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  • 1
    Electronic Resource
    Electronic Resource
    Palo Alto, Calif. : Annual Reviews
    Annual Review of Cell and Developmental Biology 16 (2000), S. 221-241 
    ISSN: 1081-0706
    Source: Annual Reviews Electronic Back Volume Collection 1932-2001ff
    Topics: Biology , Medicine
    Notes: Abstract Stomatal guard cells are unique as a plant cell model and, because of the depth of present knowledge on ion transport and its regulation, offer a first look at signal integration in higher plants. A large body of data indicates that Ca2+ and H+ act independently, integrating with protein kinases and phosphatases, to control the gating of the K+ and Cl- channels that mediate solute flux for stomatal movements. Oscillations in the cytosolic-free concentration of Ca2+ contribute to a signaling cassette, integrated within these events through an unusual coupling with membrane voltage for solute homeostasis. Similar cassettes are anticipated to include control pathways linked to cytosolic pH. Additional developments during the last two years point to events in membrane traffic that play equally important roles in stomatal control. Research in these areas is now adding entirely new dimensions to our understanding of guard cell signaling.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 100 (1997), S. 0 
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Multiple signalling pathways and their messengers – entailing changes in cytosolic-free Ca2+([Ca2−]). pH (pH) and protein phosphorylation – underpin K+and anion channel control during stomatal movements. This redundancy is wholly consistent with the ability of the guard cells to integrate the wide range of environmental and hormonal stimuli that affect stomatal aperture. Signal redundancy effects a spectrum of graded responses by linking pathways to gate signal transmission, and so boosts or mutes the final ‘integrated signal’ that reaches each ion channel. All evidence supports a role for the AB11 protein phosphatase and protein kinase elements in gating K+channel sensitivity to pH and ABA. Changes in [Ca2+]I. in turn, are demonstrably sensitive to pH1. Because each of these signal elements modulate and, in turn, are influenced by the activity of different sets of ion channels, the additional couplings engender a remarkably complex network, layering positive and negative controls with the ion channels that facilitate ion fluxes for stomatal movement.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 124 (1991), S. 95-112 
    ISSN: 1432-1424
    Keywords: stomatal guard cell ; abscisic acid ; ion channel activation ; signal transduction ; transport control ; H+-ATPase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 102 (1988), S. 235-246 
    ISSN: 1432-1424
    Keywords: stomatal guard cell ; K+ channel ; outward rectifier ; voltage-depending gating ; tetraethylammonium ; cyanide ; Vicia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Guard cells of higher plants control transpirational water loss and gas exchange for photosynthesis by opening and closing pores in the epidermis of the leaf. To power these turgordriven movements, guard cells accumulate (and lose) 200 to 400mm (1 to 3 pmol/cell) K+, fluxes thought to pass through K+ channels in the guard cells plasma membrane. Steady-state current-voltage (I–V) relations of intactVicia guard cells frequently show large, outward-going currents at potentials approaching 0 mV. Since this current could be carried by K+ channels, its pharmacology and dependence on external K+ (K v + ) has been examined under voltage clamp over an extended potential range. Measurements were carried out on cells which showed little evidence of primary “electrogenic” transport, thus simplifying analyses. Clamping these cells away from the free-running membrane potential (V m ) revealed an outward-rectifying current with instantaneous and time-dependent components, and sensitive to the K+ channel blocker tetraethylammonium chloride. The current declined also under metabolic blockade with NaCN and in the presence of diethylstilbesterol, responses which were attributed to secondary effects of these inhibitors. The putative K+ current rose with voltage positive toV m but it decayed over two voltage ranges, one negative toV m and one near +100 mV, to give steady-stateI–V relations with two regions of negative (slope) conductance. Voltage-dependent and kinetic characteristics of the current were affected by K v + and followed the K+ equilibrium potential. Against a (presumably) low background of primary ion transport, the K+ current contributed appreciably to charge balance atV m in 0.1mm as well as in 1 to 10mm K v + . Thus, gating of these K+ channels compensates for the prevailing K+ conditions to ensure net K+ movement out of the cell.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 72 (1983), S. 223-234 
    ISSN: 1432-1424
    Keywords: Neurospora ; microelectrode KCl leakage ; intracellular K+/Cl− ; Cl− permeability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Microcapillary electrodes filled with a variety of salt solutions, including 1m KCl, have been used to measure the membrane potentials and resistances of spherical cells from the mycelial fungusNeurospora (cell diameters 15–25 μm, cell volumes 3–8 pl). During impalements with electrodes containing 0.3–1.0m KCl, membrane potential and resistance decayed over a period of 3–10 min. In contrast, electrodes filled with 0.1m KCl gave stable membrane potentials of −180 mV and membrane resistivities of 40 kΩ cm2, values comparable to earlier results from the fungal hyphae. Salt leakage from 1.0m KCl-filled electrodes (tip diameters 0.2–0.3 μm, resistances 50–75 MΩ) occurred at rates of 4–5 fmol sec−1, as indicated by direct intracellular measurements with ion-sensitive microelectrodes. Depending on cell size, such leakage rates could elevate cytoplasmic KCl content at initial rates of 30–170 mM min−1, and actual values as high as 70mm min−1 were observed. Salt leakage and changes in cytoplasmic KCl concentration were reduced five- to sevenfold when impalements were made with electrodes containing 0.1m KCl. The effects on cell membrane parameters of salt leakage from microelectrodes could be attributed to chloride ions. Substitution of the KCl electrolyte with half-molar K2SO4 or Na2SO4 and molar concentrations of K- and Na-MES [potassium and sodium 2-(N-morpholino)ethanesulfonate] gave stable membrane potentials in excess of −200 mV and membrane resistivities greater than 50 kΩ cm2, while the permeant anions NO 3 − and SCN− depressed the membrane parameters in a manner similar to that observed with 1m KCl. Furthermore, modest elevation of cytoplasmic chloride concentration (below ca. 50 mM) affected both membrane potential and resistance in direct proportion to the concentration, and could be quantitatively described by the Constant Field Theory with a fixed membrane permeability (P Cl∼4×10−8 cm sec−1). Higher cytoplasmic chloride levels produced a collapse of the membrane resistance and drastic depolarization in a fashion requiring large changes of membrane permeability. At least for cells with volumes of 10 pl or less, the standard practice of filling electrodes with 1 or 3m KCl should be abandoned. Half-molar (and lower) concentrations of K2SO4 or Na2SO4 are suggested as satisfactory replacements.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-1424
    Keywords: Chara ; reaction kinetic carrier model ; H+-ATP ; (difference) current-voltage analysis ; nonlinear leak ; ATP ; intracellular pH ; extracellular pH
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary It is generally agreed that solute transport across theChara plasma membrane is energized by a proton electrochemical gradient maintained by an H+-extruding ATPase. Nonetheless, as deduced from steady-state current-voltage (I-V) measurements, the kinetic and thermodynamic constraints on H+-ATPase function remain in dispute. Uncertainties necessarily surround long-term effects of the relatively nonspecific antagonists used in the past; but a second, and potentially more serious problem has sprung from the custom of subtracting, across the voltage spectrum, currents recorded following pump inhibition from currents measured in the control. This practice must fail to yield the trueI-V profile for the pump when treatments alter the thermodynamic pressure on transport. We have reviewed these issues, using rapid metabolic blockade with cyanide and fitting the resultant whole-cellI-V and difference-current-voltage (dI-V) relations to a reaction kinetic model for the pump and parallel, ensemble leak. Measurements were carried out after blocking excitation with LaCl3, so that steady-state currents could be recorded under voltage clamp between −400 and +100 mV. Exposures to 1mm NaCN (CN) and 0.4mm salicylhydroxamic acid (SHAM) depolarized (positive-going)Chara membrane potentials by 44–112 mV with a mean half time of 5.4±0.8 sec (n=13). ATP contents, which were followed in parallel experiments, decayed coincidently with a mean half time of 5.3±0.9 sec ([ATP] t=0, 0.74±0.3mm; [ATP] t=x , 0.23±0.02mm). Current-voltage response to metabolic blockade was described quantitatively in context of these changes in ATP content and the consequent reduction in pump turnover rate accompanied by variable declines in ensemble leak conductance. Analyses ofdI-V curves (±CN+SHAM) as well as of families ofI-V curves taken at times during CN+SHAM exposures indicated a stoichiometry for the pump of one charge (H+) transported per ATP hydrolyzed and an equilibrium potential near −420 mV at neutral external pH; under these conditions, the pump accounted for approximately 60–75% of the total membrane conductance nearV m. Complementary results were obtained also in fitting previously publishedI-V data gathered over the external pH range 4.5–7.5 Kinetic features deduced for the pump were dominated by a slow step preceding H+ unloading outside, and by recycling and loading steps on the inside which were in rapid equilibrium. These characteristics predict, in marked contrast to the situation forNeurospora, that cytoplasmic acid loads inChara should shift the pumpI-V curve negative-going along the voltage axis with little change in maximum current output at positive voltages.
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  • 7
    ISSN: 1432-1424
    Keywords: stomatal guard cell ; (difference-) current-voltage relation ; conductance-voltage relation ; H+ pump ; kinetic carrier model ; K+ transport ; Vicia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The steady-state, current-voltage (I–V) characteristics of stomatal guard cells fromVicia faba L. were explored by voltage clamp using conventional electrophysiological techniques, but with double-barrelled microelectrodes containing 50mm K+-acetate. Attention was focused, primarily, on guard cell response to metabolic blockade. Exposures to 0.3–1.0mm NaCN and 0.4mm salicylhydroxamic acid (SHAM) lead consistently to depolarizing (positive-going) shifts in guard cell potentials (V m ), as large as +103 mV, which were generally complete within 60–90 sec (mean response half-time, 10.3±1.7 sec); values forV m in NaCN plus SHAM were close or positive to −100 mV and well removed from the K+ equilibrium potential. Guard cell ATP content, which was followed in parallel experiments, showed a mean half-time for decay of 10.8±1.9 ([ATP] t=0, 1.32±0.28mm; [ATP] t=60−180sec, 0.29±0.40mm). In respiring cells, theI–V relations were commonly sigmoid aboutV m or gently concave to the voltage axis positive toV m . Inward- and outward-rectifying currents were also observed, especially near the voltage extremes (nominally −350 and +50 mV). Short-circuit currents (atV=0 mV) were typically about 200–500 mA m−2. The principal effect of cyanide early on was to linearize theI–V characteristic while shifting it to the right along the voltage axis, to decrease the membrane conductance, and to reduce the short-circuit current by approx. 50–75%. The resulting difference-current-voltage (dI–V) curves (±cyanide) showed a marked sensitivity to voltages negative from −100 mV and, when clamp scans had been extended sufficiently, they revealed a distinct minimum near −300 mV before rising at still more negative potentials. The difference currents, along with changes in guard cell potential, conductance and ATP content are interpreted in context of a primary, ATP-consuming ion pump. FittingdI–V curves to reaction kinetic model for the pump [Hansen, U.-P., et al. (1981)J. Membrane Biol. 63:165; Blatt, M.R. (1986)J. Membrane Biol. 92:91] implicates a stoichiometry of one (+) charge transported outward for each ATP hydrolyzed, with pump currents as high as 200 mA m−2 at the free-running potential. The analysis indicates that the pump can comprise more than half of the total membrane conductance and argues against modulations of pump activity alone, as an effective means to controlling K+ transport for stomatal movements.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 92 (1986), S. 91-110 
    ISSN: 1432-1424
    Keywords: electrogenic transport ; reaction kinetic model ; conductance-voltage curve ; equilibrium potential ; equilibrium binding ; solute/substrate concentration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary A problem often confronted in analyses of chargecarrying transport processesin vivo lies in identifying porterspecific component currents and their dependence on membrane potential. Frequently, current-voltage (I–V)—or more precisely, difference-current-voltage (dI-V)—relations, both for primary and for secondary transport processes, have been extracted from the overall membrane current-voltage profiles by subtracting currents measured before and after experimental manipulations expected to alter the porter characteristics only. This paper examines the consequences of current subtraction within the context of a generalized kinetic carrier model for Class I transport mechanisms (U.-P. Hansen, D. Gradmann, D. Sanders and C.L. Slayman, 1981,J. Membrane Biol. 63:165–190). Attention is focused primarily ondI-V profiles associated with ion-driven secondary transport for which external solute concentrations usually serve as the experimental variable, but precisely analogous results and the same conclusions are indicated in relation to studies of primary electrogenesis. The model comprises a single transport loop linkingn (3 or more) discrete states of a carrier ‘molecule.’ State transitions include one membrane chargetransport step and one solute-binding step. Fundamental properties ofdI-V relations are derived analytically for alln-state formulations by analogy to common experimental designs. Additional features are revealed through analysis of a “reduced” 2-state empirical form, and numerical examples, computed using this and a “minimum” 4-state formulation, illustratedI-V curves under principle limiting conditions. Class I models generate a wide range ofdI-V profiles which can accommodate essentially all of the data now extant for primary and secondary transport systems, including difference current relations showing regions of negative slope conductance. The particular features exhibited by the curves depend on the relative magnitudes and orderings of reaction rate constants within the transport loop. Two distinct classes ofdI-V curves result which reflect the relative rates of membrane charge transit and carrier recycling steps. Also evident in difference current relations are contributions from ‘hidden’ carrier states not directly associated with charge translocation in circumstances which can give rise to observations of counterflow or exchange diffusion. Conductance-voltage relations provide a semi-quantitative means to obtaining pairs of empirical rate parameters. It is demonstrated thatdI-V relationscannot yield directly meaningful transport reversal potentials in most common experimental situations. Well-defined arramgements of reaction constants are shown to givedI-V curves which exhibit little or no voltage sensitivity and finite currents over many tens to hundreds of millivoltsincluding the true reversal potential. Furthermore, difference currents show apparent Michaelian kinetics with solute concentration atall membrane potentials. These findings bring into question several previous reports of reversal potentials, stoichiometries and apparent current-source behavior based primarily on difference current analysis. They also provide a coherent explanation for anomolous and shallow conductances and paradoxical situations in which charge stoichiometry varies with membrane potential.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 98 (1987), S. 169-189 
    ISSN: 1432-1424
    Keywords: voltage-dependent cotransport ; H+-K+ symport ; Neurospora ; current-voltage analysis ; ordered-binding models ; vanadate inhibition ; competitive activation ; linear mixed activation ; glucose-inhibited transport ; fungi
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Active transport of potassium in K+-starvedNeurospora was previously shown to resemble closely potassium uptake in yeast,Chlorella, and higher plants, for which K+ pumps or K+/H+-ATPases had been proposed. ForNeurospora, however, potassium-proton cotransport was demonstrated to operate, with a coupling ratio of 1 H+ to 1 K+ taken inward so that K+, but not H+, moves against its electrochemical gradient (Rodriguez-Navarro et al.,J. Gen. Physiol. 87:649–674). In the present experiments, the current-voltage (I–V) characteristic of K+−H+ cotransport in spherical cells ofNeurospora has been studied with a voltage-clamp technique, using difference-current methods to dissect it from other ion-transport processes in theNeurospora plasma membrane. Addition of 5-200 μM K+ to the bathing medium causes 10–150 mV depolarization of the unclamped membrane, and yields a sigmoidI–V curve with a steep slope (maximal conductance of 10–30 μS/cm2) for voltages of −300 to −100 mV, i.e., in the normal physiologic range. Outside that range the apparentI–V curve of the K+-H+ symport saturates for both hyperpolarization and depolarization. It fails to cross the voltage axis at its predicted reversal potential, however, an effect which can be attributed to failure of theI–V difference method under reversing conditions. In the absence of voltage clamping, inhibitors—such as cyanide or vanadate—which block the primary proton pump inNeurospora also promptly inhibit K+ transport and K+-H+ currents. But when voltage clamping is used to offset the depolarizing effects of pump blockade, the inhibitors have no immediate effect on K+-H+ currents. Thus, the inhibition of K+ transport usually observed with these agents reflects the kinetic effect of membrane depolarization rather than any direct chemical action on the cotransport system itself. Detailed study of the effects of [K+]o and pHo on theI–V curve for K+-H+ symport has revealed that increasing membrane potential systematicallydecreases the apparent affinity of the transporter for K+, butincreases affinity for protons (K m range: for [K+]o, 15–45 μM; for [H+]o, 10–35 nM). This behavior is consistent with two distinct reaction-kinetic models, in which (i) a neutral carrier binds K+ first and H+ last in the forward direction of transport, or (ii) a negatively charged carrier (−2) binds H+ first and K+ last.
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Planta 150 (1980), S. 354-356 
    ISSN: 1432-2048
    Keywords: Actin ; Chloroplast movement ; Heavy meromyosin ; Mougeotia ; Protoplast (Mougeotia)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The cell wall of the green alga Mougeotia was enzymatically digested by macerase and cellulysin. Released protoplasts were spread on poly-L-ornithine, formvar-carbon-coated grids, and cell fragments were collected for structural characterization. Large numbers of 5–7 nm filaments are seen which may be decorated with heavy meromyosin (HMM), a digest product of muscle myosin that binds specifically to actin, supporting the hypothesis that the phytochrome-mediated chloroplast movements in these algae are driven by a contractile complex of actomyosin.
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