Separation of K+-and Cl --selective ion channels from rye roots on a continuous sucrose density gradient

Microsomal membranes from rye (Secale cereale L.) roots wereseparated by isopycnic sucrose density gradient centrifugation.The ion channels present in gradient fractions were assayedby reconstitution into planar 1-palmitoyl-2-oleoyl phosphatidylethanolaminebilayers (PLB) and the distributions of ion channel activitieswere compared with membrane markerenzyme activities. A numberof ion channel activities were observed and could be distinguishedon the combined bases of their conductance, selectivity, kineticsand pharmacology. A voltage-dependent maxi (498 pS) cation-channel,a voltage-dependent 199-pS cationchannel, 48-pS and 18-pS K⁺channels, and a 148-pS Cl^– channel (all unitary conductancesdetermined in asymmetrical cis trans 325:100mM KCl) colocalizedwith the plasma membrane marker-enzyme, vanadatesensitive ATPase.A weakly K ⁺-selective (108 pS) channel, a 1249-pS cation-channeland a 98-pS K ⁺ channel colocalized with the tonoplast markerenzyme,nitrate-sensitive ATPase. A 706-pS K⁺ channel colocalized withthe expected distribution of intact plastids and a 38-pS Cl^–channel colocalized with either plastid or ER membranes. Themembrane location of several other channels including a hypervoltage-sensitivemaxi (497 pS) cation-channel, a 270-pS K⁺ channel, an 8-pS K⁺channel and a 4-pS K⁺ channel was equivocal, but they were tentativelyassigned to the Golgi. Thus, the plasma membrane and tonoplastorigin of ion channels previously characterized following theincorporation of plasma membrane prepared by aqueous-polymertwo-phase partitioning or tonoplast derived from isolated vacuolesinto PLB was confirmed and the ion channel complement of previouslyunassayed membranes was defined. This demonstrates the usefulnessof PLB in identifying and characterizing ion channels from plantcell membranes, in particular, those of membranes which areinaccessible to patch-clamp electrodes. Key words: Chloride (Cl^–) channel, potassium (K⁺) channel, planar lipid bilayer, root, rye, Secale cerealeL.     

Cation-permeable vacuolar ion channels in the moss Physcomitrella patens: a patch-clamp study

Patch-clamp studies carried out on the tonoplast of the moss Physcomitrella patens point to existence of two types of cation-selective ion channels: slowly activated (SV channels), and fast-activated potassium-selective channels. Slowly and instantaneously saturating currents were observed in the whole-vacuole recordings made in the symmetrical KCl concentration and in the presence of Ca²⁺ on both sides of the tonoplast. The reversal potential obtained at the KCl gradient (10 mM on the cytoplasmic side and 100 mM in the vacuole lumen) was close to the reversal potential for K⁺ (E _K), indicating K⁺ selectivity. Recordings in cytoplasm-out patches revealed two distinct channel populations differing in conductance: 91.6 ± 0.9 pS (n = 14) at ?80 mV and 44.7 ± 0.7 pS (n = 14) at +80 mV. When NaCl was used instead of KCl, clear slow vacuolar SV channel activity was observed both in whole-vacuole and cytoplasm-out membrane patches. There were no instantaneously saturating currents, which points to impermeability of fast-activated potassium channels to Na⁺ and K⁺ selectivity. In the symmetrical concentration of NaCl on both sides of the tonoplast, currents have been measured exclusively at positive voltages indicating Na⁺ influx to the vacuole. Recordings with different concentrations of cytoplasmic and vacuolar Ca²⁺ revealed that SV channel activity was regulated by both cytoplasmic and vacuolar calcium. While cytoplasmic Ca²⁺ activated SV channels, vacuolar Ca²⁺ inhibited their activity. Dependence of fast-activated potassium channels on the cytoplasmic Ca²⁺ was also determined. These channels were active even without Ca²⁺ (2 mM EGTA in the cytosol and the vacuole lumen), although their open probability significantly increased at 0.1 μM Ca²⁺ on the cytoplasmic side. Apart from monovalent cations (K⁺ and Na⁺), SV channels were permeable to divalent cations (Ca²⁺ and Mg²⁺). Both monovalent and divalent cations passed through the channels in the same direction—from the cytoplasm to the vacuole. The identity of the vacuolar ion channels in Physcomitrella and ion channels already characterised in different plants is discussed.     

Thermodynamics of Malate Transport across the Tonoplast of Leaf Cells of CAM Plants

The electrochemical potential difference for each dissociationstate of malic acid across the tonoplast of leaf cells was examinedin two CAM plants, Graptopetalum paraguayense and Kalanchoëdaigremontiana. The concentration of malic acid in each dissociationstate was estimated from an analysis of pH and concentrationsof ionic species that included calcium, malate and isocitrate.The vacuoles contained 30–40 mM isocitrate and 50–70mM calcium in G. paraguayense, and 20–30 mM isocitrateand 70–100 mM calcium in K. daigremontiana. For the calculationof the pattern of dissociation of malic acid, the formationof chelates of calcium with malate and isocitrate, which havedifferent stability constants depending on the dissociationof the acids, were also taken into consideration. The vacuolarconcentrations of the divalently dissociated form of malic acid(mal^2– were 4–7 mM and 1-3 mM in G. paraguayenseand in K. daigremontiana, respectively. To obtain informationabout the cytoplasmic concentration of malate, the apparentinhibition constant for malate of phosphoenolpyruvate carboxylasewas measured. It was about 330 µM in the dark period and60 µM in the light period. Considering an inside-positivemembrane potential, we conclude that mal^2– can be takenup passively into the vacuole during the dark period and canbe released passively from the vacuole during the light period.Two types of channel (the "SV-type" channel and a novel "MU-type"channel) which we found recently in G. paraguayense [Iwasakiet al. (1992) Plant Physiol. 98: 1494] are probably involvedin the uptake and the release of malate in the diurnal CAM rhythm.The existence of a large pH-buffering capacity due to isocitricacid in the vacuole allows the accumulation of a large amountof malic acid during the diurnal CAM rhythm. (Received February 12, 1992; Accepted July 10, 1992)     

Ca Fluxes and Membrane Potentials in Nitella translucens

The concentrations of Ca have been measured in the flowing cytoplasmand the vacuole of the single cells of Nitella translucens,the cells being immersed in an artificial pond Water (composition:NaCl, 1.0 mM; KCl, 0.1 mM; CaCl₂, 0. mM). In the flowing cytoplasmthe total concentration is 8 mM and in the vacuole 12 mM. Measurementsof the electrical potential differences across the plasmalemmaand tonoplast membranes show that the cytoplasm is at a potentialof —134 mV with respect to the bathing medium and —24mV with respect to the vacuole. An attempt has been made tomeasure the tracer fluxes of Ca and it is shown that the cellsare not in flux equilibrium. The influx is 0.046 µµmoles cm^–2 sec^–1; the efflux was too small to measurewith any degree of accuracy. The observed potential differences across both membranes arecompared with the Nernst potentials for Ca. This analysis showsthat Ca is not in electrochemical equilibrium across eithermembrane and that the driving forces on Ca are directed fromthe bathing medium and the vacuole into the cytoplasm. It issuggested that there is no necessity for a metabolically drivenCa pump at the plasmalemma because the low cytoplasmic Ca contentcould be due to the low permeability of the plasmalemma; theGoldman flux equation gives a value of P_Ca = 4.3x10^–8cm sec^–1. A Ca pump at the tonoplast appears to be necessaryto explain the steep electrochemical potential gradient fromthe vacuole to the cytoplasm. The efflux of Ca from the isolated cell wall has been measured.From these measurements it was possible to estimate the concentrationof indiffusible anions in the Donnan Free Space; the value obtainedwas 0.74 equiv. 1.^–1.     

Patch-Clamp Study on a Ca2+-Regulated K+ Channel in the Tonoplast of the Brackish Characeae Lamprothamnium succinctum

Cytoplasmic drops were prepared from internodal cells of thebrackish Characeae Lamprothamnium succinctum. Applying the patch-clamptechnique to single drops covered with tonoplast, we demonstratedthe presence of Ca²⁺-regulated K⁺ channels in the tonoplast.In a cell-attached mode, the selectivity of such channels forK⁺ was about 50 times that for Na⁺. This channel showed a tendencyto rectify in an outward direction. In the negative region ofthe pipette voltage, the conductance of this channel was 50pS, while it was 100 pS in the positive voltage region. Whenthe pipette voltage was increased above 50 mV, two conductancelevels were found in the cell-attached mode as well as in theexcised patch (cytoplasmic-side-out patch), which was obtainedby pulling the patch pipette from the cytoplasmic drop underconditions of low levels of Ca²⁺. Using the excised patch, wecontrolled the level of Ca²⁺ on the cytoplasmic side of thechannels. At a low level of Ca²⁺ (pCa=8) on the cytoplasmicside, the open frequency was very low and the opening time wasshort. An increase in Ca²⁺ on the cytoplasmic side (pCa = 5)increased both the frequency and the duration of opening. However,the conductance of the channels did not change. This regulationby Ca²⁺ of the K⁺ channels was reversible, that is, additionof EGTA on the cytoplasmic side inactivated the channels. Thepresent study demonstrates a direct action of Ca²⁺ on the K⁺channels. The physiological role of the K⁺ channel in the regulationof turgor in Lamprothamnium is discussed. (Received January 9, 1989; Accepted March 8, 1989)     

Ion channels and ATP-driven pumps involved in ion transport across the tonoplast of sugarbeet vacuoles

The electrical properties of the tonoplast of mature sugarbeet root vacuoles have been studied using the patch-clamp technique. In whole-vacuole recordings, the addition of 5 mM Mg-ATP to the external solution activated a proton-translocating ATPase which produced inward currents of up to 65 pA. Furthermore, we identified a voltage-dependent membrane conductance which activated at hyperpolarized (inside-negative) potentials and decreased at positive potentials. Outside-out membrane patches predominantly contained a channel which showed an increasing probability of opening at potentials more negative than about –20 mV. These channels can account for the macroscopic currents recorded in whole vacuoles. The permeability sequence of the channel for cations and anions was: P_K^staggered+ = P_Na⁺ >P_Ac⁻ >P_NO3⁻ >P_Mal²⁻ >P_Cl⁻. The unit conductance of this channel was about 70 pS in symmetrical 50 mM KCl and 180 pS in symmetrical 200 mM KCl solutions. Another channel type of smaller conductance (15 pS in 50 mM KCl) was also present, but its properties have not yet been studied. The permeability sequence of the nonselective channel corresponds to that found by tracer measurements in vacuole suspensions, implying that the channel studied may present the molecular pathway for the movement of ions across the tonoplast.     

Outward-Rectifying K+ Channels in Stomatal Guard Cell Protoplasts

Ion channels in stomatal guard cell protoplasts from Vicia fabawere examined using the patch-clamp technique. Most ion channelshaving unit conductance ranging between 10 and 30 pS showedclear outward-rectification in symmetrical 50mM KCl. The largeinside-out membranes contained these outward-rectifiers as themajor and relatively stable channels. The channels were K⁺ ion-selective.Kinetic analysis revealed that the channels have three conductancestates: open, closed and inactivated. The rates of transitionto and from the inactivated state were highly voltage-dependent. (Received April 6, 1988; Accepted May 25, 1988)     

Cholesterol-modulated ion channel activity in tonoplast vesicles: a planar lipid bilayer study

The reconstitution of native tonoplast vesicles from Commelinacommunis guard cell protoplasts into cholesterol-free planarlipid bilayers revealed single ion channel activity. An 8.1pS ion channel was found to have a low selectivity for cationsover anions (P_K: P_Cl = 1: 0.83) in asymmetrical conditions (100cis: 10 trans mM KCI). Addition of cholesterol (50 mol%) tothe bilayer induced instantaneous and reversible closure ofthe channel. Key words: Cholesterol, ion channel, planar lipid bilayer, tonoplast     

Inhibition of inward rectifying tonoplast channels by a vacuolar factor: Physiological and kinetic implications

Summary Regulation of ion-channel activity must take place in order to regulate ion transport. In case of tonoplast ion channels, this is possible on both the cytoplasmic and the vacuolar side. Isolated vacuoles of youngVigna unguiculata seedlings show no or hardly any channel activity at tonoplast potentials >80 mV, in the vacuole-attached configuration. When the configuration is changed to an excised patch or whole vacuole, a fast (excised patch) or slow (whole vacuole) increase of inward rectifying channel activity is seen. This increase is accompanied by a shift in the voltage-dependent gating to less hyperpolarized potentials. In the whole vacuole configuration the level of inward current increases and also the activation kinetics changes. Induction of channel activity takes up to 20 min depending on the age of the plants used and the diameter of the vacuole. On the basis of the estimated diffusion velocities, it is hypothesized that a compound with a mol wt of 20,000 to 200,000 is present in vacuoles of young seedlings, which shifts the population of channels to a less voltage-sensitive state.Ecotrans publication no. 27.     

The heterogeneity of ion channels in chromaffin granule membranes

Chromaffin granules are involved in catecholamine synthesis and traffic in the adrenal glands. The transporting membrane proteins of chromaffin granules play an important role in the ion homeostasis of these organelles. In this study, we characterized components of the electrogenic ⁸⁶Rb⁺ flux observed in isolated chromaffin granules. In order to study single channel activity, chromaffin granules from the bovine adrenal medulla were incorporated into planar lipid bilayers. Four types of cationic channel were found, each with a different conductance. The unitary conductances of the potassium channels are 360 ± 10 pS, 220 ± 8 pS, 152 ± 8 pS and 13 ± 3 pS in a gradient of 450/150 mM KCl, pH 7.0. A multiconductance potassium channel with a conductivity of 110 ± 8 pS and 31 ± 4 pS was also found. With the exception of the 13 pS conductance channel, all are activated by depolarizing voltages. One type of chloride channel was also found. It has a unitary conductance of about 250 pS in a gradient of 500/150 mM KCl, pH 7.0.     

Ionic Channels of the Sugar Beet Tonoplast are Regulated by a Multi-ion Single-file Permeation Mechanism

Ionic channels of the sugar beet tonoplast were studied using the patch-clamp technique. At micromolar concentrations of cytosolic calcium, several (at least four) distinct single-channel current levels were routinely identified. On the basis of channel voltage dependence, kinetic properties and conductance of single openings, the largest channel (103 ± 2 pS in symmetric 150 mm KCl) corresponds to the slow vacuolar (SV) channel already identified by Hedrich and Neher (1987). The majority of the whole-vacuole current was ascribed to this time-dependent slow-activating channel elicited by positive vacuolar potentials. The channel of intermediate amplitude (41 ± 1 pS in 150 mm KCl) did not show any voltage dependence and delay in the activation upon the application of voltage steps to both positive and negative transmembrane potentials. Owing to its voltage independence this channel was denominated FV1. The opening probability of the SV-type channel increased by increasing the cytoplasmic calcium concentration, while the activity of the FV1 channel did not increase appreciably by changing the calcium concentration in the range from 6 μm to 1 mm. All the channels identified showed a linear current-voltage characteristic in the range ±100 mV and at least the three most conductive ones displayed potassium selectivity properties. Substitution of potassium with tetramethylammonium (TMA) on the cytosolic side demonstrated that both the SV and FV1 channels are impermeable to TMA influx into the vacuole and support the potassium selectivity properties of these two channels. Moreover, the single channel conductances of all the channels identified increased as a function of the potassium concentration and reached a maximum conductivity at [K⁺] ∼0.5 m. This behavior can be explained by a multi-ion occupancy single-file permeation mechanism. Received: 26 December 1995/Revised: 10 July 1996     

NaCl Uptake in Roots of Zea mays Seedlings: Comparison of Root Pressure Probe and EDX Data

The extent by which salinity affects plant growth depends partlyon the ability of the plant to exclude NaCl. To study the uptakeof NaCl into excised roots of Zea mays L. cv. ‘Tanker’,two different techniques were applied. A root pressure probewas used to record steady state as well as transient valuesof root (xylem) pressure upon exposure of the root to mediacontaining NaCl and KCl as osmotic solutes. In treatments withNaCl, pressure/time responses of the root indicated a significantuptake of NaCl into the xylem. NaCl induced kinetics were completelyreversible when the NaCl solution was replaced by an isosmoticKCl solution. This indicated a passive movement of Na⁺-saltsacross the root cylinder. Root samples were taken at differenttimes of exposure to NaCl and prepared for X-ray microanalysis(EDX analysis). Radial profiles of ion concentrations (Na⁺,K⁺, Cl^–) were measured in cell vacuoles and xylem vesselsalong the root axis. Na⁺ appeared rapidly in mature xylem (earlymetaxylem) and living xylem (late metaxylem) before it was detectablein vacuoles of the root cortex. EDX results confirmed that thekinetics observed by the pressure probe technique correspondedmainly to an influx of Na⁺-salts into early metaxylem. In latemetaxylem, the uptake of Na⁺ was associated with a decline ofK⁺. The Na⁺/K⁺ exchange indicated a mechanism to reduce sodiumfrom the transpiration stream. Ion localization, ion transport, maize, root pressure, salinity, water relations, X-ray microanalysis, Zea mays     

TREK-2 (K2P10.1) and TRESK (K2P18.1) are major background K+ channels in dorsal root ganglion neurons

Dorsal root ganglion (DRG) neurons express mRNAs for many two-pore domain K⁺ (K_2P) channels that behave as background K⁺ channels. To identify functional background K⁺ channels in DRG neurons, we examined the properties of single-channel openings from cell-attached and inside-out patches from the cell bodies of DRG neurons. We found seven types of K⁺ channels, with single-channel conductance ranging from 14 to 120 pS in 150 mM KCl bath solution. Four of these K⁺ channels showed biophysical and pharmacological properties similar to TRESK (14 pS), TREK-1 (112 pS), TREK-2 (50 pS), and TRAAK (73 pS), which are members of the K_2P channel family. The molecular identity of the three other K⁺ channels could not be determined, as they showed low channel activity and were observed infrequently. Of the four K_2P channels, the TRESK-like (14 pS) K⁺ channel was most active at 24°C. At 37°C, the 50-pS (TREK-2 like) channel was the most active and contributed the most (69%) to the resting K⁺ current, followed by the TRESK-like 14-pS (16%), TREK-1-like 112-pS (12%), and TRAAK-like 73-pS (3%) channels. In DRG neurons, mRNAs of all four K_2P channels, as well as those of TASK-1 and TASK-3, were expressed, as judged by RT-PCR analysis. Our results show that TREKs and TRESK together contribute >95% of the background K⁺ conductance of DRG neurons at 37°C. As TREKs and TRESK are targets of modulation by receptor agonists, they are likely to play an active role in the regulation of excitability in DRG neurons. two-pore domain K⁺ channel; conductance; excitability     

Salt Tolerance in the Succulent, Coastal Halophyte, Sarcocornia natalensis

The effects of 0, 50, 100, 200, 300, 400 and 500 mol m^–3NaCl on growth and ion accumulation in the succulent, coastalhalophyte Sarcocornia natalensis (Bunge ex Ung.-Sternb.) A.J. Scott were investigated. Increase in salinity from 0 to 300 mol m^–3 NaCl stimulatedproduction of fresh, dry, and organic dry mass, increased succulenceand shifted resource allocation from roots to shoots. Growthwas optimal at 300 mol m^–3 and decreased with furtherincrease in salinity. Water contributed to a large proportion of the increase in freshmass. Inorganic ions, especially Na⁺ and Cl– contributedsubstantially to the dry mass. At 300 mol m^–3 NaCl inorganicions contributed to 37% of total dry mass and NaCl concentrationin the shoots was 482 mol m^–3. Expressed sap osmotic potentialsdecreased from –2.10 to –3.95 MPa as salinity increasedfrom 0 to 300 mol m^–3 NaCl. Massive accumulation of inorganicions, especially Na⁺ and Cl^–, accounted for 86% of theosmotic adjustment at 300 mol m^–3 NaCl. Salinity treatments decreased the concentrations of K+ in shoots.Plant Na+ :K+ ratios increased steadily with salinity and reacheda maximum of 16.6 at 400 mol m3 NaCl. It is suggested that the exceptional salt tolerance of S. natalensisis achieved by massive inorganic ion accumulation which providessufficient solutes for osmoregulation, increased water fluxand turgor-induced growth. Key words: Sarcocornia natalensis, salt tolerance, halophyte     

Ion-channels reconstituted into lipid bilayer from human sperm plasma membrane

Ion environment and ionic fluxes through membrane are thought to be important in the spermatozoa's maturation, capacitation, and the initiating process of gamete interaction. In this work, the membrane proteins isolated from human sperm plasma membrane were reconstituted into planar lipid bilayers via fusion, and the ion channels activities were observed under voltage clamp mode. In cis 200 // trans 100 mM KCl solution, a TEA-sensitive cation-selective channel with a unit conductance of 40 pS was recorded. In a gradient of 200//100 mM NaCl solutions, a Na⁺-selective channel with a unit conductance of 26 pS was recorded. In both cases, reversal potential was about −18 mV, which is close to the predicated value of a perfect Nernst K⁺ or Na⁺ electrode. In 50//10 mM CaCl₂ solution, a cation channel activity with a unit conductance of 40 pS and reversal potential of about −20 mV was usually observed. In 200//100 mM NMDG(N-methyl-D-glucamine)-Cl solution, where the cation ions were substituted with NMDG, a 30-pS anion-selective channel activity was also detected. The variety in the types of ion channels observed in human spermatozoa plasma membrane suggests that ion channels may play a range of different roles in sperm physiology and gamete interaction. Mol. Reprod. Dev. 50:354–360, 1998. © 1998 Wiley-Liss, Inc.     

Reconstitution of vacuolar ion channels into planar lipid bilayers.

Vacuolar ion channels were characterized after reconstitution into planar lipid bilayers. (1) Channel activity was observed after incorporation of tonoplast-enriched microsomal membranes, purified tonoplast membranes or of solubilized tonoplast proteins. (2) Channels of varying single-channel conductances were detected after reconstitution. In symmetrical 100 mmol l-1 KCl, conductances between 1 and 110 pS were frequently measured; the largest number of independent reconstitution events was seen for single-channel conductances of 16-25 pS (28 experiments), 30-42 pS (26), 49-56 pS (15) and 64-81 pS (15). Channel current usually increased linearly with voltage. (3) In asymmetrical solutions, cation-, non-selective and, for the first time for the tonoplast, anion-selective channels were detected. Ca(2+)-dependent regulation of channel opening was not observed in our reconstitution system. (4) Permeability was also observed for Cl-, NO3-, SO4(2-) and phosphate. (5) After fractionation of tonoplast proteins by size exclusion chromatography, ion channel activity was recovered in specific fractions. (6) Some of these fractions catalyzed sulfate transport after reconstitution into liposomes. The results suggest that different channels are active at the tonoplast membrane at a larger number than has been concluded from previous work.     

Current-Voltage Curves of Single CI- Channels which Coexist with Two Types of K+ Channel in the Tonoplast of Chara corallina

A Cl^– channel and two types of K⁺ channel have been observed,by the use of the patch-clamp technique, in the membrane surroundingcytoplasmic droplets from Chara corallina. Measurements on cell-attachedpatches showed that the channel selective for Cl^– hada chord conductance of 21 pS at the resting membrane p.d. (mean= 11 mV, n = 19) and when open, passed an outward current of1.4 pA (n = 24 patches) at the resting p.d., with reversal ofthe direction of current at –54 mV (130 mol m^–3Cl^– in the external solution). The Cl^– concentrationin the cytoplasmic droplet calculated from the reversal p.d.was 15 mol m^–3. The channel strongly rectified outwardcurrent flow, but this rectification disappeared with symmetricalCl^– concentrations across detached patches of membrane.It is concluded that rectification observed in cell-attachedpatches is primarily due to asymmetric ^Cl– concentrationsrather than an asymmetry in energy barriers to Cl^– permeationin the channel or any voltage-dependent kinetics of the channel.The channel was rarely observed in detached patches despitebeing commonly observed in cell-attached patches. However, theabsence of Ca²⁺ at the cytoplasmic face of the membrane allowedobservation of the channel in detached patches for brief periods,during which ion substitution experiments revealed a permeabilitysequence of aspartate (76:33:1). A 100 pS K⁺ channel previously described by Luhring (1986) wasfrequently observed, in some instances simultaneously, witha channel having a conductance of 60 pS and displaying outwardrectification. This rectification was due to the channel remainingopen almost continuously for positive membrane potential differences(p.d.) and remaining shut almost continuously for negative p.d.'s.The 60 pS channel, like the 100 pS K⁺ channel, reversed currentflow at the resting p.d., suggesting that it was also permeableto K⁺. Key words: Plant ion-channels, chloride channel, potassium channel, patch-clamp     

Partial purification of a potassium channel with low permeability for sodium from tonoplast membranes of Hordeum vulgare cv. Gerbel

Summary A potassium-specific tonoplast channel was identified by reconstitution of tonoplast polypeptides into planar lipid bilayer membranes. Highly purified tonoplast membranes were solubilized in Triton X-100-containing buffer and fractionated by size-exclusion chromatography. The protein fractions were assayed for ion channel activity in a planar bilayer system, and the potassium channel was routinely recovered in specific fractions corresponding to an apparent molecular mass of 80 kDa. In symmetrical electrolyte solutions of 100 mM potassium chloride, the potassium channel had a single-channel conductance of 72 pS. Substates of the channel with conductances of 17, 33 and 52 pS were frequently observed. After identification of the channel in low or high KCl, addition of sodium acetate or sodium chloride caused only insignificant conductance changes. This result suggested that the channel was not or little permeable for sodium or chloride, whereas it had similar single-channel conductance for rubidium and caesium ions as compared with potassium ions. The channel is presumably responsible for the equilibration of potassium between the vacuole and the cytosol. The role of the channel in the physiology of the barley cell under salt stress is discussed.The authors would like to thank U. Heber for many helpful discussions. This work was supported by grants of the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 176, projects B3 and B7) and by the Fonds der Chemischen Industrie.     

Ion Transport in Suaeda maritima: Its Relation to Growth and Implications for the Pathway of Radial Transport of Ions across the Root

The ion relations of the halophytc Suaeda maritima are described.When plants grew in 340 mol m^–3 sodium chloride (—1•76MPa) leaf solute potentials decreased, and were sustained around—2•5 MPa Inorganic ion concentration (mostly of sodiumchloride) accounted for this. Comparable shoot ion concentrationsof potassium, nitrate and sulphate occurred when plants grewon different salinity types characterized by these ions. Netsodium transport and shoot sodium concentration increased dramaticallywith increases in external sodium chloride concentration upto 85 mol m^–3; thereafter, further increases in externalsodium chloride concentration had relatively little effect uponeither shoot sodium concentration or upon net transport of sodiumto the shoot. The net transport of sodium plus potassium onlydoubled when the external concentration of sodium plus potassiumincreased from 24 to 687 mol m^–3 Shoot ion concentrationswere remarkably constant with time, external concentration andsalinity type. The membrane flux rates and symplasmic ion concentrations neededto sustain the observed net transport of sodium (of some 10mmol g^–1 dry wt. of roots d^–1) are calculated fromanatomical and stereological data for the root system of thisspecies. The minimum net sodium chloride flux to load the symplasmwould be 260 nmol m^–2s^–1 if the whole cortical andepidermal plasmalemmal surface area were used uniformly, butthe flux rate required would be 3000 nmol m^–2s^–1if uptake took place only at the root surface. A flux rate ofat least 1000 nmol m^–2s^–1 is needed between symplasmand xylem. The symplasmic concentration of NaCl would be atleast 80 mol m^–3. It is argued (1), that both symplasmicand xylem loading are likely to be passive processes mediatedby ion channels rather than active carriers, (2), that net iontransport at 340 mol m^–3 sodium chloride is close to themaximum which is physiologically sustainable and (3), that growthof this halophyte is limited by NaCl supply from the root. Key words: Suaeda maritima, halophyte, salinity, roots, radial ion transport     

Patch-Clamp Study on Ion Channels in the Tonoplast of Nitellopsis obtusa

Cytoplasmic drops covered with the tonoplast were prepared frominternodal cells of Nitellopsis grown in fresh water. Applyingthe patch-clamp technique and the microinjection technique tosuch drops, we characterized the ion channels in the tonoplast.Both in cell-free patches and in the cytoplasmic-drop-attachedpatches, the tonoplast K⁺ channel was identified. The permeabilityratio between Na⁺ and K⁺ was calculated to be 0.2. This channelwould provide a molecular basis for the Na⁺/K⁺ exchange at thetonoplast. In cell-free patches, the K⁺channel was not activatedby Ca²⁺. However, in the case of attached patches, microinjectionof Ca²⁺ into a drop activated the K⁺ channel with a lag of afew seconds, suggesting that some cytoplasmic factor(s) maymediate the activation of the K⁺ channel by Ca²⁺. The conductanceof this channel was not changed by cytoplasmic Ca²⁺, but theprobability of opening increased markedly. In addition to theK⁺ channel, a second type of channel was also identified incell-free patches. This channel may be the Cl^– channel. ³ Present address: Department of Insect Physiology and Behavior,National Institute of Sericultural and Entomological Science,Tsukuba, Ibaraki, 305 Japan (Received August 6, 1990; Accepted December 6, 1990)