Characterization of ion channels in the plasma membrane of epidermal cells of expanding pea (Pisum sativum arg) leaves

Ion channels in isolated patches of the plasma membrane of pea (Pisum sativum arg) epidermal cells were studied with the patch-clamp technique. One anion and one cation channel were dominantly present in most trials. The anion channel conducts nitrate, halides and malate, with a conductance in symmetrical 100 mm Cl^– of 300 pS and can be blocked by SITS when applied to the cytoplasmic side of the membrane. The cation channel poorly discriminates between potassium, sodium and lithium, is not blocked by either TEA or Ba²⁺, and has a conductance of 35 pS in symmetrical 100 mm K⁺. The open probability of the cation channel increases with increase of the Ca²⁺ concentration on the cytoplasmic side of the membrane from 0.1 to 1 m. The possible role of these two channels in the physiology of epidermal cells is discussed.This work was supported by NSF grant DCB-890 3744 to E.V.     

Demonstration and characterization of Ca2+ channel in tonoplast-free cells ofNitellopsis obtusa

Summary The presence of a Ca²⁺ channel in the plasmalemma of tonoplast-freeNitellopsis obtusa cells was demonstrated and its characteristics were studied using current- and voltage-clamp techniques. A long-lasting inward membrane current (I _m ), recorded using a step voltage clamp, consisted of a single component without time-dependent inactivation. Increasing either [Ca²⁺] _o or [Cl^–] _o 1) enhanced the maximum amplitude of inwardI _m ((I _m ) _p ) and 2) shifted the peak voltage ((V _m ) _p ) at(I _m ) _p to more positive values under ramp-shaped voltage clamping and 3) depolarized the peak value of action potentials. This behavior is consistent with predictions based on the Nernst equation for Ca²⁺ but not for Cl^–. DIDS (4,4-diisothiocyano-2,2-disulfonic acid stilbene) did not suppress(I _m ) _p in tonoplast-free cells, in contrast with its effect on normal cells. La³⁺ and nifedipine blocked(I _m ) _p irreversibly. On the other hand, Ca²⁺ channel agonist, BAY K 8644 irreversibly enhanced(I _m ) _p . Both Sr²⁺ influx and K⁺ efflux increased upon excitation. The charge carried by Sr²⁺ influx was compensated for by K⁺ efflux. It is concluded that only the Ca²⁺ channel is activated during plasmalemma excitation in tonoplast-free cells. In terms of the magnitude of(I _m ) _p , Sr²⁺ could replace Ca²⁺, but Mn²⁺, Mg²⁺ and Ba²⁺ could not. External pH affected(I _m ) _p and the membrane conductance (g _m ) at(I _m ) _p ((g _m ) _p ). Increasing the external ionic strength caused increases in both(I _m ) _p and(g _m ) _p , and shifted(V _m ) _p to positive values. At the same time, Sr²⁺ influx increased. Thus Ca²⁺ channel activation seems to be enhanced by increasing external ionic strength. The possible involvement of surface potential is discussed.     

Ureolytic activity of Nitellopsis obtusa (Characeae)

The decomposition of urea by Nitellopsis obtusa from Characeae was investigated. The intact cells were exposed to the inhibition by two typical urease inhibitors: boric acid and fluoride ion, used as a criterion to define if urease or UAL-ase is responsible for the ureolytic activity of the algae. It was found that boric acid and fluoride ion are simple competitive and slow-binding competitive inhibitors of Nitellopsis obtusa enzyme respectively, which is the response characteristic of urease. The inhibition constants equal to 2.3 and 0.1 mM for boric acid and fluoride ion, when compared to those of jack bean urease, indicate that in the observed kinetic behaviour of Nitellopsis obtusa urease partake transport processes taking place in the intact cells.     

Distribution and habitat of Nitellopsis obtusa (Characeae) in the Laurentian Great Lakes

Nitellopsis obtusa, a macroalga (Characeae) native to Europe and Asia, was found in U.S. waters of the St. Clair-Detroit River system in 1983, thus extending the range of this taxon into the Laurentian Great Lakes about 850 km from the St. Lawrence River where it was first discovered in North America in 1978. Its occurrence only in water frequented by commercial shipping vessels suggests that it is distributed via this mechanism. In the St. Clair-Detroit River system, N. obtusa was collected with a Ponar grab at four locations, and with a grapnel at one additional location. It was the ninth most frequently found macrophyte and it was most abundant at Belle Isle in the Detroit River, where the mean dry-weight biomass in Ponar samples was 0 g m^-2 in June, 37 g m^-2 in August, and 32 g m⁻² in September. Maximum biomass of this taxon in one Ponar grab at this location was 289 g m^-2 in September. The alga occurred primarily in water of relatively low current velocity (11.3 cm s⁻¹) and in association with Vallisneria americana, Myriophyllum spicatum, Potamogeton richardsonii, Najas flexilis, and Elodea canadensis. Contribution 654, Great Lakes Fishery Laboratory, Ann Arbor, MI 48105, USA Contribution 654, Great Lakes Fishery Laboratory, Ann Arbor, MI 48105, USA     

Thiamine triphosphate activates an anion channel of large unit conductance in neuroblastoma cells

In neuroblastoma cells, the intracellular thiamine triphosphate (TTP) concentration was found to be about 0.5 m, which is several times above the amount of cultured neurons or glial cells. In inside-out patches, addition of TTP (1 or 10) m to the bath activated an anion channel of large unit conductance (350–400 pS) in symmetrical 150 mm NaCl solution. The activation occurred after a delay of about 4 min and was not reversed when TTP was washed out. A possible explanation is that the channel has been irreversibly phosphorylated by TTP. The channel open probability (P _o) shows a bell-shaped behavior as a function of pipette potential (V _p). P _o is maximal for –25 mV<V _p<10 mV and steeply decreases outside this potential range. From reversal potentials, permeability ratios of P_Cl/ P_Na = 20 and P_Cl/P_gluconate = 3 were estimated. ATP (5 mm) at the cytoplasmic side of the channel decreased the mean single channel conductance by about 50%, but thiamine derivatives did not affect unit conductance; 4,4 -diisothiocyanostilbene-2,2-disulfonic acid (0.1 mm) increased the flickering of the channel between the open and closed state, finally leading to its closure. Addition of oxythiamine (1 mm), a thiamine antimetabolite, to the pipette filling solution potentiates the time-dependent inactivation of the channel at V _p=–20 mV but had the opposite effect at +30 mV. This finding corresponds to a shift of P _o towards more negative resting membrane potentials. These observations agree with our previous results showing a modulation of chloride permeability by thiamine derivatives in membrane vesicles from rat brain.We would like to thank the National Funds for Scientific Research (Belgium) for financially supporting the stay of L.B. in Konstanz. We wish to thank A. Ngezahayo, F. Mendez and Dr. P. Wins for helpful discussions. This work was in part supported by a research grant from the Fonds special pour la Recherche à l'Université de Liege to L.B., the SFB 156 of the DFG and a grant of the Hermann and Lilly Schilling Stiftung to H.-A.K. Neuroblastoma, PC-12 and glioma cell lines were a gift from Prof. G. Moonen (Department of Human Physiology, University of Liège).     

Inhibition of anion channels derived from mitochondrial membranes of the rat heart by stilbene disulfonate—DIDS

The objective of this work was to characterize in more detail the inhibition effect of diisothiocyanatostilbene-2′,2-disulfonic acid (DIDS) on anion channels isolated from the rat heart mitochondria. The channels reconstituted into a planar lipid membrane displayed limited powers of discrimination between anions and cations and the ion conductance measured under asymmetric (250/50 mM KCl, cis/trans) and symmetric (150 mM KCl) conditions was ∼100 pS. DIDS caused a dramatic decrease in the channel activity (IC₅₀ = 11.7 ± 3.1 μM) only when it was added to the cis side of a planar lipid membrane. The inhibition was accompanied by the significant prolongation of closings and the shortening of openings within the burst as well as gaps between bursts were prolonged and durations of bursts were reduced. The blockade was complete and irreversible when concentration of DIDS was increased up to 200 μM. Our data indicate that DIDS is an allosteric blocker of mitochondrial anion channels and this specific effect could be used as a tool for reliable identification of anion channels on the functional level.     

Putative ClC-2 Chloride Channel Mediates Inward Rectification in <Emphasis Type="Italic">Drosophila </Emphasis>Retinal Photoreceptors

We report that Drosophila retinal photoreceptors express inwardly rectifying chloride channels that seem to be orthologous to mammalian ClC-2 inward rectifier channels. We measured inwardly rectifying Cl⁻ currents in photoreceptor plasma membranes: Hyperpolarization under whole-cell tight-seal voltage clamp induced inward Cl⁻ currents; and hyperpolarization of voltage-clamped inside-out patches excised from plasma membrane induced Cl⁻ currents that have a unitary channel conductance of ∼3.7 pS. The channel was inhibited by 1 mM Zn²⁺ and by 1 mM 9-anthracene, but was insensitive to DIDS. Its anion permeability sequence is Cl⁻ = SCN⁻> Br⁻>> I⁻, characteristic of ClC-2 channels. Exogenous polyunsaturated fatty acid, linolenic acid, enhanced or activated the inward rectifier Cl⁻ currents in both whole-cell and excised patch-clamp recordings. Using RT-PCR, we found expression in Drosophila retina of a ClC-2 gene orthologous to mammalian ClC-2 channels. Antibodies to rat ClC-2 channels labeled Drosophila photoreceptor plasma membranes and synaptic regions. Our results provide evidence that the inward rectification in Drosophila retinal photoreceptors is mediated by ClC-2-like channels in the non-transducing (extra-rhabdomeral) plasma membrane, and that this inward rectification can be modulated by polyunsaturated fatty acid. G. Ugarte and R. Delgado contributed equally to this work.     

Isolation of protoplasts and ion channel recording of plasma membrane patches and whole-cell recordings of the marine alga, <Emphasis Type="Italic">Ulva pertusa</Emphasis> (Chlorophyta)

Whole-cell patch-clamp techniques were used to study ion channels of a marine alga. High quality protoplasts suitable for electrophysiological studies were isolated from the green marine alga, Ulva pertusa, using enzyme mixtures consisting of cellulase and abalone power and identified by calcofluor fluorescence. The vitality of protoplasts varied depending on the alga growth stage, and those isolated from younger tissue in March maintained a high vitality with high sealing success rate compared with protoplasts isolated from mature or non-growing plants in August or November. In the whole-cell configuration, large inward currents were elicited by negative voltage pulses. The voltage-dependent component was predominantly carried by Cl⁻, as confirmed by the use of the Cl⁻ channel inhibitor DIDS and reversal potential of current-voltage plots. This evidence suggests that hyperpolarization-activated Cl⁻ permeable channels are responsible for the influx of Cl⁻ into U. pertusa cells. Voltage-dependent outward currents were also recorded in several protoplasts, and their properties need further investigation.     

Gravity-dependent polarity of cytoplasmic streaming inNitellopsis

Summary The internodal cells of the characean algaNitellopsis obtusa were chosen to investigate the effect of gravity on cytoplasmic streaming. Horizontal cells exhibit streaming with equal velocities in both directions, whereas in vertically oriented cells, the downwardstreaming cytoplasm flows ca. 10% faster than the upward-streaming cytoplasm. These results are independent of the orientation of the morphological top and bottom of the cell. We define the ratio of the velocity of the downward- to the upward-streaming cytoplasm as the polar ratio (PR). The normal polarity of a cell can be reversed (PR<1) by treatment with neutral red (NR). The NR effect may be the result of membrane hyperpolarization, caused by the opening of K⁺ channels. The K⁺ channel blocker TEA Cl^– inhibits the NR effect.External Ca²⁺ is required for normal graviresponsivness. The [Ca²⁺] of the medium determines the polarity of cytoplasmic streaming. Less than 1 M Ca²⁺ resulted in a PR<1 while greater than 1 M Ca²⁺ resulted in the normal gravity response. The voltage-dependent Ca²⁺ -channel blocker, nifedipine, inhibited the gravity response in a reversible manner, while treatment with LaCl₃ resulted in a PR<1, indicating the presence of two types of Ca²⁺ channels. A new model for graviperception is presented in which the whole cell acts as the gravity sensor, and the plasma membrane acts as the gravireceptor. This is supported by ligation and UV irradiation experiments which indicate that the membranes at both ends of the cell are required for graviperception. The density of the external medium also affects the PR ofNitellopsis. Calculations are presented that indicate that the weight of the protoplasm may provide enough potential energy to open ion channels.     

Identification of a cationic channel in synaptosomal membranes

Synaptosomal membranes were fused with liposomes using the hydration technique to produce giant proteoliposomes amenable to patch clamp recordings. Single channel currents of a cationic channel with particular properties were detected. In a solution of 150 mM NaCl, the channel displayed a unit conductance of 136 pS and a mean open state lifetime of 1.1 ms. The gating of the channel was shown to be voltage as well as calcium dependent. Pharmacological studies revealed that the channel was insensitive to a variety of channel blockers, but was inactivated by ruthenium red. Presumably, this channel may play a role in regulating the evoked release of neurotransmitters. Offprint requests to: H. Breer     

Hodgkin-Huxley analysis of a GCAC1 anion channel in the plasma membrane of guard cells

A quantitative analysis of the time- and voltage-dependent kinetics of the guard cell anion channel (GCAC1) current in guard cell protoplasts from Vicia faba was analyzed using the whole-cell patch clamp technique. The voltage-dependent steady-state activation of GCAC1 current followed a Boltzmann distribution. For the corresponding steady-state value of the activation variable a power of two was derived which yielded suitable fits of the time course of voltage-dependent current activation. The GCAC1 mediated chloride current could successfully be described in terms of the Hodgkin-Huxley equations commonly evoked for the Na channel in nerve. After step depolarizations from a potential in the range of the resting potential to potentials above the equilibrium potential for chloride an activation and also an inactivation could be described. The gating of both processes exhibited an inverse relationship on the polarity of the applied step potentials in the order of milliseconds. Deactivating tail currents decline exponentially. The presented analysis contributes to the understanding of the rising phase of the observed action potentials in guard cells of V. faba. Evidence is presented that the voltage-dependent kinetic properties of the GCAC1 current are different from those properties described for the excitable anion currents in the plasmalemma of Chara corallina (Beilby & Coster, 1979a).The authors gratefully acknowledge the encouragement of Dr. David Colquhoun to apply the Hodgkin-Huxley model to the GCAC1 channel. The work was in part supported by a grant of the Deutsche Forschungsgemeinschaft to R.H. and a grant of the Herman and Lilly Schilling Stiftung to H.-A.K.     

Effects of D2O on permeation and gating in the Ca2+-activated potassium channel fromChara

We studied the effects of H₂O/D₂O substitution on the permeation and gating of the large conductance Ca²⁺-activated K⁺ channels inChara gymnophylla droplet membrane using the patchclamp technique. The selectivity sequence of the channel was: K⁺>Rb⁺≫Li⁺, Na⁺, Cs⁺ and Cl⁻. The conductance of this channel in symmetric 100mm KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D₂O as compared to H₂O. The blockade of channel conductance by cytosolic Ca²⁺ weakened in D₂O as a result of a decrease in zero voltage Ca²⁺ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D₂O primarily due to the change in Ca²⁺ binding to the channel during the activation step. The Hill coefficient for Ca²⁺ binding was 3 in D₂O and around 1 in H₂O. The values of the Ca²⁺ binding constant in the open channel conformation were 0.6 and 6 μm in H₂O and D₂O, respectively, while the binding in the closed conformation was much less affected by D₂O. The H₂O/D₂O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1mm internal Ca²⁺.     

Cu2+, Co2+, and Mn2+ modify the gating kinetics of high-voltage-activated Ca2+ channels in rat palaeocortical neurons

The effects of three divalent metal cations (Mn2+, Co2+, and Cu2+) on high-voltage-activated (HVA) Ca2+ currents were studied in acutely dissociated pyramidal neurons of rat piriform cortex using the patch-clamp technique. Cu2+, Mn2+, and Co2+ blocked HVA currents conducted by Ba2+ ( IBa) with IC50 of approximately 920 nM, approximately 58 micro M, and approximately 65 micro M, respectively. Additionally, after application of non-saturating concentrations of the three cations, residual currents activated with substantially slower kinetics than control IBa. As a consequence, the current fraction abolished by the blocking cations typically displayed, in its early phase, an unusually fast-decaying transient. The latter phenomenon turned out to be a subtraction artifact, since none of the pharmacological components (L-, N-, P/Q-, and R-type) that constitute the total HVA currents under study showed a similarly fast early decay: hence, the slow activation kinetics of residual currents was not due to the preferential inhibition of a fast-activating/inactivating component, but rather to a true slowing effect of the blocker cations. The percent IBa-amplitude inhibition caused by Mn2+, Co2+, and Cu2+ was voltage-independent over the whole potential range explored (up to +30 mV), hence the slowing of IBa activation kinetics was not due to a mechanism of voltage- and time-dependent relief from block. Moreover, Mn2+, Co2+, and Cu2+ significantly reduced I(Ba) deactivation speed upon repolarization, which also is not compatible with a depolarization-dependent unblocking mechanism. The above results show that 1) Cu2+ is a particularly potent HVA Ca2+-channel blocker in rat palaeocortical neurons; and 2) Mn2+, Co2+, and Cu2+, besides exerting a blocking action on HVA Ca2+-channels, also modify Ca2+-current activation and deactivation kinetics, most probably by directly interfering with channel-state transitions.     

Glutamate activates cation currents in the plasma membrane of<Emphasis Type="Italic"> Arabidopsis</Emphasis> root cells

The effect of glutamate on plant plasma membrane cation transport was studied in roots of Arabidopsis thaliana (L.) Heynh. Patch-clamp experiments using root protoplasts, ²²Na⁺ unidirectional fluxes into intact roots and measurements of cytosolic Ca²⁺ activity using plants expressing cytosolically-targeted aequorin in specific cell types were carried out. It was demonstrated that low-millimolar concentrations of glutamate activate within seconds both Na⁺ and Ca²⁺ currents in patch-clamped protoplasts derived from roots. The probability of observing glutamate-activated currents increased with increasing glutamate concentration (up to 29% at 3 mM); half-maximal activation was seen at 0.2–0.5 mM glutamate. Glutamate-activated currents were voltage-insensitive, instantaneous (completely activated within 2–3 ms of a change in voltage) and non-selective for monovalent cations (Na⁺, Cs⁺ and K⁺). They also allowed the permeation of Ca²⁺. Half-maximal Na⁺ currents occurred at 20–30 mM Na⁺. Glutamate-activated currents were sensitive to non-specific blockers of cation channels (quinine, La³⁺, Gd³⁺). Although low-millimolar concentrations of glutamate did not usually stimulate unidirectional influx of ²²Na⁺ into intact roots, they reliably caused an increase in cytosolic Ca²⁺ activity in protoplasts isolated from the roots of aequorin-transformed Arabidopsis plants. The response of cytosolic Ca²⁺ activity revealed a two-phase development, with a rapid large transient increase (lasting minutes) and a prolonged subsequent stage (lasting hours). Use of plants expressing aequorin in specific cell types within the root suggested that the cell types most sensitive to glutamate were in the mature epidermis and cortex. The functional significance of these glutamate-activated currents for both cation uptake into plants and cell signaling remains the subject of speculation, requiring more knowledge about the dynamics of apoplastic glutamate in plants.Abbreviations GLR Gene in plants encoding glutamate receptor-like protein - iGluRs Ionotropic glutamate receptors     

Plasmalemma patch clamp experiments in plant root cells: procedure for fast isolation of protoplasts with minimal exposure to cell wall degrading enzymes

Summary A convenient and rapid isolation procedure for root cell protoplasts suitable for patch clamp experiments, was developed for root cells of tomato (Lycopersicon esculentum) andPlantago species, grown on hydroculture. The procedure is based on a minimal exposure of cells to cell wall degrading enzyme mixtures. After an incubation period of 30 min in a cell wall degrading enzyme mixture all free floating cells were discarded. Subsequently the root material was rinsed and a second group of cells, still present inside the tissue, was freed by application of mechanical pressure. The newly released protoplasts were filtered and collected on the glass bottom of a patch clamp dish. The bathing medium was rinsed extensively removing cellulose fibrils and protoplasts not attached to the glass. Removal of these cellulose fibrils significantly improved the seal success ratio. The isolated protoplasts were suitable for patch clamp experiments in the cell-attached patch, the whole cell and the isolated patch configuration.Abbreviations BSA bovine serum albumin - BTP bis-tris propane - CAP cell-attached patch - OOP outside out patch - PEG polyethylene glycol - WC whole cell     

Comparison of K+-channel activation and deactivation in guard cells from a dicotyledon (Vicia faba L.) and a graminaceous monocotyledon (Zea mays)

We describe and compare inward and outward whole-cell K⁺ currents across the plasma membrane surrounding guard-cell protoplasts from the dicotyledon, Vicia faba, and the graminaceous monocotyledon, Zea mays. Macrosopic whole-cell current is considered in terms of microscopic single-channel activity, which involves discrete steps between conducting (open) and nonconducting (closed) states of the channel protein. Kinetic equations are used to model the number of open and closed states for channels conducting K⁺ influx (K(in)) and K⁺ efflux (K(out)) in the two species, and to calculate the rate at which open-closed transitions occur. The opening and closure of K(in) channels in both Vicia and Zea follow single-exponential timecourses, indicating that K(in)-channel proteins in each species simply fluctuate between one open and one closed state. In both species, opening of K(in) channels is voltage-independent, but closure of K(in) channels is faster at more positive membrane potentials. In response to identical voltage stimuli, K(in) channels in Zea open and close approximately three times as fast as in Vicia. In contrast to K(in), K(out) channels in Zea open and close more slowly than in Vicia. The closure of K(out) channels follows a single-exponential timecourse in each species, indicating one open state. The kinetics of K(out)-channel opening are more complicated and indicate the presence of at least two (Vicia) or three (Zea) closed states. The authors thank Professor N.A. Walker and Dr. D.R. Laver for the use of laboratory equipment, for helpful discussion and for provision of the program, GETHH. Thanks also to Dr. R.J. Ritchie for assistance with statistical analyses and to Ms. Janet Sherwood for maintenance of Vicia and Zea plants. This work was supported by grants from the National Science Foundation (DCB-89-04041) and the McKnight Foundation (S.M.A) and by a Charles Gilbert Heydon Travelling Fellowship (K.F-G).     

Adaptation of the photosynthetic apparatus of Nitellopsis obtusa to changing light intensity at the molecular level: different pathways of a singlet excitation quenching

The effect of prolonged illumination (60 min) with photosynthetically active monochromatic radiation of low intensity (3 μmol m⁻² s⁻¹) and high intensity (60 μmol m⁻² s⁻¹), corresponding to the physiological conditions and light stress conditions, respectively, was studied in the algae Nitellopsis obtusa. Illumination of Nitellopsis obtusa cells with strong light was associated with activation of the xanthophyll cycle, manifested by the deepoxidation of violaxanthin and accumulation of antheraxanthin and zeaxanthin. At the same time, the efficient singlet excitation quenching in the photosynthetic apparatus was activated, as demonstrated by the decrease in the intensity of the chlorophyll a fluorescence emission by ca 50 %. The difference of the fluorescence excitation spectra recorded before and after the light treatment match the difference absorption spectrum of the xanthophyll cycle pigments. The illumination with low light intensity resulted also in the chlorophyll a fluorescence quenching but the effect was very small (less than 10 %). The fluorescence quenching is interpreted in terms of the energy transfer between the Q_y energy level of chlorophyll a and the 2¹ A_g ⁻ energy level of zeaxanthin. The singlet energy levels of carotenoids, corresponding to the green spectral region, are also taken into consideration in the interpretation of the excitation energy exchange between the carotenoids and chlorophylls. Possible molecular mechanisms involved in the activation of the strong and the weak excitation quenching, including violaxanthin isomerization, and possible physiological functions of such pathways of energy transfer are discussed.