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.     

Regulation and possible physiological role of the Ca2-dependent K+ channel of cortical collecting ducts of the rat

In the luminal membrane of rat cortical collecting duct (CCD) a big Ca²⁺-dependent and a small Ca²⁺-independent K⁺ channel have been described. Whereas the latter most likely is responsible for the K⁺ secretion in this nephron segment, the function of the large-conductance K⁺ channel is unknown. The regulation of this channel and its possible physiological role were examined with the conventional cell-free and the cell-attached nystatin patch-clamp techniques. Patch-clamp recordings were obtained from the luminal membrane of isolated perfused CCD segments and from freshly isolated CCD cells. Intracellular calcium was measured using the calcium-sensitive dye fura-2. The large-conductance K⁺ channel was strongly voltage- and calcium-dependent. At 3 mol/l cytosolic Ca²⁺ activity it was half-maximally activated. At 1 mmol/l it was neither regulated by cytosolic pH nor by ATP. At 1 mol/l Ca²⁺ activity the open probability (P _o) of this channel was pH-dependent. At pH 7.0 P _o was decreased to 4±2% (n=9) and at pH 8.5 it was increased to 425±52% (n=9) of the control. At this low Ca²⁺ activity the P _o of the channel was reduced by 1 mmol/l ATP to 8±4% (n=6). Cell swelling activated the large-conductance K⁺ channel (n=14) and hyperpolarized the membrane potential of the cells by 9±1 mV (n=23). Intracellular Ca²⁺ activity increased after hypotonic stress. This increase depended on the extracellular Ca²⁺ activity. A possible physiological function of the large-conductance K⁺ channel in rat CCD cells may be the reduction of the intracellular K⁺ concentration after cell swelling. Once this channel is activated by increases in the cytosolic Ca²⁺ activity it can be regulated by changes in cellular pH and ATP.Supported by DFG Schl 277/2-3     

Large conductance calcium-activated potassium channels in cultured retinal pericytes under normal and high-glucose conditions

Pericytes are considered to contribute to the regulation of retinal microcirculation which is impaired in diabetic retinopathy. Single, large-conductance, Ca²⁺-dependent K⁺ channels (BK) were studied in cultured bovine retinal capillary pericytes using the patch-clamp method. In excised patches with symmetrical 135-mmol/l K⁺ solutions a single channel conductance of 238±9.9 pS was measured. With a K⁺ gradient of 4/ 135 mmol/l (extracellular/intracellular) the slope conductance averaged 148±2.9 pS at 0 mV. The mean permeability was 4.2×10^–13 cm³/s. The channel was highly selective for K⁺ with a permeability ratio for K⁺ over Na⁺ of 1/0.02. The mean open time and the open probability (P_o) of the BK channel increased with depolarization and with increasing internal [Ca²⁺] showing a maximal sensitivity to Ca²⁺ between 10^–4 and 10^–5 mol/l Ca²⁺. Ba²⁺ (5 mmol/l), quinine (5 mmol/l), and verapamil (Michaelis constant 1.5×10^–5 mol/l) blocked from the intracellular side. Tetraethylammonium induced a dose-dependent block from the outside only with a halfmaximal blocking concentration of 2.5×10^–4 mol/l. Charybdotoxin (10^–8 mol/l) blocked completely from the extracellular side. The channel activity was not changed by either internal adenosine triphosphate (ATP, 10^–4 mol/l) or the putative opener of ATP-sensitive K⁺ channels Hoe 234 (10^–6 mol/l). In cell-attached patches channelP _o was less than 3%. After a 3-day incubation in culture medium containing an elevated glucose concentration (22.5 mmol/l) the channel activity in attached patches was markedly increased. These data indicate that cultured retinal pericytes possess a BK channel. The activity of the channel increases after incubation with elevated glucose concentrations, which could indicate altered regulation of the channel under these conditions. The implications of altered function of BK channels are discussed with respect to haemodynamic changes observed in diabetic retinopathy.     

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.     

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.     

Sodium-activated potassium current in sensory neurons: a comparison of cell-attached and cell-free single-channel activities

Single-channel currents from Na⁺-dependent K⁺ channels (K_Na) were recorded from cell-attached and inside-out membrane patches of cultured avian trigeminal ganglion neurons by means of the patchclamp technique. Single-channel properties, such as the high elementary conductance and the occurrence of subconductance levels, were unchanged after the patches had been excised from the cells, indicating that they are not under the control of soluble cytoplasmic factors. In cellattached recordings at the cell resting potential the degree of K_Na activity, measured as the probability of the channel being open, P _o, was low in most cases (around 0.01) and similar to that observed in the inside-out configuration when the bath solution contained concentrations of Na⁺ around 30 mM and of K⁺ close to the physiological intracellular levels. However, in some cell-attached patches P _o was high (around 0.2) and comparable to the values measured in cell-free recordings with high Na⁺ concentrations in the bath (100 mM). The excision of a highactivity patch in the presence of 30 mM Na⁺ resulted in a fall of P _o in about 20 s, which is consistent with the wash-out of a soluble cytoplasmic molecule. After the excision all K_Na displayed a similar Na⁺ sensitivity, irrespective of the degree of activation observed in the cellattached mode. In inside-out patches the P _o values observed in the presence of either low or high concentrations of Na⁺ in bath solutions were not modified by internal Ca²⁺ (0.8–8.5 M). The variable degree of K_Na activation observed in cell-attached recordings suggests that either internal Na⁺ concentrations reach very high levels close to the membrane, or soluble factor(s) are involved in the modulation of K_Na activity: under such conditions, the Na⁺-activated K⁺ current may contribute to the maintenance of the resting membrane potential and to control neuronal membrane excitability.     

Calcium dependent gating of thel-glutamate activated,excitatory synaptic channel on crayfish muscle

Excitatory, glutamate-activated single channel currents were measured in outside-out patches of crayfish muscle. The open time of single channel openings, and the durations and rates of bursts were evaluated. These kinetic parameters were not appreciably affected by replacement of extracellular Na⁺ by Li⁺ or choline. Changes in extracellular Ca²⁺ concentration Ca_o also did not influence the duration of single openings. However the mean burst duration decreased for Ca_o<13.5 mM and the rate of bursts declined with a power of almost 2 in low Ca_o. At Ca_o<1 mM practically no channel openings were observed in presence of glutamate. In order to exclude more rapid desensitization of the glutamate receptors in low Ca_o as the cause of disappearance of channel openings, glutamate was applied in short pulses with a liquid-filament switch. In 0 Ca_o also a glutamate pulse did not trigger channel openings. In presence of 13.5 mM Ca_o, the inorganic Ca-channel blockers La³⁺ and Cd²⁺ diminished the duration and rate of bursts of channel openings in a similar manner as low Ca_o. The effects of low Ca_o and of Cd²⁺ were tested also on quantal postsynaptic currents, EPSCs, which were recorded through a perfused macro-patch-clamp electrode. At 1.4 mM Ca_o in the perfused electrode tip, spontaneous EPSCs were reduced at least by a factor of 4, and elicited EPSCs by a factor of 16. Application of Cd²⁺ had similarly strong effects on the EPSCs. Also the decay of EPSCs was shortened substantially in 1.4 mM Ca_o or 5 mM Cd²⁺.The inhibitory Cl^–-channel of crayfish muscle, activated by glutamate or GABA, also was studied in outside-out patches. The openings of this channel persisted in 0 Ca_o solutions; the block of channel openings in low Ca_o thus is a specific property of the excitatory channel. The action of Ca_o on the excitatory channel may be described as that of a cofactor to glutamate. A possible reaction scheme is proposed.Supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 220     

K+ channels in the basolateral membrane of rat cortical collecting duct

Impalement studies in isolated perfused cortical collecting ducts (CCD) of rats have shown that the basolateral membrane possesses a K⁺ conductive pathway. In the present study this pathway was investigated at the single-channel level using the patch-clamp technique. Patch-clamp recordings were obtained from enzymatically isolated CCD segments and freshly isolated CCD cells with the conventional cell-free, cell-attached and the cell-attached nystatin method. Two K⁺ channels were found which were highly active on the cell with a conductance of 67±5 pS (n=18) and 148±4 pS (n=21) with 145 mmol/l K⁺ in the pipette. In excised patches the first channel had a conductance of 28±2 pS (n=15), whereas the second one had a conductance of 85±1 pS (n=53) at 0 mV clamp voltage with 145 mmol/l K⁺ on one side and 3.6 mmol/l K⁺ on the other side of the membrane. So far it has not been possible to characterize the smaller channel further. Excised, and with symmetrical K⁺ concentrations of 145 mmol/l, the intermediate channel had a linear conductance of 198±19 pS (n=5). After excision in the inside-out configuration the open probability (P _o) of this channel was low (0.18±0.05, n=13) whereas in the outside-out configuration this channel had a threefold higher P _o (0.57±0.04, n=12). Several inhibitors were tested in excised membranes. Ba²⁺ (1 mmol/l), tetraethylammonium (TEA⁺, 10 mmol/l) and verapamil (0.1 mmol/l) all blocked this channel reversibly. Furthermore P _o was reversibly reduced by 10 nmol/l charybdotoxin (outside-out). This K⁺ channel of the basolateral membrane was regulated by cellular pH. P _o was reduced to 26±3% at pH 6.5 (n=6) and increased to 216±18% at pH 8.5 (n=7) compared to pH 7.4. Half-maximal inhibition was reached at pH 7.0. The channel had its highest P _o at a Ca²⁺ activity of less than 10^–8 mol/l (n=13). Increasing the Ca²⁺ activity to 1 mmol/l on the cytosolic side of the membrane resulted in a reduction of P _o to 13±3% (n=11). Half-maximal inhibition was reached at a Ca²⁺ activity of 10^–5 mol/l. The high activity of both K⁺ channels of the basolateral membrane on the cell indicates that they may serve for K⁺ recirculation across the basolateral membrane.     

Ca2+-activated K+ channels in airway smooth muscle are inhibited by cytoplasmic adenosine triphosphate

Large-conductance Ca²⁺-activated K⁺ channels were studied in membranes of cultured rabbit airway smooth muscle cells, using the patch-clamp technique. In cell-attached recordings, channel openings were rare and occurred only at very positive potentials. Bradykinin (10 M), an agonist which releases Ca²⁺ from the sarcoplasmic reticulum, transiently increased channel activity. The metabolic blocker 2,4-dinitrophenol (20 M), which lowers cellular adenosine triphosphate (ATP) levels, induced a sustained increase of channel activity in cell-attached patches. In excised patches, these channels had a slope conductance of 155 pS at 0 mV, were activated by depolarization and by increasing the Ca²⁺ concentration at the cytoplasmic side above 10^–7 mol/l. ATP, applied to the cytoplasmic side of the patches, dose-dependently decreased the channel's open-state probability. An inhibition constant (K _i) of 0.2 mmol/l was found for the ATP-induced inhibition. ATP reduced the Ca²⁺ sensitivity of the channel, shifting the Ca²⁺ activation curve to the right and additionally reducing its steepness. Our results demonstrate that cytoplasmic ATP inhibits a large-conductance Ca²⁺-activated K⁺ channel in airway smooth muscle. This ATP modulation of Ca²⁺-activated K⁺ channels might serve as an important mechanism linking energy status and the contractile state of the cells.     

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

Shear stress induced membrane currents and calcium transients in human vascular endothelial cells

We have measured membrane currents induced by shear stress together with intracellular calcium signals in endothelial cells from human umbilical cord veins. In the presence of extracellular calcium (Ca²⁺]_o), shear stress induced an inward current at a holding potential of 0 mV which is accompanied by a rise in intracellular Ca²⁺ ([Ca²⁺]_i). In the absence of extracellular calcium shear stress was unable to evoke a calcium signal but still induced a membrane current. The voltage dependence of the shear stress induced current was obtained from difference currents evoked by linear voltage ramps before and during application of shear stress. Its reversal potential E_rev shifted from –2.3±0.8 mV (n=4) in a nominally Ca²⁺ free solution to +1.5±1.6 mV at 1.5 mM [Ca²⁺]_o (n=4) and to +21.9±4.4 mV (n=7) at 10 mM [Ca²⁺]_o. From our data we conclude that shear stress opens an ion channel that is 12.5±2.9 (n=7) times more permeable for calcium than for sodium or cesium.     

Large and small conductance calcium-activated potassium channels in the GH3 anterior pituitary cell line

Single Ca²⁺-activated K⁺ channels were studied in membrane patches from the GH₃ anterior pituitary cell line. In excised inside-out patches exposed to symmetrical 150 mM KCl, two channel types with conductances in the ranges of 250–300 pS and 9–14 pS were routinely observed. The activity of the large conductance channel is enhanced by internal Ca²⁺ and by depolarization of the patch membrane. This channel contributes to the repolarization of Ca²⁺ action potentials but has a Ca²⁺ sensitivity at –50 mV that is too low for it to contribute to the resting membrane conductance. The small conductance channel is activated by much lower concentrations of Ca²⁺ at –50 mV, ad its open probability is not strongly voltage sensitive. In cell-attached patches from voltage-clamped cells, the small conductance channels were found to be active during slowly decaying Ca²⁺-activated K⁺ tails currents and during Ca²⁺-activated K⁺ currents stimulated by thyrotropin-releasing hormone induced elevations of cytosolic calcium. In cell-attached patches on unclamped cells, the small conductance channels were also active at negative membrane potentials when the frequency of spontaneously firing action potentials was high or during the slow afterhyperpolarization following single spontaneous action potentials of slightly prolonged duration. The small conductance channel may thus contribute to the regulation of membrane excitability.     

A newly identified Ca2+ dependent K+ channel in the smooth muscle membrane of single cells dispersed from the rabbit portal vein

We found a new type of Ca²⁺-dependent K⁺ channel in smooth muscle cell membranes of single cells of the rabbit portal vein. A slope conductance of the current was 180 pS when 142 mM K⁺ solution was exposed to both sides of the membrane (this channel was named the K_M channel, in comparison to the known K_L and K_S channels from the same membrane patch; Inoue et al. 1985). This K_M channel was less sensitive to the cytoplasmic Ca²⁺ concentration, [Ca²⁺]_i, but was sensitive to the extracellular Ca²⁺, [Ca²⁺]_o, e.g. in the outside-out membrane patch, lowering the [Ca²⁺]_o in the bath markedly reduced the open probability of this channel, and also in cell-attached configuration, lowering of the [Ca²⁺]_o using the internally perfused patch clamp electrode device reduced the opening of K_M channel. TEA⁺ (1–10 mM) reduced the amplitude of the elementary current through the K_M channel applied from each side of the membrane, but this agent inhibited the K_M channel to a greater extent when applied to the inner than to the outer surface of the membrane. Furthermore, this K_M channel had a weak voltage dependency, and the open probability of the channel remained much the same within a wide range of potential (from –60 mV to +60 mV). Whereas most Ca²⁺-dependent K⁺ channels are regulated mainly by [Ca²⁺]_i and possess a voltage dependency, these properties of the K_M channel differed from other Ca²⁺-dependent K⁺ channels. The elucidation of this K_M channel should facilitate explanations of the actions of external Ca²⁺ or TEA⁺ on the membrane potential, in the smooth muscles of the rabbit portal vein.     

Histamine-activated,non-selective cation currents and Ca2+ transients in endothelial cells from human umbilical vein

Permeation properties and modulation of an ionic current gated by histamine were measured in single endothelial cells from human umbilical cord veins by use of the patch-clamp technique in the ruptured-whole-cell mode or using perforated patches. We combined these current measurements with a microfluorimetric method to measure concomitantly free intracellular calcium concentration ([Ca²⁺]_i). Application of histamine induced an intracellular calcium transient and an ionic current that reversed near 0 mV. The amplitude of the current ranged from –0.2 to –2nA at –100mV. The tonic rise in [Ca²⁺]_i and the ionic current are partly due to Ca²⁺ influx. This Ca²⁺ entry pathway is also permeable for Ba²⁺ and Mn²⁺. The amplitude of the histamine-activated current was also closely correlated with the amplitude of the concomitant Ca²⁺ transient, suggesting that the latter is at least partially due to Ca²⁺ influx through histamine-activated channels. The reversal potential of the histamine-induced current was 7.6±4.1 mV (n=14) when the calcium concentration in the bath solution ([Ca²⁺]_o) was 1.5mmol/l. With 10 mmol/l [Ca²⁺]_o it was –13.7±4.7 mV and shifted to +13.0±1.5 mV in nominally Ca²⁺-free solution (n=3 cells). The amplitude of the current in Ca²⁺-free solution was enhanced compared to that in 10 mmol/l [Ca²⁺]_o. The shift of the reversal potential and the concomitant change of the current amplitude suggest that the channel is permeable for calcium but has a smaller permeability for calcium than for monovalent cations. The latency between the application of histamine and the appearance of the current was voltage dependent and was much smaller at more negative potentials. This effect is unlikely to be due to desensitization, but may suggest a voltage-dependent step in the signal transduction chain. Similar histamine-induced Ca²⁺ signals were observed if the currents were measured in patches perforated with nystatin. The onset of the agonist-activated current was, however, much more delayed and its amplitude significantly lower than in ruptured patches. The histamine-induced currents and intracellular Ca²⁺-transients were largely reduced after incubation of endothelial cells with the phorbol ester TPA. H7, a blocker of protein kinase C, induced membrane currents and Ca²⁺ signals in the absence of an agonist. It is concluded that the agonist-activated Ca²⁺-entry in endothelial cells occurs through non-selective cation channels which can be down-regulated by protein kinase C activation.     

Small and intermediate conductance chloride channels in HT29 cells

Recently, it has been shown that intermediate conductance outwardly rectifying chloride channels (ICOR) are blocked by cytosolic inhibitor (C. I.) found in the cytosol of human placenta and epithelial cells. C. I. also reduced the baseline current in excised membrane patches of HT₂₉ cells. In the present study, this effect of C. I. was characterized further. Heat treated human placental cytosol was extracted in organic solvents and dissolved in different electrolyte solutions. It is shown that the reduction of baseline conductance (g _o) is caused by inhibition of small non-resolvable channels, which are impermeable to Na⁺ and SO₄ ^2–, but permeable to Cl^–. The regulation of these small Cl^–-conducting channels (g _o) and of ICOR was examined further. First, no activating effects of protein kinase A (PKA) on the open probability (P _o) of the ICOR or on the g_o) were observed. The P_o of the ICOR was reduced by 22% in a Ca²⁺-free solution. g _o was insensitive to changes in the Ca²⁺ activity. The effects of C. I. from a cystic fibrosis (CF) placenta and the CF pancreatic duct cell line CFPAC-1 were compared with the effects of corresponding control cytosols, and no significant differences between CF and control cytosols were found. We conclude that the excised patches of HT₂₉ cells contain ICOR and small non-resolvable Cl^–-conducting channels which are similarly inhibited by C. I. Apart from a weak effect of Ca²⁺ on the ICOR, g _o and the ICOR do not seem to be directly controlled by Ca²⁺ or PKA. C. I. of normal and CF epithelia have a similar inhibitory potency on Cl^– channels.     

Ion channels in isolated mouse jejunal crypts

 The patch-clamp technique was used to characterise the ion channels in cells located in the mid region of mouse jejunal crypts. Six different channels were seen. A large outwardly rectified K⁺ channel (BK) (conductance, g at 0 mV = 92 ± 6 pS), which was highly selective for K⁺ [P _K ⁺ (1) > P _Rb ⁺ (0.6) >> P _Cs ⁺ (0.09) ≈ P _Na ⁺ (0.07) > P _Li ⁺ (0.04)], had a low, voltage-independent open probability (P _o) in the on-cell (O/C) configuration and appeared in 66% of the patches. In inside-out (I/O) patches, this channel had a linear current/voltage (I/V) relationship (g = 132 ± 3 pS), P _o was voltage dependent and it was blocked by cytoplasmic Ba²⁺ (5 mmol/l). An intermediate K⁺ channel (IK) which was present in 49% of O/C patches, had a linear I/V (g = 38 ± 3 pS), ran-down in O/C patches, and was not seen in I/O patches. A number of smaller channels (SC) with conductances ranging from 5 to 20 pS were seen in 16% of O/C patches. Also present in the basolateral membrane were a Cl^– channel (ICOR) and a nonselective cation channel (NSCC). These channels were only seen in I/O patches. ICOR had an outwardly rectified conductance (g at 0 mV = 36 ± 2 pS), its P _o was independent of voltage and unaffected by variations in cytoplasmic Ca²⁺ (100 nmol/l to 1 mmol/l) or ATP (0–1 mmol/l). The NSCC had a linear conductance (20 ± 1 pS), its P _o increased with depolarisation and elevation of cytoplasmic [Ca²⁺] (≥ 10 μmol/l), but was reduced by cytoplasmic ATP. None of the basolateral channels described here were activated by cAMP-dependent secretagogues, although a Cl^– conductance was activated. This cAMP-dependent Cl^– conductance was distinct from the basolateral Cl^– channel and thus is most likely located in the apical membrane. Received: 25 June 1997 / Accepted: 14 October 1997     

Single channel Ca2+ currents inHelix pomatia neurons

Unitary Ca²⁺ currents of TEA injected Helix neurons were recorded in the Giga seal situation (6, 7) from microscopic membrane patches exposed to 50 mM [Ca²⁺]_o, O [Na⁺]_o, 20 mM [TEA⁺]_o and 2.5 M [TTX]_o. Constant field assumptions yield a channel permeability of 2.9±1.0×10^–14 cm³s^–1 corresponding to slope conductances of 5 to 15 pS between 0 and –30 mV. Frequency of occurrence of the units strongly increased with depolarization. Mean open time of the Ca²⁺ channels was about 3 ms without obvious dependence on voltage. A similar open time was seen with [Ba²⁺]_o, yielding about double the current strength when compared with [Ca²⁺]_o.     

Properties of membrane currents in isolated smooth muscle cells from guinea-pig trachea

Tracheal smooth muscle cells were enzymatically isolated from guinea-pig trachea. These cells contracted in response to acetylcholine (0.01–10 M) in a concentration-dependent fashion. Under current-clamp conditions with 140 mM K⁺ in the pipette solution, the membrane potential oscillated spontaneously at around –30 mV. Under voltage-clamp conditions, there appeared spontaneous but steady oscillations of outward current (I _o). On depolarization from a holding potential at –40 mV, three components of outward current were elicited: transient outward current (I _T), steady-state outward current (I _s) and I _o. These three components of outward current reversed around the K⁺ equilibrium potential and were abolished by Cs⁺ in the pipette, indicating that K⁺ was the major charge carrier of these outward currents. All these three components were completely suppressed by extracellular tetraethylammonium (10 mM). Both I _T and I _o were depressed by quinidine (1 mM), 4-aminopyridine (10 mM) and nifedipine (100 nM), but I _s was not affected. I _T and I _o were suppressed by a Ca²⁺-free perfusate with less than 1 nM Ca²⁺ in the pipette, while with 10 nM Ca²⁺ in the pipette, only I _o was suppressed. In both conditions, I _s was not affected by the Ca²⁺-free perfusate. Therefore, it is suggested that I _o, I _T and I _s are separate types of K⁺ current. With Cs⁺ in the pipette, K⁺ currents were almost completely suppressed and a transient inward current was observed during depolarizing pulses. The inward current was not affected by tetrodotoxin and increased when the concentration of extracellular Ca²⁺ was raised, indicating that the current is a Ca²⁺ channel current. Even with a holding potential of –80 mV, the low-threshold inward current could not be observed. The high-threshold Ca²⁺ current was abolished by nifedipine (100 nM) and was enhanced by Bay K 8644 (100 nM). The order of permeation of divalent cations through the Ca²⁺ channel was Ba²⁺ >Sr²⁺ Ca²⁺. Cd²⁺ blocked the Ca²⁺ current more effectively than Ni²⁺. These results may indicate that the Ca²⁺ current of tracheal smooth muscle cells is mainly composed of the current through an L-type Ca²⁺ channel.     

Protein kinase A increases availability of calcium channels in smooth muscle cells from guinea pig basilar artery

We studied single Ca²⁺ channels in smooth muscle cells from the basilar artery of the guinea pig using conventional patch-clamp techniques. With 40 mM or 90 mM Ba²⁺ as the charge carrier, a 23-pS inward current channel was observed in 46/187 cell-attached patches studied without the dihydropyridine, BAY K8644, in the pipette solution. At 0 mV, this channel exhibited short and long openings with time constants of 1.03 and 3.65 ms, respectively. The probability of channel opening was voltage dependent with half-activation occurring at +9.9 mV. In 14/26 patches tested, addition of 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP) to the bath increased the probability of opening at -10 mV by a factor of 2.6, from 0.0272±0.0429 to 0.0695±0.0788 (P <0.01, paired t-test). Mean data from five patches fit to a Boltzmann function indicated that at positive potentials, the probability of opening increased by a factor of 1.7, from 0.352 to 0.600, whereas the voltage dependence, the number of channels, the number of open states, the time constants of the open states, and the proportion of time spent in each open state were unchanged. When BAY K8644 was added to the pipette solution, the 23-pS channel was observed in nearly all patches (62/66), but the voltage dependence of activation was shifted –15.3 mV compared to control. In some patches studied with 90 mM Ba²⁺, a 9-pS inward current channel also was observed and its activity also was increased significantly by 8-Br-cAMP. When membrane patches were excised from the cell and studied in an inside-out configuration, single-channel activity due to the 23-pS channel lasted 1–3 min before being irreversibly lost, regardless of the presence of BAY K8644 in the pipette or of 8-Br-cAMP plus Mg · ATP and leupeptin in the bath. Subsequent addition of the catalytic subunit of protein kinase A (PKA_CS) did not restore Ca²⁺ channel activity. Conversely, when patches were excised into a solution already containing 8-Br-cAMP plus Mg · ATP, leupeptin and PKA_CS, channel activity was prominent and generally lasted until the seal was lost, or until the experiment was terminated at 30–45 min, unless protein kinase inhibitor also was present, in which case channel lifetime was short. Our findings indicate that availability of the L-type Ca²⁺ channel in basilar artery smooth muscle cells is increased by activation of cAMP-dependent protein kinase A, and that the (or one of the) phosphoprotein(s) involved may not be membrane bound.     

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