Identification of an ATP-sensitive K+ channel in spiny neurons of rat caudate nucleus

On the somata of GABAergic spiny neurons in the caudate nucleus of the rat an ATP-sensitive K⁺ channel (K_ATP-channel) was identified. The K_ATP-currents in cell-attached patches were activated both by energy-depleting conditions (200 M cyanide) and by diazoxide (300 M) and were reversibly blocked by tolbutamide (EC₅₀=5 M). In inside-out patch membranes both ATP (1 mM) and its non-hydrolyzable analog AMP-PNP (adenylylimidodiphosphate; EC₅₀=27M) reversibly inhibited channel activity. These results demonstrate that the K_ATP-channel in spiny neurons displays properties characteristic of the K_ATP-channel in hippocampal, neocortical and nigral neurons and in pancreatic ß-cells.     

Opposite effects of tolbutamide and diazoxide on the ATP-dependent K+ channel in mouse pancreatic β-cells

The influence of the antidiabetic sulphonylurea tolbutamide on K⁺ channels of mouse pancreatic -cells was investigated using different configurations of the patch clamp technique. The dominant channel in resting cells is a K⁺ channel with a single-channel conductance of 60 pS that is inhibited by intracellular ATP or, in intact cells, by stimulation with glucose. In isolated patches of -cells membrane, this channel was blocked by tolbutamide (0.1 mM) when applied to either the intracellular or extracellular side of the membrane. The dose-dependence of the tolbutamide-induced block was obtained from whole-cell experiments and revealed that 50% inhibition was attained at approximately 7 M. In cell-attached patches low concentrations of glucose augmented the action of tolbutamide. Thus, the simultaneous presence of 5 mM glucose and 0.1 mM tolbutamide abolished channel activity and induced action potentials. These were not produced when either of these substances was added alone at these concentrations. The inhibitory action of tolbutamide or glucose on the K⁺ channel was counteracted by the hyperglycaemic sulphonamide diazoxide (0.4 mM). Tolbutamide (1 mM) did not affect Ca²⁺-dependent K⁺ channels. It is concluded that the hypo- and hyperglycaemic properties of tolbutamide and diazoxide reflect their ability to induce the closure or opening, respectively, of ATP-regulated K⁺ channels.     

Properties of single potassium channels in hypothalamic neurons

In this work the cell-attached and the excised membrane patch configurations have been used to determine properties of voltage-dependent K⁺ channels in cultured rat hypothalamic neurons. With inside-out patches and 140 mM K⁺ in the bath and 5 mM K⁺ in the pipette step depolarizations, in excess of 20 mV, elicited channel activity with at least two independent current levels. The larger current level was studied presently and current-voltage plots showed the conductance of the channel to be 48 pS; measurements of the changes in reversal potential with 140 mM or 5 mM K⁺ in the bathing solution indicated the channel to be selective for K⁺. The 48 pS channel was blocked when the bathing solution contained 140 mM Cs⁺. A concentration of 10 mM TEA applied to the outside of the patch in the outside-out configuration caused a loss of channel activity whereas 4-AP had no obvious effect on channel conductance or kinetics. Changes in the bath concentration of Ca²⁺ had no apparent effect to alter the frequency or duration of channel openings for inside-out patches. In some instances obvious changes in channel kinetics were observed during the course of an experiment; these included long periods where no channel opening events occurred and on a few other occasions progressive increases in mean open time. In cell-attached patch experiments with no TTX in the bathing medium to block sodium channels, action potentials could be recorded through capacitive coupling between the cell and the pipette. In such cases the heights of spontaneous K⁺ channel currents were very similar to those associated with channel openings during the repolarization phase of the spike. The properties of the K⁺ channel studied here are consistent with those associated with the delayed rectifier K⁺ conductance (I _K) and would serve to control electrical excitability in hypothalamic neurons.     

Biophysical,pharmacological and developmental properties of ATP-sensitive K+ channels in cultured myotomal muscle cells from Xenopus embryos

Unlike mammalian muscle cells in culture, cultured myotomal muscle cells of Xenopus embryos express ATP-sensitive K⁺ (K_ATP) channels. The K_ATP channels are blocked by internal ATP (half-maximal inhibition K _0.5 = 16 M) and to a lesser extent by internal ADP, are voltage independent, have an inward rectification at positive potentials and are inhibited by glibenclamide (K _0.5 = 2 M). Surprisingly, these K_ATP channels are not sensitive to K⁺ channel openers such as cromakalim. Opening of these K_ATP channels does not occur under normal physiological conditions. It is elicited by metabolic exhaustion of the muscle cell and it precedes the development of an irreversible rigor state. Neither intracellular acidosis nor an increase of intracellular Ca²⁺ are involved in K_ATP channel opening. Different types of K⁺ channels are successively expressed after plating of myotomal muscle cells: (1) sustained delayed-rectifier K⁺ channels; (2) K_ATP channels; (3) inward-rectifier K⁺ channels; (4) transient delayed-rectifier K⁺ channels. The current density associated with K_ATP channels far exceeds that of voltage-dependent K⁺ channels. Innervation controls the expression of these K_ATP channels. Co-culture of muscle cells with neurons from the neural tube decreases the number of active K_ATP channels per patch. Similarly, in situ innervated submaxillaris muscle of tadpoles at stage 50–55 has a very low density of K_ATP channels.     

Aromatic aldehydes and aromatic ketones open ATP-sensitive K+ channels in guinea-pig ventricular myocytes

Patch-clamp techniques were used to study the effects of three carbonyl compounds, 3,4-dihydroxy-benzaldehyde, 2,3-dihydroxybenzaldehyde, and 2,4-dihydroxy-acetophenone, on the adenosine-5-triphosphate(ATP)-sensitive K⁺ channel current (I _K.ATP) in guinea-pig ventricular myocytes. 3,4-Dihydroxybenzaldehyde (0.5–1 mM) shortened the action potential duration, and this effect was inhibited by application of a specific blocker of I _K.ATP, glibenclamide. The shortening of the action potential duration was shown to be caused by a time-independent outward current. In the cell-attached patch configuration, all three compounds activated a kind of single-channel current, which showed an inward rectification at positive potentials and which had a linear current/voltage relation at negative potentials, having a conductance of 90 pS. The current reversed at about 0 mV in symmetrical K⁺ concentrations on both sides of the membrane. In excised patches this current was blocked by internal application of ATP. Thus we identified this channel as I _K.ATP. The activation effects of two aromatic aldehydes were stronger than that of the aromatic ketone. The effect of these compounds on I _K.ATP was not reduced by addition of cysteine (10 mM). In inside-out patches, 3,4-dihydroxybenzaldehyde increased the activity of I _K.ATP, which had been blocked by 0.5 mM MgATP in the presence of 0.5 mM ADP, but the activation effect was variable and much weaker than that in the cell-attached configuration, and was completely eliminated in the absence of ADP. These results suggest that these compounds: (a) modulate I _K.ATP perhaps through an intracellular mechanism, (b) bind covalently to proteins to form a Schiff base which may by responsible for the effects, and (c) may require an ADP-dependent process.     

A potassium current activated by lemakalim and metabolic inhibition in rabbit mesenteric artery

K⁺ channels which are inhibited by intracellular ATP (ATP_i) (K_ATP channels) are thought to be the physiological target site of the K⁺ channel opening drugs (2) and to underlie a variety of physiological phenomena including hypoxia induced vasodilation (3). However, electrophysiological evidence for ATP_i-regulated K⁺ currents in smooth muscle is scarce. We, therefore, investigated the effects of one K⁺ channel opener, lemakalim, and metabolic inhibition on the membrane conductance of freshly dissociated rabbit mesenteric artery smooth muscle cells, using the perforated-patch whole cell recording technique (6). The cells were metabolically inhibited with 1 mM iodoacetic acid and 50 M dinitrophenol. Both lemakalim (0.1–3 M) and metabolic inhibition activated a time-independent and glyburide sensitive K⁺ current at physiological membrane potentials. The similarities between the lemakalim and metabolic inhibition activated currents suggest that a single class of channels underlies both currents. These results are the first whole-cell current recordings to demonstrate the activation of a smooth muscle membrane conductance by metabolic inhibition, lending support to the view that hypoxia induced vasodilation arises from the activation of K_ATP channels.     

Properties of single K+ channels in the basolateral membrane of rabbit proximal straight tubules

The basolateral membrane of rabbit straight proximal tubules, which were cannulated and perfused on one side, was investigated with the patch clamp technique. Properties of inward and outward directed single K⁺ channel currents were studied in cell-attached and insideout oriented cell-excised membrane patches. In cell-attached patches with NaCl Ringer solution both in pipette and bath, outward K⁺ currents could be detected after depolarization of the membrane patch by about 20–30 mV. The current-voltage (i/V) relationship could be fitted by the Goldman-Hodgkin-Katz (GHK) current equation, with the assumption that these channels were mainly permeable for K⁺ ions. A permeability coefficientP _K of (0.17±0.04) · 10^–12 cm³/s was obtained, the single channel slope conductance at infinite positive potentialg(V ) was 50±12 pS and the single channel conductance at the membrane resting potentialg(V _bl) was 12±3 pS (n=4). In cell-excised patches, with NaCl in the pipette and KCl in the bath, the data could also be fitted to the GHK equation and yieldedP _K = (0.1 ±0.01) ·10^–12 cm³/s,g(V ) = 40 ± 4 pS andg(V _bl) = 7 ± 1 pS (n=8). In cell-attached patches with KCl in the pipette and NaCl in the bath, inward K⁺ channels occurred at clamp potentials 60 mV, whereas outward K⁺ channel current was detected at more positive voltages. The current-voltage curves showed slight inward rectification. The single channel conductance, obtained from the linear part of the i/V curve by linear regression, was 46±3 pS and the reversal potential was 59±6 mV (n=9). In cell-excised patches with KCl in the pipette and NaCl in the bath, inward directed K⁺ channel currents could again be described by the GHK equation. The single channel parameters were similar to those recorded for outward K⁺ currents (see above). In inside-out oriented cell-excised patches with NaCl in the pipette and KCl in the bath, reducing bath (i.e. cytosolic) Ca²⁺ concentration from 10^–6 mol/l to less than 10^–9 mol/l did not affect the open state probability of single channel currents. These results demonstrate that the observed channels are permeable for K⁺ ions in both directions and that these basolateral K⁺ channels in rabbit proximal straight tubule are not directly dependent on Ca²⁺ ions.     

Anion and cation channels in the basolateral membrane of rabbit parietal cells

Ion channels in the basolateral membrane of rabbit parietal cells in isolated gastric glands were studied by the patch clamp technique. Whole-cell current-clamp recordings showed that the membrane potential (E _m ) changed systematically as a function of the chloride concentrations of the basolateral bathing solution ([Cl^–]₀), and of the pipette (intracellular) solution. The relationship betweenE _m and [Cl^–]₀ was not affected by additions of histamine, dibutyryl-cAMP, 4-acetoamido-4-isothiocyanostilbene-2,2-disulfonic acid and diphenylamine-2-carboxylate. The whole-cell Cl^– conductance was insensitive to voltage. In cell-attached and cell-free patch membranes, however, single Cl^– channel opening events could not be observed. The value ofE _m depended little on the basolateral K⁺ concentration, but inward-rectifier K⁺ currents were observed in the whole-cell configuration, activated by hyperpolarizing pulses and inhibited by extracellular Ba²⁺. In cell-attached and cell-free patches, openings of single inward-rectifier K⁺ channels and non-selective cation channels were infrequently recorded. Neither cAMP nor Ca²⁺ activated these cation channels. The single K⁺ channel conductance was about 230 pS under the symmetrical high K⁺ conditions and was inhibited by intracellular tetraethylammonium ions (TEA). The non-selective cation channel had a voltage-independent single conductance of 22 pS and was not inhibited by TEA.     

Single channel recordings of reconstituted ion channel proteins: an improved technique

Single channel recording of reconstituted ion channels is possible by patch clamp measurements of giant liposomes formed by dehydration-rehydration of lipid films. This hydration technique consists of carefully controlled dehydration of a suspension of small vesicles followed by rehydration of the residue resulting in formation of large liposomes. Patch pipettes can be attached to the liposome surface, yielding stable, high resistance seals between membranes and glass pipettes. This method allows the study of the properties of reconstituted ion channels from different tissues. The hydration technique was used to characterize the reconstituted K⁺-channel of sarcoplasmic reticulum from rabbit skeletal muscle. In a solution of 100 mM KCl, the sarcoplasmic reticulum K⁺-channel studied displays a conductance _K ⁺ of 145 pS. The single channel conductance in 100 mM Rb⁺ and Na⁺ is _Rb ⁺ = 98 pS and _Na ⁺ = 65 pS respectively. A concentration of 0.5 mM decamethonium causes a flickering channel block. These properties are in good agreement with the ones found in sarcoplasmic reticulum K⁺-channels characterized by other methods. Other ion channels have also been reconstituted and studied by this technique. This improved method is compared with previous approaches and its applicability for the characterization of reconstituted ion channel proteins is discussed.     

Cation specificity and pharmacological properties of the Ca2+-dependent K+ channel of rat cortical collecting ducts

The luminal membrane of principal cells of rat cortical collecting duct (CCD) is dominated by a K⁺ conductance. Two different K⁺ channels are described for this membrane. K⁺ secretion probably occurs via a small-conductance Ca²⁺-independent channel. The function of the second, large-conductance Ca²⁺-dependent channel is unclear. This study examines properties of this channel to allow a comparison of this K⁺ channel with the macroscopic K⁺ conductance of the CCD and with similar K⁺ channels from other preparations. The channel is poorly active on the cell. It has a conductance of 263±11 pS (n=36, symmetrical K⁺ concentrations) and of 139±3 pS (n=91) with 145 mmol/l K⁺ on one side and 3.6 mmol/l K⁺ on the other side of the membrane. Its open probability is high after excision (0.71±0.03, n=85). The channel flickers rapidly between open and closed states. Its permeability in the cell-free configuration was 7.0±0.2×10^–13 cm³/s (n=85). It is inhibited by several typical blockers of K⁺ channels such as Ba²⁺, tetraethylammonium, quinine, and quinidine and high concentrations of Mg²⁺. The Ca²⁺ antagonists verapamil and diltiazem also inhibit this K⁺ channel. As is typical for the maxi K⁺ channel, it is inhibited by charybdotoxin but not by apamin. The selectivity of this large-conductance K⁺ channel demonstrates significant differences between the permeability sequence (P _K > P _Rb > P _NH4 > P _Cs=P _Li=P _Na=P _choline=0) and the conductance sequence (g _K > g _NH4 > g _Rb > g _Li=g _choline > g _Cs=g _Na=0). The only other cations that are significantly conducted by this channel besides K⁺ (g _K at V _c = is 279±8 pS, n=88) are NH ₄ ⁺ (g _NH4=127±22 pS, n=10) and Rb⁺ (g _Rb=36±5 pS, n=6). The K⁺ currents through this channel are reduced by high concentrations of choline⁺, Cs⁺, Rb⁺, and NH ₄ ⁺ . These properties and the dependence of this channel on Ca²⁺ and voltage classify it as a maxi K⁺ channel. A possible physiological function of this channel is discussed in the accompanying paper.Supported by DFG Gr 480/10, by Schl 277/2-3 and by GIF 88/II     

Tetraethylammonium ion sensitivity of a 35-pS Ca2+-activated K+ channel in GH3 cells that is activated by thyrotropin-releasing hormone

Single Ca²⁺-activated K⁺ channels were studied in membrane patches from the GH₃ anterior pituitary cell line. We have previously demonstrated the coexistence of large-conductance and small-conductance (280 pS and 11 pS in symmetrical 150 mM K⁺, respectively) Ca²⁺-activated K⁺ channels in this cell line (Lang and Ritchie 1987). Here we report the existence of a third type of Ca²⁺-activated K⁺ channel that has a conductance of about 35 pS under similar conditions. In excised inside-out patches, this channel can be activated by elevations of the internal free Ca²⁺ concentration, and the open probability increases as the membrane potential is made more positive. In excised patches, the sensitivity of this 35-pS channel to internal Ca²⁺ is low; at positive membrane potentials, this channel requires Ca²⁺ concentrations greater than 10 M for activation. However, 35-pS channels have a much higher sensitivity to Ca²⁺ in the first minute after excision (activated by 1 M Ca²⁺ at –50 mV). Therefore, it is possible that the Ca²⁺ sensitivity of this channel is stabilized by intracellular factors. In cell-attached patches, this intermediate conductance channel can be activated (at negative membrane potentials) by thyrotropin-releasing hormone-induced elevations of the intracellular Ca²⁺ concentration and by Ca²⁺ influx during action potentials. The intermediate conductance channel is inhibited by high concentrations of external tetraethylammonium ions (K _d=17 mM) and is relatively resistant to inhibition by apamin.     

Single Ca2+-activated K+ channels in excised membrane patches of hamster oocytes

The properties of the Ca²⁺-activated K⁺ channel in unfertilized hamster oocytes were investigated at the single-channel level using inside-out excised membrane patches. The results indicate a new type of Ca²⁺-activated K⁺ channel which has the following characteristics: (1) single-channel conductance of 40–85 pS for outward currents in symmetrical K⁺ (150 mM) solutions, (2) inward currents of smaller conductance (10–50 pS) than outward currents, i.e. the channel is outwardly rectified in symmetrical K⁺ solutions, (3) channel activity dependent on the internal concentration of free Ca⁺ and the membrane potential, (4) modification of the channel activity by internal adenosine 5 diphosphate (0.1 mM) producing a high open probability regardless of membrane potential.     

Demonstration of a novel apamin-insensitive calcium-activated K+ channel in mouse pancreatic B cells

The whole-cell configuration of the patchclamp technique was used to characterize the biophysical and pharmacological properties of an oscillating K⁺-current that can be induced by intracellular application of GTP[S] in mouse pancreatic B cells (Ämmälä et al. 1991). These K⁺ conductance changes are evoked by periodic increases in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and transiently repolarize the B cell, thus inhibiting action-potential firing and giving rise to a bursting pattern. GTP[S]-evoked oscillations in K⁺ conductance were reversibly suppressed by a high (300 M) concentration of carbamylcholine. By contrast, ₂-adrenoreceptor stimulation by 20 M clonidine did not interfere with the oscillatory behaviour but evoked a small sustained outward current. At 0 mV membrane potential, the oscillating K⁺-current elicited by GTP[S] was highly sensitive to extracellular tetraethylammonium (TEA; 70% block by 1 mM). The TEA-resistant component, which carried approximately 80% of the current at –40 mV, was affected neither by apamin (1 M) nor by tolbutamide (500 M). The current evoked by internal GTP[S] was highly selective for K⁺, as demonstrated by a 51-mV change in the reversal potential for a sevenfold change in [K⁺]_o. Stationary fluctuation analysis indicated a unitary conductance of 0.5 pS when measured with symmetric ( 140mM) KCl solutions. The estimated singlechannel conductance with physiological ionic gradients is 0.1 pS. The results indicate the existence of a novel Ca²⁺-gated K⁺ conductance in pancreatic B cells. Activation of this K⁺ current may contribute to the generation of the oscillatory electrical activity characterizing the B cell at intermediate glucose concentrations.     

K+ channels in PC12 cells are affected by propofol

The effect on K⁺ currents (I _K) of the general anaesthetic propofol (PR) (2,6-diisopropylphenol) was tested in undifferentiated clonal pheochromocytoma (PC12) cells using the patch-clamp technique in whole-cell and single-channel configurations. PR decreased macroscopic I _K amplitudes in a concentration-dependent way from 50 M to 1 mM. The blocking effect was unchanged by repetitive depolarizing pulses and it was independent of the holding potential. Whereas activation of I _K in control conditions was fitted by sigmoidal plus exponential time courses, only the sigmoidal time course gave an adequate fit with PR in the bath. The above effects were reversible. PR concentrations below 140 M decreased single-channel activity for K⁺ channels with unitary conductance of 22 pS, in the voltage range between –40 and 60 mV from a holding potential of –50 mV. In contrast, the anaesthetic had nearly no effect on the opening probability of a channel with conductance of 10 pS. The unitary current amplitudes were unaffected in both channel types. These results suggest that PR action on I _K may depend on the different blocking mechanisms of the K⁺ channels.     

G-protein-mediated regulation of a Ca2+-dependent K+ channel in cultured vascular endothelial cells

The purpose of the present study was to determine the mechanism by which bradykinin activates the small conductance, inwardly rectifying, Ca²⁺-activated K⁺ channel (K_Ca) found in cultured bovine aortic endothelial cells. Channel activity was studied using the patch-clamp technique in whole-cell, cell-attached, inside-out and outside-out configurations. Channel conductance at potentials positive to 0 mV was 10±2 pS and at potentials negative to 0 mV 30±3 pS (n=7) when examined in symmetrical K⁺ (150 mmol/l) solutions. The channel open probability (P _o) was only weakly voltage dependent changing approximately 0.2 units over 160 mV. In contrast, raising the intracellular Ca²⁺ concentration from 100 nmol/l to 10 mol/l at –60 mV produced a graded increase in channel P _o from 0.15 to 0.96; the concentration required for half-maximum response (apparent K_0.5) was 719 nmol/l. At a constant Ca²⁺ concentration, application of guanosine triphosphate (GTP) to the cytoplasmic surface of the patch increased channel P _o. This effect was dependent upon the simultaneous presence of both GTP and Mg²⁺, and was reversed by the subsequent application of the guanosine diphosphate (GDP) analogue, guanosine-5-O-(2-thiodiphosphate) (GDPS). The hydrolysis-resistant GTP analogue, guanosine-5-O-(3-thiotriphosphate) (GTPS), induced a long-lasting increase in channel P _o. In the presence of Mg²⁺-GTP, the apparent K_0.5 for Ca²⁺ decreased from a control value of 722 nmol/l to 231 nmol/l. Addition of bradykinin to outside-out patches previously exposed to intracellular Mg²⁺-GTP further enhanced K_Ca activity, shifting the apparent K_0.5 for Ca²⁺ from 228 nmol/l to 107 nmol/l. This activation by bradykinin was not observed in patches following prior exposure to GDPS. These results suggest that bradykinin can activate the K_Ca channel of vascular endothelial cells via a G-protein-mediated change in the sensitivity of the channel for Ca²⁺. We postulate that vasoactive agonists may use this mechanism to maintain an elevated K⁺ permeability as the intracellular Ca²⁺ concentration returns towards normal resting levels.     

Solubilisation and reconstitution of the rabbit skeletal muscle sarcoplasmic reticulum K+ channel into liposomes suitable for patch clamp studies

Sarcoplasmic reticulum (SR) membrane vesicles have been prepared from rabbit skeletal muscle and solubilised using K⁺ cholate. Solubilised membrane proteins were reconstituted into small asolectin liposomes by dialysis against cholate-free solution. Large liposomes were produced by freezing and thawing at –80°C and room temperature, respectively. The liposomes were assayed for the SR K⁺ channel using the patch clamp technique. Channel density was modulated by varying protein: lipid ratios during reconstitution. Channels inserted into the membrane with a preferred orientation. The solubilised and reconstituted channel behaves ohmically over the holding potential range ±70 mV and has a conductance of 178.4±4.4 pS (mean ± SE,n=37) in 200 mM KCl. The channel has a selectivity sequence of K⁺>NH ₄ ⁺ >Rb⁺>Na⁺ and K⁺ conductance is blocked by hexamethonium and decamethonium. The opening probability of the reconstituted channel is voltage dependent. The conductance and gating characteristics displayed by the solubilised and reconstituted channel correlate well with those previously observed following the fusion of native SR membrane vesicles with planar phospholipid bilayers.     

K+ and Cl− currents in freshly isolated rat osteoclasts

Membrane electrical properties of freshly isolated rat osteoclasts were studied using patch-clamp recording methods. Characterization of the passive membrane properties indicated that the osteoclast cell membrane behaved as an isopotential surface. The specific membrane capacitance was 1.2±0.3 F/cm² (mean ±SD), with no difference between cells plated on glass and those adhering to a permeable collagen substrate. The current/voltage (I/V) relationship of all cells showed inward rectification and I/V curves shifted 51 mV positive per tenfold increase of [K⁺]_out, indicating an inwardly rectifying K⁺ conductance. The voltage dependence of the K⁺ chord conductance (g _K) also shifted positive along the voltage axis, and the maximum conductance increased, with elevation of [K⁺]_out. g _K for cells bathed in 4.7 mM [K⁺]_out increased e-fold per 12mV hyperpolarization, and half-maximal activation was at –89 mV. Approximately 18% (50 pS/pF) of the maximum g _K was active at –70 mV. Inward single-channel currents were recorded in cell-attached patches at hyperpolarizing potentials. With symmetrical K⁺, channel conductance was 25±3 pS and reversal was close to the K⁺ equilibrium potential, consistent with this K⁺ channel underlying the whole-cell K⁺ currents. With both conventional whole-cell and perforated-patch recording, no voltage-activated Ca²⁺ current was detected. In approximately 30% of osteoclasts studied, an outwardly rectifying current was observed, which was reversibly blocked by 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene2,2-disulphonic acid (SITS). This DIDS- and SITS-sensitive current reversed direction at the chloride equilibrium potential. We conclude that an inwardly rectifying K⁺ current is present in all rat osteoclasts and that some osteoclasts also exhibit an outwardly rectifying Cl^– current. Both these membrane conductances may play an important physiological role by dissipating the potential that arises from the electrogenic transport of H⁺ across the ruffled membrane of the osteoclast.     

Characterization of the high-conductance Ca2+-activated K+ channel in adult human skeletal muscle

Ca²⁺-activated K⁺ channels of a large conductance (BK_Ca) in human skeletal muscle were studied by patch clamping membrane blebs and by using the three microelectrode voltage-clamp recording technique on resealed fibre segments. Single-channel recordings in bleb-attached and inside-out modes revealed BK_Ca conductances of 230 pS for symmetrical and 130 pS for physiological K⁺ distributions. Open probability increased with membrane depolarization and increasing internal [Ca²⁺]. The Hill coefficient was 2.0, indicating that at least two Ca²⁺ ions are required for full activation. Kinetic analysis revealed at least two open and three closed states. An additional long-lived inactivated state, lasting about 0.5–20 s, was observed following large depolarizations, when extracellular K⁺ was lowered to physiological values. BK_Ca were blocked by three means: (1) externally by tetraethylammonium which reduced single-channel amplitude (IC₅₀ approx. 0.3 mM); (2) internally by polymyxin B which decreased the open probability (IC₅₀ approx. 5 g/ml); and (3) externally by charybdotoxin which caused long-lasting periods of inactivation (IC₅₀ <10 nM). Measurements on resealed fibre segments at physiological [K⁺] were in accordance with the single-channel data: only when intracellular [Ca²⁺] was elevated did charybdotoxin (50 nM) reduce the macroscopic membrane K⁺ conductance with depolarizing voltage steps.     

Hypoxia increases the activity of Ca2+-sensitive K+ channels in cat cerebral arterial muscle cell membranes

The cellular mechanisms mediating hypoxia-induced dilation of cerebral arteries have remained unknown, but may involve modulation of membrane ionic channels. The present study was designed to determine the effect of reduced partial pressure of O₂, PO ₂, on the predominant K⁺ channel type recorded in cat cerebral arterial muscle cells, and on the diameter of pressurized cat cerebral arteries. A K⁺-selective single-channel current with a unitary slope conductance of 215 pS was recorded from excised inside-out patches of cat cerebral arterial muscle cells using symmetrical KCl (145 mM) solution. The open state probability (NP _o) of this channel displayed a strong voltage dependence, was not affected by varying intracellular ATP concentration [(ATP]_i) between 0 and 100 M, but was significantly increased upon elevation of intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Low concentrations of external tetraethylammonium (0.1–3 mM) produced a concentration-dependent reduction of the unitary current amplitude of this channel. In cell-attached patches, where the resting membrane potential was set to zero with a high KCl solution, reduction of O₂ from 21% to < 2% reversibly increased the NP _o, mean open time, and event frequency of the Ca²⁺-sensitive, high-conductance single-channel K⁺ current recorded at a patch potential of + 20 mV. A similar reduction in PO₂ also produced a transient increase in the activity of the 215-pS K⁺ channel measured in excised inside-out patches bathed in symmetrical 145 mM KCl, an effect which was diminished, or not seen, during a second application of hypoxic superfusion. Hypoxia had no effect on [Ca²⁺]_i or intracellular pH (pH_i) of cat cerebral arterial muscle cells, as measured using Ca²⁺- or pH-sensitive fluorescent probes. Reduced PO₂ caused a significant dilation of pressurized cerebral arterial segments, which was attenuated by pre-treatment with 1 mM tetraethylammonium. These results suggest that reduced PO₂ increases the activity of a high-conductance, Ca²⁺-sensitive K⁺ channel in cat cerebral arterial muscle cells, and that these effects are mediated by cytosolic events independent of changes in [Ca²⁺]_i and pH_i.     

Ion channels in the basolateral membrane of intralobular duct cells of mouse mandibular glands

We have used single-channel patch-clamp techniques to study the ion channels in the basolateral membranes of intralobular duct cells from the mouse mandibular gland. In 39% of cell-attached patches, we observed a K⁺ channel that had an inwardly rectifying current/voltage (I/V) relation with a maximum slope conductance of 123±9 pS (n=12) and a zero current potential of +49.4±3.4 mV (n=5) relative to the resting cell potential. The selectivity sequence of this channel, as estimated by zero current potential measurements, was: K⁺ (1) > Rb⁺ (0.38) > NH ₄ ⁺ (<0.34), Cs⁺ (<0.16) > Na⁺ (<0.028). The activity of the channel was not affected by changes in membrane potential, nor was it affected by changes in the free Ca²⁺ concentration on the cytosolic side of inside-out excised patches in the range 1 nmol/l to 1 mol/l. In 38% of cell-attached patches we observed a second K⁺ channel type with a maximum slope conductance of 62±3 pS (n=12) and an inwardly rectifyingI/V relation. The selectivity sequence of this channel was K⁺ (1) > Rb⁺ (<0.5) > NH ₄ ⁺ (<0.2) > Na⁺ (<0.09). The activity of this channel type was not affected by changes in membrane potential. In 18% of excised patches, we also observed a non-selective cation channel that was not demonstrable in cell-attached patches. It had a slope conductance of 22±2 pS (n=6) and was blocked by the non-selective cation channel blocker, flufenamate (10 mol/l). A fourth channel type, observed only in 5% of patches was a Cl^– channel with a slope conductance of 40 pS and a linearI/V relation. The K⁺ channels observed in this study seem likely to underlie the K⁺ conductance described in the basolateral membrane of extralobular ducts by in vitro perfusion studies. Our finding that they are inwardly rectifying suggests that they may not be the sole route of K⁺ transport across the basolateral membrane.