Michael R. Blatt

Membrane transport in stomatal guard cells: The importance of voltage control

SummaryPotassium uptake and export in the resting conditions and in response to the phytohormone abscisic acid (ABA) were examined under voltage clamp in guard cells of Vicia faba L. In 0.1 mm external K+ (with 5 mm Ca2+-HEPES, pH 7.4) two distinct transport states could be identified based on the distribution of the free-running membrane voltage(VM) data in conjunction with the respective I-V and G-V relations. One state was dominated by passive diffusion (mean VM= −143± 4 mV), the other (mean VM= −237± 10 mV) exhibited an appreciable background of primary H+ transport activity. In the presence of pump activity the free-running membrane voltage was negative of the respective K+equilibrium potential (EK+), in 3 and 10 mm external K+. In these cases VMwas also negative of the activation voltage for the inward rectifying K+ current, thus creating a strong bias for passive K+ uptake through inward-rectifying K+ channels. In contrast, when pump activity was absent VMwas situated positive of EK+and cells revealed a bias for K+ efflux. Occasionally spontaneous voltage transitions were observed during which cells switched between the two states. Rapid depolarizations were induced in cells with significant pump activity upon adding 10 μm ABA to the medium. These depolarizations activated current through outward-rectifying K+ channels which was further amplified in ABA by a rise in the ensemble channel conductance. Current-voltage characteristics recorded before and during ABA treatments revealed concerted modulations in current passage through at least four distinct transport processes, results directly comparable to one previous study (Blatt, M.R., 1990, Planta 180:445) carried out with guard cells lacking detectable primary pump activity. Comparative analyses of guard cells in each case are consistent with depolarizations resulting from the activation of an inward-going, as yet unidentified current, rather than an ABA-induced fall in H+-ATPase output. Also observed in a number of cells was an inward-directed current which activated in ABA over a narrow range of voltages positive of -150 mV; this and additional features of the current suggest that it may reflect the ABA-dependent activation of an anion channel previously characterized in Vicia guard cell protoplasts, but rule out its function as the primary mechanism for initial depolarization. The analyses also yield indirect evidence for a rise in cytoplasmic Ca2+ activity in ABA, as well as for a K+ current distinct from the dominant inward and outward-rectifying K+ channels, but neither support nor discount a role for Ca2+ influx in depolarizing the membrane. A striking similarity was found for the modulation of inward currents either in response to ABA or after spontaneous depolarizations. This renders the possibility of an agonist (i.e., ABA) activated Ca2+ current across the plasma membrane as trigger for the voltage transitions unlikely.

Potassium channel currents in intact stomatal guard cells: rapid enhancement by abscisic acid.

Evidence of a role for abscisic acid (ABA) in signalling conditions of water stress and promoting stomatal closure is convincing, but past studies have left few clues as to its molecular mechanism(s) of action; arguments centred on changes in H+-pump activity and membrane potential, especially, remain ambiguous without the fundamental support of a rigorous electrophysiological analysis. The present study explores the response to ABA of K+ channels at the membrane of intact guard cells of Vicia faba L. Membrane potentials were recorded before and during exposures to ABA, and whole-cell currents were measured at intervals throughout to quantitate the steady-state and time-dependent characteristics of the K+ channels. On adding 10 μM ABA in the presence of 0.1, 3 or 10 mM extracellular K+, the free-running membrane potential (Vm) shifted negative-going (−)4–7 mV in the first 5 min of exposure, with no consistent effect thereafter. Voltage-clamp measurements, however, revealed that the K+-channel current rose to between 1.84- and 3.41-fold of the controls in the steady-state with a mean halftime of 1.1 ± 0.1 min. Comparable changes in current return via the leak were also evident and accounted for the minimal response in Vm. Calculated at Vm, the K+ currents translated to an average 2.65-fold rise in K+ efflux with ABA. Abscisic acid was not observed to alter either K+-current activation or deactivation.These results are consistent with an ABA-evoked mobilization of K+ channels or channel conductance, rather than a direct effect of the phytohormone on K+-channel gating. The data discount notions that large swings in membrane voltage are a prerequisite to controlling guard-cell K+ flux. Instead, thev highlight a rise in membrane capacity for K+ flux, dependent on concerted modulations of K+-channel and leak currents, and sufficiently rapid to account generally for the onset of K+ loss from guard cells and stomatal closure in ABA.

Potassium-dependent, bipolar gating of K+ channels in guard cells

SummaryGuard 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+ (Kv+) 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 (Vm) 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 toVm but it decayed over two voltage ranges, one negative toVm 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 Kv+ and followed the K+ equilibrium potential. Against a (presumably) low background of primary ion transport, the K+ current contributed appreciably to charge balance atVm in 0.1mm as well as in 1 to 10mm Kv+. Thus, gating of these K+ channels compensates for the prevailing K+ conditions to ensure net K+ movement out of the cell.

Electrical characteristics of stomatal guard cells: The contribution of ATP-dependent, “Electrogenic” transport revealed by current-voltage and difference-current-voltage analysis

SummaryThe 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 (Vm), as large as +103 mV, which were generally complete within 60–90 sec (mean response half-time, 10.3±1.7 sec); values forVm 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 aboutVm or gently concave to the voltage axis positive toVm. 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.

A cytolytic δ-endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid bilayers

In order to determine the mechanism of action of the 27 kDa mosquitocidal δ‐endotoxin of Bacillus thuringiensis var. israelensis we have studied its effects on the conductance of planar lipid bilayers. The toxin formed cation‐selective channels in the bilayers, permeable to K+ and Na+ but not to N‐methylglucamine or Cl−, showing very fast, cooperative opening and closing. Channel opening was greatly reduced in the presence of divalent cations (Ca2+, Mg2+) and the effect was reversed when these ions were removed. These results are consistent with our proposal that B. thuringiensis toxins act by a mechanism of colloid‐osmotic lysis.

Electrical characteristics of stomatal guard cells: The ionic basis of the membrane potential and the consequence of potassium chlorides leakage from microelectrodes

The membrane electrical characteristics of stomatal guard cells in epidermal strips from Vicia faba L. and Commelina communis L. were explored using conventional electrophysiological methods, but with double-barrelled microelectrodes containing dilute electrolyte solutions. When electrodes were filled with the customary 1–3 M KCl solutions, membrane potentials and resistances were low, typically decaying over 2–5 min to near-30 mV and <0.2 kω·cm2 in cells bathed in 0.1 mM KCl and 1 mM Ca2+, pH 7.4. By contrast, cells impaled with electrodes containing 50 or 200 mM K+-acetate gave values of-182±7 mV and 16±2 kω·cm2 (input resistances 0.8–3.1 Gω, n=54). Potentials as high as (-) 282 mV (inside negative) were recorded, and impalement were held for up to 2 h without appreciable decline in either membrane parameter. Comparison of results obtained with several electrolytes indicated that Cl- leakage from the microelectrode was primarily responsible for the decline in potential and resistance recorded with the molar KCl electrolytes. Guard cells loaded with salt from the electrodes also acquired marked potential and conductance responses to external Ca2+, which are tentatively ascribed to a K+ conductance (channel) at the guard cell plasma membrane.Measurements using dilute K+-acetate-filled electrodes revealed, in the guard cells, electrical properties common to plant and fungal cell membranes. The cells showed a high selectivity for K+ over Na+ (permeability ratio PNa/PK=0.006) and a near-Nernstian potential response to external pH over the range 4.5–7.4 (apparent PH/PK=500–600). Little response to external Ca2+ was observed, and the cells were virtually insensitive to CO2. These results are discussed in the context of primary, charge-carrying transport at the guard cell plasma membrane, and with reference to possible mechanisms for K+ transport during stomatal movements. They discount previous notions of Ca2+-and CO2-mediated transport control. It is argued, also, that passive (diffusional) mechanisms are unlikely to contribute to K+ uptake during stomatal opening, despite membrane potentials which, under certain, well-defined conditions, lie negative of the potassium equilibrium potential likely prevailing.

Mechanisms of fusicoccin action: evidence for concerted modulations of secondary K(+) transport in a higher plant cell.

Fusicoccin (FC) has long been known to promote K+ uptake in higher plant cells, including stomatal guard cells, yet the precise mechanism behind this enhancement remains uncertain. Membrane hyperpolarization, thought to arise from primary H+ pumping stimulated in FC, could help drive K+ uptake, but the extent to which FC stimulates influx and uptake frequently exceeds any reasonable estimates from Constant Field Theory based on changes in the free-running membrane potential (Vm) alone; furthermore, unidirectional flux analyses have shown that in the toxin K+ (86Rb+) exchange plummets to 10% of the control (G.M. Clint and E.A.C. MacRobbie 1984, J. Exp. Bot.35 180–192). Thus, the activities of specific pathways for K+ movement across the membrane could be modified in FC. We have explored a role for K+ channels in mediating these fluxes in guard cells ofVicia faba L. The correspondence between FC-induced changes in chemical (86Rb+) flux and in electrical current under voltage clamp was followed, using the K+ channel blocker tetraethylammonium chloride (TEA) to probe tracer and charge movement through K+-selective channels. Parallel flux and electrical measurements were carried out when cells showed little evidence of primary pump activity, thus simplifying analyses. Under these conditions, outward-directed K+ channel current contributed appreciably to charge balance maintainingVm, and adding 10 mM TEA to block the current depolarized (positive-going)Vm; TEA also reduced86Rb+ efflux by 68–80%. Following treatments with 10 μM FC, both K+ channel current and86Rb+ efflux decayed, irreversbly and without apparent lag, to 10%–15% of the controls and with equivalent half-times (approx. 4 min). Fusicoccin also enhanced86Rb+ influx by 13.9-fold, but the influx proved largely insensitive to TEA. Overall, FC promotednet cation uptake in 0.1 mM K+ (Rb+), despite membrane potentials which were 30–60 mVpositive of the K+ equilibrium potential. These results tentatively link (chemical) cation efflux to charge movement through the K+ channels. They offer evidence of an energy-coupled mechanism for K+ uptake in guard cells. Finally, the data reaffirm early suspicions that FC alters profoundly the K+ transport capacity of the cells, independent of any changes in membrane potential.

Voltage dependence of the Chara proton pump revealed by Current-voltage measurement during rapid metabolic blockade with cyanide

SummaryIt 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 nearVm. 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.

Mechanisms of fusicoccin action: A dominant role for secondary transport in a higher-plant cell

Fusicoccin (FC) is commonly thought to promote “electrogenic” H+ extrusion through its action on the H+-ATPase of the plant plasma membrane. Nonetheless, essential support from rigorous electrophysiological analysis has remained largely absent. The present investigation surveys the effects of FC on the charge transport properties at the membrane of a higher-plant cell — stomatal guard cells of Vicia faba L. — for which the electrical geometry is defined, and from which the voltage-dependent kinetic characteristic for the pump has been identified. Current-voltage (I-V) relations of the guard cells were determined before and during treatments with FC, and during brief exposures to NaCN plus salicylhydroxamic acid. Responses of the pump and of the ensemble of secondary transport processes were identified in the whole-membrane conductance-voltage relations and in the difference-current-voltage (dI-V) characteristic for the pump. In 0.1 mM K+, exposure to 10 μM FC shifted guard-cell potentials negative by 29–61 mV. Current-and conductance-voltage profiles indicated limited changes in the pump I-V characteristic, an observation which was confirmed through explicit kinetic analysis of pump dI-V relations. However, the voltage response was accompanied by a 1.5-to 2.6-fold fall in membrane conductance. These results challenge conventional views of fusicoccin action by ascribing the electrical responses to reduced current passage through secondary transport pathways as well as to enhanced electrogenic ion pumping.

Interpretation of steady-state current-voltage curves: Consequences and implications of current subtraction in transport studies

SummaryA 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.