K+-Sensitive Gating of the K+ Outward Rectifier in Vicia Guard Cells

The effect of extracellular cation concentration and membrane voltage on the current carried by outward-rectifying K⁺ channels was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with double-barrelled microelectrodes and the K⁺ current was monitored under voltage clamp in 0.1–30 mm K⁺ and in equivalent concentrations of Rb⁺, Cs⁺ and Na⁺. From a conditioning voltage of −200 mV, clamp steps to voltages between −150 and +50 mV in 0.1 mm K⁺ activated current through outward-rectifying K⁺ channels (I _{K, out}) at the plasma membrane in a voltage-dependent fashion. Increasing [K⁺] _o shifted the voltage-sensitivity of I _{K, out} in parallel with the equilibrium potential for K⁺ across the membrane. A similar effect of [K⁺] _o was evident in the kinetics of I _{K, out} activation and deactivation, as well as the steady-state conductance- (g _K ⁻) voltage relations. Linear conductances, determined as a function of the conditioning voltage from instantaneous I-V curves, yielded voltages for half-maximal conductance near −130 mV in 0.1 mm K⁺, −80 mV in 1.0 mm K⁺, and −20 mV in 10 mm K⁺. Similar data were obtained with Rb⁺ and Cs⁺, but not with Na⁺, consistent with the relative efficacy of cation binding under equilibrium conditions (K⁺≥ Rb⁺ > Cs⁺ > > Na⁺). Changing Ca²⁺ or Mg²⁺ concentrations outside between 0.1 and 10 mm was without effect on the voltage-dependence of g _K or on I _{K, out} activation kinetics, although 10 mm [Ca²⁺] _o accelerated current deactivation at voltages negative of −75 mV. At any one voltage, increasing [K⁺] _o suppressed g _K completely, an action that showed significant cooperativity with a Hill coefficient of 2. The apparent affinity for K⁺ was sensitive to voltage, varying from 0.5 to 20 mm with clamp voltages near −100 to 0 mV, respectively. These, and additional data indicate that extracellular K⁺ acts as a ligand and alters the voltage-dependence of I _{K, out} gating; the results implicate K⁺-binding sites accessible from the external surface of the membrane, deep within the electrical field, but distinct from the channel pore; and they are consistent with a serial 4-state reaction-kinetic model for channel gating in which binding of two K⁺ ions outside affects the distribution between closed states of the channel. Received: 27 November 1996/Revised: 4 March 1997     

Allosteric Effects of Permeating Cations on Gating Currents during K+ Channel Deactivation

K⁺ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K⁺ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K⁺ or Cs⁺, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K⁺ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs⁺ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at E_Cs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K⁺ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.     

Clustered Distribution of Calcium Sensitivities: An Indication of Hetero-tetrameric Gating Components in Ca2+-activated K+ Channels Reconstituted from Avian Nasal Gland Cells

High-conductance, Ca²⁺-activated K⁺ channels from the basolateral membrane of rabbit distal colon epithelial cells were reconstituted into planar phospholipid bilayers to examine the effect of Mg²⁺ on the single-channel properties. Mg²⁺ decreases channel current and conductance in a concentration-dependent manner from both the cytoplasmic and the extracellular side of the channel. In contrast to other K⁺ channels, Mg²⁺ does not cause rectification of current through colonic Ca²⁺-activated K⁺ channels. In addition, cytoplasmic Mg²⁺ decreases the reversal potential of the channel. The Mg²⁺-induced decrease in channel conductance is relieved by high K⁺ concentrations, indicating competitive interaction between K⁺ and Mg²⁺. The monovalent organic cation choline also decreases channel conductance and reversal potential, suggesting that the effect is unspecific. The inhibition of channel current by Mg²⁺ and choline most likely is a result of electrostatic screening of negative charges located superficially in the channel entrance. But in addition to charge, other properties appear to be necessary for channel inhibition, as Na⁺ and Ba²⁺ are no (or only weak) inhibitors. Mg²⁺ and possibly other cations may play a role in the regulation of current through these channels. Received: 25 August 1995/Revised: 16 November 1995     

Na+ Sensitivity of ROMK1 K+ Channel: Role of the Na+/H+ Antiporter

To examine the extracellular Na⁺ sensitivity of a renal inwardly rectifying K⁺ channel, we performed electrophysiological experiments on Xenopus oocytes or a human kidney cell line, HEK293, in which we had expressed the cloned renal K⁺ channel, ROMK1 (Kir1.1). When extracellular Na⁺ was removed, the whole-cell ROMK1 currents were markedly suppressed in both the oocytes and HEK293 cells. Single-channel ROMK1 activities recorded in the cell-attached patch on the oocyte were not affected by removal of Na⁺ from the pipette solution. However, macro-patch ROMK1 currents recorded on the oocyte were significantly suppressed by Na⁺ removal from the bath solution. A blocker of Na⁺/H⁺ antiporters, amiloride, largely inhibited the Na⁺ removal-induced suppression of whole-cell ROMK1 currents in the oocytes. The pH-insensitive K80M mutant of ROMK1 was much less sensitive to Na⁺ removal. Na⁺ removal was found to induce a significant decrease in intracellular pH in the oocytes using H⁺-selective microelectrodes. Coexpression of ROMK1 with NHE3, which is a Na⁺/H⁺ antiporter isoform of the kidney apical membrane, conferred increased sensitivity of ROMK1 channels to extracellular Na⁺ in both the oocytes and HEK293 cells. Thus, it is concluded that the ROMK1 channel is regulated indirectly by extracellular Na⁺, and that the interaction between NHE transporter and ROMK1 channel appears to be involved in the mechanism of Na⁺ sensitivity of ROMK1 channel via regulating intracellular pH. Received: 13 April 1999/Revised: 15 July 1999     

Ion Channel Permeable for Divalent and Monovalent Cations in Native Spinach Thylakoid Membranes

A cation-selective channel was characterized in isolated patches from osmotically swollen thylakoids of spinach (Spinacea oleracea). This channel was permeable for K⁺ as well as for Mg²⁺ and Ca²⁺ but not for Cl⁻. When K⁺ was the main permeant ion (symmetrical 105 mm KCl) the conductance of the channel was about 60 pS. The single channel conductance for different cations followed a sequence K⁺ > Mg²⁺≥ Ca²⁺. The permeabilities determined by reversal potential measurements were comparable for K⁺, Ca²⁺, and Mg²⁺. The cation channel displayed bursting behavior. The total open probability of the channel increased at more positive membrane potentials. Kinetic analysis demonstrated that voltage dependence of the total open probability was determined by the probability of bursts formation while the probability to find the channel in open state within a burst of activity was hardly voltage-dependent. The cation permeability of intact spinach thylakoids can be explained on the single channel level by the data presented here. Received: 26 December 1995/Revised: 17 April 1996     

Gating Properties of Inward-Rectifier Potassium Channels: Effects of Permeant Ions

Two inward-rectifier K⁺ channels, ROMK2 (Kir1.1b) and IRK1 (Kir2.1), were expressed in Xenopus oocytes and their gating properties were studied in cell-attached membrane patches. The gating properties depended strongly on the ion being conducted (K⁺, NH₄ ⁺, Rb⁺, or Tl⁺), suggesting tight coupling between permeation and gating. Mean open times were strongly dependent on the nature of the conducted ion. For ROMK2 the order from the longest to the shortest times was K⁺ > Rb⁺ > Tl⁺ > NH₄ ⁺. For IRK1 the sequence was K⁺ > NH₄ ⁺ > Tl⁺. In both cases the open times decreased monotonically as the membrane voltage was hyperpolarized. Both the absolute values and the voltage dependence of closed times were dependent on the conducted species. ROMK2 showed a single closed state whose mean lifetimes were biphasic functions of voltage. The maxima were at various voltages for different ions. IRK1 had at least two closed states whose lifetimes decreased monotonically with K⁺, increased monotonically with Tl⁺, and were relatively constant with NH₄ ⁺ as the conducted ion. We explain the ion-dependence of gating by assuming that the ions bind to a site within the permeation pathway, resulting in a stable, ion-dependent, closed state of the channel. The patterns of voltage-dependence of closed-state lifetimes, which are specific for different ions, can be explained by variations in the rate at which the bound ions leave the pore toward the inside or the outside of the cell. Received: 18 April 2001/Revised: 28 June 2001     

Enhanced Closed-state Inactivation in a Mutant Shaker K+ Channel

Many mutations that shift the voltage dependence of activation in Shaker channels cause a parallel shift of inactivation. The I2 mutation (L382I in the Shaker B sequence) is an exception, causing a 45 mV activation shift with only a 9 mV shift of inactivation midpoint relative to the wildtype (WT) channel. We compare the behavior of WT and I2 Shaker 29-4 channels in macropatch recordings from Xenopus oocytes. The behavior of WT channels can be described by both simple and detailed kinetic models which assume that inactivation proceeds only from the open state. The behavior of I2 channels requires that they inactivate from closed states as well, a property characteristic of voltage-gated sodium channels. A detailed ``multiple-state inactivation' model is presented that describes both activation and inactivation of I2 channels. The results are consistent with the view that residue L382 is associated with the receptor for the inactivation particles in Shaker channels. Received: 16 December 1996/Revised: 5 February 1997     

Two Distinct K+ Channels in Lamprey (Lampetra fluviatilis) Erythrocyte Membrane Characterized by Single Channel Patch Clamp

Two channels, distinguished by using single-channel patch-clamp, carry out potassium transport across the red cell membrane of lamprey erythrocytes. A small-conductance, inwardly rectifying K⁺-selective channel was observed in both isotonic and hypotonic solutions (osmolarity decreased by 50%). The single-channel conductance was 26 ± 3 pS in isotonic (132 mm K⁺) solutions and 24 ± 2 pS in hypotonic (63 mm K⁺) solutions. No outward conductance was found for this channel, and the channel activity was completely inhibited by barium. Cell swelling activated another inwardly rectifying K⁺ channel with a larger inward conductance of 65 pS and outward conductance of 15 pS in the on-cell configuration. In this channel, rectification was due to the block of outward currents by Mg²⁺ and Ca²⁺ ions, since when both ions were removed from the cytosolic side in inside-out patches the conductance of the channel was nearly ohmic. In contrast to the small-conductance channel, the swelling-activated channel was observed also in the presence of barium in the pipette. Neither type of channel was dependent on the presence of Ca²⁺ ions on the cytosolic side for activity. Received: 18 July 1997/Revised: 30 January 1998     

Potassium Uptake Through the TOK1 K+ Channel in the Budding Yeast

The current through TOK1 (YKC1), the outward-rectifying K⁺ channel in Saccharomyces cerevisiae, was amplified by expressing TOK1 from a plasmid driven by a strong constitutive promoter. TOK1 so hyper-expressed could overcome the K⁺ auxotrophy of a mutant missing the two K⁺ transporters, TRK1 and TRK2. This trk1Δtrk2Δ double mutant hyperexpressing the TOK1 transgene had a higher internal K⁺ content than one expressing the empty plasmid. We examined protoplasts of these TOK1-hyperexpressing cells under a patch clamp. Besides the expected K⁺ outward current activating at membrane potential (V _m ) above the K⁺ equilibrium potential (E _K+ ), a small inward current was consistently observed when the V _m was slightly below E _K+ . The inward and the outward currents are similar in their activation rates, deactivation rates, ion specificities and Ba²⁺ inhibition, indicating that they flow through the same channel. Thus, the yeast outwardly rectifying K⁺ channel can take up K⁺ into yeast cells, at least under certain conditions. Received: 1 October 1998/Revised: 9 December 1998     

Single-Channel Analysis of a Delayed Rectifier K+ Channel in Xenopus Myelinated Nerve

Single-channel properties of a delayed rectifier voltage-gated K⁺ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between −60 and −40 mV with a potential of half-maximal activation, E₅₀, at −47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal activation decreased from 80 to 1.6 msec between −60 and +40 mV. The channel inactivated monoexponentially with a time constant of 10.9 sec at −40 mV. The time constant of deactivation was 126 msec at −80 mV and 16.9 msec at −110 mV. In symmetrical 105 mm K⁺, the single-channel conductance (γ) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13–15°C. In Na⁺-rich solution with 2.5 mm extracellular K⁺γ was 7 pS and the reversal potential was negative to −80 mV, indicating a high selectivity for K⁺ over Na⁺. γ depended on extracellular K⁺ concentration (K _D = 19.6 mm) and temperature (Q ₁₀= 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel current amplitude at all potentials tested with a half-maximal inhibiting concentration (IC₅₀) of 0.6 mm. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX, IC₅₀= 6.8 nm) and mast cell degranulating peptide (MCDP, IC₅₀= 41.9 nm). In Ringer solution the membrane potential of macroscopic I-channel patches was about −65 mV and depolarized under TEA and DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane potential. Received: 2 June 1995/Revised: 13 October 1995     

Effects of Na+ on the Predominant K+ Channel in the Tonoplast of Chara: Decrease of Conductance by Blocks in 100 Nanosecond Range and Induction of Oligo- or Poly-subconductance Gating Modes

We present three mechanisms by which Na⁺ inhibits the open channel currents of the predominant K⁺ channel in the tonoplast of Chara corallina: (i) Fast block, i.e., short (100 ns range) interruptions of the open channel current which are determined by open channel noise analysis, (ii): Oligo-subconductance mode, i.e., a gating mode which occurs preferentially in the presence of Na⁺; this mode comprises a discrete number (here 3) of open states with smaller conductances than normal, and (iii): Polysubconductance mode, i.e., a gating mode with a nondiscrete, large number (>30) of states with smaller conductances than the main open channel conductance. This novel mode has also been observed only in the presence of Na⁺. Received: 16 November 1999/Revised: 8 February 2000     

pH-Sensitive Gating Kinetics of the Maxi-K Channel in the Tonoplast of Chara australis

The most frequently observed K⁺ channel in the tonoplast of Characean giant internodal cells with a large conductance (ca. 170 pS; Lühring, 1986; Laver & Walker, 1987) behaves, although inwardly rectifying, like animal maxi-K channels. This channel is accessible for patch–clamp techniques by preparation of cytoplasmic droplets, where the tonoplast forms the membrane delineating the droplet. Lowering the pH of the bathing solution, that virtually mimicks the vacuolar environment, from an almost neutral level to values below pH 7, induced a significant but reversible decrease in channel activity, whereas channel conductance remained largely unaffected. Acidification (pH 5) on both sides of the membrane decreased open probability from a maximum of 80% to less than 20%. Decreasing pH at the cytosolic side inhibited channel activity cooperatively with a slope of 2.05 and a pK _a 6.56. In addition, low pH at the vacuolar face shifted the activating voltage into a positive direction by almost 100 mV. This is the first report about an effect of extraplasmatic pH on gating of a maxi-K channel. It is suggested that the Chara maxi-K channel possesses an S4-like voltage sensor and negatively charged residues in neighboring transmembrane domains whose S4-stabilizing function may be altered by protonation. It was previously shown that gating kinetics of this channel respond to cytosolic Ca²⁺ (Laver & Walker, 1991). With regard to natural conditions, pH effects are discussed as contributing mainly to channel regulation at the vacuolar membrane face, whereas at the cytosolic side Ca²⁺ affects the channel. An attempt was made to ascribe structural mechanisms to different states of a presumptive gating reaction scheme. Received: 8 May 1998/Revised: 18 September 1998     

Cloning and Function of the Rat Colonic Epithelial K+ Channel KVLQT1

K_VLQT1 (KCNQ1) is a voltage-gated K⁺ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K⁺ channel in the colonic epithelium. We now report cloning and expression of K_VLQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to K_VLQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of K_VLQT1 in crypt cells and surface epithelium. Expression of rK_VLQT1 in Xenopus oocytes induced a typical delayed activated K⁺ current, that was further activated by increase of intracellular cAMP but not Ca²⁺ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K⁺ conductance in the colonic mucosal epithelium and inhibited whole cell K⁺ currents in patch-clamp experiments on isolated colonic crypts. We conclude that K_VLQT1 is forming an important component of the basolateral cAMP-activated K⁺ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis. Received: 17 July 2000/Revised: 25 October 2000     

The Ach-evoked Ca2+-activated K+ Current in Mouse Mandibular Secretory Cells. Single Channel Studies

Although acetylcholine (ACh) is able to activate voltage- and Ca²⁺-sensitive K⁺ (BK) channels in mouse mandibular secretory cells, our recent whole cell studies have suggested that these channels, like those in sheep parotid secretory cells, do not contribute appreciably to the conductance that carries the ACh-evoked whole cell K⁺ current. In the present study, we have used cell-attached patch clamp methods to identify and characterize the K⁺ channel type responsible for carrying the bulk of this current. When the cells were bathed in a NaCl-rich solution the predominant channel type activated by ACh (1 μmol/l or 50 nmol/l) had a conductance only of 40 pS; it was not blocked by TEA but it was sensitive to quinine and it conducted Rb⁺ to an appreciable extent. BK channels, which could be seen in some but not all patches from resting cells, also showed increased activity when ACh was added to the bath, but they were much less conspicuous during ACh stimulation than the 40-pS channels. When the cells were bathed in a KCl-rich rather than a NaCl-rich solution, a small-conductance K⁺ channel, sensitive to quinine but not to TEA, was still the most conspicuous channel to be activated by ACh although its conductance was reduced to 25 pS. Our studies confirm that the ACh-evoked whole-cell K⁺ current is not carried substantially by BK channels and show that it is carried by a small-conductance K⁺ channel with quite different properties. Received: 28 September 1995/Revised: 26 December 1995     

Temperature Sensitivity of the Tonoplast Ca2+-Activated K+ Channel in Chara: The Influence of Reversing the Sign of Membrane Potential

A detailed temperature dependence study of a well-defined plant ion channel, the Ca²⁺-activated K⁺ channel of Chara corallina, was performed over the temperature range of their habitats, 5–36°C, at 1°C resolution. The temperature dependence of the channel unitary conductance at 50 mV shows discontinuities at 15 and 30°C. These temperatures limit the range within which ion diffusion is characterized by the lowest activation energy (E _a = 8.0 ± 1.6 kJ/mol) as compared to the regions below 15°C and above 30°C. Upon reversing membrane voltage polarity from 50 to −50 mV the pattern of temperature dependence switched from discontinuous to linear with E _a = 13.6 ± 0.5 kJ/mol. The temperature dependence of the effective number of open channels at 50 mV showed a decrease with increasing temperature, with a local minimum at 28°C. The mean open time exhibited a similar behavior. Changing the sign of membrane potential from 50 to −50 mV abolished the minima in both temperature dependencies. These data are discussed in the light of higher order phase transitions of the Characean membrane lipids and corresponding change in the lipid-protein interaction, and their modulation by transmembrane voltage. Received: 14 June 2000/Revised: 20 September 2000     

Regulation of Ca2+-activated K+ Channel from Rabbit Distal Colon Epithelium by Phosphorylation and Dephosphorylation

The Ca²⁺-activated maxi K⁺ channel is predominant in the basolateral membrane of the surface cells in the distal colon. It may play a role in the regulation of the aldosterone-stimulated Na⁺ reabsorption from the intestinal lumen. Previous measurements of these basolateral K⁺ channels in planar lipid bilayers and in plasma membrane vesicles have shown a very high sensitivity to Ca²⁺ with a K _0.5 ranging from 20 nm to 300 nm, whereas other studies have a much lower sensitivity to Ca²⁺. To investigate whether this difference could be due to modulation by second messenger systems, the effect of phosphorylation and dephosphorylation was examined. After addition of phosphatase, the K⁺ channels lost their high sensitivity to Ca²⁺, yet they could still be activated by high concentrations of Ca²⁺ (10 μm). Furthermore, the high sensitivity to Ca²⁺ could be restored after phosphorylation catalyzed by a cAMP dependent protein kinase. There was no effect of addition of protein kinase C. In agreement with the involvement of enzymatic processes, lag periods of 30–120 sec for dephosphorylation and of 10–280 sec for phosphorylation were observed. The phosphorylation state of the channel did not influence the single channel conductance. The results demonstrate that the high sensitivity to Ca²⁺ of the maxi K⁺ channel from rabbit distal colon is a property of the phosphorylated form of the channel protein, and that the difference in Ca²⁺ sensitivity between the dephosphorylated and phosphorylated forms of the channel protein is more than one order of magnitude. The variety in Ca²⁺ sensitivities for maxi K⁺ channels from tissue to tissue and from different studies on the same tissue could be due to modification by second messenger systems. Received: 28 February 1995/Revised: 22 December 1995     

Effects of Internal K+ and ABA on the Voltage- and Time-dependence of the Outward K+-rectifier in Vicia Guard Cells

One of the main effects of abscisic acid (ABA) is to induce net loss of potassium salts from guard cells enabling the stomata to close. K⁺ is released from the vacuole into the cytosol and then to the extracellular space. The effects of increasing cytosolic K⁺ on the voltage- and time-dependence of the outwardly rectifying K⁺-current (I _K,out) in guard cell protoplasts (GCP) was examined in the whole-cell configuration of the patch-clamp technique. The same quantitative analysis was performed in the presence of ABA at different internal K⁺ concentrations ([K⁺] _i ). Varying [K⁺] _i in the patch pipette from 100 to 270 mm increased the magnitude of I _K,out in a nonlinear manner and caused a negative shift in the midpoint (V _0.5) of its steady-state activation curve. External addition of ABA (10–20 μm) also increased the magnitude of I _K,out at all [K⁺] _i , but caused a shift in V _0.5 of the steady-state activation curve only in those GCP loaded with 150 mm internal K⁺ or less. Indeed, V _0.5 did not shift upon addition of ABA when the [K⁺] _i was above 150 mm and up to 270 mm, i.e., the shift in V _0.5 caused by ABA depended on the [K⁺] _i . Both increase in [K⁺] _i and external addition of ABA, decreased (by ≈ 20%) the activation time constant (τ _n ) of I _K,out. The small decrease in τ _n , in both cases, was found to be independent of the membrane voltage. The results indicate that ABA mimics the effect of increasing cytoplasmic K⁺, and suggest that ABA may increase I _K,out and alter V _0.5 of its steady-state activation curve via an enhancement in cytosolic K⁺. This report describes for the first time the effects of [K⁺] _i on the voltage- and time-dependence of I _K,out in guard cells. It also provides an explanation for the quantitative (total membrane current) and qualitative (current kinetics) differences found between intact guard cells and their protoplasts. Received: 1 December 1995/Revised: 8 May 1996