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.     

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.     

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.     

A long lasting Ca2+ -activated outward current in guinea-pig atrial myocytes

Among other characteristics, the steady-state current-voltage relationship of patch-clamped single atrial myocytes from guinea-pig hearts is defined by an outward current hump in the potential region –15 to +40mV. This hump was reversibly suppressed by Co²⁺ (3 mM) or nitrendipine (5 M) and enhanced by Bay K 8644 (5 M). The maintained outward current component suppressed by Co²⁺ extended between –15.2±1.9 mV and +39.5 ±1.7 mV (mean±SEM of 14 cells) and has an amplitude of 95.7±9.4 pA at +10 mV. In isochronal I-V curves, the hump was already visible at 400 ms with essentially the same amplitude as at 1500 ms. The Co²⁺ -sensitive outward current underlying the hump was poorly time-dependent during 1.5 s voltage pulses but slowly relaxed upon repolarization. Tail currents reversed near the K⁺ equilibrium potential under our experimental conditions. The current hump of the steady-state I-V curve was also abolished by caffeine (10 mM) or ryanodine (3 M), both drugs that interfere with sarcoplasmic reticulum function. Apamin (1 M) or quinine (100 M) but not TEA (5–50 mM) markedly reduced its amplitude. However, at similar concentrations as required to inhibit the hump, both apamin and quinine appeared to be poorly specific for Ca²⁺ -activated K⁺ currents in heart cells since they also inhibited the L-Type Ca²⁺ current. It is concluded that a long lasting Ca²⁺ -activated outward current, probably mainly carried by K⁺ ions but not sensitive to TEA, exists in atrial myocytes which is responsible for the current hump of the background I-V curve.     

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.     

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.     

Role of Na+/Ca2+ exchange in transcellular Ca2+ transport across primary cultures of rabbit kidney collecting system

Cells from connecting tubule and cortical collecting duct of rabbit kidney were isolated by immunodissection with mAb R2G9 and cultured on permeable filters. Confluent monolayers developed an amiloride-sensitive transepithelial potential difference of –50±1 mV (lumen negative) and a transepithelial resistance of 507±18 cm². Transepithelial Ca²⁺ transport increased dose-dependently with apical [Ca²⁺] and, in solutions containing 1 mM Ca²⁺, the active transcellular Ca²⁺ transport rate was 92±2 nmol h^–1 cm^–2. Transcellular Ca²⁺ transport was dependent on basolateral Na⁺ (Na _b ⁺ ). Isoosmotic substitution of Na _b ⁺ for N-methylglucamine resulted in a concentration-dependent decrease in Ca²⁺ absorption, with maximal inhibition of 67±5%. A Hill plot of the Na⁺-dependence yielded a coefficient of 1.9±0.4, indicating more than one Na⁺ site on a Na⁺-dependent Ca²⁺ transport system. In addition, the absence of Ca _b ²⁺ resulted in a significant increase in Ca²⁺ transport both in the presence and absence of Na _b ⁺ . Added basolaterally, ouabain (0.1 mM) inhibited Ca²⁺ transport to the same extent as did Na⁺-free solutions, while bepridil (0.1 mM), an inhibitor of Na⁺/Ca²⁺ exchange, reduced Ca²⁺ transport by 32±6%. Methoxyverapamil, felodipine, flunarizine and diltiazem (10 M) were without effect. Depolarisation of the basolateral membrane, by raising [K⁺]_b to 60 mM, significantly decreased transcellular Ca²⁺ transport, which is indicative of electrogenic Na⁺/Ca²⁺ exchange. In conclusion, active Ca²⁺ transport in the collecting system of rabbit kidney is largely driven by basolateral Na⁺/Ca²⁺ exchange. However, a residual Ca²⁺ absorption of about 30% was always observed, suggesting that other Ca²⁺ transport mechanisms, presumably a Ca²⁺-ATPase, participate as well.     

Actions of pinacidil on membrane currents in canine ventricular myocytes and their modulation by intracellular ATP and cAMP

We studied the effects of pinacidil (3–50 M) on the membrane currents of canine ventricular myocytes, using the whole-cell variant of the patch-clamp technique, and the modulation of these effects by intracellular environment, using the pipette perfusion technique. The following observations were obtained: (1) pinacidil induced a dosedependent outward shift in current at voltages positive to ±70 mV; (2) the pinacidil-induced current was largely timeindependent at voltages positive to ±50 mV and displayed an increase in current fluctuations at more positive voltages, resembling the kinetic properties of current through the ATP-regulated K⁺ channels; (3) elevating the extracellular potassium concentration ([K⁺]_o) caused a positive shift in the voltage where the pinacidil-induced current crossed the voltage axis and increased the slope conductance of this current; (4) the pinacidil-induced current was reduced by Ba²⁺ (0.5–1.5 mM) and abolished by intracellular Cs⁺ (125 mM); (5) glibenclamide reversibly reduced or abolished the pinacidil-induced current; (6) the action of pinacidil was decreased by elevating [ATP] in the pipette solution (from 1 to 10 mM); (7) the action of pinacidil was augmented by adding isoproterenol (1 M) to the superfusate or adding cAMP (0.1 mM) to the pipette solution; (8) elevating temperature augmented, and accelerated the onset of, pinacidil's action; (9) pinacidil reversibly decreased the Ca²⁺ -independent transient outward current (I_to1) but augmented the Ca²⁺ -dependent transient outward current (I_to2). Based on these observations, we reached the following conclusions: (1) the main effect of pinacidil is to increase an outward current through the ATP-regulated K⁺ channels; (2) pinacidil's action is modulated by an enzymatic reaction.     

Different inhibitions of the voltage-dependent K+ current by Ca2+ antagonists in the smooth muscle cell membrane of rabbit small intestine

Actions of Ca²⁺ antagonists (verapamil, nicardipine and diltiazem) on the voltage-dependent K⁺ current, obtained from the fragmented smooth muscle cell membrane (smooth muscle ball; SMB) of the rabbit small intestine, were investigated using voltage clamp techniques. To eliminate the influence of the Ca²⁺-dependent K⁺ current, the voltage-dependent K⁺ current was recorded in 2.5 mM Mn²⁺ (Ca²⁺ omitted) solution. These three Ca²⁺ antagonists inhibited the peak amplitude of the K⁺ current, in a dose-dependent manner. During application of a long command pulse (duration, 3 s), the amplitude of the voltage-dependent K⁺ current decreased slowly with time. Diltiazem inhibited the K⁺ current with a slight prolongation of the 20% decay time, while TEA (tetraethyl-ammonium), a K⁺ channel blocker, inhibited the current, without affecting the decay. By contrast, verapamil and nicardipine accelerated inactivation. In the control, the voltage-dependent inactivation was also seen in the K⁺ current. This inactivation curve below 0 mV was not modified by 10 M diltiazem, 5 M verapamil nor 3 M nicardipine. These results indicate that inhibition of the voltage-dependent K⁺ current by verapamil or nicardipine differed from that by diltiazem.An abstract of this work was reported at the 59th general meeting of Japan Pharmacological Society (Terada et al. 1986).     

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.     

Na+-Ca2+ exchange in the isolated cochlear outer hair cells of the guinea-pig studied by fluorescence image microscopy

The outer hair cell isolated from the guinea-pig was superfused in vitro and the cytosolic calcium concentration ([Ca²⁺]_i) and sodium concentration ([Na⁺]_i) were measured using fluorescence indicators. Under the resting condition, [Ca²⁺]_i and [Na⁺]_i were 91±9 nM (n = 51) and 110±5 mM (n = 12), respectively. Removal of external Na⁺ by replacing with N-methyl-D-glucamine (NMDG⁺) increased [Ca²⁺]_i by 270±79% (n = 27) and decreased [Na⁺]_i by 23±4 mM (n = 6). Both changes in [Ca²⁺]_i and [Na⁺]_i were totally reversible on returning external Na⁺ to the initial value and were inhibited by addition of 0.1 mM La³⁺ or 100 M amiloride 5-(N,N-dimethyl) hydrochloride. Elevation of external Ca²⁺ ions to 20 mM reversibly decreased [Na⁺]_i by 8±6 mM (n = 5). Moreover, the chelation of the intracellular Ca²⁺ with 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) exerted an inhibitory action on the NMDG⁺-induced reduction in [Na⁺]_i. Exposure to 5 mM NaCN for 2 min significantly and reversibly increased [Ca²⁺]_i by 290±37% (n = 5), but did not affect the [Ca²⁺]_i elevation induced by the NMDG⁺ solution. The rise in [Ca²⁺]_i induced by the NMDG⁺ solution was not enhanced by ouabain pretreatment. Addition of ouabain did not alter the [Na⁺]_i. The present results are best explained by the presence of an Na⁺-Ca²⁺ exchanger in cell membrane and indicate that the activity of Na⁺/K⁺ pump is poor in outer hair cells.     

Na+ currents in cultured mouse pancreatic B-cells

Pancreatic B-cells, kept in culture for 1–4 days, were studied in the whole-cell, cell-attached and outside-out modes of the patch clamp technique. B-cells were identified by the appearance of electrical activity in the cell-attached mode when the bath glucose was raised from 3 to 20 mM. In whole-cell, 80% of these cells showed a transient inward Na⁺ current, when depolarizing pulses were preceded by holding potentials, or prepulses to potentials more negative than –80 mV. The midpoint (E _h) of the inactivation curve (h ) was at –109 mV in 2.6 mM Ca²⁺, 1.2 mM Mg²⁺ and –120 mV in 0.2 mM Ca²⁺, 3.6 mM Mg²⁺. In 2.6 mM Ca²⁺, inactivation was fully removed atE<–150 mV. Na⁺ currents activated atE>–60 mV and were largest at around –10 mV (120 mM Na⁺). The kinetic parameters of activation (t _p) and inactivation ()_h were similar to those of other mammalian Na⁺ channels. Unitary currents with an amplitude of approximately 1 pA at –30 mV (140 mM Na⁺) with a similar voltage-dependence and time-course of mean current were recorded from outside-out patches. The results show that B-cells have a voltage-dependent Na⁺ current which, owing to the voltage-dependence of inactivation, is unlikely to play a major role in glucose-induced electrical activity.     

Potassium currents in rat colonic smooth muscle cells and changes during development and aging

In a previous study on freshly isolated single smooth muscle cells from the circular layer of the rat distal colon, we reported that the L-type Ca²⁺ current density increased during development and gradually declined with further aging [ZI Xiong, N Sperelakis, N Noffsinger, C Fenoglio-Preiser (1993) Am J Physiol 265: C617–C625]. Since K⁺ current plays a key role in controlling excitability of the cells and hence the motility of the colon, in the present study the voltage-gated K⁺ channel currents, (I _K) were investigated using the whole-cell voltage-clamp technique in colonic myocytes from rats of different ages. A Ca²⁺-sensitive K⁺ current [I _K(Ca)] and two kinds of Ca²⁺-insensitive outward K⁺ currents were identified and characterized. I _K(Ca) was recorded at potentials more positive than –40 mV in Ca²⁺-containing bath solution, and was blocked by Ca²⁺ channel antagonists and tetraethylammonium ion (TEA⁺). After removing Ca²⁺ from the bath solution and using a high ethylenebis(oxonitrilo)tetraacetate (EGTA, 4 mM) concentration in the pipette, two types of Ca²⁺-insensitive I _K were recorded. The first and faster component was usually activated at potentials more positive than –50 mV, and was more sensitive to 4-aminopyridine (4-AP). In contrast, the second and slower (delayed) component was activated at potentials more positive than –30 mV, and was more sensitive to TEA. The total density of the Ca²⁺-insensitive I _K component decreased dramatically during the neonatal period: from 32.2±3.2 pA/pF in 3-day-old rats to 17.8±2.6 pA/pF in 40-day-old rats; there was no further decline during aging (up to 480 days). This total I _K decline was due primarily to a decline in the 4-AP-sensitive component; the TEA-sensitive component remained unchanged at all ages. The possible physiological implications of the decline in density of the 4-AP-sensitive I _K component during neonatal development are discussed.     

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 of membrane excitability by intracellular pH (pHi) changers through Ca2+-activated K+ current (BK channel) in single smooth muscle cells from rabbit basilar artery

Employing microfluorometric system and patch clamp technique in rabbit basilar arterial myocytes, regulation mechanisms of vascular excitability were investigated by applying intracellular pH (pH_i) changers such as sodium acetate (SA) and NH₄Cl. Applications of caffeine produced transient phasic contractions in a reversible manner. These caffeine-induced contractions were significantly enhanced by SA and suppressed by NH₄Cl. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored in a single isolated myocyte and based the ratio of fluorescence using Fura-2 AM (R _340/380). SA (20 mM) increased and NH₄Cl (20 mM) decreased R _340/380 by 0.2 ± 0.03 and 0.1 ± 0.02, respectively, in a reversible manner. Caffeine (10 mM) transiently increased R _340/380 by 0.9 ± 0.07, and the ratio increment was significantly enhanced by SA and suppressed by NH₄Cl, implying that SA and NH₄Cl may affect [Ca²⁺]_i (p < 0.05). Accordingly, we studied the effects of SA and NH₄Cl on Ca²⁺-activated K⁺ current (IK_Ca) under patch clamp technique. Caffeine produced transient outward current at holding potential (V _h) of 0 mV, caffeine induced transient outward K⁺ current, and the spontaneous transient outward currents were significantly enhanced by SA and suppressed by NH₄Cl. In addition, IK_Ca was significantly increased by acidotic condition when pH_i was lowered by altering the NH₄Cl gradient across the cell membrane. Finally, the effects of SA and NH₄Cl on the membrane excitability and basal tension were studied: Under current clamp mode, resting membrane potential (RMP) was −28 ± 2.3 mV in a single cell level and was depolarized by 13 ± 2.4 mV with 2 mM tetraethylammonium (TEA). SA hyperpolarized and NH₄Cl depolarized RMP by 10 ± 1.9 and 16 ± 4.7 mV, respectively. SA-induced hyperpolarization and relaxation of basal tension was significantly inhibited by TEA. These results suggest that SA and NH₄Cl might regulate vascular tone by altering membrane excitability through modulation of [Ca²⁺]_i and Ca²⁺-activated K channels in rabbit basilar artery.     

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.     

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.     

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.     

DIVALENT CATION MODULATION OF A-TYPE POTASSIUM CHANNELS IN ACUTELY DISSOCIATED CENTRAL NEURONS FROM WIDE-TYPE AND MUTANT DROSOPHILA

Drosophila mutants provide an ideal model to study channel-type specificity of ion channel regulation in situ. In this study, the effects of divalent cations on voltage-gated K⁺ currents were investigated in acutely dissociated central neurons of Drosophila third instar larvae using the whole-cell patch-clamp recording. Our data showed that micromolar Cd²⁺ enhanced the peak inactivating current (I_A) without affecting the delayed component (I_K). The same results were obtained in Ca²⁺-free external solution, and from slo¹ mutation, which eliminates transient Ca²⁺-activated K⁺ current. Micromolar Cd²⁺and Zn²⁺, and millimolar Ca²⁺and Mg²⁺ all shifted the steady-state inactivation curve of I_A without affecting the voltage-dependence of I_A activation, whereas millimolar Cd²⁺markedly affected both the activation and steady-state inactivation curves for I_A. Divalent cations affected I_A with different potency; the sequence was: Zn²⁺ > Cd²⁺ > Ca²⁺ > Mg²⁺. The modulation of I_A by Cd²⁺ was partially inhibited in Sh^M, a null Shaker (one of I_A-encoding genes) mutation. Taken together, the channel-type specificity, the asymmetric effects on I_A activation and inactivation kinetics, and the diverse potency of divalent cations all strongly support the idea that physiological divalent cations modulate A-type K⁺ channels through specific binding to extracellular sites of the channels.     

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.