137

Journal of Physiology (1991), 443, pp. 137-159 With 12 figures Printed in Great Britain

CHARACTERIZATION OF A Ca2l-DEPENDENT ANION CHANNEL FROM SHEEP TRACHEAL EPITHELIUM INCORPORATED INTO PLANAR BILAYERS BY E. W. F. W. ALTON, S. D. MANNING*, P. J. SCHLATTER*, D. M. GEDDES AND A. J. WILLIAMS* From the Ion Transport Laboratory and *Department of Cardiac Medicine, National Heart and Lung Institute, London SW3

(Received 10 January 1991) SUMMARY

1. Anion-selective channels from the apical membrane of respiratory epithelia are involved in the secretion of chloride into the airway lumen. In cystic fibrosis (CF) there is an abnormality of phosphorylation-regulated chloride transport in this tissue, whilst a calcium-dependent pathway appears to function normally. 2. Using incorporation of apical membrane vesicles into planar phospholipid bilayers, we have characterized the most commonly seen anion-selective channel from sheep tracheal epithelium. 3. In symmetrical 200 mM-NaCl solutions the channel showed rectification, with a chord conductance at negative voltages of 107 pS and at positive voltages of 67 pS. The channel characteristically demonstrated subconductance states at I and 3 of the fully open level. Selectivity for chloride over sodium was approximately 6: 1. 4. The channel required a minimum of approximately 100 /M-calcium on the presumed cytoplasmic surface (cis) for opening events to be observed. Open probability (PO) of the fully open state was markedly voltage dependent, but little effect of voltage was seen on the I subconductance state. 5. The relative permeabilities of monovalent anions monitored under bi-ionic conditions gave the following sequence: NO3- > I- > Cl- = Br- > F-. The order of conductances in symmetrical solutions was Cl- = N03- > Br- > I- 0> F-. 6. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) produced a dose-related reduction in PO with a flickering block at 10-50 /1M and complete block at higher concentrations. 7. ATP produced a dose-related reduction in P. with effects at 1 /AM and complete closing at 1 mm. These effects were only seen with addition to the cis chamber. 8. The catalytic subunit of protein kinase A, either when incubated with vesicles prior to incorporation into bilayers, or when added directly to either chamber, produced no effect. 9. Channels with very similar properties were seen from transfected human tracheo-bronchial cells. 10. Recent whole-cell patch-clamp studies have suggested a distinct calciumMS 9064

C1- CHANNELS FROM SHEEP TRACHEAL EPITHELIUM activated chloride current in secretory epithelia. The described channel has properties in common with this current and may be a candidate for its single-channel basis. 138

INTRODUCTION

Airway epithelial cells transport ions, particularly sodium and chloride, across their apical and basolateral surfaces. One purpose of this movement may be to regulate the local transport of water, and hence the volume of the sol phase overlying the mucosal surface. In turn, the efficiency of ciliary beating and thus mucociliary clearance may be controlled by this volume regulation. The ions move through relatively specific channels, both sodium and chloride channels having been identified from the apical membrane of airway epithelial cells. Cystic fibrosis (CF) is characterized by the presence of thick, viscid secretions in the airways. Several abnormalities of respiratory ion transport have been identified in these patients, particularly a reduction in chloride permeability and an increase in sodium absorption (Boucher, Cotton, Gatzy, Knowles & Yankaskas, 1988). Singlechannel studies using the patch-clamp technique have identified a variety of anionselective channels from airway epithelia (Frizzell, Rechkemmer & Shoemaker, 1986; Welsh, 1986; Kunzelmann, Pavenstadt & Greger, 1989b; Duszyk, French & Man, 1990). The abnormal chloride transport is likely to result from the defective regulation of one or more of these channels. Thus, whilst non-CF chloride channels respond to stimulation of the A or C (at < 10 nM-Ca2+ concentration) kinase pathways with an increase in the open probability (P.) of channels, this does not occur in CF; normal single-channel openings are not observed in the cell-attached configuration (Schoumacher, Shoemaker, Halm, Tallant, Wallace & Frizzell, 1987; Li, McCann, Liedtke, Nairn, Greengard & Welsh, 1988; Hwang, Lu, Zeitlin, Gruenert, Huganir & Guggino, 1989; Li, McCann, Anderson, Clancy, Liedtke, Nairn, Greengard & Welsh, 1989). However, agents elevating free intracellular calcium appear to regulate both single chloride channels (Frizzell et al. 1986) and macroscopic chloride currents (Boucher, Cheng, Paradiso, Stutts, Knowles & Earp, 1989) normally in CF. Recently it has been suggested that more than one type of chloride channel may be present in airway epithelia to explain these findings (Cliff & Frizzell, 1990). Further understanding of the characteristics of these various channels may be important both in CF and its possible treatment, as well as a potential means of altering sol volume and hence mucociliary clearance, pertinent to other airway diseases. Reconstitution of membrane vesicles into planar phospholipid bilayers is an alternative means of studying single-channel behaviour to the patch clamping methods so far reported. Reconstitution offers a number of advantages over patch clamping, such as the use of native rather than cultured cell membranes and easy access to both sides of the channel for alterations in the bathing solutions or addition of pharmacological agents. Furthermore the experiments often allow longer recordings from, and more interventions to, a given channel. One previous study using this technique has described the presence of anion-selective channels from bovine airway epithelium (Valdivia, Dubinsky & Coronado, 1988), a tissue demonstrating both basal chloride secretion and sodium absorption (LangridgeSmith, Rao & Field, 1984). Sheep tracheal epithelium is readily available and, when

E. W. F. W. ALTON AND OTHERS

139

mounted in Ussing chambers, has been shown to exhibit principally sodium absorption, properties very similar to those found in human airways (Cotton, Lawson, Boucher & Gatzy, 1983). We have therefore studied the characteristics of single anion-selective channels present in membrane preparations from sheep trachea and reconstituted into planar phospholipid bilayers. Whilst several types of anionand cation-selective channels were seen, only the most commonly observed channel is described. METHODS

Materials All chemicals were obtained from Sigma Ltd or BDH Ltd and were of AnalaR or best available

grade. Preparation of membrane vesicles Sheep epithelial membranes. Membrane vesicles for reconstitution and characterization studies were prepared as previously described by Langridge-Smith, Field & Dubinsky (1983). Sheep tracheae were obtained from the local abattoir and transported in ice-cold, aerated, buffered Tyrode solution (mM): NaCl, 136-9; KCl, 2-7; MgCl2 . 6H20, 05; NaH2PO4 . 2H2O, 03; NaHCO3, 11 9; CaCl2, 1-8; D-glucose, 5X6; HEPES-NaOH buffer, 50; pH 7-4. On arrival in the laboratory they were opened longitudinally along the ventral surface, loose mucus removed with fresh ice-cold Tyrode solution and incubated for 15 min in an air-bubbled, ice-cold solution containing 250 mM-sucrose, 2 mM-EGTA, 1 mM-dithiothreitol (DTT) and 5 mM-HEPES-Tris buffer, pH 7-8. The epithelial lining was removed by scraping with the edge of a microscope slide, and placed into the ice-cold homogenization medium (50 mM-D-mannitol, 0X25 mM-MgCl2, 10 mM-dithiothreitol and 5 mMHEPES-Tris buffer, pH 7-4). The suspension was homogenized in a motor-driven Teflon-glass homogenizer for three periods of 30 s, centrifuged at 2000 g., for 8 min at 4 °C and the supernatant removed. These steps were repeated three times to increase final yield. The combined supernatants were centrifuged at 9200 9av for 10 min followed by sedimentation at 36000 9av for 40 min, both at 4 °C, to produce a combined apical and basolateral membrane pellet. Basolateral membranes were precipitated by incubating the mixed membrane pellet at 4 °C in 100 mM-D-mannitol, 10 mM-MgCl2, 5 mM-HEPES-Tris, pH 7*4, for 1 h with occasional shaking. Mixed membranes were separated by centrifugation at 1500 9av for 12 min at 4 'C. The resulting supernatant (containing apical membrane) and pellet (containing basolateral membrane) were diluted with 1 mM-EDTA before sedimentation at 104000 8g, for 15 min at 4 'C. Further purification of the basolateral membranes was obtained by incubating in ice-cold 1 M-NaBr solution for 15 min before further centrifugation at 104000 9av for 15 min. Both apical and basolateral membranes were suspended in a cryoprotectant solution consisting of 400 mM-sucrose in 5 mM-HEPES-Tris, pH 7-2, to a final protein concentration of approximately 5 mg ml-'. Aliquots were snap-frozen in liquid nitrogen and stored at -80 'C. Transfected epithelial cell membranes. The tracheobronchial cell line TBE-1, transfected using the Harvey-ras oncogene (Yoakum, Lechner, Gabrielson, Korba, Malan-Shibley, Willey, Valerio, Shamsuddin, Trump & Harris, 1985), was grown to confluence in Minimal Essential Medium (MEM) containing 1 % L-glutamine, 1 % penicillin-streptomycin and 10 % fetal calf serum (FCS). Once the cells had reached confluence, they were detached by washing in MEM containing no supplements, followed by incubation for 3 min at 37 'C with trypsin-versine. Excess Dulbecco's MEM containing 0-5 % FCS and 0 5 % penicillin-streptomycin was added to inactivate the enzymes. The cells were pelleted by centrifugation at 1000 ga, washed with MEM and pelleted again at 1000 gv. Membrane vesicles were then prepared in the same manner as for sheep tracheal epithelium. Epithelial membrane characterization To assess the efficacy of membrane separation, apical and basolateral membranes and the original homogenate were assayed for the apical marker enzyme alkaline phosphatase. Alkaline phosphatase activity (umol inorganic phosphate produced per hour per mg protein) was measured at room temperature in 2-5 ml of a solution containing 5 mM-MgCl2, 01 % Triton X-100, 100 mMHEPES-Tris, pH 9-0 with 1 mM-p-nitrophenyl phosphate and the reaction monitored by changes in absorbance at 405 nm. The extinction coefficient of 222 1 cm-2 mmol- for p-nitrophenyl

140

C1- CHANXVELS FROM 'WHEEP TRACHEAL EPITHELILUM

phosphate at 405 nm and the rate of change of absorbance with time were used in calculations of activities. Protein concentrations were determined by the Coomassie Brilliant Blue binding assay (BioRad, Herts, UK) with bovine serum albumin as standard. Single-channel technique Incorporation of membrane vesicles into planar phospholipid bilayers was carried out as previously described by Miller (1982). Briefly, the cis chamber consisted of a styrene copolymer cup of internal volume 0 8 ml containing a 200 jtm hole joining this chamber and an opposing well of volume 5 0 ml (trans chamber). KCl wells adjacent to the membrane chambers connected the cis and trans solutions via Ag/AgCl electrodes to an operational amplifier (sensitivity 10 mV/pA). External voltage commands provided by an Amstrad CPC 6128 computer linked to a digital-toanalog converter were applied to the cis chamber, the trans chamber being held at ground potential. The signal was filtered using a Krohn-Hite 4-pole low-pass filter (Krohn-Hite Corp., MA, USA) before display on an oscilloscope (Gould Electronics, UK). Unfiltered data were stored on FMI tape for further analysis. Bilayers were formed from phosphatidylethanolamine and phosphatidylserine (70:30 %) (Avanti Polar Lipids, USA) dispersed in n-decane. Chambers were filled with either 200 mM-NaCl or KCl in 10 mM-HEPES-Tris, pH 7-2. Membrane thickness was monitored by capacitance measurements during repeated + 4 mV voltage clamping. Membrane vesicles (50-100 ,ug protein) were added to the solution in the cis chamber along with 2 mM-Ca2' and fusion occurred following elevation of the osmolarity of NaCl or KCl in the cis chamber. Vesicle fusion was usually seen within 3-5 min. Following vesicle incorporation the likely number of channels was ascertained by clamping at -30 mV. At this voltage the probability of the channel being open is maximal and the presence of more than one channel was quickly obvious. All incorporations of multiple channels were discarded. In the presence of a single channel the cis chamber was perfused with the required experimental solution using a peristaltic pump.

Experimental protocol Measurement of single-channel current-voltage (I-V) relationship. For each channel an I-V relationship (+ 70 mV) was recorded prior to further experiments in the presence of a 3: 1 gradient, or in symmetrical solutions of NaCl or KCl. Clamp voltages > + 70 mV frequently caused bilayer disruption. MI1easurement of single-channel open probability (P.). All recordings were made using a 600 mm cis:200 mm trans gradient of the appropriate electrolyte solution. Twenty-five second duration recordings of channel activity at appropriate clamp voltages were made preceded by 3 s clamping at -30 mV to standardize starting P.. Data were analysed as described below. Effect of changes in cis/trans calcium concentrations. The cis chamber was clamped at -30 mV to optimize P. and 25 s recordings of channel activity made at calcium concentrations on the cis side of 1 nM-2 mm. Whenever possible, following a baseline recording at 2 mM-calcium, the chamber was perfused out with 600 mm electrolyte solution containing no added calcium (10 /tM-free calcium), 10 ,IM-EGTA (1 ,aM-free calcium) or 1 mM-EGTA (1 nM-free calcium). Aliquots of calcium were then added to either chamber and P. measured accordingly. Free calcium concentrations were calculated using Eqcal (Biosoft, Cambridge, UK). Relative anion permeabilities and monovalent anion conductances. The relative permeabilities of various anions compared to Cl- were estimated from reversal potential measurements with 500 mMNaCl in the trans chamber and either 500 mM-NaBr, Nal, NaF or NaNO3 in the cis chamber. All solutions were buffered to pH 7-2 with 10 mM-HEPES-Tris. Monovalent anion conductances were obtained from I-V relationships measured in symmetrical 500 mm solutions of the test anion. Effect of chloride channel blockers. 5-Nitro-2-(3-phenylpropylamino)-benzoate (NPPB) was added to the appropriate chamber starting at a concentration of 1 ,UM. Following measurement of P. (described below) the chamber was washed out with control solution. Increasing concentrations of blocker were then added with chamber wash-out between each concentration. NPPB was dissolved in dimethyl sulphoxide (DMSO) to produce a 100 mm stock solution. A 10 mm solution was obtained by dilution in 60 % DMSO, 30 % HEPES-Tris. Thereafter all dilutions were made using HEPES-Tris. The effects on P. of the largest volume of DMSO used (10 jul) were studied in two channels, with no changes seen in either channel. Diphenylamine-2-carboxylate (DPC) (1-6 mM), 4,4'-diisothiocyano-stilbene-2,2'-disulphonic acid (DIDS) (100 /tM) and 4-acetamido-4'-isothioeyanotostilbene-2,2'-disulphonic acid (SITS) (100 ,am) were added at the indicated concentrations. All drugs were added as 1:100 dilutions to the chambers.

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141

Effect of ATP. ATP (disodium salt) was added to the appropriate chamber to produce final concentrations of ATP of 1 am-1 mm. The chamber was washed out with control solution between the addition of each ATP concentration. Effect of catalytic subunit of protein kinase A. Following addition of magnesium (2 mM) and ATP (1 mM) to the appropriate chamber (Valdivia et al. 1988), 50,ag of purified catalytic subunit of bovine cardiac protein kinase A in 30 mM-KH2P04, 01 mM-EDTA, 10 mm-DDT, 200 mM-KCl and 40 0/4 glycerol, pH 6-7, was added to the same chamber and P. monitored for up to 1 h following kinase addition.

10 pA 2s

10 pAL 2s

Fig. 1. Current fluctuations from two separate experiments in which a single channel from sheep tracheal epithelium was incorporated into a planar phospholipid bilayer. The recordings were made at -30 mV under the following conditions: cis, 600 mM-NaCl, 2 mm-CaCI2; trans, 200 mM-NaCl, and are shown low-pass filtered at 600 Hz. The dotted lines indicate the - and 3 subconductance levels as well as fully open (0) and closed (C) levels as shown. In pre-incubation experiments with A kinase, 50 ,ug membrane vesicles were added to a 1: 200 dilution of 20 ,ug A kinase in 10 mM-CaCl2, 12-5 mM-MgCl2, 1 mM-ATP, 50 mM-piperazine-N,N'-bis2-ethane sulphonic acid, pH 68, and incubated for 15 min on ice (Valdivia et al. 1988). Five microlitre aliquots were then added to the cis side of the chamber, and following channel incorporation PO monitored at a range of holding potentials for up to 1 h. Cyclic AMP-dependent protein kinase activity was assessed by measurement of kemptide phosphorylation (Giembycz & Diamond, 1990). The specific activity of the catalytic subunit was 386-3 nM P04 min-' mg-'.

Data analysis I-V plots were constructed from recordings played onto a Lectromed Multitrace 2 chart recorder, and analysed by hand. Data were digitized (2 kHz) and PO was analysed using 'Satori' (Intracel, Cambridge, UK) run on an IBM-compatible AT. Typically the channel demonstrated two subconductance levels at approximately I and 3 of the fully open level (Fig. 1). To enable separate analysis of the 3 substate each strip of data was analysed using both 20 and 50% amplitude threshold analysis (TA) (Colquhoun & Sigworth. 1983). Using 50% TA (treating the 3 substate as a closed level) P. was calculated from open time/total recording time. P. was similarly obtained using 20 % TA, and percentage of time spent in the I substate (I %) calculated from 20% TA -50% TA for each voltage. Total time spent in the 3 substate (3 time) was calculated from 3 % x total recording time. The number of 3 substate events was obtained from

142

Cl- CHANNELS FROM SHEEP TRACHEAL EPITHELIUM

50 % TA -20 % TA event numbers. A time/' event number enabled calculation of a mean dwell time of the channel in the A substate. No attempt was made to analyse the 3 substate because of its relative infrequency compared to the A substate, and its proximity to the fully open level. Data were filtered at 100 Hz using an 8-pole Bessel filter (Frequency Devices 902) for all analysis of P,. Whilst analysis of voltages of 0 mV or more negative could be achieved by filtering less harshly (500-1100 Hz), at more positive clamp voltages the signal/noise ratio precluded even filtering at 500 Hz. Because of the need for such harsh filtering the effects of varying filter frequencies on the measured parameters were studied in three channels. Results of this analysis are discussed in the Appendix. A total of fifty-five single channels were used in the experiments analysed for this paper. RESULTS

Epithelial membrane characterization Assays for the apical membrane marker alkaline phosphatase were undertaken in order to compare the efficacy of the preparation in our hands compared with that previously reported (Langridge-Smith et al. 1983). The results of these assays are summarized in Table 1. The specific activity of alkaline phosphatase in our preparations was 8-0-fold greater in the apical fraction than in the homogenate, and 5-3-fold greater than in the basolateral fraction. Membrane vesicles harvested from the apical fraction were used in all singlechannel experiments. The most commonly observed anion channel was studied in detail. Channel conduction and selectivity Figure 2A shows representative single-channel fluctuations at a range of clamp voltages following incorporation of the channel into a planar phospholipid bilayer. The current-voltage (I-V) relationship for the fully open state in asymmetrical (600 :200 mm, cis: trans) solutions (n = 23 channels) and in symmetrical (200 mM) solutions (n = 5) is shown in Fig. 2B. The reversal potential with a 3:1 NaCl gradient across the bilayer was + 19 mV with a Cl-/Na+ selectivity ratio of approximately 6: 1 calculated from the Goldman-Hodgkin-Katz (GHK) equation. Rectification of current at positive clamp voltages was observed in both symmetrical and asymmetrical conditions. In symmetrical 200 mm-NaCl the single-channel chord conductance at negative voltages was 107 pS and at positive voltages it was 67 pS. Frequent channel run-down after 1-2 min was seen in symmetrical conditions at these salt concentrations. Figure 3 shows I-V relationships for each of the subconductance states of the channel in asymmetric conditions (600:200 mm NaCl). For the I substate the mean percentage (+ S.E.M.) of the fully open state was 36-3 ± 2 1 % (range 27-0-55-6 %) and for the - substate 780 + 1-6% (range 66-7-84-1 %). No voltage dependence of these values was noted for either substate. The reversal potential for the I substate (+ 20 mV) was not different to that of the fully open channel.

Voltage dependence of open probability The effect of the applied clamp voltage on the open probability of the channel (P.), using 50% TA, is shown in Fig. 4. P. was found to decrease markedly at positive voltages. After high positive clamp voltages the channel generally closed, but it was possible to reopen the channel following a few seconds at a holding potential of

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-60 10 pA 2s Fig. 2. A, single-channel current fluctuations at the holding potentials indicated (mV); 600:200 mM-NaCl (cis: trans), filtered at 600 Hz. Dotted lines indicate the closed channel level. Tracings at positive holding potentials have been inverted so that all channel openings are shown as upwards deflections. B, single-channel current-voltage relationships in symmetrical 200 mM-NaCl (@) (n = 5) and 600:200 mM-NaCl (cis: trans) (*) (n = 23). Points represent mean values and error bars S.E.M. (most error bars fall within the symbols). The continuous curves were drawn by eye and have no theoretical

significance. TABLE 1. Relative specific activity of alkaline phosphatase in the homogenate and apical and basolateral fractions of the sheep tracheal epithelial membranes. Results are expressed as mean values + S.E.M. for four membrane preparations. Assays were carried out as described in the Methods Total alkaline Specific activity Protein phosphatase (,umol inorganic phosphate h-' mg-') (mg) (/tmol h-1) 19-7+5-6 156-4 + 42-2 8-0+0-1 Homogenate 2-4+0-2 152-7 + 25-2 63-5 + 6-0 Apical Basolateral 10-8+0 7 127-2+7-1 12-0+1-0

-30 mV or following a series of short oscillations between clamp voltages of +30 and -50 mV. Only a slight increase in the occurrence of the I substate was seen as the clamp voltage became more positive (Fig. 4). Overall the I substate represented approximately 8 % of the total time the channel spent in the open state. The effect of voltage on PO for channels obtained from the TBE-1-transfected cell line is also

144

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shown in Fig. 4. No differences were seen between either channel population, nor when NaCl or KCI bathing solutions were compared (data not shown).

Effects of clamp voltage on channel kinetics The effects of the applied voltage on the kinetic behaviour of the channel have been studied. A summary of the results is shown in Fig. 5.

E. W. F. W. ALTON AND OTHERS

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Negative clamp voltages Using 50 % TA, with increasingly negative clamp voltages, fewer opening events (Fig. 5A) were seen of longer duration (Fig. 5E). There were also fewer closings (Fig. 5A) of unchanged duration (Fig. 5C) with little change in P. over this voltage range B

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(see Fig. 4). The fewer closing events may relate either to less full closings, or to fewer entries of the channel into the 3 substate, treated as closings at this threshold amplitude. To distinguish these possibilities, 20 % TA shows there to be little

146 C1- CHANNELS FROM SHEEP TRACHEAL EPITHELIUM alteration in the duration (Fig. 5D) of full closings, but the number of 3 events is reduced (Fig. 5B), albeit, of the same duration (Fig. 5F). Thus increasingly negative clamp voltages change channel kinetics to fewer long open events without effect on full closings. However, the channel enters the 3 substate less often, although when it does so the dwell time is unchanged. Since the I substate represents only a few per cent of the channel's total open probability (see Fig. 4), there is relatively little change in the total PO over this voltage range.

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Positive clamp voltages With increasing positive clamp voltages fewer openings (Fig. 5A) of unchanged duration (Fig. 5E), and fewer closing events (Fig. 5A) of longer duration (Fig. 5C) were seen. These were accompanied by a large change in total P. (see Fig. 4). Twenty per cent TA indicates that there are now fewer 3 events (Fig. 5B) of increased duration (Fig. 5F) as well as fewer (Fig. 5A) longer length (Fig. 5D) full closings. Thus at positive voltages both types of 'closing' events from the fully open state are reduced in number but longer in duration, accompanied by fewer openings of unchanged duration. Overall this results in a marked reduction in P. since both 3 and full closings are affected. Calcium dependence of open probability The effect of varying the calcium concentration in the cis chamber is shown in Fig. 6. No channel activity was seen until > 50 jsM-calcium was present, PO increasing steeply thereafter. Two millimolar calcium added to the trans chamber did not alter the requirement for > 50 #M-cis calcium to produce channel opening. Alterations in

E. W. F. W. ALTON AND OTHERS

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clamp voltage from -90 to + 80 mV did not overcome the inhibitory effect of < 50 JSM-cis calcium (data not shown). Relative anion permeabilities and monovalent anion conductances The relative permeabilities of various monovalent anions compared to Cl- were assessed from zero current reversal potential measurements under bi-ionic conditions; e.g. with 500 mM-NaBr in the cis chamber and 500 mM-NaCl in the trans chamber. Measurements were corrected to take into account the junction potentials

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[ATP] (AM) Fig. 8. A, single-channel current fluctuations illustrating the effect of increasing concentrations of ATP on the sheep tracheal epithelial channel. All recordings were made in asymmetric NaCl solutions (cis 600 mM-NaCl: trans 200 mM-N aCl) and are shown lowpass filtered at 600 Hz at a holding potential of -30 mV. The dotted lines indicate the closed channel level. The cis chamber was perfused back to the control solution between each concentration of ATP. In each case the effect was reversible. At 1 mm complete closing of the channel was seen which was not reversible (data not shown). B, the effect

E. W. F. W. ALTON AND OTHERS

149

(within the range +4 mV), and permeability ratios (PA/Pc1) were calculated from the GHK relationship. No single-channel fluctuations were discernible at any voltage (± 90 mV) in bi-ionic conditions with fluoride as the test anion. Table 2 summarizes the data from these experiments. The sequence of relative anion permeabilities (in the presence of Na+) is as follows: NO3- > 1- > Cl- = Br- > F-. The chord conductances at positive and negative voltages for the channel in symmetrical 500 mm solutions of the test anion are also shown in Table 2. No current flow was observed in symmetrical fluoride. The order of conductances obtained is as follows: Cl- = NO3- > Br- > 1- > F-. TABLE 2. Relative anion permeabilities and conductances Test anion Erev -ve PA/PCI G+ve

(A)

(n)

(mV)

(pS)

(pS)

(n)

1 132 0 (19) 100 (17) Chloride 78 70 (4) -2 5(5) 0 86 Bromide 27 33 (3) 1-50 11-0(8) Iodide (3) -(7) Fluoride 135 2-98 90 23 0(6) (5) Nitrate solutions. In bio-ionic to control NaCl with are respect shifts expressed Reversal potential (Erev) experiments the trans chamber contained the control 500 mM-NaCl solution whilst the cis chamber contained the sodium salt of the test anion, A, at the same concentration. Erev values have been corrected to take into account the measured junction potentials (range +4 mV) and permeability ratios (PA/Pc1) calculated from the GHK equation. Activities were taken from W. J. Hamer & Y.-C. Wu; quoted in Lobo (1989). PNa/PcI was taken as 0-17 (see Fig. 2B). Conductance measurements for each test anion were measured from channels bathed symmetrically in 500 mmNaA. The chord conductance values at positive and negative voltages are shown.

Effect of channel blockers The chloride channel blocker NPPB produced a dose-related reduction in the time the channel spent in the fully open state (Fig. 7). At concentrations up to 50 ,tM a flickering block was seen, with higher concentrations producing long periods of closings with only occasional opening events. NPPB was immediately effective when applied to the cis chamber but a blocking effect was only observed after 5 min following addition to the trans chamber. The latter effect could be temporarily reduced by perfusion of the cis chamber with control solution, suggesting that the effect of trans addition was due to diffusion into the cis chamber. Both DPC and SITS/DIDS caused complete closing of the channel at concentrations of 1-6 mm and 100 fSM respectively (data not shown). -

ATP effects ATP produced a dose-related reduction in P. with effects at 1 ,UM and complete closing of the channel at 1 mm (Fig. 8). These effects were only seen with addition to the cis chamber. The rapid flickering block was seen at negative clamp voltages with of increasing concentrations of ATP on the PO of the fully open state (El) and the j subconductance state (A). Open symbols are mean values for two or three determinations at each ATP concentration. The filled symbols represent P,O measurements for experiments in which no ATP was added to the chambers (n = 23).

C1- CHANNELS FROM SHEEP TRACHEAL EPITHELIUM no apparent effect at positive potentials. The effect was reversible at all concentrations (except 1 mM) when ATP was removed by perfusion (data not shown). Effect of ATP on channel kinetics The analysis of the effect of ATP on measured single-channel kinetic parameters is shown in Fig. 9. 150

A

n

B

1000

600

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400 j

0

6 500

z

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c

D

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80

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Fig. 9. The effect of the ATP concentration on the following measured parameters for the sheep tracheal epithelial channel. A, total number of events measured using 50 % TA; B, number of I events, C, mean closed time measured using 50 % TA; D, mean closed time measured using 20 % TA; E, mean open time measured using 50 % TA; F, dwell time in 3 subconductance state. These parameters were measured as described in the Methods. Open symbols are mean values for two or three determinations at each ATP concentration. The filled symbols represent measurements of parameters for experiments in which no ATP was added to the chambers (n = 23).

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1-10 /%M The addition of 1-10 1tM-ATP to the cis chamber produced only a small decrease in total PO using 50% TA (Fig. 8B). An increase in the number (Fig. 9A) of shorter length (Fig. 9E) openings was accompanied by more closings (Fig. 9A) of unchanged 1.0

Zc-2IN.

0.8

A

0.6

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-60

-40

-20

0

20

40

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Fig. 10. The effect of applied voltage on the probability of the channel being open in the presence and absence of the catalytic subunit of protein kinase A. Circles represent mean P. values determined using 50 % TA and squares represent mean values for I Po determined as described in the Methods section. All filled symbols are data recorded following pre-incubation of the membrane vesicles with the purified subunit prior to bilayer incorporation (n = 5). The open symbols are control values in the absence of subunit (data the same as filled symbols in Fig. 4).

duration (Fig. 90). Twenty per cent TA showed no change in the duration of full closings (Fig. 9D). However, the number of ' events increased (Fig. 9B), but the dwell time remained unchanged (Fig. 9F). Thus there was an increase in the percentage of time spent in the I state (Fig. 8A and B), but because of the relatively small contribution of this to the total Po,, little change was seen in this parameter

(Fig. 8B).

10-1000 PtM

Increasing ATP concentrations as analysed by 50 % TA produced fewer openings (Fig. 9A) of unchanged (i.e. short) duration (Fig. 9E) and fewer closings (Fig. 9A) of longer duration (Fig. 90). Twenty per cent TA shows that full closing duration increased greatly (Fig. 9D) and there was also some reduction in the number of A' events (Fig. 9B). Thus, an increase in the duration of full closings was accompanied by a marked fall in total Po (Fig. 8B). The effect of catalytic subunit of protein kinase A From membrane vesicles incubated with the purified catalytic subunit of protein kinase A prior to incorporation into bilayers, five single channels were seen.

Cl- CHANNELS FROM SHEEP TRACHEAL EPITHELIUM Comparison of the open probability of the fully open and 3 substates of these with the twenty-three previously described channels is shown in Fig. 10. No differences were seen in either comparison. In four channels the subunit was added directly to the cis chamber. The concentration of ATP was chosen to try to minimize the blocking effects of ATP whilst providing sufficient substrate for phosphorylation. No effect of kinase was seen in addition to the blocking effect of ATP under these conditions (data not shown) for up to 1 h at a range of holding potentials. In four channels the subunit was added directly to the trans chamber under the same conditions as for cis chamber addition. No effect was seen on the described channel, although on three occasions other types of channels were activated by kinase addition (data not shown). These phosphorylated channels were not studied in detail. 152

DISCUSSION

A number of anion-selective channels have been described from patch-clamp studies of airway epithelium. The single-channel conductances (approximately 20-55 pS; Frizzell et al. 1986; Welsh, 1986; Jetten, Yankaskas, Stutts, Willumsen & Boucher, 1989; Kunzelmann et al. 1989b; Duszyk et al. 1990) and calcium independence (Welsh, 1986; Kunzelmann et al. 1989b; Duszyk et al. 1990) suggest that the channel described in this paper differs from those previously reported. This is further illustrated by their voltage-independent P. (Welsh, 1986; Duszyk et al. 1990) and differences in halide selectivity (Welsh, 1986; Frizzell, 1987; Kunzelmann et al. 1989b; Duszyk et al. 1990). A 50 pS channel requiring 180 nM-cytoplasmic calcium for channel opening, with strong voltage dependence and a halide selectivity sequence following Eisenman's first sequence, has been described (Frizzell et al. 1986; Frizzell, 1987). However, this channel is upregulated by A kinase and no inhibitory effects of ATP have been reported. One previous study using membrane reconstitution described a channel of similar size to that described in the present study, but with no calcium dependence nor obvious subconductance states and a different halide selectivity (Valdivia et al. 1988). The reasons for the current apparent diversity in airway epithelial channels is unclear but may reflect differences of technique or experimental conditions. Patchclamp studies have been carried out using cells obtained from primary culture. Reconstitution allows the use of native membranes, although the described channel was also seen in cultured cells. Sheep airway epithelium was the predominant tissue used in this study; however, the channel was also seen in native human airway (data not shown) as well as in the human TBE-1 cell line. Preparation of membrane vesicles for reconstitution studies may produce vesicles containing channels from regions of the apical membrane not accessible to the patch pipette. Membrane vesicles were prepared by the previously described method (LangridgeSmith et al. 1983). Epithelial cells were scraped from the tracheal surface and apical and basolateral fractions analysed for the apical enzyme marker alkaline phosphatase. Our apical:basolateral ratio of 5-3 (Table 1) compares with 10-7 noted for bovine trachea. It is likely that apical contamination of the basolateral fraction

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accounts for the increased basolateral total activity and hence the slightly lower apical/basolateral ratio. That the channel is of epithelial origin is also suggested by our comparison of data obtained from the TBE-1 epithelial cell line (Fig. 4). We cannot be certain of the orientation of the channel within the bilayer. The cisonly calcium dependence strongly suggests that this represents the cytosolic surface. We know of no channel requiring extracellular calcium for activation, and the effects of ATP on the cis surface are compatible with this orientation. However effects of ATP on the extracellular surface of airway epithelial cells have been described recently (Mason, Paradiso, Brown, Harden & Boucher, 1990). Our assumption is in contrast to that for the bovine airway chloride channel reconstituted into planar bilayers, in which it was considered likely that the trans chamber represented the cytosolic surface (Valdivia et al. 1988). Evidence for this was based upon phosphorylation by purified A kinase subunit having an effect only from the trans surface. However an effect of pre-incubation with kinase was also seen following subsequent channel incorporation into the bilayer. Since it is unlikely that the kinase could phosphorylate a channel within the membrane vesicle, it is likely that the cytosolic surface was on the external vesicle surface, and hence would have incorporated into the bilayer as the cis side being cytosolic. If our assumption is correct, the sheep airway epithelial channel described here displays outward rectification as do the kinase-dependent channels reported in previous patch-clamp studies.

Conductance and selectivity The described epithelial channel has a reversal potential of + 19 mV in 600:200 mM asymmetric solutions indicating a selectivity for chloride over sodium of approximately 6:1. A similar anion selectivity is described for the outwardly rectifying phosphorylation-dependent epithelial chloride channel (Welsh, 1986). The mean conductance of approximately 87 pS at 0 mV in symmetrical 200 mm solutions includes this channel in the intermediate-sized chloride channels described in various epithelia (Greger, Gerlach & Kunzelmann, 1989). Rectification at positive voltages was seen both in asymmetric and symmetric bathing solutions. Halide selectivity as measured by permeability ratios corresponds to Eisenman's first sequence, indicating interaction of the anion with a channel site of relatively low field strength. Selectivity monitored by relative conductances differs from this permeability sequence. Further information relating to the interpretation of this conductance sequence requires analysis of conductance/saturation and mole fraction studies to determine the degree of ion occupancy of the channel.

Voltage dependence A prominent feature of the described channel is the presence of substates (Fig. 1). These have been previously noted or alluded to in other chloride channels (Li, McCann & Welsh, 1990), but to our knowledge there has been no previous attempt to assess their contribution to channel kinetics (see Appendix). Decreasing clamp voltages from negative values towards zero increased entry into the I substate, with little effect on total P. (Fig. 4) and only a small increase in I P. (Fig. 12) amongst channels with < 10% contribution to total PO of the - substate.

154

C1- CHANNELS FROM SHEEP TRACHEAL EPITHELIUM

The channel showed marked voltage dependence of the fully open state with long closings at positive clamp voltages (Figs 2A and 4). Thus within the likely physiological range of the apical membrane voltage (-25 to -40 mV), relatively large changes in P. were seen. Inhibition by amiloride of sodium absorption, the principal mode of ion transport in these tissues, causes significant alterations in apical membrane voltage (Willumsen, Davis & Boucher, 1989). Whether an 'endogenous amiloride' is present to regulate such absorption in vivo, is at present only speculation, although candidates such as 5-HT and melatonin have been proposed (Legris, Will & Hopfer, 1982). Effect of calcium The channel described here required a minimum of > 50 /LM-calcium on the cis side for channel opening, P. rising steeply over a narrow range (Fig. 6). If this is also the case in intact cells, the described channel may contribute to the apical chloride conductance seen following ionophore-induced elevation of free calcium. Thus in the T84 cell line whole-cell currents were stimulated by ionomycin in the presence of 1 mM-external calcium, but not with bath calcium concentrations of 1 ,tM (Cliff & Frizzell, 1990). This ionomycin-induced current could be differentiated from a cyclic AMP-induced current, suggesting a separate pathway to that activated by phosphorylation, and thus a possible route for therapeutic intervention in CF. Bradykinin has been shown to activate chloride secretion in CF as well as normal tissues, but increases free intracellular calcium to < 1IJtM (Boucher et al. 1989); however, bradykinin has several effects on intracellular second messengers (Smith, McCann & Welsh, 1990). Whether these changes are synergistically involved in channel regulation is unknown. Furthermore several intracellular calcium stores may be present, and may be able to release calcium with differing spatial and temporal characteristics (Berridge & Irvine, 1989). Thus comparison between macroscopic current and single-channel regulation by various calcium concentrations is presently difficult.

Effects of ATP, NPPB and protein kinase A During preliminary studies to examine the effects of protein kinase A on channel activity it became clear that addition of the Mg2+-ATP cocktail caused a decrease in PO. The presence or absence of Mg2+ in concentrations up to 2 mm had no effect, whilst ATP, alone or in combination with Mg2+, produced the noted changes. A very similar blocking effect of ATP on an anion-selective channel from platelet membranes has been described (Manning & Williams, 1989). At voltages at which the channel is predominantly open, low concentrations of ATP increased the relative time spent in the I subconductance state (Figs 8A and 9), a similar effect to increasing clamp voltage towards zero from negative voltages (Fig. 5). At higher concentrations ATP caused long full closings (Figs 8A and 9), again similar to the effects of increasingly positive clamp voltages. The present data do not allow us to describe a detailed model for the effects of ATP and voltage. Since the latter acts on channel gating we speculate that ATP may also alter gating, the voltage-related effect allowing ATP access to a regulatory site. However ATP could also be acting directly as a voltage-dependent blocker. If a

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similar regulatory process was present in vivo a chloride channel tonically inhibited by ATP would be analogous to the basolateral potassium channel present in airway epithelia (Kunzelmann, Pavenstadt & Greger, 1989a). Thus an increase in Na+-K+ATPase activity, which is likely to occur during chloride secretion, could favour chloride channel opening at the apical membrane. As for the potassium channel 1 mM-ATP caused complete inhibition of chloride channel activity. Such ATP concentrations are likely to be lower than intracellular levels. Similar apparent decreases in ATP sensitivity have been described for potassium channels in several cell types when comparison is made between studies using excised patches or wholecell voltage clamp (Kunzelmann et al. 1989a). As in the case of patch excision, reconstitution removes the channel from its normal regulatory processes and may induce similar alterations in ATP sensitivity. The chloride channel blocker NPPB typically produces a flickering block at micromolar concentrations in epithelial chloride channels (Dreinhofer, G6gelein & Greger, 1988) with complete block at higher concentrations (Greger, Schlatter & Gogelein, 1987). This effect was also seen with the described channel, 100 JtM-NPPB producing a complete block (Fig. 7). Interestingly, NPPB appeared to cause an increase in the 3 substate at low concentrations with flickering between fully open and closed states at higher concentrations. Under the given conditions, the catalytic subunit of protein kinase A, whether added directly to either chamber, or following pre-incubation with the membrane vesicles, produced no change in PO in a total of thirteen channels. Both kemptide phosphorylation and the activation of channels other than that described in this study indicated subunit activity. In the presence of an inhibitory effect of ATP it is perhaps not surprising that the kinase does not reverse this process, and it is likely that this channel is not upregulated by phosphorylation. This is in marked contrast to the well-described smaller conductance chloride channel(s), which appear to be abnormally regulated in CF. We have described the basic characteristics of a calcium-dependent chloride channel, down-regulated by ATP and unaffected by A kinase. Frizzell has recently proposed that three types of chloride channel are present in airway epithelia, i.e. kinase dependent, calcium dependent and volume regulated. The calcium-induced current shows outward rectification, time-dependent voltage activation at depolarizing voltages, inactivation at hyperpolarizing voltages and sensitivity to DIDS (Cliff & Frizzell, 1990). These properties are shared by the described channel and we suggest that it may be a candidate for the single-channel basis of the calcium-induced current. We are presently attempting to ascertain the role of this channel in the macroscopic chloride current of sheep trachea mounted in Ussing chambers. If it is possible to include a function for this channel in the physiological chloride current of a sodium-absorbing epithelium, comparison with CF airways would be of interest. APPENDIX

The presence of rectification at positive voltages and the proximity of the prominent substate to the baseline necessitated relatively harsh filtering to overcome baseline noise. Since the prominent substate is at the 3 open level we undertook a

C1- CHANNELS FROM SHEEP TRACHEAL EPITHELIUM comparison of P. at 20 % TA as well as the conventional 50 % level. The effect of filter frequency on measured parameters is shown in Fig. 11. No change in PO of the fully open state using either 20 or 50% TA was seen, nor of the I substate (Fig. I IA). However the mean open time using 20 % TA decreased more markedly than using 156

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Fig. 11. The effect of filter frequency on the following measured parameters from three channels. A, open probability of the fully open state, using 50% TA (0) and 20% TA ( O ), and the I subconductance state (AL). B, mean open time measured using 50 % TA (0) and 20% TA (A). C, dwell time in the I subconductance state (El) and mean closed time using 20% TA (0).

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50 % TA (Fig. 11 B). Thus it is likely that full closing events are of shorter duration than 3 dwell times. Filter frequency had little effect on the 3 substate dwell time or mean closed time using 20% TA (Fig. 11C). Using 20 % TA, even with 100 Hz filtering, at positive clamp voltages it was clear that baseline noise on occasions crossed the 20 % analysis line. An indication of the

j

0.20

*

0-15 0.10

0.05

-60

-40

-20

0 20 40 60 V (mV) Fig. 12. The effect of voltage on the PO of the 3 subconductance state where this state represents > 10% (@) and < 10% (A) of the totalP.. Points are mean values+s.E.M. for two to fourteen separate determinations at each applied voltage.

degree of this problem is provided by analysis of the number of open/closed events at either threshold. If the event number at 20 % TA exceeds that at 50 % TA either the channel opened only to the 3 substate from the closed level, or baseline noise accounted for the increased event number at 20 % TA. The effect of voltage on channels in which the - substate represented > 10 % of total PO is shown in Fig. 12 and compared to channels with < 10 % 3 substate P.. Little influence was seen at positive voltages where a maximum effect would expected. Several lines of evidence suggest that our results were not substantially altered by these methods of analysis: (a) When channels with 20% TA exceeding 50% TA event number were excluded, no effect on PO was seen. (b) Event number using 50 % TA was unaffected by baseline noise under the described conditions, even at positive voltages. Since the I substate only contributes approximately 8% of total event number, the voltage dependence of the 20 % TA event number would be predicted to substantially resemble that seen for 50 %. However if baseline noise contributed significantly, the shape of the 20% TA event number would deviate significantly from 50 % TA at positive voltages, an effect that was not seen. (c) At increasing positive voltages it was clear from examination of channel recordings that event number using 20 % TA decreased as clamp voltage became more positive, in agreement with the obtained analysis. (d) At 0 mV, interference of baseline noise was not seen during inspection of data entered into Satori for analysis. Therefore the large increase in event number at this clamp voltage is likely to be representative of

158

Cl- CHANNELS FROM SHEEP TRACHEAL EPITHELIlUM

channel kinetics. (e) Although the channel generally entered the 3 substate from the fully open level, longer runs were seen in which this was achieved from the closed level. These events will then be recorded only at the 20 % TA, and will have a similar effect to artifactual membrane noise. Separate analysis of channels demonstrating > 10 % of PO due to the 33 substate showed little difference in voltage dependence

(Fig. 12). XVe are grateful to Professor R. Greger for the NPPB, Professor I. Levitan for the catalytic subunit, Dr C. Harris for providing the TBE-1 cell line, Mr S. Smith and Dr P. Jeffery for help with maintenance of the cell culture and Dr M. Giembycz for carrying out the kemptide phosphorylation and for useful discussion. E. A. was supported by an MIRC Training Fellowship, P.S. by the Wellcome Trust and the studies by the CF Research Trust and the British Lung Foundation. REFERENCES

BERRIDGE, M. J. & IRVINE, R. F. (1989). Inositol phosphates and cell signalling. Nature 341, 197-205. BOUCHER, R. C., CHENG, E. H., PARADISO, A. M., STUTTTS, M. J., KNOWLES, M. R. & EARP, H. S. (1989). Chloride secretory response of cystic fibrosis human airway epithelia. Preservation of calcium but not protein kinase C- and A-dependent mechanisms. Journal of Clinical Investigation 84, 1424-1431. BOUCHER, R. C., COTTON, C. U., GATZY, J. T., KNOWLES, M. R. & YANKASKAS, J. R. (1988). Evidence for reduced Cl- and increased Na' permeability in cystic fibrosis human primary cell cultures. Journal of Physiology 405, 77-103. CLIFF, W. H. & FRIZZELL, R. A. (1990). Separate Cl- conductances activated by cAMP and Ca2" in Cl--secreting epithelial cells. Proceedings of the National Academy of Sciences of the USA 87, 4956-4960. COLQUHOUN, D. & SIGWORTH, F. J. (1983). Fitting and statistical analysis of single channel records. In Single-Channel Recording, ed. SAKMANN, B. & NEHER, E., pp. 191-263. Plenum Press, New York. COTTON. C. U., LAWSON. E. E., BOUCHER. R. C. & GATZY, J. T. (1983). Bioelectric properties and ion transport of airways excised from adult and fetal sheep. Journal of Applied Physiology 55, 1542-1549. DREINHOFFER, J., G6GELEIN, H. & GREGER, R. (1988). Blocking kinetics of Cl- channels in colonic carcinoma cells (HT29) as revealed by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Biochimica et Biophysica Acta 946, 135-142. DUSZYK, M., FRENCH, A. S. & MAN. S. F. (1990). The 20-pS chloride channel of the human airway epithelium. Biophysical Journal 57, 223-230. FRIZZELL, R. A. (1987). Cystic fibrosis: a disease of ion channels? Trends in Neurosciences 10, 190-193. FRIZZELL, R. A., RECHKEMMER, G. & SHOEMAKER. R. L. (1986). Altered regulation of airway epithelial cell chloride channels in cystic fibrosis. Science 233, 558-560. GIEMBYCZ, M. A. & DIAMOND, J. (1990). Evaluation of kemptide, a synthetic serine-containing heptapeptide, as a phosphate acceptor for the estimation of cyclic AMP-dependent protein kinase activity in respiratory tissues. Biochemical Pharmacology 39, 271-283. GREGER, R., GERLACH, L. & KUNZELMANN, K. (1989). Epithelial chloride channels: properties and regulation. In Ion Transport, ed. KEELING. D. & BENHAM, C.. pp. 237-259. Academic Press Ltd, London. GREGER, R., SCHLATTER, E. & G6GELEIN, H. (1987). Chloride channels in the luminal membrane of the rectal gland of the dogfish (Squalus acanthias). Properties of the "larger" conductance channel. Pflugers Archiv 409. 114-121. HWANG, T. C., Lu, L., ZEITLIN, P. L., GRUENERT, D. C., HUGANIR, R. & GUGGINO, W. B. (1989). Cl- channels in CF: lack of activation by protein kinase C and cAMP-dependent protein kinase. Science 244, 1351-1353.

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159

JETTEN, A. M., YANKASKAS, J. R., STUTTS, M. J., WILLUMSEN, N. J. & BOUCHER, R. C. (1989).

Persistence of abnormal chloride conductance regulation in transformed cystic fibrosis epithelia. Science 244, 1472-1475. KUNZELMANN, K., PAVENSTXDT, H. & GREGER, R. (1989a). Characterization of potassium channels in respiratory cells. II. Inhibitors and regulation. Pftugers Archiv 414, 297-303. KUNZELMANN, K., PAVENSTXDT, H. & GREGER, R. (1989b). Properties and regulation of chloride channels in cystic fibrosis and normal airway cells Pfliugers Archiv 415, 172-182. LANGRIDGE-SMITH, J. E., FIELD, M. & DUBINSKY, W. P. (1983). Isolation of transporting plasma membrane vesicles from bovine tracheal epithelium. Biochimica et Biophysica Acta 731, 318-328. LANGRIDGE-SMITH, J. E., RAO, M. C. & FIELD, M. (1984). Chloride and sodium transport across bovine tracheal epithelium: effects of secretagogues and indomethacin. Pftugers Archiv 402, 42-47. LEGRIS, G. J., WILL, P. C. & HOPFER, U. (1982). Inhibition of amiloride-sensitive sodium conductance by indoleamines. Proceedings of the National Academy of Sciences of the USA 79, 2046-2050. Li, M., MCCANN, D., ANDERSON, M. P., CLANCY, J. P., LIEDTKE, C. M., NAIRN, A. C., GREENGARD, P. & WELSH, M. J. (1989). Regulation of chloride channels by protein kinase C in normal and cystic fibrosis airway epithelia. Science 244, 1353-1356. Li, M., MCCANN, J. D., LIEDTKE, C. M., NAIRN, A. C., GREENGARD, P. & WELSH, M. J. (1988). Cyclic AMP-dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium. Nature 331, 358-360. Li, M., MCCANN, J. D. & WELSH, M. J. (1990). Apical membrane C1- channels in airway epithelia: anion selectivity and effect of an inhibitor. American Journal of Physiology 259, C295-301. LOBO, V. M. M. (1989). Handbook of Electrolyte Solutions. Elsevier, Amsterdam. MANNING, S. D. & WILLIAMS, A. J. (1989). Conduction and blocking properties of a predominantly anion selective channel from human platelet surface membrane reconstituted into planar phospholipid bilayers. Journal of Membrane Biology 109, 113-122. MASON, S. J., PARADISO, A. M., BROWN, A., HARDEN, T. K. & BOUCHER, R. C. (1990). Extracellular adenosine triphosphate (ATP) induces calcium release, inositol phosphate (IP) production and chloride secretion by cystic fibrosis and the normal human airway epithelium. Pediatric Pulmonology, suppl. 5, 221. MILLER, C. (1982). Open-state substructure of single chloride channels from Torpedo electroplax. Philosophical Transactions of the Royal Society B 299, 401-411. SCHOUMACHER, R. A., SHOEMAKER, R. L., HALM, D. R., TALLANT, E. A., WALLACE, R. W. & FRIZZELL, R. A. (1987). Phosphorylation fails to activate chloride channels from cystic fibrosis airway cells. Nature 330, 752-754. SMITH, J. J., MCCANN, J. D. & WELSH, M. J. (1990). Bradykinin stimulates airway epithelial Cl secretion via two second messenger pathways. American Journal of Physiology 258, L369-377. VALDIVIA, H. H., DUBINSKY, W. P. & CORONADO, R. (1988). Reconstitution and phosphorylation of chloride channels from airway epithelium membranes. Science 242, 1441-1444. WELSH, M. J. (1986). An apical-membrane chloride channel in human tracheal epithelium. Science 232, 1648-1650. WILLUMSEN, N. J., DAVIS, C. W. & BOUCHER, R. C. (1989). Intracellular Cl- activity and cellular Cl- pathways in cultured human airway epithelium. American Journal of Physiology 256, C1033-1044. YOAKUM, G. H., LECHNER, J. F., GABRIELSON, E. W., KORBA, B. E., MALAN-SHIBLEY, L., WILLEY, J. C., VALERIO, M. G., SHAMSUDDIN, A. M., TRUMP, B. F. & HARRIS, C. C. (1985). Transformation of human bronchial epithelial cells transfected by Harvey ras oncogene. Science 227, 1174-1179.