45Ca2+ efflux from preloaded islets perifused with Ca2+-free medium, even in the complete absence of extracellular Na+. Studies of the uptake of 14C-labelled 5,5-dimethyloxazolidine-2,4-dione showed that 0.1 microM-monensin increased net intracellular pH from 7.05 to 7.13. 7 Monensin has widespread, complex, effects on the secretory responses and ion handling by the B cells, which are difficult to interpret in terms solely of actions as a Na+ ionophore.     

Role of Ba2+-resistant K+ channels in endothelium-dependent hyperpolarization of rat small mesenteric arteries

In rat small mesenteric arteries, the influence of modulation of basal smooth muscle K+ efflux on the mechanism of endothelium-dependent hyperpolarization was investigated. The membrane potentials of the vascular smooth muscle cells were measured using conventional microelectrode techniques. Incubation of resting arteries with the gap junction uncoupler carbenoxolone (20 micro M) decreased the endothelium-dependent hyperpolarization elicited by a submaximal concentration of acetylcholine (3 micro M) to about 65% of the control. In the presence of Ba2+ (200 micro M), which depolarized the membrane potential by 10 mV, the acetylcholine-induced membrane potential response was doubled in magnitude, reaching values not different from control. Moreover, the hyperpolarization was more resistant to carbenoxolone in these conditions. Finally, both in the absence and in the presence of carbenoxolone, the combined application of Ba2+ and ouabain (0.5 mM) did not abolish the acetylcholine response. These results suggest that gap junctional coupling plays a role in endothelium-dependent hyperpolarization of smooth muscle cells of resting rat small mesenteric arteries. Additionally, these findings show that the hyperpolarization does not rely on activation of inward rectifying K+ channels. Although a minor contribution of Na-K pumping cannot be excluded, the Ba2+ experiments show that the membrane electrical response is mediated by activation of a Ba2+-resistant K+ conductance.     

Membrane potential and bicarbonate secretion in isolated interlobular ducts from guinea-pig pancreas

The interlobular duct cells of the guinea-pig pancreas secrete HCO(3)(-) across their luminal membrane into a HCO(3)(-)-rich (125 mM) luminal fluid against a sixfold concentration gradient. Since HCO(3)(-) transport cannot be achieved by luminal Cl-/HCO(3)(-) exchange under these conditions, we have investigated the possibility that it is mediated by an anion conductance. To determine whether the electrochemical potential gradient across the luminal membrane would favor HCO(3)(-) efflux, we have measured the intracellular potential (V(m)) in microperfused, interlobular duct segments under various physiological conditions. When the lumen was perfused with a 124 mM Cl- -25 mM HCO(3)(-) solution, a condition similar to the basal state, the resting potential was approximately -60 mV. Stimulation with dbcAMP or secretin caused a transient hyperpolarization (approximately 5 mV) due to activation of electrogenic Na+-HCO(3)(-) cotransport at the basolateral membrane. This was followed by depolarization to a steady-state value of approximately -50 mV as a result of anion efflux across the luminal membrane. Raising the luminal HCO(3)(-) concentration to 125 mM caused a hyperpolarization (approximately 10 mV) in both stimulated and unstimulated ducts. These results can be explained by a model in which the depolarizing effect of Cl- efflux across the luminal membrane is minimized by the depletion of intracellular Cl- and offset by the hyperpolarizing effects of Na+-HCO(3)(-) cotransport at the basolateral membrane. The net effect is a luminally directed electrochemical potential gradient for HCO(3)(-) that is sustained during maximal stimulation. Our calculations indicate that the electrodiffusive efflux of HCO(3)(-) to the lumen via CFTR, driven by this gradient, would be sufficient to fully account for the observed secretory flux of HCO(3)(-).     

Physiological characterization of Saccharomyces cerevisiae kha1 deletion mutants

Maintenance of intracellular K+ homeostasis is one of the crucial requisites for the survival of yeast cells. In Saccharomyces cerevisiae, the high K+ content corresponds to a steady state between simultaneous influx and efflux across the plasma membrane. One of the transporters formerly believed to extrude K+ from the yeast cells (besides Ena1-4p and Nha1p) was named Kha1p and presumed as a putative plasma membrane K+/H+ antiporter. We prepared kha1 and tok1-kha1 deletion strains in the B31 and MAB 2d background. Both the strains contain the ena1-4 and nha1 deletions; that means they lack the main active sodium and potassium efflux systems. MAB 2d has additional trk1 and trk2 deletions, i.e. is impaired in active K+ uptake as well. We performed a large physiological study with these strains to specify the phenotype of kha1 deletion. In our experiments, no difference in K+ content or efflux was observed in strains lacking the KHA1 gene compared with control strains. Two main phenotype manifestations of the kha1 deletion were growth defect on high external pH and hygromycin sensitivity. The correlation between these phenotypes and the kha1 deletion was confirmed by plasmid complementation. Fluorescence microscopy of green fluorescent protein (GFP)-tagged Kha1p showed that this antiporter is localized preferentially intracellularly (in contrast to the plasma membrane Na+/H+ antiporter Nha1p). Based on these findings, Kha1p is probably not localized in plasma membrane and does not mediate efflux of alkali metal cations from cells, but is important for the regulation of intracellular cation homeostasis and optimal pH control, similarly as the Nhx1p.     

Effect of magnesium-dependent cell membrane alterations on the transport of K+ in Ehrlich ascites tumour cells

Mg-deficiency or Mg-loading of tumour cells changes the permeability of the cell membrane. The influence of this change on the K+ transport across the membrane was investigated using 86Rb+ and K+ analog. The time course of the influx and efflux rates were estimated by means of a mathematical approach for a two-compartment system with inconstant pool sizes. The comparison of the two states of the cells demonstrates that in Mg-deficient cells the passive K+ efflux is significantly enhanced (40%). This in turn stimulates the active counter transport mediated by the (Na+-K+)-ATPase, raising the ATP consumption by about 30%. However, the enzyme is not able to maintain the cellular K+ content under these conditions. After a short transient increase due to the initially enhanced influx the passive net efflux prevails. Differences in the electrophoretic mobility of the two states of the cells confirm Mg-dependent changes of the cell membrane structure.     

Modulation of the voltage-sensitive Na+/H+ exchange in sea urchin spermatozoa through membrane potential changes induced by the egg peptide speract

Sea urchin sperm motility can be activated by alkalinization of the internal pH, and previous studies have shown that the internal pH can be regulated by a voltage-sensitive Na+/H+ exchanger present in the flagellar plasma membrane. In this study, the effects of speract, a peptide purified from egg conditioned media, on the Na+/H+ exchange were investigated. Evidence presented indicates that speract activates K+ channels in the flagellar membrane and modulates the Na+/H+ exchange activity through resultant changes in membrane potential. In the presence of tetraphenylphosphonium, a lipophilic ion, or high external Na+, the isolated flagella were depolarized, and Na+/H+ exchanger was inhibited. Speract and valinomycin, a K+ ionophore, were able to reactivate 22Na+ uptake, H+ efflux, and alkalinization of intraflagellar pH under either of the depolarizing conditions. Membrane potential measurements using 3,3'-dipropylthiodicarbocyanide iodide indicated repolarization by either speract or valinomycin. The speract-induced voltage changes did not require Na+ but were sensitive to [K+]. Thus, speract induced a slight depolarization in Na+-free seawater with 10 mM K+ but a hyperpolarization with 2 mM K+. Further support for the activation of K+ channels in the flagella was the 2-5-fold stimulation of K+ efflux induced by speract as measured with a K+ electrode. The ionic selectivity of the speract-activated channel assessed by voltage measurements was K+ greater than Rb+ greater than Cs+. The half-maximally effective concentration of speract was about 0.2 nM. That the H+ and K+ efflux in response to peptide was receptor-mediated was confirmed by the use of speract or resact on intact sea urchin spermatozoa, where the peptides were found to stimulate K+ efflux and to reverse the tetraphenylphosphonium inhibition on H+ efflux only in the homologous spermatozoa. Modulation of the voltage-sensitive Na+/H+ exchange by egg peptides, therefore, appears to be indirect and is coupled through its action on membrane potential.     

The sodium/proton antiport system in a newly isolated alkalophilic Bacillus sp.

The pH homeostasis and the sodium/proton antiport system have been studied in the newly isolated alkalophilic Bacillus sp. strain N-6, which could grow on media in a pH range from 7 to 10, and in its nonalkalophilic mutant. After a quick shift in external pH from 8 to 10 by the addition of Na2CO3, the delta pH (inside acid) in the cells of strain N-6 was immediately established, and the pH homeostatic state was maintained for more than 20 min in an alkaline environment. However, under the same conditions, the pH homeostasis was not observed in the cells of nonalkalophilic mutant, and the cytoplasmic pH immediately rose to pH 10. On the other hand, the results of the rapid acidification from pH 9 to 7 showed that the internal pH was maintained as more basic than the external pH in a neutral medium in both strains. The Na+/H+ antiport system has been characterized by either the effect of Na+ on delta pH formation or 22Na+ efflux in Na+-loaded right-side-out membrane vesicles of strain N-6. Na+- or Li+-loaded vesicles exhibited a reversed delta pH (inside acid) after the addition of electron donors (ascorbate plus tetramethyl-p-phenylenediamine) at both pH 7 and 9, whereas choline-loaded vesicles generated delta pHs of the conventional orientation (inside alkaline). 22Na+ was actively extruded from 22Na+-loaded vesicles whose potential was negative at pH 7 and 9. The inclusion of carbonyl cyanide m-chlorophenylhydrazone inhibited 22Na+ efflux in the presence of electron donors. These results indicate that the Na+/H+ antiport system in this strain operates electrogenically over a range of external pHs from 7 to 10 and plays a role in pH homeostasis at the alkaline pH range. The pH homeostasis at neutral ph was studied in more detail. K+ -depleted cells showed no delta pH (acid out) in the neutral conditions in the absence of K+, whereas these cells generated a delta pH if K+ was present in the medium. This increase of internal pH was accompanied by K+ uptake from the medium. These results suggest that electrogenic K+ entry allows extrusion of H+ from cells by the primary proton pump at neutral pH.     

Potassium movement during sodium-induced motility initiation in the rat caudal epididymal spermatozoa

The sodium-induced sperm motility initiation of the rat cauda epididymal sperm has been studied in different potassium concentrations. High K+ inhibited motility initiation. At a K+ concentration of 50 mM (concentration found in the rat cauda epididymidis), sperm motility was inhibited by 80%. K+ movement across the sperm membrane has been followed by using 86Rb+ as a marker for K+. When the 86Rb+ preloaded sperm were suspended in a sodium-free medium, there was a little efflux of 86Rb+. However, if they were suspended in a sodium-containing medium, the efflux rate was greatly increased. This increase in 86Rb+ efflux rate was associated with an initiation of sperm motility. Both 86Rb+ efflux and motility initiation were triggered by a K+ ionophore 18-crown-6 (2 X 10(-3)M). However, the ionophore-induced 86Rb+ efflux and motility initiation only occurred in the presence of extracellular Na+. Tetraethylammonium (TEA) chloride, which blocks K+ channels, inhibited motility initiation in a dose-dependent manner. Changes in the membrane potential of sperm have been followed using the fluorescent dye diO-C6-(3) whose fluorescence in sperm suspension changes markedly with changes in sperm membrane potential. During motility initiation, there was a fall in fluorescence of the dye due to increased partition into sperm cells. This observation may indicate a hyperpolarization of the sperm membrane during motility initiation. It was concluded that sperm motility initiation is associated with a complex ionic event. Na+ enters sperm cells in exchange with H+ and K+. This change in the permeability of the sperm membrane to ions is reflected by a change in the sperm membrane potential.