GABA Concentration Sets the Conductance of Delayed GABAA Channels in Outside-out Patches from Rat Hippocampal Neurons

GABA_A channels were activated by GABA in outside-out patches from rat cultured hippocampal neurons. They were blocked by bicuculline and potentiated by diazepam. In 109 of 190 outside-out patches, no channels were active before exposure to GABA (silent patches). The other 81 patches showed spontaneous channel activity. In patches containing spontaneous channel activity, rapid application of GABA rapidly activated channels. In 93 of the silent patches, channels could be activated by GABA but only after a delay that was sometimes as long as 10 minutes. The maximum channel conductance of the channels activated after a delay increased with GABA concentration from less than 10 pS (0.5 μm GABA) to more than 100 pS (10 mm GABA). Fitting the data with a Hill-type equation gave an EC ₅₀ value of 33 μm and a Hill coefficient of 0.6. The channels showed outward rectification and were chloride selective. In the presence of 1 μm diazepam, the GABA EC ₅₀ decreased to 0.2 μm but the maximum conductance was unchanged. Diazepam decreased the average latency for channel opening. Bicuculline, a GABA antagonist, caused a concentration-dependent decrease in channel conductance. In channels activated with 100 μm GABA the bicuculline IC ₅₀ was 19 μm. The effect of GABA on channel conductance shows that the role of the ligand in GABA_A receptor channel function is more complex than previously thought. Received: 23 October 2000/Revised: 27 February 2001     

Two Distinct K+ Channels in Lamprey (Lampetra fluviatilis) Erythrocyte Membrane Characterized by Single Channel Patch Clamp

Two channels, distinguished by using single-channel patch-clamp, carry out potassium transport across the red cell membrane of lamprey erythrocytes. A small-conductance, inwardly rectifying K⁺-selective channel was observed in both isotonic and hypotonic solutions (osmolarity decreased by 50%). The single-channel conductance was 26 ± 3 pS in isotonic (132 mm K⁺) solutions and 24 ± 2 pS in hypotonic (63 mm K⁺) solutions. No outward conductance was found for this channel, and the channel activity was completely inhibited by barium. Cell swelling activated another inwardly rectifying K⁺ channel with a larger inward conductance of 65 pS and outward conductance of 15 pS in the on-cell configuration. In this channel, rectification was due to the block of outward currents by Mg²⁺ and Ca²⁺ ions, since when both ions were removed from the cytosolic side in inside-out patches the conductance of the channel was nearly ohmic. In contrast to the small-conductance channel, the swelling-activated channel was observed also in the presence of barium in the pipette. Neither type of channel was dependent on the presence of Ca²⁺ ions on the cytosolic side for activity. Received: 18 July 1997/Revised: 30 January 1998     

III. Ion Channel Expression in PMA-differentiated Human THP-1 Macrophages

Ion channel expression was studied in THP-1 human monocytic leukemia cells induced to differentiate into macrophage-like cells by exposure to the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Inactivating delayed rectifier K⁺ currents, I _DR, present in almost all undifferentiated THP-1 monocytes, were absent from PMA-differentiated macrophages. Two K⁺ channels were observed in THP-1 cells only after differentiation into macrophages, an inwardly rectifying K⁺ channel (I _IR) and a Ca²⁺-activated maxi-K channel (I _BK). I _IR was a classical inward rectifier, conducting large inward currents negative to E _K and very small outward currents. I _IR was blocked in a voltage-dependent manner by Cs⁺, Na⁺, and Ba²⁺, block increasing with hyperpolarization. Block by Na⁺ and Ba²⁺ was time-dependent, whereas Cs⁺ block was too fast to resolve. Rb⁺ was sparingly permeant. In cell-attached patches with high [K⁺] in the pipette, the single I _IR channel conductance was ∼30 pS and no outward current could be detected. I _BK channels were observed in cell-attached or inside-out patches and in whole-cell configuration. In cell-attached patches the conductance was ∼200–250 pS and at potentials positive to ∼100 mV a negative slope conductance of the unitary current was observed, suggesting block by intracellular Na⁺. I _BK was activated at large positive potentials in cell-attached patches; in inside-out patches the voltage-activation relationship was shifted to more negative potentials by increased [Ca²⁺]. Macroscopic I _BK was blocked by external TEA⁺ with half block at 0.35 mm. THP-1 cells were found to contain mRNA for Kv1.3 and IRK1. Levels of mRNA coding for these K⁺ channels were studied by competitive PCR (polymerase chain reaction), and were found to change upon differentiation in the same direction as did channel expression: IRK1 mRNA increased at least 5-fold, and Kv1.3 mRNA decreased on average 7-fold. Possible functional correlates of the changes in ion channel expression during differentiation of THP-1 cells are discussed. Received: 19 September 1995/Revised: 14 March 1996     

Effects of Mutating Leucine to Threonine in the M2 Segment of α1 and β1 Subunits of GABAAα1β1 Receptors

The conserved leucine residues at the 9′ positions in the M2 segments of α₁ (L264) and β₁ (L259) subunits of the human GABA_A receptor were replaced with threonine. Normal or mutant α₁ subunits were co-expressed with normal or mutant β₁ subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a higher ``resting' chloride conductance than cells expressing wild-type α₁β₁ receptors. This chloride conductance was blocked by 10 mm penicillin, a recognized blocker of GABA_A channels, but not by bicuculline (100 μm) or picrotoxin (100 μm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 μm). In cells expressing wild-type β₁ with mutated α₁ subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type α₁β₁ receptors. In cells expressing mutated β₁ subunits with wild-type or mutated α₁ subunits (αβ(L9′T) and α(L9′T)β(L9′T)), no response to GABA could be elicited: this was not due to an absence of GABA_A receptors in the plasmalemma because the cells bound [³H]-muscimol. It was concluded that in GABA_A channels containing the L9′T mutation in the β₁ subunit, GABA-binding does not cause opening of channels, and that the L9′T mutation in either or both subunits gives an open-channel state of the GABA_A receptor in the absence of ligand. Received: 17 April 1996/Revised: 5 July 1996     

Identification of Anion-selective Channels in the Basolateral Membrane of Mitochondria-rich Epithelial Cells

Epithelial cells of toad (Bufo bufo) skin were isolated by treatments of the epidermis with collagenase and trypsin. Cl^- channels in the basolateral membrane from soma or neck of mitochondria-rich cells were studied in cell-attached and excised inside-out configurations. Of a total of 87 sealed patches only 28 (32%) were electrically active, and in these we identified four different types of Cl^- channels. The two major populations constituted Ohmic Cl^- channels with limiting conductance (γ_125/125) of 10 pS and 30 pS, respectively. A much rarer 150 pS Ohmic Cl^- channel was also characterized. From i/V relationships of individual channels the following Goldman-Hodgkin-Katz permeabilities were calculated, 2.2 (±0.1) × 10^-14, 5.7 (±0.7) × 10^-14, and 32 (±2) × 10^-14 cm³/sec, for the 10, 30 and 150 pS Cl^- channels, respectively. The 30 pS channel was activated by hyperpolarization. The gating kinetics of the 150 pS channel was complex with burstlike closures within openings of long duration. The fourth type of Cl^- channel was studied in patches generating `noisy currents' with no discrete single-channel events, but with vanishing fluctuations at pipette potentials near E _Cl. Noise analysis revealed a power spectrum with cutoff frequencies of 1.2 and 13 Hz, indicating that resolution of kinetic steps was limited by small channel currents rather than fast channel gating. From the background noise level we estimated the channel conductance to be less than 1.7 pS. Despite the fact that the majority of patches did not contain electrically active Cl^- channels, patches being active, generally, contained more than a single active channel. Thus, for the above three types of resolvable channels, the mean number of active channels per patch amounted to 2.1, 1.4, and 2.0, respectively. This observation, like the finding of few patches with several unresolvable channels, indicates that electrically active Cl^- channels are organized in clusters. Received: 10 October 1996/Revised: 8 January 1997     

Opening of Cardiac Sarcolemmal KATP Channels by Dinitrophenol Separate from Metabolic Inhibition

Opening of ATP-sensitive K⁺ (K_ATP) channels by the uncoupler of oxidative phosphorylation, 2,4 dinitrophenol (DNP), has been assumed to be secondary to metabolic inhibition and reduced intracellular ATP levels. Herein, we present data which show that DNP (200 μm) can induce opening of cardiac K_ATP channels, under whole-cell and inside-out conditions, despite millimolar concentrations of ATP (1–2.5 mm). DNP-induced currents had a single channel conductance (71 pS), inward rectification, reversal potential, and intraburst kinetic properties (open time constant, τ_open: 4.8 msec; fast closed time constant, τ_closed(f): 0.33 msec) characteristic of K_ATP channels suggesting that DNP did not affect the pore region of the channel, but may have altered the functional coupling of the ATP-dependent channel gating. A DNP analogue, with the pH-titrable hydroxyl replaced by a methyl group, could not open K_ATP channels. The pH-dependence of the effect of DNP on channel opening under whole-cell, cell-attached, and inside-out conditions suggested that transfer of protonated DNP across the sarcolemma is essential for activation of K_ATP channels in the presence of ATP. We conclude that the use of DNP for metabolic stress-induced K_ATP channel opening should be reevaluated. Received: 10 September 1996/Revised: 27 December 1996     

CFTR Channels Expressed in CHO Cells Do Not Have Detectable ATP Conductance

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated, ATP-dependent chloride channel which may have additional functions. Recent reports that CFTR mediates substantial electrodiffusion of ATP from epithelial cells have led to the proposal that CFTR regulates other ion channels through an autocrine mechanism involving ATP. The aim of this study was to determine the ATP conductance of wild-type CFTR channels stably expressed in Chinese hamster ovary cells using patch clamp techniques. In the cell-attached configuration with 100 mm Mg · ATP or Tris · ATP solution in the pipette and 140 mm NaCl in the bath, exposing cells to forskolin caused the activation of a low-conductance channel having kinetics resembling those of CFTR. Single channel currents were negative at the resting membrane potential (V _m ), consistent with net diffusion of Cl from the cell into the pipette. The transitions decreased in amplitude, but did not reverse direction, as V _m was clamped at increasingly positive potentials to enhance the driving force for inward ATP flow (>+80 mV). In excised patches, single channel currents did not reverse under essentially biionic conditions (Cl_in/ATP_out or ATP_in/Cl_out), although PKA-activated currents were clearly visible in the same patches at voltages where they would be carried by chloride ions. Moreover, with NaCl solution in the bath and a mixture of ATP and Cl in the pipette, the single channel I/V curve reversed at the predicted equilibrium potential for chloride. CFTR channel currents disappeared when patches were exposed to symmetrical ATP solutions and were restored by reexposure to Cl solution. Finally, in the whole-cell configuration with NaCl in the bath and 100 mm MgATP or TrisATP in the pipette, cAMP-stimulated cells had time-independent, outwardly rectifying currents consistent with CFTR selectivity for external Cl over internal ATP. Whole-cell currents reversed near V _m =−55 mV under these conditions, however the whole cell resistance measured at −100 mV was comparable to that of the gigaohm seal between the plasma membrane and glass pipette (7 GΩ). We conclude that CFTR does not mediate detectable electrodiffusion of ATP. Received: 8 November 1995/Revised: 23 January 1996     

Characterization of Single Inward Rectifier Potassium Channels from Embryonic Xenopus laevis Myocytes

Single inward rectifier K⁺ channels were studied in Xenopus laevis embryonic myocytes. We have characterized in detail the channel which is most frequently observed (Kir) although we routinely observe three other smaller current levels with the properties of inward rectifier K⁺ channels (Kir_(0.3), Kir_(0.5) and Kir_(0.7)). For Kir, slope conductances of inward currents were 10.3, 20.3, and 27.9 pS, in 60, 120 and 200 mM [K⁺] _o respectively. Extracellular Ba²⁺ blocked the normally high channel activity in a concentration-dependent manner (K _A = 7.8 μm, −90 mV). In whole-cell recordings of inward rectifier K⁺ current, marked voltage dependence of Ba²⁺ block over the physiological range of potentials was observed. We also examined current rectification. Following step depolarizations to voltages positive to E _K , outward currents through Kir channels were not observed even when the cytoplasmic face of excised patches were exposed to Mg²⁺-free solution at pH 9.1. This was probably also true for Kir_(0.3), Kir_(0.5) and Kir_(0.7) channels. We then examined the possibility of modulation of Kir channel activity and found neither ATP nor GTP-γS had any effect on Kir channel activity when added to the solution perfusing the cytoplasmic face of a patch. Kinetic analysis revealed Kir channels with a single open state (mean dwell time 72 msec) and two closed states (time constants 1.4, 79 msec). These results suggest that the native Kir channels of Xenopus myocytes have similar properties to the cloned strong inward rectifier K⁺ channels, in terms of conductance, kinetics and barium block but does show some differences in the effects of modulators of channel activity. Furthermore, skeletal muscle may contain either different inward rectifier channels or a single-channel type which can exist in stable subconductance states. Received: 16 September 1996/Revised: 14 March 1997     

Bimodal Kinetics of a Chloride Channel from Human Fibroblasts

Excised patches were used to study the kinetics of a Cl channel newly identified in cultured human fibroblasts (L132). The conductance of ca. 70 pS in 150 mm symmetrical Cl, and the marked outward rectification ascribe this channel to the ICOR family. Long single-channel recordings (>30 min) revealed that the channel spontaneously switches from a kinetic mode characterized by high voltage dependence (with activity increasing with depolarization; mode 1), into a second mode (mode 2) insensitive to voltage, and characterized by a high activity in the voltage range ±120 mV. On patch excision the channel always appeared in mode 1, which was maintained for a variable time (5–20 min). In most instances the channels then switched into mode 2, and never were seen to switch back, in spite of the eight patches that cumulatively dwelled in this mode 2.33-fold as compared to mode 1. Stability plots of long recordings showed that the channel was kinetically stable in both modes, allowing standard analysis of steady-state kinetics to be performed. Open and closed time distributions of mode 1 and mode 2 revealed that the apparent number of kinetic states of the channel was the same in the two modes. The transition from mode 1 into mode 2 was not instantaneous, but required a variable time in the range 5–60 sec. During the transition the channel mean open time was intermediate between mode 1 and mode 2. The intermediate duration in the stability plot however is not to be interpreted as if the channel, during the transition, rapidly switches between mode 1 and mode 2, but represents a distinct kinetic feature of the transitional channel. Received: 31 December 1998/Revised: 13 April 1999     

Single K+ Channels in Embryonic Leech Ganglion Cells

We investigated the properties of single K⁺ channels in the soma membrane of embryonic leech ganglion cells using the patch-clamp technique. We compared these K⁺ channels with the K⁺ channels found previously in Retzius neurons of the adult leech. In ganglion cells of 9- to 15-day-old embryos we characterized eight different types of K⁺ channels with mean conductances of 21, 55, 84, 111, 122, 132, 149 and 223 pS. The 55 pS and 84 pS channels showed flickering and were active for less than 2 min after excising the patch. The 111 pS channel was an outward rectifier, and the open state probability (p _o ) decreased in the inside-out configuration when the Ca²⁺ concentration was raised from pCa 7 to pCa 3. The 122 pS channel also showed outward rectification. This type of channel was activated after changing from the cell-attached to the inside-out configuration and it did not inactivate during more than 30 min. The p _o was Ca²⁺- and voltage-insensitive. One hundred μm glibenclamide reversibly reduced p _o . The 132 pS channel was an outward rectifier and was Ca²⁺-insensitive. The 149 pS channel inactivated in the inside-out configuration. The 149- and the 223 pS channel showed inward rectification. The 111 pS channel had similar properties to the Ca²⁺-dependent K⁺ channel and the 122 pS channel resembled the ATP-inhibited K⁺ channel found previously in Retzius neurons of the adult leech. Received: 20 April 1995/Revised: 18 January 1996     

Anion Channels in Chara corallina Tonoplast Membrane: Calcium Dependence and Rectification

Tonoplast K⁺ channels of Chara corallina are well characterized but only a few reports mention anion channels, which are likely to play an important role in the tonoplast action potential and osmoregulation of this plant. For experiments internodal cells were isolated. Cytoplasmic droplets were formed in an iso-osmotic bath solution according to a modified procedure. Ion channels with conductances of 48 pS and 170 pS were detected by the patch-clamp technique. In the absence of K⁺ in the bath solution the 170 pS channel was not observed at negative pipette potential values. When Cl⁻ on either the vacuolar side or the cytoplasmic side was partly replaced with F⁻, the reversal potential of the 48 pS channel shifted conform to the Cl⁻ equilibrium potential with similar behavior in droplet-attached and excised patch mode. These results showed that the 48 pS channel was a Cl⁻ channel. In droplet-attached mode the channel rectified outward current flow, and the slope conductance was smaller. When Chara droplets were formed in a bath solution containing low (10⁻⁸ m) Ca²⁺, then no Cl⁻ channels could be detected either in droplet-attached or in inside-out patch mode. Channel activity was restored if Ca²⁺ was applied to the cytoplasmic side of inside-out patches. Rectification properties in the inside-out patch configuration could be controlled by the holding pipette potential. Holding potential values negative or positive to the calculated reversal potential for Cl⁻ ions induced opposite rectification properties. Our results show Ca²⁺-activated Cl⁻ channels in the tonoplast of Chara with holding potential dependent rectification. Received: 30 March 1999/Revised: 10 August 1999     

Characterization of Angiotensin II-regulated K+ Conductance in Rat Adrenal Glomerulosa Cells

Nystatin perforated-patch clamp and single-channel recording methods were used to characterize macroscopic and single-channel K⁺ currents and the effects of angiotensin II (AngII) in cultured rat adrenal glomerulosa cells. Two basic patterns of macroscopic current-voltage relationships were observed: type 1 exhibited a rapidly activating, noninactivating, voltage-dependent outward current and type 2 exhibited an inactivating voltage-dependent outward current attributed to charybdotoxin sensitive Ca⁺⁺-dependent K⁺ channels. Most cells exhibited the type 1 pattern and experiments focused on this cell type. Cell-attached and inside-out patches were dominated by a single K⁺ channel class which exhibited an outward conductance of 12 pS (20 mm K⁺ pipette in cell-attached and inside-out configurations, 145 mm K⁺ _in), a mean open time of 2 msec, and a weakly voltage-dependent low open probability that increased with depolarization. Channel open probability was reversibly inhibited by bath stimulation with AngII. At the macroscopic level, type 1 cell macroscopic K⁺ currents appeared comprised of two components: a weakly voltage-dependent current controlling the resting membrane potential (−85 mV) which appeared mediated by the 12 pS K⁺ channel and a rapidly activating, noninactivating voltage-dependent current activated above −50 mV. The presence of the second voltage-dependent K⁺ channel class was suggested by the effects of AngII, the blocking effects of quinidine and Cs⁺, and the properties of the weakly voltage-dependent K⁺ channel described. The K⁺ selectivity of the macroscopic current was demonstrated by the dependence of current reversal potentials on the K⁺ equilibrium potential and by the effects of K⁺ channel blockers, Cs⁺ and quinidine. AngII (10 pm to 1 nm) reversibly inhibited macroscopic K⁺ currents and this effect was blocked by the AT₁ receptor antagonist losartin. Received: 6 August 1996/Revised: 15 November 1996     

Activation of Maxi-K Channels by Parathyroid Hormone and Prostaglandin E2 in Human Osteoblast Bone Cells

Patch clamp experiments were performed on two human osteosarcoma cell lines (MG-63 and SaOS-2 cells) that show an osteoblasticlike phenotype to identify and characterize the specific K channels present in these cells. In case of MG-63 cells, in the cell-attached patch configuration (CAP) no channel activity was observed in 2 mm Ca Ringer (control condition) at resting potential. In contrast, a maxi-K channel was observed in previously silent CAP upon addition of 50 nm parathyroid hormone (PTH), 5 nm prostaglandin E₂ (PGE₂) or 0.1 mm dibutyryl cAMP + 1 μm forskolin to the bath solution. However, maxi-K channels were present in excised patches from both stimulated and nonstimulated cells in 50% of total patches tested. A similar K channel was also observed in SaOS-2 cells. Characterization of this maxi-K channel showed that in symmetrical solutions (140 mm K) the channel has a conductance of 246 ± 4.5 pS (n = 7 patches) and, when Na was added to the bath solution, the permeability ratio (P_K/P_Na) was 10 and 11 for MG-63 and SaOS-2 cells respectively. In excised patches from MG-63 cells, the channel open probability (P _o ) is both voltage- (channel opening with depolarization) and Ca-dependent; the presence of Ca shifts the P _o vs. voltage curve toward negative membrane potential. Direct modulation of this maxi-K channel via protein kinase A (PKA) is very unlikely since in excised patches the activity of this channel is not sensitive to the addition of 1 mm ATP + 20 U/ml catalytic subunit of PKA. We next evaluated the possibility that PGE₂ or PTH stimulated the channel through a rise in intracellular calcium. First, calcium uptake (⁴⁵Ca⁺⁺) by MG-63 cells was stimulated in the presence of PTH and PGE₂, an effect inhibited by Nitrendipine (10 μm). Second, whereas PGE₂ stimulated the calcium-activated maxi-K channel in 2 mm Ca Ringer in 60% of patches studied, in Ca-free Ringer bath solution, PGE₂ did not open any channels (n = 10 patches) nor did cAMP + forskolin (n = 3 patches), although K channels were present under the patch upon excision. In addition, in the presence of 2 mm Ca Ringer and 10 μm Nitrendipine in CAP configuration, PGE₂ (n = 5 patches) and cAMP + forskolin (n = 2 patches) failed to open K channels present under the patch. As channel activation by phosphorylation with the catalytic subunit of PKA was not observed, and Nitrendipine addition to the bath or the absence of calcium prevented the opening of this channel, it is concluded that activation of this channel by PTH, PGE₂ or dibutyryl cAMP + forskolin is due to an increase in intracellular calcium concentration via Ca influx. Received: 17 September 1995/Revised: 7 December 1995     

Photofrin II Sensitized Modifications of Ion Transport Across the Plasma Membrane of an Epithelial Cell Line: II. Analysis at the Level of Membrane Patches

In the first part of this study, photofrin II sensitized membrane modifications of OK-cells were investigated at the level of macroscopic membrane currents. In this second part, the inside-out configuration of the patch-clamp technique is applied to analyze the phenomena at the microscopic level. It is shown that the characteristic single channel fluctuations of the electric current disappear after the start of illumination of membrane patches in the presence of photofrin II. This holds for all three types of ion channels investigated: the large-conductance Ca²⁺-dependent K⁺ channel (maxi-K_Ca), a K⁺ channel of small conductance (sK), and a stretch-activated nonselective cation channel (SA-cat). Part of the experiments show a transient activation of the channels (indicated by an increase of the probability in the open-channel state) before the channels are converted into a closed nonconductive state. Inactivation of all three channel types proceeds by a continuous reduction of their open probability, while the single channel conductance values are not affected. The process of photodynamically induced channel inactivation is followed by a pronounced increase of the leak conductance of the plasma membrane. The latter process — after light-induced initiation — is found to continue in the dark. The ionic pathways underlying the leak conductance also allow permeation of Ca²⁺ ions. The resulting Ca²⁺-flux may contribute to the photodynamically induced increase of the intracellular Ca²⁺ concentration observed in various cell lines. Received: 26 May 1998/Revised: 8 September 1998     

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 17β-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 μm) reversibly suppressed the amplitude of K⁺ outward currents. The IC ₅₀ value of the 2-methoxyestradiol-induced decrease in outward current was 3 μm. Evans blue (30 μm) or niflumic acid (30 μm), but not diazoxide (30 μm), reversed the 2-methoxyestradiol-induced decrease in outward current. In the inside-out configuration, application of 2-methoxyestradiol (3 μm) to the bath did not modify the single-channel conductance of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels; however, it did suppress the channel activity. 2-Methoxyestradiol (3 μm) produced a shift in the activation curve of BK_Ca channels to more positive potentials. Kinetic studies showed that the 2-methoxyestradiol-induced inhibition of BK_Ca 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 17β-estradiol (10 μm) nor estriol (10 μm) affected BK_Ca channel activity, whereas 2-hydroxyestradiol (10 μm) slightly suppressed it. Under current-clamp condition, 2-methoxyestradiol (10 μm) caused membrane depolarization and Evans blue (30 μm) reversed 2-methoxyestradiol-induced depolarization. The present study provides evidence that 2-methoxyestradiol can suppress the activity of BK_Ca channels in endothelial cells. These effects of 2-methoxyestradiol on ionic currents may contribute to its effects on functional activity of endothelial cells. Received: 27 November 2000/Revised: 13 April 2001     

Large Conductance Ca2+-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 IC₅₀= 5.3 mm). Raising the internal Ca²⁺ 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 Ca²⁺-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 μm Ca²⁺, 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 Ca²⁺ in the pipette. Application of 20 μm 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 Ca²⁺ but is also controlled by the cytoskeleton. Received 18 June 1999/Revised: 18 January 2000     

Membrane Potassium Channels and Human Bladder Tumor Cells. I. Electrical Properties

These experiments were conducted to determine the membrane K⁺ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K⁺ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca²⁺-dependent outward K⁺ current, 0.57 ± 0.13 nS/pF at −70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca²⁺-activated, voltage-dependent K⁺ channel (K_Ca) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the K_Ca activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K⁺ channel, 21 ± 1 pS at positive transmembrane potential (V _m ) and 145 ± 13 pS at negative V _m . Glibenclamide (50 μm), Ba²⁺ (1 mm) and quinine (100 μm) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K⁺ channels in inside/out patches in a dose-dependent manner, and the IC₅₀= 46 μm. The identity of this K⁺ channel with an ATP-sensitive K⁺ channel (K_ATP) was confirmed by its inhibition with ATP (2 mm) and by its activation with diazoxide (100 μm). We conclude that plasma membranes of HTB-9 cells contain the K_Ca and a lower conductance K⁺ channel with properties consistent with a sulfonylurea receptor-linked K_ATP. Received: 12 June 1997/Revised: 21 October 1997     

The Antifungal Imidazole Clotrimazole and its Major In Vivo Metabolite are Potent Blockers of the Calcium-Activated Potassium Channel in Murine Erythroleukemia Cells

Clotrimazole (CLT), a member of the antifungal imidazole family of compounds, has been found to inhibit both calcium (Ca²⁺)-activated ⁸⁶Rb and potassium (K) fluxes of human red cells and to inhibit red cell binding of ¹²⁵I-charybdotoxin (ChTX) [11]. We have now used patch-clamp techniques to demonstrate reversible inhibition of whole cell K_Ca2+ currents in murine erythroleukemia (MEL) cells by submicromolar concentrations of CLT. Inhibition was equivalent whether currents were elicited by bath application of the Ca²⁺ ionophore A23187 or by dialyzing cells with a pipette solution containing micromolar concentrations of free Ca²⁺. The extent of inhibition of whole cell MEL K_Ca2+ currents was voltage-dependent, decreasing with increasing test potential. We also determined the single channel basis of the CLT inhibition in MEL cells by demonstrating the inhibition of a calcium-activated, ChTX-sensitive K channel by CLT in outside-out patches. The channel was also blocked by the des-imidazolyl metabolite of CLT, 2-chlorophenyl-bisphenyl-methanol (MET II) [15], thus demonstrating that the imidazole ring is not required for the inhibitory action of CLT. Single K_Ca2+ channels were also evident in inside-out patches of MEL cells. Block of K current by CLT was not unique to MEL cells. CLT also inhibited a component of the whole cell K current in PC12 cells. Channel specificity of block by CLT was determined by examining its effects on other types of voltage-sensitive currents. CLT block showed the following rank order of potency: K currents in PC12 cells > Ca²⁺ currents in PC12 cells ≫ Na currents in sympathetic neurons. These results demonstrate that direct inhibition of single K_Ca2+ by CLT can be dissociated from inhibition of cytochrome P-450 in MEL cells. Received: 10 September 1996/Revised: 12 December 1996     

Expression of Cyclic Nucleotide-Gated Cation Channels in Airway Epithelial Cells

Using the whole-cell patch-clamp technique, the selectivity and pharmacology of 8-Br-cGMP-stimulated currents in the human alveolar cell line A549 was compared to 8-Br-cGMP-stimulated currents in HK293 cells transfected with hαCNC1. Whole cell currents stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 or A549 cells are carried by inward sodium and outward potassium with nearly the same selectivity. The whole-cell inward currents that are stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 are inhibited by l-cis-diltiazem with an IC₅₀ of 154 μm, by 2′,4′-dichlorobenzamil with an IC₅₀ of 50 μm and by amiloride with an IC₅₀ of 133 μm. The whole-cell inward currents in A549 cells that are stimulated by 8-Br-cGMP, are inhibited by l-cis-diltiazem with an IC₅₀ of 87 μm, by 2′4′-dichlorobenzamil with an IC₅₀ of 38 μm and by amiloride with an IC₅₀ of 32 μm suggesting that these airway cells contain cyclic nucleotide-gated cation channels. RT-PCR data suggest that mRNA of both αCNC1 and βCNC subunits are present in A549 cells and the presence of the βCNC subunit, may as previously reported, increase the affinity of these channel blockers compared to the hαCNC1 subunit alone. The mRNA of two other isoforms of this channel, CNC2 and CNC3, are also expressed in the A549 cell line. This study documents the IC₅₀ of externally applied channel blockers that can be used for in vitro or in vivo experiments to document sodium absorption via cyclic nucleotide-gated cation channels in airway cells. Received: 24 February/Revised: 28 May 1999     

Crayfish sensory terminals and motor neurones exhibit two distinct types of GABA receptors

Motor neurones of the crayfish walking system display inhibitory responses evoked either by γ-amino butyric acid (GABA) or glutamate, possibly involving the same receptor (Pearlstein et al. 1994). In order to test if this sensibility to both GABA and glutamate was a specific property of crayfish GABA receptors, pharmacological characteristics of GABA-evoked responses in both sensory terminals from CB chordotonal organ and motor neurones of the walking system have been compared. Both receptors are GABA-gated Cl⁻ channels activated by specific GABA_A (muscimol, isoguvacine), GABA_B (3-aminopropyl phosphinic acid), and GABA_C (cis-4-amino crotonic acid) agonists, and blocked by competitive (β-guanidino propionic acid) and non-competitive (picrotoxin) antagonists. They were insensitive to specific GABA_A (bicuculline, SR-95531) and GABA_B (phaclofen) antagonists. Furthermore, in both cases, nipecotic acid and the modulatory drug diazepam had no effect. However, our results demonstrate that GABA receptors of sensory terminals are different from those of motor neurones. GABA-induced desensitisation only occurred in sensory terminals. Moreover, glutamate was shown to activate GABA-gated Cl⁻ channels in motor neurones, but not in sensory terminals. Therefore, GABA is likely to be the endogenous neurotransmitter of presynaptic inhibition in sensory terminals, whereas inhibition between antagonistic motor neurones would be achieved by glutamate. Accepted: 10 July 1996