A mutation that increases a novel calcium-activated potassium conductance ofParamecium tetraurelia

Summary Under two-electrode voltage clamp, a mutant ofP. tetraurelia, restless (rst/rst), showed a large increase in induced current and an outward tail current when compared to the wildtype cell for hyperpolarizing voltage steps. An increase in the induced and tail currents is also observed for depolarizing voltage steps. The larger current during voltage steps and tail in the mutant were eliminated by the use of CsCl-filled electrodes and tetraethylammonium ion (TEA⁺) in the bath solution, characterizing the lesion as affecting a K⁺ conductance. Ionophoretic injection of ethylene glycol bis-(beta-aminoethyl ether) n,n,n,n-tetraacetic acid (EGTA) to buffer internal Ca²⁺ concentration reduced the increased K⁺ current and tail of therestless cell, indicating Ca²⁺ activation of the K⁺ current. Time course and amplitude of remaining currents after blockage of K⁺ conductances with Cs⁺ and TEA⁺ were similar in wild-type andrestless cells suggesting norestless defect in entry of calcium. The Ca²⁺-activated sodium current was similar in the mutant to that in wild type arguing against a defect in calcium regulation activating the K⁺ channel in therestless cell. We conclude that therestless mutation alters a Ca²⁺-activated potassium conductance other than the one previously described. The multiplicity of Ca²⁺-activated potassium conductances inParamecium is discussed.     

K<Superscript>+</Superscript>-induced Ca<Superscript>2+</Superscript> Conductance Responsible for the Prolonged Backward Swimming in K<Superscript>+</Superscript>-agitated Mutant of <Emphasis Type="Italic">Paramecium caudatum</Emphasis>

The K⁺-agitated (Kag) mutant of Paramecium caudatum shows prolonged backward swimming in K⁺-rich solution. To understand the regulation mechanisms of the ciliary motility in P. caudatum, we examined the membrane electrical properties of the Kag mutant. The duration of the backward swimming of the Kag in K⁺-rich solution was about 10 times longer than that of the wild type. In response to an injection of the outward current, the wild type produced an initial action potential and a subsequent membrane depolarization due to I-R potential drop, while the Kag exhibited repetitive action potentials during the depolarization. Under voltage-clamp conditions, the depolarization-activated transient inward current exhibited by the Kag was slightly smaller than that exhibited by the wild type. In response to an application of K⁺-rich solution, both the wild type and the Kag exhibited a depolarizing afterpotential representing the activation of the K⁺-induced Ca²⁺ conductance. The inactivation time course of the K⁺-induced Ca²⁺ conductance of Kag was about 10 times longer than that of the wild type. This difference corresponds well with the difference in behavioral responses between Kag and wild type to K⁺-rich solution. We conclude that the overreaction of the Kag mutant to the K⁺-rich solution is caused by slowing down of the inactivation of the K⁺-induced Ca²⁺ conductance.     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

Relationship between the F0F1-ATPase and the K+-transport system within the membrane of anaerobically grownEscherichia coli. N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity in mutants with defects in K+-transport

A considerable (2-fold) stimulation of the DCCD-sensitive ATPase activity by K⁺ or Rb⁺, but not by Na⁺, over the range of zero to 100mM was shown in the isolated membranes ofE. coli grown anaerobically in the presence of glucose. This effect was observed only in parent and in thetrkG, but not in thetrkA, trkE, ortrkH mutants. ThetrkG or thetrkH mutant with anunc deletion had a residual ATPase activity not sensitive to DCCD. A stimulation of the DCCD-sensitive ATPase activity by K⁺ was absent in the membranes from bacteria grown anaerobically in the presence of sodium nitrate. Growth of thetrkG, but not of othertrk mutants, in the medium with moderate K⁺ activity did not depend on K⁺ concentration. Under upshock, K⁺ accumulation was essentially higher in thetrkG mutant than in the othertrk mutant. The K⁺-stimulated DCCD-sensitive ATPase activity in the membranes isolated from anaerobically grownE. coli has been shown to depend absolutely on both the F₀F₁ and theTrk system and can be explained by a direct interaction between these transport systems within the membrane of anaerobically grown bacteria with the formation of a single supercomplex functioning as a H⁺-K⁺ pump. ThetrkG gene is most probably not functional in anaerobically grown bacteria.This study was performed at the Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637.     

Ion permeation and rectification in ATP-sensitive channels from insulin-secreting cells (RINm5F): Effects of K+, Na+ and Mg2+

Summary Patch-clamp techniques were used to study the permeability to ions of an ATP-sensitive channel in membranes from the pancreatic B-cell line (RINm5F). With patches in the outside-out configuration, theI-V curves for different Na⁺–K⁺ mixtures in the bath and 140 mM K⁺ in the pipette were almost linear, and crossed the zero-current axis at voltages that indicated a variable permeability ratio. When K⁺ was added symmetrically, the plot of the conductancevs. K⁺ activity exhibited saturation, with aG _max of about 160 pS and a half-maximal activity of 216 mM. TheI-V behavior for different K⁺–Na⁺ mixtures in the bath could be accurately described with a model based on Eyring theory, assuming two sites and one-ion occupancy. For K⁺, the dissociation constants (K_K) of the two sites were 290 and 850 mM, the lower value pertaining to the site close to the intracellular medium. In experiments with inside-out patches, both Na⁺ and Mg²⁺, when present in the bath, induced a voltagedependent block of the outward current. Fitting the data with the model suggested that for these ions only one of the two sites binds significantly, the corresponding dissociation constants being (mM): 46 for Na⁺ and 34 for Mg²⁺. Blocking by Na⁺ and Mg²⁺ may account for the low outward current seen in intact cells. This hypothesis is consistent with the observation that such current is further reduced by addition of 2,4-DNP, since metabolism inhibitors are expected to lower the ATP level, thereby liberating Mg²⁺ from the Mg²⁺-ATP complex, as well as inducing accumulation of Na⁺ by decreasing the rate of the Na⁺–K⁺ pump.     

Reduced transmitter release conferred by mutations in theslowpoke-encoded Ca2+-activated K+ channel gene ofDrosophila

Potassium channels control the repolarization of nerve terminals and thus play important roles in the control of synaptic transmission. Here we describe the effects of mutations in theslowpoke gene, which is the structural gene for a calcium activated potassium channel, on transmitter release at the neuromuscular junction inDrosophila melanogaster. Surprisingly, we find that theslowpoke mutant exhibits reduced transmitter release compared to normal. Similarly, theslowpoke mutation significantly suppresses the increased transmitter release conferred either by a mutation inShaker or by application of 4-aminopyridine, which blocks theShaker-encoded potassium channel at theDrosophila nerve terminal. Furthermore, theslowpoke mutation suppresses the striking increase in transmitter release that occurs following application of 4-aminopyridine to theether a go-go mutant. This suppression is most likely the result of a reduction of Ca²⁺ influx into the nerve terminal in theslowpoke mutant. We hypothesize that the effects of theslowpoke mutation are indirect, perhaps resulting from increased Ca²⁺ channel inactivation, decreased Na⁺ or Ca²⁺ channel localization or gene expression, or by increases in the expression or activity of potassium channels distinct fromslowpoke.     

Cloning of two SOS1 transporters from the seagrass <Emphasis Type="Italic">Cymodocea nodosa</Emphasis>. SOS1 transporters from <Emphasis Type="Italic">Cymodocea</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis> mediate potassium uptake in bacteria

Two cDNAs isolated from Cymodocea nodosa, CnSOS1A, and CnSOS1B encode proteins with high-sequence similarities to SOS1 plant transporters. CnSOS1A expressed in a yeast Na⁺-efflux mutant under the control of a constitutive expression promoter mimicked AtSOS1 from Arabidopsis; the wild type cDNA did not improve the growth of the recipient strain in the presence of Na⁺, but a cDNA mutant that expresses a truncated protein suppressed the defect of the yeast mutant. In similar experiments, CnSOS1B was not effective. Conditional expression, under the control of an arabinose responsive promoter, of the CnSOS1A and CnSOS1B cDNAs in an Escherichia coli mutant defective in Na⁺ efflux was toxic, and functional analyses were inconclusive. The same constructs transformed into an E. coli K⁺-uptake mutant revealed that CnSOS1A was also toxic, but that it slightly suppressed defective growth at low K⁺. Truncation in the C-terminal hydrophilic tail of CnSOS1A relieved the toxicity and proved that CnSOS1A was an excellent low-affinity K⁺ and Rb⁺ transporter. CnSOS1B mediated a transient, extremely rapid K⁺ or Rb⁺ influx. Similar tests with AtSOS1 revealed that it was not toxic and that the whole protein exhibited excellent K⁺ and Rb⁺ uptake characteristics in bacteria.     

The isolation and characterization of a mutant allele at a new X-linked locus,mex, affecting NADP+-dependent enzymes inDrosophila melanogaster

The isolation and characterization of mutant alleles in a regulatory gene affecting NADP⁺-dependent enzymes are described. The locus,mex, is at position 26.5 ± 0.74 on the X chromosome ofDrosophila melanogaster. The newly isolated mutant allele,mex ¹, is recessive to either themex allele found in Oregon-R wild-type individuals or that found in thecm v parental stock in which the new mutants were induced. Themex ¹ mutant allele is associated with statistically significant decreases in malic enzyme (ME) specific activity and ME specific immunologically cross-reacting material (ME-CRM) in newly emerged adult males. During this same developmental stage in males, the NADP⁺-dependent isocitrate dehydrogenase specific activity increases to statistically significant levels. Females of themex ¹ mutant strain show statistically significant elevated levels of the pentose phosphate shunt enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Isoelectric focusing and thermolability comparisons of the active ME from mutant and control organisms indicate that the enzyme is the same. Developmental profiles ofmex ¹ and control strains indicate that this mutant allele differentially modulates the levels of ME enzymatic activity and ME-CRM during development. This work was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to M.M.B.     

Electrophysiological evidence suggests a defective Ca2+ control mechanism in a newParamecium mutant

Summary A new mutant ofParamecium tetraurelia, k-shyA, was characterized behaviorally and electrophysiologically. The mutant cell exhibited prolonged backward swimming episodes in response to depolarizing conditions. Electrophysiological comparison of k-shyA with wild type cells under voltage clamp revealed that the properties of three Ca²⁺-regulated currents were altered in the mutant. (i) The voltage-dependent Ca²⁺ current recovered from Ca²⁺-dependent inactivation two- to 10-fold more slowly than wild type. Ca²⁺ current amplitudes were also reduced in the mutant, but could be restored by EGTA injection. (ii) The decay of the Ca²⁺-dependent K⁺ tail current was slower in the mutant. (iii) The decay of the Ca²⁺-dependent Na⁺ tail current was also slower in the mutant. All other membrane properties studied, including the resting membrane potential and resistance and the voltage-sensitive K⁺ currents, were normal in k-shyA. Considered together, these observations are consistent with a defect in the ability of k-shyA to reduce the free intracellular Ca²⁺ concentration following stimulation. The possible targets of the genetic lesion and alternative explanations are discussed. The k-shy mutants may provide a useful tool for molecular and physiological analyses of the regulation of Ca²⁺ metabolism inParamecium.     

Effect of aPMR1 disruption on the processing of heterologous glycoproteins secreted in the yeastSaccharomyces cerevisiae

TheSaccharomyces cerevisiae PMR1 gene encodes a Ca²⁺-ATPase localized in the Golgi. We have investigated the effects ofPMR1 disruption inS. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human α₁-antitrypsin (α₁-AT), human antithrombin III (ATHIII), andAspergillus niger glucose oxidase (GOD). Thepmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite a lower growth rate of thepmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from thepmr1 mutant compared to that of the wild-type strain. Thepmr1 mutant strain secreted α₁-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the wild-type strain. Furthermore, the core-glycosylated forms secreted in thepmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in themnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-α1,3-mannose antibody revealed that GOD secreted in thepmr1 mutant did not have terminal α1,3-linked mannoses unlike those secreted in themnn9 mutant and the wild type strains. The present results indicate that thepmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose outer chains.     

Factors controlling the K+ conductance inChara

Summary Previous current/voltage (I/V) investigations of theChara K⁺ state have been extended by increasing the voltage range (up to +200 mV) through blocking the action potential with La³⁺. A region of negative slope was found in theI/V characteristics at positive PD's, similar to that already observed at PD's more negative than the resting level. These decreases in membrane currents at PD's more negative than –150 mV and at PD's close to 0 or positive are thought to arise from the K⁺ channel closure. Both the negative slope regions could be reversibly abolished by 0.1mm K⁺, 20mm Na⁺, more than 10mm Ca²⁺ or 5mm tetraethylammonium (TEA). The K⁺ channels are therefore blocked by TEA, closed by low [K⁺] _o or high [Ca²⁺] _o and are highly selective to K⁺ over Na⁺. With the K⁺ channels closed, the remainingI/V profile was approximately linear over the interval of 400 mV (suggesting a leakage current), but large rectifying currents were observed at PD's more positive than +50 mV. These currents showed a substantial decrease in high [Ca²⁺] _o , sometimes displayed a slight shift to more positive PD's with increasing [K⁺] _o and were unaffected by TEA or changes in [Na⁺] _o . The slope of the linear part of theI/V profile was steeper in low [K⁺] _o than in TEA or high [Na⁺] _o (indicating participation of K⁺, but not Na⁺, in the leak current). Diethylstilbestrol (DES) was employed to inhibit the proton pump, but it was found that the leakage current and later the K⁺ channels were also strongly affected.     

K outward rectifying channels as targets of phosphatase inhibitor deltamethrin inVicia faba guard cells

The dominant outward rectifier K⁺ currents were examined in protoplasts from Vicia faba guard cells. In whole-cell patch-clamp recordings, we generally observed that the conductance of the K⁺ inward and the outward rectifier gradually decreases with a half time in the order of 2.3 ± 0.7 min. As a consequence of this rundown, a new steady state was achieved which was 90 ± 5 percnt; lower than that obtained at the beginning of the recording. The rundown of the outward rectifier could be greatly reduced by pre-treating protoplasts either with the membrane permeable drug deltamethrin or by perfusing protoplasts with a pipette solution containing 5 μmol/L cyclosporine A. Furthermore, after the rundown, the conductance of the outward rectifier could be partially restored upon addition of 5 μmol/L deltamethrin to the bath medium. Since deltamethrin and cyclosporine A are established inhibitors of the calcium sensitive phosphatase calcineurin, the data argue for a participation of this type of phosphatase in the control of the activity of K⁺ outward rectifier channels in guard cells.     

Inhibition of Mg2+ current by single-gene mutation in Paramecium

Eccentric is a newly-isolated mutant of Paramecium tetraurelia that fails to swim backwards in response to Mg²⁺. In the wild type, this backward swimming results from Mg²⁺ influx via a Mg²⁺-specific ion conductance (I _Mg. Voltage-clamp analysis confirmed that, as suspected, step changes in membrane potential over a physiological range fail to elicit I _Mg from eccentric. Further electrophysiological investigation revealed a number of additional ion-current defects in eccentric: (i) The Ca²⁺ current activated upon depolarization inactivates more slowly in eccentric than in the wild type, and it requires longer to recover from this inactivation. (ii) The Ca²⁺-dependent Na⁺ current deactivates significantly faster in the mutant, (iii) The two K⁺ currents observed upon hyperpolarization are reduced by >60% in eccentric. It is difficult to envision how these varied pleiotropic effects could result from loss of a single ion current. Rather, they suggest that the eccentric mutation affects a global regulatory system. Two plausible hypotheses are discussed.We are grateful to Dr. Yoshiro Saimi for his comments and suggestions on this work, and for the support of the Lucille P. Markey Charitable trust and the National Institutes of Health (GM22714 and GM38646).     

Ca2+-activated K+ conductance of the human red cell membrane: Voltage-dependent Na+ block of outward-going currents

Summary Human red cells were prepared with various cellular Na⁺ and K⁺ concentrations at a constant sum of 156mm. At maximal activation of the K⁺ conductance,g _K(Ca), the net efflux of K⁺ was determined as a function of the cellular Na⁺ and K⁺ concentrations and the membrane potential,V _m , at a fixed [K⁺]_ex of 3.5mm.V _m was only varied from (V _m –E _K)25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988).g _K(Ca) as a function of cellular Na⁺ and K⁺ concentrations atV _m =–40, 0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na⁺. Since the present Ca²⁺-activated K⁺ channels have been shown to be of the multi-ion type, the experimental data from each set of Na⁺ and K⁺ concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na⁺ is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na⁺ concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101mm. Data from a previous study of voltage dependence as a function of the degree of Ca²⁺ activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence ofg _K(Ca) for outward currents at (V _m –E _K)>25 25 mV reflects a voltage-dependent Na⁺ block of the Ca²⁺-activated K⁺ channels.     

Voltage-gated K+ channels in the mouse interleukin 3-dependent cell line,FDC-P2

Summary The electrical properties of a mouse interleukin (IL)-3-dependent cell line, FDC-P2, were examined using the tightseal whole-cell clamp technique. Under current clamp conditions with 140mM K⁺ in the pipette, the cells had a resting potential of –30 mV. Under voltage-clamp conditions, a transient outward current was elicited upon depolarization from a holding potential of –80 mV. The current was activated at potentials more positive than –10 mV and had a delayed-rectifying property. It showed rapid activation and slow inactivation during command steps. The current was abolished by Cs⁺ in the pipette, indicating that K⁺ is the charge carrier. The K⁺ current was suppressed by tetraethylammonium withK _i of <0.1mM and was not affected by scorpion toxin. Recovery from inactivation was steeply voltage dependent: As the holding potential was more hyperpolarized, the recovery became faster. Thus, with a holding potential of –80 mV, the current showed slight use-dependent inactivation, while the current decreased prominently by repetitive depolarization at a holding potential of –40 mV. These properties of the K⁺ current are similar to those of thel-type K⁺ channel current in mature T lymphocytes. The K⁺ current in FDC-P2 cells was dramatically reduced after culture in the IL-3-free medium for 1–2 days. When IL-3 was re-added to the medium, the current was re-expressed. These observations suggest that expression of the K⁺ current depends on extracellular IL-3, and that the current may play some roles in proliferation of these cells.     

Potassium channels and different states ofChara plasmalemma

Summary The current-voltage (I/V) technique was employed to investigate the different electrophysiological states of theChara plasmalemma and their interaction under a range of conditions. In K⁺ state the membrane became very permeable (conductances >20 S m 2) as [K⁺]₀ increased to 10mm. As the cells were then easily damaged by the voltage-clamp procedures, it was difficult to determine the saturation K⁺ conductance. TEA (tetraethylammonium chloride) reversibly blocked the K⁺ channels, but had no effect on theI/V curve of the pump state, indicating that the K⁺ channels were not participating in this state. Acid pH₀ (4.5) diminished the K⁺ conductance, but did not alter the response of the K⁺ channels to change in [K⁺]₀. Alkaline pH₀ (11.0) madeChara resting PD bistable: the PD either stayed near the estimatedE _K and theI/V curve showed a negative conductance region typical of the K⁺ state, or it hyperpolarized and the near-linearI/V profile of the proton-permeable state was observed.     

Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops fromChara australis

Summary Patch-clamp studies of cytoplasmic drops from the charophyteChara australis have previously revealed K⁺ channels combining high conductance (170 pS) with high selectivity for K⁺, which are voltage activated. The cation-selectivity sequence of the channel is shown here to be: K⁺>Rb⁺>NH ₄ ⁺ Na⁺ and Cl^–. Divalent cytosolic ions reduce the K⁺ conductance of this channel and alter its K⁺ gating in a voltage-dependent manner. The order of blocking potency is Ba²⁺>Sr²⁺>Ca²⁺>Mg²⁺. The channel is activated by micromolar cytosolic Ca²⁺, an activation that is found to be only weakly voltage dependent. However, the concentration dependence of calcium activation is quite pronounced, having a Hill coefficient of three, equivalent to three bound Ca²⁺ needed to open the channel. The possible role of the Ca²⁺-activated K⁺ channel in the tonoplast ofChara is discussed.     

Blockade of potassium channels in the plasmalemma ofChara corallina by tetraethylammonium,Ba2+, Na+ and Cs+

Summary The outer membranes of plant cells contain channels which are highly selective for K⁺. In the giant-celled green algaChara corallina, K⁺ currents in the plasmalemma were measured during the action potential and when the cell was depolarized to the K⁺ equilibrium potential in high external K⁺ concentrations. Currents in both conditions were reduced by externally added tetraethylammonium (TEA⁺), Ba²⁺, Na⁺ and Cs⁺. In contrast to inhibition by TEA⁺, the latter three ions inhibited inward K⁺ current in a voltage-dependent manner, and reduced inward current more than outward. Ba²⁺ and Na⁺ also appeared to inhibit outward current in a strongly voltage-dependent manner. The blockade by Cs⁺ is studied in more detail in the following paper. TEA⁺ inhibited both inward and outward currents in a largely voltage-independent manner, with an apparentK _D of about 0.7 to 1.1mm, increasing with increasing external K⁺. All inhibitors reduced current towards a similar linear leak, suggesting an insensitivity of the background leak inChara to these various K⁺ channel inhibitors. The selectivity of the channel to various monovalent cations varied depending on the method of measurement, suggesting that ion movement through the K⁺-selective channel may not be independent.     

Current-voltage relationships of a sodium-sensitive potassium channel in the tonoplast ofChara corallina

Summary The membrane of mechanically prepared vesicles ofChara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1mm ATP to the bath medium induced a membrane current of about 3.2 mA·m^–2 creating a voltage across the tonoplast of about –7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K⁺-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K⁺ with limiting currents of about +15 and –30 pA, respectively, as measured in symmetric 250mm KCl solutions. The channel is virtually impermeable to Na⁺ and Cl^–. However, addition of Na⁺ decreases the K⁺ currents. TheI–V relationships of the open channel as measured at various K⁺ concentrations with or without Na⁺ added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data.     

NhaK,a novel monovalent cation/H<Superscript>+</Superscript> antiporter of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Four Na⁺/H⁺ antiporters, Mrp, TetA(L), NhaC, and MleN have so far been described in Bacillus subtilis 168. We identified an additional Na⁺/H⁺ antiporter, YvgP, from B. subtilis that exhibits homology to the cation: proton antiporter-1 (CPA-1) family. The yvgP-dependent complementation observed in a Na⁺(Ca²⁺)/H⁺ antiporter-defective Escherichia coli mutant (KNabc) suggested that YvgP effluxed Na⁺ and Li⁺. In addition, effects of yvgP expression on a K⁺ uptake-defective mutant of E. coli indicated that YvgP also supported K⁺ efflux. In a fluorescence-based assay of everted membrane vesicles prepared from E. coli KNabc transformants, YvgP-dependent Na⁺ (K⁺, Li⁺, Rb⁺)/H⁺ antiport activity was demonstrated. Na⁺ (K⁺, Li⁺)/H⁺ activity was higher at pH 8.5 than at pH 7.5. Mg²⁺, Ca²⁺ and Mn²⁺ did not serve as substrates but they inhibited YvgP antiport activities. Studies of yvgP expression in B. subtilis, using a reporter gene fusion, showed a significant constitutive level of expression that was highest in stationary phase, increasing as stationary phase progressed. In addition, the expression level was significantly increased in the presence of added K⁺ and Na⁺.