Characterization of a high-conductance,voltage-dependent cation channel from the plasma membrane of rye roots in planar lipid bilayers

Plasma-membrane vesicles were purified by aqueous-polymer two-phase partitioning of a microsomal membrane fraction from rye (Secale cereale L.) roots and incorporated into planar 1-palmitoyl-2-oleoyl phosphatidylethanolamine bilayers. A high-conductance cation channel (a maxi cation channel) was characterized from single-channel electrical recordings. The channel was incorporated into the bilayer with its cytoplasmic surface facing the trans compartment and voltages were referenced cis with respect to trans. The channel was permeable to both monovalent and divalent cations. The unitary conductance was 451 pS in symmetrical 100 mM KCl and 213 pS in symmetrical 100 mM BaCl₂. The permeability ratio P_KP_Ba was 1.002.56. Unitary conductances declined in the order K⁺Rb⁺>Cs⁺>Na⁺> Li⁺ (monovalent cations) and Ba²⁺>Sr²⁺>Ca²⁺> Mg²⁺>Co²⁺>Mn²⁺ (divalent cations). The relative permeabilities of monovalent cations mirrored their conductivity sequence, whereas the permeabilities of all divalent cations were similar. The maxi cation channel showed complex kinetics, exhibiting both voltage- and time-dependent inactivation and voltage-dependent gating. The voltage dependence of the kinetics shifted in parallel with changes in the reversal potential of the channel. In symmetrical 100 mM KCl, following a voltage step from zero to the test voltage, the channel inactivated and the active-channel lifetime ( _i) shortened exponentially as the test voltage was increased. The channel always opened immediately upon depolarization to zero volts, indicating that inactivation of the channel did not result from the loss of any intrinsic factor. The probability of finding an active channel in the open state (P₀) exhibited a bell-shaped relationship with membrane potential. At voltages between -40 and 80 mV, P₀ exceeded 0.99, but p₀ declined abruptly at more extreme voltages. Under ionic conditions which approximated physiological conditions, in the presence of 100 mM KCl on the trans (cytoplasmic) side and 1 mM KCl plus 2 mM CaCl₂ on the cis (extracellular) side, the reversal potential was 15.6 mV and the kinetics approximated those observed in symmetrical 100 mM KCl. Thus, the channel would open upon depolarization of the plasma membrane in vivo. If the channel functioned physiologically as a Ca²⁺ channel it might be involved in intracellular signalling: the channel could open in response to a variety of environmental, developmental and pathological stimuli which depolarize the plasma membrane, allowing Ca²⁺ into the cytoplasm and thereby initiating a physiological response.Abbreviations E_K Nernst (equilibrium) potential for potassium - E_rev zero-current (reversal) potential - I/V current/voltage - _c apparent mean lifetime of the activated-channel closed state - _i apparent mean lifetime of the activated channel following a voltage step from zero volts - ₀ apparent mean lifetime of the activated-channel open state - PE 1-palmitoyl-2-oleoyl phosphatidylethonlamine - P₀ probability of finding the activated channel in an open state - TEA⁺ tetraethylammonium This work was supported by the Agriculture and Food Research Council and by a grant from the Science and Engineering Research Council Membrane Initiative (GR/F 33971) to Prof. E.A.C. MacRobbie (University of Cambridge, UK).     

Characterization of a voltage-dependent cation-channel from the plasma membrane of rye (Secale cereale L.) roots in planar lipid bilayers

Plasma-membrane vesicles were purified by aqueous-polymer two-phase partitioning of a microsomal membrane fraction from rye (Secale cereale L.) roots and incorporated into planar 1-palmitoyl-2-oleoyl phosphatidylethanolamine bilayers. A voltage-dependent cation-channel became incorporated into the bilayer with its cytoplasmic surface facing the trans compartment (which was grounded) and was characterized from single-channel recordings. The channel had a unitary conductance of 174 pS in symmetrical 100 mM KCl. The selectivity towards monovalent cations, determined from both conductance measurements in symmetrical 100 mM cation chloride and from permeability ratios in the presence of (cis: trans) 100 mM cation chloride: 100 mM KCl, was CsKRb>Na. The channel was also permeable to both Ba²⁺ and Ca²⁺. Although the unitary conductances in symmetrical 100 mM BaCl₂ and CaCl₂ were only 46 pS and 40 pS, respectively, the apparent permeabilities of the divalent cations relative to K⁺ were greater than expected (P_KP_BaP_Ca, 1.001.662.60). This anomaly might result from competition between divalent and monovalent cations for an intrapore binding site. The channel exhibited complex gating kinetics, which were modulated in response to changes in the zero-current (reversal) potential of the channel (E_rev). In symmetrical 100 mM KCl the channel inactivated at positive voltages greater than 100 mV and the activated channel exhibited a high probability of being in an open-state (P₀>0.90) at all voltages between ±100 mV. Channel P₀ approximated unity at voltages in the range -60 to +20 mV. As more-negative voltages were applied, P₀ decreased gradually. In contrast, as more positive voltages were applied, P₀ decreased initially to a local minimum (approaching P₀=0.90), then increased as the voltage was further increased before declining at extreme positive voltages. Under physiologically relevant ionic conditions, with 100 mM KCl plus contaminant Ca²⁺ on the trans (cytoplasmic) side and 1 mM KCl plus 2 mM CaCl₂ on the cis (extracellular) side of the channel, E_rev was 25.2 mV and the relative permeability P_Ca/P_K was 7.45. Thus, the channel would be activated by plasma-membrane depolarization in vivo and facilitate Ca²⁺ influx and net K⁺ efflux. A role in intracellular signalling is proposed for this channel. It could open in response to stimuli which depolarize the plasma membrane, allowing Ca²⁺ into the cytoplasm and, thereby, initiating a cellular response. The outward K⁺ current would act to stabilize the trans-plasma membrane voltage, preventing excessive depolarization during Ca²⁺ influx.Abbreviations and Symbols E_K Nernst (equilibrium) potential for potassium ions - E_rev zero-current (reversal) potential of the channel - _c apparent mean lifetime of the activated-channel closed-state - _o apparent mean lifetime of the activated-channel open-state - PE dephosphatidylethanolamine - P_O probability of finding the activated channel in an open-state This work was supported by the Agriculture and Food Research Council and by a grant from the Science and Engineering Research Council Membrane Initiative (GR/F 33971) to Prof. E.A.C. MacRobbie (University of Cambridge).     

The K+ channel in the plasma membrane of rye roots has a multiple ion residency pore

The permeation of K⁺ and Na⁺ through the pore of a K⁺ channel from the plasma membrane of rye roots was studied in planar 1-palmitoyl-2-oleoyl phosphatidylethanolamine bilayers. The pore contains at least two ion-binding sites which can be occupied simultaneously. This was indicated by: (i) biphasic relationships with increasing cation concentration of both channel conductance at the zero-current (reversal) potential of the channel (E _rev) and unitary-current at a specified voltage and (ii) a decline in E _rev in the presence of equimolar Na⁺ (cis):K⁺ (trans) as the cation concentration was increased. To determine the spatial characteristics and energy profiles for K⁺ and Na⁺ permeation, unitary-current/ voltage data for the channel were fitted to a three energy-barrier, two ion-binding site (3B2S) model. The model allowed for simultaneous occupancy of binding sites and ionic repulsion within the pore, as well as surface potential effects. Results suggested that energy peaks and energy wells (ion binding sites) were situated asymmetrically within the electrical distance of the pore, the trans energy-well being closer to the center of the pore than its cis counterpart; that the energy profile for K⁺ permeation differed significantly from that of Na⁺ in having a higher cis energy peak and a deeper cis energy well; that cations repelled each other within the pore and that vestibule surface charge was negligible. The model successfully simulated various aspects of K⁺ and Na⁺ permeation including: (i) the complexities in current rectification over a wide range of contrasting ionic conditions; (ii) the biphasic relationships with increasing cation concentration of both channel conductance at E _rev and unitary-current at a specified voltage; (iii) the decline in E _rev in equimolar Na⁺ (cis):K⁺ (trans) as cation concentrations were increased and (iv) the complex relationships between mole fraction and E _rev at total cation concentrations of 100 and 300 mm.We thank Prof. O. Alvarez (Universidad de Chile, Santiago, Chile) for supplying the computer program AJUSTE and Prof. D. Sanders (University of York, UK), Prof. D. Gradmann and Dr. G. Thiel (University of Göttingen, Germany) for stimulating ideas. This work was supported by the Agricultural and Food Research Council.     

Potassium channels

The atomic structures of K+ channels have added a new dimension to our understanding of K+ channel function. I will briefly review how structures have influenced our views on ion conduction, gating of the pore, and voltage sensing.     

Lepidopteran-specific crystal toxins from Bacillus thuringiensis form cation- and anion-selective channels in planar lipid bilayers

Summary Previous studies in our laboratory have shown that CryIC, a lepidopteran-specific toxin from Bacillus thuringiensis, triggers calcium and chloride channel activity in SF-9 cells (Spodoptera frugiperda, fall armyworm). Chloride currents were also observed in SF-9 membrane patches upon addition of CryIC toxin to the cytoplasmic side of the membrane. In the present study the ability of activated CryIC toxin to form channels was investigated in a receptor-free, artificial phospholipid membrane system. We demonstrate that this toxin can partition in planar lipid bilayers and form ion-selective channels with a large range of conductances. These channels display complex activity patterns, often possess subconducting states and are selective to either anions or cations. These properties appeared to be pH dependent. At pH 9.5, cation-selective channels of 100 to 200 pS were most frequently observed. Among the channels recorded at pH 6.0, a 25–35 pS anion-selective channel was often seen at pH 6.0, with permeation and kinetic properties similar to those of the channels previously observed in cultured lepidopteran cells under comparable pH environment and for the same CryIC toxin doses. We conclude that insertion of CryIC toxin in SF-9 cell native membranes and in artificial planar phospholipid bilayers may result from an identical lipid-protein interaction mechanism.The assistance of A. Mazza and G.A.R. Mealing is gratefully acknowledged. The trypsin-activated, HPLC-purified CryIC toxin isolated from B. thuringiensis var. entomocidus crystal was a kind gift from M. Pusztai, Institute for Biological Sciences, NRC, Ottawa.     

A chloride-permeable channel from Phaseolus vulgaris roots incorporated into planar lipid bilayers

Ion channels are key participants in physiological processes of plant cells. Here, we report the first characterization of a high conductance, Cl(-)-permeable channel, present in enriched fractions of plasma membranes of bean root cells. The Cl(-) channel was incorporated into planar lipid bilayers and its activity was recorded under voltage clamp conditions. The channel is voltage-dependent, excludes the passage of cations (K(+), Na(+), and Ca(2+)), and is inhibited by micromolar concentrations of Zn(2+). The Cl(-) conductance here characterized represents a previously undescribed channel of plant cells.     

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^–.     

Outer membrane protein A of Escherichia coli forms temperature-sensitive channels in planar lipid bilayers

The temperature dependence of single-channel conductance and open probability for outer membrane protein A (OmpA) of Escherichia coli were examined in planar lipid bilayers. OmpA formed two interconvertible conductance states, small channels, 36-140 pS, between 15 and 37 degrees C, and large channels, 115-373 pS, between 21 and 39 degrees C. Increasing temperatures had strong effects on open probabilities and on the ratio of large to small channels, particularly between 22 and 34 degrees C, which effected sharp increases in average conductance. The data infer that OmpA is a flexible temperature-sensitive protein that exists as a small pore structure at lower temperatures, but refolds into a large pore at higher temperatures.     

Characterization of a voltage-dependent Ca2+-selective channel from wheat roots

A new mechanism for calcium flux in wheat (Triticum aestivum L.) root cells has been characterized. Membrane vesicles were enriched in plasma membrane using aqueous-polymer two-phase partitioning and incorporated into artificial lipid bilayers, allowing characterization of single channels under voltage-clamp conditions. Membrane marker activities showed 74% and 83% purity in plasma membrane when expressed in terms of membrane area and activity, respectively. Since membrane vesicles obtained by aqueous-polymer two-phase partitioning yield a population of membrane vesicles of regular orientation, and vesicle fusion into planar lipid bilayers occurs in a defined manner, the orientation of the channel upon vesicle incorporation could be determined. Thus ionic activities and potentials could be controlled appropriately on what we propose to be the cytosolic (trans) and extracellular (cis) faces of the channel. The unitary conductance in symmetrical 1 mM CaCl₂ was 27±0.4 (pS). The correlation between the theoretical and observed reversal potentials in asymmetrical conditions showed that the channel was highly selective for Ca²⁺ over Cl^–. Experiments simulating physiological ionic conditions showed a P_Ca ²⁺/P_K ⁺ of 17–26, decreasing in this range as the extracellular CaCl₂ concentration increased from 0.1 to 1 mM. The channel was also permeable to the essential nutrient ions, Mg²⁺ and Mn²⁺. The open probability of the channel was strongly dependent on the membrane potential. Inactivation with time was observed at more negative membrane potentials, and was immediately reversed as soon as the membrane potential was decreased. At membrane potentials more negative than -130mV, the channel remained mainly in the closed state, suggesting that in vivo the channel would remain largely closed and would open only upon membrane depolarization. The channel was blocked by micromolar concentrations of extracellular verapamil and trivalent cations, Al³⁺ being the most effective of those tested. Exposure of the cytosolic and extracellular sides of the channel to inositol 1,4,5-trisphosphate had no effect on the channel activity. We suggest a plasma-membrane origin for the channel as shown by biochemical and electrophysiological evidence, and discuss possible physiological roles of this channel, both in Ca²⁺ uptake into roots and in signal transduction.Abbreviations IP₃ 1,4,5-trisphosphate - PM plasma membrane We wish to thank Dr. Christa Niemietz, Dr. Robert Reid and Prof. Andrew Smith for valuable discussions. This work was supported by the Australian Research Council and an OPRS award to M.P.     

A high conductance cationic channel from Phaseolus vulgaris roots incorporated into planar lipid bilayers

A previously undescribed plasma membrane cation channel from Phaseolus vulgaris bean roots was studied after its incorporation into planar lipid bilayers. The channel allows the passage of monovalent cations excluding the flux of both anions (Cl-) and divalent cations (Ca2+). The channel presents a high ( approximately 213 pS) conductance in (300 mM Kcis+)/ (150 mMKtrans+) conditions. The probability of opening (Po) is low at all the tested voltages, but it increases significantly at trans-negative potentials. Permeability ratios (Pcation/PK+) under bi-ionic conditions follow the sequence: K+ (1.0)>NH4+ (0.86)>Na+ (0.78). Under the same conditions, the conductance ratios (gamma cation/gamma K+) follow the sequence: NH4+ (1.1) > or = K+ (1.0)>Na+ (0.80). The low probability of opening exhibited by the channel upon its incorporation into a lipid bilayer makes it a candidate to regulation by (and therefore participation in) cellular signalling networks.     

Conduction and blocking properties of a predominantly anion-selective channel from human platelet surface membrane reconstituted into planar phospholipid bilayers

We have investigated the basic properties of a predominantly anion-selective channel derived from highly purified human platelet surface membrane. Single channels have been reconstituted into planar phospholipid bilayers by fusion of membrane vesicles and recorded under voltage-clamp conditions. The channel is found to have the following properties: (i) Channel activity occurs in bursts of openings separated by long closed periods. (ii) The current-voltage relationship is nonlinear. Channel current is seen to rectify, with less current flowing at positive than at negative voltages. Rectification may be due to asymmetric block by HEPES/Tris buffers. In 450 mM KCl, 5 mM HEPES/Tris, pH 7.2, the single channel conductance at -40 mV is approximately 160 pS and at +40 mV is approximately 90 pS. (iii) The conductance-concentration relationship follows a simple saturation curve. Half maximal conductance is achieved at a concentration of approximately 1000 mM KCl, and the curve saturates at a conductance of approximately 500 pS. (iv) Reversal potentials interpreted in terms of the Goldman-Hodgkin-Katz equation indicate a Cl: K permeability ratio of 4:1. (v) The channel accepts all of the halides as well as a number of other anions. The following sequence of relative anion permeabilities (in the presence of K+) is obtained: F- less than acetate- less than gluconate- less than Cl- less than Br- less than I- less than NO3- less tha SCN-.(vi) Cations as large as TEA+ are permeant. (vii) Current through the channel is blocked in the presence of DIDS, SITS and ATP, but not by Zn2+.     

Aminoglycoside blockade of Ca2+-activated K+ channel from rat brain synaptosomal membranes incorporated into planar bilayers

Summary Ca²⁺-activated K⁺ channels from rat brain synaptosomal membranes were incorporated into planar lipid bilayers, and the effects of aminoglycoside antibiotics on the single channel conductance (258±13 pS at 100mm K⁺) were investigated. Aminoglycosides reduced the single channel conductance from the cis (cytoplasmic) side in a dose- and voltage-dependent manner. Voltage dependence of the blockade indicated an interaction between positively charged amino residues of aminoglycoside antibiotics and a binding site located within the electric field of the ion-conducting pathway. The order of blocking potency was consistent with that of the number of amino residues of aminoglycosides (neomycin (6)>dibekacin (5)>ribostamycin (4)=kanamycin (4)), while the electrical distance (z=0.46–0.49) of the binding site kept almost constant for each drug. Thesezs were almost the same with those (0.46–0.51) of alkyldiamine blockers with two amino residues (total net charge of +2) and approximately twice of those (0.25–0.26) of alkylmonoamine blockers (total net charge of +1). Assuming that amino residues of aminoglycosides and alkylamines shared the same binding site located at 25% voltage drop from the cytoplasmic surface of the channel, the site would have to be at least large enough to accommodate one diamino sugar residue of the aminoglycoside in order to simultaneously interact with two positively charged amino groups. Dose- and voltage-dependent blockade of the channel by gallamine, an extremely bulky trivalent organic cation, supported the picture that the channel has a wide mouth on the cytoplasmic side and its pore region, where voltage drop occurs, may also be quite wide and nonselective, suddenly tapering to a constriction where most charged cations block the channel by occluding the K⁺-conducting pathway.     

Cytoplasmic calcium affects the gating of potassium channels in the plasma membrane ofChara corallina: a whole-cell study using calcium-channel effectors

The action of a wide range of drugs effective on Ca²⁺ channels in animal tissues has been measured on Ca²⁺ channels open during the action potential of the giant-celled green alga,Chara corallina. Of the organic effectors used, only the 1,4-dihydropyridines were found to inhibit reversibly Ca²⁺ influx, including, unexpectedly, Bay K 8644 and both isomers of 202–791. Methoxyverapamil (D-600), diltiazem, and the diphenylbutylpiperidines, fluspirilene and pimozide were found not to affect the Ca²⁺ influx. Conversely, bepridil greatly and irreversibly stimulated Ca²⁺ influx, and with time, stopped cytoplasmic streaming (which is sensitive to increases in cytoplasmic Ca²⁺). By apparently altering the cytoplasmic Ca²⁺ levels with various drugs, it was found that (with the exception of the inorganic cation, La³⁺) treatments likely to lead to an increase in cytoplasmic Ca²⁺ levels caused an increase in the rate of closure of the K⁺ channels. Similarly, treatments likely to lead to a decrease in cytoplasmic Ca²⁺ decreased the rate of K⁺ channel closure. The main effect of bepridil on the K⁺ channels was to increase the rate of voltage-dependent channel closure. The same effect was obtained upon increasing the external concentration of Ca²⁺, but it is likely that this was due to effects on the external face of the K⁺ channel. Addition of any of the 1,4-dihydropyridines had the opposite effect on the K⁺ channels, slowing the rate of channel closure. They sometimes also reduced K⁺ conductance, but this could well be a direct effect on the K⁺ channel; high concentrations (50 to 100 μM) of bepridil also reduced K⁺ conductance. No effect of photon irradiance or of abscisic acid could be consistently shown on the K⁺ channels. These results indicate a control of the gating of K⁺ channels by cytoplasmic Ca²⁺, with increased free Ca²⁺ levels leading to an increased rate of K⁺-channel closure. As well as inhibiting Ca²⁺ channels, it is suggested that La³⁺ acts on a Ca²⁺-binding site of the K⁺ channel, mimicking the effect of Ca²⁺ and increasing the rate of channel closure.     

Reconstitution of a calcium-activated potassium channel in basolateral membranes of rabbit colonocytes into planar lipid bilayers

Summary A highly enriched preparation of basolateral membrane vesicles was isolated from rabbit distal colon surface epithelial cells employing the method described by Wiener, Turnheim and van Os (Weiner, H., Turnheim, K., van Os, C.H. (1989)J. Membrane Biol.110:147–162) and incorporated into planar lipid bilayers. With very few exceptions, the channel activity observed was that of a high conductance, Ca²⁺-activated K⁺ channel. This channel is highly selective for K⁺ over Na⁺ and Cl^–, displays voltage-gating similar to maxi K(Ca) channels found in other cell membranes, and kinetic analyses are consistent with the notion that K⁺ diffusion through the channel involves either the binding of a single K⁺ ion to a site within the channel or single-filling (multi-ion occupancy). Channel activity is inhibited by the venom from the scorpionLeiurus quinquestriatus, Ba²⁺, quinine, and trifluoperazine. The possible role of this channel in the function of these cells is discussed.     

Genes for calcium-permeable channels in the plasma membrane of plant root cells

In plant cells, Ca²⁺ is required for both structural and biophysical roles. In addition, changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) orchestrate responses to developmental and environmental signals. In many instances, [Ca²⁺]_cyt is increased by Ca²⁺ influx across the plasma membrane through ion channels. Although the electrophysiological and biochemical characteristics of Ca²⁺-permeable channels in the plasma membrane of plant cells are well known, genes encoding putative Ca²⁺-permeable channels have only recently been identified. By comparing the tissue expression patterns and electrophysiology of Ca²⁺-permeable channels in the plasma membrane of root cells with those of genes encoding candidate plasma membrane Ca²⁺ channels, the genetic counterparts of specific Ca²⁺-permeable channels can be deduced. Sequence homologies and the physiology of transgenic antisense plants suggest that the Arabidopsis AtTPC1 gene encodes a depolarisation-activated Ca²⁺ channel. Members of the annexin gene family are likely to encode hyperpolarisation-activated Ca²⁺ channels, based on their corresponding occurrence in secretory or elongating root cells, their inhibition by La³⁺ and nifedipine, and their increased activity as [Ca²⁺]_cyt is raised. Based on their electrophysiology and tissue expression patterns, AtSKOR encodes a depolarisation-activated outward-rectifying (Ca²⁺-permeable) K⁺ channel (KORC) in stelar cells and AtGORK is likely to encode a KORC in the plasma membrane of other Arabidopsis root cells. Two candidate gene families, of cyclic-nucleotide gated channels (CNGC) and ionotropic glutamate receptor (GLR) homologues, are proposed as the genetic correlates of voltage-independent cation (VIC) channels.     

Acclimation of potassium influx in rye (Secale cereale) to low root temperatures

The influx of K⁺(⁸⁶Rb⁺) into intact roots of rye (Secale cereale L. cv. Rheidal) exposed to a differential temperature (DT) between the root (8° C) and shoot (20° C) is initially reduced compared with warm-grown (WG) controls with both shoot and root maintained at 20° C. Over a period of 3 d, however, K⁺-influx rates into DT plants are restored to levels similar to or greater than those of the WG controls, the absolute rates of K⁺ influx being strongly dependent upon the shoot/root ratio. Acclimation in DT plants results in a reduction of K⁺ influx into the apical (0–2 cm) region of the seminal root which is associated with a compensatory increase in K⁺ influx into the more mature, basal regions of the root. Values of V _max and apparent K _m for K⁺ influx into DT plants were similar to those for WG plants at assay temperatures of 8° C and 20° C except for an increase in the apparent K _m at 8° C. The influx of K⁺ from solutions containing 0.6 mol·m^-3 K⁺ into both WG and DT plants was found to be linearly related to assay temperature over the range 2–27° C, and the temperature sensitivity of K⁺ influx to be dependent upon shoot/root ratio. At high shoot/root ratios, the ratio of K⁺ influx at 20° C:K⁺ influx at 8° C for WG plants approached a minimum value of 1.9 whereas that for DT plants approached unity indicating that K⁺ influx into DT plants has a large temperature-insensitive component. Additionally, when plants were grown in solutions of low potassium concentration, K⁺ influx into DT plants was consistently greater than that into WG plants, in spite of having a greater root potassium concentration ([K⁺]int). This result indicates some change in the regulation of K⁺ influx by [K⁺]int in plants exposed to low root temperatures. We suggest that K⁺ influx into rye seedlings exposed to low root temperatures is regulated by the increased demand placed on the root system by a proportionally larger shoot and that the acclimation of K⁺ influx to low temperatures may be the result of an increased hydraulic conductivity of the root system.Abbreviations DT differential temperature pretreatment - [K⁺]int root potassium concentration - [K⁺]ext potassium concentration of nutrient medium - WG warm-grown pretreatment     

Involvement of polyamines in the inhibiting effect of injury caused by cutting on K+ uptake through the plasma membrane

The inhibition of K⁺ uptake through the plasma membrane resulting from injury caused by cutting, or from application of polyamines (PAs), has been investigated in root segments of maize (Zea mays L.) and pea (Pisum sativum L.). It was found, for both treatments, that K⁺ uptake recovered if the segments were washed for 2 h. The K⁺ uptake inhibited by cutting and that inhibited by spermidine treatment were stimulated to the same extent by fusicoccin. In addition, there was a correlation between the extent of the recovery of K⁺ uptake caused by washing and the distribution, along the root axis, of both PAs and the activities of enzymes responsible for PA degradation. In apical segments of maize, where the PA content and the activity of the degradative enzyme polyamine oxidase (EC 1.5.3.3) were higher than in the more distal segments, the recovery of K⁺ uptake caused by washing was also higher. On the other hand, the opposite trend was observed in root segments of pea, where the PA content and the activity of the degradative enzyme diamine oxidase (EC 1.4.3.6) were higher in distal segments in which K⁺ uptake was greatly stimulated by washing. The effect of the amine-oxidase inhibitor, aminoguanidine, indicates that the degradation products of PAs are involved in the mechanism of inhibition of K⁺ uptake by PAs. The data also seem to indicate that PAs and their degradation products are responsible for the inhibition of K⁺ uptake occurring as a result of injury sustained by cutting roots into segments.Abbreviations DAO diamine oxidase - FC fusicoccin - PA polyamine - PAO polyamine oxidase - PUT putrescine - SPD spermidine     

Ionic channels in the plasma membrane ofSchizosaccharomyces pombe: Evidence from patch-clamp measurements

Patch-clamp studies of the yeastSchizosaccharomyces pombe reveal that the plasma membrane contains a voltage-gated channel mildly selective for potassium over sodium, lithium, and chloride. The channel exhibits several conductances with a maximum of 153 pS. The channel gates in the region of physiologically relevant voltages, being closed at hyperpolarizing and open at depolarizing voltages. It is not inhibited by tetraethylammonium, quinine, or quinidine applied from the cytoplasmic side of the membrane; similarly, ATP and stretch have no effect. The frequency of its occurrence in patches implies that about 35 channels of this kind are present in the plasma membrane of a single cell.