Mg2+ block and inward rectification of mechanosensitive channels in Xenopus oocytes

 The effects of Mg²⁺ on single mechanosensitive (MS) channel currents recorded from Xenopus oocytes were studied using cell-attached and inside-out patch configurations. Mg²⁺ both permeates and blocks MS channels. Under symmetrical ionic conditions, the blocking effects of Mg²⁺ can be described by a Hill coefficient of 0.9 at ±100 mV and IC₅₀s of 0.12 mM (–100 mV) and 0.60 mM at (+100 mV). Although block by intracellular Mg²⁺ may contribute to inward MS channel rectification, significant current rectification is retained even under symmetrical KCl concentrations and in the complete absence of Mg²⁺. The observed voltage dependencies of the IC₅₀ for Mg²⁺ block and the K_m for K⁺ current saturation indicate asymmetries in the MS channel pore. In addition, the absence of K⁺ self block and anomalous mole fraction effects with K⁺/Tl⁺ mixtures indicate a single site pore model. Received: 11 September 1997 / Received after revision: 21 November 1997 / Accepted: 24 November 1997     

Reciprocal effects of Ca2+ and Mg-ATP on the ‘run-down’ of the K+ channels in opossum kidney cells

Using the patch clamp technique, we identified an inwardly rectifying K⁺ channel in the membrane of opossum kidney cells. The single channel conductance was about 90 pS for inward currents and 30 pS for outward currents under a symmetrical high-K⁺ condition. The activity of the channel was found to decrease with time during recording from inside-out patches. In the solution with submicromolar Ca²⁺, the activity disappeared within 4–20 min. Intracellular Ca²⁺ promoted the run-down of the channel activity at 0.1–1 mM, whereas millimolar Mg-ATP restored the activity after run-down. The run-down channels could never be reactivated by ATP in the absence of Mg²⁺, or by a nonhydrolyzable ATP analog, AMPPNP, even in the presence of Mg²⁺.     

A Na+-activated K+ current (I K,Na) is present in guinea-pig but not rat ventricular myocytes

 The effects of removing extracellular Ca²⁺ and Mg²⁺ on the membrane potential, membrane current and intracellular Na⁺ activity (a ⁱ _Na) were investigated in guinea-pig and rat ventricular myocytes. Membrane potential was recorded with a patch pipette and whole-cell membrane currents using a single-electrode voltage clamp. Both guinea-pig and rat cells depolarize when the bathing Ca²⁺ and Mg²⁺ are removed and the steady-state a ⁱ _Na increases rapidly from a resting value of 6.4± 0.6 mM to 33±3.8 mM in guinea-pig (n=9) and from 8.9±0.8 mM to 29.3±3.0 mM (n=5) in rat ventricular myocytes. Guinea-pig myocytes partially repolarized when, in addition to removal of the bathing Ca²⁺ and Mg²⁺, K⁺ was also removed, however rat cells remained depolarized. A large diltiazem-sensitive inward current was recorded in guinea-pig and rat myocytes, voltage-clamped at –20 mV, when the bathing divalent cations were removed. When the bathing K⁺ was removed after Ca²⁺ and Mg²⁺ depletion, a large outward K⁺ current developed in guinea-pig, but not in rat myocytes. This current had a reversal potential of –80±0.7 mV and was not inhibited by high Mg²⁺ or glybenclamide indicating that it is not due to activation of non-selective cation or adenosine triphosphate (ATP)-sensitive K channels. The current was not activated when Li⁺ replaced the bathing Na⁺ and was blocked by R-56865, suggesting that it was due to the activation of K_Na channels. Received: 15 October 1998 / Received after revision: 22 January 1999 / Accepted: 5 February 1999     

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.     

Inwardly rectifying K+ currents in fetal alveolar type II cells: regulation by protein kinase A and protein phosphatases

Fetal guinea-pig lung alveolar type II (ATII) cells have inwardly rectifying (IR) K⁺ currents that display Mg²⁺- and G-protein-dependent run-down. We have used the whole-cell patch-clamp technique to investigate further the regulation of these currents. Under control conditions [KCl-rich pipette solution (1 mM free Mg²⁺, 10 nM free Ca²⁺) and KCl-rich bath solution], we found that IR K⁺ currents diminished with a t _1/2 of 7.6 min and were absent by 30 min. Experimental manoeuvres designed to inhibit phosphorylation increased the rate of current run-down. Thus, intracellular addition of 100 μM H-7, a general kinase inhibitor, reduced the t _1/2 to 4.7 min and the currents were absent by 16 min. Similarly, protein kinase A (PKA) inhibitor peptide (50 nM) also accelerated run-down. Agents known to increase phosphorylation, such as db-cAMP (0.5 mM) and forskolin (10 μM), resulted in a significant slowing of run-down (t _1/2>16 min) as did intracellular addition of the catalytic subunit of PKA (100 nM). Similarly, inhibition of dephosphorylation by either 1 μM okadaic acid [protein phosphatase 1/2A (PP-1/2A) inhibitor] or anti-human protein phosphatase 2Cα (PP2C) antiserum decreased the rate of run-down. These results indicate that the phosphorylation-dependent activation state of the fetal ATII cell IR K⁺ channel is regulated by a complex interplay of kinases and phosphatases involving PKA (activation), and PP2C and PP-1/2A (inactivation). Received: 10 February 1999 / Received after revision and accepted: 31 March 1999     

Patch-clamp studies of K+ and Cl− channel currents in canine pancreatic acinar cells

K⁺ and Cl^– channel currents in the plasma membrane of isolated canine pancreatic acinar cells were studied by patch-clamp single-channel and whole-cell current recording techniques. In excised inside-out patches, we found a Ca⁺-activated (control range 0.01–0.4 M) and voltage-activated K²⁺-selective channel with a unit conductance of approximately 40 pS in symmetrical K⁺-rich solutions. In intact cells, addition of acetylcholine (1 M) or bombesin tetradecapeptide (0.1 nM) to the bath evoked an increase in frequency of K⁺ channel opening. In whole-cell recordings on cells dialyzed with K⁺-rich and Ca²⁺-free solution containing 0.5 mM EGTA, the resting potential was about –40mV. Depolarizing voltage pulses activated outward K⁺ currents, which were blocked by 10 mM tetraethylammonium, whereas hyperpolarizing pulses evoked smaller inward currents. Acetylcholine or bombesin activated the K⁺ current and enhanced the inward current, which was reduced by a low Cl^– (10 mM) intracellular solution at –90 mV holding potential. These results suggest that both Ca²⁺- and voltage-activated K⁺ channels and Ca²⁺-activated Cl^– channels exist in the plasma membrane of canine pancreatic acinar cells.     

Electrophysiological study on the high K+ sensitivity of rat glomerulosa cells

 Elevation of extracellular potassium concentration by as little as some tenth of mM activates rat adrenal glomerulosa cells. In the present study some factors responsible for this high K⁺ sensitivity were examined. Using whole-cell voltage-clamp technique we found that both T-type and L-type voltage-dependent Ca²⁺ channels have very low threshold potential (–71 and –58 mV, resp.). By means of patch-clamp technique combined with single-cell fluorimetry we also provided evidence that the activation of I_gl, a K⁺-activated inward rectifying current is associated with Ca²⁺ influx. Both the low activation threshold of voltage-dependent Ca²⁺ channels and the function of I_gl contribute to the exceptional K⁺ sensitivity of the glomerulosa cells. Received: 30 September 1997 / Accepted: 4 November 1997     

Human Kir2.1 channel carries a transient outward potassium current with inward rectification

We have previously reported a depolarization-activated 4-aminopyridine-resistant transient outward K⁺ current with inward rectification (I _to.ir) in canine and guinea pig cardiac myocytes. However, molecular identity of this current is not clear. The present study was designed to investigate whether Kir2.1 channel carries this current in stably transfected human embryonic kidney (HEK) 293 cells using whole-cell patch-clamp technique. It was found that HEK 293 cells stably expressing human Kir2.1 gene had a transient outward current elicited by voltage steps positive to the membrane potential (around −70 mV). The current exhibited a current–voltage relationship with intermediate inward rectification and showed time-dependent inactivation and rapid recovery from inactivation. The half potential (V _0.5) of availability of the current was −49.4 ± 2.1 mV at 5 mM K⁺ in bath solution. Action potential waveform clamp revealed two components of outward currents; one was immediately elicited and then rapidly inactivated during depolarization, and another was slowly activated during repolarization of action potential. These properties were similar to those of I _to.ir observed previously in native cardiac myocytes. Interestingly, inactivation of the I _to.ir was strongly slowed by increasing intracellular free Mg²⁺ (Mg²⁺ _i , from 0.03 to 1.0, 4.0, and 8.0 mM). The component elicited by action potential depolarization increased with the elevation of Mg²⁺ _i . Inclusion of spermine (100 μM) in the pipette solution remarkably inhibited both the I _to.ir and steady-state current. These results demonstrate that the Mg²⁺ _i -dependent current carried by Kir2.1 likely is the molecular identity of I _to.ir observed previously in cardiac myocytes.     

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.     

Low-amiloride-affinity Na+ channel in the epithelial cells isolated from the endolymphatic sac of guinea-pigs

 By using the whole-cell patch-clamp technique, an amiloride-sensitive Na⁺-selective conductance was found in epithelial cells from the endolymphatic sac (ES) epithelia of guinea-pigs. In the current-clamp configuration, the average resting membrane potential was –41.7±8.4 mV (n = 22). Application of amiloride at a concentration of 20 μM elicited a decrease in cation conductance that was responsible for a membrane hyperpolarization by 17.9±6.0 mV (n = 22). Substitution of N-methyl d-glucamine chloride (NMDG-Cl) for external NaCl led to a more significant membrane hyperpolarization by 28.4±8.3 mV (n = 22). At holding potential of –70 mV, amiloride and ethylisopropylamiloride (EIPA) blocked the inward current in a concentration-dependent manner over the range of concentrations of between 0.1 μM and 50 μM, with an inhibitory constant (K _i) of 1.3±0.4 μM (n = 7) and 1.5±0.3 μM (n = 5), respectively. In the voltage-clamp configuration, substitution of NMDG-Cl for external NaCl significantly reduced the inward current (n = 9), indicating that the whole-cell conductance has a high permeability for Na⁺. Superfusion with 20 μM amiloride induced a significant reduction of the inward current, shifted the reversal potential from –39.4±8.8 mV to –60.4±10.5 mV (n = 12), and decreased the inward conductance from 5.0±1.3 nS to 3.7±1.5 nS (n = 12). The permeability ratio of Na⁺ over K⁺, calculated from the difference in reversal potential between the currents before and after application of amiloride, was approximately 5:1. Additionally, the conductance was not activated by application of forskolin, 3-isobutyl-1-methylxanthine (IBMX) and 8-bromo-cAMP (8-Br-cAMP). These findings suggest that a low-amiloride-affinity Na⁺ channel localized in the ES epithelial cells may be involved in uptake of Na⁺ in the ES. Received: 29 May 1996 / Received after revision: 1 August 1996 / Accepted: 2 August 1996     

Na+-dependent regulation of the free Mg2+ concentration in neuropile glial cells and P neurones of the leech Hirudo medicinalis

 To investigate the Mg²⁺ regulation in neuropile glial (NG) cells and pressure (P) neurones of the leech Hirudo medicinalis the intracellular free Mg²⁺ ([Mg²⁺]_i) and Na⁺ ([Na⁺]_i) concentrations, as well as the membrane potential (E _m), were measured using Mg²⁺- and Na⁺-selective microelectrodes. The mean steady-state values of [Mg²⁺]_i were found to be 0.91 mM (mean E _m=–63.6 mV) in NG cells and 0.20 mM (mean E _m=–40.6 mV) in P neurones with a [Na⁺]_i of 6.92 mM (mean E _m=–61.6 mV) and 7.76 mM (mean E _m=–38.5 mV), respectively. When the extracellular Mg²⁺ concentration ([Mg²⁺]_o) was elevated, [Mg²⁺]_i in P neurones increased within 5–20 min whereas in NG cells a [Mg²⁺]_i increase occurred only after long-term exposure (6 h). After [Mg²⁺]_o was reduced back to 1 mM, a reduction of the extracellular Na⁺ concentration ([Na⁺]_o) decreased the inwardly directed Na⁺ gradient and reduced the rate of Mg²⁺ extrusion considerably in both NG cells and P neurones. In P neurones Mg²⁺ extrusion was reduced to 15.4% in Na⁺-free solutions and to 6.0% in the presence of 2 mM amiloride. Mg²⁺ extrusion from NG cells was reduced to 6.2% in Na⁺-free solutions. The results suggest that the major [Mg²⁺]_i-regulating mechanism in both cell types is Na⁺/ Mg²⁺ antiport. In P neurones a second, Na⁺-independent Mg²⁺ extrusion system may exist. Received: 11 August 1998 / Received after revision: 14 October 1998 / Accepted: 15 October 1998     

Acetylcholine stimulates a Ca2+-dependent Cl− conductance in mouse lacrimal acinar cells

Patch-clamp whole-cell current recordings under voltage-clamp conditions were carried out on isolated mouse exorbital lacrimal acinar cells. Acetylcholine evoked outward current at a membrane potential of –20 mV whereas an inward current was observed at –80 mV. The outward current is due to the well-known calcium-activated K⁺ channels whereas the inward current was Cl^– dependent. The acetylcholine-evoked Cl^– current was abolished when the intracellular Ca²⁺ concentration was clamped at very low levels by a high intracellular EGTA concentration. Acetylcholine therefore activates a Ca²⁺-dependent Cl^– conductance in mouse lacrimal acinar cells.     

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.     

Patch-clamp analysis of voltage-gated currents in intermediate lobe cells from rat pituitary thin slices

Ionic currents of hypophyseal intermediate lobe cells were studied using a thin-slice preparation of the rat pituitary in conjunction with conventional and perforated whole-cell patch-clamp recording techniques. A majority (89%) of the cells studied generated Na⁺, Ca²⁺ and K⁺ currents upon depolarizing voltage steps and responded to bath application of -aminobutyric acid (GABA; 20–50 M) with inward currents (in symmetrical chloride, holding potential –80 mV). A small percentage of cells (11%) did not display inward membrane currents upon depolarization and was unresponsive to GABA. In the first type of cells, Ca²⁺ and K⁺ currents were further studied in isolation. Ca²⁺ tail currents showed a biphasic time course upon repolarization, with time constants and amplitudes of 2.07±0.29 ms, 123±22 pA (for the slowly deactivating component) and 0.14±0.06 ms, 437±33 pA (for the fast-deactivating component; means±SD of n=4 cells). Slowly and fast-deactivating conductances were half-maximally activated at around –10 mV and +10 mV respectively. Depolarizing voltage steps elicited two types of K⁺ current, which were separated using a prepulse protocol. A fast-activating, transient component showed half-maximal steadystate inactivation between –65 mV and –45 mV depending on the divalent cation composition of the external solution. Its decay was fitted by single-exponential functions with time constants of 36±11 ms and 3.9±0.9 ms at –20 mV and +40 mV respectively (mean±SD; n=4 cells). Whereas the peak current amplitudes of the transient K⁺ current component remained stable, the amplitude of the second, delayed component increased progressively throughout the course of whole-cell experiments. In cells recorded with the perforated whole-cell technique, bath application of dopamine (10 nM–1 M) induced large hyperpolarizations from a spontaneous membrane potential of –40 mV, but did not consistently affect the amplitude of the voltage-gated K⁺ conductances. These data are compared to previous studies using other preparations of the intermediate lobe, and differences are discussed, thus helping to extend our knowledge of electrical excitability of hypophyseal cells.     

Adrenaline diminishes K+ contractures and Ba2+-current in chicken slow skeletal muscle fibres

The effects of adrenaline and the β-adrenergic agonist isoprenaline on K⁺-evoked tension (K⁺-contracture) and Ba²⁺ current were investigated in chicken slow (anterior latissimus dorsi (ald)) muscle using isometric-tension measurements and current recording. Addition of adrenaline (10⁻⁷–10⁻⁵M) or isoprenaline (10⁻⁶–10⁻⁵ M) to the bath reduced the amplitude of the K⁺-contractures. These effects were blocked by the β-antagonist propranolol (5 × 10⁻⁶ M). External application of a cAMP analogue (8-bromo cyclic AMP; 1 × 10⁻⁴ M) also decreased the amplitude of the K⁺-contractures. To analyze the possible relationship between the induced tension reduction and effects on sarcolemmal Ca²⁺ channels, a slow action potential and a slow inward membrane current were studied in intact ald chicken muscle fibres. When the ald muscle was immersed in a Na⁺- and Cl⁻-free solution containing Ba²⁺ and depolarizing pulses were delivered from a −80 mV holding potential, the muscle fibres exhibited a small, slow Ba²⁺-dependent potential (observed at about −26 mV, peak amplitude, around −10 mV). The response was blocked by the addition of Co²⁺ (5 mM) or Cd²⁺ (2 mM). Using the three-microelectrode voltage-clamp technique, a slow inward membrane current underlying the Ba²⁺ potential could be discerned. The current had a mean threshold of −60 mV, reached maximum at about −5 mV and ranged from ca. 9 to 19 μA/cm² (depending on the external Ba²⁺ concentration). It had a mean reversal potential of +45 mV. The Ba²⁺ inward current was diminished when adrenaline or isoprenaline was added to the bath (1 × 10⁻⁵ M); however, this decrease did not occur when propranolol was present (5 × 10⁻⁶ M). These results suggest that the decreases in the tension of K⁺-contractures induced by adrenaline and isoprenaline may occur through β-adrenergic effects on sarcolemmal Ca²⁺ channels in ald chicken slow muscle fibres. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effects of nucleotides and potassium channel openers on the SUR2A/Kir6.2 complex K+ channel expressed in a mammalian cell line, HEK293T cells

 The effects of potassium channel opening drugs and intracellular nucleotides on the ATP-sensitive K+ (K_ATP) channel composed of SUR2A and Kir6.2 in HEK293T cells were examined using the patch-clamp technique. The SUR2A/Kir6.2 channel was activated effectively by pinacidil, marginally by nicorandil but not by diazoxide. The pinacidil-activated channel currents were inhibited by glibenclamide with a K _i value of 160 nM. Upon formation of inside-out (I-O) patches, spontaneous openings of the channels appeared, which were inhibited by intracellular ATP (ATP_i) equipotently in the presence and in the absence of intracellular Mg²⁺ (Mg²⁺ _i). The channel activity ran-down gradually in I-O patches. The run-down channels could be reactivated by ATP_i only in the presence of Mg²⁺ _i. Uridine 5’-diphosphate (UDP) antagonized the ATP_i-mediated inhibition of the channel activity before run-down. After run-down, UDP activated the channel without antagonizing ATP_i-mediated channel inhibition. Thus, the SUR2A/Kir6.2 reproduced the major properties of the native cardiac K_ATP channel well in terms of nucleotide regulation and pharmacology, and therefore can be a useful tool with which to elucidate the molecular mechanisms characterizing the K_ATP channel. Received: 24 October 1997 / Received after revision and accepted: 4 December 1997     

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).     

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.     

Contribution of BK channels to action potential repolarisation at minimal cytosolic Ca2+ concentration in chromaffin cells

BK channels modulate cell firing in excitable cells in a voltage-dependent manner regulated by fluctuations in free cytosolic Ca²⁺ during action potentials. Indeed, Ca²⁺-independent BK channel activity has ordinarily been considered not relevant for the physiological behaviour of excitable cells. We employed the patch-clamp technique and selective BK channel blockers to record K⁺ currents from bovine chromaffin cells at minimal intracellular (about 10 nM) and extracellular (free Ca²⁺) Ca²⁺ concentrations. Despite their low open probability under these conditions (V₅₀ of +146.8 mV), BK channels were responsible for more than 25% of the total K⁺ efflux during the first millisecond of a step depolarisation to +20 mV. Moreover, BK channels activated about 30% faster (τ = 0.55 ms) than the rest of available K⁺ channels. The other main source of fast voltage-dependent K⁺ efflux at such a low Ca²⁺ was a transient K⁺ (I_A-type) current activating with V ₅₀ = −14.2 mV. We also studied the activation of BK currents in response to action potential waveforms and their contribution to shaping action potentials both in the presence and the absence of extracellular Ca²⁺. Our results show that BK channels activate during action potentials and accelerate cell repolarisation even at minimal Ca²⁺ concentration, and suggest that they could do so also in the presence of extracellular Ca²⁺, before Ca²⁺ entering the cell facilitates their activity.     

ATP reduces voltage-activated K+ current in cultured rat hippocampal neurons

Effects of extracellular ATP were investigated in cultured rat hippocampal neurons using whole-cell voltage-clamp techniques. When a depolarizing step to +10 mV was applied from a holding potential of -60 mV, an outward K⁺ current was activated. ATP (3 to 300 μM) reduced the K⁺ current. Among adenosine derivatives, ADP (100 μM) slightly inhibited the K⁺ current, and AMP or adenosine (100 μM) was ineffective. UTP was as potent as ATP and α,β-methylene ATP was less effective than ATP. The inhibition by ATP of the K⁺ current was abolished by inclusion of 2 mM GDPβS in the intracellular solution. The results indicate that ATP inhibits K⁺ channels in rat hippocampal neurons through UTP-responsive P₂-purinoceptors coupled with GTP-binding proteins.