Inside-out Configuration Research Articles

Calcium influx, arachidonic acid,and control of endothelialcell proliferation

Recent evidences suggest a role for arachidonic acid (AA) in the triggering of store-independent, ornon-capacitative, calcium entry in different cell types. Here, using patch clamp and fluorimetric single-cell calcium measurements, we provide evidence for AA-activated calcium influx in bovine aortic endothelial cells (BAEC).AA-activated calcium entry is independent from intracellular calcium stores depletion at low doses of the fatty acid (≥ 5μM) and insensitive to a decrease of pH to 6.7.Single-channel analysis in inside-out configuration reveals the presence of a family of AA-activated calcium-permeable channels, with different conductances and reversal potentials.Treatment with AA or ETYA induces a proliferative effect, significantly affected by external EGTA application during the early period (up to 2h) of stimulation with the agonists.We conclude that low concentrations of arachidonic acid are able to evoke a store-independent calcium influx, exerting a mitogenic role in BAECs.

Spontaneous transient outward currents: modulation by nociceptin in murine dentate gyrus granule cells

Spontaneous transient outward currents have been found in peripheral neurons and smooth muscle cells, but rarely in central neurons. Using a nystatin-perforated patch clamp technique, we succeeded in recording spontaneous transient outward currents in mouse dentate gyrus granule cells. Nociceptin/orphanin FQ increased the amplitude and frequency of transient outward currents. We consider modulation of spontaneous transient outward currents to be a new means to regulate cell activity in central neurons, and studied their characteristics and mechanism of augmentation. The whole-cell current–voltage relationship showed outward rectification and the reversal potential was close to the equilibrium potential for K +. The frequency of spontaneous transient outward currents increased at depolarized potentials. Tetraethylammonium, iberiotoxin and a Ca 2+ chelator BAPTA-AM inhibited spontaneous transient outward currents. These results suggest the involvement of large-conductance Ca 2+-activated K + channels. Single-channel recordings in the inside-out configuration revealed Ca 2+-activated K + channels with a conductance ranging from 82 to 352 pS. The augmenting effect of nociceptin/orphanin FQ was cancelled by [Phe 1ψ(CH 2-NH)Gly 2]Nociceptin(1–13)NH 2. Cd 2+ did not affect the transient outward currents or augmentation by nociceptin/orphanin FQ. Whereas nociceptin/orphanin FQ, theophylline and cyclic ADP ribose induced transient outward currents with short duration observed under control conditions, inositol 1,4,5-trisphosphate induced transient outward currents with long duration, in addition to those with short duration. Ryanodine inhibited nociceptin/orphanin FQ from augmenting spontaneous transient outward currents. Our data suggest that Ca 2+ sparks transiently activate large-conductance Ca 2+-activated K + channels to induce transient outward currents. Nociceptin/orphanin FQ probably sensitizes ryanodine receptors and increases transient outward currents to reduce cell excitability.

Diadenosine tetraphosphate-gating of cardiac K(ATP) channels requires intact actin cytoskeleton.

Diadenosine polyphosphates (ApnA) have been recently discovered in the heart, and their levels found to be regulated by ischemia. These signaling molecules are believed to regulate cellular processes that alarm a cell to metabolic stress. In particular, changes in cardiac diadenosine polyphosphates (ApnA) levels may contribute to the regulation of ATP-sensitive K+ (K(ATP)) channel activity, an ion channel that couples the cellular metabolic state with membrane excitability. A feature of myocardial ischemia is the disruption of the actin cytoskeleton which critically regulates the behavior of K(ATP) channels. Whether the integrity of actin microfilaments regulates the interaction of ApnA with K(ATP) channels is not known. The inside-out configuration of the patch-clamp technique was applied to cardiomyocytes isolated from guinea-pig heart. Following patch excision, the prototype dinucleotide, diadenosine tetraphosphate (Ap4A), inhibited K(ATP) channel opening. Treatment of the internal side of membrane patches with either cytochalasin B or DNase I, disrupters of the actin cytoskeleton, prevented Ap4A-induced inhibition of K(ATP) channel opening. Application of purified actin to DNase-treated membrane patches restored the ability of Ap4A to close K(ATP) channels. This study shows that inhibition of cardiac K(ATP) channel by Ap4A, a putative alarmone, requires intact subsarcolemmal actin network. Such interaction between K(ATP) channels, the cardiomyocyte cytoskeleton and intracellular Ap4A could affect different channel-dependent functions.

O(2) modulates large-conductance Ca(2+)-dependent K(+) channels of rat chemoreceptor cells by a membrane-restricted and CO-sensitive mechanism.

Hypoxic inhibition of large-conductance Ca(2+)-dependent K(+) channels (maxiK) of rat carotid body type I cells is a well-established fact. However, the molecular mechanisms of such inhibition and the role of these channels in the process of hypoxic transduction remain unclear. We have examined the mechanisms of interaction of O(2) with maxiK channels exploring the effect of hypoxia on maxiK currents recorded with the whole-cell and the inside-out configuration of the patch-clamp technique. Hypoxia inhibits channel activity both in whole-cell and in excised membrane patches. This effect is strongly voltage- and Ca(2+)-dependent, being maximal at low [Ca(2+)] and low membrane potential. The analysis of single-channel kinetics reveals a gating scheme comprising three open and five closed states. Hypoxia inhibits channel activity increasing the time the channel spends in the longest closed states, an effect that could be explained by a decrease in the Ca(2+) sensitivity of those closed states. Reducing maxiK channels with dithiothreitol (DTT) increases channel open probability, whereas oxidizing the channels with 2,2'-dithiopyridine (DTDP) has the opposite effect. These results suggest that hypoxic inhibition is not related with a reduction of channel thiol groups. However, CO, a competitive inhibitor of O(2) binding to hemoproteins, fully reverts hypoxic inhibition, both at the whole-cell and the single-channel level. We conclude that O(2) interaction with maxiK channels does not require cytoplasmic mediators. Such interaction could be mediated by a membrane hemoprotein that, as an O(2) sensor, would modulate channel activity.

A TREK-1-like potassium channel in atrial cells inhibited by beta-adrenergic stimulation and activated by volatile anesthetics.

Many members of the two-pore-domain potassium (K(+)) channel family have been detected in the mammalian heart but the endogenous correlates of these channels still have to be identified. We investigated whether I(KAA), a background K(+) current activated by negative pressure (stretch) and by arachidonic acid (AA) and sensitive to intracellular acidification, could be the native correlate of TREK-1 in adult rat atrial cells. Using the inside-out configuration of the patch-clamp technique, we found that I(KAA), like TREK-1, was outwardly rectifying in physiological K(+) conditions, with a conductance of 41 pS at +50 mV. Like TREK-1, I(KAA) was reversibly activated by clinical concentrations of volatile anesthetics (in mmol/L, chloroform 0.18, halothane 0.11, and isoflurane 0.69). In cell-attached experiments, I(KAA) was inhibited by chlorophenylthio-cAMP (500 micromol/L) and also by stimulation of beta-adrenergic receptors with isoproterenol (1 micromol/L). In addition, TREK-1 mRNAs were detected in all cardiac tissues, and the TREK-1 protein was immunolocalized in isolated atrial myocytes. Such a background potassium channel might contribute to the positive inotropic effects produced by beta-adrenergic stimulation of the heart. It might also be involved in the regulation of the atrial natriuretic peptide secretion.

Inhibition of large-conductance calcium-activated potassium channel by 2-methoxyestradiol in cultured vascular endothelial (HUV-EC-C) cells.

2-Methoxyestradiol, an endogenous metabolite of 17beta-estradiol, is known to have antitumor and antiangiogenic actions. The effects of 2-methoxyestradiol on ionic currents were investigated in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein. In the whole-cell patch-clamp configuration, 2-methoxyestradiol (0.3-30 microm) reversibly suppressed the amplitude of K+ outward currents. The IC50 value of the 2-methoxyestradiol-induced decrease in outward current was 3 microm. Evans blue (30 microm) or niflumic acid (30 microm), but not diazoxide (30 microm), reversed the 2-methoxyestradiol-induced decrease in outward current. In the inside-out configuration, application of 2-methoxyestradiol (3 microm) to the bath did not modify the single-channel conductance of large-conductance Ca2+-activated K+ (BKCa) channels; however, it did suppress the channel activity. 2-Methoxyestradiol (3 microm) produced a shift in the activation curve of BKCa channels to more positive potentials. Kinetic studies showed that the 2-methoxyestradiol-induced inhibition of BKCa channels is primarily mediated by a decrease in the number of long-lived openings. 2-Methoxyestradiol-induced inhibition of the channel activity was potentiated by membrane stretch. In contrast, neither 17beta-estradiol (10 microm) nor estriol (10 microm) affected BKCa channel activity, whereas 2-hydroxyestradiol (10 microm) slightly suppressed it. Under current-clamp condition, 2-methoxyestradiol (10 microm) caused membrane depolarization and Evans blue (30 microm) reversed 2-methoxyestradiol-induced depolarization. The present study provides evidence that 2-methoxyestradiol can suppress the activity of BKCa channels in endothelial cells. These effects of 2-methoxyestradiol on ionic currents may contribute to its effects on functional activity of endothelial cells.

Anomalous mole-fraction effects in recombinant and native cyclic nucleotide-gated channels in rat olfactory receptor neurons.

Anomalous mole-fraction effects (AMFE) were studied, using the inside-out configuration of the patchclamp technique, in both recombinant wild-type alpha-homomeric rat olfactory adenosine 3',5'-cyclic monophosphate (cAMP)-gated channels (rOCNC1) expressed in human embryonic kidney cells (HEK 293) and native cyclic nucleotide-gated (CNG) channels in acutely isolated rat olfactory receptor neurons. Single-channel and macroscopic currents were activated by 200 microM and 500 microM cAMP, respectively. Macroscopic currents, measured with mixtures of Na(+)-NH(4)(+) or Cs(+)-Li(+) in the cytoplasmic bathing solution, displayed AMFE in the rOCNC1 channels at both positive and negative membrane potentials. The rOCNC1 single-channel conductance showed a distinct minimum (or maximum) in an 80% Na(+)-20% NH(4)(+) mixture (or a 60% Cs(+)-40% Li(+) mixture), but only at positive membrane potentials. Macroscopic measurements in native olfactory CNG channels with mixtures of Na(+)-NH(4)(+) indicated similar AMFE. These results suggest that both native CNG channels and recombinant alpha-homomeric channels allow several ions to be present simultaneously within the channel pore. They also further validate the dominant role of the alpha-subunit in permeation through these channels, provide the first evidence to suggest that rOCNC1 channels have multi-ion properties and further justify the use of the rOCNC1 channel as an effective model for structure-function studies of ion permeation and selectivity in olfactory CNG channels.

The effects of flecainide on ATP-sensitive K(+) channels in pig urethral myocytes.

The effects of the antiarrhythmic drug flecainide on levcromakalim-induced hyperpolarization, macroscopic and unitary K(+) currents in pig urethra were investigated using patch-clamp techniques. The effects of flecainide were also examined on currents in inside-out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K(+) channel (Kir6.2) subunits (i.e. Kir6.2DeltaC36) which form ATP-sensitive K(+) channels (K(ATP) channels). In current-clamp mode, application of flecainide (> or =100 microM) caused a significant depolarization after the membrane potential had been hyperpolarized by levcromakalim. In voltage-clamp experiments, the levcromakalim-induced outward current was suppressed by 300 microM flecainide in quasi-physiological K(+) conditions (K(i)=51 microM). In contrast, approximately 20% of the levcromakalim-induced inward current still remained even after application of 300 microM flecainide in symmetrical 140 mM K(+) conditions (K(i)=51 microM). In contrast, approximately 20% of the levcromakalim-induced inwar=126 microM). In cell-attached configuration, the channel activity of the levcromakalim-induced K(ATP) channels was reversibly inhibited by flecainide (> or =30 microM) at -50 mV. Their activity was also suppressed by either disopyramide or cibenzoline. Flecainide reversibly inhibited the channel activity of Kir6.2DeltaC36 expressed in COS7 cells using inside-out configuration. Inhibitory effects of flecainide on the levcromakalim-induced currents became more potent when the value of external pH increased, although this slightly reduced the proportion of drug molecules carrying a positive charge. These results suggest that flecainide inhibits channel activity through blocking the pore site of the K(ATP) channel in pig urethra.

Diphenylamine-2-carboxylic acid (DPC), Usually an inhibitor of Cl- and non-selective cation channels, inhibits Cl-/HCO3- exchange and opens Cl- and cation conductances in rabbit gallbladder epithelium.

In the apical plasma membrane of rabbit gallbladder epithelium various drugs (hydrochlorothiazide, phlorizin, phenylglyoxal) inhibit Cl-/HCO3- exchange and probably enhance the almost negligible intrinsic anion conductance of the exchanger. By radiochemical measurements of apical Cl- influx, the anion exchange is shown here to be directly and immediately inhibited by diphenylamine-2-carboxylic acid (DPC) too. Using conventional microelectrode techniques in intact tissue, DPC, with same dose/response curve, is shown to activate an apical anion conductance (GCl) that has similar properties and amplitude to the GCl activated by the other exchange inhibitors so far tested; the actions are not additive. Patch-clamp methods (cell-attached and excised inside-out patch configurations) reveal that GCl is due to anion channels that are non-rectifying, cytoplasm independent, sensitive to stilbene and dipyridamole and have conductance of a few picosiemens. All this strengthens the correlation between inhibition of anion exchange and the activation of GCl and channels with features similar to those of the almost negligible intrinsic anion conductance of the exchanger. Among the drugs tested, the effects of DPC and hydrochlorothiazide are even more similar, such that even their dose/response curves overlap. Moreover, both drugs also directly activate some verapamil-sensitive Ca2+ channels and consequently apamin-sensitive, Ca2+-activated K+ channels. Thus DPC, usually an inhibitor of Cl- and non-selective cation channels, is shown here to be capable of activating Cl- and cation conductances.

Dual action of ZD6169, a novel K+ channel opener, on ATP‐sensitive K+ channels in pig urethral myocytes

1. The effects of ZD6169, a novel K(+) channel opener, on both membrane and unitary currents in pig urethra were investigated using patch-clamp techniques. Its effect was also examined on currents in inside-out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K(+) channel (Kir6.2) subunits (Kir6.2C36) which form ATP-sensitive K(+) channels (K(ATP) channels). 2. In current-clamp mode, ZD6169 (< or = 10 microM) induced a concentration-dependent membrane hyperpolarization. Higher concentrations (> or = 30 microM) caused a transient membrane hyperpolarization, followed by a gradual membrane depolarization. On removal of ZD6169, an after hyperpolarization was observed. 3. In conventional voltage-clamp configuration, at -50 mV in symmetrical 140 mM K(+) conditions, ZD6169 (100 microM) caused a transient inward current which gradually decayed. Removal of ZD6169 evoked a much larger amplitude K(+) current with a similar time course. 4. ZD6169 produced an inward glibenclamide-sensitive K(+) current, demonstrating a bell-shaped concentration-response relationship. 5. In cell-attached configuration in symmetrical 140 mM K(+) conditions, ZD6169 (< or = 30 microM) activated an K(ATP) channel which was reversibly suppressed by application of glibenclamide. In contrast, ZD6169 (100 microM) inhibited the activity of the levcromakalim-induced K(ATP) channels. 6. ZD6169 (100 microM) had no significant effect on the channel activity of Kir6.2C36 in inside-out configuration, although cibenzoline greatly suppressed the channel activity. 7. These results demonstrate that ZD6169 possesses a dual effect on the activity of the K(ATP) channel; activating at low concentration and inhibiting at higher concentration.

Toosendanin-induced inhibition of small-conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus

The effect of toosendanin (TSN) on small-conductance calcium-activated potassium channels (SK Ca) in pyramidal neurons of rat hippocampal CA1 region was observed using the inside-out configuration of patch-clamp technique. The results showed that TSN (1.7∼170 μM) inhibited the SK Ca activity by reducing the open probability and open frequency significantly in a concentration-dependent manner, and the effects were partially reversible. Elevating Ca 2+ concentration at the intracellular side of the patch ([Ca 2+] i ) from 1 to 10 μM decreased the inhibitory efficacy. Analysis of the channel kinetics indicated that TSN increased the slow closed time constant significantly, while open time and unitary conductance of channel did not change. These data provide a further explanation for TSN-induced facilitation of neurotransmitter release and antibotulismic effects of the drug.

Vinpocetine-induced stimulation of calcium-activated potassium currents in rat pituitary GH 3 cells

The effects of vinpocetine, an inhibitor of cyclic GMP phosphodiesterase, on ionic currents were examined in rat pituitary GH 3 lactotrophs with the aid of the patch-clamp technique. In GH 3 cells bathed in normal Tyrode’s solution, vinpocetine (10 μM) reversibly increased the amplitude of Ca 2+-activated K + current ( I K(Ca)) with an ec 50 value of 4 μM. When the recording pipettes were filled with 10 mM EGTA, vinpocetine also stimulated I K(Ca). In the cell-attached configuration, application of vinpocetine to the bath increased the activity of large-conductance Ca 2+-activated K + (BK Ca) channels. In excised membrane patches, application of vinpocetine (10 μM) to the bath did not change the single-channel conductance of BK Ca channels; however, it did increase channel activity. In the inside-out configuration, neither 8-bromo cyclic GMP nor YC-1 applied intracellularly affected BK Ca channel activity. The vinpocetine-induced change in the kinetic behavior of BK Ca channels was due to an increase in mean open time and a decrease in mean closed time. Vinpocetine (10 μM) caused a leftward shift in the midpoint for the voltage-dependent opening. Under the current-clamp mode, vinpocetine (10 μM) decreased the firing rate of spontaneous action potentials induced by thyrotropin-releasing hormone (10 μM) in GH 3 cells. In pheochromocytoma PC12 cells, vinpocetine (10 μM) applied intracellularly also enhanced the activity of BK Ca channels without altering single-channel conductance. Thus, the present study suggests that vinpocetine-mediated stimulation of I K(Ca) may result from the direct activation of BK Ca channels and indirectly from elevated cytosolic Ca 2+.

Diadenosine tetraphosphate-gating of recombinant pancreatic ATP-sensitive K(+) channels.

Diadenosine tetraphosphate (Ap4A) has been recently discovered in the pancreatic beta cells where targets ATP-sensitive K(+) (K(ATP)) channels, depolarizes the cell membrane and induces insulin secretion. However, whether Ap4A inhibit pancreatic K(ATP) channels by targeting protein channel complex itself was unknown. Therefore, we coexpressed pancreatic K(ATP) channel subunits, Kir6.2 and SUR1, in COS-7 cells and examined the effect of Ap4A on the single channel behavior using the inside-out configuration of the patch-clamp technique. Ap4A inhibited channel opening in a concentration-dependent manner. Analysis of single channels demonstrated that Ap4A did not change intraburst kinetic behavior of K(ATP) channels, but rather decreased burst duration and increased between-burst duration. It is concluded that Ap4A antagonizes K(ATP) channel opening by targeting channel subunits themselves and by keeping channels longer in closed interburst states.

Inhibitors of the Cl-/HCO3- exchanger activate an anion channel with similar features in the epithelial cells of rabbit gallbladder: patch-clamp analysis.

The stilbene- and dipyridamole-sensitive Cl-conductance (GCl), non-additively activated by some inhibitors of the Cl-/HCO3- exchanger (hydrochlorothiazide, phlorizin, phenylglyoxal) after the exchanger inhibition in the apical plasma membrane of rabbit gallbladder epithelium, has been investigated by patch-clamp technique with cell-attached and inside-out configurations. No Cl- channels were observed under basal conditions or after treatment with 2.5 x 10(-4) mol/l 8-Br-cAMP or hydrochlorothiazide (HCTZ) on the cytosolic side. Conversely, with 2.5 x 10(-4) mol/l HCTZ or 2 mmol/l phlorizin in the pipette, a non-rectifying Cl- channel with about 5 pS conductance and 0.3-0.4 voltage-independent open probability was observed; it was inhibited by 10(-4) - 5 x 10(-4) mol/l SITS, 10(-4) mol/l furosemide or 0.6 x 10(-4) mol/l dipyridamole; the effects of HCTZ and phlorizin were not additive. Open probability increased from 0 (after seal formation) to a maximum of 0.3-0.4 reached in 7-9 min. Similar results were obtained with both configurations. With the cell-attached configuration, HCTZ added to the bath did not activate Cl- channels in the patch. The channel was shown to exclude cations, to be selective for Cl-, but also conductive for gluconate (PGluc/PCl = 0.18). On this basis, it is concluded that: (1) GCl, activated either by HCTZ or phlorizin, has the same underlying anion channels, (2) the channels can be activated only on the external side of the membrane, also in the absence of cytoplasm, without cellular mediations or effects at a distance along the membrane, (3) the channels are inhibited by the same drugs which inhibit the very small intrinsic anion conductance of the exchanger, (4) they are either related to a slow conversion of inhibited exchangers into channels or, less probably, they are parallel to the exchanger and slowly activated by intra-membrane (or membrane-bound) mediators in their turn activated by near, inhibited exchangers.

Diversity of K&lt;sup&gt;+&lt;/sup&gt; channels in circular smooth muscle of opossum lower esophageal sphincter

We previously demonstrated that a balance of K+ and Ca2+-activated Cl- channel activity maintained the basal tone of circular smooth muscle of opossum lower esophageal sphincter (LES). In the current studies, the contribution of major K+ channels to the LES basal tone was investigated in circular smooth muscle of opossum LES in vitro. K+ channel activity was recorded in dispersed single cells at room temperature using patch-clamp recordings. Whole-cell patch-clamp recordings displayed an outward current beginning to activate at -60 mV by step test pulses lasting 400 ms (-120 mV to +100 mV) with increments of 20 mV from holding potential of -80 mV ([K+]I = 150 mM, [K+]o = 2.5 mM). However, no inward rectification was observed. The outward current peaked within 50 ms and showed little or no inactivation. It was significantly decreased by bath application of nifedipine, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and iberiotoxin (IBTN). Further combination of TEA with 4-AP, nifedipine with 4-AP, and IBTN with TEA, or vice versa, blocked more than 90% of the outward current. Ca2+-sensitive single channels were recorded at asymetrical K+ gradients in cell-attached patch-clamp configurations (100.8+/-3.2 pS, n = 8). Open probability of the single channels recorded in inside-out patch-clamp configurations were greatly decreased by bath application of IBTN (100 nM) (Vh = -14.4+/-4.8 mV in control vs. 27.3+/-0.1 mV, n = 3, P < 0.05). These data suggest that large conductance Ca2+-activated K+ and delayed rectifier K+ channels contribute to the membrane potential, and thereby regulate the basal tone of opossum LES circular smooth muscle.

Characterization of Inhibition by Risperidone of the Inwardly Rectifying K+ Current in Pituitary GH3 Cells

The effects of risperidone on ionic currents in rat pituitary GH3 cells were investigated with the aid of the patch-clamp technique. Hyperpolarization-activated K+ currents in GH3 cells bathed in high-K+ Ca2+-free solution were studied to determine the effect of risperidone and other related compounds on the inwardly rectifying K+ current (IK(IR)). Risperidone (0.1–10 μM) suppressed the amplitude of IK(IR) in a concentration-dependent manner. The IC50 value for the risperidone-induced inhibition of IK(IR) was 1 μM. Risperidone (3 μM) was found to slow the rate of activation. An increase in current deactivation by the presence of risperidone was also observed. Haloperidol (10 μM) and thioridazine (10 μM) inhibited the amplitude of IK(IR) effectively, and clozapine slightly suppressed it; however, metoclopramide (10 μM) had no effect on it. Risperidone (10 μM) had no effect on voltage-dependent K+ and L-type Ca2+ currents. However, in the inside-out configuration, risperidone (10 μM) did not alter the single-channel conductance, but reduced the activity of large-conductance Ca2+-activated K+ (BKCa) channels. Under the current-clamp mode, risperidone (3 μM) depolarized the membrane potential and increased the firing rate. With the aid of the spectral analysis, cells that exhibited an irregular firing pattern were also converted to those displaying a regular firing pattern after addition of risperidone (3 μM). The present study provides evidence that risperidone, in addition to the blockade of dopamine receptors, can produce a depressant effect on IK(IR) and BKCa channels, and implies that the blockade of these ionic currents by risperidone may affect membrane excitability and prolactin secretion in GH3 cells.

Characterization of ATP-Sensitive Potassium Channels Functionally Expressed in Pituitary GH 3 Cells

ATP-sensitive K+ (KATP) channels have been characterized in pituitary GH3 cells with the aid of the patch-clamp technique. In the cell-attached configuration, the presence of diazoxide (100 microM) revealed the presence of glibenclamide-sensitive KATP channel exhibiting a unitary conductance of 74 pS. Metabolic inhibition induced by 2,4-dinitrophenol (1 mM) or sodium cyanide (300 microM) increased KATP channel activity, while nicorandil (100 microM) had no effect on it. In the inside-out configuration, Mg-ATP applied intracellularly suppressed the activity of KATP channels in a concentration-dependent manner with an IC50 value of 30 microM. The activation of phospholipase A2 caused by mellitin (1 microM) was found to enhance KATP channel activity and further application of aristolochic acid (30 microM) reduced the mellitin-induced increase in channel activity. The challenging of cells with 4,4'-dithiodipyridine (100 microM) also induced KATP channel activity. Diazoxide, mellitin and 4,4'-dithiodipyridine activated the KATP channels that exhibited similar channel-opening kinetics. In addition, under current-clamp conditions, the application of diazoxide (100 microM) hyperpolarized the membrane potential and reduced the firing rate of spontaneous action potentials. The present study clearly indicates that KATP channels similar to those seen in pancreatic beta cells are functionally expressed in GH3 cells. In addition to the presence of Ca(2+)-activated K+ channels, KATP channels found in these cells could thus play an important role in controlling hormonal release by regulating the membrane potential.

The role of anion and cation channels in volume regulatory responses in trout red blood cells

(1) An outwardly rectifying chloride channel (ORCC) of large conductance has been detected under isotonic conditions (320 mosM l −1) in the plasma membrane of trout red blood cells (RBCs) using the excised inside-out configuration. The channel, with a permeability ratio P Cl/ P cation of 12, was inhibited by the Cl − channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) (50 μM), and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) (100 μM) in the bathing solution. (2) In hypotonic conditions (215 mosM l −1), 44% of cell-attached patches showed spontaneous single channel activity identified as nonselective cationic (NSC) channels. A second group, corresponding to 7% of cell-attached patches, showed spontaneous activity corresponding to a channel type presenting outward rectification and anionic selectivity. Finally, 49% of patches displayed a complex spontaneous signal corresponding to the superimposition of inward and outward currents probably due to activation of different channel types. (3) Giga-seals obtained without suction in intact cells under isotonic conditions possessed NSC channels that were quiescent but which could be activated either by mechanical deformation of cell membrane or by hypotonic cell swelling. (4) Hypotonically swollen RBCs exhibited regulatory volume decrease (RVD) over 3 h, which was linked to a fivefold to sixfold increase in unidirectional fluxes of K +, a net loss of intracellular K + and net gain of extracellular Na +. RVD and the hypotonically activated, unidirectional K + influx continued after replacement of Cl − by methylsulfonate (MeSF) albeit more slowly. (5) The NSC channel inhibitor, barium, and the Cl − channel inhibitor, NPPB, both inhibited the RVD response by ∼50% in Cl − containing saline. When Cl − was replaced by MeSF, the inhibition was >90% suggesting that NSC channels and ORCC play key roles in the chloride-independent component of RVD.

Enhancement in activities of large conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia

It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization (fAHP) increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance Ca 2+-activated potassium (BK Ca) channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by using inside-out configuration of patch clamp techniques. (1) The unitary conductance of BK Ca channels in post-ischemic neurons (295 pS) was higher than that in control neurons (245 pS) in symmetrical 140/140 mM K + in inside-out patch; (2) the membrane depolarization for an e-fold increase in open probability ( P o) showed no significant differences between two groups while the membrane potential required to produce one-half of the maximum P o was more negative after ischemia, indicating no obvious changes in channel voltage dependence; (3) the [Ca 2+] i required to half activate BK Ca channels was only 1 μM in post-ischemic whereas 2 μM in control neurons, indicating an increase in [Ca 2+] i sensitivity after ischemia; and (4) BK Ca channels had a longer open time and a shorter closed time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BK Ca channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal excitability and increase in fAHP.

Potassium channels in primary cultures of seawater fish gill cells. I. Stretch-activated K(+) channels.

Previous studies using the patch-clamp technique demonstrated the presence of a small conductance Cl(-) channel in the apical membrane of respiratory gill cells in primary culture originating from sea bass Dicentrarchus labrax. We used the same technique here to characterize potassium channels in this model. A K(+) channel of 123 +/- 3 pS was identified in the cell-attached configuration with 140 mM KCl in the bath and in the pipette. The activity of the channel declined rapidly with time and could be restored by the application of a negative pressure to the pipette (suction) or by substitution of the bath solution with a hypotonic solution (cell swelling). In the excised patch inside-out configuration, ionic substitution demonstrated a high selectivity of this channel for K(+) over Na(+) and Ca(2+). The mechanosensitivity of this channel to membrane stretching via suction was also observed in this configuration. Pharmacological studies demonstrated that this channel was inhibited by barium (5 mM), quinidine (500 microM), and gadolinium (500 microM). Channel activity decreased when cytoplasmic pH was decreased from 7.7 to 6.8. The effect of membrane distension by suction and exposure to hypotonic solutions on K(+) channel activity is consistent with the hypothesis that stretch-activated K(+) channels could mediate an increase in K(+) conductance during cell swelling.