Potassium channels in Eremosphaera viridis

The dependence of the membrane potential of Eremosphaera viridis on different external concentrations of potassium, sodium, calcium, and protons was compared with the diffusion potential measured in the dark and in the presence of NaN₃. In contrast to some other algae, the membrane potential in the light as well as in the dark seemed to be predominantly determined by the calculated diffusion potential and less by an electrogenic pump which, however, seemed to be involved at potassium concentrations >1 mol·m^-3 and at higher pH_os (>pH 6). Furthermore, some characteristics of an action-potential-like response (CAP) triggered by light-off, and independent of the membrane-potential threshold value, were determined. The CAP had a delay period of 5.4 s and needed 4.5 s for polarization to a plateau. On average, the plateau held for 8.8 s and the CAP lasted 37.7 s. The peak amplitudes of CAP (P _AP) exactly followed the Nernst potential of potassium. Other cations like sodium, calcium and protons did not appreciably affect the peak amplitudes of CAP. From these and other results it can be assumed that the CAP is caused by a temporary opening of potassium channels in the plasma membrane of Eremosphaera (Köhler et al., 1983, Planta 159, 165–171). The release of a CAP by light-off has been partly explained by the participation of a transient increase of proton concentration in the cytoplasm. It was possible to trigger a CAP by external pH changes and by the addition of sodium acetate, thus supporting the hypothesis that a pH decrease in the cytoplasm may be one element of the signal transfer from the photosynthetic system to the potassium channels in the plasmalemma. Calcium also seemed to have an influence on triggering the CAP.Abbreviations and symbols CAP chemical-induced action-potential-like response - E _D calculated diffusion potential (mV) - E _D ^* measured diffusion potential (mV) - E _K potassium equilibrium potential (mV) - E _m membrane potential (mV) - P _AP peak of action potential (mV) Part II will appear in Planta, Vol. 167, No. 1, 1986     

The biotin-dependent sodium ion pump glutaconyl-CoA decarboxylase from Fusobacterium nucleatum (subsp. nucleatum)

Membrane preparations of Fusobacterium nucleatum grown on glutamate contain glutaconyl-CoA decarboxylase at a high specific activity (13.8 nkat/mg protein). The enzyme was solubilized with 2% Triton X-100 in 0.5M NaCl and purified 63-fold to a specific activity of 870 nkat/mg by affinity chromatography on monomeric avidin-Sepharose. The activity of the decarboxylase was strictly dependent on Na⁺ (K _m=3 mM) and was stimulated up to 3-fold by phospholipids. The glutaconyl-CoA decarboxylases from the gram-positive bacteria Acidaminococcus fermentans and Clostridium symbiosum have a lower apparent K _m for Na⁺ (1 mM) and were not stimulated by phospholipids. In addition only the fusobacterial decarboxylase required sodium ion for stability and was inactivated by potassium ion. By incorporation of this purified enzyme into phospholipids an electrogenic sodium ion pump was reconstituted. The enzyme consists of four subunits, (m=65 kDa), (33 kDa), (19 kDa), and (16 kDa) with the functions of a carboxy transferase (), a carboxy lyase ( and probably ) and a biotin carrier (). The subunits are very similar to those of the glutaconyl-CoA decarboxylases from the gram-positive bacteria. With an antiserum directed against the decarboxylase from A. fermentans the - and the biotin containing subunits of the three decarboxylases and that from Peptostreptoccus asaccharolyticus could be detected on Western blots.     

Changes in membrane potential and resistance caused by transient increase of potassium conductance in the unicellular green alga Eremosphaera viridis

The electrophysiological membrane parameters of the unicellular green alga Eremosphaera viridis were determined using an improved computer-supported single-microelectrode technique. These cells developed an average membrane potential of-150 mV in the light and a specific resistance of 1 Ω m² with an external potassium concentration of 1.1 mM and pH 5.5. In the dark, many cells showed a less polarized potential of 30–40 mV and a smaller membrane resistance. At potassium concentrations in the external medium higher than 1 mM, the membrane potential strongly depends on the external potassium content apart from a small electrogenic component. At concentrations lower than 1 mM K⁺, a dependence of the membrane potential upon external potassium concentrations could not be verified. Inserting the internal ion activities in the Goldmann equation shows that, in this range, the proton conductance seems to be predominant over the potassium conductance. Transient changes in the membrane potential and in the membrane resistance were observed after switching off the light, after addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea or N,N′-dicyclohexylcarbodiimide, after a sudden decrease in temperature, and after current pulses. These changes resemble the action potentials (AP) found in other plant cells (Chara, Acetabularia). On average, the AP has a delay period of 5.1 s and a duration of 43.8 s showing a sudden decrease and a slower regeneration. The voltage peak during an AP followed exactly the Nernst potential of potassium over a range of external potassium concentrations from 5 μM to 0.2 M. This is true for depolarization or hyperpolarization, depending on the external K⁺-concentration. Tetraethylammonium-hydrogensulphate, a rather specific inhibitor of K⁺ channels in nervous cells, suppressed the AP. The correlation of the appearance of the AP with a short-term opening of potassium channels in the membrane of Eremosphaera is discussed.     

Vacuolar ion currents in the primitive green algaEremosphaera viridis: The electrical properties are suggestive of both the Characeae and higher plants

Summary The electrical properties of the vacuolar membrane of the primitive green algaEremosphaera virdis were investigated using the patch-clamp technique. In whole vacuole measurements two types of transport systems with long activation time-constants were identified. The first, showing marked outward rectification, was activated by an increase in the cytosolic calcium concentration. Furthermore, it displayed sensitivity to micromolar concentrations of the anion channel blocker Zn²⁺ and to acidification of the cytosol. In contrast, the second time-activated current component was almost insensitive to changes in cytosolic pH and was blocked by the potassium channel inhibitor TEA. In addition to these slowly activating current components, the vacuolar membrane contained at least two further transport systems, responsible for an instantaneous current. These two current components were distinguished by their different sensitivity to protons, cytosolic calcium, and TEA. Comparing these electrical properties to those observed in vacuoles of higher plants or in cytoplasmic droplets from characean algae, respectively, it seems thatEremosphaera is intermediate, corresponding to the systematic position of this simple green alga.Abbreviations [Ca²⁺]_cyt cytosolic free calcium concentration - EGTA ethyleneglycol-bis(-aminoethylether)N,N,N,N-tetraacetic acid - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] - I electric current - IRC inward rectifying current - MES 2-[N-morpholino]ethanesulfonic acid - ORC outward rectifying current - pH_cyt cytosolic pH - pH_vac vacuolar pH - P_o open probability - P_x permeability coefficient of ion species X - TEA tetraethylammonium chloride - Tris tris[hydroxymethyl]aminomethane - V voltage     

Potassium Channels in Eremosphaera viridis: Modulation of Channel Opening,Conductance and Inhibition*

I/V relationships were performed by the voltage clamp technique in the sphaerical green alga Eremosphaera viridis de Bary. We focused on the course of the transient potential (TP) found in light-off experiments when specific potassium channels open. I/V measurements done during this period show a N-shaped curve. The shape depended on external potassium. TPs could be released by light-off and by addition of barium, strontium, or α-naphthyl phosphate. We calculated the number of specific potassium channels to be between 1750 and 34 825 channels per cell. Barium (1 mol × m^?3) and tetraethylammonium (10 mol × m^?3) inhibited TPs. I/V relationships demonstrated, when N-shaped, that potassium channels start to close at voltages more negative than ?195 mV (0.1 mol × m^?3 K⁺), ?160 mV (1 mol × m^?3 K⁺) and ?148 mV (10 mol × m^?3 K⁺). In the region of ?300 mV conditions similar to those at rest are reached. External sodium suppressed the development of N-shaped I/V relationships and reduced the membrane conductance during a TP between 8 % and 29 %. This indicates an influence of external sodium on potassium channels.     

Transfer of germ plasm from the secondary to the primary gene pool in pennisetum

Summary Germ plasm from the A-genome of Pennisetum purpureum Schum. (AABB) of the secondary gene pool was transferred to cultivated pearl millet (AA) [P. glaucum (L.) R. Br.] by pollinating cytoplasmicnuclear male-sterile (cms) pearl millet with fertile allohexaploid pearl millet x P. purpureum hybrids (AAAABB). Certain allohexaploids used as pollinators on cms pearl millet resulted in 14-chromosome diploid pearl millet progenies. Three types of diploid pearl millet plants were produced in addition to the expected 28-chromosome AAAB-genome plants: (1) cms plants with only the A-genome, (2) cms plants with the A- and A-genomes, and (3) fertile plants with the A- and A-genomes. The latter group has allowed the utilization of genes for fertility restoration, stiff stalk, maturity, height, and morphological characteristics from the A-genome of P. purpureum in the pearl millet breeding program. Production of monoploid gametes by the allohexaploids appeared to be genetically controlled.     

The Ca2+-induced leak current in Xenopus oocytes is indeed mediated through a Cl− channel

Defolliculated oocytes of Xenopus laevis responded to removal of external divalent cations with large depolarizations and, when voltage clamped, with huge currents. Single channel analysis revealed a Cl^– channel with a slope conductance of about 90 pS at positive membrane potentials with at least four substates. Single channel amplitudes and mean channel currents had a reversal potential of approximately –15 mV as predicted by the Nernst equation for a channel perfectly selective for Cl^–. Readdition of Ca²⁺ immediately inactivated the channel and restored the former membrane potential or clamp current. The inward currents were mediated by a Ca²⁺ inactivated Cl^– channel (CaIC). The inhibitory potency of Ca²⁺ was a function of the external Ca²⁺ concentration with a half maximal blocker concentration of about 20 m.These channels were inhibited by the Cl^– channel blockers flufenamic acid, niflumic acid and diphenylamine-2-carboxylate (DPC). In contrast, 4,4-acetamido-4-isothiocyanatostilbene-2,2-disulfonicacid (SITS), another Cl^– channel blocker, led to activation of this Cl^– channel. Like other Cl^– channels, the CaIC was activated by cytosolic cAMP. Extracellular ATP inhibited the channel while ADP was without any effect. Injection of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activating phorbol ester, stimulated the Cl^– current. Cytochalasin D, an actin filament disrupting compound, reversibly decreased the clamp current demonstrating an influence of the cytoskeleton.The results indicate that removal of divalent cations activates Cl^– channels in Xenopus oocytes which share several features with Cl^– channels of the CLC family. The former so-called leak current of oocytes under divalent cation-free conditions is nothing else than an activation of Cl^– channels.The microelectrode measurements are part of the PhD thesis of K. Liebold; the patch clamp contributions are part of the PhD thesis of F.W. Reifarth. This study was supported by the Deutsche Forschungsgemeinschaft (We1858/2-l) and by Sonderforschungsbereich 249.     

Genetic and environmental factors in the resistance of Drosophila subobscura adults to high temperature shock

Summary We have carried out two equivalent selection experiments to increase and decrease heat shock resistance of Drosophila subobscura adults, using an indirect selection method that avoids excessive consanguinity. Heat shock was 33±0.5 °C at saturation humidity. Control lines showed a rapid change of the physiological trait as a consequence of laboratory culture conditions, expressed as a decrease both in heat shock resistance and in the initial population variability for heat shock resistance. Thus, this reduction of variability seems to consist in the loss of those combinations of genes that confer high resistance to heat shock. After eight generations of selection, the selected lines were differentiated from their respective control lines, and the selection response obtained was similar in resistant and sensitive lines. Differences in survival of progeny of reciprocal crosses between selected lines suggest that inheritance of heat resistance may depend in part on the origin of egg cytoplasm.     

Development of shoots and roots in anther-derived callus of Azadirachta indica A. Juss.—a medicinal tree

Callus originated in microsporangial wall layers and connective tissues of anthers containing uninucleate microspores on Nitsch's or Murashige and Skoog's medium supplemented with growth regulators. A higher percentage of cultures (43) produced callus on Nitsch's medium containing 10 M indole-3-acetic acid + 1 M 6-benzyladenine. After 13–15 weeks, green nodular structures and prominent roots developed in 25% of the cultures on Murashige and Skoog's medium + 10 M -naphthaleneacetic acid + 1 M kinetin. Multiple shoots were induced in this anther-derived callus when subcultured on Murashige and Skoog's medium augmented with 4.44 M 6-benzyladenine + 0.53 M -naphthaleneacetic acid along with 18.75 M polyvinylpyrrolidone. The excised shoots formed roots after subculturing on Murashige and Skoog's medium + 4.90 M indole-3-butyric acid + 18.75 M polyvinylpyrrolidone, thus developing complete plantlets. Examination of callusing anthers also revealed two- to multi-celled pollen masses with intact exine.Abbreviations BA 6-benzyladenine - CW coconut water - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - HCl hydrochloric acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KMnO₄ potassium permanganate - MS Murashige & Skoog's medium - NAA -naphthaleneacetic acid - NB Nitsch's medium - PVP polyvinylpyrrolidone     

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.     

KATP channels of mouse skeletal muscle: mechanism of channel blockage by AMP-PNP

Single ATP-sensitive potassium channels (K _ATP channels) were studied in inside-out membrane patches excised from mouse skeletal muscle. Channel blockage by the non-hydrolysable ATP analogue AMP-PNP was investigated in the absence or presence of 1 mM MgCl₂ with K⁺-rich solutions bathing the internal membrane surface. Currents through single. K _ATP channels were recorded at –40 and +40 mV AMP-PNP (5 to 500 M; Li salt) reduced the open-probability p_o of K _ATP channels and decreased the single-channel currents at high nucleotide concentrations by approximately 10%. Half maximal reduction of p_o at –40 mV was observed at nucleotide concentrations of 29 M in the absence and of 39 M in the presence of Mg²⁺. The steepness of the AMP-PNP concentration-response curves was strongly affected by Mg²⁺, the Hill coefficients of the curves were 0.6 in the absence and 1.6 in the presence of 1 mM MgCl₂. The efficacies of channel blockage by AMP-PNP at –40 and ⁺40 mV were not significantly different. The results indicate that a K _ATP channel can bind more divalent Mg²⁺-complexes of AMP-PNP than trivalent protonated forms of the nucleotide and that channel blockage is hardly affected by the membrane electric field. To estimate the contribution of lithium ions to the observed results, we studied the effects of LiCl (0.8 to 10 mM) in the Mg²⁺-free solution on the single channel current i. At a Li⁺ concentration of 10 mM, i was hardly affected at –40 mV but reduced by a factor of 0.75 at +40 mV. The results are interpreted by a fast, voltage-dependent blockage of K _ATP channels by internal Li⁺ ions. Correspondence to: B. Neumcke     

Potassium-39 NMR of K+ interaction with the gramicidin channel and NMR-derived conductance ratios for Na+, K+ and Rb+

Summary A potassium-39 NMR study of potassium ion interaction with the gramicidin transmembrane channel in phospholipid bilayers at high ion activity is reported which allows determination of a weak binding constant, K _b ^w 8.3/m, and an off-rate constant for the weak site,k _off ^w 2.6×10⁷/sec. These values are interpreted with the aid of additional NMR data as the binding constant for formation of the doubly occupied channel state and the rate constant for an ion leaving the doubly occupied state. Considering the singly occupied channel state for the potassium ion to be electrically silent at 1 molar ion activity, as with the sodium ion, the single-channel conductance for 100 mV and 30°C calculated to be 29 pS, and using the same approximation with previous NMR results on the sodium and rubidium ions, reasonable conductance ratios were calculated. Further experimental estimates of the other three constants with the experimental location of binding sites and Eyring rate theory to introduce voltage dependence allowed a more complete calculation of the two-site channel. The single-channel conductance for potassium ion is calculated to be 24 pS at 1m activity and 26 pS at 0.6m activity, which compares for diphytanoyl phosphatidylcholine membranes to an experimental most probable single-channel conductance of 25 pS and a mean channel conductance of 20 pS. The calculated conductance ratios using NMR-derived constants were (K)/(Na)=2.0 and (Rb)/(Na)=4.3. These results are close to the experimental values and provide further basis for the use of NMR of quadrupolar ions to provide information on the ionic mechanism of channel transport.     

β-Oxidation of fatty acids in algae: Localization of thiolase and acyl-CoA oxidizing enzymes in three different organisms

In the algae Mougeotia, Bumilleriopsis and Eremosphaera, recently shown to possess the enzymes hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and enoyl-CoA hydratase (EC 4.2.1.17), the presence of thiolase (EC 2.3.1.9) and acyl-CoA-oxidizing enzymes can also be demonstrated, indicating that -oxidation of fatty acids is possible in these organisms. The compartmentation of enzymes is different in the various algae. In Mougeotia, both thiolase and the acyl-CoA-oxidizing enzyme are located exclusively in the peroxisomes. The latter enzyme was found to be an oxidase using molecular oxygen as an electron acceptor. On the other hand, in Bumilleriopsis all enzymes of the fatty-acid -oxidation pathway tested are constituents only of the mitochondria, and acyl-CoA is oxidized by a dehydrogenase incapable of reducing oxygen. Finally, in Eremosphaera thiolase and acyl-CoA-oxidizing enzymes were found in the peroxisomes as well as in the mitochondria. In the peroxisomes, oxidation of acyl-CoA is catalyzed by an oxidase, whereas the corresponding enzyme in the mitochondria is a dehydrogenase. The acyl-CoA oxidases/dehydrogenases of the three algae differ not only by their capability for oxidation of acyl-CoA of different chain lengths but also with regard to their K_m values and substrate specificities. Indications were obtained that the oxygen is reduced to water rather than to H₂O₂ by the algal acyl-CoA oxidases. When cells of Eremosphaera were cultured with hypolipodemic substances in the growth medium the activities of the peroxisomal enzymes, but not those of the mitochondrial enzymes of the fatty-acid -oxidation pathway, were increased by a factor of two to three.Abbreviations DPIP 2,6-dichlorophenol indophenol - INT p-iodonitrotetrazolium violet - MEHP monoethylhexylphthalate     

Induction,polarity and spatial control of cytokinesis in some abnormal subsidiary cell mother cells ofZea mays

Summary Cytokinesis in the subsidiary cell mother cells (SMCs) ofZea mays leaves grown in the presence of 5 mM of caffeine solution is usually partially inhibited. A continuous wall strip, resembling a portion of the subsidiary cell (SC) wall, is laid down in the preprophase microtubule band (PMB) cortical zone. Sometimes, the incomplete SC (SC) wall grows centripetally in the absence of a phragmoplast and the gap becomes smaller or closes. The SC nucleus escapes through the SC wall gap into the larger SMC compartment and may fuse with the other nucleus.The aberrant SMCs (a-SMCs) pass through another division cycle, reattempting to produce a SC. A typical PMB is found in the SC space, in the site of the previous PMB. Moreover, in some preprophase SMCs, the cytoplasm adjacent to the SC wall is traversed by a small number of microtubules. The preprophase nuclei are partly or totally separated from the PMB by the perforated SC wall and may lie far from the latter.Usually, one mitotic spindle is assembled. The cycling paired polarized nuclei appear to synchronize and their chromosomes line up together on a single metaphase plate. Although the mitotic spindle axis is diversely oriented, one of its poles tends to be stabilized in the proximity of the SC wall gap. These divisions separate abnormal cells. Most or all the cell plate edges fuse with wall regions far from the PMB cortical zone. However, when some of them approach the SC wall strips, they are attracted and intersect their rims. In rare occasions the cell plate, invading the SC space is guided by the PMB cortical zone to create a SC-like curved wall portion, in absence of a daughter nucleus.Observations show that the cell plate arrangement in redividing aberrant SMCs is not subjected to a strict spatial control. The disorder of polarization sequence generated by the SC wall ring and especially the perturbation of the spatial (and functional?) relationship between PMB-PMB cortical zone and the nucleus—mitotic spindle is a causal factor of the variable cell plate arrangements.     

Ion channels in the membrane ofChara inflata

Summary Voltage-clamped steps in the electric potential difference (PD) across the membrane in cells of the green alga,Chara inflata, cause voltage- and time-dependent current flows, interpreted to arise from opening and closing of various types of ion channel in the membrane. With cells in the light, these channels are normally closed, and the resting PD is probably determined by the operation of an H⁺ efflux pump. Positive steps in PD from the resting level often caused the opening of K⁺ channels with sigmoid kinetics. The channels began to show opening when the PD–120 mV for an external concentration of K⁺ of 1.0mm. Return of the PD to the resting level caused closing of the channels with complex kinetics. Various treatments of the cell could cause these K⁺ channels to open, and remain open continuously, with the PD then lying closer to the Nernst PD for K⁺. The K⁺ channels have been identified by the blocking effects of TEA⁺. Another group of channels, probably Cl^– and Ca²⁺ associated with the action potential open when the PD is stepped to values less negative than –50 mV. Negative steps from the resting PD cause the slow opening, with a time course of seconds, of yet another type of channel, probably Cl^–.     

Potassium accumulation in growing Methanobacterium thermoautotrophicum and its relation to the electrochemical proton gradient

Cultures of Methanobacterium thermoautotrophicum (Marburg) growing on media low in potassium accumulated the cation up to a maximal concentration gradient ([K⁺]_{intracellular}/[K⁺]_{extracellular}) of approximately 50,000-fold. Under these conditions, the membrane potential was determined by measuring the equilibrium distribution of the lipophilic cation (¹⁴C) tetraphenylphosphonium (TPP⁺). This cation was accumulated by the cells 350-to 1,000-fold corresponding to a membrane potential (inside negative) of 170–200 mV. The pH gradient, as measured by equilibrium distribution of the weak acid, benzoic acid, was found to be lower than 0.1 pH units (extracellular pH=6.8). The addition of valinomycin (0.5–1 nmol/mg cells) to the culture reduced the maximal concentration gradient of potassium from 50,000-to approximately 500-fold, without changing the membrane potential. After dissipation of the membrane potential by the addition of ¹²C-TTP⁺ (2 mol/mg cells) or tetrachlorosalicylanilide (3 nmol/mg cells), a rapid and complete efflux of potassium was observed.These data indicate that potassium accumulation in the absence of valinomycin is not in equilibrium with the membrane potential. It is concluded that at low extracellular K⁺ concentrations potassium is not accumulated by M. thermoautotrophicum via an electrogenic uniport mechanism.Non-common abbreviations TPP⁺ Tetra phenylphosphonium bromide - DTE Dithioerythritol - TCS 3,5,3,4-Tetrachlorosalycylanilide     

Sulphate influx in wheat and barley roots becomes more sensitive to specific protein-binding reagents when plants are sulphate-deficient

When young wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) plants were deprived of an external sulphate supply (-S plants), the capacity of their roots to absorb sulphate, but not phosphate or potassium, increased rapidly (derepression) so that after 3–5 d it was more than tenfold that of sulphate-sufficient plants (+S plants). This increased capacity was lost rapidly (repression) over a 24-h period when the sulphate supply was restored. There was little effect on the uptake of L-methionine during de-repression of the sulphate-transport system, but S input from methionine during a 24-h pretreatment repressed sulphate influx in both+S and-S plants.Sulphate influx of both+S and-S plants was inhibited by pretreating roots for 1 h with 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) at concentrations > 0.1 mol · m^-3. This inhibition was substantially reversed by washing for 1 h in DIDS-free medium before measuring influx. Longer-term pretreatment of roots with 0.1 mol·m^-3 DIDS delayed de-repression of the sulphatetransport system in-S plants but had no influence on+S plants in 3 d.The sulphydryl-binding reagent, n-ethylmaleimide, was a very potent inhibitor of sulphate influx in-S roots, but was much less inhibitory in +S roots. Its effects were essentially irreversible and were proportionately the same at all sulphate concentrations within the range of operation of the high-affinity sulphate-transport system. Inhibition of influx was 85–96% by 300 s pretreatment by 0.3 mol·m^-3 n-ethylmaleimide. No protection of the transport system could be observed by including up to 50 mol·m^-3 sulphate in the n-ethylmaleimide pre-treatment solution. A similar differential sensitivity of-S and+S plants was seen with p-chloromercuriphenyl sulphonic acid.The arginyl-binding reagent, phenylglyoxal, supplied to roots at 0.25 or 1 mol·m^-3 strongly inhibited influx in-S wheat plants (by up to 95%) but reduced influx by only one-half in+S plants. The inhibition of sulphate influx in-S plants was much greater than that of phosphate influx and could not be prevented by relatively high (100 mol·m^-3 sulphate concentrations accompanying phenylglyoxal treatment. Effects of phenylglyoxal pretreatment were unchanged for at least 30 min after its removal from the solution but thereafter the capacity for sulphate influx was restored. The amount of new carrier appearing in-S roots was far greater than in+S roots over a 24-h period.The results indicate that, in the de-repressed state, the sulphate transporter is more sensitive to reagents binding sulphydryl and arginyl residues. This suggests a number of strategies for identifying the proteins involved in sulphate transport.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NEM n-ethylmaleimide - PCMBS p-chloromercuriphenyl sulphonic acid     

Different photoreceptors within the same retina express unique combinations of potassium channels

Single electrode current and voltage clamp recordings in Calliphora, and whole-cell voltage clamp recordings in Drosophila were used to characterise the voltage-gated K channels in both major classes of photoreceptors, R7/8 (long visual fibres, LVFs) and R1-6 (short visual fibres, SVFs). R7/8 were identified by their unique spectral properties, ca. 3–4 fold higher input resistances and 3–4 fold lower cell capacitance. In Calliphora SVFs possess both fast and slow activating delayed rectifier potassium conductances. Drosophila SVFs possess a slowly inactivating delayed rectifier (I_Ks), a very rapidly inactivating A channel encoded by the Shaker gene (I_A), and, in a minority of cells, a third K conductance with intermediate kinetics (I_Kf). In both specs the LVFs lack the slowest component, but exhibit the faster K conductance(s) with properties indistinguishable from those in the SVFs. These findings add to established evidence demonstrating the significant role played by potassium channels in tuning the photoreceptor membrane. The results also suggest that R1-6 photoreceptors and R7/8 form inputs to visual subsystems tuned to different temporal frequencies.Abbreviations LVF long visual fibre - SVF short visual fibre - R1-6 retinular cells 1 to 6 inclusive - R7/8 retinular cell 7 and 8 - I _A rapidly inactivating A type potassium conductance; channel coded by Shaker gene - I _Kf rapidly activating, slowly inactivating delayed rectifier-like potassium conductance - I _Ks slowly activating, slowly inactivating delayed rectifier-like potassium conductance - I _KDs slowly activating delayed rectifier potassium conductance - I _KDf rapidly activating delayed rectifier potassium conductance     

Channel-mediated K+ flux in barley aleurone protoplasts

Gibberellic acid (GA₃) stimulates K⁺ efflux from the barley (Hordeum vulgare L. cv. Himalaya) aleurone. We investigated the mechanism of K⁺ flux across the plasma membrane of aleurone protoplasts using patch-clamp techniques. Potassium-ion currents, measured over the entire surface of the protoplast plasma membrane, were induced when the electrochemical gradient for K⁺ was inward (into the cytoplasm). The magnitude and voltage-dependence of this inward current were the same in protoplasts treated with GA₃ and in control protoplasts (no GA₃). Inward currents activated by negative shifts in the membrane potential (E_M) from the Nernst potential for K⁺ (E_K) showed membrane conductance to be a function of the electrochemical gradient (i.e. E_M-E_K). Single-channel influx currents of K⁺ were recorded in small patches of the plasma membrane. These channels had a single-channel conductance of 5–10 pS with 100 mM K⁺ on the inside and 10 mM K⁺ on the outside of the plasma membrane. Single-channel currents, like whole-cell currents, were the same in protoplasts treated with GA₃ and control protoplasts. Voltage-gated efflux currents were found only in protoplasts tha thad been incubated without GA₃. We conclude that K⁺ influx in the aleurone is mediated by channels and these membrane proteins are not greatly effected by GA₃.Abbreviations and symbols F_K Nernst potential for K⁺ - E_M membrane potential - E_rev reversal potential - GA₃ gibberellic acid - K_i concentration of K⁺ inside the cell - K_o concentration of K⁺ outside the cell - R gas constant - S conductance (siemens) - T temperature (^oK) - _i ionic activity coefficient for internal (cytoplasmic) solution - _o ionic activity coefficient for external medium