Inside-out Patch Recordings Research Articles

Giant excised patch recordings of recombinant ion channel currents expressed in mammalian cells

The giant excised patch variant of patch clamp recording combines microsecond time resolution of macroscopic currents with rapid exchange of the experimental solutions at the intracellular membrane surface. This technique has been applied to a limited number of cell types, including Xenopus oocytes, muscle cells, and photoreceptors. We have applied this technique to recording recombinant ion channel currents expressed in membrane patches excised from HEK293 cell lines. Giant inside-out patch recordings of Na + channels and SK Ca type calcium-activated potassium channels show high temporal resolution and excellent signal to noise characteristics. This technique will facilitate the study of recombinant ion channels expressed in mammalian cells.

State-dependent modification of ATP-sensitive K+ channels by phosphatidylinositol 4,5-bisphosphate.

With inside-out patch recordings in ventricular myocytes from the hearts of guinea pigs, we studied ATP-sensitive K+ (K(ATP)) channels activated by phosphatidylinositol 4,5-bisphosphate (PIP2) with respect to sensitivity to ATP when in either a rundown state (RS) or a non-rundown state (NRS). Rundown of K(ATP) channels was induced by exposure either to ATP-free solution or to ATP-free solution containing 19 microM Ca2+. Exposure of membrane patches to 10 microM PIP2 reactivated channels with both types of rundown. The reactivation by PIP2 did not require ATP in the bath. The IC50 of channels recovered from RS and before the rundown was 37.1 and 31.1 microM, respectively. PIP2 irreversibly increased the mean current when the channel was in the NRS. This was associated with a shift of IC50 to 250.6 microM after PIP2 exposure. PIP2 activates NRS K(ATP) channels by decreasing their sensitivity to ATP, whereas PIP2 reactivates RS-K(ATP) channels independently of ATP without changing ATP sensitivity.

Voltage-dependent K + currents in spiral prominence epithelial cells of rat cochlea

It has been suggested that spiral prominence is associated with ion transport, but the characterization of ion channels has not been explored so far. We studied the electrical properties and ion conductances of the spiral prominence epithelial cells (SPECs), which are epithelial cells covering the luminal side of spiral prominence, in the upper turn of neonatal rat cochlea using a whole-cell variant patch clamp technique. The cell capacitance was 16.3±2.1 pF ( n=33) and the resting membrane potential was −68.9±2.5 mV ( n=14) in perilymph-like bath solution. It was found that those SPECs possess a large voltage-activated, outwardly rectifying K + current and a small inwardly rectifying K + current. The outward K + current was activated by depolarizing pulses more positive than −30 mV, and sensitive to tetraethylammonium chloride (20 mM), 4-aminopyridine (10 mM), but not to Ba 2+ (0.5 mM). Tail current analysis revealed that it was primarily K +-selective. The time course of activation was well fitted by an exponential function raised to second power. The small inwardly rectifying K + current was sensitive to Ba 2+ (0.5 mM), and the Ba 2+-sensitive current was K +-selective. In cell-attached or inside-out patch recordings, no discernible K + channel currents were found in the apical membrane of SPECs. Based on these results, we conclude that SPECs have two types of voltage-dependent K + currents, which are most likely located in the basolateral membrane.

Large conductance Ca(2+)-activated K(+) channels in human meningioma cells.

Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures. In whole cells, depolarization to voltages higher than +80 mV evoked a large K(+) outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC(50) = 5.3 mm). Raising the internal Ca(2+) from 10 nm to 2 mm shifted the voltage of half-maximum activation (V(1/2)) of the K(+) current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca(2+)-activated (BK(Ca)) K(+) channel with a conductance of 296 pS (130 mm K(+) at both sides of the patch). V(1/2) of single-channel currents was +6, -12, -46, and -68 mV in the presence of 1, 10, 100, and 1000 microm Ca(2+), respectively, at the internal face of the patch. In cell-attached patches the open probability (P(o)) of BK(Ca) channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K(+) currents with 10 nm Ca(2+) in the pipette. Application of 20 microm cytochalasin D increased P(o) of BK(Ca) channels in cell-attached patches within minutes. These data suggest that the activation of BK(Ca) channels in meningioma cells does not only depend on voltage and internal Ca(2+) but is also controlled by the cytoskeleton.

Nonselective cation and BK channels in apical membrane of outer sulcus epithelial cells.

The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K(Ca)) channel and (iii) a small K(+) channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear current-voltage (I-V) relationship (27 pS) and was equally conductive for Na(+) and K(+), but not permeable to Cl(-) or N-methyl-d-glucamine. Channel activity required the presence of Ca(2+) at the cytosolic face, but was detected at Ca(2+) concentrations as low as 10(-7) m (open probability (P(o)) = 0.11 +/- 0.03, n = 8). Gadolinium decreased P(o) of the NSC channel from both the external and cytosolic side (IC(50) approximately 0.6 microm). NSC currents were decreased by amiloride (10 microm - 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K(+) could be fitted to the I-V relationship in asymmetrical K(+) and Na(+) solutions. The channel was impermeable to Cl(-) and N-methyl-d-glucamine. P(o) of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca(2+). TEA (20 mm), charybdotoxin (100 nm) and Ba(2+) (1 mm) but not amiloride (1 mm) reduced P(o) from the extracellular side. In contrast, external flufenamic acid (100 microm) increased P(o) and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward current. We conclude that the NSC channel is Ca(2+) activated, voltage-insensitive and involved in both constitutive K(+) and Na(+) reabsorption from endolymph while the BK channel might participate in the K(+) pathway under stimulated conditions that produce an elevated intracellular Ca(2+) or depolarized membrane potential.

Estrogen modulates a large conductance chloride channel in cultured porcine aortic endothelial cells.

Estrogen is known to exert a protective effect on cardiovascular disease, but the mechanism for this effect is unclear. It has, however, been reported that estrogen and antiestrogen modify ionic currents and membrane potential in various cells. The aim of this study was to clarify whether the chloride channel of aortic endothelial cells was, in fact, modified by estrogen and antiestrogen with inside-out patch and cell-attached patch recording methods. Tamoxifen activated a large-conductance (368 +/- 23 pS, n = 6, in symmetric 150 mM Cl- solution) chloride channel of endothelial cells grown in the presence of 1 microg/ml colchicine. The channels were activated mainly between +/-40 mV, but were inactivated at more extreme potentials. The open probability of channels in cell-attached patches increased from <0.01 to 0.37 +/- 0.08 (n = 8) when cells were treated with 15 microM tamoxifen. This effect can be blocked by 17beta-estradiol, but not by progesterone. The results showed that tamoxifen increased chloride channel activity in the presence of colchicine in cultured endothelial cells, and this action was suppressed by 17beta-estradiol but not by progesterone. This rapid effect by estrogens suggests that these hormones exert nongenomic, short-term activity and do not appear to affect the nuclear estrogen receptor. With these effects, estrogen and antiestrogen bind to the endothelial cells plasma membrane site and subsequently may activate an intracellular second messenger pathway.

Inactivation of large-conductance, calcium-activated potassium channels in rat cortical neurons

Inside-out patch recordings from rat acutely dissociated cerebral cortical neurons revealed time and voltage-dependent activity of a large-conductance calcium-activated potassium channel. Channel activity inactivated within minutes following a depolarizing voltage step, and was recovered from inactivation by membrane hyperpolarization. Inactivation rate was not influenced by internal calcium or membrane voltage; however, reducing channel activity with intracellular calcium destabilized inactivation. Channel inactivation was abolished by intracellular trypsin treatment, suggesting that an associated inactivating particle was responsible for inactivation. Application of alkaline phosphatase to the internal aspect of the patch membrane increased channel activity and abolished channel inactivation, without affecting its voltage and calcium dependence. Internal application of Mg-ATP, but not Mg-5′-adenylylamidodiphosphate, retarded recovery of channel activity from inactivation, whereas internal application of protein phosphatase-1α enhanced recovery from inactivation. The abolition of channel inactivation by alkaline phosphatase was prevented by prior internal tetraethylammonium treatment, indicating that the alkaline phosphatase site is closely associated with the channel pore. These results demonstrate that cortical large-conductance calcium-activated potassium channel inactivation is probably mediated by an endogenous, trypsin-sensitive, inactivation particle. This particle appears to inactivate the open channel and requires a critical phosphate group for stable block. The slow time-course of channel inactivation may have some pathophysiological significance in maintenance of epileptiform activity.

Reduced glutathione inhibits beta-NAD+-activated non-selective cation currents in the CRI-G1 rat insulin-secreting cell line.

1. Whole-cell voltage-clamp recordings were used to study the characteristics of a non-selective cation current, activated by intracellular beta-NAD+, present in CRI-G1 insulin-secreting cells. The monovalent cations Na+, K+ and Cs+ were equally permeant through this channel. 2. The magnitude of the beta-NAD+ current was dependent on the concentration of both beta-NAD+ and Ca2+ in the cell. The properties of the beta-NAD+-activated macroscopic current are similar to those of the beta-NAD+-activated non-selective cation channel (NSNAD) examined at the single channel level in this cell line. 3. The presence of intracellular reduced glutathione (GSH) inhibited the beta-NAD+-activated macroscopic current and the activity of the NSNAD channel in inside-out patch recordings. 4. The inhibition of beta-NAD+-activated currents by GSH is mimicked by its analogue ophthalmic acid but not by another thiol reducing agent dithiothreitol, indicating the presence of a specific GSH binding site present on the NSNAD channel or associated protein.

Dual actions of the metabolic inhibitor, sodium azide on K(ATP) channel currents in the rat CRI-G1 insulinoma cell line.

1. The effects of various inhibitors of the mitochondrial electron transport chain on the activity of ATP-sensitive K+ channels were examined in the Cambridge rat insulinoma G1 (CRI-G1) cell line using a combination of whole cell and single channel recording techniques. 2. Whole cell current clamp recordings, with 5 mM ATP in the pipette, demonstrate that the mitochondrial uncoupler sodium azide (3 mM) rapidly hyperpolarizes CRI-G1 cells with a concomitant increase in K+ conductance. This is due to activation of K(ATP) channels as the sulphonylurea tolbutamide (100 microM) completely reversed the actions of azide. Other inhibitors of the mitochondrial electron transport chain, rotenone (10 microM) or oligomycin (2 microM) did not hyperpolarize CRI-G1 cells or increase K+ conductance. 3. In cell-attached recordings, bath application of 3 mM sodium azide (in the absence of glucose) resulted in a rapid increase in K(ATP) channel activity, an action readily reversible by tolbutamide (100 microM). Application of sodium azide (3 mM), in the presence of Mg-ATP, to the intracellular surface of excised inside-out patches also increased K(ATP) channel activity, in a reversible manner. 4. In contrast, rotenone (10 microM) or oligomycin (2 microM) did not increase K(ATP) channel activity in either cell-attached, in the absence of glucose, or inside-out membrane patch recordings. 5. Addition of sodium azide (3 mM) to the intracellular surface of inside-out membrane patches in the presence of Mg-free ATP or the non-hydrolysable analogue 5'-adenylylimidodiphosphate (AMP-PNP) inhibited, rather than increased, K(ATP) channel activity. 6. In conclusion, sodium azide, but not rotenone or oligomycin, directly activates K(ATP) channels in CRI-G1 insulin secreting cells. This action of azide is similar to that reported previously for diazoxide.

Tandospirone-induced K+ current in acutely dissociated rat dorsal raphe neurones.

1. The effects of tandospirone (TDS) on dissociated rat dorsal raphe neurones were investigated using the patch-clamp method. 2. Under current-clamp conditions, TDS hyperpolarized the cell membrane, resulting in the reduction of firing rates. 3. Under voltage-clamp conditions, TDS induced an inward rectifying K+ current in a concentration-dependent manner. 4. The TDS-induced K+ currents (I(TDS)) were mimicked by 8-OH-DPAT, a 5-HT1A agonist. The I(TDS) was blocked by spiperone, a 5-HT1A receptor antagonist, in a concentration-dependent manner. 5. N-Ethylmaleimide, an agent which uncouples between the receptor and the G-protein, irreversibly blocked the I(TDS). 6. In neurones perfused intracellularly with a pipette-solution containing GTP using the conventional whole-cell patch recording, the I(TDS) showed a gradual rundown. When the neurones were perfused with GTPgammaS, TDS activated the inwardly rectifying K+ current in an irreversible manner. 7. In the inside-out patch recording mode, TDS-activated single K+ channel currents (i(TDS)) which also showed an inward rectification. When the GDP in cytosolic side was completely replaced with GTP, the open probability of i(TDS) significantly increased. 8. These results indicate that the activation of 5-HT1A receptors by TDS directly opens the inward rectifying K+ channels via a G-protein mediated process.

Effect of nitric oxide on calcium-activated potassium channels in colonic smooth muscle of rabbits.

Nitric oxide (NO) hyperpolarizes intestinal smooth muscle cells. This study was designed to determine the mechanism whereby NO activates KCa channels of circular smooth muscle of the rabbit colon. Transmural biopsies of the rabbit colon were stained for NADPH-diaphorase. Freshly dispersed circular smooth muscle cells were studied in the whole cell configuration, as well as in on-cell and excised inside-out patch recording configurations, while KCa current and the activity of KCa channels, respectively, were monitored. NADPH-diaphorase-positive nerve fibers were found in both muscle layers. NO (1%) increased whole cell net outward current by 79% and hyperpolarized resting membrane voltage from -59 to -73 mV (n = 8 cells, P < 0.01). In the on-cell patch recording configuration. NO (0.5% or 1%) in the bath increased NPo of KCa channels; charybdotoxin (125 nM) in the pipette solution blocked this effect. In the excised inside-out patch recording configuration, NO (1%) had no effect on NPo of KCa channels. In the on-cell patch recording configuration, methylene blue (1 microM) or cystamine (5 mM) in the bath solution decreased the effect of NO (1%) on NPo of KCa channels. NPo was increased by 8-bromo-cGMP (8-BrcGMP; 1 mM), a cGMP analog, and zaprinast (100 microM), an inhibitor of cGMP phosphodiesterase. These data suggest that NO increased whole cell outward K+ current by activating KCa channels through a cGMP pathway.

Alpha2-Adrenoceptor-mediated potassium currents in acutely dissociated rat locus coeruleus neurones.

1. The noradrenaline (NA)-activated response was investigated in neurones acutely dissociated from the rat locus coeruleus (LC) using nystatin-perforated, conventional whole-cell and inside-out patch recording modes under current- and voltage-clamp conditions. 2. Under current-clamp conditions, NA hyperpolarized the LC neurones, abolishing the spontaneous action potentials. In voltage-clamp studies, NA induced an inwardly rectifying K+ current (INA) in a concentration-dependent manner with a half-maximum effective concentration of 2.2 x 10(-7) M. 3. INA was mimicked by the alpha2-agonist UK14304 but was inhibited by either the alpha2B/alpha2C antagonist ARC239 or the alpha1- and alpha2B/alpha2C antagonist prazosin, suggesting the contribution of alpha2B/alpha2C adrenoceptors. 4. INA was inhibited by the intracellular application of GDPbetaS but fully activated by intracellular perfusion of GTPgammaS. 5. In the inside-out recording mode, the application of GTP to the cytoplasmic side of the patch membrane markedly enhanced the open probability of the NA-activated single channels which represented the inwardly rectifying properties. 6. These results indicate that the activation of alpha2B/alpha2C adrenoceptors coupled with GTP-binding protein directly activates the inwardly rectifying K+ currents in rat LC neurones, thus resulting in a decrease in the spontaneous firing activities.

A method for direct patch-clamp recording from smooth muscle cells embedded in functional brain microvessels.

The aim of this project was to develop a method to enable routine application of all patch-clamp configurations to smooth muscle cells while they remain embedded in blood vessels. Small blood vessels were isolated from rabbit brain using an enzymatic and mechanical procedure. Vessels were identified under a microscope and the majority were small arterioles with a mean external diameter, in Ca2+-containing (1.5 mM) solution, of 29 microm and variable lengths of 100 microm or more. Arterioles excluded trypan blue, constricted in response to 60 mM K+ and dilated in response to levcromakalim. Patch-clamp gigaOhm seals were made regularly on smooth muscle cells embedded in arterioles. The membrane potential recorded using amphotericin-B-containing patch pipettes averaged -72 mV. Short arteriolar segments could be voltage-clamped. Injection of depolarising current or bath application of 10 mM Ba2+ induced constriction of the entire arteriolar segment. Cell-attached patch, inside-out patch and outside-out patch recordings were made readily and K+ channel unitary currents were studied. The method is readily applied and has several advantages over previous methods for the study of ion channels in smooth muscle cells. Notably, avoidance of single-cell isolation means that enzymatic treatment is minimised and cells can be studied within their normal environment of the blood vessel wall.

A vesicle preparation for resolving single-channel currents in tegument of male Schistosoma mansoni.

A tegumental vesicle preparation from adult male Schistosoma mansoni was developed that allows the resolution of single ion-channel currents. Adult male schistosomes were exposed to a low pH (3.75) medium for a period of approximately 30 min at 37 degrees C. During this period smooth vesicles formed from the tegument. Fluorescence microscopy following staining of the tegument with the dye, 5-N-[octadecanoyl]aminofluorescein (AF-18), transmission electron microscopy and scanning electron microscopy revealed that the vesicles were produced from the outer tegumental membrane. The fluorescence studies showed the presence of the double bilayer structure of the outer membrane in > 41% of the vesicles. These studies suggested that the preparation is suitable for single-channel recording with the patch-clamp technique. Cell-attached and isolated inside-out patch recordings of ion-channel activity were obtained with giga-ohm resistance seals. Different types of ion-channel were recorded from tegumental vesicles from male schistosomes, illustrating the potential of the technique. The channels observed included: a non-selective cation channel (360 pS); a K+ channel (with a conductance of 115 pS in high bath-K conditions); and a Cl- selective channel (20 pS). The currents of these ion-channels may cross the double bilayer of the outer tegumental membrane.

Endothelin activates large-conductance K+ channels in rat lactotrophs: reversal by long-term exposure to dopamine agonist.

Endothelin-1 (ET-1) inhibits PRL secretion from cultured rat lactotrophs. However, ET-1 stimulates PRL secretion after cultured lactotrophs have been exposed for 48 h to dopamine or D2 dopamine agonists. In the present study, we have used cell-attached and inside-out patch recordings to establish an ionic basis for these effects. Bath application of 20 nM ET-1 to untreated lactotrophs evoked a robust and persistent activation of large-conductance K+ channels in cell-attached patches. This effect of ET-1 had a long latency to onset, was maintained for as long as ET-1 was present, and required at least 10 min of washing in control saline before complete recovery was achieved. The stimulatory effect of 20 nM ET-1 on these channels was markedly attenuated in the presence of the selective ET(A) receptor antagonist BQ-610 (200 nM), or after pertussis toxin (200 ng/ml, 16 h) pretreatment. The unitary slope conductance of the ET-1 activated channels in cell attached patches was 165 and 95 pS when the recording electrodes contained 150 and 5.4 mM KCl, respectively. These channels were voltage-sensitive and their activity increased upon patch depolarization. Previously activated channels in cell-attached patches became quiescent immediately upon patch excision into Ca2+-free bath saline. Exposure of the intracellular surface to 0.1 microM Ca2+ restored the activity of these channels similar to the level seen before patch excision. In addition, preincubating the cells with the membrane-permeable Ca2+-chelator BAPTA-AM, or using Ca2+-free solution in the recording pipettes, prevented the activation of these channels by ET-1. The ET-1 activated large-conductance Ca2+-dependent K+ (BK(Ca)) channels were blocked by 20 mM tetraethylammonium but were insensitive to the K+ channel blockers apamin (1 microM), charybdotoxin (200 nM), or iberiotoxin (200 nM). Acute application of 10 microM dopamine and 20 nM ET-1 caused activation of BK(Ca) channels with indistinguishable kinetic properties, although the effect of dopamine occurred with shorter latency. After 48-h exposure to the specific D2 dopamine receptor agonist (+/-)-2-(N-phenyl-N-propyl) amino-5-hydroxytetralin hydrochloride (PPHT, 500 nM), bath application of 20 nM ET-1 resulted in inhibition of spontaneously active BK(Ca) channels. These data suggest that both the stimulatory and inhibitory effects of ET-1 on PRL secretion are mediated, at least in part, by actions on BK(Ca) channels, and that long term exposure to dopamine or D2 agonists alters the signaling pathways from the ET(A) receptor to BK(Ca) channels.

Endothelin Activates Large-Conductance K+ Channels in Rat Lactotrophs: Reversal by Long-Term Exposure to Dopamine Agonist

Endothelin-1 (ET-1) inhibits PRL secretion from cultured rat lactotrophs. However, ET-1 stimulates PRL secretion after cultured lactotrophs have been exposed for 48 h to dopamine or D2 dopamine agonists. In the present study, we have used cell-attached and inside-out patch recordings to establish an ionic basis for these effects. Bath application of 20 nm ET-1 to untreated lactotrophs evoked a robust and persistent activation of large-conductance K+ channels in cell-attached patches. This effect of ET-1 had a long latency to onset, was maintained for as long as ET-1 was present, and required at least 10 min of washing in control saline before complete recovery was achieved. The stimulatory effect of 20 nm ET-1 on these channels was markedly attenuated in the presence of the selective ETA receptor antagonist BQ-610 (200 nm), or after pertussis toxin (200 ng/ml, 16 h) pretreatment. The unitary slope conductance of the ET-1 activated channels in cell attached patches was 165 and 95 pS when the recording electrodes contained 150 and 5.4 mm KCl, respectively. These channels were voltage-sensitive and their activity increased upon patch depolarization. Previously activated channels in cell-attached patches became quiescent immediately upon patch excision into Ca2+-free bath saline. Exposure of the intracellular surface to 0.1 μm Ca2+ restored the activity of these channels similar to the level seen before patch excision. In addition, preincubating the cells with the membrane-permeable Ca2+-chelator BAPTA-AM, or using Ca2+-free solution in the recording pipettes, prevented the activation of these channels by ET-1. The ET-1 activated large-conductance Ca2+-dependent K+ (BKCa) channels were blocked by 20 mm tetraethylammonium but were insensitive to the K+ channel blockers apamin (1μ m), charybdotoxin (200 nm), or iberiotoxin (200 nm). Acute application of 10 μm dopamine and 20 nm ET-1 caused activation of BKCa channels with indistinguishable kinetic properties, although the effect of dopamine occurred with shorter latency. After 48-h exposure to the specific D2 dopamine receptor agonist (±)-2-(N-phenyl-N-propyl) amino-5-hydroxytetralin hydrochloride (PPHT, 500 nm), bath application of 20 nm ET-1 resulted in inhibition of spontaneously active BKCa channels. These data suggest that both the stimulatory and inhibitory effects of ET-1 on PRL secretion are mediated, at least in part, by actions on BKCa channels, and that long term exposure to dopamine or D2 agonists alters the signaling pathways from the ETA receptor to BKCa channels.

Arachidonic acid activation of potassium channels in rat visual cortex neurons

We investigated the effects of arachidonic acid on K + channels in freshly dissociated neurons of 10- to 20-day-old rat visual cortex, using a perforated and conventional whole-cell patch-clamp and inside-out excised patch configurations. Arachidonic acid at 5–30 μM induced an outward current in 88.1% of the neurons in whole-cell mode, and evoked channel opening with a conductance of 170–238 pS in 90.5% of neurons under inside-out patch recording. Arachidonic acid-activated K + channels were partially blocked by extracellular administration of 1 mM tetraethylammonium and 100 nM charybdotoxin. However, Ba 2+ completely blocked the channel in all cases. None of the other K + channel blockers, including 4-aminopyridine, quinidine, apamin and glibenclamide, inhibited the arachidonic acid-activated channels. Intracellular perfusion with Ca 2+-free and 5 mM BAPTA in Ca 2+-free extracellular perfusate containing 2 mM EGTA in conventional whole-cell recording did not inhibit the K + channel, implying that the channel is not Ca 2+ dependent. Neither guanosine 5′- O-(2-thiodiphosphate) nor staurosporine applied in inside-out mode affected the arachidonic acid-activated channels, indicating that G-protein and protein kinase C are not involved in this phenomenon. In addition, neither indomethacin nor nordihydroguaiaretic acid blocked the channel currents, demonstrating that it is arachidonic acid itself but not its metabolites that induced the effect. Among the fatty acids tested, only cis-unsaturated fatty acids, having more than two double bonds, such as arachidonic acid, docosahexaenoic acid and linolenic acid, activated the K + channels. These findings suggest that there exists a novel type of K + channel activated by arachidonic acid which may play a critical role in modulating neuronal excitability in cortical neurons.

Two types of BK channels in immature rat neocortical pyramidal neurons.

1. The properties of large conductance Ca(2+)-activated K+ channels (BK channels) were investigaed in neocortical infragranular pyramidal neurons by the use of inside-out patch recordings. Neurons were acutely isolated from slices of newborn to 28-day-old rats (P0-P28) by using minimal protease exposure followed by trituration with a vibrating glass probe. Two types of BK channels, slow-gating and fast-gating, were observed in immature neurons (P0-P5), whereas only slow-gating BK were found in more mature neurons. Fast-gating BK channels differed in conductance, voltage dependence, and kinetics from the slow-gating ones. 2. The properties of fast-gating channels included a conductance of 145 +/- 12.9 (SE) pS; frequent openings with short mean open times that were relatively voltage-independent, mean closed times that showed a voltage-dependent increase, a voltage-dependent decrease in open probability (Po). The properties of slow-gating channels contrasted with those of the fast-gating ones, in that the former had a conductance of 181 +/- 3.9 pS, longer mean open times that showed a voltage-dependent increase, mean closed times that showed a marked voltage-dependent decrease, a voltage-dependent increase in Po, and slight inward rectification. The significance of these developmental variations in channel properties is discussed.

Inhibition of K ATP channel activity by troglitazone in CRI-G1 insulin-secreting cells

Patch-clamp recording techniques were used to examine the effect of troglitazone on K ATP channel activity in Cambridge rat insulinoma-G1 (CRI-G1) insulin-secreting cells. In both inside-out and outside-out patch recordings, bath application of troglitazone reduced K ATP channel activity. This inhibition was independent of the membrane voltage and was poorly reversible. In whole-cell studies, troglitazone inhibited K ATP channel currents with an IC 50 of 697 ± 92 nM and an associated Hill coefficient of 1.2 ± 0.2. In current clamp recordings 10 μM troglitazone depolarised the CRI-G1 cell membrane by 36.8 ± 3.9 mV with a concomitant decrease in membrane conductance. However, in contrast to the rapid depolarisation produced by tolbutamide, the effects of troglitazone developed more slowly, usually taking 15–20 min to develop.

Voltage-activated and spontaneous Cl −-channel activity in cultured human melanoma cell-line A2058

Voltage-activated and spontaneous chloride-channel activity was studied in melanoma cell-line A2058 by patch-clamp technique. Whole-cell and inside-out patch recordings carried out with leak subtraction show voltage-activated chloride-conductance. In addition, a large leak-type conductance typical of epithelial cells was found in whole-cell experiments. This current was carried mostly by chloride-ions but also a leak-type potassium conductance was found showing KCl fluxes to be possible. Cell-attached and inside-out patch recordings showed at least two types of spontaneous chloride-channel activity. Bursting, flickering-type channels were found only in cell-attached recordings. That led to the conclusion that some intracellular factors are needed for that kind of activity. A second spontaneous, 30 pS chloride-channel with slow kinetics was found both in cell-attached and inside-out patch configuration. A voltage-activated chloride-channel found had a conductance of approximately 25 pS. In our experiments these channels did not need external calcium for activation. Voltage ramp recordings in cell-attached configuration gives the intracellular chloride concentration of 163 mM on the basis of chloride reversal potential (extracellular 146 mM in our experiments). Intracellular chloride concentration thus seems to be maintained slightly higher than the extracellular one.