Characterization of ATP-Sensitive Potassium Channels Functionally Expressed in Pituitary GH3 Cells

ATP-sensitive K⁺ (K_ATP) 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 μm) revealed the presence of glibenclamide-sensitive K_ATP channel exhibiting a unitary conductance of 74 pS. Metabolic inhibition induced by 2,4-dinitrophenol (1 mm) or sodium cyanide (300 μm) increased K_ATP channel activity, while nicorandil (100 μm) had no effect on it. In the inside-out configuration, Mg-ATP applied intracellularly suppressed the activity of K_ATP channels in a concentration-dependent manner with an IC₅₀ value of 30 μm. The activation of phospholipase A₂ caused by mellitin (1 μm) was found to enhance K_ATP channel activity and further application of aristolochic acid (30 μm) reduced the mellitin-induced increase in channel activity. The challenging of cells with 4,4′-dithiodipyridine (100 μm) also induced K_ATP channel activity. Diazoxide, mellitin and 4,4′-dithiodipyridine activated the K_ATP channels that exhibited similar channel-opening kinetics. In addition, under current-clamp conditions, the application of diazoxide (100 μm) hyperpolarized the membrane potential and reduced the firing rate of spontaneous action potentials. The present study clearly indicates that K_ATP channels similar to those seen in pancreatic β cells are functionally expressed in GH₃ cells. In addition to the presence of Ca²⁺-activated K⁺ channels, K_ATP channels found in these cells could thus play an important role in controlling hormonal release by regulating the membrane potential. Received: 19 June 2000/Revised: 13 September 2000     

Membrane Potassium Channels and Human Bladder Tumor Cells: II. Growth Properties

These experiments were done to determine the effect of glibenclamide and diazoxide on the growth of human bladder carcinoma (HTB-9) cells in vitro. Cell growth was assayed by cell counts, protein accumulation, and ³H-thymidine uptake. Glibenclamide added at 75 and 150 μm for 48 hr reduced cell proliferation. Dose-inhibition curves showed that glibenclamide added for 48 hr reduced cell growth at concentrations as low as 1 μm (IC₅₀= 73 μm) when growth was assayed in the absence of added serum. This μM-effect on cell growth was in agreement with the dose range in which glibenclamide decreased open probability of membrane K_ATP channels. Addition of glibenclamide for 48 hr also altered the distribution of cells within stages of the cell cycle as determined by flow cytometry using 10⁻⁵ m bromodeoxyuridine. Glibenclamide (100 μm) increased the percentage of cells in G₀/G₁ from 33.6% (vehicle control) to 38.3% (P < 0.05), and it reduced the percentage of cells in S phase from 38.3% to 30.6%. On the other hand, diazoxide, which opens membrane K_ATP channels in HTB-9 cells, stimulated growth measured by protein accumulation, but it did not increase the cell number. We conclude that the sulfonylurea receptor and the corresponding membrane K_ATP channel are involved in mechanisms controlling HTB-9 cell growth. However, K_ATP is not rate-limiting among the signaling mechanisms or molecular switches that regulate the cell cycle. Received: 12 June 1997/Revised: 21 October 1997     

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     

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     

State-Dependent Inhibition of Inactivation-Deficient CaV1.2 and CaV2.3 Channels By Mibefradil

The structural determinants of mibefradil inhibition were analyzed using wild-type and inactivation-modified Ca_V1.2 (α1C) and Ca_V2.3 (α1E) channels. Mibefradil inhibition of peak Ba²⁺ currents was dose- and voltage-dependent. An increase of holding potentials from −80 to −100 mV significantly shifted dose-response curves toward higher mibefradil concentrations, namely from a concentration of 108 ± 21 μm (n= 7) to 288 ± 17 μm (n= 3) for inhibition of half of the Ca_v1.2 currents (IC ₅₀) and from IC ₅₀= 8 ± 2 μm (n= 9) to 33 ± 7 μm (n= 4) for Ca_V2.3 currents. In the presence of mibefradil, Ca_V1.2 and Ca_V2.3 experienced significant use-dependent inhibition (0.1 to 1 Hz) and slower recovery from inactivation suggesting mibefradil could promote transition(s) to an absorbing inactivated state. In order to investigate the relationship between inactivation and drug sensitivity, mibefradil inhibition was studied in inactivation-altered Ca_V1.2 and Ca_V2.3 mutants. Mibefradil significantly delayed the onset of channel recovery from inactivation in CEEE (Repeat I + part of the I–II linker from Ca_V1.2 in the Ca_V2.3 host channel), in EC(AID)EEE (part of the I–II linker from Ca_V1.2 in the Ca_V2.3 host channel) as well as in Ca_V1.2 E462R, and Ca_V2.3 R378E (point mutation in the β-subunit binding motif) channels. Mibefradil inhibited the faster inactivating chimera EC(IS1-6)EEE with an IC ₅₀= 7 ± 1 μm (n= 3), whereas the slower inactivating chimeras EC(AID)EEE and CEEE were, respectively, inhibited with IC ₅₀= 41 ± 5 μm (n= 4) and IC ₅₀= 68 ± 9 μm (n= 5). Dose-response curves were superimposable for the faster EC(IS1-6)EEE and Ca_V2.3, whereas intermediate-inactivating channel kinetics (CEEE, Ca_V1.2 E462R, and Ca_V1.2 E462K) were inhibited by similar concentrations of mibefradil with IC ₅₀≈ 55–75 μm. The slower Ca_V1.2 wild-type and Ca_V1.2 Q473K channels responded to higher doses of mibefradil with IC ₅₀≈ 100–120 μm. Mibefradil was also found to significantly speed up the inactivation kinetics of slower channels (Ca_V1.2, CEEE) with little effect on the inactivation kinetics of faster-inactivating channels (Ca_V2.3). A open-channel block model for mibefradil interaction with high-voltage-activated Ca²⁺ channels is discussed and shown to qualitatively account for our observations. Hence, our data agree reasonably well with a ``receptor guarded mechanism' where fast inactivation kinetics efficiently trap mibefradil into the channel. Received: 14 March 2001/Revised: 25 June 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     

Reconstitution in Lipid Bilayers of an ATP-Sensitive K+ Channel from Pig Coronary Smooth Muscle

A K⁺ channel with a main conductance of 29 pS was recorded after the incorporation of coronary artery membrane vesicles into lipid bilayers. This channel was identified as an ATP-sensitive K⁺ channel (K_ATP) because its activity was diminished by the internal application of 50–250 μm ATP-Na₂. Moreover, it was opened when 10–50 μm pinacidil was externally applied. Single-channel records revealed the existence of several (sub)conductance states. At 0 mV and with a 5/250 KCl gradient, the main conductance of the K_ATP channel was 29 pS. The other (sub)conductance states were less frequent and had discrete values of 12, 17 and 22 pS. Pinacidil stabilized the channel open state primarily in the 29 pS conductance level; whereas ATP inhibited all the conductance levels. In general, K_ATP channels were characterized by brief openings followed by long closings (open probability, P _o ≈ 0.02); only occasionally (3 out of 12 experiments) did the K_ATP channels have a high open probability (P _o ≥ 0.7). Channel activity could be increased or rescued by adding 2.5–10 mm UDP-TRIS and 0.5–2 mm MgCl₂ to the internal side of the channel. Received: 7 November 1995/Revised: 10 June 1996     

Single-Channel Analysis of a Delayed Rectifier K+ Channel in Xenopus Myelinated Nerve

Single-channel properties of a delayed rectifier voltage-gated K⁺ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between −60 and −40 mV with a potential of half-maximal activation, E₅₀, at −47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal activation decreased from 80 to 1.6 msec between −60 and +40 mV. The channel inactivated monoexponentially with a time constant of 10.9 sec at −40 mV. The time constant of deactivation was 126 msec at −80 mV and 16.9 msec at −110 mV. In symmetrical 105 mm K⁺, the single-channel conductance (γ) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13–15°C. In Na⁺-rich solution with 2.5 mm extracellular K⁺γ was 7 pS and the reversal potential was negative to −80 mV, indicating a high selectivity for K⁺ over Na⁺. γ depended on extracellular K⁺ concentration (K _D = 19.6 mm) and temperature (Q ₁₀= 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel current amplitude at all potentials tested with a half-maximal inhibiting concentration (IC₅₀) of 0.6 mm. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX, IC₅₀= 6.8 nm) and mast cell degranulating peptide (MCDP, IC₅₀= 41.9 nm). In Ringer solution the membrane potential of macroscopic I-channel patches was about −65 mV and depolarized under TEA and DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane potential. Received: 2 June 1995/Revised: 13 October 1995     

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     

Activation of inwardly rectifying potassium channels by muscarinic receptor-linked G protein in isolated human ventricular myocytes

Muscarinic receptor-linked G protein, G _i , can directely activate the specific K⁺ channel (I _K(ACh)) in the atrium and in pacemaker tissues in the heart. Coupling of G _i to the K⁺ channel in the ventricle has not been well defined. G protein regulation of K⁺ channels in isolated human ventricular myocytes was examined using the patch-clamp technique. Bath application of 1 μm acetylcholine (ACh) reversibly shortened the action potential duration to 74.4 ± 12.1% of control (at 90% repolarization, mean ±sd, n= 8) and increased the whole-cell membrane current conductance without prior β-adrenergic stimulation in human ventricular myocytes. The ACh effect was reversed by atropine (1 μm). In excised inside-out patch configurations, application of GTPγS (100 μm) to the bath solution (internal surface) caused activation of I _K(ACh) and/or the background inwardly-rectifying K⁺ channel (I _K1) in ventricular cell membranes. I _K(ACh) exhibited rapid gating behavior with a slope conductance of 44 ± 2 pS (n= 25) and a mean open lifetime of 1.8 ± 0.3 msec (n= 21). Single channel activity of GTPγS-activated I _K1 demonstrated long-lasting bursts with a slope conductance of 30 ± 2 pS (n= 16) and a mean open lifetime of 36.4 ± 4.1 msec (n= 12). Unlike I _K(ACh), G protein-activated I _K1 did not require GTP to maintain channel activity, suggesting that these two channels may be controlled by G proteins with different underlying mechanisms. The concentration of GTP at half-maximal channel activation was 0.22 μm in I _K(ACh) and 1.2 μm in I _K1. Myocytes pretreated with pertussis toxin (PTX) prevented GTP from activating these channels, indicating that muscarinic receptor-linked PTX-sensitive G protein, G _i , is essential for activation of both channels. G protein-activated channel characteristics from patients with terminal heart failure did not differ from those without heart failure or guinea pig. These results suggest that ACh can shorten the action potential by activating I _K(ACh) and I _K1 via muscarinic receptor-linked G _i proteins in human ventricular myocytes. Received: 23 September 1996/Revised: 18 December 1996     

Kinetic Analysis of the Inhibitory Effect of Glibenclamide on KATP Channels of Mammalian Skeletal Muscle

We investigated the block of K_ATP channels by glibenclamide in inside-out membrane patches of rat flexor digitorum brevis muscle. (1) We found that glibenclamide inhibited K_ATP channels with an apparent K _i of 63 nm and a Hill coefficient of 0.85. The inhibition of K_ATP channels by glibenclamide was unaffected by internal Mg²⁺. (2) Glibenclamide altered all kinetic parameters measured; mean open time and burst length were reduced, whereas mean closed time was increased. (3) By making the assumption that binding of glibenclamide to the sulphonylurea receptor (SUR) leads to channel closure, we have used the relation between mean open time, glibenclamide concentration and K _D to estimate binding and unbinding rate constants. We found an apparent rate constant for glibenclamide binding of 9.9 × 10⁷ m ⁻¹ sec⁻¹ and an unbinding rate of 6.26 sec⁻¹. (4) Glibenclamide is a lipophilic molecule and is likely to act on sulfonylurea receptors from within the hydrophobic phase of the cell membrane. The glibenclamide concentration within this phase will be greater than that in the aqueous solution and we have taken this into account to estimate a true binding rate constant of 1.66 × 10⁶ m ⁻¹ sec⁻¹. Received: 7 July 1996/Revised: 4 October 1996     

An L-type Calcium Channel in Renal Epithelial Cells

A voltage-activated Ca⁺⁺ channel has been identified in the apical membranes of cultured rabbit proximal tubule cells using the patch-clamp technique. With 105 mm CaCl₂ solution in the pipette and 180 NaAsp in the bath, the channel had a conductance of 10.4 ± 1.0 pS (n= 8) in on-cell patches, and 9.8 ± 1.1 pS (n= 8) in inside-out patches. In both on-cell and inside-out patches, the channel is active by membrane depolarization. For this channel, the permeation to Ba⁺⁺ and Ca⁺⁺ is highly selective over Na⁺ and K⁺ (P_Ca(Ba):P_Na(K) >200:1). The sensitivity to dihydropyridines is similar to that for L-type channels where the channel was blocked by nifedipine (10 μm), and activated by Bay K 8644 (5 μm). When activated by Bay K 8644, the channel showed subconductance levels. Treatment with forskolin (12.5 μm), phorbol ester (1 μm), or stretching (40 cm water) did not activate this channel. These results indicate that this Ca⁺⁺ channel is mostly regulated by membrane voltage, and appears to be an epithelial class of L-type Ca⁺⁺ channel. As such, it may participate in calcium reabsorption during periods of enhanced sodium reabsorption, or calcium signaling in volume regulation, where membrane depolarization occurs for prolonged periods. Received: 1 April 1996/Revised: 5 August 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     

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     

Characteristics of Single, Large-Conductance Calcium-Dependent Potassium Channels (BKCa) from Smooth Muscle Cells Isolated from the Rabbit Mesenteric Artery

Smooth muscle cells isolated from the secondary and tertiary branches of the rabbit mesenteric artery contain large Ca²⁺-dependent channels. In excised patches with symmetrical (140 mm) K⁺ solutions, these channels had an average slope conductance of 235 ± 3 pS, and reversed in direction at −6.1 ± 0.4 mV. The channel showed K⁺ selectivity and its open probability (P _o ) was voltage-dependent. Iberiotoxin (50 nm) reversibly decreased P _o , whereas tetraethylammonium (TEA, at 1 mm) reduced the unitary current amplitude. Apamin (200 nm) had no effect. The channel displayed sublevels around 1/3 and 1/2 of the mainstate level. The effect of [Ca²⁺] on P _o was studied and data fitted to Boltzmann relationships. In 0.1, 0.3, 1.0 and 10 μm Ca²⁺, V _1/2 was 77.1 ± 5.3 (n= 18), 71.2 ± 4.8 (n= 16), 47.3 ± 10.1 (n= 11) and −14.9 ± 10.1 mV (n= 6), respectively. Values of k obtained in 1 and 10 μm [Ca²⁺] were significantly larger than that observed in 0.1 μm [Ca²⁺]. With 30 μm NS 1619 (a BK_Ca channel activator), V _1/2 values were shifted by 39 mV to the left (hyperpolarizing direction) and k values were not affected. TEA applied intracellularly, reduced the unitary current amplitude with a K _d of 59 mm. In summary, BK_Ca channels show a particularly weak sensitivity to intracellular TEA and they also display large variation in V _1/2 and k. These findings suggest the possibility that different types (isoforms) of BK_Ca channels may exist in this vascular tissue. Received: 22 December 1997/Revised: 27 March 1998     

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     

Mg-Dependent, Zn-ATPase: Enzymatic Characteristics, Ion Specificities and Tissue Distribution

Mucosal crude microsomes, prepared from proximal rat small intestine, exhibited significant Mg-dependent, Zn-ATPase activity; V _max = 23 μmoles Pi/mg protein/hr, K _m = 160 nm, and Hill Coefficient, n= 1.5. Partial purification (∼10-fold) was achieved by detergent extraction, and centrifugation through 250 mm sucrose: V _max = 268 units, K _m = 1 nm, and n= 6. In partially purified preparations, the assay was linear with time to 60 min, and with protein concentration to 1 μg/300 μl. Activities at pH 8 and 8.5 were higher than at pH 7.2. The ATP K _m was 0.7 mm, with an optimal ATP/Mg ratio of ∼2. Ca elicited ATPase activity but did not augment the Zn-dependent activity. In partially purified preparations, the homologous salts of Co, Cd, Cu, and Mn exhibited no detectable activity. Vanadate inhibition studies yielded two component kinetics with a K _i of 12 μm for the first component, and 96 μm for the second component, in partially purified preparations. Tissue distribution analyses revealed gradients of activity. In the proximal half of the small intestine, Mg/Zn activity increased progressively from crypt to villus tip. In long axis studies, this activity decreased progressively from proximal to distal small bowel. Received: 12 September 2000/Revised 6 January 2001     

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 Ca²⁺ at the cytosolic face, but was detected at Ca²⁺ 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 (IC₅₀∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 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 Ca²⁺. TEA (20 mm), charybdotoxin (100 nm) and Ba²⁺ (1 mm) but not amiloride (1 mm) reduced P _o from the extracellular side. In contrast, external flufenamic acid (100 μm) 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 Ca²⁺ 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 Ca²⁺ or depolarized membrane potential. Received: 14 October 1999/Revised: 10 December 1999     

Are B-type Ca2+ Channels of Cardiac Myocytes Akin to the Passive Ion Channel in the Plasma Membrane Ca2+ Pump?

The present study demonstrates that B-type Ca²⁺ channels observed in rat ventricular myocytes markedly reacted to agents known to affect the ion-motive plasma membrane Ca²⁺-ATPase (PMCA) pump. Chlorpromazine (CPZ)-activated B-type Ca²⁺ channels were completely blocked by internal application of PMCA pump inhibitors, namely La³⁺ (100 μm), eosin (10 μm) and AIF₃ (100 μm). Calmodulin (50 U/ml), the main endogenous positive regulator of PMCA, was unable to activate but significantly reduced CPZ-activated B-type channel activity. In the same manner, ATP (1 and 4 mm), the main energizing substrate of PMCA, was able to reversibly and significantly reduce this activity in a dose-dependent manner. Interestingly, anti-PMCA antibody 5F10, but not anti-Na/K ATPase antibody (used as a negative control) induced a marked Ba²⁺-conducting channel activity that shared the same characteristics with that of CPZ-activated B-type channels. 5F10-Activated channels were mostly selective towards Ba²⁺, mainly had three observed conductance levels (23, 47 and 85 pS), were observed with a frequency of about 1 out of 5 membrane patches and were completely blocked by 10 μm eosin. These results suggest that B-type Ca²⁺ channels are some form of the PMCA pump. Received: 24 July 2000/Revised: 5 October 2000     

Studies on H+-ATPase in Cultured Rabbit Nonpigmented Ciliary Epithelium

Studies were conducted to examine the influence of the H⁺-ATPase inhibitor bafilomycin A₁ on cultured rabbit nonpigmented ciliary epithelial cells (NPE). Cytoplasmic pH and sodium concentrations were measured by digital fluorescence microscopy using BCECF and SBFI respectively. In some experiments, cell sodium content was measured by atomic absorption spectroscopy. Added alone, bafilomycin A₁ (100 nm) failed to change cytoplasmic pH but it caused an increase of cytoplasmic sodium concentration which occurred within 10 min. It is likely that the rise of cytoplasmic sodium concentration was responsible for the stimulation of active sodium-potassium transport which occurred in bafilomycin A₁-treated cells as judged by a 50% increase of ouabain sensitive potassium (⁸⁶Rb) uptake. In bafilomycin A₁-treated cells, but not in control cells, dimethylamiloride (DMA) inhibited ouabain-sensitive potassium (⁸⁶Rb) uptake in a dose-dependent manner with an IC₅₀ of ∼2 μm. DMA (10 μm) also prevented the increase of cytoplasmic sodium caused by bafilomycin A₁. Added alone, DMA (10 μm) failed to change cytoplasmic sodium content but reduced cytoplasmic pH by ∼0.4 pH units. In cells that first received 10 μm DMA, the subsequent addition of bafilomycin A₁ (100 nm) caused a further cytoplasmic pH reduction of ∼0.3 pH units. Taken together, the results suggest H⁺-ATPase might contribute to the regulation of basal cytoplasmic pH in cultured NPE. In the presence of bafilomycin A₁, Na-H exchanger activity appears to be stimulated, so stabilizing cytoplasmic pH but resulting in an increase of cytoplasmic sodium concentration and consequent stimulation of active sodium-potassium transport. Received: 19 March 1999/Revised: 20 September 1999