Inactivation of membrane-bound (Na+ +K+)-ATPase of Yoshida sarcoma cells and cobra venom cytotoxin complex with the glycolipid components of the enzyme system.

The molecular mechanisms involved in the inactivation of (Na+ + K+)-stimulated ATPase of Yoshida sarcoma cells by a cytotoxic protein (P6) from cobra venom have been examined. The overall data obtained using purified (Na+ + K+)-stimulated ATPase of Yoshida sarcoma cells suggest that cytotoxin P6 combines with phosphatidyl serine and a glycolipid which are closely associated with (Na+ + K+)-stimulated ATPase which in turn may lead to the inactivation of the enzyme in this cell system.     

Influence of charge on the inactivation of membrane bound (Na+ + K+)-ATPase of Yoshida sarcoma cells by inhibitor proteins from cobra venom.

Inactivation of (Na+ + K+)-ATPase of Yoshida sarcoma cells and beef brain microsomes by phospholipase A2 and a cytotoxin P6 from snake venom has been examined in relation to their activity to degrade phospholipids. Cytotoxin P6 which was most basic and devoid of phospholipase activity was most effective in inhibiting the (Na+ + K+)-ATPase of Yoshida sarcoma cells. Phospholipase A2 from Naja naja which was most active in degrading phospholipids was least effective in inhibiting (Na+ + K+)-ATPase in Yoshida sarcoma cells or in beef brain microsomes. Addition of trace amounts of cytotoxin P6 to the phospholipase considerably enhanced the inactivation of (Na+ + K+)-ATPase. The evidence suggests that the charge of the inhibitor protein and its specific structure play an important role in the inactivation of (Na+ + K+)-ATPase.     

Modulation of C-type inactivation by K+ at the potassium channel selectivity filter.

With prolonged or repetitive activation, voltage-gated K+ channels undergo a slow (C-type) inactivation mechanism, which decreases current flow through the channel. Previous observations suggest that C-type inactivation results from a localized constriction in the outer mouth of the channel pore and that the rate of inactivation is controlled by the-rate at which K+ leaves an unidentified binding site in the pore. We have functionally identified two K+ binding sites in the conduction pathway of a chimeric K+ channel that conducts Na+ in the absence of K+. One site has a high affinity for K+ and contributes to the selectivity filter mechanism for K+ over Na+. Another site, external to the high-affinity site, has a lower affinity for K+ and is not involved in channel selectivity. Binding of K+ to the high-affinity binding site slowed inactivation. Binding of cations to the external low-affinity site did not slow inactivation directly but could slow it indirectly, apparently by trapping K+ at the high-affinity site. These data support a model whereby C-type inactivation involves a constriction at the selectivity filter, and the constriction cannot proceed when the selectivity filter is occupied by K+.     

Solvent substitution as a probe of channel gating in Myxicola. Differential effects of D2O on some components of membrane conductance.

Careful examination of effects of solvent substitution on excitable membranes offers the theoretical possibility of identifying those aspects of the gating and translocation processes which are associated with significant changes in solvent order. Such information can then be used to develop or modify moire detailed models. We have examined the effects of heavy water substitution in Cs+-and K+-dialyzed Myxicola giant axons. At temperatures of 4-6 degrees C, the rates of Na+, K+, and Na+ inactivation during a maintained depolarization were all showed by approximately 50% in the presence of D2O. In contrast, the effects of solvent substitution on the time-course of prepulse inactivation and reactivation were much larger, with slowing averaging 160%. Studies at higher temperatures yielded Q10's for Na+ activation and K+ activation which were essentially comparable (0.72) and slightly but significantly smaller than that for inactivation during a maintained depolarization (0.84). In contrast, the Q10 for the D2O effect on prepulse inactivation was approximately 0.48. Heavy water substitution decrease Gk to a significantly greater extent than G(Na), while the decrease in the conductance of the Na+ channel caused by D2O was independent of whether the current-carrying species was Na+ or Li+. Sodium channel selectivity to the alkali metal cations and NH4+ was not changed by D2O substitution.     

Studies on (K+ + H+)-ATPase. I. Essential arginine residue in its substrate binding center

1. A membrane vesicle fraction containing a high (K+ + H+)-ATPase activity was isolated from porcine gastric mucosa. The enzyme has a pH optimum of 7.0 and is stimulated by T1+, K+, Rb+ and NH4+ with KA values of 0.13, 2.7, 7.6 and 26 mM, respectively, at this pH. 2. Incubation of the isolated membrane fraction with butanedione leads to inactivation of the (K+ + H+)-ATPase activity. The pH-dependence of the (K+ + H+)-ATPase activity. The pH-dependence of the inactivation and the reversibility of the reaction, observed after removal of excess butanedione and borate, indicate that modification of arginine is involved. 3. The inactivation of (K+ + H+)-ATPase activity by butanedione is time-dependent and follows second-order kinetics. From the dependence of the inactivation rate on the reagent concentration it appears that a single arginine residue is involved in the inactivation of the (K+ + H+)-ATPase activity. 4. ATP, deoxy-ATP, ADP and adenylyl imidodiphosphate (AMPPNP), but not CTP, GTP and ITP which are poor substrates, protect the enzyme against butanedione inactivation, suggesting that the essential arginine residue is located in the ATP binding centre. 5. In the presence of Mg2+ the butanedione inactivation is increased, and the protection by ATP, deoxy-ATP and ADP (but not that by AMPPNP) is less pronounced. This suggests that Mg2+ induces a conformational change in the enzyme, exposing the arginine group and coinciding with phosphorylation and subsequent release of ADP from its binding site.     

(Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421), calcium ions and ethylenediamine as the K(+)-site directed probe for Na+/K(+)-ATPase

Recently, we have shown that a hydrophobic amine (AU-1421) produces an irreversible inactivation of Na+/K(+)-ATPase activity. This inactivation was prevented by K+ and its congeners. In this study, we examined the possibility of Ca2+ or ethylenediamine as a probe of the K+ occlusion center of Na+/K(+)-ATPase. The inactivation by AU-1421 was prevented by Ca2+ with an apparent high affinity (approximately 0.1 mM). Ca2+ protection was cancelled by high concentrations of ATP, ADP or Mg2+. Ca2+ and K+ were similar in these respects. Kinetic analyses of the above data indicated the presence of two AU-1421 occlusion sites on the enzyme, either one of which is susceptible to Ca2+ occlusion. Ethylenediamine also prevented the inactivation by AU-1421 or by C12E8 solubilization of the enzyme, suggesting that ethylenediamine, like K+, stabilized the enzyme. However, an apparent affinity of ethylenediamine (approximately 1.4 mM) was one order of magnitude lower than that of K+ (approximately 0.2 mM), and the protective manner did not show a simple competition. In addition, ethylenediamine binding was unaffected by ATP or ADP at a low affinity site, and antagonized K+ binding. From these results we concluded that ethylenediamine does not act like K+ or Ca2+ in protecting AU-1421 inactivation, since it can't stabilize the enzyme conformation as an E2 (K(+)-bound form).     

Ionic events during the volume response of human peripheral blood lymphocytes to hypotonic media. II. Volume- and time-dependent activation and inactivation of ion transport pathways

Hypotonic dilution of human peripheral blood lymphocytes (PBL) induces large conductive permeabilities for K+ and Cl-, associated with the capacity of the cells to regulate their volumes. When rapid cation leakage is assured by the addition of the ionophore gramicidin, the behavior of the anion conductance pathway can be independently examined. Using this technique it is demonstrated that the volume- induced activation of Cl- transport is triggered at a threshold of approximately 1.15 X isotonic cell volume. If the volume of a cell is increased to this level or above, the Cl- transport system is activated, whereas if the volume of a swollen cell is decreased below the threshold value, the Cl- transport is inactivated. Activation and inactivation are independent of the relative volume changes and of the actual cellular Na+, K+, or Cl- concentrations, as well as of the changes in membrane potential in PBL. When net salt movement and thus volume change are inhibited by specific blockers of K+ transport (e.g., quinine, or Ca2+ depletion), volume-induced Cl- conductance shows a time-dependent inactivation, with a half-time of 5-8 min. The Cl- conductance, when activated, appears to involve an all-or-none response. In contrast, volume-induced K+ conductance is a graded response, with the increase in K+ flux being roughly proportional to the hypotonicity-induced increase in cell volume. The data indicate that during lymphocyte volume response in hypotonic media, anion conductance increases by orders of magnitude, exceeding the K+ conductance, so that the rate of the volume decrease (KCl efflux) is determined by a graded alteration in K+ conductance. When the cell volume approaches the isotonic value, it is stabilized by the inactivation of the anion conductance pathway.     

K+ channel inactivation mediated by the concerted action of the cytoplasmic N- and C-terminal domains.

We have examined the molecular mechanism of rapid inactivation gating in a mouse Shal K+ channel (mKv4.1). The results showed that inactivation of these channels follows a complex time course that is well approximated by the sum of three exponential terms. Truncation of an amphipathic region at the N-terminus (residues 2-71) abolished the rapid phase of inactivation (r = 16 ms) and altered voltage-dependent gating. Surprisingly, these effects could be mimicked by deletions affecting the hydrophilic C-terminus. The sum of two exponential terms was sufficient to describe the inactivation of deletion mutants. In fact, the time constants corresponded closely to those of the intermediate and slow phases of inactivation observed with wild-type channels. Further analysis revealed that several basic amino acids at the N-terminus do not influence inactivation, but a positively charged domain at the C-terminus (amino acids 420-550) is necessary to support rapid inactivation. Thus, the amphipathic N-terminus and the hydrophilic C-terminus of mKv4.1 are essential determinants of inactivation gating and may interact with each other to maintain the N-terminal inactivation gate near the inner mouth of the channel. Furthermore, this inactivation gate may not behave like a simple open-channel blocker because channel blockade by internal tetraethylammonium was not associated with slower current decay and an elevated external K+ concentration retarded recovery from inactivation.     

Voltage-dependent K+ currents and underlying single K+ channels in pheochromocytoma cells

Properties of the whole-cell K+ currents and voltage-dependent activation and inactivation properties of single K+ channels in clonal pheochromocytoma (PC-12) cells were studied using the patch-clamp recording technique. Depolarizing pulses elicited slowly inactivating whole-cell K+ currents, which were blocked by external application of tetraethylammonium+, 4-aminopyridine, and quinidine. The amplitudes and time courses of these K+ currents were largely independent of the prepulse voltage. Although pharmacological agents and manipulation of the voltage-clamp pulse protocol failed to reveal any additional separable whole-cell currents in a majority of the cells examined, single-channel recordings showed that, in addition to the large Ca++-dependent K+ channels described previously in many other preparations, PC-12 cells had at least four distinct types of K+ channels activated by depolarization. These four types of K+ channels differed in the open-channel current-voltage relation, time course of activation and inactivation, and voltage dependence of activation and inactivation. These K+ channels were designated the Kw, Kz, Ky, and Kx channels. The typical chord conductances of these channels were 18, 12, 7, and 7 pS in the excised configuration using Na+-free saline solutions. These four types of K+ channels opened in the presence of low concentrations of internal Ca++ (1 nM). Their voltage-dependent gating properties can account for the properties of the whole-cell K+ currents in PC-12 cells.     

An essential arginine residue in the ATP-binding centre of (Na+ + K+)-ATPase.

1. Incubation of purified (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3) from rabbit kidney outer medulla with butanedione in borate buffer leads to reversible inactivation of the (Na+ + K+)-ATPase activity. 2. The reaction shows second-outer kinetics, suggesting that modification of a single amino acid residue is involved in the inactivation of the enzyme. 3. The pH dependence of the reaction and the effect of borate ions strongly suggest that modification of an arginine residue is involved. 4. Replacement of Na+ by K+ in the butanedione medium decreases inactivation. 5. ATP, ADP and adenylyl imido diphosphate, particularly in the presence of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid to complex Mg2+, protect the enzyme very efficiently against inactivation by butanedione. 6. The (Na+ + Mg2+)-dependent phosphorylation capacity of the enzyme is inhibited in the same degree as the (Na+ + K+)-ATPase activity by butanedione. 7. The K+-stimulated p-nitrophenylphosphatase activity is much less inhibited than the (Na+ + K+)ATPase activity. 8. The ATP stimulation of the K+-stimulated p-nitrophenylphosphatase activity is inhibited by butanedione to the same extent as the (Na+ + K+)-ATPase activity. 9. Modification of sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoic acid) protects partially against the inactivating effect of butanedione. 10. The results suggest that an arginine residue is present in the nucleotide binding centre of the enzyme.     

Purification and characterization of (Na+, K+)-ATPase. V. Conformational changes in the enzyme Transitions between the Na-form and the K-form studied with tryptic digestion as a tool.

1. Purified (Na+, K+)-ATPase consisting of membrane fragments was digested with trypsin. The time course of enzyme inactivation was related to the electrophoretic pattern of native and cleaved proteins remaining in the membrane. 2. Differences in both the inactivation kinetics and the cleavage of the large chain (mol. wt 98 000) allow distinction of two patterns of tryptic digestion of (Na+, K+)-ATPase seen with Na+ or K+ in the medium. 3. With K+, the inactivation of (Na+, K+)-ATPase is linear with time in semilogarithmic plots and the activity is lost in parallel with cleavage of the large chain to fragments with molecular weights 58 000 and 48 000. 4. With Na+, the inactivation curves are biphasic. In the initial phase of rapid inactivation, 50% of the activity is lost with minor changes in the composition of the large chain. In the final phase, the large chain is cleaved at a low rate to a fragment with a molecular weight of 78 000. 5. It is concluded that the regions of the large chain exposed in the presence of K+ are distinct from the regions exposed in presence of Na+ and that two conformations of (Na+, K+)-ATPase can be sensed with trypsin, a (t)K-form and a (t)Na-form. 6. Reaction of the (t)K-form with ATP cause transition to the (t)Na-form. Relatively high concentrations of ATP are required and Mg2+ is not necessary. Phosphorylation of (Na+, K+)-ATPase is accompanied by transition from the (t)Na-form to the (t)K-form. Previous kinetic data suggest that these conformational changes are accompanied by shifts in the affinities of the enzyme for Na+ and K+.     

Purification from brain of an intrinsic membrane protein fraction enriched in (Na+ + K+)-ATPase.

A microsomal fraction from canine brain gray matter has been extracted with the detergent sodium dodecyl sulfate to partially purify the membrane-bound (Na+ + K+)-stimulated adenosine triphosphatase. Phospholipid, glycolipid, and a family of other glycoproteins are also enriched by the procedure; it is proposed that the product is an intrinsic membrane protein fraction. 6--8-fold purification of (Na+ + K+)-ATPase is obtained without solubilizing the enzyme and without irreversibly altering its turnover number. Final specific activities are 350--400 mumol of ATP hydrolyzed/h per mg protein. The stimulation and reversible inactivation of the (Na+ + K+)-ATPase by dodecyl sulfate were examined for information relevant to the mechanism of action of the detergent.     

A delayed rectifier potassium current in Xenopus oocytes.

A delayed voltage-dependent K+ current endogenous to Xenopus oocytes has been investigated by the voltage-clamp technique. Both activation and inactivation of the K+ current are voltage-dependent processes. The K+ currents were activated when membrane potential was depolarized from a holding potential of -90 to -50 mV. The peak current was reached within 150 ms at membrane potential of +30 mV. Voltage-dependent inactivation of the current was observed by depolarizing the membrane potential from -50 to 0 mV at 10-mV increments. Voltage-dependent inactivation was a slow process with a time constant of 16.5 s at -10 mV. Removal of Ca2+ from the bath has no effect on current amplitudes, which indicates that the current is Ca2+)-insensitive. Tail current analysis showed that reversal potentials were shifted by changing external K+ concentration, as would be expected for a K(+)-selective channel. The current was sensitive to quinine, a K+ channel blocker, with a Ki of 35 microM. The blockade of quinine is voltage-independent in the range of -20 to +60 mV. Whereas oocytes from the same animal have a relatively homogeneous current distribution, average amplitude of the K+ current varied among oocytes from different animals from 30 to 400 nA at membrane potential of +30 mV. Our results indicate the presence of the endogenous K+ current in Xenopus oocytes with characteristics of the delayed rectifier found in some nerve and muscle cells.     

Single channel studies of the phosphorylation of K+ channels in the squid giant axon. II. Nonstationary conditions

The effects of phosphorylation on the properties of the 20-pS channel of the squid giant axon were studied using the cut-open axon technique. Phosphorylation of the channel was achieved by photoreleasing caged ATP (inside the patch pipette) in the presence of the catalytic subunit of the protein kinase A. An inverted K+ gradient (500 K+ external parallel 5 K+ internal) was used to study the activation process. Phosphorylation decreased the frequency of openings of the channel at most potentials by shifting the probability vs. voltage curve toward more positive potentials. The mean open times showed no voltage dependence and were not affected by phosphorylation. The distribution of first latencies, on the other hand, displayed a sharp voltage dependence. Phosphorylation increased the latency to the first opening at all potentials, shifting the median first latency vs. voltage curve toward more positive potentials. The slow inactivation process was studied in the presence of a physiological K+ gradient (10 K+ external parallel 310 K+ internal). Pulses to 40 mV from different holding potentials were analyzed. Phosphorylation increases the overall ensemble probability by decreasing the number of blank traces. A single channel inactivation curve was constructed by computing the relative appearance of blank traces at different holding potentials before and after photoreleasing caged ATP. As determined in dialyzed axons, the effect of phosphorylation consisted in a shift of the inactivation curve toward more positive potentials. The 20-pS channel has the same characteristics as the delayed rectifier current in activation kinetics, steady-state inactivation, and phosphorylation effects.     

The reaction of sulfhydryl groups of sodium and potassium ion-activated adenosine triphosphatase with N-ethylmaleimide. The relationship between ligand-dependent alterations of nucleophilicity and enzymatic conformational states

The reaction between N-ethylmaleimide and (Na+ + K+)-ATPase, performed under ligand conditions which produce each of the kinetic states of the enzyme and their associated conformational forms, was examined through an analysis of the inhibition of enzymatic activity and the incorporation of radiolabeled reagent into the enzyme. The inactivation reactions displayed pseudo-first order kinetics with respect to the concentration of active enzyme, indicating that the loss of activity is associated with the alkylation of a unique sulfhydryl group. In the absence of enzyme phosphorylation, the nucleophilicity of this sulfhydryl group is affected primarily by the nature of the monovalent cation present and does not correlate with the conformational state. A method for determining the actual concentration and specific radioactivity of radiolabeled N-ethylmaleimide during the reaction with (Na+ + K+)-ATPase was developed, allowing the measurement of the total reactive sulfhydryl groups of native (Na+ + K+)-ATPase under conditions identical with those of the inactivation studies. The labeling of the enzyme complex is associated almost exclusively with the large polypeptide, which contains four sulfhydryl groups which react with this reagent. One of these residues is presumably the sulfhydryl responsible for inactivation of the enzyme. Two react stoichiometrically and rapidly with N-ethylmaleimide under all conditions. The nucleophilicity of the fourth sulfhydryl group is governed by the conformational state of the enzyme, but the alkylation of this residue does not result in loss of enzymatic activity.     

A novel hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine, as a probe of the K+ occlusion center of Na+/K(+)-ATPase

A hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421), was examined as a probe of the K+ occlusion center of Na+/K(+)-ATPase. Treatment of the enzyme with AU-1421 at 37 degrees C and pH 7.0 produced irreversible inactivation of the enzyme. This inactivation was prevented, with simple competitive kinetics, by K+ or its congeners in the order of Tl+ greater than Rb+ greater than NH+4 greater than Cs+. The concentrations of these cations required for the protection, were consistent with the affinities for transport and ATPase activity. The apparent binding constant for K+ was calculated to be 0.03 mM, from the competition with AU-1421. This protection was cancelled by a high concentration of ATP or ADP. A high concentration of Na+ (Kd = 6.5-6.9 mM), as a substitute for K+, also prevented the inactivation by AU-1421. Thus, the enzyme was protected from AU-1421 when the occlusion center was occupied by a monovalent cation, irrespective of the enzyme conformation, E1 (Na(+)-bound form) or E2 (K(+)-bound form). On the other hand, the enzyme was most sensitive to AU-1421 in the presence of low concentration of Na+ (0.4-0.8 mM) or a high concentration of ATP. Tris, imidazole or choline, which favors the E1 state, also accelerated the inactivation by AU-1421. These suggest that AU-1421 reacts with the occlusion center through the E1 state.     

External pore collapse as an inactivation mechanism for Kv4.3 K+ channels

Kv4 channels are thought to lack a C-type inactivation mechanism (collapse of the external pore) and to inactivate as a result of a concerted action of cytoplasmic regions of the channel. To investigate whether Kv4 channels have outer pore conformational changes during the inactivation process, the inactivation properties of Kv4.3 were characterized in 0 mM and in 2 mM external K+ in whole-cell voltage-clamp experiments. Removal of external K+ increased the inactivation rates and favored cumulative inactivation by repetitive stimulation. The reduction in current amplitude during repetitive stimulation and the faster inactivation rates in 0 mM external K+ were not due to changes in the voltage dependence of channel opening or to internal K+ depletion. The extent of the collapse of the K+ conductance upon removal of external K+ was more pronounced in NMG+-than in Na+-containing solutions. The reduction in the current amplitude during cumulative inactivation by repetitive stimulation is not associated with kinetic changes, suggesting that it is due to a diminished number of functional channels with unchanged gating properties. These observations meet the criteria for a typical C-type inactivation, as removal of external K+ destabilizes the conducting state, leading to the collapse of the pore. A tentative model is presented, in which K+ bound to high-affinity K+-binding sites in the selectivity filter destabilizes an outer neighboring K+ modulatory site that is saturated at approximately 2 mM external K+. We conclude that Kv4 channels have a C-type inactivation mechanism and that previously reported alterations in the inactivation rates after N- and C- termini mutagenesis may arise from secondary changes in the electrostatic interactions between K+-binding sites in the selectivity filter and the neighboring K+-modulatory site, that would result in changes in its K+ occupancy.     

The cytosolic inactivation domains of BKi channels in rat chromaffin cells do not behave like simple, open-channel blockers.

Most BK-type voltage- and Ca(2+)-dependent K+ channels in rat chromaffin cells exhibit rapid inactivation. This inactivation is abolished by brief trypsin application to the cytosolic face of membrane patches. Here we examine the effects of cytosolic channel blockade and pore occupancy on this inactivation process, using inside-out patches and whole-cell recordings. Occupancy of a superficial pore-blocking site by cytosolic quaternary blockers does not slow inactivation. Occupancy of a deeper pore-blocking site by cytosolic application of Cs+ is also without effect on the onset of inactivation. Although the rate of inactivation is relatively unaffected by changes in extracellular K+, the rate of recovery from inactivation (at -80 and -140 mV with 10 microM Ca2+) is faster with increases in extracellular K+ but is unaffected by the impermeant ion, Na+. When tail currents are compared after repolarization, either while channels are open or after inactivation, no channel reopening is detectable during recovery from inactivation. BK inactivation appears to be mechanistically distinct from that of other inactivating voltage-dependent channels. Although involving a trypsin-sensitive cytosolic structure, the block to permeation does not appear to occur directly at the cytosolic mouth or inner half of the ion permeation pathway.     

Characterization of a mammalian cDNA for an inactivating voltage-sensitive K+ channel.

A cDNA clone encoding a K+ channel polypeptide with 72% amino acid sequence identity to Drosophila Shal was isolated from rat hippocampus. Functional expression of the cDNA in Xenopus oocytes generated 4-amino-pyridine-sensitive K+ channels displaying rapid inactivation kinetics. The fastest component of inactivation was slowed by the deletion of 3 basic residues in the amino-terminal region. Northern blots revealed that the mRNA encoding this K+ channel polypeptide was expressed at a similar level in the brain and in the heart. In situ hybridization revealed that the mRNA encoding this K+ channel appeared concentrated in the hippocampus, dentate gyrus, and habenular nucleus in the brain. Thus, this K+ channel polypeptide is likely to form some of the A-type K+ channels expressed in the mammalian nervous system and heart.     

Transient outward K+ currents across the plasma membrane of laticifer from Hevea brasiliensis.

Non-inactivating outward rectifying K+ channel currents have been identified in a variety of plant cell types and species. The present study of laticifer protoplasts from Hevea brasiliensis, cells which are specialized for stress response, has revealed, through a switch-clamp method, an outward rectifying current displaying rapid inactivation. The inactivation depended on the external K+ concentration and on the voltage. This current inactivation appeared clearly different from all those previously described in plant cells and it shared homology with current kinetics of animal Shaker family channels. These results, given the recent cloning of plant K+ channel beta-subunits, shed new light on possible plant K+ channel regulation.