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R Hedrich

Publications and source records attributed to R Hedrich.

At least 55 records · Page 3Linked to original sources
PubMed1997

Receptor-mediated activation of a plant Ca2+-permeable ion channel involved in pathogen defense.

Pathogen recognition at the plant cell surface typically results in the initiation of a multicomponent defense response. Transient influx of Ca2+ across the plasma membrane is postulated to be part of the signaling chain leading to pathogen resistance. Patch-clamp analysis of parsley protoplasts revealed a novel Ca2+-permeable, La3+-sensitive plasma membrane ion channel of large conductance (309 pS in 240 mM CaCl2). At an extracellular Ca2+ concentration of 1 mM, which is representative of the plant cell apoplast, unitary channel conductance was determined to be 80 pS. This ion channel (LEAC, for large conductance elicitor-activated ion channel) is reversibly activated upon treatment of parsley protoplasts with an oligopeptide elicitor derived from a cell wall protein of Phytophthora sojae. Structural features of the elicitor found previously to be essential for receptor binding, induction of defense-related gene expression, and phytoalexin formation are identical to those required for activation of LEAC. Thus, receptor-mediated stimulation of this channel appears to be causally involved in the signaling cascade triggering pathogen defense in parsley.

Journal Article↗
PubMed1997

Plant K+ channel alpha-subunits assemble indiscriminately.

In plants a large diversity of inwardly rectifying K+ channels (K(in) channels) has been observed between tissues and species. However, only three different types of voltage-dependent plant K+ uptake channel subfamilies have been cloned so far; they relate either to KAT1, AKT1, or AtKC1. To explore the mechanisms underlying the channel diversity, we investigated the assembly of plant inwardly rectifying alpha-subunits. cRNA encoding five different K+ channel alpha-subunits of the three subfamilies (KAT1, KST1, AKT1, SKT1, and AtKC1) which were isolated from different tissues, species, and plant families (Arabidopsis thaliana and Solanum tuberosum) was reciprocally co-injected into Xenopus oocytes. We identified plant K+ channels as multimers. Moreover, using K+ channel mutants expressing different sensitivities to voltage, Cs+, Ca2+, and H+, we could prove heteromers on the basis of their altered voltage and modulator susceptibility. We discovered that, in contrast to animal K+ channel alpha-subunits, functional aggregates of plant K(in) channel alpha-subunits assembled indiscriminately. Interestingly, AKT-type channels from A. thaliana and S. tuberosum, which as homomers were electrically silent in oocytes after co-expression, mediated K+ currents. Our findings suggest that K+ channel diversity in plants results from nonselective heteromerization of different alpha-subunits, and thus depends on the spatial segregation of individual alpha-subunit pools and the degree of temporal overlap and kinetics of expression.

Arabidopsis Proteins↗
PubMed1996

Changes in voltage activation, Cs+ sensitivity, and ion permeability in H5 mutants of the plant K+ channel KAT1.

KAT1 is a voltage-dependent inward rectifying K+ channel cloned from the higher plant Arabidopsis thaliana [Anderson, J. A., Huprikar, S. S., Kochian, L. V., Lucas, W. J. & Gaber, R. F. (1992) Proc. Natl. Acad. Sci. USA 89, 3736-3740]. It is related to the Shaker superfamily of K+ channels characterized by six transmembrane spanning domains (S1-S6) and a putative pore-forming region between S5 and S6 (H5). The 115 region between Pro-247 and Pro-271 in KAT1 contains 14 additional amino acids when compared with Shaker [Aldrich, R. W. (1993) Nature (London) 362, 107-108]. We studied various point mutations introduced into H5 to determine whether voltage-dependent plant and animal K+ channels share similar pore structures. Through heterologous expression in Xenopus oocytes and voltage-clamp analysis combined with phenotypic analysis involving a potassium transport-defective Saccharomyces cerevisiae strain, we investigated the selectivity filter of the mutants and their susceptibility toward inhibition by cesium and calcium ions. With respect to electrophysiological properties, KAT1 mutants segregated into three groups: (i) wild-type-like channels, (ii) channels modified in selectivity and Cs+ or Ca2+ sensitivity, and (iii) a group that was additionally affected in its voltage dependence. Despite the additional 14 amino acids in H5, this motif in KAT1 is also involved in the formation of the ion-conducting pore because amino acid substitutions at Leu-251, Thr-256, Thr-259, and Thr-260 resulted in functional channels with modified ionic selectivity and inhibition. Creation of Ca2+ sensitivity and an increased susceptibility to Cs+ block through mutations within the narrow pore might indicate that both blockers move deeply into the channel. Furthermore, mutations close to the rim of the pore affecting the half-activation potential (U1/2) indicate that amino acids within the pore either interact with the voltage sensor or ion permeation feeds back on gating.

Amino Acid Sequence↗
PubMed1996

Functional expression of the plant K+ channel KAT1 in insect cells.

Following the biophysical analysis of plant K+ channels in their natural environment, three members from the green branch of the evolutionary tree of life KAT1, AKT1, and KST1 have recently been identified on the molecular level. Among them, we focussed on the expression and characterization of the Arabidopsis thaliana K+ channel KAT1 in the insect cell line Sf9. The infection of Sf9 cells with KAT1-recombinant baculovirus resulted in functional expression of KAT1 channels, which was monitored by inward-rectifying, K+-selective (impermeable to Na+ and even NH4+) ionic conductance in whole-cell patch-clamp recordings. A voltage threshold as low as -60 to -80mV for voltage activation compared to other plant inward rectifiers in vivo, and to in vitro expression of KAT1 in Xenopus oocytes or yeast, may be indicative for channel modulation by the expression system. A rise in cytoplasmic Ca2+ concentration (up to 1 mM), a regulator of the inward rectifier in Vicia faba guard cells, did not modify the voltage dependence of KAT1 in Sf9 cells. The access to channel function on one side and channel protein on the other make Sf9 cells a suitable heterologous system for studies on the biophysical properties, post-traditional modification and assembly of a green inward rectifier.

Animals↗
PubMed1995

Cloning and electrophysiological analysis of KST1, an inward rectifying K+ channel expressed in potato guard cells.

Potassium uptake by guard cells represents part of the osmotic motor which drives stomatal opening. Patch-clamp measurements have identified inward rectifying K+ channels capable of mediating K+ uptake in guard cells and various other plant cell types. Here we report the molecular cloning and characterization of a voltage-dependent K+ channel (KST1) from potato (Solanum tuberosum L.) guard cells. In situ hybridization shows expression of kst1 in guard cells. Two-electrode voltage-clamp and patch-clamp studies of the gene product after cRNA injection into Xenopus oocytes identified KST1 as a slowly activating, voltage-dependent, inward rectifying K+ channel. The single channel current voltage curve was linear in the range -160 to +20 mV, with a deduced single channel conductance of 7 pS in symmetrical 100 mM K+. This channel type, modulated by pH changes within the physiological range, required ATP for activation. In line with the properties of a K(+)-selective channel, KST1 was permeable to K+, Rb+ and NH4+ and excluded Na+ and Li+. Cs+ at submillimolar concentrations blocked the channel in a voltage-dependent manner. Related studies on potato guard cell protoplasts confirmed the biophysical characteristics of the kst1 gene product (KST1) in the heterologous expression system. Therefore, KST1 represents a major K+ uptake channel in potato guard cells.

Amino Acid Sequence↗
PubMed1995

Inward rectifier potassium channels in plants differ from their animal counterparts in response to voltage and channel modulators.

We have investigated the electrophysiological basis of potassium inward rectification of the KAT1 gene product from Arabidopsis thaliana expressed in Xenopus oocytes and of functionally related K+ channels in the plasma membrane of guard and root cells from Vicia faba and Zea mays. The whole-cell currents passed by these channels activate, following steps to membrane potentials more negative than -100 mV, with half activation times of tens of milliseconds. This voltage dependence was unaffected by the removal of cytoplasmic magnesium. Consequently, unlike inward rectifier channels of animals, inward rectification of plant potassium channels is an intrinsic property of the channel protein itself. We also found that the activation kinetics of KAT1 were modulated by external pH. Decreasing the pH in the range 8.5 to 4.5 hastened activation and shifted the steady state activation curve by 19 mV per pH unit. This indicates that the activity of these K+ channels and the activity of the plasma membrane H(+)-ATPase may not only be coordinated by membrane potential but also by pH. The instantaneous current-voltage relationship, on the other hand, did not depend on pH, indicating that H+ do not block the channel. In addition to sensitivity towards protons, the channels showed a high affinity voltage dependent block in the presence of cesium, but were less sensitive to barium. Recordings from membrane patches of KAT1 injected oocytes in symmetric, Mg(2+)-free, 100 mM-K+, solutions allowed measurements of the current-voltage relation of single open KAT1 channels with a unitary conductance of 5 pS. We conclude that the inward rectification of the currents mediated by the KAT1 gene product, or the related endogenous channels of plant cells, results from voltage-modulated structural changes within the channel proteins. The voltage-sensing or the gating-structures appear to interact with a titratable acidic residue exposed to the extracellular medium.

Animals↗
PubMed1995

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).

Chloride Channels↗
PubMed1994

Coordination of plasma membrane and vacuolar membrane ion channels during stomatal movement.

Transduction of extracellular signals in guard cells requires coupling of ion fluxes across the vacuolar and plasma membrane. Changes in cytoplasmic proton and Ca2+ concentrations are important messages during stomatal movement. Here we summarize the different biophysical properties of voltage- and ligand-dependent ion channels in both the vacuolar and plasma membranes of guard cells, focusing on how cytoplasmic Ca2+ and proton concentrations direct ion fluxes towards stomatal opening or closure. Particular attention is given to the patch clamp analysis of plasma membrane cation channels (GCKCin, GCKCout and Ca2+ channels), the anion channel (GCAC1) and the vacuolar SV-type channels. A working model is presented of how the ion channels of both membrane systems could communicate to synchronize potassium salt release during stomatal closure.

Animals↗
PubMed1993

Malate-induced feedback regulation of plasma membrane anion channels could provide a CO2 sensor to guard cells.

Plants have developed strategies to circumvent limitations in water supply through the adjustment of stomatal aperture in relation to the photosynthetic capacity (water-use efficiency). The CO2 sensor of guard cells, reporting on the metabolic status of the photosynthetic tissue, is, however, as yet unknown. We elucidated whether extracellular malate has the capability to serve as a signal metabolite in regulating the membrane properties of guard cells. Patch-clamp studies showed that slight variations in the external malate concentration induced major alterations in the voltage-dependent activity of the guard cell anion channel (GCAC1). Superfusion of guard cell protoplasts with malate solutions in the physiological range caused the voltage-gate to shift towards hyperpolarized potentials (Km(mal) = 0.4 mM elicits a 38 mV shift). The selectivity sequence of the anion channel NO3- (4.2) > or = I- (3.9) > Br- (1.9) > Cl- (1) >> mal (0.1) indicates that malate is able to permeate GCAC1. The binding site for shifting the gate is, however, located on the extracellular face of the channel since cytoplasmic malate proved ineffective. Single-channel analysis indicates that extracellular malate affects the voltage-dependent mean open time rather than the unitary conductance of GCAC1. In contrast to malate the rise in the extracellular Cl- concentration increases the unit conductance of the anion efflux channel. We suggest that stomata sense changes in the intercellular CO2 concentration and thus the photosynthetic activity of the mesophyll via feedback regulation of anion efflux from guard cells through malate-sensitive GCAC1.

Anions↗
PubMed1992

A new scheme of symbiosis: ligand- and voltage-gated anion channels in plants and animals.

Anion channels in the plasma membrane of both plant and animal cells participate in a number of important cellular functions such as volume regulation, trans-epithelial transport, stabilization of the membrane potential and excitability. Only very recently attention has turned to the presence of anion channels in higher plant cells. A dominant theme among recent discoveries is the role of Ca2+ in activating or modulating channel current involved in signal transduction. The major anion channel of stomatal guard cell protoplasts is a 32-40 pS channel which is highly selective for anions, in particular NO3-, Cl- and malate. These channels are characterized by a steep voltage dependence. Anion release is elicited upon depolarization and restricted to a narrow voltage span of -100 mV to the reversal potential of anions. During prolonged activation the current slowly inactivates. A rise in cytoplasmic calcium in the presence of nucleotides evokes activation of the anion channels. Following activation they catalyse anion currents 10-20 times higher than in the inactivated state thereby shifting the resting potential of the guard cell from a K(+)-conducting to an anion-conducting state. Patch-clamp studies have also revealed that growth hormones directly affect voltage-dependent activity of the anion channel in a dose-dependent manner. Auxin binding resulted in a shift of the activation potential towards the resting potential. Auxin-dependent gating of the anion channel is side- and hormone-specific. Its action is also channel-specific as K+ channels coexisting in the same membrane patch were insensitive to this ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Identification and modulation of a voltage-dependent anion channel in the plasma membrane of guard cells by high-affinity ligands.

Guard cell anion channels (GCAC1) catalyze the release of anions across the plasma membrane during regulated volume decrease and also seem to be involved in the targeting of the plant growth hormones auxins. We have analyzed the modulation and inhibition of these voltage-dependent anion channels by different anion channel blockers. Ethacrynic acid, a structural correlate of an auxin, caused a shift in activation potential and simultaneously a transient increase in the peak current amplitude, whereas other blockers shifted and blocked the voltage-dependent activity of the channel. Comparison of dose-response curves for shift and block imposed by the inhibitor, indicate two different sites within the channel which interact with the ligand. The capability to inhibit GCAC1 increases in a dose-dependent manner in the sequence: probenecid less than A-9-C less than ethacrynic acid less than niflumic acid less than IAA-94 less than NPPB. All inhibitors reversibly blocked the anion channel from the extracellular side. Channel block on the level of single anion channels is characterized by a reduction of long open transitions into flickering bursts, indicating an interaction with the open mouth of the channel. IAA-23, a structural analog of IAA-94, was used to enrich ligand-binding polypeptides from the plasma membrane of guard cells by IAA-23 affinity chromatography. From this protein fraction a 60 kDa polypeptide crossreacted specifically with polyclonal antibodies raised against anion channels isolated from kidney membranes. In contrast to guard cells, mesophyll plasma membranes were deficient in voltage-dependent anion channels and lacked crossreactivity with the antibody.

Benzoates↗
PubMed1991

Stretch-activated chloride, potassium, and calcium channels coexisting in plasma membranes of guard cells of Vicia faba L.

Mechanosensitive ion channels in the plasma membrane of Vicia faba guard cell protoplasts were studied by use of the patch clamp technique. Stretch-activated (SA) channels in outside-out patches were analyzed for channel conductance, kinetics and ion selectivity. We found three distinct SA channels, permeable to Cl-, K+ and Ca2+ and distinguishable from spontaneous (non-SA) channels for these ions on the basis of conductance, kinetics, and voltage-dependence, as well as sensitivity to membrane stretch. In the attached patch configuration, light suction (2 to 10 kPa) reversibly induced channel opening with multiple amplitudes and complex kinetics. The open probability for SA channels increased nonlinearly with pipette suction. In guard cells in situ, these SA channels may mediate ion transport across the plasma membrane directly, as well as influence the activity of non-SA channels via effects on membrane voltage and cytoplasmic calcium. Through such effects, SA channels likely influence volume and turgor regulation of guard cells, and thereby control of leaf gas exchange.

Calcium Channels↗
PubMed1990

Ca2+ and nucleotide dependent regulation of voltage dependent anion channels in the plasma membrane of guard cells.

Using the patch-clamp technique we discovered that the voltage dependent anion channels in the plasma membrane of guard cells are activated by a rise in cytoplasmic Ca2+ in the presence of nucleotides. Upon activation, these anion channels catalyse anion currents 10-20 times higher than in the inactivated state, thus shifting the plasma membrane from a K+ conducting state to an anion conducting state. Prolonged stimulation by depolarizing voltages results in the inactivation of the anion current (t1/2 = 10-12 s). We suggest that activation of the anion channel by Ca2+ and nucleotides is a key event in the regulation of salt efflux from guard cells during stomatal closure.

Adenosine Triphosphate↗
PubMed1990

Malate Dehydrogenases in Guard Cells of Pisum sativum.

Guard cell protoplasts of Pisum sativum show considerable NADP-dependent malate dehydrogenase (MDH) activity in darkness which can be enhanced severalfold by illumination or treatment with dithiothreitol (DTT). The question arose whether guard cells possess an NADP-MDH different from that present in the chloroplasts of the mesophyll (which is inactive in darkness or in the absence of DTT). MDH activities were determined in extracts of isolated protoplasts from mesophyll and epidermis, and in mechanically prepared epidermal pieces (with guard cells as the only living cells and no interference from proteases originating from the cell wall digesting enzymes). Guard cells possessed NAD-dependent MDHs of high activity and incomplete exclusion of NADP as a coenzyme. This NADP-dependent activity of the NAD-MDH(s) could not be stimulated by DTT or, inferentially, by light. The DTT- (and light-) dependent NADP-MDH represented 0.05% of the total protein of the guard cells and had a specific activity of 0.1 unit per milligram protein; both values are in the same range as the corresponding ones of the mesophyll cells. Agreement was also found in the extent of light activation, in subunit molecular weight, immunological cross-reactions, and in the behavior on an ion exchange column. The activity of the chloroplastic NADP-MDH in guard cells barely suffices to meet the malate requirement for stomatal opening in the light. It is therefore likely that NAD-MDHs residing in other compartments of the guard cells supplement the activity of the chloroplastic NADP-MDH particularly during stomatal opening in darkness.

Journal Article↗
PubMed1989

Comparative studies on the electrical properties of the H+ translocating ATPase and pyrophosphatase of the vacuolar-lysosomal compartment.

The electrical properties of the vacuolar-lysosomal H+ pumps were studied by direct measurement of the pump currents using the whole-cell configuration of the patch-clamp technique. Both pumps, the proton-translocating ATPase and pyrophosphatase, when activated by MgATP or inorganic Mg pyrophosphate (MgPP(i)), transport protons into the vacuole and polarize the membrane potential (positive inside the vacuole). Accumulation of protons in the lumen of vacuole vesicles was monitored by absorbance changes of the pH probe, acridine orange. The electrochemical gradient provided by both the ATPase and pyrophosphatase stimulates effectively the uptake of various metabolites such as malate, citrate and sucrose. The maximal current density produced by the ATPase was about 2.5 microA/cm2 and about 0.5 microA/cm2 for the pyrophosphatase. K(m)ATP was 0.6 mM; K(m)PPi was 15-20 microM with progressive inhibition above 150 microM. At a cytoplasmic pH of 7.5 both enzymes were capable of pumping protons against a 10,000-fold concentration gradient (pH 3.5 inside the vacuole). Proton current produced by the ATPase was blocked reversibly by extravacuolar NO(3)- only.

Adenosine Triphosphate↗
PubMed1989

Involvement of ion channels and active transport in osmoregulation and signaling of higher plant cells.

The transport of inorganic and organic ions across the plasma membrane and organelle membranes of higher plants by ion channels, electrogenic pumps and co-transporters is essential to vital processes such as osmoregulation, growth, development, signal transduction and the storage of solutes. Recent studies have led to the identification of specialized transport proteins in the plasma membrane and vacuolar membrane of higher plant cells. Here we have integrated the functional aspects of these membrane proteins into a model which proposes a novel basis for ion transport processes involved in the regulation of gas exchange in leaves.

Ion Channels↗