A Tight-Seal Whole Cell Study of the Voltage-Dependent Gating Mechanism of K-Channels of Protoplasmic Droplets of Chara corallina

The biophysical properties of voltage-dependent K⁺-channels of protoplasmic droplets of Chara corallina Klein ex Willd., em, R.D.W. were investigated using the tight-seal whole cell method. Two potassium currents were observed in voltage-clamp mode and they can be used to explain the transient membrane potential time course observed in current-clamp mode. The K⁺-channels are identified by the effect of tetraethylammonium chloride which blocks both currents. A two-state, constant dipole moment model is used to fit the voltage-conductance curve. From this model the minimum equivalent gating charge involved in the gating mechanism of K⁺-channels of Chara can be estimated.     

Changing Val-76 towards Kir channels drastically influences the folding and gating properties of the bacterial potassium channel KcsA

All K⁺-channels are stabilized by K⁺-ions in the selectivity filter. However, they differ from each other with regard to their selectivity filter. In this study, we changed specific residue Val-76 in the selectivity filter of KcsA to its counterpart Ile in inwardly rectifying K⁺-channels (Kir). The tetramer was exclusively converted into monomers as determined by conventional gel electrophoresis. However, by perfluoro-octanoic acid (PFO) gel electrophoresis mutant channel was mostly detected as tetramer. Tryptophan fluorescence and acrylamide quenching experiments demonstrated significant alteration in channel folding properties via increase in hydrophilicity of local environment. Furthermore, in planar lipid bilayer experiments V76I exhibited drastically lower conductance and decreased channel open time as compared to the unmodified KcsA. These studies suggest that V76I might contribute to determine the stabilizing, folding and channel gating properties in a selective K⁺-channel.     

Single voltage-dependent and outward rectifying K+-channels in isolated rat heart cells

Studies on single K⁺-channel currents recorded from isolated rat heart muscle cells, in which early repolarization is known to be exceptionally fast, are reported here. A K⁺-channel which is blocked by TEA (tetraethylammonium) from the inside only has been found.The total open time of the channel, measured in steady-state after activation, indicated outward rectifying properties. The single channel conductance increases with depolarization from 25 pS at-70 mV to 75 pS at+70 mV.Selectivity of the channel has also been measured and it was found that only Rb⁺ and K⁺ can permeate the channel, whereas the permeability (P) for Li⁺, Na⁺, Cl^-, Mg²⁺, and Ca²⁺ is less than 0.05 times .Ba²⁺ and Cs⁺ block the channel activity.These results clearly demonstrate the existence of K⁺-selective outward rectifying conductance pathways in rat ventricular myocytes.     

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

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

Potassium Channels in Motor Cells of Samanea saman: A Patch-Clamp Study

Leaflet movements in Samanea saman are driven by the shrinking and swelling of cells in opposing (extensor and flexor) regions of the motor organ (pulvinus). Changes in cell volume, in turn, depend upon large changes in motor cell content of K⁺, Cl⁻ and other ions. We performed patch-clamp experiments on extensor and flexor protoplasts, to determine whether their plasma membranes contain channels capable of carrying the large K⁺ currents that flow during leaflet movement. Recordings in the “whole-cell” mode reveal depolarization-activated K⁺ currents in extensor and flexor cells that increase slowly (t_½ = ca. 2 seconds) and remain active for minutes. Recordings from excised patches reveal a single channel conductance of ca. 20 picosiemens in both cell types. The magnitude of the K⁺ currents is adequate to account quantitatively for K⁺ loss, previously measured in vivo during cell shrinkage. The K⁺ channel blockers tetraethylammonium (5 millimolar) or quinine (1 millimolar) blocked channel opening and decreased light- and dark-promoted movements of excised leaflets. These results provide evidence for the role of potassium channels in leaflet movement.     

K+ Channels in the Plasma Membrane of Lily Pollen Protoplasts

Using the patch-clamp technique K⁺ channels could be observed in the plasma membrane of protoplasts from pollen grains of Lilium longiflorum. With depolarizing membrane potentials the open probability of the different K⁺ channels increased. Two K⁺ channel populations occurring occasionally had a single channel conductance of 120 pS and 42 pS, respectively. The most often observed K⁺ channel had a single channel conductance of 19 pS which showed an increase of channel activity with increasing free cytoplasmic Ca²⁺ concentration. This channel population might be involved in the pathway of endogenous transcellular K⁺ currents which are activated during pollen tube tip extension.     

Potassium Channels in Chara corallina: CONTROL AND INTERACTION WITH THE ELECTROGENIC H PUMP

Plasmalemma electrical properties were used to investigate K⁺ transport and its control in internodal cells of Chara corallina Klein ex Willd., em R.D.W. Cell exposure to solutions containing 10 mm KCl caused the potential, normally −250 millivolts (average), to depolarize in two steps. The first step was a 21 millivolt depolarization that lasted from 1 to 40 minutes. The second step started with an action potential and left the membrane potential at −91 millivolts, with a 10-fold reduction in resistance. We suggest that the second step was caused by the opening of K⁺ -channels in the membrane. This lowered the resistance and provided a current pathway that partially short-circuited the electrogenic pump. Although largely short-circuited, the electrogenic pump was still operating as indicated by: (a) the depolarized potential of −91 millivolts was more negative than Ek (=−42 millivolts in 10 mm K⁺); (b) a large net K⁺ uptake occurred while the cell was depolarized; (c) both the electrogenic pump inhibitor, diethylstilbestrol, and the sulfhydryl-reagent N-ethylmaleimide (which increased the passive membrane permeability) further depolarized the potential in 10 mm KCl.A two-phase recovery back to normal cell potentials occurred upon lowering the K⁺ concentration from 10 to 0.2 mm. The first phase was an apparent Nernst potential response to the change in external K⁺ concentration. The second phase was a sudden hyperpolarization accompanied by a large increase in membrane resistance. We attribute the second phase to the closing of K⁺ -channels and the removal of the associated short-circuiting effect on the electrogenic pump, thereby allowing the membrane to hyperpolarize. Further experiments indicated that the K⁺ -channel required Ca²⁺ for normal closure, but other ions could substitute, including: Na⁺, tetraethylammonium, and 2,4,6-triaminopyrimidine. Apparently, K⁺ -channel conductance is determined by competition between Ca²⁺ and K⁺ for a control (gating?) binding site.     

Mutations in the K+-Channel KcsA Toward Kir Channels Alter Salt-Induced Clusterization and Blockade by Quaternary Alkylammonium Ions

Protein aggregation is a result of malfunction in protein folding, assembly, and transport, caused by protein mutation and/or changes in the cell environment, thus triggering many human diseases. We have shown that bacterial K⁺-channel KcsA, which acts as a representative model for ion channels, forms salt-induced large conductive complexes in a particular environment. In the present study, we investigated the effects of point mutations in the selectivity filter of KcsA on intrinsic stability, aggregation, and channel blocking behavior. First, we found that a low sodium chloride concentration in potassium-containing media induced fast transfer of single channels to a planar lipid bilayer. Second, increasing the sodium chloride concentration drastically increased the total channel current, indicating enhanced vesicle fusion and transfer of multiple channels to a planar lipid bilayer. However, such complexes exhibited high conductance as well as higher open probability compared to the unmodified KcsA behavior shown previously. Interestingly, the affinity of aggregated complexes for larger symmetric quaternary alkylammonium ions (QAs) was found to be much higher than that for tetraethylammonium, a classical blocker of the K⁺ channel. Based on these findings, we propose that mutant channel complexes exhibit larger pore dimensions, thus resembling more the topological properties of voltage-gated and inwardly rectifying K⁺ channels.     

Ion channels in the plasma membrane of protoplasts from the halophytic angiosperm Zostera muelleri

Patch clamp studies show that there may be as many as seven different channel types in the plasma membrane of protoplasts derived from young leaves of the halophytic angiosperm Zostera muelleri. In whole-cell preparations, both outward and inward rectifying currents that activate in a timeand voltage-dependent manner are observed as the membrane is either depolarized or hyperpolarized. Current voltage plots of the tail currents indicate that both currents are carried by K⁺. The channels responsible for the outward currents have a unit conductance of approximately 70 pS and are five times more permeable to K⁺ than to Na⁺. In outside-out patches we have identified a stretch-activated channel with a conductance of 100 pS and a channel that inwardly rectifies with a conductance of 6 pS. The reversal potentials of these channels indicate a significant permeability to K⁺. In addition, the plasma membrane contains a much larger K⁺ channel with a conductance of 300 pS. Single channel recordings also indicate the existence of two Cl^– channels, with conductances of 20 and 80 pS with distinct substates. The membrane potential difference of perfused protoplasts showed rapid action potentials of up to 50 mV from the resting level. The frequency of these action potentials increased as the external osmolarity was decreased. The action potentials disappeared with the addition of Gd³⁺, an effect that is reversible upon washout.We would like to thank K. Morris and D. McKenzie for technical assistance and the Australian Research Council for financial support.     

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     

Properties of Single K and Cl Channels in Asclepias tuberosa Protoplasts

Potassium and chloride channels were characterized in Asclepias tuberosa suspension cell derived protoplasts by patch voltage-clamp. Whole-cell currents and single channels in excised patches had linear instantaneous current-voltage relations, reversing at the Nernst potentials for K⁺ and Cl⁻, respectively. Whole cell K⁺ currents activated exponentially during step depolarizations, while voltage-dependent Cl⁻ channels were activated by hyperpolarizations. Single K⁺ channel conductance was 40 ± 5 pS with a mean open time of 4.5 milliseconds at 100 millivolts. Potassium channels were blocked by Cs⁺ and tetraethylammonium, but were insensitive to 4-aminopyridine. Chloride channels had a single-channel conductance of 100 ± 17 picosiemens, mean open time of 8.8 milliseconds, and were blocked by Zn²⁺ and ethacrynic acid. Whole-cell Cl⁻ currents were inhibited by abscisic acid, and were unaffected by indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology and development.     

K Channels Are Responsible for an Inwardly Rectifying Current in the Plasma Membrane of Mesophyll Protoplasts of Avena sativa

In whole-cell recording, the conductance of the plasma membrane of protoplasts isolated from mesophyll cells of leaves of oat (Avena sativa) was greater for inward than outward current. The inward current in both the whole-cell mode and with isolated patches was dependent on [K⁺]_o. When the membrane voltage was more positive than −50 millivolts, the membrane conductance in the whole-cell mode was low, and K⁺ channels in cell-attached or outside-out patches had a low probability of being open. At a membrane voltage more negative than −50 millivolts, the membrane conductance increased by sevenfold in the whole-cell mode, and the probability of the channels being open increased. The inward current was highly selective for K⁺ compared with Cs⁺, Na⁺, choline or Cl⁻. Low concentrations of [Cs⁺]_o or [Na⁺]_o blocked the inward current in a strongly voltage-dependent fashion. Comparison of single-channel with the macroscopic current yields an estimate of about 200 inwardly rectifying K⁺ channels per cell at a density of 0.035 per square micrometer. At physiological membrane voltages and [K⁺]_o about 10 millimolar, the influx through these channels is sufficient to increase the internal [K⁺] by 2 millimolar per minute. These K⁺ channels are activated by membrane voltages in the normal physiological range and could contribute to K⁺ uptake whenever the membrane is more negative than the K⁺ equilibrium potential.     

Identification and characterization of inward K+ -channels in plasma membranes ofArabidopsis root cortex cells

Patch clamping whole-cell recording techniques were applied to study the inward K⁺ -channels inArabidopsis root cortex cells. The inward K⁺ -channels in the plasma membranes of the root cortex cell protoplasts were activated by hyperpolarized membrane potentials. The channels were highly selective for K⁺ ions over Na⁺ ions. The channel activity was significantly inhibited by the external TEA⁺ or Ba²⁺. The changes in cytoplasmic Ca²⁺ concentrations did not affect the whole-cell inward K⁺ -currents. The possible association between the channel selectivity to K⁺ and Na⁺ ions and plant salt-tolerance was also discussed.     

Expression of a genomic clone encoding a brain potassium channel in mammalian cells using lipofection

A genomic clone encoding a mouse brain K⁺ channel (MBK1) was isolated, characterized and expressed in COS cells using the lipofection technique. Transfected COS cells expressed voltage-dependent K⁺ currents that activated within 20 ms at 0 mV and showed less than 10% inactivation during 250 ms depolarizing pulses at 60 mV. Expressed K⁺ currents were reversibly blocked by 4-aminopyridine and tetraethylammonium, and were moderately sensitive to dendrotoxin, but insensitive to charybdotoxin. Thus MBK1, expressed transiently in a mammalian cell line, exhibits features characteristic of non-inactivating K⁺ channels with a conspicuous insensitivity to charybdotoxin. Lipofection is, therefore, a valuable strategy for expression of channel proteins in mammalian cells.Abbreviations 4-AP 4 aminopyridine - TEA tetraethylammonium - CTX charybdotoxin - DTX dendrotoxin - V applied voltage - V_rev reversal potential - I current - G conductance - MBK1 mouse brain potassium channel 1 - TES N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid Correspondence to: M. Montal.     

Coupling of K+-gating and permeation with Ca2+ block in the Ca2+-Activated K+ channel inChara australis

Summary The patch-clamp technique is used here to investigate the kinetics of Ca²⁺ block in single high-conductance Ca²⁺-activated K⁺ channels. These channels are detected in the membrane surounding cytoplasmic drops fromChara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (–300 to 200 mV). Ca²⁺ on either side of the channel reduces its K⁺ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca²⁺-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca²⁺ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca²⁺ indicates that (i) external Ca²⁺ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca²⁺ or K⁺, and (iii) the kinetics of Ca²⁺ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca²⁺ indicates that more than one Ca²⁺ is involved.     

Selectivity and permeation of alkali metal ions in K+-channels

Ion conduction in K⁺-channels is usually described in terms of concerted movements of K⁺ progressing in a single file through a narrow pore. Permeation is driven by an incoming ion knocking on those ions already inside the protein. A fine-tuned balance between high-affinity binding and electrostatic repulsive forces between permeant ions is needed to achieve efficient conduction. While K⁺-channels are known to be highly selective for K⁺ over Na⁺, some K⁺ channels conduct Na⁺ in the absence of K⁺. Other ions are known to permeate K⁺-channels with a more moderate preference and unusual conduction features. We describe an extensive computational study on ion conduction in K⁺-channels rendering free energy profiles for the translocation of three different alkali ions and some of their mixtures. The free energy maps for Rb⁺ translocation show at atomic level why experimental Rb⁺ conductance is slightly lower than that of K⁺. In contrast to K⁺ or Rb⁺, external Na⁺ block K⁺ currents, and the sites where Na⁺ transport is hindered are characterized. Translocation of K⁺/Na⁺ mixtures is energetically unfavorable owing to the absence of equally spaced ion-binding sites for Na⁺, excluding Na⁺ from a channel already loaded with K⁺.     

Inward and outward K+-selective currents in the plasma membrane of protoplasts from maize root cortex and stele

In an attempt to understand the processes mediating ion transport within the root, the patch clamp technique was applied to protoplasts isolated from the cortex and stele of maize roots and their plasma membrane conductances investigated. In the whole-cell configuration, membrane hyperpolarization induced a slowly activating inwardly rectifying conductance in most protoplasts isolated from the root cortex. In contrast, most protoplasts isolated from the stele contained a slowly activating outwardly rectifying conductance upon plasma membrane depolarization. The reversal potential of the inward current indicated that it was primarily due to the movement of K⁺; the outwardly rectifying conductance was comparatively less selective for K⁺. Membrane hyperpolarization beyond a threshold of about ?70 mV induced inward currents. When E_K was set negative of this threshold, inward currents activated negative of E_K and no outward currents were observed positive of E_K. Outward currents in the stelar protoplasts activated at potentials positive of ?85 mV. However, when E_K was set positive of ?85 mV a small inward current was also observed at potentials negative (and slightly positive) of the equilibrium potential for K⁺. Inwardly and outwardly rectifying K⁺ channels were observed in outside-out patches from the plasma membrane of cortical and stelar cells, respectively. Characterization of these channels showed that they were likely to be responsible for the macroscopic ‘whole-cell’ currents. Inward and outward currents were affected differently by various K⁺ channel blockers (TEA⁺, Ba²⁺ and Cs⁺). In addition, Ca²⁺ above 1 mM partially blocked the inward current in a voltage-dependent manner but had little effect on the outward current. It is suggested that the inwardly rectifying conductance identified in protoplasts isolated from the cortex probably represents an important component of the low-affinity K⁺ uptake mechanism (mechanism II) identified in intact roots. The outwardly rectifying conductance identified in protoplasts isolated from the stele could play a role in the release of cations into the xylem vessels for transport to the shoot.     

Demonstration of Voltage-Dependent and TTX-Sensitive Na+-Channels in Human Melanocytes

The electrophysiological properties of cultured human melanocytes were investigated using the whole-cell configuration of the patch-clamp technique. Depolarizations to membrane potentials more positive than -30 mV resulted in the rapid development (<1 ms to peak) of an inward current. The maximum peak current was observed at +10 mV and reached an average amplitude of about 270 pA. During the depolarizations, the current inactivated with a time constant of about 2 ms. The current was abolished by the addition of 0.3 μM tetrodotoxin, a blocker of voltage-gated Na⁺-channels, and disappeared when Na⁺ was omitted from the extracellular medium. In addition, the melanocytes contain at least two types of outward K⁺-current. The first type, observed in every cell, was highly sensitive (K_i 1 mM) to the K⁺-channel blocker TEA, required depolarizations beyond zero to be activated and did not inactivate. The second type was less regularly observed (10% of the cells). This current activated at more negative voltages (–20 mV), was resistant to TEA (20 mM) but was blocked by 2 mM 4-aminopyridine and inactivated rapidly during depolarizations. We conclude that human melanocytes are equipped with voltage-dependent Na⁺-channels, a delayed rectifying K⁺-current and a K⁺-current similar to the A-current in neurones.     

Characterization of Angiotensin II-regulated K+ Conductance in Rat Adrenal Glomerulosa Cells

Nystatin perforated-patch clamp and single-channel recording methods were used to characterize macroscopic and single-channel K⁺ currents and the effects of angiotensin II (AngII) in cultured rat adrenal glomerulosa cells. Two basic patterns of macroscopic current-voltage relationships were observed: type 1 exhibited a rapidly activating, noninactivating, voltage-dependent outward current and type 2 exhibited an inactivating voltage-dependent outward current attributed to charybdotoxin sensitive Ca⁺⁺-dependent K⁺ channels. Most cells exhibited the type 1 pattern and experiments focused on this cell type. Cell-attached and inside-out patches were dominated by a single K⁺ channel class which exhibited an outward conductance of 12 pS (20 mm K⁺ pipette in cell-attached and inside-out configurations, 145 mm K⁺ _in), a mean open time of 2 msec, and a weakly voltage-dependent low open probability that increased with depolarization. Channel open probability was reversibly inhibited by bath stimulation with AngII. At the macroscopic level, type 1 cell macroscopic K⁺ currents appeared comprised of two components: a weakly voltage-dependent current controlling the resting membrane potential (−85 mV) which appeared mediated by the 12 pS K⁺ channel and a rapidly activating, noninactivating voltage-dependent current activated above −50 mV. The presence of the second voltage-dependent K⁺ channel class was suggested by the effects of AngII, the blocking effects of quinidine and Cs⁺, and the properties of the weakly voltage-dependent K⁺ channel described. The K⁺ selectivity of the macroscopic current was demonstrated by the dependence of current reversal potentials on the K⁺ equilibrium potential and by the effects of K⁺ channel blockers, Cs⁺ and quinidine. AngII (10 pm to 1 nm) reversibly inhibited macroscopic K⁺ currents and this effect was blocked by the AT₁ receptor antagonist losartin. Received: 6 August 1996/Revised: 15 November 1996