Ammonia,tetraethylammonium, barium and amiloride induce metamorphosis in the marine hydroid Hydractinia

Summary In Hydractinia metamorphosis from the swimming larval stage to the sessile polyp stage has been found to be inducible by several agents, including Li⁺, K⁺, Cs⁺, Rb⁺, diacylglycerol (DG), tetradecanoyl-phorbol-acetate (TPA) and some other tumour-promoting phorbol esters. Induction is antagonized by ouabain and compounds which are able to increase the internal level of S-adenosylmethionine (SAM). Based on the finding that Hydractinia larvae contain such compounds in a stored form, including N-methylpicolinic acid, N-methylnicotinic acid and N-trimethylglycine, as well as on the results of experiments with antagonists of SAM production and transmethylation, it has been argued that regulation of the internal SAM level plays a key role in the control of metamorphosis. However, it remains to be clarified whether the inducing agents act by decreasing the SAM level or by via different pathways. In the present study, substances chemically related to the substances known to induce or inhibit metamorphosis were tested for their metamorphosis-inducing abilities. Some were found to be effective, including NH₄ ⁺, methylamine, tetraethylammonium ions (TEA⁺), ethanolamine, Ba²⁺, Sr²⁺ and the diuretic, amiloride. It is of particular interest that in many organisms TPA and DG increase cytoplasmic pH while amiloride prevents a rise in pH_i. Several of the substances known to trigger metamorphosis may increase the internal NH₄ ⁺ concentration by hindering the export of the constantly produced NH₄ ⁺ through K⁺ channels or through the Na⁺-H⁺ antiport. Treatment with Cs⁺ for 1 h increases the internal level of NH₄ ⁺. Produced and applied ammonia, as well as applied methylamine and ethanolamine, may act by accepting methyl groups, thus reducing the SAM level.     

Evidence for the involvement of PI-signaling and diacylglycerol second messengers in the initiation of metamorphosis in the hydroid Hydractinia echinata Fleming

Metamorphosis of the planula larvae into polyps does not occur spontaneously but depends on the reception of external trigger stimuli. Artificially, metamorphosis can be initiated by a pulse-type application of Cs⁺ or tumor-promoting phorbol esters (W. A. Müller (1985) Differentiation 29, 216–222). In the present study we examined the putative involvement of the phosphatidylinositol system in signal transduction. Planulae of Hydractinia echinata were preincubated with [³H]-inositol. Upon exposure of the larvae to Cs⁺ the label in inositol trisphosphate (InsP₃) increased twofold as early as 15 sec after addition of Cs⁺. Within the first 60 sec the levels of inositol monophosphate (InsP₁) and inositol bisphosphate (InsP₂) were also elevated compared to the values in nonstimulated larvae. After 1 and 3 hr, respectively, of incubation with Cs⁺, only the label in InsP₂ was increased. When applied to saponin-permeabilized larvae, InsP₃ did not induce metamorphosis. But 1,2-dioctanoyl-rac-glycerol (diC₈) was effective in inducing metamorphosis with a half-maximal effective concentration of 9 μM. The percentage of metamorphosed animals after the application of 5 μM diC₈ (30 mM Cs⁺) was increased by the simultaneous application of 1 μM (0.1 μM) of the diacylglycerol kinase inhibitor R 59022. The results are interpreted as evidence for the involvement of the PI-signaling/diacylglycerol transduction system in the initiation of metamorphosis of planula larvae of H. echinata.     

Metamorphosis ofHydractinia echinata Insights into pattern formation in Hydroids

Summary Hydractinia echinata is a marine, colony-forming coelenterate. Fertilized eggs develop into freely swimming planula larvae, which undergo metamorphosis to a sessile (primary) polyp. Metamorphosis can be triggered by means of certain marine bacteria and by Cs⁺. Half a day after this treatment a larva will have developed into a polyp. The induction of metamorphosis can be prevented by addition of inhibitor I, a substance partially purified from tissue ofHydra. The larvae ofH. echinata also appear to contain this substance. Inhibitor I appliedafter the onset of metamorphosis blocks its continuation as long as it remains in the culture medium. Cs⁺ applied within the same period of time also blocks the continuation of metamorphosis. However, these two agents have opposite effects on the body pattern of the resultant polyps. The experiments indicate that application of Cs⁺ triggers the generation of the pre-pattern. Inhibitor I appears to be a factor of this prepattern. A model is proposed which describes the basic features of head and foot/stolon formation not only forHydractinia but also for other related hydroids.     

Cesium,Na+-K+ pump and pacemaker potential in cardiac purkinje fibers

The mechanisms of the hyperpolarizing and depolarizing actions of cesium were studied in cardiac Purkinje fibers perfused in vitro by means of a microelectrode technique under conditions that modify either the Na⁺-K⁺ pump activity or I_f. Cs⁺ (2 mM) inconsistently increased and then decreased the maximum diastolic potential (MDP); and markedly decreased diastolic depolarization (DD). Increase and decrease in MDP persisted in fibers driven at fast rate (no diastolic interval and no activation of I_f). In quiescent fibers, Cs⁺ caused a transient hyperpolarization during which elicited action potentials were followed by a markedly decreased undershoot and a much reduced DD. In fibers depolarized at the plateau in zero [K⁺]_o (no I_f), Cs⁺ induced a persistent hyperpolarization. In 2 mM [K⁺]_o, Cs⁺ reduced the undershoot and suppressed spontaneous activity by hyperpolarizing and thus preventing the attainment of the threshold. In 7 mM [K⁺]_o, DD and undershoot were smaller and Cs⁺ reduced them. In 7 and 10 mM [K⁺]_o, Cs⁺ caused a small inconsistent hyperpolarization and a net depolarization in quiescent fibers; and decreased MDP in driven fibers. In the presence of strophanthidin, Cs⁺ hyperpolarized less. Increasing [Cs⁺]_o to 4, 8 and 16 mM gradually hyperpolarized less, depolarized more and abolished the undershoot. We conclude that in Purkinje fibers Cs⁺ hyperpolarizes the membrane by stimulating the activity of the electrogenic Na⁺-K⁺ pump (and not by suppressing I_f); and blocks the pacemaker potential by blocking the undershoot, consistent with a Cs⁺ block of a potassium pacemaker current.     

Heat shock as inducer of metamorphosis in marine invertebrates

Summary In most sessile marine invertebrates, metamorphosis is dependent on environmental cues. Here we report that heat stress is capable of inducing metamorphosis in the hydroid Hydractinia echinata. The onset of heat-induced metamorphosis is correlated with the appearance of heat-shock proteins. Larvae treated with the metamorphosis-inducing agents Cs⁺ or NH₄ ⁺ also synthesize heat-shock proteins. In heat-shocked larvae, the internal NH₄ ⁺-concentration increases. This fits the hypothesis that methylation plays a central role in control of metamorphosis. In the tunicate Ciona intestinalis, a heat shock is able to induce metamorphosis too. Offprint requests to: M. Walther     

The voltage-dependent potassium-uptake channel of corn coleoptiles has permeation properties different from other K+ channels

The initial response of coleoptile cells to growth hormones and light is a rapid change in plasma-membrane polarization. We have isolated protoplasts from the cortex of maize (Zea mays L.) coleoptiles to study the electrical properties of their plasma membrane by the patch-clamp techniqueUsing the whole-cell configuration and cell-free membrane patches we could identify an H⁺-ATPase, hyperpolarizing the membrane potential often more negative than -150 mV, and a voltage-dependent, inward-rectifying K⁺ channel (unit conductance 5–7 pS) as the major membrane conductan-ces Potassium currents through this channel named CKC1_in (for Coleoptile K ⁺ Channel inward rectifier) were elicited upon voltage steps negative to -80 mV, characterized by a half-activation potential of -112 mV. The kinetics of activation, well described by a double-exponential process, were strongly dependent on the degree of hyperpolarization and the cytoplasmic Ca²⁺ level. Whereas at nanomolar Ca²⁺ concentrations K⁺ currents increased with a t_1/2=16 ms (at -180 mV), higher calcium levels slowed the activation process about fourto fivefoldUpon changes in the extracellular K⁺ concentration the reversal potential of the K⁺ channel followed the Nernst potential for potassium with a 56-mV shift for a tenfold increaseThe absence of a measurable conductance for Na⁺, Rb⁺, Cs⁺ and a permeability ratio P_{NH 4} ⁺ /P_K+ around 0.25 underlines the high selectivity of CKC1_in for K⁺In contrast to Cs⁺, which at submillimolar concentration blocks the channel in a voltage-dependent manner, Rb⁺, often used as a tracer for K+, does not permeate this type of K⁺ channelThe lack of Rb⁺ permeability is unique with respect to other K⁺ transporters. Therefore, future molecular analysis of CKC1_in, considered as a unique variation of plant inward rectifiers, might help to understand the permeation properties of K⁺ channels in general.Abbreviations CKC1_in Coleoptile K ⁺ Channel inward rectifier - U membrane voltage - I_ss steady-state currents - I_tail tail currents Experiments were conducted in the laboratory of F.G. during the stay of RHas a guest professor sponsored by Special Project RAISA, subproject N2.1, paper N2155.     

Induction of metamorphosis from the larval to the polyp stage is similar in Hydrozoa and a subgroup of Scyphozoa (Cnidaria, Semaeostomeae)

Larvae of cnidarians need an external cue for metamorphosis to start. The larvae of various hydrozoa, in particular of Hydractinia echinata, respond to Cs⁺, Li⁺, NH₄ ⁺ and seawater in which the concentration of Mg²⁺ ions is reduced. They further respond to the phorbolester, tetradecanoyl-phorbol-13-acetate (TPA) and the diacylglycerol (DAG) diC8, which both are argued to stimulate a protein kinase C. The only well-studied scyphozoa, Cassiopea spp., respond differently, i.e. to TPA and diC8 only. We found that larvae of the scyphozoa Aurelia aurita, Chrysaora hysoscella and Cyanea lamarckii respond to all the compounds mentioned. Trigonelline (N-methylnicotinic acid), a metamorphosis inhibitor found in Hydractinia larvae, is assumed to act by delivering a methyl group for transmethylation processes antagonising metamorphosis induction in Chrysaora hysoscella and Cyanea lamarckii. The three species tested are scyphozoa belonging to the subgroup of semaeostomeae, while Cassiopea spp. belong to the rhizostomeae. The results obtained may contribute to the discussion concerning the evolution of cnidarians and may help to clarify whether the way metamorphosis can be induced in rhizostomeae as a whole is different from that in hydrozoa and those scyphozoa belonging to the subgroup semaeostomeae. Electronic Publication     

Potassium channels in the plasmalemma ofChara corallina are multi-ion pores: Voltage-dependent blockade by Cs+ and anomalous permeabilities

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 when the cell was depolarized to the K⁺ equilibrium potential in relatively high external K⁺ concentrations. K⁺ current was reduced by externally added Cs⁺. Cs⁺ mainly inhibited inward K⁺ current, in a strongly voltage-dependent manner; the effective valence of the blocking reaction was often greater than 1, increasing with higher external Cs⁺ concentrations and with lower K⁺ concentrations. This is consistent with the channels being single-file, multi-ion pores. Outward current could also be inhibited by Cs⁺, when external K⁺ concentrations were low relative to Cs⁺ concentrations. As the ratio of K⁺/Tl⁺ was changed (keeping the sum of the two ions equal), both the resting potential and plasmalemma conductance went through minimums; this is the so-called anomalous mole fraction effect, and is consistent with a channel whose pore can be multiply occupied. These effects together strongly suggest that the K⁺ channels found in the plasmalemma ofChara are multi-ion pores.     

Cloning and functional expression in <Emphasis Type="Italic">Saccharomyces cereviae</Emphasis> of a K<Superscript>+</Superscript> transporter,AlHAK, from the graminaceous halophyte, <Emphasis Type="Italic">Aeluropus littoralis</Emphasis>

High-affinity K⁺ transporters play an important role in K⁺ absorption of plants. We isolated a HAK gene from Aeluropus littoralis, a graminaceous halophyte. The amino acid sequence of AlHAK showed high homology with HAK transporters obtained from Oryza sativa (82%) and Hordeum vulgare (82%). When expressed in Saccharomyces cereviae WΔ3, AlHAK performed high-affinity K⁺ uptake with a K_m value of 8 μM, and the growth of transformants was dramatically inhibited by 150 mM Rb⁺ and 150 mM Cs⁺ but less affected by 300 mM Na⁺. AlHAK may thus improve the capacity of plants to maintain a high cytosolic K⁺/Na⁺ ratio at high salinity.     

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.     

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.     

Contrasting roles in ion transport of two K+-channel types in root cells of Arabidopsis thaliana

Plant roots accumulate K⁺ over a range of external concentrations. Root cells have evolved at least two parallel plasma-membrane K⁺ transporters which operate at millimolar and micromolar external [K⁺]: high-affinity K⁺ uptake is energised by symport with H⁺, while low-affinity uptake is assumed to occur via ion channels. To determine the role of ion channels in low-affinity K⁺ uptake, a characterisation of the principal K⁺-selective ion channels in the plasma membrane of Arabidopsis thaliana (L.) Heynh. cv. Columbia roots was undertaken. Two classes of K⁺-selective channels were frequently observed: one inward (IRC) and one outward (ORC) rectifying with unitary conductances of 5 pS, 20 pS (IRCs) and 15 pS (ORC), measured in symmetrical 10 mM KCl. The dominant IRC (5 pS) and ORC (15 pS) were highly cation-selective (P_Cl P_K < 0.025) but less selective amongst monovalent cations (P_NaP_K0.17–0.3). Both the IRC and the ORC were blocked by Ba²⁺, Cs⁺ and tetra-ethyl-ammonium, whereas 4-aminopyridine and quinidine selectively inhibited the ORC. The ORC open probability was steeply voltage-dependent and ORC activation potentials were close to the potassium equilibrium potential (E_K+), enabling ORCs to conduct mainly outward, but occasionally inward, K⁺ current. By contrast, gating of the 5-pS IRC was weakly voltageependent and IRC gating was invariably restricted to membrane potentials more negative than E_K+, ensuring K⁺ transport was always inwardly directed. Studies on channel activity were conducted for a large number of root cells grown at two levels of external [K⁺], one where K⁺ uptake is likely to be principally through channels (6 mM K⁺) and one where it must be energised (100 M K⁺). Shifting growth conditions from high to low K⁺ did not affect single-channel properties such as conductance and selectivity, nor the manifestation of the ORC and 20-pS IRC, but led to enhanced activity of the 5-pS IRC. The enhanced activity of the 5-pS IRC was mirrored by a parallel increase in unidirectional ⁸⁶Rb⁺ influx after low-K⁺ growth, clearly indicating a dominant role of this particular channel in K⁺ uptake at supra millimolar external [K⁺].Abbreviations E_K+ potassium equilibrium potential - E_m membrane potential - HK high [K⁺] - IRC inward rectifying channel - LK low [K⁺] - ORC outward rectifying channel - TEA tetra-ethyl-ammonium Financial support was provided by the Biotechnology and Biological Sciences Research Council (Grant PG87/529) and by the European Union (Framework III, Biotechnology Programme).     

Contribution of a H+ pump in determining the resting potential of neuroblastoma cells

The aim of this work was to examine the effects of changes in external K⁺ concentration (K _o ) around its physiological value, of various K⁺ channels blockers, including internal Cs⁺, of vacuolar H⁺-ATPase inhibitors and of the protonophore CCCP on the resting potential and the voltage-dependent K⁺ current of differentiated neuroblastoma x glioma hybrid NG108-15 cells using the whole-cell patch-clamp technique. The results are as follows: (i) under standard conditions (K _o =5 mm) the membrane potential was –60±1 mV. It was unchanged when K _o was decreased to 1 mm and was depolarized by 4±1 mV when K_o was increased to 10 mm. (ii) Internal Cs⁺ depolarized the membrane by 21±3 mV. (iii) The internal application of the vacuolar H⁺-ATPase inhibitors N-ethylmaleimide (NEM), NO ₃ ^– and bafilomycin A1 (BFA) depolarized the membrane by 15±2, 18±2 and 16±2 mV, respectively, (iv) When NEM or BFA were added to the internal medium containing Cs⁺, the membrane was depolarized by 45±1 and 42±2 mV, respectively. (v) The external application of CCCP induced a transient depolarization followed by a prolonged hyperpolarization. This hyperpolarization was absent in BFA-treated cells. The voltage-dependent K⁺ current was increased at negative voltages and decreased at positive voltages by NEM, BFA and CCCP. Taken together, these results suggest that under physiological conditions, the resting potential of NG108-15 neuroblastoma cells is maintained at negative values by both voltage-dependent K⁺ channels and an electrogenic vacuolar type H⁺-ATPase.This work was supported by a grant from INSERM (CRE 91 0906).     

Stimulation of metamorphosis in Hydractinia echinata involves generation of lysophosphatidylcholine

Summary Whilst the significance of the phosphoinositide cycle in the activation of developmental events by extra-cellular signals is well established, the involvement of the phosphatidylcholine (PC) cycle is a matter just emerging. In the present study, the metabolism of phosphatidylcholine in early metamorphosis of Hydractinia echinata (Coelenterata; Hydrozoa) was investigated by incubation of planula larvae with ³H-choline, extraction of the metabolites and isolation of the metabolites by thin-layer chromatography (TLC). Phosphatidylcholine (PC), lysophosphatidylcholine (LPC), acetylcholine and glycerophosphocholine were the labelled metabolites. Induction of metamorphosis did not stimulate an increased incorporation of choline into PC. In larvae preincubated with ³H-choline to a steady state level of incorporation, a significant transient elevation of the radioactive label in LPC was observed 90 min after addition of metamorphosis stimulating agents. LPC probably derived from PC by the action of a phospholipase A₂ (PLA₂). LPCs from bovine and soybean origin as well as isolated larval LPC did not influence metamorphosis. PLA₂ from bee venom promoted Cs⁺-induced metamorphosis but did not influence phorbol ester-induced metamorphosis. The data suggest that a PLA₂ is activated during metamorphosis. This PLA₂ activation does not occur in those putative receptor cells which receive the primary external inducing stimulus but in the many larval cells which resume proliferation or differentiation in response to a second, internally propagated signal. Offprint requests to: T. Leitz     

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.     

Bradykinin-induced potassium current in cultured bovine aortic endothelial cells

Summary Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca²⁺ concentration, [Ca²⁺] _i , accompanied by an increase in surface membrane K⁺ permeability. In this study, electrophysiological measurement of K⁺ current was combined with⁸⁶Rb⁺ efflux measurements to characterize the K⁺ flux pathway in BAECs. Bradykinin- and Ca²⁺-activated K⁺ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin,noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca²⁺ activation is in the range of 10 to 100nm [Ca²⁺] _i . The whole-cell current measurement show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K⁺ current present in control conditions, an inwardly rectifying K⁺ current, was blocked by Ba²⁺ and was not affected bynoxiustoxin or tetrabutylammonium chloride. Efflux of⁸⁶Rb^– from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and bynoxiustoxin, but not by apamin or furosemide. Thus,⁸⁶Rb⁺ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca²⁺-activated membrane currents. The results confirm that bradykinin-stimulated⁸⁶Rb⁺ efflux occurs via Ca²⁺-activated K⁺ channels. The blocking agents identified may provide a means for interpreting the role of the Ca²⁺-activated K⁺ current in the response of BAECs to bradykinin.     

Calcium-dependent potassium channel inParamecium studied under patch clamp

Summary We have studied a class of Ca _i ²⁺ -dependent K channels in inside-out excised membrane patches fromParamecium under patch clamp. Single channels had a conductance of 72 ±9.0 pS in a solution containing 100mM K⁺. The channels were selective for K⁺ over Rb⁺ with the permeability ratio of 1 0.56. and over Na⁺, Cs⁺ or NH ₄ ⁺ with a ratio 1<0.1. The channel activity was dependent on Ca _i ²⁺ , which was applied to the cytoplasmic side; the Ca _i ²⁺ concentration for the half maximal activation was 2 m. The Hill coefficient for the Ca _i ²⁺ dependence of the channel activity was 2.58, indicating that more than two Ca _i ²⁺ bindings are necessary for full activation. Unlike most Ca _i ²⁺ -dependent K channels in other organisms, the channels inParamecium were slightly more active upon hyperpolarization than upon depolarization. The voltage dependence was fitted to a Boltzmann curve with 41.2 mV pere-fold change in channel activity. While a high Ca _i ²⁺ concentration activated the channels, it also irreversibly reduced the channel activity over time. The decay of channel activity occurred faster at higher Ca _i ²⁺ concentrations. Quaternary ammonium ions suppressed ion passage through the channel; more highly alkylated quaternary ammonium ions were more efficient in blocking. Ba _i ²⁺ and Ca _i ²⁺ were relatively ineffective in blockage. It was concluded that these Ca _i ²⁺ -dependent K channels inParamecium are different from the previously described Ca _i ²⁺ -dependent K channels, and are perhaps of a novel class.     

The influence of pH and external H+ concentration on caesium toxicity and accumulation inEscherichia coli andBacillus subtilis

Summary Toxicity screening ofEscherichia coli NCIB 9484 andBacillus subtilis 007, NCIB 168 and NCIB 1650 has shown Cs⁺ to be the most toxic Group 1 metal cation. However, toxicity and accumulation of Cs⁺ by the bacteria was affected by two main external factors; pH and the presence of other monovalent cations, particularly K⁺. Over the pH range 6–9 bothE. coli andB. subtilis showed increasing sensitivity towards caesium as the pH was raised. The presence of K⁺ and Na⁺ in the laboratory media used lowered caesium toxicity and lowered acumulation of the metal. In order to assess accurately Cs⁺ toxicity towards the bacterial strains it was therefore necessary to define the K⁺:Cs⁺ ratio in the external medium. The minimum inhibitory K⁺:Cs⁺ concentration ratio for theBacillus strains tested was in the range 12–13 whileE. coli had a minimum inhibitory K⁺:Cs⁺ concentration ratio of 16.     

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.     

Potassium channels in plasmalemma ofNitella cells at rest

Summary Cation channels of passive transport in the plasmalemma ofNitella flexilis cells at rest were studied by the voltageclamp technique using microelectrodes. Two types of potassium channels have been identified. They are activated at different voltages: over –100 to –80 mV (D-channels) and below –100 mV (H-channels). The zero-current potential of instantaneous voltage-current curves (IVCC's) for both types of channels shifts by 50 to 55 mV in response to a 10-fold increase of K⁺ concentration in the solution. Ion movement in D-channels follows the free diffusion mechanism; in H-channels the independence principle is violated. The channel selectivity (in the order of decreasing permeability) is: K⁺>Rb⁺>NH ₄ ⁺ >Na⁺Li⁺>Cs⁺>TEA⁺ choline⁺. It has been found that D-channel Cs⁺ block is potential dependent while tetraethylammonium (TEA⁺) blocks H-channels in a potential-independent manner, but H⁺ ions do not affect the inward potassium current of the channels. Two types of potassium channels appear to be located in different parts of the membrane and their entrance parts are of different structure.