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)     

Slo3 K+ channels: voltage and pH dependence of macroscopic currents

The mouse Slo3 gene (KCNMA3) encodes a K(+) channel that is regulated by changes in cytosolic pH. Like Slo1 subunits responsible for the Ca(2+) and voltage-activated BK-type channel, the Slo3 alpha subunit contains a pore module with homology to voltage-gated K(+) channels and also an extensive cytosolic C terminus thought to be responsible for ligand dependence. For the Slo3 K(+) channel, increases in cytosolic pH promote channel activation, but very little is known about many fundamental properties of Slo3 currents. Here we define the dependence of macroscopic conductance on voltage and pH and, in particular, examine Slo3 conductance activated at negative potentials. Using this information, the ability of a Horrigan-Aldrich-type of general allosteric model to account for Slo3 gating is examined. Finally, the pH and voltage dependence of Slo3 activation and deactivation kinetics is reported. The results indicate that Slo3 differs from Slo1 in several important ways. The limiting conductance activated at the most positive potentials exhibits a pH-dependent maximum, suggesting differences in the limiting open probability at different pH. Furthermore, over a 600 mV range of voltages (-300 to +300 mV), Slo3 conductance shifts only about two to three orders of magnitude, and the limiting conductance at negative potentials is relatively voltage independent compared to Slo1. Within the context of the Horrigan-Aldrich model, these results indicate that the intrinsic voltage dependence (z(L)) of the Slo3 closed-open equilibrium and the coupling (D) between voltage sensor movement are less than in Slo1. The kinetic behavior of Slo3 currents also differs markedly from Slo1. Both activation and deactivation are best described by two exponential components, both of which are only weakly voltage dependent. Qualitatively, the properties of the two kinetic components in the activation time course suggest that increases in pH increase the fraction of more rapidly opening channels.     

Extracellular protons inhibit the activity of inward-rectifying potassium channels in the motor cells of Samanea saman pulvini.

The intermittent influx of K+ into motor cells in motor organs (pulvini) is essential to the rhythmic movement of leaves and leaflets in various plants, but in contrast to the K+ influx channels in guard cells, those in pulvinar motor cells have not yet been characterized. We analyzed these channels in the plasma membrane of pulvinar cell protoplasts of the nyctinastic legume Samanea saman using the patch-clamp technique. Inward, hyperpolarization-activated currents were separated into two types: time dependent and instantaneous. These were attributed, respectively, to K+ -selective and distinctly voltage-dependent K(H) channels and to cation-selective voltage-independent leak channels. The pulvinar K(H) channels were inhibited by external acidification (pH 7.8-5), in contrast to their acidification-promoted counterparts in guard cells. The inhibitory pH effect was resolved into a reversible decline of the maximum conductance and an irreversible shift of the voltage dependence of K(H) channel gating. The leak appeared acidification insensitive. External Cs (10 mM in 200 mM external K+) blocked both current types almost completely, but external tetraethylammonium (10 mM in 200 mM external K+) did not. Although these results do not link these two channel types unequivocally, both likely serve as K+ influx pathways into swelling pulvinar motor cells. Our results emphasize the importance of studying multiple model systems.     

ABA depolarizes guard cells in intact plants, through a transient activation of R- and S-type anion channels

During drought, the plant hormone abscisic acid (ABA) induces rapid stomatal closure and in turn reduces transpiration. Stomatal closure is accompanied by large ion fluxes across the plasma membrane, carried by K+ and anion channels. We recorded changes in the activity of these channels induced by ABA, for guard cells of intact Vicia faba plants. Guard cells in their natural environment were impaled with double-barrelled electrodes, and ABA was applied via the leaf surface. In 45 out of 85 cells tested, ABA triggered a transient depolarization of the plasma membrane. In these cells, the membrane potential partially recovered in the presence of ABA; however, a full recovery of the membrane potentials was only observed after removal of ABA. Repetitive ABA responses could be evoked in single cells, but the magnitude of the response varied from one hormone application to the other. The transient depolarization correlated with the activation of anion channels, which peaked 5 min after introduction of the stimulus. In guard cells with a moderate increase in plasma membrane conductance (DeltaG < 5 nS), ABA predominantly activated voltage-independent (slow (S)-type) anion channels. During strong responses (DeltaG > 5 nS), however, ABA activated voltage-dependent (rapid (R)-type) in addition to S-type anion channels. We conclude that the combined activation of these two channel types leads to the transient depolarization of guard cells. The nature of this ABA response correlates with the transient extrusion of Cl- from guard cells and a rapid but confined reduction in stomatal aperture.     

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)     

Two Voltage-Gated,Calcium Release Channels Coreside in the Vacuolar Membrane of Broad Bean Guard Cells

Voltage-gated, Ca2+ release channels have been characterized at the vacuolar membrane of broad bean guard cells using patch clamps of excised, inside-out membrane patches. The most prevalent Ca2+ release channel had a conductance of 27 pS over voltages negative of the reversal potential (Erev) (cytosol referenced to vacuole), with 5,10, or 20 mM Ca2+ as the charge carrier on the vacuolar side and 50 mM K+ on the cytosolic side. The single-channel current saturated at ~2.6 pA. The relative permeability of the channel was in the range of a Pca2+:Pk+ ratio of 6:1. Divalent cations could act as charge carriers on the vacuolar side with a conductance series of Ba2+ > Mg2+ > Sr2+ > Ca2+ and a selectivity sequence of Ca2+ [approximately equals to] Ba2+ [approximately equals to] Sr2+ > Mg2+. The channel was gated open by cytosol-negative (physiological) transmembrane voltages, increases in vacuolar Ca2+ concentration, and increases in the vacuolar pH. The channel was potently inhibited by the Ca2+ channel blockers Gd3+ (half-maximal inhibition at 10.3 [mu]M) and nifedipine (half-maximal inhibition at 77 [mu]M). The stilbene derivative 4,4[prime]-diisothiocyano-2,2[prime]-stilbene disulfonate was also inhibitory (half-maximal inhibition for a 4-min incubation period at 6.3[mu]M). The 27-pS channel coresides in individual guard cell vacuoles with a less frequently observed 14-pS Ca2+ release channel that had similar, although not identical, voltage dependence and gating characteristics and a lower selectivity for Ca2+ over K+. The requirement for two channels with a similar function at the vacuolar membrane of guard cells is discussed.     

Single guard cell recordings in intact plants: light-induced hyperpolarization of the plasma membrane

Guard cells are electrically isolated from other plant cells and therefore offer the unique possibility to conduct current- and voltage-clamp recordings on single cells in an intact plant. Guard cells in their natural environment were impaled with double-barreled electrodes and found to exhibit three physiological states. A minority of cells were classified as far-depolarized cells. These cells exhibited positive membrane potentials and were dominated by the activity of voltage-dependent anion channels. All other cells displayed both outward and inward rectifying K+-channel activity. These cells were either depolarized or hyperpolarized, with average membrane potentials of -41 mV (SD 16) and -112 mV (SD 19), respectively. Depolarized guard cells extrude K+ through outward rectifying channels, while K+ is taken up via inward rectifying channels in hyperpolarized cells. Upon a light/dark transition, guard cells that were hyperpolarized in the light switched to the depolarized state. The depolarization was accompanied by a 35 pA decrease in pump current and an increase in the conductance of inward rectifying channels. Both an increase in pump current and a decrease in the conductance of the inward rectifier were triggered by blue light, while red light was ineffective. From these studies we conclude that light modulates plasma membrane transport through large membrane potential changes, reversing the K+-efflux via outward rectifying channels to a K+-influx via inward rectifying channels.     

Mechanism of Ba2+ block of M-like K channels of rod photoreceptors of tiger salamanders

IKx is a voltage-dependent K+ current in the inner segment of rod photoreceptors that shows many similarities to M-current. The depression of IKx by external Ba2+ was studied with whole-cell voltage clamp. Ba2+ reduced the conductance and voltage sensitivity of IKx tail currents and shifted the voltage range over which they appeared to more positive potentials. These effects showed different sensitivities to Ba2+: conductance was the least sensitive (K0.5 = 7.6 mM), voltage dependence intermediate (K0.5 = 2.4 mM) and voltage sensitivity the most sensitive (K0.5 = 0.2 mM). Ca2+, Co2+, Mn2+, Sr2+, and Zn2+ did not have actions comparable to Ba2+ on the voltage dependence or the voltage sensitivity of IKx tail currents. In high K+ (100 mM), the voltage range of activation of IKx was shifted 20 mV negative, as was the tau-voltage relation. High K+ did not prevent the effect of Ba2+ on conductance, but abolished its ability to affect voltage dependence and voltage sensitivity. Ba2+ also altered the apparent time-course of activation and deactivation of IKx. Low Ba2+ (0.2 mM) slowed both deactivation and activation, with most effect on deactivation; at higher concentrations (1-25 mM), deactivation and activation time courses were equally affected, and at the highest concentrations, 5 and 25 mM Ba2+, the time course became faster than control. Rapid application of 5 mM Ba2+ suggested that the time dependent currents in Ba2+ reflect in part the slow voltage-dependent block and unblock of IKx channels by Ba2+. This blocking action of Ba2+ was steeply voltage- dependent with an apparent electrical distance of 1.07. Ba2+ appears to interact with IKx channels at multiple sites. A model which assumes that Ba2+ has a voltage-independent and a voltage-dependent blocking action on open or closed IKx channels reproduced many aspects of the data; the voltage-dependent component could account for both the Ba(2+)- induced shift in voltage dependence and reduction in voltage sensitivity of IKx tail currents.     

Calcium-Activated K+ Channels and Calcium-Induced Calcium Release by Slow Vacuolar Ion Channels in Guard Cell Vacuoles Implicated in the Control of Stomatal Closure

Stomatal closing requires the efflux of K+ from the large vacuolar organelle into the cytosol and across the plasma membrane of guard cells. More than 90% of the K+ released from guard cells during stomatal closure originates from the guard cell vacuole. However, the corresponding molecular mechanisms for the release of K+ from guard cell vacuoles have remained unknown. Rises in the cytoplasmic Ca2+ concentration have been shown to trigger ion efflux from guard cells, resulting in stomatal closure. Here, we report a novel type of largely voltage-independent K+-selective ion channel in the vacuolar membrane of guard cells that is activated by physiological increases in the cytoplasmic Ca2+ concentration. These vacuolar K+ (VK) channels had a single channel conductance of 70 pS with 100 mM KCI on both sides of the membrane and were highly selective for K+ over NH4+ and Rb+. Na+, Li+, and Cs+ were not measurably permeant. The Ca2+, voltage, and pH dependences, high selectivity for K+, and high density of VK channels in the vacuolar membrane of guard cells suggest a central role for these K+ channels in the initiation and control of K+ release from the vacuole to the cytoplasm required for stomatal closure. The activation of K+-selective VK channels can shift the vacuolar membrane to more positive potentials on the cytoplasmic side, sufficient to activate previously described slow vacuolar cation channels (SV-type). Analysis of the ionic selectivity of SV channels demonstrated a Ca2+ over K+ selectivity (permeability ratio for Ca2+ to K+ of ~3:1) of these channels in broad bean guard cells and red beet vacuoles, suggesting that SV channels play an important role in Ca2+-induced Ca2+ release from the vacuole during stomatal closure. A model is presented suggesting that the interaction of VK and SV channel activities is crucial in regulating vacuolar K+ and Ca2+ release during stomatal closure. Furthermore, the possibility that the ubiquitous SV channels may represent a general mechanism for Ca2+-induced Ca2+ release from higher plant vacuoles is discussed.     

The Role of Potassium Channels in the Temperature Control of Stomatal Aperture

We used the patch-clamp technique to examine the effect of temperature (13-36[deg]C) on the depolarization-activated K channels (KD channels) and on the hyperpolarization-activated channels (KH channels) in the plasma membrane of Vicia faba guard-cell protoplasts. The steady-state whole-cell conductance of both K channel types increased with temperature up to 20[deg]C. However, whereas the whole-cell conductance of the KH channels increased further and saturated at 28[deg]C, that of KD channels decreased at higher temperatures. The unitary conductance of both channel types increased with temperature like the rate of diffusion in water (temperature quotient of approximately 1.5), constituting the major contribution to the conductance increase in the whole cells. The mean number of available KH channels was not affected significantly by temperature, but the mean number of available KD channels increased significantly between 13 and 20[deg]C and declined drastically above 20[deg]C. This decrease and the reduced steady-state voltage-dependent probability of opening of the KD channels above 28[deg]C (because of a shift of voltage dependence by +21 mV) account for the depression of the whole-cell KD conductance at the higher temperatures. This may be a basic mechanism by which leaves of well-watered plants keep their stomata open during heat stress to promote cooling by transpiration.     

Interactions of protons with single open L-type calcium channels. Location of protonation site and dependence of proton-induced current fluctuations on concentration and species of permeant ion

We further investigated the rapid fluctuations between two different conductance levels promoted by protons when monovalent ions carry current through single L-type Ca channels. We tested for voltage dependence of the proton-induced current fluctuations and for accessibility of the protonation site from both sides of the membrane patch. The results strongly suggest an extracellular location of the protonation site. We also studied the dependence of the kinetics of the fluctuations and of the two conductance levels on the concentration of permeant ion and on external ionic strength. We find that saturation curves of channel conductance vs. [K] are similar for the two conductance levels. This provides evidence that protonation does not appreciably change the surface potential near the entry of the permeation pathway. The proton-induced conduction change must therefore result from an indirect interaction between the protonation site and the ion-conducting pathway. Concentration of permeant ion and ionic strength also affect the kinetics of the current fluctuations, in a manner consistent with our previous hypothesis that channel occupancy destabilizes the low conductance channel conformation. We show that the absence of measurable fluctuations with Li and Ba as charge carriers can be explained by significantly higher affinities of these ions for permeation sites. Low concentrations of Li reduce the Na conductance and abbreviate the lifetimes of the low conductance level seen in the presence of Na. We use whole-cell recordings to extrapolate our findings to the physiological conditions of Ca channel permeation and conclude that in the presence of 1.8 mM Ca no proton-induced fluctuations occur between pH 7.5 and 6.5. Finally, we propose a possible physical interpretation of the formal model of the protonation cycle introduced in the companion paper.     

Apical K+ channels in Necturus taste cells. Modulation by intracellular factors and taste stimuli

The apically restricted, voltage-dependent K+ conductance of Necturus taste receptor cells was studied using cell-attached, inside-out and outside-out configurations of the patch-clamp recording technique. Patches from the apical membrane typically contained many channels with unitary conductances ranging from 30 to 175 pS in symmetrical K+ solutions. Channel density was so high that unitary currents could be resolved only at negative voltages; at positive voltages patch recordings resembled whole-cell recordings. These multi-channel patches had a small but significant resting conductance that was strongly activated by depolarization. Patch current was highly K+ selective, with a PK/PNa ratio of 28. Patches containing single K+ channels were obtained by allowing the apical membrane to redistribute into the basolateral membrane with time. Two types of K+ channels were observed in isolation. Ca(2+)-dependent channels of large conductance (135-175 pS) were activated in cell-attached patches by strong depolarization, with a half-activation voltage of approximately -10 mV. An ATP-blocked K+ channel of 100 pS was activated in cell-attached patches by weak depolarization, with a half-activation voltage of approximately -47 mV. All apical K+ channels were blocked by the sour taste stimulus citric acid directly applied to outside-out and perfused cell-attached patches. The bitter stimulus quinine also blocked all channels when applied directly by altering channel gating to reduce the open probability. When quinine was applied extracellularly only to the membrane outside the patch pipette and also to inside-out patches, it produced a flickery block. Thus, sour and bitter taste stimuli appear to block the same apical K+ channels via different mechanisms to produce depolarizing receptor potentials.     

Gating of maxi K+ channels studied by Ca2+ concentration jumps in excised inside-out multi-channel patches (myocytes from guinea pig urinary bladder)

Currents through maxi K+ channels were recorded in inside-out macro-patches. Using a liquid filament switch (Franke, C., H. Hatt, and J. Dudel. 1987. Neurosci, Lett. 77:199-204) the Ca2+ concentration at the tip of the patch electrode ([Ca2+]i) was changed in less than 1 ms. Elevation of [Ca2+]i from less than 10 nM to 3, 6, 20, 50, 320, or 1,000 microM activated several maxi K+ channels in the patch, whereas return to less than 10 nM deactivated them. The time course of Ca(2+)-dependent activation and deactivation was evaluated from the mean of 10-50 sweeps. The mean currents started a approximately 10-ms delay that was attributed to diffusion of Ca2+ from the tip to the K+ channel protein. The activation and deactivation time courses were fitted with the third power of exponential terms. The rate of activation increased with higher [Ca2+]i and with more positive potentials. The rate of deactivation was independent of preceding [Ca2+]i and was reduced at more positive potentials. The rate of deactivation was measured at five temperatures between 16 and 37 degrees C; fitting the results with the Arrhenius equation yielded an energy barrier of 16 kcal/mol for the Ca2+ dissociation at 0 mV. After 200 ms, the time-dependent processes were in a steady state, i.e., there was no sign of inactivation. In the steady state (200 ms), the dependence of channel openness, N.P(o), on [Ca2+]i yielded a Hill coefficient of approximately 3. The apparent dissociation constant, KD, decreased from 13 microM at -50 mV to 0.5 microM at +70 mV. The dependence of N.P(o) on voltage followed a Boltzmann distribution with a maximal P(o) of 0.8 and a slope factor of approximately 39 mV. The results were summarized by a model describing Ca2+- and voltage-dependent activation and deactivation, as well as steady-state open probability by the binding of Ca2+ to three equal and independent sites within the electrical field of the membrane at an electrical distance of 0.31 from the cytoplasmic side.     

The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient

Voltage-activated H+ currents were studied in rat alveolar epithelial cells using tight-seal whole-cell voltage clamp recording and highly buffered, EGTA-containing solutions. Under these conditions, the tail current reversal potential, Vrev, was close to the Nernst potential, EH, varying 52 mV/U pH over four delta pH units (delta pH = pHo - pHi). This result indicates that H+ channels are extremely selective, PH/PTMA > 10(7), and that both internal and external pH, pHi, and pHo, were well controlled. The H+ current amplitude was practically constant at any fixed delta pH, in spite of up to 100-fold symmetrical changes in H+ concentration. Thus, the rate-limiting step in H+ permeation is pH independent, must be localized to the channel (entry, permeation, or exit), and is not bulk diffusion limitation. The instantaneous current- voltage relationship exhibited distinct outward rectification at symmetrical pH, suggesting asymmetry in the permeation pathway. Sigmoid activation kinetics and biexponential decay of tail currents near threshold potentials indicate that H+ channels pass through at least two closed states before opening. The steady state H+ conductance, gH, as well as activation and deactivation kinetic parameters were all shifted along the voltage axis by approximately 40 mV/U pH by changes in pHi or pHo, with the exception of the fast component of tail currents which was shifted less if at all. The threshold potential at which H+ currents were detectably activated can be described empirically as approximately 20-40(pHo-pHi) mV. If internal and external protons regulate the voltage dependence of gH gating at separate sites, then they must be equally effective. A simpler interpretation is that gating is controlled by the pH gradient, delta pH. We propose a simple general model to account for the observed delta pH dependence. Protonation at an externally accessible site stabilizes the closed channel conformation. Deprotonation of this site permits a conformational change resulting in the appearance of a protonation site, possibly the same one, which is accessible via the internal solution. Protonation of the internal site stabilizes the open conformation of the channel. In summary, within the physiological range of pH, the voltage dependence of H+ channel gating depends on delta pH and not on the absolute pH.     

Neutralization of gating charges in domain II of the sodium channel alpha subunit enhances voltage-sensor trapping by a beta-scorpion toxin

beta-Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3-S4 loop at the extracellular end of the S4 voltage sensor in domain II of the alpha subunit. Here, we probe the role of gating charges in the IIS4 segment in beta-scorpion toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances beta-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the beta-scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from -80 to -140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound toxin. Our results are consistent with a voltage-sensor-trapping model in which the beta-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances toxin-induced channel activation.     

Sensitivity of the sodium and potassium channels of Myxicola giant axons to changes in external pH

Myxicola giant axons were studied using standard voltage-clamp techniques in solutions whose pH values ranged from 3.9 to 10.2. Buffer concentrations of 50 mM or greater were necessary to demonstrate the full effect of pH. In acidic solutions the axon underwent a variable depolarization, and both the sodium and potassium conductances were reversibly depressed with approximate pKa's of 4.8 and 4.4, respectively. The voltage dependence of GNa was only slightly altered by acidic conditions, whereas there occurred large shifts in GK along the voltage axis consistent with a substantial decrease in net negative surface charge in the vicinity of the K+ channels. The sodium and potassium activation rate constants were decreased by acidic conditions, but the results could not be described as a simple translation along the voltage axis.     

Effect of external magnesium and calcium on human connexin46 hemichannels

One of the most striking features of hemi-gap-junctional channels is that they are dramatically modulated by extracellular divalent cations. In this study, we characterized the effects of external divalent cations and voltage on macroscopic human connexin46 (hCx46) hemi-gap-junctional currents using the two-electrode voltage-clamp technique. Increasing extracellular magnesium resulted in a shift of the voltage dependence of activation to more positive potentials, a decrease in the maximum conductance, an acceleration of deactivation, and a slowing of activation. Hyperpolarizing the membrane potential could mimic the effect of raising external magnesium on the activation kinetics and maximum conductance. These results could be interpreted in terms of a sequential model of channel activation with two independent divalent cation binding sites. This model could also explain the effects of external calcium on hCx46 hemichannels. However, the apparent binding affinities for calcium were significantly higher than for magnesium. In addition, we identified a mutation in the first extracellular domain of hCx46 (hCx46*N63S) that resulted in hemichannels that showed increased sensitivity to magnesium blockade.     

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)     

A Cationic Channel in the Guard Cell Tonoplast of Allium cepa

Stomatal movements depend on an osmoregulation process in which swelling or shrinking of the guard cells opens or closes the stomatal pore. Ions and water fluxes are an essential aspect of guard cell osmoregulation. Thus far, studies of these fluxes have focused on the guard cell plasma membrane. Guard cells, however, are a multi-compartment system that includes a prominent vacuole, which has a primary role in turgor regulation. This study reports on a detailed characterization of an ion channel at the guard cell tonoplast of Allium cepa (onion). We used patch-clamp methodology with isolated tonoplast patches to study conduction and gating at the single channel level. A voltage-dependent outward-rectifying cationic channel (210 picosiemens) was the dominant conductance. In symmetrical solutions the channel displayed an ohmic behavior in its current-voltage relationship. It also showed a very large rectification in the open probability. The channel was predominantly cationic and its sequence of ionic selectivity was weak (Na+ > K+ > Rb+ > Cs+). The channel conductance was not affected by intravacuolar pH. Analysis of membrane patches with multiple channels showed that the probability of a channel to open was independent of the opening of the other channels present in the patch and that there was a conservation of the open probability for different channels. Ensemble records generated using a pulse protocol showed slow activation and deactivation kinetics. A first-latency analysis of single-channel records in response to protocols with different prepulse duration indicated that this channel has more than one closed state.