Blockade of potassium channels in the plasmalemma ofChara corallina by tetraethylammonium,Ba2+, Na+ and Cs+

Summary The outer membranes of plant cells contain channels which are highly selective for K⁺. In the giant-celled green algaChara corallina, K⁺ currents in the plasmalemma were measured during the action potential and when the cell was depolarized to the K⁺ equilibrium potential in high external K⁺ concentrations. Currents in both conditions were reduced by externally added tetraethylammonium (TEA⁺), Ba²⁺, Na⁺ and Cs⁺. In contrast to inhibition by TEA⁺, the latter three ions inhibited inward K⁺ current in a voltage-dependent manner, and reduced inward current more than outward. Ba²⁺ and Na⁺ also appeared to inhibit outward current in a strongly voltage-dependent manner. The blockade by Cs⁺ is studied in more detail in the following paper. TEA⁺ inhibited both inward and outward currents in a largely voltage-independent manner, with an apparentK _D of about 0.7 to 1.1mm, increasing with increasing external K⁺. All inhibitors reduced current towards a similar linear leak, suggesting an insensitivity of the background leak inChara to these various K⁺ channel inhibitors. The selectivity of the channel to various monovalent cations varied depending on the method of measurement, suggesting that ion movement through the K⁺-selective channel may not be independent.     

Pharmacology of K+ channels in the plasmalemma of the green algaChara corallina

Summary The outer membranes of plant cells contain channels which are highly selective for K⁺. However, many of their properties and their similarities to K⁺ channels found in animal cells had not previously been established. The channels open when the cells are depolarized in solutions with a high K⁺/Ca²⁺ ratio. In this work, the pharmacology of a previously identified plant K⁺ channel was examined. This survey showed that the channels have many properties which are similar to those of high-conductance Ca²⁺-activated K⁺ channels (highG K⁺(Ca²⁺)). K⁺ currents inChara were reduced by TEA⁺, Na⁺, Cs⁺, Ba²⁺, decamethonium and quinine, all inhibitors of, among other things, highG K⁺(Ca²⁺) channels. Tetracaine also inhibited K⁺ currentsChara, but its effect on most types of K⁺ channels in animal tissues is unknown. The currents were not inhibited by 4-aminopyridine (4AP), caffeine, tolbutamide, dendrotoxin, apamin or tubocurarine, which do not inhibit highG K⁺(Ca²⁺) channels, but affect other classes of K⁺ channels. The channels were locked open by 4AP, in a remarkably similar manner to that reported for K⁺(Ca²⁺) channels of a molluscan neuron. No evidence for the role of the inositol cycle in channel behavior was found, but its role in K⁺ channel control in animal cells is obscure. Potassium conductance was slightly decreased upon reduction of cytoplasmic ATP levels by cyanide + salicylhydroxamic acid (SHAM), consistent with channel control by phosphorylation. The anomalously strong voltage dependence of blockade by some ions (e.g. Cs⁺) is consistent with the channels being multiion pores. However, the channels also demonstrate some differences from the highG K⁺(Ca²⁺) channels found in animal tissues. The venom of the scorption,Leiurus quinquestriatus (LQV), and a protein component, charybdotoxin (CTX), an apparently specific inhibitor of highG K⁺(Ca²⁺) channels in various animal tissues, had no effect on the K⁺ channels in theChara plasmalemma. Als,, pinacidil, an antihypertensive drug which may increase highG K⁺(Ca²⁺) channel activity had no effect on the channels inChara. Although the described properties of theChara K⁺ channels are most similar to those of high conductance K⁺(Ca²⁺) in animal cells, the effects of CTX and pinacidil are notably different; the channels are clearly of a different structure to those found in animal cells, but are possibly related.     

Selective block by α-dendrotoxin of the K+ inward rectifier at the Vicia guard cell plasma membrane

The efficacy and mechanism of -dendrotoxin (DTX) block of K⁺ channel currents in Vicia stomatal guard cells was examined. Currents carried by inward- and outward-rectifying K⁺ channels were determined under voltage clamp in intact guard cells, and block was characterized as a function of DTX and external K⁺ (K⁺) concentrations. Added to the bath, 0.1-30 nM DTX blocked the inward-rectifying K⁺ current (I_K,in), but was ineffective in blocking current through the outward-rectifying K⁺ channels (I_K,out) even at concentrations of 30 nM. DTX block was independent of clamp voltage and had no significant effect on the voltage-dependent kinetics for I_K,in, neither altering its activation at voltages negative of –120 mV nor its deactivation at more positive voltages. No evidence was found for a use dependence to DTX action. Block of I_K,in followed a simple titration function with an apparent K_1/2 for block of 2.2 nM in 3 mm K _o ⁺ . However, DTX block was dependent on the external K⁺ concentration. Raising K⁺ from 3 to 30 mm slowed block and resulted in a 60–70% reduction in its efficacy (apparent K _i = 10 mm in 10 nm DTX). The effect of K⁺ in protecting I _K,in was competitive with DTX and specific for permeant cations. A joint analysis of I_K,in block with DTX and K⁺ concentration was consistent with a single class of binding sites with a K _d for DTX of 240 pm. A K _d of 410 m for extracellular K⁺ was also indicated. These results complement previous studies implicating a binding site requiring extracellular K⁺ (K_1/2 1 mm) for I_K,in activation; they parallel features of K⁺ channel block by DTX and related peptide toxins in many animal cells, demonstrating the sensitivity of plant plasma membrane K⁺ channels to nanomolar toxin concentrations under physiological conditions; the data also highlight one main difference: in the guard cells, DTX action appears specific to the K⁺ inward rectifier.We thank J.O. Dolly (Imperial, London) and S.M. Jarvis (University of Kent, Canterbury) for several helpful discussions. This work was supported by SERC grant GR/H07696 and was aided by equipment grants from the Gatsby Foundation, the Royal Society and the University of London Central Research Fund. G.O. was supported by an Ausbildungsstipendium (OB 85/1-1) from the Deutsche Forschungsgemeinschaft. F.A. holds a Sainsbury Studentship.     

Ca2+-Activated K+ channel from human erythrocyte membranes: Single channel recification and selectivity

Summary The Ca²⁺-activated K⁺ channel of the human red cell membranes was characterized with respect to rectification and selectivity using the patch-clamp technique. In inside-out patches exposed to symmetric solutions of K⁺, Rb⁺, and NH ₄ ⁺ , respectively, inward rectifyingi-V curves were obtained. The zero current conductances were: K⁺ (23.5 pS±3.2)>NH ₄ ⁺ (14.2 pS±1.2)>Rb⁺ (11.4 pS±1.8). With low extracellular K⁺ concentrations (substitution with Na⁺) the current fluctuations reversed close to the Nernst potential for the K ion and the rectification as well as thei-V slopes decreased. With mixed intracellular solutions of K⁺ and Na⁺ enhanced rectification were observed due to a Na⁺ block of outward currents. From bi-ionic reversal potentials the following permeability sequence (P _K/P _X) was calculated: K⁺ (1.0)>Rb⁺ (1.4±0.1)>NH ₄ ⁺ (8.5±1.3)>Li⁺(>50); Na⁺ (>110); Cs⁺ (5). Li⁺, Na⁺, and Cs⁺ were not found to carry any current, and only minimum values of the permeability ratios were estimated. Tl⁺ was permeant, but the permeability and conductance were difficult to quantify, since with this ion the single channel activity was extremely low and the channels seemed to inactivate. The inward rectification in symmetric solutions indicate an asymmetric open channel structure, and the different selectivity sequences based on conductances and permeabilities reflect interionic interactions in the permeation process.     

Potassium channels in plasmalemma ofNitella cells at rest

Summary Cation channels of passive transport in the plasmalemma ofNitella flexilis cells at rest were studied by the voltageclamp technique using microelectrodes. Two types of potassium channels have been identified. They are activated at different voltages: over –100 to –80 mV (D-channels) and below –100 mV (H-channels). The zero-current potential of instantaneous voltage-current curves (IVCC's) for both types of channels shifts by 50 to 55 mV in response to a 10-fold increase of K⁺ concentration in the solution. Ion movement in D-channels follows the free diffusion mechanism; in H-channels the independence principle is violated. The channel selectivity (in the order of decreasing permeability) is: K⁺>Rb⁺>NH ₄ ⁺ >Na⁺Li⁺>Cs⁺>TEA⁺ choline⁺. It has been found that D-channel Cs⁺ block is potential dependent while tetraethylammonium (TEA⁺) blocks H-channels in a potential-independent manner, but H⁺ ions do not affect the inward potassium current of the channels. Two types of potassium channels appear to be located in different parts of the membrane and their entrance parts are of different structure.     

Ca2+-activated K+ conductance of the human red cell membrane: Voltage-dependent Na+ block of outward-going currents

Summary Human red cells were prepared with various cellular Na⁺ and K⁺ concentrations at a constant sum of 156mm. At maximal activation of the K⁺ conductance,g _K(Ca), the net efflux of K⁺ was determined as a function of the cellular Na⁺ and K⁺ concentrations and the membrane potential,V _m , at a fixed [K⁺]_ex of 3.5mm.V _m was only varied from (V _m –E _K)25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988).g _K(Ca) as a function of cellular Na⁺ and K⁺ concentrations atV _m =–40, 0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na⁺. Since the present Ca²⁺-activated K⁺ channels have been shown to be of the multi-ion type, the experimental data from each set of Na⁺ and K⁺ concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na⁺ is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na⁺ concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101mm. Data from a previous study of voltage dependence as a function of the degree of Ca²⁺ activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence ofg _K(Ca) for outward currents at (V _m –E _K)>25 25 mV reflects a voltage-dependent Na⁺ block of the Ca²⁺-activated K⁺ channels.     

Current Transients Associated with BK Channels in Human Glioma Cells

We have previously demonstrated the expression of BK channels in human glioma cells. There was a curious feature to the whole-cell currents of glioma cells seen during whole-cell patch-clamp: large, outward current transients accompanied repolarization of the cell membrane following an activating voltage step. This transient current, I _transient, activated and inactivated rapidly (1 ms). The I-V relationship of I _transient had features that were inconsistent with simple ionic current through open ion channels: (i) I _transient amplitude peaked with a –80 mV voltage change and was invariant over a 200 mV range, and (ii) I _transient remained large and outward at –140 mV. We provide evidence for a direct relationship of I _transient to glioma BK currents. They had an identical time course of activation, identical pharmacology, identical voltage-dependence, and small, random variations in the amplitude of the steady-state BK current and I _transient seen over time were often perfectly in phase. Substituting intracellular K⁺ with Cs⁺, Li⁺, or Na ⁺ ions reversibly reduced I _transient and BK currents. I _transient was not observed in recordings of other BK currents (hbr5 expressed in HEK cells and BK currents in rat neurons), suggesting I _transient is unique to BK currents in human glioma cells. We conclude that I _transient is generated by a mechanism related to the deactivation, and level of prior activation, of glioma BK channels. To account for these findings we propose that K⁺ ions are trapped within glioma BK channels during deactivation and are forced to exit to the extracellular side in a manner independent of membrane potential.     

Effects of ADP upon the ATP-sensitive K+ channel in rat ventricular myocytes

Summary The effects of ADP upon the gating of ATP-sensitive K⁺ channels from rat ventricular myocytes have been investigated by patch-clamp single-channel current recording experiments. ADP was applied to the internal surface of excised insideout membrane patches and depending upon the experimental protocol and the concentration it was found that ADP could either inhibit or stimulate openings of ATP-sensitive K⁺ channels. In the absence of inactivation, ATP-sensitive K⁺ channels were inhibited by ADP in a dose-dependent manner. Partially inactivated channels, on the other hand, were stimulated by low (10 to 250 M) and inhibited by high (>250 M) concentrations of ADP. ATP-sensitive K⁺ channels which were being inhibited by ATP (<1 mM) could be opened by the simultaneous application of ADP (50 M to 1 mM). ADP had no effect upon channels inhibited by mM concentrations of ATP. The situation was further complicated when it was found that inhibition evoked by ADP was strongly attenuated by the presence of Mg²⁺ ions whilst channel stimulation, whether of partially inactivated channels or channels inhibited by ATP, required the presence of Mg²⁺ ions. The analog of ADP, ADPS, always evoked inhibition of ATP-sensitive K⁺ channels which was not affected by the presence or absence of Mg²⁺ ions.     

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

Summary Guard cells of higher plants control transpirational water loss and gas exchange for photosynthesis by opening and closing pores in the epidermis of the leaf. To power these turgordriven movements, guard cells accumulate (and lose) 200 to 400mm (1 to 3 pmol/cell) K⁺, fluxes thought to pass through K⁺ channels in the guard cells plasma membrane. Steady-state current-voltage (I–V) relations of intactVicia guard cells frequently show large, outward-going currents at potentials approaching 0 mV. Since this current could be carried by K⁺ channels, its pharmacology and dependence on external K⁺ (K _v ⁺ ) has been examined under voltage clamp over an extended potential range. Measurements were carried out on cells which showed little evidence of primary electrogenic transport, thus simplifying analyses. Clamping these cells away from the free-running membrane potential (V _m ) revealed an outward-rectifying current with instantaneous and time-dependent components, and sensitive to the K⁺ channel blocker tetraethylammonium chloride. The current declined also under metabolic blockade with NaCN and in the presence of diethylstilbesterol, responses which were attributed to secondary effects of these inhibitors. The putative K⁺ current rose with voltage positive toV _m but it decayed over two voltage ranges, one negative toV _m and one near +100 mV, to give steady-stateI–V relations with two regions of negative (slope) conductance. Voltage-dependent and kinetic characteristics of the current were affected by K _v ⁺ and followed the K⁺ equilibrium potential. Against a (presumably) low background of primary ion transport, the K⁺ current contributed appreciably to charge balance atV _m in 0.1mm as well as in 1 to 10mm K _v ⁺ . Thus, gating of these K⁺ channels compensates for the prevailing K⁺ conditions to ensure net K⁺ movement out of the cell.     

Evidence for a K+ channel in bovine chromaffin granule membranes: single-channel properties and possible bioenergetic significance

A K⁺ channel was incorporated into voltage-clamped planar lipid bilayers from bovine chromaffin granules and resealed granule membranes (ghosts). It was not incorporated from plasma membrane-rich fractions from the adrenal medulla. The channel had a conductance of 400 pS in symmetric 450 mM KCI, with the permeability sequence K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺, and was insensitive to both Ca²⁺ and charybdotoxin. It exhibited complex gating kinetics, consistent with the presence of multiple open and closed states, and its gating was voltage-dependent. The channels appeared to incorporate into bilayers with the same orientation, and were blocked from one side (the side of vesicle addition) by 0.2-1 mM TEA'. The block was slightly voltage-dependent. Acidification of resealed granule membranes in response to external ATP (which activated the vacuolartype ATPase) was significantly reduced in the presence of 1 mM intralumenal TEACI (with 9 mM KCl), and parallel measurements with the potential-sensitive dye Oxonol V showed that such vesicles tended to develop higher internal-positive membrane potentials than control vesicles containing only 10 mM KCI. 1 mM TEA⁺ had no effect on proton-pumping activity when applied externally, and did not directly affect either the proton-pumping or ATP hydrolytic activity of the partially-purified ATPase. These results suggest that chromaffin granule membranes contain a TEA⁺-sensitive K⁺ channel which may have a role in regulating the vesicle membrane potential. Correspondence to: R. H. Ashley     

Inwardly rectifying potassium current in rabbit osteoclasts: A whole-cell and single-channel study

Summary Ionic conductances of rabbit osteoclasts were investigated using both whole-cell and cell-attached configurations of the patch-clamp recording technique. The predominant conductance found in these cells was an inwardly rectifying K⁺ conductance. Whole-cell currents showed an N-shaped current-voltage (I–13;V) relation with inward current activated at potentials negative to E_K. When external K⁺ was varied, I-V curves shifted 53 mV/10-fold change in [K⁺]_out, as predicted for a K⁺-selective channel. Inward current was blocked by Ba²⁺ and showed a time-dependent decline at negative potentials, which was reduced in Na⁺-free external solution. Inward single-channel currents were recorded in the cell-attached configuration. Single-channel currents were identified as inward-rectifier K⁺ channels based on the following observations: (i) Unitary I-V relations rectified, with only inward current resolved. (ii) Unitary conductance () was 31 pS when recorded in the cell-attached configuration with 140 mm K⁺ in the pipette and was found to be dependent on [K⁺]. (iii) Addition of Ba²⁺ to the pipette solution abolished single-channel events. We conclude that rabbit osteoclasts possess inwardly rectifying K⁺ channels which give rise to the inward current recorded at negative potentials in the whole-cell configuration. This inwardly rectifying K⁺ current may be responsible for setting the resting membrane potential and for dissipating electrical potential differences which arise from electrogenic transport of protons across the osteoclast ruffled border.This work was supported by The Arthritis Society and the Medical Research Council of Canada. M.E.M.K. was supported by a fellowship, S.J.D. a development Grant and S.M.S. a scholarship from the Medical Research Council. We thank Dr. Zu Gang Zheng for help with scanning microscopy.     

K+ current stimulation by Cl- in the midgut epithelium of tobacco hornworm (Manduca sexta)

Summary Chloride-stimulated K⁺ secretion by Manduca sexta midgut (5th-instar larvae) was measured as K⁺-carried short-circuit current of the tissue mounted in an Ussing chamber. Microscopic parameters, such as single-channel current and channel density for the rate-determining passive transport step across the basolateral goblet cell membrane (i.e. K⁺ channels), were estimated by means of current-fluctuation analysis of the K⁺ channel blockade by haemolymph-side Ba²⁺ ions. Ba²⁺ was equally effective with Cl^- or gluconate (Glu^-) as the principal ambient anion. The Ba²⁺-induced K⁺ channel conduction noise is reflected by a Lorentzian, or relaxation, noise component in the power spectrum of the K⁺ current fluctuations. A reduced Lorentzian plateau value, but an unchanged corner frequency, were observed when Cl^- was replaced by Glu^-. The results from the analysis of a two-state model of K⁺ channel block by Ba²⁺, with respect to the anion-replacement effects, suggest that the observed changes in K⁺ current and Lorentzian plateau value mirror a complex change of the underlying parameters: Cl^- omission reduces single channel current but increases channel density so that the product of single channel current and channel density is smaller in Glu^- than in Cl^-. It seems likely that basolateral K⁺ channels (1) are subject to anionic gating ligands, and (2) depend on anions with respect to the rate of K⁺ transfer through and open K⁺ channel.Abbreviations a.c. alternating current - single-channel conductance - E _K K⁺ Nernst potential - f frequency contained in current noise - f _c corner frequency - Glu^- gluconate - G _t transepithelial conductance - I _sc short-circuit current - I _K K⁺ current - I _K(max) maximal K⁺ current - i single-channel current - K _Ba barium inhibition constant - K _m Michaelis constant of saturating K⁺ current - k ₀₁ and k ₁₀ barium association and dissociation rate constant, respectively - M K⁺ channel density - S _f power density - S _o Lorentzian plateau value - P _o channel-open probability - P _K K⁺ permeability - V _sc cellular potential at short-circuit These results have already been presented in part, at the 1989 joint meeting of the German and Israel Physiological Societies in Jerusalem (Zeiske et al. 1990).     

The gating of nucleotide-sensitive K+ channels in insulin-secreting cells can be modulated by changes in the ratio ATP4−/ADP3− and by nonhydrolyzable derivatives of both ATP and ADP

Summary The³¹P-NMR technique has been used to assess the intracellular ratios and concentrations of mobile ATP and ADP and the intracellular pH in an insulin-secreting cell line, RINm5F. The single-channel current-recording technique has been used to investigate the effects of changes in the concentrations of ATP and ADP on the gating of nucleotide-dependent K⁺ channels. Adding ATP to the membrane inside closes these channels. However, in the continued presence of ATP adding ADP invariably leads to the reactivation of ATP-inhibited K⁺ channels, even at ATP^4–/ADP^3– concentration ratios greater than 71. Interactions between ATP^4– and ADP^3– seem competitive. An increase in the concentration ratio ATP^4–/ADP^3– consistently evoked a decrease in the open-state probability of K⁺ channels; conversely a decrease in ATP^4–/ADP^3– increased the frequency of K⁺ channel opening events. Channel gating was also influenced by changes in the absolute concentrations of ATP^4– and ADP^3–, at constant free concentration ratios. ADP-evoked stimulation of ATP-inhibited channels did not result from phosphorylation of the channel, as ADP--S, a nonhydrolyzable analog of ADP, not only stimulated but enhanced ADP-induced activation of K⁺ channels, in the presence of ATP. Similarly, ADP was able to activate K⁺ channels in the presence of two nonhydrolyzable derivatives of ATP, AMP-PNP and methylene ATP.     

Pump and K+ inward rectifiers in the plasmalemma of wheat root protoplasts

An electrogenic pump, a slowly activating K⁺ inward rectifier and an intermittent, spiky, K⁺ inward rectifier, have been identified in the plasmalemma of whole protoplasts from root cortical cells of wheat (Triticum) by the use of patch clamping techniques. Even with high external concentrations of K⁺ of 100 m m, the pump can maintain the membrane potential difference (PD) down to –180 mV, more negative than the electrochemical equilibrium potentials of the various ions in the system. The slowly activating K⁺ inward rectifier, apparent in about 23% of protoplasts, allows inward current flow when the membrane PD becomes more negative than the electrochemical equilibrium potential for K⁺ by about 50 mV. The current usually consists of two exponentially rising components, the time constant of one about 10 times greater than the other. The longer time constant is voltage dependent, while the smaller time constant shows little voltage dependence. The rectifier deactivates, on return of the PD to less negative levels, with a single exponential time course, whose time constant is strongly voltage dependent. The spiky K⁺ inward rectifier, present in about 68% of protoplasts, allows intermittent current, of considerable magnitude, through the plasmalemma at PDs usually more negative than about –140 mV. Patch clamp experiments on detached outside-out patches show that a possibly multi-state K⁺ channel, with maximum conductance greater than 400 pS, may constitute this rectifier. The paper also considers the role of the pump and the K⁺ inward rectifiers in physiological processes in the cell.We thank Don Mackenzie and Kay Morris for their valuable technical assistance, particularly in the preparation of protoplasts. The project is funded by the Australian Research Council.     

Contrasting roles in ion transport of two K+-channel types in root cells of Arabidopsis thaliana

Plant roots accumulate K⁺ over a range of external concentrations. Root cells have evolved at least two parallel plasma-membrane K⁺ transporters which operate at millimolar and micromolar external [K⁺]: high-affinity K⁺ uptake is energised by symport with H⁺, while low-affinity uptake is assumed to occur via ion channels. To determine the role of ion channels in low-affinity K⁺ uptake, a characterisation of the principal K⁺-selective ion channels in the plasma membrane of Arabidopsis thaliana (L.) Heynh. cv. Columbia roots was undertaken. Two classes of K⁺-selective channels were frequently observed: one inward (IRC) and one outward (ORC) rectifying with unitary conductances of 5 pS, 20 pS (IRCs) and 15 pS (ORC), measured in symmetrical 10 mM KCl. The dominant IRC (5 pS) and ORC (15 pS) were highly cation-selective (P_Cl P_K < 0.025) but less selective amongst monovalent cations (P_NaP_K0.17–0.3). Both the IRC and the ORC were blocked by Ba²⁺, Cs⁺ and tetra-ethyl-ammonium, whereas 4-aminopyridine and quinidine selectively inhibited the ORC. The ORC open probability was steeply voltage-dependent and ORC activation potentials were close to the potassium equilibrium potential (E_K+), enabling ORCs to conduct mainly outward, but occasionally inward, K⁺ current. By contrast, gating of the 5-pS IRC was weakly voltageependent and IRC gating was invariably restricted to membrane potentials more negative than E_K+, ensuring K⁺ transport was always inwardly directed. Studies on channel activity were conducted for a large number of root cells grown at two levels of external [K⁺], one where K⁺ uptake is likely to be principally through channels (6 mM K⁺) and one where it must be energised (100 M K⁺). Shifting growth conditions from high to low K⁺ did not affect single-channel properties such as conductance and selectivity, nor the manifestation of the ORC and 20-pS IRC, but led to enhanced activity of the 5-pS IRC. The enhanced activity of the 5-pS IRC was mirrored by a parallel increase in unidirectional ⁸⁶Rb⁺ influx after low-K⁺ growth, clearly indicating a dominant role of this particular channel in K⁺ uptake at supra millimolar external [K⁺].Abbreviations E_K+ potassium equilibrium potential - E_m membrane potential - HK high [K⁺] - IRC inward rectifying channel - LK low [K⁺] - ORC outward rectifying channel - TEA tetra-ethyl-ammonium Financial support was provided by the Biotechnology and Biological Sciences Research Council (Grant PG87/529) and by the European Union (Framework III, Biotechnology Programme).     

Voltage dependence of the Ca2+-activated K+ conductance of human red cell membranes is strongly dependent on the extracellular K+ concentration

Summary The conductance of the Ca²⁺-activated K⁺ channel (g _K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V _m ) and extracellular K⁺ concentration ([K⁺]_ex). ATP-depleted cells, with fixed values of cellular K⁺ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K⁺ channels, to buffer-free salt solutions with given K⁺ concentrations. Outward-current conductances were calculated from initial net effluxes of K⁺, correspondingV _m , monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E _K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of⁴²K at (V _m –E _K)0, using a single-file flux ratio exponent of 2.7. Within a [K⁺]_ex range of 5.5 to 60mm and at (V _m –E _K) 20 mV a basic conductance, which was independent of [K⁺]_ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm² between 0 and –100 mV. As (V _m –E _K) decreased from 20 towards zero mVg _K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm². The zero-current conductance was not significantly dependent on [K⁺]_ex (30 to 156mm) corresponding toV _m (–50 mV to 0). A further increase ing _K(Ca) symmetrically aroundE _K is suggested as (V _m –E _K) becomes positive. Increasing the extracellular K⁺ concentration from zero and up to 3mm resulted in an increase ing _K(Ca) from 50 to 70 S/cm². Since the driving force (V _m –E _K) was larger than 20 mV within this range of [K⁺]_ex this was probably a specific K⁺ activation ofg _K(Ca). In conclusion: The Ca²⁺-activated K⁺ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.     

Whole-cell currents in macrophages: I. Human monocyte-derived macrophages

Summary We examined the variability of occurrence and frequency of voltage-dependent whole-cell currents in human peripheral blood monocyte-derived macrophages (HMDM) maintained in culture for up to three weeks. An increase in cell capacitance from an average value of 9 pF on the day of isolation to 117 pF at 14 days accompanied growth and differentiation in culture. The average resting potential was approximately –34 mV for cells beyond two days in culture. Cells exhibited a voltage-and time-dependent outward current upon membrane depolarization above approximately –30 mV, which appeared to be composed of a number of separate currents with variable expression from donor to donor. Three of these currents are carried by K⁺. The frequency of each outward current type was calculated for 974 cells obtained from 36 donors. The HMDMs in these studies exhibited two 4-aminopyridine (4-AP) sensitive, time-dependent outward currents (I _A andI _B ) that could be differentiated on the basis of the presence or absence of steady-state inactivation in the physiological potential range, time course of inactivation during maintained depolarization, as well as threshold of activation. The 4-AP-insensitive outward current activated at approximately 10 mV. One component of the 4-AP insensitive-outward current (I _C ) could be blocked by external TEA and by the exchange of internal Cs⁺ or Na⁺ for K⁺. The probability of observingI _B andI _C appeared to be donor dependent. Following total replacement of internal K⁺ with Cs⁺, two additional currents could be identified (i) a delayed component of outward current (I _D ) remained which could be blocked by low concentrations of external Zn²⁺ (4 m) and was insensitive to anion replacement in the external solution and (ii) a Cl^– current with a reversal potential which shifted in the presence of external anion replacement and which was irreversibly inhibited by the stilbene SITS. The activation of a prominent time-independent inward currents was often observed with increasing hyperpolarization. This inward current was blocked by external Ba²⁺ and corresponded to the inwardly rectifying K⁺ current. Neither inward nor outward current expression appeared dependent on whether cells were differentiated in adherent or suspension culture nor was there demonstrable differential current expression observed upon transition from suspension to adherent form.     

L-type Ca<Superscript>2+</Superscript> channel and Ca<Superscript>2+</Superscript>-permeable nonselective cation channel as a Ca<Superscript>2+</Superscript> conducting pathway in myocytes isolated from the cricket lateral oviduct

The Ca²⁺-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at –40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs²⁺-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca²⁺ with Ba²⁺ slowed the current decay. Increasing the external Ca²⁺ or Ba²⁺ concentration increased the amplitude of the inward current and the current–voltage (I–V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na⁺ in the absence of extracellular Ca²⁺. Current carried by Na⁺ (I _Na) was almost completely blocked by the dihydropyridine Ca²⁺ channel antagonist, nifedipine, suggesting that the I _Na is through voltage-dependent L-type Ca²⁺ channels. The other inward current is voltage-independent and its I–V relationship was linear between –100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs⁺-aspartate and 140 mM Na⁺-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na⁺ was replaced with an equimolar amount of K⁺ or Cs⁺, or 50 mM Ca²⁺ or Ba²⁺. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I–V relationship for the current evoked by the hypotonic solution also showed a linear relationship between –100 mV to 0 mV. Bath application of Gd³⁺ (10 M) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca²⁺-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca²⁺ channel and stretch-activated Ca²⁺-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.Abbreviations I _Ca Ca²⁺ current - I _Na Na⁺ current - I–V current–voltage - NSCC nonselective cation channel Communicated by G. Heldmaier     

Modulation of HERG channel inactivation by external cations

We investigated the effect of external cations on the permeability characteristics and gating kinetics of the human ether-à-go-go-related gene (HERG) current using the whole-cell patch-clamp technique. Inward HERG currents were recorded on hyperpolarization in 140 mM external Cs⁺ and Rb⁺, as well as K⁺. The permeability ratios of Rb⁺ and Cs⁺ relative to K⁺ were 1.25 and 0.56, respectively. Biphasic outward currents were recorded on depolarization in 140 mM Cs⁺ and in Rb⁺ with much smaller amplitude. The voltage dependence of inactivation was affected by external cations, such that the half-inactivation voltage shifted from –69.4±3.7 mV in K⁺ to –30.7±1.6 mV in Cs⁺ and to –35.8±1.9 mV in Rb⁺ (n=5). The time constants of inactivation were also changed significantly by external cations; of inactivation at +40 mV was 16.4±2.2 ms in 140 mM K⁺, 181±20.3 ms in Cs⁺, and 94.1±7.6 ms in Rb⁺ (n=5). Voltage dependence of activation was not altered significantly. The inhibition of the rapid inactivation mechanism by large cations may suggest that the foot-in-the-door model of gating is involved in HERG channel inactivation.     

Ca2+-activated K+ channels in the apical membrane ofNecturus choroid plexus

Summary The properties of Ca²⁺-activated K⁺ channels in the apical membrane of theNecturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K⁺ over Na⁺ and K⁺ over Cs⁺. They were blocked by high cytoplasmic Na⁺ concentrations (110mm). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca²⁺ concentrations. Increasing Ca²⁺ from 5 to 500nm, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca²⁺ was 500nm. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba²⁺ reduced both channel-open probability and conductance. The sites for the action of Ba²⁺ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117mm were calculated as the apparent Ba²⁺ dissociation constants (K _d (0 mV) for the effects on probability and conductance, respectively. TEA⁺ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with aK _d (0 mV)=5.6mm. A second site, with a higher affinity,K _d (0 mV)=0.23mm, may account for the near total block of chanel conductance by 2mm TEA⁺ applied to the outside of the membrane. It is concluded that the channels inNecturus choroid plexus exhibit many of the properties of maxi Ca²⁺-activated K⁺ channels found in other tissues.