Parallel control of the inward-rectifier K+ channel by cytosolic free Ca2+ and pH inVicia guard cells

The influence of cytosolic pH (pH_i) in controlling K⁺-channel activity and its interaction with cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K⁺-channel currents were recorded under voltage clamp while pH_i or [Ca²⁺]_i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K⁺ concentration, current through inward-rectifying K⁺ channels (I_K,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na⁺-acetate outside imposed acid loads, lowering pH_i from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pH_i was independent of the weak acid used, and indicated a H⁺-buffering capacity which rose from 90 mM H⁺/pH unit near 7.5 to 160 mM H⁺/pH unit near pH_i 7.0. With acid-going pH_i, (I_K,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pH_i lasting 3–5 min and was accompanied by a reduction in (I_K,in) before recovery of the initial resting pH_i and current amplitude. The pH_i-sensitivity of the current was consistent with a single, titratable site for H⁺ binding with a pK_a near 6.3. Acid pH_i loads also affected current through the outward-rectifying K⁺ channels (I_K,out) in a manner antiparallel to (I_K,in) The effect on I_K, out was also scalar, but showed an apparent pK_a of 7.4 and was best accommodated by a cooperative binding of two H⁺. Parallel measurements showed that Na⁺-butyrate loads were generally without significant effect on [Ca²⁺]_i, except when pH_i was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca²⁺]_i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pH_i changes and K⁺-channel responses. The action on [Ca²⁺]_i coincided with a greater variability in (I_K,in) stimulation evident at pH_i values around 7.0 and below, and with negative displacements in the voltage-dependence of (I_K,in) gating. These results distinguish the actions of pH_i and [Ca²⁺]_i in modulating (I_K,in) they delimit the effect of pH_i to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pH_i as a second messenger capable of acting in parallel with, but independent of [Ca²⁺]_i in controlling the K⁺ channels.Abbreviations BCECF 2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - g_K ensemble (steady-state) K⁺-channel conductance - I_K,out, I_K,in outward-, inward-rectifying K⁺ channel (current) - IN current-voltage (relation) - Mes 2-(N-morpholinolethanesulfonic acid - pH_i cytosolic pH - V membrane potential     

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

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.     

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

Potassium 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 VM was 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 VM was 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 microM 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.(ABSTRACT TRUNCATED AT 400 WORDS)     

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 ofVicia 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 M ABA in the presence of 0.1, 3 or 10 mM extracellular K⁺, the free-running membrane potential (V _m) 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 inV _m. Calculated atV _m, 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 membranecapacity 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.     

Permeation of Ca2+ through K+ channels in the plasma membrane of Vicia faba guard cells

Summary The whole-cell patch-clamp method has been used to measure Ca²⁺ influx through otherwise K⁺-selective channels in the plasma membrane surrounding protoplasts from guard cells of Vicia faba. These channels are activated by membrane hyperpolarization. The resulting K⁺ influx contributes to the increase in guard cell turgor which causes stomatal opening during the regulation of leaf-air gas exchange. We find that after opening the K⁺ channels by hyperpolarization, depolarization of the membrane results in tail current at voltages where there is no electrochemical force to drive K⁺ inward through the channels. Tail current remains when the reversal potential for permeant ions other than Ca²⁺ is more negative than or equal to the K⁺ equilibrium potential (–47 mV), indicating that the current is due to Ca²⁺ influx through the K⁺ channels prior to their closure. Decreasing internal [Ca²⁺] (Ca _i ) from 200 to 2 nm or increasing the external [Ca²⁺] (Ca _o ) from 1 to 10 mm increases the amplitude of tail current and shifts the observed reversal potential to more positive values. Such increases in the electrochemical force driving Ca²⁺ influx also decrease the amplitude of time-activated current, indicating that Ca²⁺ permeation is slower than K⁺ permeation, and so causes a partial block. Increasing Ca _o also (i) causes a positive shift in the voltage dependence of current, presumably by decreasing the membrane surface potential, and (ii) results in a U-shaped current-voltage relationship with peak inward current ca. –160 mV, indicating that the Ca^2– block is voltage dependent and suggesting that the cation binding site is within the electric field of the membrane. K⁺ channels in Zea mays guard cells also appear to have a Ca _i -, and Ca _o -dependent ability to mediate Ca²⁺ influx. We suggest that the inwardly rectiying K⁺ channels are part of a regulatory mechanism for Ca _i . Changes in Ca _o and (associated) changes in Ca _i regulate a variety of intracellular processes and ion fluxes, including the K⁺ and anion fluxes associated with stomatal aperture change.This work was supported by grants to S.M.A. from NSF (DCB-8904041) and from the McKnight Foundation. K.F.-G. is a Charles Gilbert Heydon Travelling Fellow. The authors thank Dr. R. MacKinnon (Harvard Medical School) and two anonymous reviewers for helpful comments.     

K outward rectifying channels as targets of phosphatase inhibitor deltamethrin inVicia faba guard cells

The dominant outward rectifier K⁺ currents were examined in protoplasts from Vicia faba guard cells. In whole-cell patch-clamp recordings, we generally observed that the conductance of the K⁺ inward and the outward rectifier gradually decreases with a half time in the order of 2.3 ± 0.7 min. As a consequence of this rundown, a new steady state was achieved which was 90 ± 5 percnt; lower than that obtained at the beginning of the recording. The rundown of the outward rectifier could be greatly reduced by pre-treating protoplasts either with the membrane permeable drug deltamethrin or by perfusing protoplasts with a pipette solution containing 5 μmol/L cyclosporine A. Furthermore, after the rundown, the conductance of the outward rectifier could be partially restored upon addition of 5 μmol/L deltamethrin to the bath medium. Since deltamethrin and cyclosporine A are established inhibitors of the calcium sensitive phosphatase calcineurin, the data argue for a participation of this type of phosphatase in the control of the activity of K⁺ outward rectifier channels in guard cells.     

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 k·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 k·cm² (input resistances 0.8–3.1 G, 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 P_Na/P_K=0.006) and a near-Nernstian potential response to external pH over the range 4.5–7.4 (apparent P_H/P_K=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.Abbreviations and symbols EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 2-(N-morpholino) propanesulfornic acid - E equilibrium potential - G_m membrane conductance - R_in input resistance - V_m membrane potential     

Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops fromChara australis

Summary Patch-clamp studies of cytoplasmic drops from the charophyteChara australis have previously revealed K⁺ channels combining high conductance (170 pS) with high selectivity for K⁺, which are voltage activated. The cation-selectivity sequence of the channel is shown here to be: K⁺>Rb⁺>NH ₄ ⁺ Na⁺ and Cl^–. Divalent cytosolic ions reduce the K⁺ conductance of this channel and alter its K⁺ gating in a voltage-dependent manner. The order of blocking potency is Ba²⁺>Sr²⁺>Ca²⁺>Mg²⁺. The channel is activated by micromolar cytosolic Ca²⁺, an activation that is found to be only weakly voltage dependent. However, the concentration dependence of calcium activation is quite pronounced, having a Hill coefficient of three, equivalent to three bound Ca²⁺ needed to open the channel. The possible role of the Ca²⁺-activated K⁺ channel in the tonoplast ofChara is discussed.     

K+ channels of stomatal guard cells. Characteristics of the inward rectifier and its control by pH

Intracellular microelectrode recordings and a two-electrode voltage clamp have been used to characterize the current carried by inward rectifying K+ channels of stomatal guard cells from the broadbean, Vicia faba L. Superficially, the current displayed many features common to inward rectifiers of neuromuscular and egg cell membranes. In millimolar external K+ concentrations (Ko+), it activated on hyperpolarization with half-times of 100-200 ms, showed no evidence of time- or voltage-dependent inactivation, and deactivated rapidly (tau approximately 10 ms) on clamping to 0 mV. Steady-state conductance-voltage characteristics indicated an apparent gating charge of 1.3-1.6. Current reversal showed a Nernstian dependence on Ko+ over the range 3-30 mM, and the inward rectifier was found to be highly selective for K+ over other monovalent cations (K+ greater than Rb+ greater than Cs+ much greater than Na+). Unlike the inward rectifiers of animal membranes, the current was blocked by charybdotoxin and alpha-dendrotoxin (Kd much less than 50 nM), as well as by tetraethylammonium chloride (K1/2 = 9.1 mM); gating of the guard cell K+ current was fixed to voltages near -120 mV, independent of Ko+, and the current activated only with supramillimolar K+ outside (EK+ greater than -120 mV). Most striking, however, was inward rectifier sensitivity to [H+] with the K+ current activated reversibly by mild acid external pH. Current through the K+ inward rectifier was found to be largely independent of intracellular pH and the current reversal (equilibrium) potential was unaffected by pHo from 7.4 to 5.5. By contrast, current through the K+ outward rectifier previously characterized in these cells (1988. J. Membr. Biol. 102:235) was largely insensitive to pHo, but was blocked reversibly by acid-going intracellular pH. The action of pHo on the K+ inward rectifier could not be mimicked by extracellular Ca2+ for which changes in activation, deactivation, and conductance were consonant with an effect on surface charge ([Ca2+] less than or equal to 1 mM). Rather, extracellular pH affected activation and deactivation kinetics disproportionately, with acid-going pHo raising the K+ conductance and shifting the conductance-voltage profile positive-going along the voltage axis and into the physiological voltage range. Voltage and pH dependencies for gating were consistent with a single, titratable group (pKa approximately 7 at -200 mV) residing deep within the membrane electric field and accessible from the outside.(ABSTRACT TRUNCATED AT 400 WORDS)     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

Oscillations of cytoplasmic concentrations of Ca2+ and K+ in fused L cells

Summary Using Ca²⁺- and K⁺-selective microelectrodes, the cytosolic free Ca²⁺ and K⁺ concentrations were measured in mouse fibroblastic L cells. When the extracellular Ca²⁺ concentration exceeded several micromoles, spontaneous oscillations of the intracellular free Ca²⁺ concentration were observed in the submicromolar ranges. During the Ca²⁺ oscillations, the membrane potential was found to oscillate concomitantly. The peak of cyclic increases in the free Ca²⁺ level coincided in time with the peak of periodic hyperpolarizations. Both oscillations were abolished by reducing the extracellular Ca²⁺ concentration down to 10^–7 m or by applying a Ca²⁺ channel blocker, nifedipine (50 m). In the presence of 0.5mm quinine, an inhibitor of Ca²⁺-activated K⁺ channel, sizable Ca²⁺ oscillations still persisted, while the potential oscillations were markedly suppressed. Oscillations of the intracellular K⁺ concentration between about 145 and 140mm were often associated with the potential oscillations. The minimum phase of the K⁺ concentration was always 5 to 6 sec behind the peak hyperpolarization. Thus, it is concluded that the oscillation of membrane potential results from oscillatory increases in the intracellular Ca²⁺ level, which, in turn, periodically stimulate Ca²⁺-activated K⁺ channels.     

Ca2+-activated K+ permeability in human erythrocytes: Modulation of single-channel events

Elevated levels of intracellular Ca²⁺ activate a K⁺-selective permeability in the membrane of human erythrocytes. Currents through single channels were analysed in excised inside-out membrane patches. The effects of several ions that are known to inhibit K⁺ fluxes are described with respect to the single-channel events. The results suggest that the blocking ions can partly move into the channels (but cannot penetrate) and interact with other ions inside the pore. The reduction of single-channel conductance by Cs⁺, tetraethylammonium and Ba²⁺ and of single-channel activity by quinine and Ba²⁺ is referred to different rates of access to the channel. The concentration- and voltage-dependent inhibition by ions with measurable permeability (Na⁺ and Rb⁺) can be explained by their lower permeability, with single-file movement and ionic interactions inside the pore.     

Voltage and Ca2+ activation of single large-conductance Ca2+-activated K+ channels described by a two-tiered allosteric gating mechanism

The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, q(eff), of 2.3 +/- 0.6 e(o)). Estimates of q(eff) were little changed for intracellular Ca2+ (Ca2+(i)) ranging from 0.0003 to 1,024 microM. Increasing Ca2+(i) from 0.03 to 1,024 microM shifted the voltage for half maximal activation (V(1/2)) 175 mV in the hyperpolarizing direction. V(1/2) was independent of Ca2+(i) for Ca2+(i) < or = 0.03 microM, indicating that the channel can be activated in the absence of Ca2+(i). Open and closed dwell-time distributions for data obtained at different Ca2+(i) and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (q(eff) = -0.5 e(o)) and an increase in the mean opening rate (q(eff) = 1.8 e(o)), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+(i) (approximately 0 through 1,024 microM), voltage (+80 to -80 mV), and Po (10(-4) to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.     

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).     

Spontaneous inactivation of the Ca-sensitive K channels of human red cells at high intracellular Ca activity

Ionophore A23187-mediated Ca²⁺-induced oscillations in the conductance of the Ca²⁺-sensitive K⁺ channels of human red cells were monitored with ion specific electrodes. The membrane potential was continuously reflected in CCCP-mediated pH changes in the buffer-free medium, changes in extracellular K⁺ activity were followed with a K⁺-selective electrode, and changes in the intracellular concentration of ionized calcium were calculated on the basis of cellular ⁴⁵Ca content. An increased cellular ⁴⁵Ca content at the successive minima of the oscillations where the K⁺ channels are closed indicates that the activation of the channels might be a (dCa²⁺/dt)-sensitive process and that accommodation to enhanced levels of intracellular free calcium may occur. An incipient inactivation of the K⁺ channels at intracellular ionized calcium levels of about 10 μM and a concurrent membrane potential of about −65 mV was observed. At a membrane potential of about −70 mV and an intracellular concentration of about 2·10⁻⁴M no inactivation of K⁺ channels took place. Inactivation of the K⁺ channels is suggested to be a compound function of the intracellular level of free calcium and the membrane potential. The observed sharp peak values in cellular ⁴⁵Ca content support the notion that a necessary component of the oscillatory system is a Ca²⁺ pump operating with a significant delay in the activation/inactivation process in response to changes in cellular concentration of ionized calcium.     

Characterization of the plasma-membrane H+-ATPase from Vicia faba guard cells

Stomatal movement is controlled by external and internal signals such as light, phytohormones or cytoplasmic Ca²⁺. Using Vicia faba L., we have studied the dose-dependent effect of auxins on the modulation of stomatal opening, mediated through the activity of the plasma-membrane H⁺-ATPase. The patch-clamp technique was used to elucidate the electrical properties of the H⁺-ATPase as effected by growth regulators and seasonal changes. The solute composition of cytoplasmic and extracellular media was selected to record pump currents directly with high resolution. Proton currents through the ATPase were characterized by a voltage-dependent increase in amplitude, positive to the resting potential, reaching a plateau at more depolarized values. Upon changes in extracellular pH, the resting potential of the cell shifted with a non-Nernst potential response (±21 mV), indicating the contribution of a depolarizing ionic conductance other than protons to the permeability of the plasma membrane. The use of selective inhibitors enabled us to identify the currents superimposing the H⁺-pump as carried by Ca²⁺. Auxinstimulation of this electroenzyme resulted in a rise in the outwardly directed H⁺ current and membrane hyperpolarization, indicating that modulation of the ATPase by the hormone may precede salt accumulation as well as volume and turgor increase. Annual cycles in pump activity (1.5–3.8 μA · cm^-2) were expressed by a minimum in pump current during January and February. Resting potentials of up to -260 mV and plasmamembrane surface area, on the other hand, did not exhibit seasonal changes. The pump activity per unit surface area was approximately 2- to 3-fold higher in guard cells than in mesophyll cells and thus correlates with their physiological demands.     

The Ca2+-extruding ATPase of the human platelet creates and responds to cytoplasmic pH changes,consistent with a 2 Ca2+/nH+ exchange mechanism

The Ca²⁺-extruding ATPase pump of the human platelet was studiedin situ by measuring Ca²⁺ extrusion from quin2-overloaded platelets (Johansson, J.S., Haynes, D.H. 1988.J. Membrane Biol. 104:147–163). Cytoplasmic pH (pH_cyt) was measured by BCECF fluorescence in parallel experiments. The pump was studied by raising the cytoplasmic free Ca²⁺ to 2.5 μM and monitoring active Ca²⁺ extrusion into a Ca²⁺-free medium. The pump was shown to perturb pH_cyt, to not respond to changes in membrane potential and to respond to imposed changes in pH_cyt in a manner consistent with the Ca²⁺ pump acting as a 2 Ca²⁺/nH⁺ exchanger. (i) Raising the external pH (pH_ext) from 7.40 to 7.60 lowers the V_max of the pump in basal condition (V_max,1) from 110±18 to 73±12 μM/min (=μmol/liter cell volume/min). (ii) Lowering pH_ext to 7.13 raised V_max,1 to 150±15 μM/min. (iii) In an N-methyl-d-glucamine (NMDG⁺) medium, the pump operation against high [Ca²⁺]_cyt acidifies the cytoplasm by −0.36±0.10 pH units, and the pump becomes self-inhibited. (iv) Use of nigericin to drive pH_cyt down to 6.23 reduces the V_max,1 to 18±11 μM/min. (v) Alkalinization of the cytoplasm by monensin in the presence of Na⁺ raises the V_max,1 (basal state withK _m,1=80 nM) to 136±24 μM/min, but also activates the pump fourfold (V_max,2=280±28 μM/min;K _m,2=502±36 nM). (vi) Transient elevation of pH_cyt by NH₄Cl at high [Ca²⁺]_cyt activates the pump eightfold (V_max,2≥671±350 μM/min). The large activation by alkaline pH_cyt at high [Ca²⁺]_cyt can be explained by Ca²⁺-calmodulin activation of the pump (Valant, P.A., Adjei, P.N., Haynes, D.H. 1992.J. Membrane Biol. 130:63–82) and by increased Ca²⁺ affinity of calmodulin at high pH.     

Recording of single K+ channels in the membrane of cytoplasmic drop ofChara australis

Summary The cytoplasmic drop formed of effused cytoplasm fromChara internodes is enclosed by a membrane. Patch clamp experiments have been carried out on this membrane, revealing a K⁺ channel as the most frequently detected ion translocator. The K⁺ channel is saturated at a level of about 20 pA inward and 10 pA outward current. The channel conductance is dependent on the accessability of K⁺ ions, its maximum value amounts to about 165 pS. The discrimination of Na⁺ and Cl^– is significant, permeability ratios P_Na/P_K and P_Cl/P_K were estimated to be 0.01 either. Binding experiments with the fluorescent probe concanavalin A/FITC suggest that the membrane is derived from the tonoplast.Abbreviations E_K K⁺ equilibrium potential - FITC fluorescein isothiocyanat - V_m membrane voltage - V_pip pipette clamp voltage - V_r reversal voltage