Small-conductance Ca2+-activated K+ channels in bovine chromaffin cells

Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca²⁺]_i measurements were used to characterize Ca²⁺-activated K⁺ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 M) induced a rise of [Ca²⁺]_i concomitantly with an outward current at holding potentials positive to –80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range –80 mV to –40 mV. Increasing the extracellular K⁺ concentration to 20 mM at the holding potential of –78 mV was associated with inwardly directed currents during the [Ca²⁺]_i elevations induced either by histamine (10 M) or short voltage-clamp depolarizations. The current reversal potential was close to the K⁺ equilibrium potential, being a function of external K⁺ concentration. Current fluctuation analysis suggested a unit conductance of 3–5 pS for the channel that underlies this K⁺ current. The current could be blocked by apamin (1 M). Whole-cell current-clamp recordings snowed that histamine (10 M) application caused a transient hyperpolarization, which evolved in parallel with the [Ca²⁺]_i changes. It is proposed that a small-conductance Ca²⁺-activated K⁺ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species.     

Changes of membrane currents in cardiac cells induced by long whole-cell recordings and tolbutamide

Single isolated myocytes were obtained from the ventricles of adult guinea pig hearts. The whole-cell recording configuration of the patch-clamp technique was used to measure membrane currents. A decrease (run-down) of the Ca²⁺ inward current and an increase of a time-independent K⁺ outward current were observed during long lasting (1–3 h) recordings. The time at which the outward current developed depended on the intracellular ATP concentration in the pipette, suggesting that this current is identical to the ATP-dependent K⁺ current described by Noma and Shibasaki (1985). However, the maximum outward current reached in the experiments was independent of the ATP concentration indicating a limited diffusion of ATP in the cell interior. In single-channel experiments on isolated patches of cell membrane and in whole-cell recordings the ATP-dependent K⁺ current could be blocked by the hypoglycaemic sulphonylurea tolbutamide. The IC₅₀ of 0.38 mM was about 50 times higher than that reported for pancreatic -cells (Trube et al. 1986). The Ca²⁺ inward current and the inwardly retifying K⁺ current were not affected by tolbutamide (3 mM).     

Electrophysiological properties of neurones in cultures from postnatal rat dentate gyrus

Electrophysiological properties of neurofilament-positive neurones in dissociated cell cultures were prepared at postnatal days 4–5 from rat dentate gyrus and studied using the whole-cell patch-clamp technique. These cells expressed a fast-inactivating, 0.5 M tetrodotoxin-sensitive Na⁺ current; a high-voltage-activated (HVA) Ca²⁺ current, which was 30 M Cd²⁺- and partially 2 M nicardipine-sensitive; and an inward rectifier current, which was sensitive to extracellularly applied 1 mM Cs⁺. The outward current pattern was composed of a delayed rectifier-like outward current sensitive to 20 mM tetraethylammonium (TEA) and a fast-inactivating, Ca²⁺-dependent outward current. This transient Ca²⁺-dependent K⁺ outward current was identified by a subtraction procedure. K⁺ currents recorded under conditions of blocked Ca²⁺ currents (after rundown of the HVA Ca²⁺ current or blocked by extracellularly applied Cd²⁺) were subtracted from control currents. By comparison with the current pattern of identified dentate granule cells, it is concluded that the investigated cell type originated from interneurones or projection neurones of the dentate hilus.     

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.     

Electrophysiological properties of membrane currents in single myometrial cells isolated from pregnant rats

The whole-cell voltage-clamp method was applied to single smooth muscle cells prepared from the longitudinal layer of the pregnant rat myometrium (17–20 days of gestation). It was found that the transient inward current mainly consists of Ca²⁺ current, because the removal of Ca²⁺ ions from the external medium and 10 M nifedipine eliminated this inward current. Its steady-state inactivation curve was obtained by the standard method, in which the membrane potential of half inactivation and the slope factor were estimated to be –58.0±4.9 mV (n=11) and 8.9±1.4 mV (n=11), respectively. In a small number of preparations (in 2 out of 30 preparations), there remained a very fast inward current in Ca²⁺-free medium containing Mg²⁺. Tetrodotoxin (TTX, 10 M) can abolish this current, suggesting that the channel for this current is equivalent to the Na⁺ channel in nerve cells. Two major phases of outward currents were identified by voltage jumps from negative holding levels to more positive levels. The first phase was a fast transient outward current. This current remained intact after external tetraethylammonium (TEA, 20 mM) was added. Following the transient current, a large delayed rectified outward current reached its peak over a period of 50 ms and then decayed. The reversal potential for this outward current was determined by observing the change of polarity of the tail currents with the change in extracellular K⁺ concentration ([K⁺]₀). The slope for the change of reversal potential per ten-fold change in [K⁺]₀ is 57.7 mV at more than 23.2 mM [K⁺]_o, indicating that this current is mostly carried by K⁺ ions. Voltage-dependent inactivation of the delayed rectified outward current was determined by the standard method. The membrane potential for half inactivation and the slope factor were estimated to be –42.8±3.9 mV (n=3) and 10.1±1.5 mV (n=3), respectively. External TEA (20 mM) effectively eliminated the delayed rectified outward currents. Nifedipine (10 M) suppressed not only Ca²⁺ current but also outward K⁺ currents.     

Intracellular Ca2+ inactivates an outwardly rectifying K+ current in human adenomatous parathyroid cells

We have used whole-cell patch-clamp techniques to study the conductances in the plasma membranes of human parathyroid cells. With a KCl-rich pipette solution containing Ca²⁺ buffered to a concentration of 0.1 mol/l, the zero current potential was –71.1±0.5 mV (n=19) and the whole-cell current/ voltage (I/V) relation had an inwardly rectifying and an outwardly rectifying component. The inwardly rectifying current activated instantaneously on hyperpolarization of the plasma membrane to potentials more negative than –80 mV, and a semi-logarithmic plot of the reversal potential of the inward current (estimated by extrapolation from the range in which it was linear) as a function of extracellular K⁺ concentration ([K⁺]_o) revealed a linear relation with a slope of 64 mV per decade change in [K⁺]_o, which is not significantly different from the Nernstian slope, demonstrating that the current was carried by K⁺ ions. The conductance exhibited a square root dependence on [K⁺]_o as has been observed for inward rectifiers in other tissues. The current was blocked by the presence of Ba²⁺ (1 mmol/l) or Cs⁺ (1.5 mmol/l) in the bath. The outwardly rectifying current was activated by depolarization of the membrane potential to potentials more positive than –20 mV. It was inhibited by replacement of pipette K⁺ with Cs⁺, indicating that it also was a K⁺ current: it was partially (42%) blocked when tetraethylammonium (TEA⁺, 10 mmol/l) was added to the bath. The outwardly rectifying, but not the inwardly rectifying K⁺ current, was regulated by intracellular free Ca²⁺ concentration ([Ca²⁺]_i) such that increasing [Ca²⁺]_i above 10 nmol/l inhibited the outwardly rectifying current, the half-maximum effect being seen at 1 mol/l. Since it is known that increases in [Ca²⁺]_o produce increases in [Ca²⁺]_i, and that they depolarize parathyroid cells by reducing the membrane K⁺ conductance, we suggest that it is the reduction of the outwardly rectifying K⁺ conductance by increases in [Ca²⁺]_i which is responsible for the reduction in K⁺ conductance seen when [Ca²⁺]_o is increased.     

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.     

Transient Ca2+-channel current characterized by a low-threshold voltage in zona glomerulosa cells of rat adrenal cortex

Voltage-gated Ca²⁺-current was identified in single isolated cells of the zona glomerulosa of adrenal cortex and its properties were studied by the tight-seal whole cell recording technique. The Ca²⁺-channel current was dissected from the net current by dialyzing the cells with CsCl. The identified Ca²⁺-current was found to be activated by a relatively small depolarization only when the cells were held at a large negative holding potential, but it was inactivated within 10–30 ms. The time course of activation and inactivation was voltage-dependent and became faster when the amplitude of depolarization was increased. The transmembrane potential of the glomerulosa cells was highly sensitive to [K⁺]_e, the slope of the potential change per tenfold change in [K⁺]_e being 48 mV. An increase in [K⁺]_e from 4.7 to 10 mM induce a membrane depolarization by 15 mV, which was sufficient to cause the membrane to reach the threshold potential (–60 mV) for activation of the Ca²⁺-current at physiological concentration of [Ca²⁺]_e (2.5 mM –CaCl₂). The observed properties of the Ca²⁺-current and K⁺-dependence of the membrane potential may give reasonable explanation for the mechanism of Ca²⁺-uptake and consequent aldosterone secretion induced by a small increase in [K⁺]_e, which is known to be one of the major stimulations for aldosterone secretion.     

Electrogenic Na+/K+-transport in human endothelial cells

Na⁺/K⁺ pump currents were measured in endothelial cells from human umbilical cord vein using the whole-cell or nystatin-perforated-patch-clamp technique combined with intracellular calcium concentration ([Ca²⁺]_i) measurements with Fura-2/AM. Loading endothelial cells through the patch pipette with 40 mmol/l [Na⁺] did not induce significant changes of [Ca²⁺]_i. Superfusing the cells with K⁺-free solutions also did not significantly affect [Ca²⁺]_i. Reapplication of K⁺ after superfusion of the cells with K⁺-free solution induced an outward current at a holding potential of 0 mV. This current was nearly completely blocked by 100 mol/l dihydroouabain (DHO) and was therefore identified as a Na⁺/K⁺ pump current. During block and reactivation of the Na⁺/K⁺ pump no changes in [Ca²⁺]_i could be observed. Pump currents were blocked concentration dependently by DHO. The concentration for half-maximal inhibition was 21 mol/l. This value is larger than that reported for other tissues and the block was practically irreversible. Insulin (10–1000 U/l) did not affect the pump currents. An increase of the intracellular Na⁺ concentration ([Na⁺]_i) enhanced the amplitude of the pump current. Half-maximal activation of the pump current by [Na⁺]_i occurred at about 60 mmol/l. The concentration for half-maximal activation by extracellular K⁺ was 2.4±1.2 mmol/l, and 0.4±0.1 and 8.7±0.7 mmol/l for Tl⁺ and NH₄ ⁺ respectively. The voltage dependence of the DHO-sensitive current was obtained by applying linear voltage ramps. Its reversal potential was more negative than –150 mV. Pump currents measured with the conventional whole-cell technique were about four times smaller than pump currents recorded with the nystatin-perforated-patch method. If however 100 mol/l guanosine 5-O-(3-thiotriphosphate) (GTPS) were added to the pipette solution, the currents measured in the ruptured-whole-cell-mode were not significantly different from the currents measured with the perforated-patch technique. We suppose that the use of the perforated-patch technique prevents wash out of a guanine nucleotide-binding protein (G-protein)-connected intracellular regulator that is necessary for pump activation.     

The apamin-sensitive potassium current in frog skeletal muscle: its dependence on the extracellular calcium and sensitivity to calcium channel blockers

Slow outward potassium currents were recorded in isolated frog skeletal muscle fibres using the double mannitol-gap voltage-clamp technique.Detubulated fibres failed to generate a slow outward current, and apamin had no effect on the remaining current.The maximum blocking effect of organic and inorganic Ca²⁺-channel blockers on the slow outward channels of intact fibres was larger than that of apamin. Apamin failed to induce an additional block when applied after Ca²⁺-channel blockers.In a low-Ca²⁺ solution (OCa, EGTA 1 mM) the slow outward current was slightly increased and the blocking effect of apamin was enhanced. A Ca²⁺-rich solution (Ca²⁺×10) increased the slow outward current and the blocking effect of apamin was drastically reduced.It is concluded that the apamin-sensitive current which is a component of the slow outward K⁺ current is located in the tubular membrane. Its activation seems barely dependent on the Ca²⁺ influx via the slow inward Ca²⁺ current. Apamin-receptor binding appears to be dependent on the extracellular Ca²⁺ concentration. Blockade of slow outward current by Ca²⁺-channel blockers is likely to be the result of a direct action on the slow K⁺ permeability rather than a consequence of Ca²⁺ channel inhibition.     

Caffeine and histamine-induced oscillations of K(Ca) current in single smooth muscle cells of rabbit cerebral artery

In the present experiment, we characterized the intracellular Ca²⁺ oscillations induced by caffeine (1 mM) or histamine (1–3 M) in voltage-clamped single smooth muscle cells of rabbit cerebral (basilar) artery. Superfusion of caffeine or histamine induced periodic oscillations of large whole-cell K⁺ current with fairly uniform amplitudes and intervals. The oscillatory K⁺ current was abolished by inclusion of ethylenebis(oxonitrilo)tetraacetate (EGTA, 5 mM) in the pipette solution. Caffeine- and histamine-induced periodic activation of the large-conductance Ca²⁺-activated K⁺ [K_(Ca)] channel was recorded in the cell-attached patch mode. These results suggest that the oscillations of K⁺ current are carried by the K_(Ca) channel and reflect the oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Ryanodine (1–10 M) abolished both caffeine- and histamine-induced oscillations. Caffeine- induced oscillations were abolished by the sarcoplasmic reticulum Ca²⁺-adenosine 5-triphosphatase (Ca²⁺-ATPase) inhibitor, cyclopiazonic acid (10 M), and a high concentration of caffeine (10 mM). Inclusion of heparin (3 mg/ml) in the pipette solution blocked histamine-induced oscillations, but did not block caffeine-induced oscillations. By the removal of extracellular Ca²⁺, but not by the addition of verapamil and Cd²⁺, the caffeine-induced oscillations were abolished. Increasing Ca²⁺ influx rate increased the frequencies of caffeine-induced oscillations. Spontaneous oscillations were also observed in cells that were not superfused with agonists, and had similar characteristics to the caffeine-induced oscillations. From the above results, it is concluded, that in smooth muscle cells of the rabbit cerebral (basilar) artery, ryanodine-sensitive Ca²⁺-induced Ca²⁺ release pools play key roles in the generation of caffeine- and histamine-induced intracellular Ca²⁺ oscillations.     

Effects of intracellular Ca2+ chelating compounds on inward currents caused by Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum (SR) of mammalian cardiac myocytes occuring either due to activation by a depolarization or the resulting transmembrane Ca²⁺ current (I _Ca), or spontaneously due to Ca²⁺ overload has been shown to cause inward current(s) at negative membrane potentials. In this study, the effects of different intracellular Ca²⁺ chelating compounds on I _Ca-evoked or spontaneous Ca²⁺-release-dependent inward currents were examined in dialysed atrial myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. As compared to dialysis with solutions containing only a low concentration of a high affinity ethylene glycol-bis(-aminoethylether) N,N,N,N-tetraacetic acid (EGTA) like chelator (50–200 M), inward membrane currents (at –50 mV) due to evoked Ca²⁺ release, spontaneous Ca²⁺ release or Ca²⁺ overload following long-lasting depolarizations to very positive membrane potentials are prolonged if the dialysing fluid contains a high concentration of a low affinity Ca²⁺ chelating compound such as citrate or free adenosine 5-triphosphate (ATP). Without such a non-saturable Ca²⁺ chelator in the dialysing fluid, Ca²⁺-release-dependent inward currents are often oscillatory and show an irregular amplitude. With a low affinity chelator in a non-saturable concentration, discrete inward currents with constant properties can be recorded. We conclude that the variability in Ca²⁺-release-dependent inward current seen in single cells arises from spatial inhomogeneities of intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to localized saturation of endogenous and exogenous high affinity Ca²⁺ buffers (e.g. [2]). This can be avoided experimentally by addition of a non-saturable buffer to the intracellular solution. This condition might be useful, if properties of Ca²⁺ release from the SR and/ or the resulting membrane current, like for example arrhythmogenic transient inward current, are to be investigated on the single cell level.     

Activation of Ca2+-dependent K+ and Cl− currents by UTP and ATP in CFPAC-1 cells

Activation of Cl^– and K⁺ conductances by nucleotide receptor-operated mobilization of intracellular Ca²⁺ was investigated in CFPAC-1 cells with the perforated-patch technique. Adenosine 5-triphosphate (ATP) and uridine 5-triphosphate (UTP) caused a dose-dependent fast and transient membrane hyperpolarization. UTP was more effective than ATP. In voltageclamped cells, two currents with different ionic permeability and kinetics were activated by the nucleotides. The first one was carried by Cl^– ions, peaked in the first few seconds after addition of nucleotides, and lasted for 1±0.3 min. Its amplitude was about 2.7 nA at –100 mV with 100 mol/l of either ATP or UTP. The second current was carried by K⁺ ions and was blocked by Cs⁺. This current peaked more slowly and had a mean duration of 4.6±0.7 min. Its amplitude was 0.9 nA and 0.5 nA at –20 mV with 100 umol/l UTP and ATP, respectively. Activation of the nucleotide receptor caused a transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) that was similar in the presence or absence of extracellular Ca²⁺. The ED₅₀ for UTP was 24 umol/l and that for ATP was 94 mol/l. Depletion of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ store by thapsigargin prevented both the nucleotide-induced [Ca²⁺]_i increase and the activation of membrane currents. Addition of 2 mmol/l Ca²⁺ to thapsigargin-treated cells produced a sustained increase of Cl^– and K⁺ currents, which was reversed by Ca²⁺ removal. The present study demonstrates that CFPAC-1 cells respond to nucleotide receptor activation with a transient increase in [Ca²⁺]_i that stimulates Ca²⁺-dependent Cl^– and K⁺ currents. This phenomenon is probably mediated by inositol 1,4,5-trisphosphate-dependent Ca²⁺ stores.     

Direct measurements of Ca2+-activated K+ currents in inner hair cells of the guinea-pig cochlea using photolabile Ca2+ chelators

Intracellular photorelease of Ca²⁺ from caged Ca²⁺ (DM-nitrophen or nitr5) and the patch-clamp technique in the whole-cell configuration were used to investigate Ca²⁺-activated currents in inner hair cells (IHCs) of the mammalian cochlea. Photoliberation of intracellular Ca²⁺ activated outward currents with a mean amplitude of 260±110 pA when IHCs were voltage-clamped, near the resting membrane potential, at –50 mV. The photoactivated currents were reversibly blocked by extracellular application of tetraethylammonium (TEA, 10 mM), neomycin (1 mM) and charybdotoxin (1 M), but not by apamin. The voltage dependence of membrane currents activated by photolysis of DM-nitrophen demonstrated a reversal potential near the K⁺ equilibrium potential (E _k) and saturation near 0 mV. The presence of Ca²⁺-activated currents was further confirmed by the effects of extracellular adenosine 5-triphosphate (ATP, 10 M) and the Ca²⁺ ionophore ionomycin (10 M). Both agents raised intracellular Ca²⁺ and simultaneously activated outward currents when IHCs were voltage-clamped near the resting membrane potential. In experiments where currents were activated by depolarizing voltage steps, nifedipine (50 M) and Cd²⁺ (1 mM) reduced significantly (20–50%) the whole-cell outward currents, suggesting the presence of L-type Ca²⁺ currents activating K⁺ currents. These results are the first direct evidence for Ca²⁺-activated K⁺ currents in mammalian IHCs, these currents being potentially important for cell repolarization during sound-induced depolarization and synaptic transmission.     

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.     

Effects of dopamine on ion transport across the rat distal colon

Dopamine (5·10^–6–5·10^–4 M) induced a concentration-dependent decrease in short-circuit current (I_sc) across the rat distal colon. This response was preceded by a transient and inconsistent increase in I_sc. The -adrenoceptor blocker phentolamine and the inhibitors of dopamine-2-like (D₂-like) receptors L-741,626 and L-745,870 inhibited the dopamine response, suggesting a contribution of adrenergic and dopaminergic receptors. The decrease in I_sc evoked by dopamine was inhibited by bumetanide, an inhibitor of the basolateral Na⁺-K⁺-2 Cl^– cotransporter responsible for the uptake of K⁺, and by quinine, a blocker of apical K⁺ channels, indicating that stimulation of K⁺ secretion contributes to the measured change in I_sc. In patch-clamp experiments dopamine hyperpolarized the membrane and increased cellular K⁺ current. This response was not concomitant with a change in the intracellular [Ca²⁺] as demonstrated in parallel fura-2 experiments. These results demonstrate that dopamine, like other catecholamines, stimulates colonic K⁺ secretion.     

Mechanisms of calcium transport across the basolateral membrane of the rabbit cortical thick ascending limb of Henle's loop

Although net Ca²⁺ absorption takes place in the thick ascending limb of Henle's loop, detailed mechanisms are unknown. Because it has been reported that the Ca²⁺ entry step across the luminal membrane is mediated by Ca²⁺ channels inserted by stimulation with parathyroid hormone, we studied the mechanism of Ca²⁺ transport across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) perfused in vitro by using microscopic fluorometry of cytosolic Ca²⁺ ([Ca²⁺]_i) with fura-2. The resting [Ca²⁺]_i in this segment was 49.8±4.5 nmol/l. Neither Na⁺ removal from the bathing solution nor addition of ouabain (0.1 mmol/l) to the bath increased [Ca²⁺]_i, indicating that a Na⁺/Ca²⁺ exchanger in the basolateral membrane may not contribute in any major way to [Ca²⁺]_i of CTAL. To confirm our technical accuracy, similar protocols were conducted in the connecting tubule, where the existence of a Na⁺/Ca²⁺ exchanger has been reported. In this segment, Na⁺ removal from the bath increased cell Ca²⁺ from 148.6 ±6.4 nmol/l to 647.6±132.0 nmol/l, confirming the documented fact. [Ca²⁺]_i in the CTAL was markedly increased when 1 mmol/l NaCN was added to the bath in the absence of glucose. Calmodulin inhibitors (trifluoperazine or W-7) increased [Ca²⁺]_i. When the bath pH was made alkaline, [Ca²⁺]_i was also increased. This response was abolished when Ca²⁺ was eliminated from the bath, indicating that the Ca²⁺ entry across the basolateral membrane is dependent on bath pH. Increase in [Ca²⁺]_i induced by an alkaline bath was inhibited by increased the bath K⁺ from 5 nmol/l to 50 mmol/l, suggesting that the Ca²⁺ entry system is voltage-dependent. However, the pH-dependent [Ca²⁺]_i increase was unaffected by 0.1–10 mol/l nicardipine in the bath. We conclude that Ca²⁺ transport across the basolateral membrane of CTAL is mediated by a pump-and-leak system of Ca²⁺ rather than a Na⁺/Ca²⁺ exchanger secondarily linked to a Na⁺, K⁺ pump.     

Characterisation of Ca2+-dependent inwardly rectifying K+ currents in HeLa cells

The whole-cell configuration of the patch-clamp technique was used to examine K⁺ currents in HeLa cells. Under quasi-physiological ionic gradients, using an intracellular solution containing 10^–7 mol/l free Ca²⁺, mainly outward currents were observed. Large inwardly rectifying currents were elicited in symmetrical 145 mmol/l KCl. Replacement of all extracellular K⁺ by isomolar Na⁺, greatly decreased inward currents and shifted the reversal potential as expected for K⁺ selectivity. The inwardly rectifying K⁺ currents exhibited little or no apparent voltage dependence within the range of from –120 mV to 120 mV. A square-root relationship between chord conductance and [K⁺]₀ at negative potentials could be established. The inwardly rectifying nature of the currents was unaltered after removal of intracellular Mg²⁺ and chelation with ATP and ethylenediaminetetraacetic acid (EDTA). Permeability ratios for other monovalent cations relative to K⁺ were: K⁺ (1.0)>Rb⁺ (0.86)>Cs⁺ (0.12)>Li⁺ (0.08)>Na⁺ (0.03). Slope conductance ratios measured at –100 mV were: Rb⁺ (1.66)>K⁺ (1.0)>Na⁺ (0.09)>Li⁺ (0.08)>Cs⁺ (0.06). K⁺ conductance was highly sensitive to intracellular free Ca²⁺ concentration. The relationship between conductance at 0 mV and Ca²⁺ concentration was well described by a Hill expression with a dissociation constant, K _D, of 70 nmol/l and a Hill coefficient, n, of 1.81. Extracellular Ba²⁺ blocked the currents in a concentration- and voltage-dependent manner. The dependence of the K _D for the blockade was analysed using a Woodhull-type treatment, locating the ion interaction site at 19 % of the distance across the electrical field of the membrane and a K _D (0 mV) of 7 mmol/l. Tetraethylammonium and 4-aminopyridine were without effect whilst quinine and quinidine blocked the currents with concentrations for half-maximum effects equal to 7 mol/l and 3.5 mol/l, respectively. The unfractionated venom of the scorpion Leiurus quinquestriatus (LQV) blocked the K⁺ currents of HeLa cells. The toxins apamin and scyllatoxin had no detectable effect whilst charybdotoxin, a component of LQV, blocked in a voltage-dependent manner with half-maximal concentrations of 40 nmol/l at –120 mV and 189 nmol/l at 60 mV; blockade by charybdotoxin accounts for the effect of LQV. Application of ionomycin (5–10 mol/l), histamine (1 mmol/l) or bradykinin (1–10 mol/l) to cells dialysed with low-buffered intracellular solutions induced K⁺ currents showing inward rectification and a lack of voltage dependence.     

Features of 4-aminopyridine sensitive outward current observed in single smooth muscle cells from the rabbit pulmonary artery

The 4-aminopyridine (4AP) sensitive outward current of enzymatically dispersed single smooth muscle cells of the rabbit main pulmonary artery were investigated using the voltage clamp method. When the cell was exposed to physiological salt solution (PSS) in the bath and high K⁺ in the pipette no inward current was generated by depolarization of the membrane, but when 4AP was present in the bath or when Cs⁺ with tetraethylammonium⁺ (Cs⁺-TEA⁺) in the pipette, an inward current was generated. This current was enhanced by Ba²⁺ or high Ca²⁺ and was blocked by inorganic or organic Ca²⁺ channel blockers.The outward current was partly inhibited by the Ca²⁺ channel blockers, Ca²⁺-free or Mn²⁺ containing solution. The residual outward current was blocked by external application of 10 mM 4AP, whereas it was inhibited by half with 100 mM TEA⁺. To investigate further natures of 4AP sensitive outward current, the following experiments were done in the bath solution containing 2.5 mM Mn²⁺. The reversal potential of this outward current, estimated from the tail current, remained the same in Na⁺-deficient solution, but shifted to near the K⁺-equilibrium potential in Cl^– deficient solution. Thus, the main current carrier for the outward current seems to be K⁺, but Cl^– may participate to some extent. The amplitude of the outward current decreased slowly. However, the reversal potential was not changed, suggesting the reduction in amplitude of the outward current was not due to the accumulation of K⁺ on the outer surface of the membrane. As 4AP inhibited the outward current to a greater extent at lower than higher membrane potential levels, 4AP bound to the channel may be dislodged at higher levels. When pH of the bath solution was modified from 7.3 to 8.0, inhibitory actions of 4AP were enhanced (pKa value of 4AP=9.17). Thus, a non-ionized form of 4AP may act as a channel blocker. We conclude that in smooth muscle cells of the pulmonary artery, lack of an action potential in physiological solution may partly be due to a small inward current as well as a large contribution of the 4AP sensitive outward current.     

Ca2+ dependence of small Ca2+-activated K+ channels in cultured N1E-115 mouse neuroblastoma cells

Single-channel properties of Ca²⁺-activated K⁺ channels have been investigated in excised membrane patches of N1E-115 mouse neuroblastoma cells under asymmetric K⁺ concentrations at 0 mV. The SK channels are blocked by 3 nM external apamin, are unaffected by 20 mM external tetraethylammonium (TEA) and have a single-channel conductance of 5.4 pS. The half-maximum open probability and opening frequency of SK channels are observed at 1 M internal Ca²⁺. Concentration/effect curves of these parameters are very steep with exponential slope factors between 7 and 13. Opentime distributions demonstrate the existence of at least two open states. The mean short open time increases with [Ca²⁺]_i, whereas the mean long open time is independent of [Ca²⁺]_i. At low [Ca²⁺]_i the short-lived open state predominates. At saturating [Ca²⁺]_i the number of longlived openings is more enhanced than the number of short-lived openings and both open states occur equally frequently. The opening frequency as well as the open times of SK channels are independent of the membrane potential in the range of –16 to +40 mV. The results indicate that activation of K⁺ current through SK channels is mainly determined by the Ca²⁺-dependent single-channel opening frequency. BK channels in N1E-115 cells are insensitive to 100 nM external apamin, are sensitive to external TEA in the millimolar range and have a single-channel conductance of 98 pS. Half-maximum open probability and opening frequency of the BK channel are observed at 7.5–21 M internal Ca²⁺. The slope factors of concentration/effect curves range between 1.7 and 2.9. As the BK channel open time is markedly enhanced at raised [Ca²⁺]_i, the Ca²⁺ dependence of the current through BK channels is determined by the single-channel opening frequency as well as the open time. SK as well as BK channels appear to be clustered and interact in a negative cooperative manner in multiple channel patches. The differences in Ca²⁺ dependence suggest that BK channels are activated by a local high [Ca²⁺]_i associated with Ca²⁺ influx, whereas SK channels may be activated by Ca²⁺ released from internal stores as well.