Large conducting potassium from airway smooth muscle

channel

reconstituted

DIANE SAVARIA, CHANTAL LANOUE, ALAIN CADIEUX, AND ERIC ROUSSEAU Departments of Physiology and Biophysics and of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec Jl H 5N4, Canada Savaria, Diane, Chantal Lanoue, Alain Cadieux, and Eric Rousseau. Large conducting potassium channel reconstituted from airway smooth muscle. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L327-L336,1992.-Microsomal fractions were prepared from canine and bovine airway smooth muscle (ASM) by differential and gradient centrifugations. Surface membrane vesicles were characterized by binding assays and incorporated into planar lipid bilayers. Single-channel activities were recorded in symmetric or asymmetric K+ buffer systems and studied under voltage and Ca2’ clamp conditions. A large-conductance K+-selective channel (>220 pS in 150 mM K+) displaying a high Ca2’, low Ba2+, and charybdotoxin (CTX) sensitivity was identified. Time analysis of single-channel recordings revealed a complex kinetic behavior compatible with the previous schemes proposed for Ca2+-activated K+ channels in a variety of biological surface membranes. We now report that the open probability of the channel at low Ca2+ concentration is enhanced on in vitro phosphorylation, which is mediated via an adenosine 3’,5’-cyclic monophosphate-dependent protein kinase. In addition to this characterization at the molecular level, a second series of pharmacological experiments were designed to assess the putative role of this channel in ASM strips. Our results show that 50 nM CTX, a specific inhibitor of the large conducting Ca2+ -dependent K+ channel, prevents norepinephrine transient relaxation on carbamylcholine-precontracted ASM strips. It was also shown that CTX reversed the steady-state relaxation induced by vasoactive intestinal peptide and partially antagonized further relaxation induced by cumulative doses of this potent bronchodilatator. Thus it is proposed that the Ca2+-activated K+ channels have a physiological role because they are indirectly activated on stimulation of various membrane receptors via intracellular mechanisms. planar lipid bilayers; calcium-activated canine trachealis muscle

potassium

channels;

THE SURFACE MEMBRANE of the airway smooth muscle (ASM) supports various types of ionic currents that have

been studied indirectly with microelectrode measurements on tissue strips (19) or directly with the patchclamp methods on isolated enzymatically dissociated cells (15, 21, 22, 28). The regulation as well as the physiological role of these currents has not been fully elucidated, and little information regarding ionic processes is available. However, it has been suggested that unitary currents through Ca2’ -selective voltage-sensitive channels could play a role in excitation-contraction coupling in mammalian ASM because in vitro assays have shown that Ca2+ channel antagonists inhibit the positive myotropic effects of different substances known as powerful bronchoconstrictors (4, 16). Alternatively, the bronchodilation (or relaxation episodes) might be due to K+ conductance increases that would be associated with a surface membrane hyperpolarization, thus inactivating Ca2+-selective channels from the surface membrane (28, 30). On the other hand, a 1040-0605/92

$2.00

Copyright

decrease in K+ conductance would induce a slight depolarization of the ASM cells as demonstrated for other tissues such as vascular smooth muscles and ,&cells (32), thus facilitating Ca2+ entry (4) and subsequent Ca2’ release from internal stores mediated by various intracellular mechanisms (23, 29, 32). Recently, studies involving whole cell patch-clamp measurements on isolated canine, guinea pig, and human ASM cells have identified voltage-dependent Ca2+ and K+ currents (21, 22, 25, 28). Furthermore, several types of K+ channels have been identified in smooth muscle cells and are potential candidates to modulate changes in the surface membrane conductance on stimulation of various receptors (9) or during in vitro voltage-clamp experiments (3,6,8,9, 28). The present study was undertaken to examine the functional properties and to clarify the physiological role of single channel protein derived from the surface membrane of ASM cells. To achieve this goal, microsomal fractions from ASM were prepared, characterized, and, for the first time, fused into planar lipid bilayers (PLBs). Previously, PLB measurements have essentially been used to study the electrical properties of channels from intracellular membranes (2, 35). Despite its limited time resolution due to the low cutoff frequency (500 Hz) used, such an approach represents an alternative to patchclamp experiments performed on isolated myocytes. This report confirms the presence of a functional Ca2+-activated K channel in structures derived from ASM surface membranes. Furthermore, pharmacological experiments were designed to investigate the putative role of this specific pathway in trachealis muscle strips. Some of these results have been communicated elsewhere in abstract form (37). MATERIALS

AND

METHODS

Preparation of the microsomaZ fractions. Microsomal fractions derived from ASM membranes were prepared either from canine or from bovine tracheae. Mongrel dogs weighing 20-25 kg were anesthetized with pentobarbital sodium (40 mg/kg) and exsanguinated. The trachea was removed intact and placed in cold Krebs solution. Bovine tracheae were obtained from the local slaughterhouse. Smooth muscle leaflets were dissected from the epithelium and the serosal layer of the tissue, rinsed in Krebs solution (4”C), and homogenized (1 g/10 ml) in a buffer containing (in mM) 300 sucrose, 20 K-piperazine-N,N’-bis( 2-ethanesulfonic acid) (PIPES), 4 K-ethylene glycol-bis(P-aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA), and 2.5 ml lima bean trypsin inhibitor, 0.1 diisopropyl-fluoro-phosphate, 0.01 indomethacin, 0.5 dithiothreitol, pH 7.0. The homogenate was centrifuged at 8,500 rpm for 20 min, at 4”C, in a type 35 rotor (Beckman). The supernatant was filtered through two layers of cheesecloth and centrifuged at 35,000 rpm for 1 h (90,000 g) in a Ti 42.1 rotor (Beckman). The pellets were resuspended

0 1992 the American

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and pooled in a buffer containing (in mM) 300 sucrose, 100 KCl, 0.1 MgCl,, 1 EGTA, and 5 K-PIPES, pH 7.0. This crude fraction was either stored at -85°C or centrifuged at 26,000 rpm through either a discontinuous Percoll (20,25,30,40,55% vol/vol) or sucrose (20, 25, 30, 35, 40% wt/wt) gradient for 25 min or 3 h (14), respectively, in a Beckman SW 60 rotor. Four fractions (MT-M& were recovered from the gradients, diluted in seven volumes of a solution containing (in mM) 100 KCl, 5 K-PIPES, 0.1 K-EGTA, 0.1 CaC12, pH 7.0, and sedimented by centrifugation for 1.5 h at 35,000 rpm in a Ti 42.1 rotor. Finally, the pellets were resuspended in 300 mM sucrose and 5 mM KPIPES, pH 7.0, quickly frozen in liquid nitrogen, and stored at -85°C (14,27,35). Biochemical assays. Protein concentrations were determined by the Lowry method with bovine serum albumin as a standard. Free Ca2+ concentrations ( [Ca2’]) were adjusted using a CaEGTA buffer and were calculated using binding constants and a computer program published by Fabiato (11). 1251-labeled Tyrbradykinin (27) and C3H]ouabain (18) binding was determined in the various microsomal fractions with optimal binding conditions as previously described. Ryanodine binding assays were performed using the procedure described by Anderson et al. (2) with slight modifications. Briefly, [3H]ryanodine (with or without nonradioactive ryanodine) was incubated with the various vesicle populations (250 pug of protein) from each fraction for 2 h at 37°C. The mixture was then centrifuged, and pellet radioactivity was determined by liquid scintillation. Specific ryanodine binding was calculated by subtracting nonspecific binding (10 mM [3H]ryanodine + 10 nM ryanodine) from total binding (10 nM [3H]ryanodine alone). Cold ryanodine was a gift from Dr. L. Ruest, Department of Chemistry, University of Sherbrooke. Because the presence of dihydropyridine binding sites and sensitive channels have been reported in surface membrane of ASM cells (20, 40), dihydropyridine binding experiments were also performed using [3H]nitrendipine as specific ligand on the various microsomal fractions as previously described w B’ilczyer formation and vesicle fusion. The PLB were formed from a lipid mixture containing phosphatidylethanolamine, 1,2bis(oleoyloxy)-3(trimethylammonio)propane (DOTAP), and phosphatidylcholine in a ratio of 5:3:2. The final lipid concentration was 25 mg/ml dissolved in decane. A 250~pm diam hole, drilled in polyvinyliden-difluoride or Delrin cups, was pretreated with the same lipid mixture dissolved in chloroform (35). With use of a Teflon stick, a drop of the decane lipid mixture was gently spread across the hole to obtain an artificial membrane. Membrane thinning was assayed by applying a triangular wave test pulse, and typical capacitance values were 250-400 pF. Aliquots of vesicle suspensions (IO-60 pg of protein) were added to the cis chamber in the proximity of the bilayer. In our standard conditions both chambers contained (in mM) 150 K-gluconate, 1 CaC12, 5 K-N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), pH 7.2. The fusions were either spontaneous, induced by stirring, or obtained by applying positive holding potentials across the bilayer. They were monitored as discrete K’ conductance increments due to the presence of K-+-selective channels in the vesicles derived from the surface membrane (15, 22, 25). In most of our experiments K-gluconate was substituted for KC1 to eliminate the contaminating electrical activity due to the simultaneous incorporation of Cl-selective channels. All bilayer experiments were performed at room temperature (20 t 2°C). Recording instrumentation and signal analysis. The currents were recorded using a low noise operational amplifier Dagan 8900. The currents were then filtered (cutoff frequency 10 kHz) and recorded on a video cassette recorder through a modified pulse code modulation device (DAS/VCR 900, Unitrade). The currents were simultaneously displayed on-line on a chart

FROM

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recorder (DASH II MT, Astro Med.) and an oscilloscope (Kikusui 5040). Current recordings were played back, filtered at 500 Hz, and sampled at 1.5 kHz for storage on hard disk and for further analysis with an HP-Vectra computer with programs kindly provided by Dr. M. Nelson, University of Vermont. The open probability values (PO) and time histograms were determined from data stored in 40-s duration files unless specified otherwise. Applied voltages are defined with respect to the trans chamber, which was held at virtual ground. Tissue preparation for pharmacological experiments. Guinea pigs (Hartley strain) weighing 250-400 g were killed by exsanguination after intraperitoneal injection of pentobarbital sodium (50 mg/kg). The trachea and the lungs were quickly removed and placed in cold Krebs solution. The trachea was separated at its junction with the main bronchi and cut helically as previously described (5). Each preparation was mounted in a 5-ml jacketed organ bath containing Krebs solution gassed with 95% 02-5% Co2 and maintained at 37°C. Contractions and relaxations were measured isometrically with a Grass FT03 force displacement transducer and recorded on a Grass polygraph (model 7D) as tension changes. All tissues were subjected to an initial loading tension of 1 g and allowed to equilibrate for 60 min (with changes of bath medium every 15 min) before starting the experiments (5, 24). Chemical reagents and solutions. Trizma base [tris(hydroxymethyl)aminomethane (Tris)], HEPES, PIPES, carbamylcholine chloride, arterenol hydrochloride (norepinephrine), bradykinin, vasoactive intestinal polypeptide (VIP), a-catalytic subunit of protein kinase A, and indomethacin were obtained from Sigma Chemical (St. Louis, MO). Phospholipids were purchased from Avanti Polar Lipids (Birmingham, AL). The radioligands used for binding assays were purchased from New England Nuclear (Mississauga, Ontario). All other chemicals were of reagent grade. Glass bidistilled deionized water was used for preparing all buffer solutions. The Krebs bicarbonate solution was prepared as follows (in mM): 118.1 NaCl, 4.7 KCl, 1.2 MgS0,=7H20, 1.2 KH2P04, 25 NaHC03, 2.5 CaC12, and 11 glucose, pH 7.4. Purified charybdotoxin was a gift of Dr. M. Garcia from Merck (Rahway, NJ). RESULTS

Characterization of the microsomal fractions. Four microsomal fractions (MI--Mrv) were recovered from the gradient centrifugations. They were characterized by binding assays using three different markers of cell membranes. Figure 1 shows the results of binding experiments performed on these ASM membrane populations. The largest number of lz51-Tyr-bradykinin binding sites was observed in fraction M Iv, suggesting that this fraction was slightly enriched in vesicles derived from the surface membranes of ASM (Fig. IA) because the bradykinin receptor is considered a marker of the plasma membrane (27). With use of [3H]ouabain as a specific marker of the surface membrane Na+-K+-adenosinetriphosphatase, a similar binding profile was obtained (Fig. 1B). On the other hand, [3H]ryanodine binding sites were enriched in the Mrrr fraction (Fig. lC), which suggest that this microsomal fraction mainly contains vesicles derived from intracellular membranes. Because ryanodine specifically binds to the Ca2+-release channel, the ryanodine receptor is a recognized marker for the sarcoplasmic reticulum (SR) membrane (2). Complementary binding experiments involving a specific ligand for the dihydropyridine binding sites, another cell surface marker, were performed on the various frac-

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somal fractions recovered from the sucrose gradients and those from the Percoll gradient, except that the fractions isolated on Percoll were more fusogenic into PLB. PLB measurements. Since we were mostly interested in studying the electrical properties of the cha nnels from the ASM surface, membrane-only vesicles from the Miy fraction were fused into PLBs in the presence of 1 mM Ca2’. Single-channel activity was initially recorded in a symmetric 150 mM K-gluconate buffer system as a function of voltage. Figure 2 shows the typical current fluctuations of a single channel. No trace superimposition was observed despite the high P, (>0.85 for positive voltages) Unit current amplitudes were directly proportional to the ho lding potentials applied across the bilayer . Many of the close events were so brief in duration that they were not fully resolvable at the cutoff frequency (500 Hz) used in this illustration. At a given voltage, the channel showed no sign of ti me-dependent inactivation. Howeve r, at negative holding potentials, the P, decreased slightly and the channel activity displayed infrequent but long closed events. This typical behavior was currently detected during PLB experiments because 80% of fusion events resulted in single-channel recordings. Because most of the vesicles were right-side out, the cytoplasmic side of the channels automatically faces toward the trans chamber on fusion into PLB. Conductance and selectivity. The conductance and the HP

150

mM

KCluc

CIS / TRANS

+ 60 mV

6

+ 50 mV

MI

M II

MIII

MIV

Fig. 1. Biochemical characterization of microsomal fractions. A: bar histograms show that higher level of 1251-Tyr-bradykinin binding was observed in fraction M iv. Data shown represent means t SE of 3 experiments performed in triplicate. B: [3H]ouabain binding profile for various microsomal fractions shows that the Na’-K+-ATPase, a specific plasma membrane marker, is enriched in fraction Miv. C: [3H]ryanodine receptor complexes were concentrated in subcellular fraction Mrir. Histogram values represent means t SE of 3 experiments performed in triplicate on 3 different membrane preparations. 12?-Tyr-bradykinin and [ 3H] ryanodine bindings were performed as described in Biochemical assays.

tions. However, the [3H]nitrendipine binding assay was not sufficiently discriminative between the different fractions (data not shown). It was also suspected that during the homogenization process, a number of vesicles could have been sealed in the inside-out configuration. Consequently, parallel binding assays were designed and performed in the absence and presence of 50 pg/ml saponin to destabilize cholesterol-containing membrane. No additional specific binding was detected on saponin treatment. This observation supported the view that most of the vesicles of the various fractions were right-side out. No major differences were detected between the micro-

+ 30 mV

1 5 PA 1 s -1OmV

- 20 mV

Fig. 2. Single-channel recordings as function of voltage applied across planar lipid bilayer. Unitary currents were obtained in symmetric 150 mM K-gluconate buffer solutions plus 1 mM CaC12 and 5 mM KHEPES, pH 7.2, after fusion of vesicle from Miv fraction. Holding potentials (HP) are specified on left of each trace. In this case electrical signals, recorded on videotape, were replayed and illustrated on slow time scale using chart recorder (cutoff frequency 500 Hz). Closed and open arrows correspond to closed and open levels, respectively.

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18 Fig. 3. Current-voltage curves obtained in various buffer systems and typical amplitude histogram. Triangles, experiment performed in symmetric 150 mM K-gluconate. Average unit conductance was 225 pS (n = 4). Squares, average data points from experiments performed in asymmetric K-gluconate (300 mM cis150 mM trans). Average unit conductance was 264 pS (n = 5). Reversal potential was -17 mV. Circles, data from experiment in asymmetric KC1 (250 mM cis-50 mM trans); unit conductance calculated for voltages above the reversal potential (-40 mV) was 195 pS. All data were obtained in presence of 1 mM Ca2+. Inset: amplitude histogram from current trace obtained at HP of +30 mV in asymmetric K-gluconate (300 mM cis-150

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selectivity of this reconstituted pathway from ASM membrane were assessed under various experimental conditions. Figure 3 shows current-voltage relationships obtained with different K+ concentrations. In symmetric 150 mM cis-trans K-gluconate, the unit conductance was 225 t 12 pS (n = 4), and the reversal potential (RP) was found at 0 t 4 mV. In the presence of asymmetric K+ concentrations (300 mM K+ cis and 150 mM K+ trans) the unit conductance was 265 t 15 pS (n = 5), and the apparent RP (-17 mV) was very close to the theoretical equilibrium potential (-17.8 mV) calculated from the Nernst equation for K+ concentration ([K+]). The third curve represents data from an experiment in asymmetric (250 mM cis-50 mM trans) KC1 where the RP was close to -40 mV and the conductance, calculated for voltage above the RP, was 195 pS. These shifts of the reversal potential with changing [K’] prove the K+ selectivity of the channel. An alternative approach in assessing the K+ selectivity of the channel was to perfuse both chambers with K+-free solution. For instance, perfusion with choline chloride reduced the current to background level (not shown). Voltage dependence. The voltage dependence of this large conducting K+-selective channel was quantified, as reported in Fig. 4. The average steady-state PO values were plotted as a function of the holding potential for two [Ca”‘]. In presence of 1 mM Ca2+ on both sides of the channel, the PO values remained high over a large voltage range (-40 to +60 mV). At lower [Ca”‘] (~10 PM) on the cytoplasmic face of the channel, P, values were decreased over the physiological voltage range and the voltage dependency was greatly affected. This behavior was very consistent from one recording to the other. Ca2+ sensitiuity. The reconstituted K+ channel was markedly dependent on trans free [Ca”‘] as illustrated in Fig. 5. Step decreases of free [Ca”‘] in the trans chamber (cytoplasmic face of the channel), produced by cumulative addition of Tris-EGTA, induced a Ca2+-dependent channel deactivation (Fig. 5A). At submicro-

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Fig. 4. Voltage dependence of K+ channel activity. Open probabilities (PO) were calculated from amplitude histograms obtained from data stored in 40-s data files. Closed symbols, data obtained from experiments in symmetric 150 mM K-gluconates in presence of 1 mM Ca2+. Open symbols, data determined from experiments in lower Ca2+ (-10 PM free trans-Ca2+). Note shift of voltage sensitivity and absence of P, values at 0 mV, which correspond to reversal potential in symmetric K+ buffer.

molar free [Ca”‘] (CO.1 PM) the channel activity was very low (P, < 0.01). However, raising the free [Ca”‘] to 500 PM in the trans chamber fully restored the channel activity attesting the reversibility of the effect of the divalent cation on the gating behavior of this channel (Fig. 5A, bottom trace). The Ca2+ sensitivity was quantified and reported in Fig. 5B. A sigmoidal relationship was obtained by plotting steady-state P, values against the pCa values in the trans chamber. The channel was maximally activated (PO > 0.9) at free [Ca”‘] higher than 100 PM (pCa 4). The half-maximal activation was obtained for a free [Ca”‘] close to 10 PM (pCa 5). Note that changes in free [Ca”‘] in the cis chamber, ranging from 1 mM down to 10 PM, have no effect on the singlechannel activity. Time analysis. The distribution of all open and closed intervals from a single-channel recording are shown in Fig. 6. A large number of intervals were collected at +30 mV in the presence of 10 PM free trans [Ca”‘] when the

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PCs Fig. 5. Ca2+ sensitivity of large conducting K+ channel. A: single K+ channel currents, shown as upward deflections, as function of trans Ca2+ concentration ([Ca”‘]). Current traces were obtained in asymmetric K-gluconate buffer (300 mM cis-150 mM trans) plus 1 mM CaC12 and 5 mM K-HEPES, pH 7.2, in both chambers (top trace). HP was +30 mV. On cumulative addition of EGTA, free [Ca”‘] in trans chamber was decreased to 12 and 0.3 PM, 2nd and 3rd traces, respectively. P, was reduced at submicromolar [Ca”‘]. On addition of CaC12 in trans chamber (500 PM free Ca2+), channel reactivated (bottom trace). Recordings were filtered at 500 Hz and sampled at 2 kHz. B: Ca2+ doseresponse curve for channel P,. Fraction of channel open time was determined on single-channel recordings from 2 independent experiments (open and closed circles) obtained as described above. HP, +30 mV. Free [Ca”‘] in trans chamber calculated by computer program (10). Direct measurement of free Ca2’ was not feasible because of sequential addition of EGTA and Ca2’ during experiments. P, values were calculated from amplitude histogram analysis.

channel was half-maximally activated (P, = 0.53) (Fig. 6A). Open and closed time histograms were both fitted with a double exponential (Fig. 6, B and C). Two open time constants (70Pl10.5 and 70p261.9 ms) and two closed time constants ( 7cll 8.5 and 7~12 43.6 ms) were determined under such experimental conditions. Furthermore, both distributions were characterized by a majority of short duration intervals. Note that the maximal value for the longer classified events was set at maximal time of 400

x102

3

4

[ ms ]

Fig. 6. Time histogram of open and closed states of Ca2+-activated K+ channel. A: time analysis was performed from half-maximal activated single channel recorded in asymmetrical K-gluconate (150 mM trans300 mM cis) plus 10 PM trans free [Ca”‘] (pCa 5). Open time (B) and closed time (C) distributions were fitted with double exponential, which indicated time constants (7). NB, number.

ms and that no long event was missing. However, at lower trans free [Ca”‘] (as shown above, Fig. 5A) or in the presence of inhibitory compounds [as illustrated below with charybdotoxin (CTX)], long-lasting closed events (>l s) were also detected during single-channel recordings. The time analysis of such low P, recordings, when performed on several minute duration file to resolve an adequate number of events, revealed that the closed time distribution might be fitted with the sum of three exponentials. The occurrence of a third closed time constant was ascribed to the presence of an inactivated state as previously described (34-39). This inactivated state was responsible for the typical bursting behavior of this channel. On the other hand, the distributions of the open and closed time histograms of a fully activated channel, as obtained in the presence of high [Ca”‘], were fitted with two open and one closed time constant, respectively. Accordingly, time analysis of the reconstituted Ca2+-activated K+ channel revealed that the minimum kinetic model would involve two open states, two closed states, and at least one inactivated state. Inhibition by CTX and divalent cation. The channel described herein displayed several features of a typical Gk(c+ This view was further supported by evaluating the

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sensitivity of this channel to CTX and Ba2’. CTX is a small polypeptide, found in the venom of the Israeli scorpion Leiurus quinquestriatus. This toxin is known to be a specific inhibitor of high-conductance, Ca2+-activated K+ channels (38). In Fig. 7, we have illustrated the effect of this toxin on channel activity. Figure 7A was obtained in control conditions, for a holding potential of +30 mV. Within a few seconds after the addition of 10 nM CTX in the cis chamber, external face of the channel, long lasting nonconducting states were observed (Fig. 7, B and C). As the CTX concentration was increased (up to 50 nM), channel inhibition was more pronounced (Fig. 70) involving very long “blocked” or inactivated state and shorter bursts of open events. This behavior was previously described as-the typical mode of action of CTX on Gk(Ca) in native or artificial membranes (38). The CTX inhibition was also characterized by its lack of reversibility, even after extensive washout of the contaminated chamber. As a matter of fact, CTX inhibition was utilized at the end of the experiments involving multiple channel activities to ascertain their identity and the number of Gktca) present in the PLB. We have also obtained evidences that the activity of the K+ channel under scrutiny could be inhibited by Ba2+ when this divalent cation was present on the cytoplasmic face of the channel . Figure 8, A and B, / shows the channel activity in the absence-and in the presence of 1 mM Ba2’ in the trans chamber. Addition of Ba2+ induced an obvious decrease in the fraction of channel open time. This was essentially due to the long closed time and to the reduced duration of the open events within a burst. Accordingly, it was postulated that Ba2’ might interfere with the Ca2+-activating binding site(s) of the K+ channel. However, because the apparent amplitude of the open events was reduced and despite the fact that this reduction of the amplitude could be explained by the use of the low cutoff frequency (500 Hz), a putative ionic block of the aqueous pore by the divalent cations cannot be ruled out. Modulation of reconstituted K+ channel. In various preparations, patch-clamp studies have suggested that 300

mM

KGluc

cis \ 150

mM

KGluc

trans

HP = + 30

AIRWAY

SMOOTH

Ca2+-activated K+ channels can be modulated by intracellular second messengers (8, 9, 25) other than Ca2+. An attempt was made in our experimental setup to visualize the putative effect of adenosine 3’,5’-cyclic monophosphate-dependent protein phosphorylation of the reconstituted K+ channel. Figure 9 illustrates the results obtained from a multiple-channel recording with use of bovine ASM vesicles. Three large-conductance K+ channels were activated in the presence of 1 mM Ca2+ (Fig. 9A). On cumulative additions of Tris-EGTA, the free [Ca”‘] in the trans chamber was sequentially decreased to 10 and 1 PM, respectively, resulting in a marked inhibition of channel openings (Fig. 9, B and C). Only one open current level was observed after the final addition of EGTA (pCa 6). However, a few minutes after the simultaneous addition of CAMP, ATP, and the catalytic subunit of the protein kinase A in the trans chamber, the channel partially reactivated without an increase in free [Ca”‘]. The overall behavior of the channels was quantified on corresponding current density histograms (Fig. 9, A ‘, B ‘, C’, and 0’). After the addition of the phosphorylation cocktail, a relative increase in channel activity was consistently observed. For instance, a fivefold increase was detected in the presence of 1 PM free Ca2+ (Fig. 9D ’ compared with C’) and a threefold increase was detected in the presence of 0.5 PM free Ca2+ (data not illustrated). Note that the phosphorylation did not modify the unitary current amplitude and the conductance of the channels. These results strongly suggest that the K+ channels reconstituted from bovine ASM vesicles are sensitive to Ca2+ changes and that a direct phosphorylation of the channel proteins, or closely associated subunits, induces an increase of the P,. Physiological implication. One of our objectives was to demonstrate the putative implication of Ca2+-activated K+ channels in modulating the contractile behavior of the ASM strips. Consequently, we tested the effect of CTX, the specific inhibitor of this pathway, during a conventional tension measurement. All the experiments were performed on guinea pig trachealis muscle strips as described in MATERIALS AND METHODS. In Fig. 10,the

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Fig. 7. Inhibitory effect of charybdotoxin (CTX) on reconstituted K+ channel. Unitary currents were recorded in asymmetric K-gluconate buffers in presence of 1 mM Ca2+ and 5 mM K-HEPES, pH 7.2. A: control conditions (P, 0.90). B: 30 s after addition of 10 nM CTX. C: 3 min (P, 0.22) after addition of 10 nM CTX. D: steady-state inhibition induced by 50 nM CTX. Note long nonconducting states and short bursts (PO 0.02).

50 nM CTX

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A 150 mM KGIuc CIS / TRANS Fig. 8. Ba2+ inhibition of high-conductance K channel. Data taken from experiment performed in symmetric 150 mM K-gluconate buffer plus 0.1 mM Ca2+ and 5 mM K’ HEPES, pH 7.2. HP +30 mV. A: control condition. B: after addition of 1 mM BaC12 in trans chamber (cytoplasmic face). Channel activity was strongly inhibited; only residual bursts of open events were detected.

a 4

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Buz+ a

top recording demonstrates the contractile responses, triggered by the addition of 0.1 PM carbachol. At the plateau level a large transient relaxation was induced after the addition of 1 PM norepinephrine (NE), still in the presence of the muscarinic agonist (Fig. 10A). This control test, involving exogenous agonists, was performed on each strip used in our study. It was followed by successive washings of the experimental chamber with fresh drug-free Krebs solution. After the relaxation process was completed, the same strip was contracted by addition of 0.1 pM carbachol. Addition of CTX at the plateau level had no effect. Furthermore, in the presence of CTX, 1 PM NE was virtually ineffective in producing a relaxation, when compared with control conditions. Taken together, these results suggest that during muscarinic stimulation, the Ca2+-activated K+ channels are closed because CTX has no effect. However, Gk(ca) might be activated on ,&agonist stimulation because NE induce a large relaxation, which is blocked by CTX treatment. Separate experiments revealed that CTX (up to 100 nM) had no effect on the resting tension and did not affect

carbachol responses but consistently abolished NE relaxations (data not illustrated). VIP induced a relaxation of the resting tension of a guinea pig ASM strip (Fig. 10B). The steady-state relaxation was reversed by two successive washouts to remove the active peptide. Ten minutes after recovery of the resting tension, the same strip was challenged for another relaxation trial with the same (0.1 PM) concentration of VIP. The addition of 50 nM CTX consistently reversed this relaxation. Furthermore, the presence of CTX prevented or at least reduced the subsequent relaxations when the muscle strip was challenged with cumulative doses of VIP. Such results attest that a CTX-sensitive pathway could be activated during the relaxation process. It can be assumed that the opening of large-conducting Ca2+-activated K+ channels would facilitate the hyperpolarization of cell membrane, thus inducing inactivation of surface membrane Ca2+ channels. This plausible mechanism would prevent further Ca2+ entry (through sarcolemma Ca2’ channel) and/or Ca2+ release from internal stores, thus allowing ASM fiber relaxation.

A 1 mM Ca2+

A’ 30

10 200 u 0

8’ 1OpM

10

20

30

B’

Ca2+

8 6 4

rc) 0

2

0 0

X

P C’ g Id k

C 1 FM Ca2+ I

-l 11s

I

5 PA

g

I,

10 CURRENT

20 [ pA

30 ]

4 3

t

I

2 1 0 0

D + 20 pM ,AMP

+ 20 U/ml

PKA + 100 NM ATP

10

20

30

Fig. 9. Modulation of bovine K+ channels activity by Ca2+ and CAMP-dependent protein kinase. Sequential traces were obtained after fusion of vesicle derived from bovine trachea (fraction MI”) in asymmetric K+ buffer as described in Fig. 5. HP +30 mV. A: control condition, with 1 mM CaC12 in both chambers; 3 channels were completely activated. B and C: on cumulative addition of EGTA, free [Ca”‘] in trans chamber was decreased to 10 and 1 PM Ca2+, respectively. Channel activity was reduced. D: 10 min after addition of a-catalytic subunit of CAMP-dependent protein kinase (20 IU/ml) in presence of 20 PM CAMP and 100 PM ATP. Channels reactivated. A ‘, B’, C’, and D’: corresponding amplitude histograms.

D’

b

Downloaded from www.physiology.org/journal/ajplung at Macquarie Univ (137.111.162.020) on February 14, 2019.

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A NE 1 pM NE 1 pM

1.4 A

CARB

g

1.2 .1 pM 4 min

’

CTX 50nM Fig. 10. CTX sensitivity of norepinephrine (NE)and vasoactive intestinal polypeptide (VIP)-induced relaxation on guinea pig trachealis muscle strips. A: airway smooth muscle (ASM) strip was contracted on addition of 0.1 PM carbachol (Carb) and transiently relaxed by 1 PM NE. This control was followed by successive washouts (W) of experimental chamber with fresh Krebs solution. Then, same strip was contracted by addition of 0.1 PM carbachol and challenged for relaxation with 1 PM NE 3 min after addition of CTX, specific inhibitor of large conducting Ca2+sensitive K’ channels. Note that relaxation induced by NE (1 PM) on carbachol-induced tone was highly reduced in presence of CTX. B: VIP induced relaxation of resting tension of guinea pig ASM (loading tension 1 g). This relaxation was reversed by 2 successive washouts of ASM strip. Second application of 0.1 FM VIP induced similar relaxation. However, addition of 50 nM CTX before plateau was reached reversed VIP-induced relaxation. Tone returned to pre-VIP tension level. Furthermore, presence of CTX prevented relaxation of ASM strip on addition of 0.1 PM VIP. Further 4-fold increase of initial dose of VIP induced slight relaxation. Both sets of results support hypothesis that CTX-sensitive pathway could be activated during relaxation process in ASM. DISCUSSION

In the present study, four microsomal fractions were isolated from ASM membranes as a function of their buoyant density. Because the isolation procedure described herein did not abolish the binding properties, the vesicle populations were characterized according to their capacity to bind specific surface and SR membrane markers. The results shown indicate that the Miv fraction was slightly enriched in vesicle population derived from the surface membrane (Fig. 1, A and B), whereas the Miii fraction was enriched in intracellular membranes (Fig. 1C). Although a thorough comparison of binding capacity between native membranes and isolated vesicles was not performed, due to obvious technical difficulties, the absence of enzymatic treatment during the isolation procedure may explain the preservation of the bradykinin receptors. The ryanodine receptors that were shown to be equivalent to the SR Ca2+ release channel (2) were preserved by the addition of protease inhibitors. Their accessibility was certainly improved by the vesiculation procedure because these receptors are localized on the large cytoplasmic domain of an intracellular transmembrane protein (2). Other studies have shown that it was possible to prepare membrane vesicles from various types of smooth muscles (14, 26). Our observations correlate well with a previous report by Kwam et al. (26), showing that a subfraction F3 derived from rat vas deferens smooth muscle was enriched in specific enzymatic marker of the endoplasmic reticulum membrane. We now demonstrate that ASM vesicles can be fused into PLB to study unitary currents through specific channel pro-

teins under voltage-clamp conditions. This approach represents an alternative to the patch-clamp method for studying the electrical properties of this small smooth muscle cell (6 pm in diam). Because the aim of this work was to investigate the functional properties of surface membrane channels from ASM preparations, the fraction MIv was currently used for our reconstitution experiments, thus allowing a direct access to both sides of the incorporated channels. The channel activity recorded in our standard experimental conditions (Figs. 2 and 5) shares several features with the well-characterized Gkcca). The reconstituted channels retained the same cation selectivity (Fig. 3), similar unit conductance values (~220 pS in 150 mM K+), and the same CTX sensitivity as the typical Gk(Ca) observed in native surface membranes from various tissues (30, 31, 38, 41) including smooth muscle cells (1, 6) studied either with the patch-clamp or the PLB technique (25, 39). However, in our system, the [Ca”‘] required to reach the half-maximal activation of the channel, 10 ,uM (Fig. 5B), was relatively high when compared with typical values ranging from 0.5 to 1 PM, determined for Gktca) (39) and other channels such as the cardiac SR Ca2+ release channel (35). Thus we assume that this discrepancy in the Ca2+ sensitivity of the channel is related to the composition of our lipid matrix, which contains 30% DOTAP, a positively charged lipid used to facilitate vesicle fusion, which affects the microenvironment in the vicinity of the reconstituted K+ channels. Earlier studies reporting lower half-maximal activation values of 0.6-l PM for the Ca2+-activation curves of Gktca) were performed on negatively charged membranes that

Downloaded from www.physiology.org/journal/ajplung at Macquarie Univ (137.111.162.020) on February 14, 2019.

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were excised patches of native membrane (13, 33, 34) or PLB containing 30% of phosphatidylserine, a negatively charged phospholipid at physiological pH (39). However, under our experimental conditions, the kinetic behavior described for the ASM Ca2+-dependent K+ channel (Fig. 6) is identical with the kinetic pattern proposed for other large-conductance Ca2+-activated K+ channels when either studied in patch of native membranes (33, 34) or in negatively charged PLB (39). In all cases, the minimum kinetic scheme involves two open, two closed, and one long-lasting inactivated state. Consequently, the composition of the lipid matrix could affect the channel’s sensitivity to Ca2+ but not its intrinsic gating behavior, its conductance, or its selectivity. We have also shown that the addition of Ba2’ to the cytoplasmic fate of the Gk(oa) ( trans side in this case) dramatically modified its gating behav ior. This blockage induced by Ba2’ represents an .other hallmark of Gk(ca) as previously reported on fusi .on of inverted transverse tubule membrane vesicles from rabbit skeletal muscle (39). At the present time, the exact role of these Gk(ca) is not clear. They may contribute to the modulation of the membrane potential in trachealis muscle and bronchi. Various intracellular agents might control the K+ permeability through the Ca2’ -activated channels. A likely candidate susceptible of activating this pathway would be free intracellular Ca2+ concentration because changes in [Ca2+li could result either from Ca2+ entry via sarcolemma1 Ca2+ selective channels (22) or from Ca2+ release from internal stores (21, 36). In ASM and other smooth muscle tissues, drug-induced variations of free Ca2+ occur in a very narrow range; 0.1 < [ Ca2+]i < 0.3 (Zl), suggesting that [Ca2+]i is not the only activator of this channel. For instance it was shown that isopren .aline, a p-adrenergic agonist, as well as okadai .c acid, a protein-phosphatase inhibitor, and protein kinase A (in the presence of 100 PM CAMP, 5 mM Na2ATP, and 0.1 PM Ca2+) were able to activate Ca2+-dependent K+ channels in membrane patches from isolated tracheal myocytes (25). Data presented in Fig. 9 support these observations because the activity of the reconstituted Ca2+-dependent K+ channels was enhanced under conditions susceptible to yield in vitro phosphorylation. Altogether, these results emphasize the hypothesis that a phosphorylation, or a lack of dephosphorylation, of the channel proteins modulates their gating behavior by shifting their Ca2+ sensitivity toward lower [Ca”‘] as already reported for other tissues (10). In ASM tissues, a CAMP-dependent phosphorylation of Ca2+- activated K+ channels could occur on NE and VIP stimulation because both bronchodilating agents are known to increase the level of intracellular CAMP (25). The resulting activation of this pathway would support a membrane repolarization on NE addition after the carbachol stimulation or a slight hyperpolarization on VIP stimulation and thus facilitate cell relaxation. Although there is still controversy in the literature about the exact mechanism that controls the VIP-induced relaxation (7, l7), the results from our pharmacomechanical experiments have clearly demonstrated that CTX prevented or reversed NE- and VIPinduced relaxation, respectively (Fig. 10). Consequently, the mechanism described is likely to occur in vivo as well

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as under our experimental conditions. One has to consider that ,&receptor-mediated relaxation is very effective after cholinergic or histaminic stimulation, both of which are known to raise tone and cytoplasmic free [Ca”‘] (21, 29). In HeLa cells the activation of lowerconductance K+ channels has been reported to be strictly dependent on [Ca2+]i on stimulation of the histamine H1 receptor (36). These observations do not preclude the fact that, in ASM cells, the activity of Gk(oa) K+ channels could be negatively controlled by other molecular mechanisms. For example, CTX has no effect on the resting tension of an ASM strip. In colonic smooth muscle cells, recent studies have clearly demonstrated that acetylcholine appears to suppress Ca2+ -dependent K+ currents via a G protein-mediated process (8, 9). This K+ current is thought to play an important role in the regulation of colonic smooth muscle membrane potentials during slow waves. Despite the fact that the pharmacological profiles of ASM and intestinal smooth muscle are to some extent similar, the occurrence of such a process will have to be ascertained in ASM cells, known for their electrical stability (15, 23). In summary, our results demonstrate for the first time that ASM microsomal fractions can be fused into artificial membranes to study their ionic permeabilities. The electrical and biochemical properties of a Ca2+-activated channel were studied at the molecular level. Considering that channel activity as well as the NE- and VIP-induced relaxations were inhibited by similar concentrations of CTX, we have established that the activation of this channel occurs during relaxation processes induced by pharmacological means. Furthermore, the mechanism underlying the activation of this specific pathway by neurophysiological agents could involve the phosphorylation of this channel that would in turn shift its Ca2+ sensitivity towards lower Ca2+ concentrations. In light of these results, it is conceivable that specific activators of this type of channel could represent selective compounds that mi ght be utilized i n the treatment of bronchoconstrictive disorders. The authors thank Dr. M. D. Payet for reading the manuscript; Pierre Pothier, Majda T. Benchekroun, and Catherine Beaudry for their technical assistance; as well as Christiane Ducharme for typing the manuscript. E. Rousseau is a scholar of the Canadian Heart and Stroke Foundation, A. Cadieux is a scholar of Fonds de la Recherche en Sante du Quebec (FRSQ). This laboratory is supported by the Medical Research Council of Canada and by the FRSQ (establishment grant). Address reprint requests to E. Rousseau. Received

9 April

1991; accepted

in final

form

27 September

1991.

REFERENCES 1. Akbarali, H., T. Nakajima, D. G. Wipe, and W. Giles. Ca2+activated K+ currents in smooth muscle. Can. J. Physiol. Pharmacol. 68: 1489-1494, 1990. 2. Anderson, K., A. F. Lay, Q.-Y. Liu, E. Rousseau, H. P. Erickson, and G. Meissner. Structural and functional characterization of purified cardiac ryanodine receptor Ca2+ release channel complex. J. BioZ. Chem. 264: 1329-1335,1989. 3. Benham, D. C., and T. B. Bolton. Patch-clamp studies of slow potential-sensitive potassium channels in longitudinal smooth muscle cells of rabbit jejunum. J. Physiol. Lond. 340: 469-486,1983.

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4. Black, J. L., C. L. Armour, P. R. Johnson, and K. S. Vincenc. The calcium dependence of histamine, carbachol and potassium chloride-induced contraction in human airways in vitro. Eur. J. Pharmacol. 125: 159-168, 1986. 5. Cadieux, A., C. Lanoue, P. Sirois, and J. Barabe. Carbamylcholine and &hydroxytryptamine-induced contraction in rat isolated airways: inhibition by calcitonin gene-related peptide. Br. J. PharmacoZ. 101: 193-199, 1990. 6. Carl, A., W. G. McHale, N. G. Publicover, and K. M. Sanders. Participation of Ca2+- activated K+ channels in electrical activity of canine gastric smooth muscle. J. PhysioZ. Lond. 429: 205221, 1990. 7. Coburn, R. F., and C. B. Baron. Coupling mechanisms in airway smooth muscle. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L119-L133,1990. 8. Cole, C. W., A. Carl, and K. M. Sanders. Muscarinic suppression of Ca2+-dependent K+ current in colonic smooth muscle. Am. J. Physiol. 257 (Cell Physiol. 26): C481-C487, 1989. 9. Cole, C. W., and K. M. Sanders. G proteins mediate suppression of Ca2+-activated K+ current by acetylcholine in smooth muscle cells. Am. J. Physiol. 257 (Cell PhysioZ. 26): C596-C600, 1989. 10. Ewald, D. A., A. Williams, and I. B. Levitan. Modulation of single Ca2+-dependent K+-channel activity by protein phosphorylation. Nature Lond. 315: 503-506, 1985. 11. Fabiato, A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solution containing multiple metals and ligands. In: Methods in Enzymology Biomembrane, edited by S. Fleischer and B. Fleischer. Orlando, FL: Academic, 1989, chapt. 31, p. 378-417. 12. Fosset, M., E. Jaimowich, E. Delpont, and M. Lazdunski. [3H]nitrendipine receptors in skeletal muscle. J. BioZ. Chem. 258: 6085-6092,1983. 13. Groschner, K., S. D. Silberberg, C. M. Gelband, and C. Van Breemen. Ca2+-activated K+ channels in airway smooth muscle are inhibited by cytoplasmic adenosine triphosphate. PfZuegers Arch. 417: 517-522,199l. 14. Grover, A. K., M. S. Kannan, and E. E. Daniel. Canine trachealis membrane fractionation and characterization. CeZZ CaZcium 1: 135-146, 1980. 15. Hisada, T., Y. Kurachi, and T. Sugimoto. Properties of membrane currents in isolated smooth muscle cells from guinea-pig trachea. Pfluegers. Arch. 416: 151-161, 1990. 16. Ito, M., K. Baba, K. Takagi, T. Satake, and T. Tomita. Some properties of calcium-induced contraction in the isolated human and guinea pig tracheal smooth muscle. Respir. Physiol. 59: 143153, 1985. 17. Ito, Y ., and K. Takeda. Non-adrenergic inhibitory nerves and putative transmitters in the smooth muscle of cat trachea. J. Physiol. Lond. 330: 497-511, 1982. 18. Jaimovich, E., P. Donoso, J. L. Liberona, and C. Hidalgo. Ion pathways in transverse tubules: quantification of receptors in membranes isolated from frog and rabbit skeletal muscle. Biochim. Biophys. Acta 855: 89-98, 1986. 19. Kirkpatrick, T. C. Excitation and contraction in bovine tracheal smooth muscle. J. Physiol. Lond. 244: 263-281, 1974. 20. Kohrogi, H., S. Horio, M. Ando, M. Sygimoto, I. Mondu, and S. Araki. Nifedipine inhibits human bronchial smooth muscle contraction induced by leukotriene Cq and Dq, prostaglandins F2, and potassium. Am. Rev. Respir. Dis. 132: 299-304, 1983. 21. Kotlikoff, M. I. Calcium currents in isolated canine airway smooth muscle cells. Am. J. PhysioZ. 254 (CeZZ Physiol. 23): C793C801,1988. 22. Kotlikoff, M. I. Potassium currents in canine smooth muscle cells. Am. J. Physiol. 259 (Lung CeZZ. Mol. Physiol. 3): L384-L395,

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1990. 23. Kotlikoff, M. I., R. K. Murray, and E. E. Reynolds. Histamine-induced calcium release and phorbol antagonism in cultured airway smooth muscle cells. Am. J. Physiol. 253 (CeZZ Physiol. 22): C561-C566,1987 24. Krampezt, K. I., and R. Bose. Relaxant of amiloride on canine tracheal smooth muscle. J. Pharmacol. Exp. Ther. 246: 641-648, 1988. 25. Kume, H., A. Takai, H. Tokumo, and T. Tomita. Regulation of Ca2+-dependent K+-channel activity in tracheal myocytes by phosphorylation. Nature Lond. 341: 152-154, 1989. 26. Kwan, C. Y., R. M. Lee, and A. K. Grover. Intracellular membrane fractionation of rat vas deferens smooth muscle. Mol. Physiol. 3: 53-69, 1983. 27. Manning, C. D., R. Varrek, J. M. Stewart, and S. H. Snyder. Two bradykinin binding sites with picomolar affinities. J. Pharmacol. Exp. Ther. 237: 504-512, 1986. 28. Marthan, R., C. Martin, T. Amedee, and J. Mironneau. Calcium channel currents in isolated smooth muscle cells from human bronchus. J. AppZ. Physiol. 66: 1706-1714, 1989. 29. Marthan, R., J. P. Savineau, and J. Mironneau. Acethylcholine induced contraction in human isolated bronchial smooth muscle: role in intracellular calcium store. Respir. Physiol. 67: 127-135, 1987. 30. McCaan, J. D., and M. J. Welsh. Calcium activated potassium channels in canine airway smooth muscle. J. Physiol. Lond. 372: 113-127,1986. 31. Moczydlowsky, E., and R. Latorre. Gating kinetics of activated K+ channels from rat muscle incorporated into planar lipid bilayers. J. Gen. PhysioZ. 82: 511-542, 1983. 32. Nelson, T. M., J. B. Patlak, J. F. Worley, and N. B. Standen. Calcium channels, potassium channels and voltage dependence of arterial smooth muscle tone. Am. J. Physiol. 259 (CeZZ Physiol. 28): c3-C18,1990. 33. Palotta, S. B. Calcium-activated potassium channels in rat muscle inactivate from a short-duration open state. J. Physiol. Lond. 363: 501-506,1985. 34. Palotta, S. B., J. R. Hepler, S. A. Oglesky, and T. K. Harden. A comparison of calcium-activated potassium channel currents in cell-attached and excised patches. J. Gen. Physiol. 89: 985-997, 1987. 35. Rousseau, E., and G. Meissner. Single cardiac sarcoplasmic reticulum Ca2+-release channel: activation by caffeine. Am. J. Physiol. 256 (Heart Circ. Physiol. 25): H328-H333, 1989. 36. Sauve, R., C. Simoneau, L. Parent, R. Monette, and G. Roy. Oscillatory activation of calcium-dependent potassium channels in Hela cells induced by histamine H, receptor stimulation: a singlechannel study. J. Membr. BioZ. 96: 199-208, 1987. 37. Savaria, D., and E. Rousseau. Reconstitution of Cl- and K+ channels from airway smooth muscles into artificial membranes made of cationic lipid (Abstract). Proc. Int. Biophysics Congr. 10th. Vancouver, Canada, 1990, p. 397. 38. Smith, C., M. Phillips, and C. Miller. Purification of charybdotoxin, a specific inhibitor of the high-conductance Ca2+-activated K+ channel. J. BioZ. Chem. 231: 14607-14613,1986. 39. Vergara, C., and R. Latorre. Kinetics of Ca2+-activated K+ channels from rabbit muscle into planar bilayers. J. Gen. Physiol. 82: 543-568,1983. 40. Worley, J. F., III, and M. I. Kotlifoff. Dihydropyridine sensitive single calcium channels in airway smooth muscle cells. Am. J. Physiol. 259 (Lung CeZZ. MOL. Physiol. 3): L468-L480, 1990. 41. Yellen, G. Ionic permeation and blockade in Ca2+-activated K+ channels of bovine chromaffin cells. J. Gen. Physiol. 84: 157-186, 1984.

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