single-channel events was very similar to that of the whole-cell, delayed rectifier (Kotlikoff, 1990).

Transcription

1 Journal of Physiology (1992), 447, pp With 11 figures Printed in Great Britain DELAYED RECTIFIER POTASSIUM CHANNELS IN CANINE AND PORCINE AIRWAY SMOOTH MUSCLE CELLS BY J. P. BOYLE*, M. TOMASIC AND M. I. KOTLIKOFFt From the Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, PA, USA (Received 22 January 1991) SUMMARY 1. In order to define the ion channels underlying the inactivating, calciuminsensitive current in airway smooth muscle cells, unitary potassium currents were recorded from canine and porcine trachealis cells, and compared with macroscopic currents. On-cell and inside-out single-channel currents were compared with wholecell recordings made in dialysed cells. 2. Depolarizing voltage steps evoked outward unitary currents. In addition to a large conductance, calcium-activated potassium channel (Kca), a lower conductance potassium channel was identified. This channel has a conductance of 12-7 ps (on-cell; 1 mm-k+ in the pipette). 3. The lower conductance channel (Kdr) was not sensitive to cytosolic Ca2+ concentration and unitary current openings occurred following a delay after the voltage step. The time course of activation of the current composed of averaged single-channel events was very similar to that of the whole-cell, delayed rectifier potassium current (VdK), recorded under conditions of low intracellular calcium (Kotlikoff, 1990). 4. Kdr channels also inactivated with kinetics similar to those of the macroscopic current. Averaged single-channel records revealed a current that inactivated with kinetics that could be described by two exponentials (T1 = 0-14 s, r2 = 1H1 s; at 5 mv). These values corresponded well with previously determined values for timedependent inactivation of IdK. Inactivation of Kdr channels was markedly voltage dependent, and was well fitted by a Boltzmann equation with V50 = -53 mv; this was similar to measurements of the macroscopic current, although the V50 value was shifted to more positive potentials in whole-cell measurements. When only the inactivating component of the macroscopic current was considered, the voltage dependence of inactivation of the single-channel current and macroscopic current were quite similar. 5. Single-channel kinetics indicated that Kdr channels occupy one open and two closed states. The mean open time was 1-7 ms. Inactivation results in a prominent * Present address: Department of Physiology, University of Leicester, PO Box 138, University Road, Leicester LEI 9HN. t To whom all correspondence should be sent at the Department of Animal Biology, University of Pennsylvania, 3900 Spruce Street, Philadelphia, PA , USA. MS 9093

2 330 J. P. BOYLE, M. TOMASIC AND M. I. KOTLIKOFF increase in the long closed time, with little effect on the mean open time or short closed time. 6. The Kdr channel was not blocked by tetraethylammonium (TEA; 1 mm), charybdotoxin (ChTX; 100nm) or glibenclamide (20,/M), but was blocked by 4- aminopyridine (4-AP; 1 mm). Similarly, 4-AP blocked the inactivating component of the macroscopic current, but a non-inactivating current remained. KCa currents were blocked by TEA (0-5-1 mm) and charybdotoxin (40 nm), but were insensitive to 4-AP (1 mm) and glibenclamide (20,M). 7. Since the activation and inactivation kinetics, voltage dependence of inactivation, and the pharmacology of Kdr channels are the same as those of the inactivating component of the delayed rectifier current, IdK, it is suggested that these channels mediate the inactivating component of the calcium-insensitive macroscopic current and account for a substantial component of the outward rectification that is characteristic of airway smooth muscle. INTRODUCTION The ionic processes that account for spontaneous electrical activity and action potential firing in some smooth muscles, but quiescent or graded electrical activity in others, are incompletely understood. Although much work has been done to elucidate the cellular and subcellular processes involved in the control of activity in spontaneously active smooth muscles such as jejunum and portal vein, relatively little is known about such processes in airway smooth muscle. Electrophysiological studies performed on strips of airway smooth muscle have shown that contractile agonists produce a graded, concentration-dependent depolarization that does not result in action potential firing, and that depolarizing current injections evoke a marked increase in potassium conductance (Kirkpatrick, 1975; Kroeger & Stephens, 1975; Coburn, 1979; Ahmed, Foster, Small & Weston, 1984). In the presence of potassium channel blockers such as tetraethylammonium (TEA') or procaine, however, the graded depolarization is replaced by phasic activity, action potentials, and an increase in tension (Kroeger & Stephens, 1975; Foster, Small & Weston, 1983). A voltage-dependent potassium current therefore appears to be a critical determinant of the quiescent, non-spiking electrical behaviour of airway smooth muscle tissue strips. Recent whole-cell, patch-clamp measurements have demonstrated outward rectification in single, disaggreggated myocytes isolated from canine trachealis (Kotlikoff, 1989; Muraki, Imaizumi, Kojima, Kawai & Watenabe, 1990; Kotlikoff, 1990), and have characterized the voltage-dependent potassium currents that underlie this behaviour. Studies of voltage-dependent K+ currents in airways and other smooth muscle have demonstrated outward potassium currents that are independent of calcium influx (Mironneau & Savineau, 1980; Okabe, Kitamura & Kuriyama, 1987; Beech & Bolton, 1989a, b; Lang, 1989; Kotlikoff, 1990). Other studies, however, have demonstrated outward currents that are activated by increases in cytosolic calcium (Ohya, Kitamura & Kuriyama, 1987; Walsh & Singer, 1987; Cole & Saunders, 1989). The outward current that is sensitive to the cytosolic calcium concentration is probably carried by the large conductance, calcium-activated K± channel (KCa), which has been extensively characterized in airways (McCann & Welsh, 1986; Kume,

3 DELAYED RECTIFIER CHANNELS Takagi, Satake, Tokuno & Tomita, 1990) and other smooth muscles (Walsh & Singer, 1983; Inoue, Kitamura & Kuriyama, 1985; Benham, Bolton, Lang & Takewaki, 1986). The channel underlying the voltage-dependent calcium-insensitive potassium current has not been well characterized, however. We report the single-channel properties of a 12-7 ps conductance, voltage-dependent delayed rectifier potassium channel in tracheal smooth muscle cells. This channel is calcium insensitive and displays activation an inactivation characteristics that are equivalent to macroscopic delayed rectifier currents in these cells. Moreover, the channel demonstrates pharmacological properties that correspond to the whole-cell current, and are distinct from those of the calcium-activated potassium channel. 331 METHODS Cell dissociation Cell dissociation methods were similar to those described previously (Kotlikoff, 1988). Trachea from adult swine were collected into ice-cold, HEPES-buffered physiological salt solution (HBSS) as soon as possible after slaughter and transported, on ice, to the laboratory. Alternatively, mongrel dogs of either sex were killed with pentobarbitone, and the trachea rapidly removed and placed in ice-cold HBSS. The trachea was washed in HBSS, transferred to a nominally Ca2+free buffered salt solution, and dissected free of mucosa and connective tissue. Approximately 1 g wet weight of trachealis was finely minced and transferred into 5 ml of oxygenated digestion medium (Medium M199, GIBCO) containing 1-7 ftm-egta, collagenase D (Boehringer Mannheim, 300 U/ml), elastane (Worthington, 8-5 U/ml) and Soy Bean Trypsin Inhibitor (Sigma type 1, 1 mg/ml), and incubated in a shaking water bath at 37 C until the tissue fragments began to break up (20-30 min). In some experiments DNAase (Sigma type IV, 200 U/ml) was included in the digestion mixture. The digestion medium was then diluted with medium M199, the tissue pieces greatly agitated, the slurry filtered through 100,um Nytex mesh, and the filtrate centrifuged at slow speeds for 5 min at 4 'C. The pellets were resuspended in medium M199 (Sigma) and the cell suspensions stored at 4 'C. Cells were used within 12 h of isolation. No differences in ion channel activity or conductance were observed between porcine and canine tracheal myocytes. Solutions and reagent The normal bath solution used was (mm): 125 NaCl, 5 KCl, 1V8 CaCl2, 1 KH2PO4, 1 MgSO2, 10 HEPES, 15 glucose, at ph 7-4 (NaOH). Calcium concentration was varied as stated in the text. In order to null the cell membrane potential, a high-potassium bath solution was used (mm): 126 KCl, 5 NaCl, 1 MgCl2, 10 HEPES, 15 glucose, at ph 7-4 (KOH). Unless otherwise stated, the pipette solution used was: 125 NaCl, 1 KCl, 1 MgCl2, 10 HEPES, at ph 7-4 (NaOH). Whole-cell experiments were performed with 60 KCl, 60 potassium gluconate, 5 NaCl, 2 MgATP, 10 HEPES, 11 EGTA/1 CaCl2, at ph 7-2 (KOH). In a number of experiments the Ca2+ concentration of the bath solution was clamped by adding CaCl2 and EGTA in fixed amounts, as indicated in the text. In experiments in which the potassium concentration was varied, NaCl was substituted for KCl to achieve the desired final concentration. As stated in the text, TEA (500 /SM), charybdotoxin (100 nm) or EGTA (1 mm) were added to pipette solutions in many experiments to block KC8 activity. This proved necessary, since the amplitude of the delayed rectifier unitary current was quite small, and test steps to positive potentials were often used to determine activity. 3,4-Diaminopyridine and 4-aminopyridine were dissolved in the bath solution, the ph corrected to 7-4 using HCl, and the bath solution completely changed to test drug effect. Other drugs were added to the recording chamber at a dilution of 100 x. 3,4-Diaminopyridine, 4-aminopyridine, tetraethylammonium chloride (TEA), ethyleneglycol-bis-(,8-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA), M199 and (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulphonic acid]) (HEPES) were obtained from Sigma. Charybdotoxin was supplied by Receptor Research Chemicals Inc. All reagents and chemicals used were of analytical grade. Recording and analysis Recordings were made using whole-cell, on-cell and inside-out patch configurations of the patchclamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) at room temperature

4 332 J. P. BOYLE, M. TOMASIC AND M. I. KOTLIKOFF (20-24 C) as previously described (Kotlikoff, 1990; Worley & Kotlikoff, 1990). Patch pipettes were pulled from borosilicate capillary glass (A-M Systems Inc); the resistances of whole-cell patch pipettes were 5-10 and MQ for on-cell or inside-out recordings, respectively. For singlechannel measurements pipette tips were coated with industrial sticky wax, and the wax subsequently burned away from the electrode tip during fire-polishing. The cells were prepared for recording by placing a small quantity of the cell suspension on the glass base of a recording chamber and allowing time for them to adhere. Cell debris and nonadherent cells were removed by pumping bath solution through the chamber for min. Only cells that appeared to be undamaged and that were either partially or fully relaxed were selected for recording. Voltage-clamp protocols were generated using a PDP11/23 computer (DEC) interfaced to a digital oscilloscope (Model 1345A, Hewlett-Packard; Laboratory Display System, Indec Systems Inc.). Currents were recorded using a List-EPC7 amplifier (List Electronics) and either stored on computer disc or on videotape. In all cases patch potential was expressed as if the patch were attached to the cell, with the external cell surface at virtual-ground potential. Current signals from whole-cell recording experiments were low-pass filtered at 3 khz (-3 db), sampled at the appropriate rate, digitized and stored on computer. Leak subtraction was performed by subtraction of an appropriately scaled, ensemble-averaged current evoked by a 10 mv hyperpolarizing voltage step from -70 mv after determining that no conductances were activated in this voltage range. Current signals from the on-cell and inside-out patch experiments were lowpass filtered at 10 khz and recorded on videotape using a pulse code modulator (CRC VR-1O0A, Instrutech Corp.). Single-channel data were subsequently digitized off-line using a Labmaster TL- 1 interface and Axotape software (Axon Instruments). Data were filtered at 1 khz (8-pole Bessel), digitized at 7-10 khz, and stored on a 45 Mbyte removable cartridge (Iomega Corp.). Singlechannel records were subsequently analysed using a single-channel analysis package kindly provided by Dr M. Nelson. Activation kinetics were determined by a least-squares fit (Sigmaplot, Jandel Scientific) of the data to a Hodgkin and Huxley activation model (Hodgkin & Huxley, 1952) of the form: I = Imax(--et/ )2, where Imax is the maximum current and r is the activation time constant. Time-dependent single-channel current inactivation was fitted by a dual exponential decay process: I = if e-t/7 +Is e-1/', where If and I. are the amplitudes of the fast and slow current components, and r, and r2 are the respective decay constants. Estimates of probability have been expressed as np0. Since np. varied between experiments, average voltage-dependent behaviour between experiments was determined by normalizing npo in terms of the maximum observed probability in a given patch (npmax) Channel activation data were fitted to a Boltzmann equation of the form: npo = npm,x/(1 + ev6o-v)k), where V50 is the potential at which the open probability is half-maximal, k is the slope factor of the relationship, and V is the test potential. Voltage-dependent inactivation was fitted by a Boltzmann equation of the form: I = Ima/(1 + ev-v5o)k), where Imax is the maximum observed current, V60 is the voltage at which the current is halfinactivated, and k is the slope factor of the relationship. Single-channel data were fitted to the analogous expression, with npo and npmax substituted for I and Imax, respectively. RESULTS Unitary voltage-dependent potassium currents On-cell measurements of voltage-dependent unitary currents revealed two prominent outward current amplitudes in canine and porcine myocytes. Figure 1 shows a typical on-cell experiment in which a single porcine airway smooth muscle cell was depolarized in high-potassium bath solution (140 mm-kcl), and the

5 DELAYED RECTIFIER CHANNELS membrane patch was stepped from a holding potential of -60 to + 40 mv. Under these conditions, very large outward unitary currents are commonly elicited at depolarized potentials. In experiments like that shown, the unitary conductance of this channel was 193 ps (on-cell; 60 mm-k+ in the pipette). The single-channel pa 100 ms Fig. 1. Single, voltage-dependent potassium channels in on-cell recordings from a single porcine airway smooth muscle cell. Depolarizing steps from a holding potential of -60 mv to 40 mv activated large amplitude and small amplitude channel activity. For clarity traces in which small amplitude channel activity is prominent are grouped (right). As can be seen, under on-cell conditions with high potassium in the pipette, the small conductance channel is difficult to resolve and often appears only as noise in the baseline. Traces in which KCa channel activity predominates have a flat, quiet baseline (left). Pipette solution as in Methods except 60 mm-nacl, 60 mm-kcl. Bath solution was highpotassium solution. conductance and calcium dependence (see below) of these channels corresponded very well with the large conductance calcium-activated potassium channel (Kca) that has previously been described in airway smooth muscle (McCann & Welsh, 1986; Kume et al. 1990). In addition to these very large amplitude current fluctuations, much smaller unitary current transitions, which sometimes appeared as increased baseline noise, could also be observed in these experiments (Fig. 1, right). The small amplitude channel had a high open probability shortly after stepping to positive test potentials, and might account for the low noise, calcium-insensitive delayed rectifier current (Vdk) observed in our previous whole-cell studies (Kotlikoff, 1990). In order to define the single-channel properties of this small amplitude channel, and to compare those properties with the macroscopic current, experiments were designed to isolate the small conductance channel. As shown in Fig. 1, depolarizations to very positive potentials evoked substantial KCa activity on-cell; under conditions of relatively high concentrations of K+ ions in

6 334 J. P. BOYLE, M. TOMASIC AND M. I. KOTLIKOFF A -70 Test 0- C - C- ; 30 mv ~~~~~~~ ~~~~~ 20 C- 2 pa C -10 C - A #* > ms Fig. 2. For legend see facing page. the pipette, current amplitudes of the small conductance channel were very low at these potentials. We therefore performed experiments under conditions of low potassium at the external membrane surface to maximize the amplitude of the current at lower step potentials, and low bath and pipette calcium to minimize KCa open probability. Under these conditions, steps to depolarizing potentials from hyperpolarized holding potentials routinely elicited only the low-conductance outward unitary currents. Figure 2A shows an example of this type of experiment, in which the small conductance channel was isolated from KCa channel activity. The current-voltage characteristics of this channel were investigated in on-cell and inside-out experiments, and the conductance compared with that of the large amplitude Kca channel (Fig. 2B). The small amplitude channel openings had a slope conductance of 12-7 ps in on-cell experiments (5 mm-k' in pipette; n = 7). Under identical conditions, the conductance of the large amplitude Kca channel was 137 ps;

7 DELAYED RECTIFIER CHANNELS 335 B /(pa) ( / Test potential (mv) Fig. 2. Current-voltage relationship of small amplitude potassium channel. A, depolarizing steps from a holding potential of -70 mv to the indicated test potential show voltage dependence of the small amplitude channel. Traces shown were obtained by recording from a cell-attached patch from a canine tracheal myocyte with 100 nmcharybdotoxin and 2 mm-egta added to the pipette solution, and 1 mm-k' in the pipette. Traces chosen are representative of open probability at each potential. B, averaged experiments (n = 7) similar to that shown in A. Linear regression (continuous line) yields a slope conductance (g) of 12-7 ps, and a reversal potential of -48 mv. n = 7). The reversal potential extrapolated from an extension of the linear regression was -48 mv for both channels. Voltage dependence and calcium sensitivity Figure 2A reveals prominent voltage-dependent activity of the small conductance potassium channel. This voltage dependence was investigated and compared to whole-cell recordings of the delayed rectifier current obtained under similar conditions. On-cell patches were stepped to test potentials of -40 to 40 mv from a holding potential of -70 mv, and the open-state probability determined at each potential. Recordings were performed using 100 nm-chtx in the pipette and 2 mm- EGTA in the bath to reduce the KCa activity routinely observed at the more positive test potentials in on-cell experiments. The relationship between test potential and npo of the small conductance channel is shown in Fig. 3, under conditions in which the cell membrane potential was nulled using the high-potassium bath solution. The threshold of current activation in these experiments was about -30 mv, and a typical sigmoidal dependence of open probability on membrane potential was observed. A Boltzmann fit to the channel activation data yielded a V50 value of mv, and a slope factor of the relationship of 6 (see Methods). The activation threshold of the single-channel measurements corresponds well with that of the macroscopic current (Kotlikoff, 1990).

8 336 J. P. BOYLE, M. TOMASIC AND M. I. KOTLIKOFF The calcium sensitivity of the small conductance potassium channel was examined in off-cell experiments. Figure 4 illustrates a typical experiment from a canine tracheal myocyte, in which initial recordings were made from inside-out patches under nominally calcium-free conditions (normal bath solution with no EGTA or Step potential (mv) Fig. 3. Voltage dependence of opening of small amplitude potassium channel. Depolarizing voltage steps from a holding potential of -70 mv to varying test potentials were performed in on-cell experiments. Equivalent number of steps at each potential were performed and the total open channel probability (npo) calculated from amplitude histograms. For each experiment (n = 7), np. was normalized by the maximum observed open probability in that experiment (npmax), and plotted versus voltage. The Boltzmann fit (straight line) yields a voltage of half-maximal activation of 5-5 mv. Experiments were performed in the presence of either 100 nm-chtx, or 1 mm-tea. CaCl2 added, free calcium approximately 10,tM), followed by the addition of EGTA to lower free calcium at the cytosolic surface of the patch. Initial voltage-clamp steps to depolarized potentials resulted in the opening of up to ten large conductance, K+selective channels. Addition of 2 mm-egta to the bath solution, lowering the free calcium concentration to approximately 1 nm, almost completely eliminated KCa channel activity. The small conductance channel was not inhibited under these conditions; rather, substantial underlying channel activity was uncovered by blockade of KCa. Experiments were also performed in which Kca activity was inhibited by the addition of 100 nm-chtx, and the cytosolic calcium concentration altered by successive additions of EGTA to the bath solution. These experiments did not reveal any sensitivity to the channel to cytosolic calcium concentration. Moreover, in similar experiments, addition of Ca2+ to nominally calcium-free bath solutions did not alter the activity of the small amplitude channel.

9 DELAYED RECTIFIER CHANNELS 337 Activation kinetics Whole-cell recordings indicate that a calcium-insensitive outward current activates with a clear delay following depolarizing voltage steps applied to single airway smooth muscle cells. In order to compare the activation kinetics of the small A 5mV -70 mv j B 5 mv -70 mv 0 C- 5 pa I 1 pa Fig. 4. Calcium insensitivity of the small amplitude potassium channel. A, consecutive recordings from an inside-out patch of a canine trachealis cell. Initial recordings made in the absence of EGTA show high channel activity. Single-channel openings are difficult to distinguish, but single-channel amplitudes are greater than 2 pa. B, consecutive traces from the same patch following the addition of EGTA (3 mm final concentration) to the bath. Large amplitude events are rare (note change in scale). The smaller amplitude events can now easily be resolved, and reveal a relatively high open probability under conditions of less than 10 nm-free calcium. Bath solution was high-potassium solution; pipette solution contained 1 mm-kcl. conductance K+ channels with these macroscopic currents, a series of depolarizing voltage steps were averaged and the time course of the current activation examined. Figure 5 shows an example of an experiment in which the activation kinetics of the

10 338 J. P. BOYLE, M. TOMASIC AND M. I. KOTLIKOFF small conductance channel were investigated. A cell-attached patch was stepped from a holding potential of -70 mv to a test potential of 5 mv for 140 ms under conditions similar to those shown in Fig. 2. Six consecutive current traces are shown (Fig. 5A). Examination of the individual traces indicates that openings of the small A J 5 mv -70 mv C 1 pal 20 ms n = 87 B 0.2 pa 4 ms 0-1 pal 5 ms Fig. 5. Activation kinetics of the delayed rectifier potassium channel. A, on-cell current records following step depolarization of 5 mv reveal that the small conductance channel usually opens following a delay. B, individual current records from the first 25 ms of the step were ensemble-averaged from all recordings to examine the kinetics of current activation. The averaged current shows a distinct time-dependent activation. C, traces in which only small conductance channel activity was observed in the first 25 ms of the step were averaged separately and fitted to a second-order exponential activation curve (dashed line). The time constant for this curve is 13-6 ms. The pipette solution contained 60 mm-kcl/60 mm-nacl with 1 mm-tea, and high-potassium bath solution with 1 mm- EGTA, 0 CaCl2; porcine myocyte. conductance K+ channel occur with higher frequency following a delay after the depolarizing step, i.e. transitions to the open state are much more likely later in the traces. Similar behaviour can be observed in Fig. 4B. In the experiment shown in

11 DELAYED RECTIFIER CHANNELS Fig. 5, Kc. activity was reduced by the addition of 2 mm-egta to the normal bath and pipette solutions, and by including 1 mm-tea in the pipette solution. Traces 1, 2 and 6 (Fig. 5A) show that although Kca activity has been reduced it has not been abolished. To examine the time-dependent activation behaviour of the small conductance channel, traces in which no KCa openings occurred were ensemble averaged. Figure 5B illustrates that the average single-channel behaviour results in a net current that activates with a distinct time dependence, which is similar to the time dependence of activation of the macroscopic delayed rectifier current (Kotlikoff, 1990). A further similarity to the whole-cell data is that the ensemble average could be well fitted by n2 activation kinetics (Hodgkin & Huxley, 1952), as shown in Fig. 5 C. The time constant of activation was 13-6 ms (step to 5 mv), which compares well with the n2 time constant of 10-6 ms (step to 15 mv) for the macroscopic current (Kotlikoff, 1990). Since the small conductance channel clearly displays delayed activation kinetics, we refer to it as a delayed rectifier channel, Kdr. In contrast to the small conductance, delayed rectifier potassium channels, Kca channels activated with very rapid kinetics. Experiments similar to those described above were performed using 1-8 mm- Ca2+ in the bath and pipette solutions, and without ChTX or TEA in the pipette. Ensemble-averaged traces of currents with substantial Kca activity indicated that the activation of this current occurred in less time than could be resolved in these experiments (data not shown), indicating an activation rate considerably faster than that of the inactivating outward current seen in whole-cell recordings from this tissue. Kinetics and voltage dependence of inactivation of Kdr A prominent feature of voltage-dependent K+ currents in airway smooth muscle is a time- and voltage-dependent inactivation of the current. Single-channel recordings of the small conductance channel, Kdrv in both on-cell and off-cell recording modes demonstrated similar behaviour. Experiments were performed to examine the kinetics of channel inactivation and to compare this behaviour with the inactivation kinetics of the macroscopic current. Long (3 s) voltage-clamp steps were performed in on-cell patches in which channel activity was high, and channel activity from these traces was averaged. Figure 6A shows six consecutive steps to 5 mv; leak currents have been subtracted from these traces. A prominent time-dependent current inactivation can be seen in the individual traces, and in ensemble averages of the single-channel behaviour (Fig. 6B). The inactivation kinetics of the average current are quite similar to those seen in whole-cell experiments. Current inactivation does not occur as a single exponential decay process, but was well described by a biexponential decay function (Fig. 6C, Methods); the two time constants for this decay were 0-14 and 1F1 s for the experiment shown. These time constants are in good agreement with previously published inactivation kinetics for the inactivating component of the whole-cell current (Kotlikoff, 1990), where the analogous results were 0-15 and 0-91 s (30 mv). Steady-state inactivation protocols were also performed to examine the voltage dependence of Kdr inactivation. Whole-cell and single-channel experiments were performed in acutely dissociated porcine myocytes and directly compared under 339

12 340 J. P. BOYLE, M. TOMASIC AND M. I. KOTLIKOFF similar recording conditions. On-cell experiments were performed in high-potassium bath solution to null the cell potential and allow a direct comparison between experiments. Whole-cell experiments were performed under conditions clamping intracellular calcium to low concentrations in order to eliminate any effect of KCa A 5 mv 0 mv C 2 pa 0.25 pa 8 ~~~~~~~~~~~~~0.5 s n =16 0*4pA 1 s Fig. 6. Time-dependent inactivation of Kdr channels. A, six consecutive traces from an oncell patch displaying only Kdr activity are shown at longer time scale to illustrate the time-dependent inactivation of channel activity. Openings are clustered in the first half of the traces, and by the end of the step open probability is relatively low. B, the ensemble average of sixteen leak-subtracted traces shows that the average current inactivates almost completely over the course of the test pulse. C, the average current was fitted to an exponential decay curve consisting of two exponential decay processes (dashed line). The parameters for the fit were rl = 0-14 s and r2 = 11 s. Recording conditions as in Fig. 5. channels on the outward current. As shown in Fig. 7, conditioning pulses to depolarizing potentials resulted in a marked inhibition of channel activity and wholecell current. Open-state probability and outward current were markedly decreased by depolarizing test potentials in the range of -80 to -30 mv. By contrast, Kca

13 DELAYED RECTIFIER CHANNELS mv 20 mv 20 mv %-O. 1 pa[ - 25 ms Events Current (pa x 10-1) Events Current (pa x 10 1) 3 Events 2 10L Current (pa x 10-1) 200 pa [250 ms Fig. 7. Voltage-dependent inactivation of Kdr channels and IdK current. Ten second conditioning pulses to potentials from -90 to -20 mv were imposed before stepping to 20 mv to activate Kd, channels in an on-cell (above), or whole-cell experiment (below) from porcine tracheal cells. Above, single-channel activity is markedly diminished by positive conditioning potentials, whereas Kca activity (large amplitude channels) is not affected. Amplitude histograms showing only the small amplitude channel current and the zero current levels illustrate the progressive decrease in np. with depolarizing prepulses. Below, whole-cell recordings at a slower time scale demonstrate the equivalent effect on the activating component of the macroscopic current. Solutions for on-cell experiment were 60 mm-kcl/60 mm-nacl pipette solution with 1 mm-tea and highpotassium bath solution containing 1 mm-egta. Whole-cell solutions were whole-cell pipette solution, and normal bath solution, as listed in Methods.

14 342 J. P. BOYLE, M. TOMASIC AND M. I. KOTLIKOFF channel activity was not affected by conditioning potential, and did not display time-dependent inactivation. Comparison of the averaged single-channel records (Fig. 6C) with the macroscopic current (Fig. 7) indicates that unlike the average single-channel behaviour, macroscopic currents do not completely inactivate. The \'7 E c Conditioning potential (mv) Fig. 8. Comparison of voltage-dependent inactivation in single-channel and whole-cell recordings. Single-channel experiments (n = 10) similar to that shown in Fig. 7 were averaged and the normalized open probability or current were plotted as a function of prepulse potential (0). The data show a typical sigmoidal dependence, and were well fitted by a Boltzmann equation (continuous line). The parameters of the line shown are VJ5o =- 53*8 mv, and kc = The results from a single whole-cell experiment at the same potential are plotted for comparison. Values shown (V) are the difference current between peak and steady-state currents. These data were also fitted to a Boltzmann equation (dashed line), with parameters of V,5 = -46-6, and kc = 77.7 inactivating current varied from approximately 20 to 50% of the peak outward current in these experiments. These data suggest that Kdrechannels underlie only the inactivating component of IdKi. To determine whether the voltage dependence of inactivation of Kdr channels and the inactivating component of IdK were similar, single-channel experiments (n = 10) were normalized to maximum open probability and compared to the inactivating component IdK (Fig. 8). Since 'dk inactivates to a steady-state level, the inactivating current was obtained by subtracting the steadystate level from the peak current at each potential. The potential at which halfinactivation of the single-channel currents occurred (50 % of maximum np0) was mv. Although the slope of the relationship is similar to previously reported data in canine myocytes (Kotlikoff, 1990), the inactivation curve is markedly leftshifted. When the inactivating component of the whole-cell current is plotted as a function of conditioning potential, the voltage dependencies of Kdr and IdKi are quite similar (Fig. 8), suggesting that Kdr channels underlie the inactivating component of 'dik I

15 DELAYED RECTIFIER CHANNELS 343 Single-channel kinetics of Kdr The single-channel kinetics of Kdr were determined from recordings in which all KCa activity was abolished with charybdotoxin (100 nm). Open- and closed-time analysis demonstrated that the open times could be fitted by a single exponential, A Open times 400 T= 1-7 a) z B 0 ~~~~~~~~~~ Time (ms) 500 Closed times 20 - U) 400 rl = r2 = = z I I~~~~~~~~~~0 II I Time (ms) Conditioning potential (mv) Fig. 9. Open- and closed-time distribution of Kdr. A, single-channel data were idealized and open- and closed-time analysis performed. A, open-time distributions from a holding potential of -90 mv (step potentials 5 mv) were well fitted by a single exponential (above), whereas closed times required two exponentials. B, the effect of holding potential on channel kinetics was examined by analysing current records from three separate experiments following pre-conditioning pulses (10 s) to -60 and -40 mv. The major effect of holding potential was to increase the mean closed time. This was mainly seen as an effect on the long closed time. A, Topen; 0, Tl,closed; *0 72,closedand that closed-time distributions were best fitted by two exponentials, consistent with a channel occupying two closed states and one open state. The mean open-time duration was 1P7 ms, whereas the time constants for the closed times weret1 = 1P6 ms

16 344 J. P. BOYLE, M. TOMASIC AND M. I. KOTLIKOFF and r2 = 10 0 ms (Fig. 9A). To examine the effect of holding potential on channel kinetics, open and closed times were determined from three steady-state inactivation experiments, and the relationship between the mean open and closed time constants and conditioning voltage plotted (Fig. 9B). The mean channel open time was not Control 1 mm-4-ap Wash 20 mv 20 mv 20 mv pa L125 ms Fig Aminopyridine blocks the inactivating whole-cell current. Voltage-clamp depolarization evoked outward IdK currents under control conditions, following incubation of 3 min with 1 mm-4-ap added to the bath, and following wash-out of 4-AP for 5 min. 4-AP completely eliminated the inactivating current, and this effect was only partially reversed after bath wash. Additionally, the non-inactivating (or very slowly inactivating) current was decreased. This current returned to approximate control levels following wash-out. Experiment shown is a porcine trachealis cell; identical experiments were performed in canine myocytes. affected by holding potential under conditions in which the open probability was markedly decreased, indicating that voltage-dependent inactivation did not result from shorter dwell times in the open state. Conversely, a decrease in the frequency of channel opening (seen as an increase in closed times) was observed with more depolarized holding potentials. This was reflected by a marked increase in the magnitude of the long closed time constant, r2 with little or no effect on r1. This result is consistent with a voltage-dependent transition to an inactivated state that results in long closed-time durations and a markedly lower probability of opening. The lack of effect on r1 suggests that this may reflect the dwell time of the closed, rather than inactivated, state. Pharmacology of Kdr and IdK We have previously shown that IdK is completely insensitive to charybdotoxin and relatively insensitive to TEA (Kotlikoff, 1990). In the present study we examined the sensitivity of the macroscopic current and unitary currents to other potassium channel blockers. 4-Aminopyridine (1 mm), which blocks the transient outward current in a number of tissues (see Hille, 1984), completely abolished the inactivating component of the outward current when applied to the bath solution (Fig. 10), indicating the pharmacological similarity of this current to other delayed rectifier currents. Conversely, glibenclamide (20,SM), a sulphonylurea which has been reported to specifically block the ATP-sensitive K+ channel (Schmid-Antomarchi, de Weille, Fosset & Lazdunski, 1987), had no effect on either transient or sustained phases of the outward current (not shown).

17 DELAYED RECTIFIER CHANNELS 345 The sensitivity of Kdr to these potassium channel blocking agents was also examined in on-cell and inside-out patches. On-cell experiments in which the pipette contained 100 nm-chtx resulted in high activity of Kdr (Fig. lla), but little or no KCa activity. In off-cell experiments, TEA had no effect on Kdr when added to the A 100 mm-chtx B 1 mm-tea C 1 mm-4-ap L 114*4 +,^tt_ 5mV -70 mv 7 7 rw~4~i 1 pa 50 ms Fig. 11. Kdr channels are blocked by 4-aminopyridine. A, traces show an on-cell experiment with 100 nm-charybdotoxin in the pipette. All KCa channel activity is blocked; however, Kdr channels are active. B and C, current traces from an inside-out patch are shown in the presence of 1 mm-tea, and following addition of 1 mm-4-ap to the bath. In the presence of 1 mm-tea, several Kdr channels are present. These are almost completely blocked following addition of 4-AP. All traces have been leak subtracted. Conditions for both experiments were normal pipette solution, high-potassium bath solution; canine trachealis cell. bath at concentrations up to 5 mm, but caused a prominent flickering block of Kca (Fig. lib). Further addition of 1 mm-4-aminopyridine (Fig. IlC) or 3,4-diaminopyridine (not shown) resulted in a % inhibition of Kdr activity. Conversely, neither 4-AP nor 3,4-diaminopyridine (up to 5 mm) had any effect on the amplitude or open probability of Kca. Additionally, the sulphonylurea glibenclamide had no inhibitory effect on either Kca or Kdr at a concentration that is known to cause almost total inhibition of ATP-sensitive potassium channels in smooth muscle (Standen, Quayle, Davies, Brayden, Huang & Nelson, 1989). DISCUSSION The electrophysiological behaviour of tracheal smooth muscle is characterized by the marked outwardly rectifying properties of the membrane, associated with a voltage-dependent potassium conductance. This has been demonstrated at the level of the tissue (Kirkpatrick, 1975; Kroeger & Stephens, 1975), and more recently in single cells from the guinea-pig (Hisada, Kurachi & Sugimoto, 1990) and dog

18 346 J. P. BOYLE, M. TOMASIC AND M. I. KOTLIKOFF (Kotlikoff, 1990; Muraki et al. 1990). We have previously reported a calciuminsensitive, delayed rectifier potassium current, IdK' in canine tracheal myocytes (Kotlikoff, 1990). In single cells, this voltage-dependent potassium current consists of at least two components, one of which inactivates over a period of seconds and a second sustained, or very slowly in activating component. A similar, calciumindependent, delayed rectifier current has been reported in smooth muscle cells from other tissues (Beech & Bolton, 1989a, b; Yamamoto, Hu & Kao, 1989; Cole & Sanders, 1989; Hume & LeBlanc, 1989; Lang, 1989), although in some cells this current does not inactivate (Hume & LeBlanc, 1989) or inactivates only slightly (Cole & Sanders, 1989). In addition, this current was TEA insensitive in the cells from rabbit portal vein and guinea-pig ureter. The present study has attempted to determine the single-channel events underlying the transient, calcium-insensitive outward current. We report here that a 12-7 ps potassium channel, Kdr, underlies the inactivating component of IdK. This conclusion is based on similar voltage dependence, activation and inactivation kinetics, and pharmacology between Kdr and IdK. In cells from trachealis and many other tissues the predominant activity recorded from inside-out patches in physiological salt solution is due to the large conductance, calcium-activated potassium channel, Kca. In the present study the activity of Kca was sufficient to interfere with attempts to record from smaller conductance channels in on-cell experiments, since these channels required steps to positive potentials to achieve adequate current amplitudes. Since IdK is not calcium sensitive, and is not blocked by TEA or charybdotoxin, we isolated Kdr channels by adding EGTA to the bath or by including charybdotoxin (100 nm) in the pipette solution. A less effective means of isolation was achieved using 1 mm-tea in the pipette. This resulted in a lower amplitude, flickering block of the channel, previously described by McCann & Welsh (1986). Single-channel properties of Kdr The voltage dependence of activation and conductance of Kdr channels were consistent with IdK. The channel has a relatively low conductance (12-7 ps), which is expected from the low noise of the macroscopic current. No difference in activation threshold or conductance was observed in single-channel recordings of Kdr from dog and pig tracheal myocytes, and the porcine whole-cell currents reported here were not distinguishable from those described in dog cells (Kotlikoff, 1990). Beech & Bolton (1989b) have reported a 5-8 ps channel (chord conductance) that displays similar voltage dependence, calcium insensitivity, and voltage-dependent activation as the macroscopic delayed rectifier current in the rabbit portal vein cells. This channel closely resembles Kdr since it has a low conductance, a similar activation threshold, and similar activation and inactivation kinetics. We suggest that the difference in recording conditions and conductance determination (outside-out versus on-cell; chord versus slope conductance) may account for the difference in conductances. The conductance of Kdr is in good agreement with single-channel measurements of delayed rectifier channels from other tissues (Clapham & Logothetis, 1988; Yue & Marban, 1988; Aldrich, Solc, Zagotto & Brainard, 1989; Duchatelle-Gourdon & Hartzell, 1990).

19 DELA YED RECTIFIER CHANNELS 347 Activation and inactivation of Kdr The slow activation kinetics and slow time-dependent inactivation of the channel described in this study directly match the behaviour of the delayed rectifier K+ current, IdK, described in cells from portal vein (Beech & Bolton, 1989b) and trachealis (Kotlikoff, 1990). The kinetics of activation of the whole-cell current are quite similar to those reported by Beech & Bolton (1989b), who reported n4 or n2 activation. In this study, we were able to directly compare the activation kinetics of the single-channel data with whole-cell records. Both processes were well described by n2 kinetics of activation, and the time constants of averaged single-channel currents were similar to those previously reported for IdK (Kotlikoff, 1990). Both the delayed rectifier current IdK and the single-channel Kdr displayed prominent time-dependent inactivation that could best be fitted by the sum of two exponentials. This behaviour is similar to that described in portal vein cells (Beech & Bolton, 1989b). Moreover, in both cases, the inactivation fits describe a biexponential decay to some non-zero steady-state current level. Time-dependent inactivation of the delayed rectifier current has also been described in nerve cells (Schwartz & Vogel, 1971; Dubois, 1981, 1983). Since both whole-cell and singlechannel records display two components, it is possible that this represents complex inactivation kinetics rather than a heterogeneous population of Kdr as described by Dubois (1981, 1983) in nerve cells. It should also be noted that non-inactivating, delayed rectifier currents (Hume & LeBlanc, 1989; Cole & Sanders, 1989), and a slowly inactivating, calcium-sensitive current (Hume & LeBlanc, 1989) have been described in other smooth muscle cells. Although similar voltage dependence of inactivation was observed between Kdr and IdK, the voltage-dependent inactivation curve for the single-channel measurements was shifted in a hyperpolarized direction relative to the whole-cell data. One explanation for this result is that the single-channel recordings underlie only the inactivating component of the outward current (Figs 7 and 10). Figure 8 shows that if only the inactivating component of IdK is considered, the voltage dependencies of current and channel inactivation compare well. This explanation is rendered more plausible by the fact that the single channels appear to completely inactivate over a similar time frame as the inactivating component of IdK. Another possible explanation is that the hyperpolarizing shift in the single-channel measurements is due to the very low levels of divalent cations in the bathing solution in the singlechannel experiments. Beech & Bolton (1989 a) have shown that the surface potential effect of divalent cations will cause substantial shifts in the activation and inactivation curves for the transient outward current. Such a shift could also contribute to the differences in V50 for inactivation between the present study (V50 for single channel data = mv, Ca2l < 1 nm), and that of Beech & Bolton (1989b) (V50 for whole-cell data = -30 mv, Ca2+ = 1-7 mm). However, the lack of complete inactivation of the macroscopic currents is unlikely to be explained on the basis of surface charge, since at positive conditioning potentials the macroscopic current does not display time-dependent inactivation (Fig. 7).

20 348 J. P. BOYLE, M. TOMASIC AND M. I. KOTLIKOFF Pharmacology of Kdr and IdK We have previously reported that IdK is relatively insensitive to blockade by TEA' ions (IC50 (concentration giving 50% of maximal inhibition) = 51 mm; Kotlikoff, 1990). Our measurements indicate that Kdr is similarly insensitive, although a complete dose-response relationship was not obtained. Inside-out recordings under conditions of up to 8 mm-tea' in the bath solution yielded active Kdr channels, and no effect on channel amplitude was observed. By contrast, concentrations of TEA+ as low as 0'5 mm resulted in a marked flickering block of KCa channel currents (Fig. 7). Similarly, Kdr channels were not blocked by concentrations of charybdotoxin as high as 100 nm in the pipette, whereas this effectively inhibited KCa channel activity. The effect of 4-aminopyridine was tested on both whole-cell currents and single Kdr channels, since this compound effectively antagonizes a component of the outward current in several smooth muscle cell types (Okabe et al. 1987; Beech & Bolton, 1989b; Muraki et al. 1990), and has prominent effects in airway smooth muscle (Kannan, Jager, Daniel & Garfield, 1983). 4-Aminopyridine and 3,4-diaminopyridine effectively inhibited Kdr channels at concentrations that abolished the calciuminsensitive IdK (Figs 10 and 11). At the single-channel level, this block occurred by a prominent decrease in the open probability, with no evidence of an effect on singlechannel amplitude. Thus Kdr and KCa channels can be easily separated pharmacologically, by the use of charybdotoxin or TEA' on the one hand (to isolate Kdr activity), or 4-AP on the other hand (to inhibit Kdr without affecting KCa). These results are very similar to the findings of Beech & Bolton (1989 b), who reported that IdK in portal vein cells was insensitive to TEA+ (4 mm), and charybdotoxin (100 nm), but inhibited by 4-AP (5 mm). Neither KCa nor Kdr were affected by the sulphonylurea, glibenclamide (20 AM). In airway smooth muscle from the guinea-pig this concentration of glibenclamide completely abolished the hyperpolarization induced by K+ channel opening drugs such as cromakalim (Murray, Boyle & Small, 1989). Similar observations have been made in other tissues (Schmid-Antomarchi et al. 1987; Standen et al. 1989). This lack of effect of glibenclamide suggests that the target of K+ channel openers is not Kca or Kdr, We are grateful to Brandt Feuerstein for excellent technical assistance, and Rosemarie Cohen for excellent secretarial support. This work was supported by National Institutes of Health grant HL J. P. B. was supported by a travel grant from the Wellcome Trust. We also thank Dr Tom Hamilton and Smith Kline Beecham for support. REFERENCES AHMED, F., FOSTER, R. W., SMALL, R. C. & WESTON, A. H. (1984). Some features of the spasmogenic actions of acetylcholine and histamine in guinea-pig isolated trachealis. British Journal of Pharmacology 83, ALDRICH, R. W., SOLC, C. K., ZAGOTTA, W. N. & BRAINARD, M. S. (1989). Single potassium channels in Drosophila nerve and muscle. In Ion Transport, ed. KEELING, D. & BENHAM, C., pp Academic Press, London. BEECH, D. J. & BOLTON, T. B. (1989a). A voltage-dependent outward current with fast kinetics in single smooth muscle cells isolated from rabbit portal vein. Journal of Physiology 412,

21 DELA YED RECTIFIER CHANNELS BEECH, D. J. & BOLTON, T. B. (1989b). Two components of potassium current activated by depolarization of single smooth muscle cells from the rabbit portal vein. Journal of Physiology 418, BENHAM, C. D., BOLTON, T. B., LANG, R. J. & TAKEWAKI, T. (1986). Calcium-activated K-channels in single dispersed smooth muscle cells of rabbit jejunum and guinea-pig mesenteric artery. Journal of Physiology 371, CLAPHAM, D. E. & LOGOTHETIS, D. E. (1988). Delayed rectifier K+ current in embryonic chick heart ventricle. American Journal of Physiology 254, H COBURN, R. F. (1979). Electromechanical coupling in canine trachealis muscle: acetylcholine contractions. American Journal of Physiology 236, C COLE, W. C. & SANDERS, K. M. (1989). Characterization of macroscopic outward currents of canine colonic myocytes. American Journal of Physiology 257, C DUBOIS, J. M. (1981). Evidence for the existence of three types of potassium channels in the frog Ranvier node membrane. Journal of Physiology 318, DUBOIs, J. M. (1983). Potassium currents in the frog node of Ranvier. Progress in Biophysics and Molecular Biology 42, DUCHATELLE-GOURDON, I. & CRISS HARTZELL, H. (1990). Single delayed rectifier channels in frog atrial cells. Biophysical Journal 57, FOSTER, R. W., SMALL, R. C. & WESTON, A. H. (1983). Evidence that the spasmogenic actions of tetraethylammonium in guinea-pig trachealis is both direct and dependent on the cellular influx of Ca2+ ion. British Journal of Pharmacology 79, HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. J. (1981). Improved patchclamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflugers Archiv 391, HILLE, B. (1984). Ionic Channels in Excitable Membranes. Sinauer Inc., Sunderland MA, USA. HISADA, T., KURACHI, Y. & SUGIMOTO, T. (1990). Properties of membrane currents in isolated smooth muscle cells from guinea-pig trachea. Pftilgers Archiv 416, HODGKIN, A. & HUXLEY, A. (1952). A quantitative description of membrane current and its application to conduction and excitation. Journal of Physiology 117, HUME, J. R. & LEBLANC, N. (1989). Macroscopic K+ currents in single smooth muscle cells of the rabbit portal vein. Journal of Physiology 413, INOUE, R., KITAMURA, K. & KURIYAMA, H. (1985). Two Ca-dependent K channels classified by tetraethylammonium distribute on smooth muscles of the rabbit portal vein. Pfluigers Archiv 405, KANNAN, M. S., JAGER, L. P., DANIEL, E. E. & GARFIELD, R. E. (1983). Effects of 4-aminopyridine and tetraethylammonium chloride on the electrical activity and cable properties of canine tracheal smooth muscle. Journal of Pharmacology and Experimental Therapeutics 227, KIRKPATRICK, C. T. (1975). Excitation and contraction in bovine tracheal smooth muscle. Journal of Physiology 244, KOTLIKOFF, M. I. (1988). Calcium currents in isolated canine airway smooth muscle cells. American Journal of Physiology 254, C KOTLIKOFF, M. I. (1989). Ion channels in airway smooth muscle. In Airway SmoothMuscle in Health and Disease, ed. COBURN, R., pp Plenum Press, New York. KOTLIKOFF, M. I. (1990). Potassium currents in canine airway smooth muscle cells. American Journal of Physiology 259, L KROEGER, E. A. & STEPHENS, N. L. (1975). Effects of tetraethylammonium on tonic airway smooth muscle: initiation of phasic electrical activity. American Journal of Physiology 228, KUME, H., TAKAGI, K., SATAKE, T., TOKUNO, H. & TOMITA, T. (1990). Effects of intracellular ph on calcium-activated potassium channels in rabbit tracheal smooth muscle. Journal ofphysiology 424, LANG, R. J. (1989). Identification of the major membrane currents in freshly dispersed singled smooth muscle cells of guinea-pig ureter. Journal of Physiology 412, MCCANN, J. D. & WELSH, M. J. (1986). Calcium-activated potassium channels in canine airway smooth muscle. Journal of Physiology 372, MIRONNEAU, J. & SAVINEAU, J. P. (1980). Effects of calcium ions on outward membrane currents in rat uterine smooth muscle. Journal of Physiology 320,

22 350 J. P. BOYLE, M. TOMASIC AND M. I. KOTLIKOFF MURAKI, K., IMAIZUMI, Y., KOJIMA, T., KAWAI, T. & WATANABE, M. (1990). Effects of tetraethylammonium and 4-aminopyridine on outward currents and excitability in canine tracheal smooth muscle. British Journal of Pharmacology 100, MURRAY, M. A., BOYLE, J. P. & SMALL, R. C. (1989). Cromakalim-induced relaxation of guinea-pig trachealis: antagonism by glibenclamide and by phentolamine. British Journal of Pharmacology 98, OHYA, Y., KITAMURA, K. & KURIYAMA, H. (1987). Cellular calcium regulates outward currents in rabbit intestinal smooth muscle cell. American Journal of Physiology 252, C OKABE, E., KITAMURA, K. & KURIYAMA, H. (1987). Features of 4-aminopyridine sensitive outward current observed in single smooth muscle cells from the rabbit pulmonary artery. Pflugers Archiv 409, SCHMID-ANTOMARCHI, H., DE WEILLE, J. R., FOSSET, M. & LAZDUNSKI, M. (1987). The receptor for antidiabetic sulphonylureas controls the activity of the ATP-modulated K'-channel in insulin secreting cells. Journal of Biological Chemistry 262, SCHWARTZ, W. & VOGEL, W. (1971). Potassium inactivation in single myelinated nerve fibre of Xenopus laevis. Pfluigers Archiv 330, STANDEN, N. B., QUAYLE, J. M., DAVIES, N. W., BRAYDEN, J. E., HUANG, Y. & NELSON, M. T. (1989). Hyperpolarizing vasodilators activate ATP-sensitive K' channels in arterial smooth muscle. Science 245, WALSH, J. V. & SINGER, J. J. (1983). Ca2l activated K' channels in vertebrate smooth muscle cells. Cell Calcium 4, WALSH, J. V. & SINGER, J. J. (1987). Identification and characteristics of major ionic currents in isolated smooth muscle cells using the voltage-clamp technique. Pfluigers Archiv 408, WORLEY, J. F. III & KOTLIKOFF, M. I. (1990). Dihydropyridine-sensitive single calcium channels in airway smooth muscle cells. American Journal of Physiology 259, L YAMAMOTO, Y., Hu, S. L. & KAO, C. Y. (1989). Outward currents in single smooth muscle cells of the guinea-pig taenea coli. Journal of General Physiology 93, YUE, D. T. & MARBAN, E. (1988). A novel cardiac potassium channel that is active and conductive at depolarized potentials. Pfliigers Archiv 413,