Proc. Natl. Acad. Sci. USA Vol. 89, pp. 10623-10627, November 1992 Medical Sciences

GTP-binding proteins inhibit cAMP activation of chloride channels in cystic fibrosis airway epithelial cells (cystic fibrosis I

regulator/patch damp)

ERIK M. SCHWIEBERT*, NEIL KIZER*, DIETER C. GRUENERTt, AND BRUCE A. STANTON* *Department of Physiology, Dartmouth Medical School, Hanover, NH 03755-3836; and Cardiovascular Research Institute, and Department of Laboratory Medicine, University of California, San Francisco, CA 94143

Communicated by Gerhard Giebisch, July 15, 1992

ABSTRACT Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of C1- secretion by airway epithelial cells. In CF, cAMP does not activate Cl1 channels in the apical membrane of airway epithelial cells. We report here whole-cell patch-clamp studies demonstrating that pertussis toxin, which uncouples heterotrimeric GTP-binding proteins (G proteins) from their receptors, and guanosine 5'-[L3-thioldiphosphate, which prevents G proteins from interacting with their effectors, increase Cl currents and restore cAMP-activated C1- currents in airway epithelial cells isolated from CF patients. In contrast, the G protein activators guanosine 5'-[y-thioltriphosphate and AIF4 reduce Cl- currents and inhibit cAMP from activating Cl currents in normal airway epithelial cells. In CF cells treated with pertussis toxin or guanosine 5'-{(-thioldiphosphate and in normal cells, cAMP activates a Cl- conductance that has properties simOlar to CF transmembrane-conductance regulator Cl- channel. We conclude that heterotrimeric G proteins inhibit cAMP-activated Cl currents in airway epithelial cells and that modulation of the inhibitory G protein signaling pathway may have the therapeutic potential for improving cAMP-activated Cl- secretion in CF.

Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl- transport in several organs, including lung, pancreas, sweat gland, and intestine (1). In airway epithelial cells isolated from normal patients, cAMP activates Cl- channels in the apical membrane, whereas cAMP fails to activate Cl- channels in airway epithelial cells isolated from patients with CF (2-4). The cellular defects in CF result from mutations in the CF transmembraneconductance regulator (CFTR) gene (5, 6). Although the precise function of the CFTR gene product is controversial, recent evidence suggests that CFTR encodes a cAMPactivated Cl- channel (3-9). The CFTR Cl- channel has a conductance of 8-10 pS, a linear current-voltage (I-V) relation, a permeability sequence of PBr- 2 Pc1- > PI-, and time-independent currents (9-14). Furthermore, the CFTR Cl- channel is inhibited by the Cl- channel blocker diphenylamine-2-carboxylic acid (DPC) and is activated by protein kinase A and protein kinase C (9, 13, 14). CFTR may have other functions, including a role in regulating intracellular vesicle acidification, protein processing, and membrane trafficking (15, 16). GTP-binding proteins (G proteins) regulate a variety of ion channels, including Cl- channels, directly by membranedelimited pathways, and indirectly by cytoplasmic pathways involving second messengers and protein kinases (17-21). Regulation by G proteins of cAMP-activated Cl- channels in airway epithelial cells, however, has not, to our knowledge, been reported. Therefore, the objective of this study was to The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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determine whether G proteins regulate cAMP-activated C1channels in normal and CF airway epithelial cells.

MATERIALS AND METHODS Cell Culture. We studied several cell lines originally isolated from human airway epithelia (Table 1). Normal and CF cells were transformed with simian virus 40 large T antigen and grown in culture, as described (22, 26). Nasal polyp epithelial cells were isolated from CF patients and grown in primary culture, as described (23) with the modification that 5% fetal bovine serum was added to the culture medium for the first 24 hr of culture. Cells were plated on tissue culture flasks or glass coverslips (for patch-clamp studies) that had been coated with an LHC-8 basal medium (Biofluids, Rockville, MD) containing human fibronectin (1 mg/ml; Collaborative Research), Vitrogen 100 (1 ml/100 ml; Collagen Corporation), and bovine serum albumin (100 gg/ml; Biofluids)

(23).

Whole-Cell Patch Clamp. The patch-clamp technique was used to form Gfl seals between patch electrodes and the apical-cell membrane (27). Average seal resistance was 1.2 ± 0.1 GM. The intrinsic capacitance of the electrode and the membrane patch in the cell-attached configuration was electronically nulled by a Warner Instruments (Hamden, CT) patch-clamp amplifier equipped with a 100-Mfl head stage. Subsequent rupture of the membrane patch by suction was recognized as an abrupt drop in resistance and the appearance of a capacitance transient during application of a test voltage step (20 mV). To generate I-V plots the membrane was clamped to a holding voltage of 0 mV and then stepped in 10-mV increments between ± 100 mV for 137.5 ms by using PCLAMP 5.51 software (Axon Instruments, Burlingame, CA). Three I-V plots were averaged every minute during control and experimental periods to yield a mean I-V plot for each minute. In control periods, I-V plots were recorded for a minimum of 5 consecutive min. During experimental maneuvers, I-V plots were recorded consecutively each minute until a steady state was observed (steady state is defined as a minimum of three consecutive periods in which the slope of the I-V plot (i.e., conductance) did not vary by >10%). Whole-cell conductance was calculated from the slope of the I-V plots, which were linear as determined by linear regression. The membrane voltage (Vm) was calculated as Vm = AV X [Rm/(Rm + R.)] (see Eqs. 1 and 2 below for determination of Rm and R.). Unfiltered current records were stored on the hard drive of an Everex AT computer and analyzed by using Abbreviations: CF, cystic fibrosis; CFTR, CF transmembraneconductance regulator; G proteins, GTP-binding proteins; DPC, diphenylamine-2-carboxylic acid; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; CPT-cAMP, 8-(4-chlorophenylthio)adenosine 3',5'-monophosphate; PTX, pertussis toxin; I-V, currentvoltage; GTP[yS], guanosine 5'-[y-thio]triphosphate.

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Proc. Natl. Acad Sci. USA 89 (1992)

Table 1. Characterization of cells Ref. CFTR genotype Cell type Phenotype 22 Wild type 9HTEoNormal 22 Wild type Normal 56FHTE8o23 AF508 homozygous CF Nasal* 24 AF508/unknown mutation CF 2CFSMEo25 AF508 homozygous CF XCFTE-29o*Nasal polyp epithelial cells were isolated from a CF patient homozygous for the AF508 CFTR mutation, as determined by the PCR by Walter Noll, Dartmouth Medical School.

PCLAMP 5.51 routines. Current magnitude was measured during the last 20 ms of each 137.5-ms voltage pulse. As described in detail (28, 29), we measured series resistance (R.), which is largely a function ofthe pipette resistance (including the resistance between the electrode and the cell interior) and cell membrane resistance (Rm) by simultaneous solution of Eqs. 1 and 2 (see below) using the following empirically derived quantities: a, 13, and I(oo), where a is the current calculated as the difference between steady state (-5 ms after a voltage pulse) and at the peak of the capacitive spike, j3 is the rate constant of the exponential current decay after the capacitive spike, I(Xo) is the current calculated as the difference between the current at 0 mV and the steady-state current during a voltage pulse, and AVis the magnitude of the voltage pulse.

I(o) =

AV Rm + Rs

AV Rm a= ~x-. Rm+Rs Rs

[1] [2]

Membrane capacitance (Cm) was calculated from [I(X)) + a]2

Cm

=

ap AV

[3]

To provide the maximum resolution of currents during the capacitive transient, and thereby to assure accurate measures of a, 13, and I(oo), currents were digitized at 20,000 points per s during the first 12.5 ms of each voltage pulse (capacitive transients 10%o during the recording, the experiment was not included in data analysis. At the end of each experiment, the patch electrode was withdrawn from the cell to form the outside-out recording configuration, and electrode-membrane seal resistance was measured and generally found to be >1 GQl. In cases where the seal resistance was PI- (1.1/1.0/0.55; n = 5). The CPT-cAMP-stimulated Cl- conductance was insensitive to the Cl- channel blocker 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid (DIDS) (100 ,uM). In contrast, the Cl- channel blocker DPC (500 ,uM) (11) reduced the C1- conductance by 53% (Fig. 2b; P < 0.05: n = 4). Cl- currents showed no appreciable time-dependence during the voltage pulse, and the I-V plots were linear (Fig. 2). The properties of these cAMP-activated Cl- currents are similar to cAMP-activated Cl- currents seen in cells infected with wild-type CFTR, to currents thought to mediate cAMP-stimulated Cl- secretion in airway epithelial cells in vivo, and to Cl- currents generated by purified CFTR-reconstituted into proteoliposomes (3, 4, 7-9, 13, 34). 100 ,uM CPT- cAMP

30

20-

10

Control 0 0

5

10

15

_

20

Minutes FIG. 1. Time course of CPT-cAMP activation of whole-cell Clconductance (Gca-). At time = 0 the whole-cell patch-clamp configuration was obtained. In one group of cells CPT-cAMP was added to the bath solution at time = 5 min (o; n = 4 cells). Control cells were not exposed to cAMP (o; n = 4 cells). Gcl- was calculated from the slope of the linear I-V plot. This figure illustrates the constancy of the basal Cl- conductance under our experimental conditions. Data are presented as means + SEMs.

Medical Sciences: Schwiebert

et

al.

Table 2. Summary of whole-cell Cl- conductances Chloride conductance, nS n Basal CPT-cAMP P value Normal cells (9HTEo-) 0.001 18 19.0 ± 1.8 40.1 ± 5.2 Control 11 15.6 ± 1.1* 17.2 ± 1.4* 0.025 GTP[yS] 5 12.0 ± 1.0* 11.7 ± 0.7* NS AlFj PTX: acute 11 39.6 4.9* 65.6 ± 6.2* 0.001 PTX: chronic 6 53.5 ± 6.9* 90.6 ± 12.1* 0.01 7 15.5 ± 0.9 22.7 ± 2.4* 0.025 PTX: inactive 6 39.7 ± 2.5* 58.0 ± 6.8* 0.05 GDPLBS] CF cells (2CFSMEo-) 11.9 ± 1.2 NS Control 16 11.6 ± 1.1 5 9.3 ± 1.8 8.0 ± 1.3 NS GTP[yS] PTX: acute 10 18.4 ± 2.2* 31.0 ± 3.8* 0.001 9 27.2 ± 2.8* 57.7 ± 3.3* 0.001 PTX: chronic NS 10.2 ± 1.1 PTX: inactive 4 9.8 ± 0.9 5 47.9 ± 11.0* 52.9 ± 9.6* NS GDP[LS] CF cells (XCFTE-29o-) 9 13.6 ± 1.2 12.0 ± 1.0 NS Control 2 7.3 ± 3.0 7.7 ± 3.2 GTP[yS] PTX: acute 6 32.3 ± 2.7* 41.7 ± 3.0* 0.05 4 24.6 ± 3.4* 30.9 ± 2.7* 0.02 GDP[PS] Nasal polyp cells 5 10.7 ± 0.5 10.3 ± 1.0 NS Control 5 9.3 ± 0.4 NS 10.4 ± 0.9 GTP[yS] NS 5 21.3 ± 4.9* 27.9 ± 5.9* PTX: chronic P values indicate level of significance comparing basal means with CP1-cAMP (100 AM) means by paired t test. Asterisks indicate values significantly different from control (P < 0.05) in same column by analysis of variance and Neuman-Keuls test. Cells were exposed to pertussis toxin (PTX) for 24 hr [PTX: chronic; PTX holotoxin at 1 jig/ml (List Biological Laboratories, Campbell, CA) in culture medium] or for -5 min (acute PTX: preactivated A protomer at 100 ng/ml in pipette) before Cl- conductance was measured. AlFZ was formulated by diluting a stock solution containing 100 mM NaF and 0.3 mM AIC13 by 1000-fold into the pipette solution (33). In control, whole-cell conductances in normal and CF cells were referable primarily to Cl- because Cl- is the only permeable ion in the pipette and bath solutions. With a seal resistance of -1 GfQ, -1 nS of whole-cell current can be attributed to a nonspecific leak. This value was not subtracted from the data reported here.

To determine whether G proteins regulate C1- currents in normal airway epithelial cells we examined the effects of guanosine 5'-[y-thio]triphosphate (GTP[yS]), a compound that activates monomeric and heterotrimeric G proteins. GTP[yS] (100 ,uM) decreased the C1- conductance and prevented CPT-cAMP activation of the C1- conductance (Table 2). To determine whether GTP[yS] inhibited C1- channels by activating monomeric or heterotrimeric G proteins, we used A1FZ, a compound that specifically activates heterotrimeric G proteins (33). A1F- (100 jM) also decreased the Clconductance and prevented CPT-cAMP activation of the C1conductance (Table 2). In contrast, PTX (100 ng/ml), a compound that specifically uncouples heterotrimeric G proteins of the Gt, Go, and G, subclasses from receptors (19) and thereby prevents G protein activation of effectors, increased the Cl- conductance (Table 2). Furthermore, CPT-cAMP reversibly increased the Cl- conductance in PTX-treated cells (Table 2). Heat-inactivated PTX had no effect on the C1conductance and did not block cAMP activation of C1channels (Table 2). Finally, guanosine 5'-[f3-thioldiphosphate (GDP[,S]) (100 ,uM), which prevents G proteins from activating their effector, also increased Cl- conductance, and CPT-cAMP reversibly increased Cl- conductance in the presence of GDP[1S] (Table 2). Similar results were observed with PTX, GDP[/1S], and GTP[yS] on both normal cell lines studied (i.e., 9HTEo- and 56FHTE8o-). These results indicate that heterotrimeric G

Proc. Natl. Acad. Sci. USA 89 (1992)

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Control

A

100p 1000 pA 10 Ms

cAMP

1000 pA 10 Ms

cAMP

F

+

__

1000

DPC

___

-.

__________

pA

10 Ms

(nA) 4 T

B

3

-

cAMP

2 1

-80

/:

+

Control 40

-1

-2

cAMP + DPC

80 V (mV)

+

-3 + -4

1

FIG. 2. Representative whole-cell patch-clamp experiments characterizing CPT-cAMP-activated Cl- currents in normal human airway epithelial cells (9HTEo-). (a) Current records illustrating effects of CPIT-cAMP and DPC on the Cl- currents. CPT-cAMP (100 AiM; Boehringer Mannheim) in the bath solution increased Clcurrents. Diphenylamine-2-carboxylic acid (DPC; 500 AiM; Fluka) in the bath inhibited CPT-cAMP-activated Cl- currents. Currents were filtered at 1 kHz. At this frequency, both capacitive spikes are attenuated. (b) I-V relations of experiments depicted in a.

proteins inhibit cAMP-activated Cl- channels in airway epithelial cells and that PTX and GDP[fS], by preventing G proteins from interacting with their effector, prevent this inhibition. Because human airway epithelial cells do not express detectable levels of mRNA for Gt, Go, or Gajil but express mRNA for Gi-2 and Gasi3 (B.A.S., E.M.S., D.C.G.,

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and Louis Ercolani, unpublished data), we conclude that

Gaji2 or Gaji3 inhibit cAMP-activated Cl- channels in human

airway epithelial cells. Heterotrimeric G proteins also inhibit calcium, sodium, and potassium channels in other cell types (21, 35-37). The Cl- currents activated by PTX, GDP[I3S], and CPTcAMP had a linear I-V relation and were not time-dependent. In addition, the currents were not inhibited by DIDS; however, DPC reduced the currents by 64% (n = 3; P < 0.05). Furthermore, the halide permeability of PTX- and GDP[3S]treated cells was PBr- 2 Pci- > PI- (1.1/1.0/0.6; n = 4). The characteristics of the PTX- and GDP[,fS]-stimulated Clcurrents are similar to the Cl- currents attributed to CFTR (3, 4, 7, 8, 13). We next examined the role of G proteins in regulating C1channels in two CF cell lines and CF nasal epithelial cells in primary culture (Table 2). PTX dramatically increased the Cl- conductance of CF cells. Moreover, CPT-cAMP increased the Cl- conductance in CF cells treated with PTX but not in untreated CF cells. GDP[/3S] also increased the Clconductance and made the Cl- conductance in both CF cell lines sensitive to CPT-cAMP-activation. In contrast, GTP[yS] had no effect on the Cl- conductance. The Clcurrents activated by PTX and GDP[,BS] in CF cells had a linear I-V relation and were not time-dependent during the voltage pulse. In addition, the currents were not inhibited by DIDS (n = 4); however, DPC reduced the currents by 60%o (n = 6; P < 0.01). The reversal potential of the I-V plots was near 0 (-0.5 ± 0.2 mV), in agreement with the value predicted by the Nernst equation for a Cl--selective conductance. Reduction of the bath Cl concentration from 140 to 14 mM shifted the reversal potential by 49 ± 5 mV, in close agreement with the value predicted by the Nernst equation for Cl (n = 4). Furthermore, the halide permeability of PTXand GDP[/3S]-treated cells was PBr- 2 Pci- > PI- (1.1/1.0/ 0.6, n = 4). The PTX-, GDP[IJS]-, and cAMP-activated C1l currents in both CF cell lines and in the CF nasal epithelial cells were similar to Cl- currents activated by these agents in normal airway epithelial cells and to CFTR Cl- channels.

DISCUSSION We report that PTX-sensitive heterotrimeric G proteins, most likely Gi2 or Gai3, regulate cAMP-activated Clchannels in several normal and CF airway epithelial cell lines, as well as CF nasal epithelial cells in primary culture. GTP['yS] and A1F4, activators of G proteins, blocked cAMP stimulation of Cl- channels in normal airway epithelial cells. In contrast, PTX and GDP[IIS], which uncouple G proteins from their effectors, made Cl- channels in CF airway epithelia sensitive to cAMP activation. In both CF cell lines, including the XCFTE-29o- cells, which are homozygous for the AF508 CFTR mutation, the combination of PTX and CPT-cAMP increased Cl- conductance to levels observed in cells expressing wild-type CFTR. These observations suggest that heterotrimeric G proteins inhibit cAMP-activated Cl- channels in airway epithelial cells. In our experiments, the cAMP-activated whole-cell C1conductance in normal cells and in CF cells treated with PTX or GDP1L3S] had properties identical to CFTR Cl- channels (9-14, 34). The Cl- currents had a linear I-V relation, were time- and voltage-independent, were inhibited by DPC, but were not inhibited by DIDS, and had a halide permeability sequence of PBr- 2 PcI- > PI-. Thus, it is reasonable to conclude that heterotrimeric G proteins can inhibit CFTR Clchannels. Single-channel patch-clamp studies are required to provide direct support for this conclusion. At least four other types of Cl- channels, in addition to CFTR Cl- channels, have been identified in the apical membrane of airway

epithelial cells, including 30/60-pS outwardly rectifying

Proc. Natl. Acad. Sci. USA 89 (1992)

channels (38-42), 10-pS channels (34, 43, 44), 20-pS channels (34, 42, 44), and 200- to 400-pS channels (39, 41, 42, 45, 46). Because the characteristics of these channels, including anion selectivity and sensitivity to pharmacological agents, differ from CFTR Cl- channels, we conclude that they do not underlie the major portion of the whole-cell Cl- currents measured in the present study. Nevertheless, we cannot exclude the possibility that the 30/60-pS, 10-pS, 20-pS, or 300- to 400-pS Cl- channels are active and contribute to a small fraction of the whole-cell Cl- currents seen in the present study, nor can we exclude the possibility that cAMP and G proteins regulate these channels. Indeed, in a preliminary report we show that G proteins located in the apical membrane of airway epithelial cells regulate the 30/60-pS outwardly rectifying Cl- channel and a 380-pS Cl- channel

(39, 41).

We have not yet examined whether heterotrimeric G

proteins regulate cAMP-activated CFTR Cl- channels directly, by membrane-delimited pathways, or indirectly, by cytoplasmic pathways involving second messengers and protein kinases (19-21). It is unlikely that PTX or GDP[,3S] increased the Cl- conductance in CF cells by elevating cAMP levels because 100 1LM CPT-cAMP had no effect on conductance and because GDP[,8S] inhibits both inhibitory (Go) and stimulatory (GJ) G proteins coupled to adenylyl cyclase. We speculate that mutations make CFTR function more susceptible to the inhibitory action of G proteins or their second messengers and thereby block activation of CFTR Cl- channels by cAMP, protein kinase A, and ATP. Inactivation of G proteins by PTX or GDP[,3S] in CF cells may relieve G protein inhibition, allowing cAMP, protein kinase A, and ATP to activate mutant forms of CFTR Cl- channels (47, 48). Thus, we propose that phosphorylation of CFTR in normal

cells is sufficient to overcome C1- channel inhibition by G proteins, whereas, in CF cells, phosphorylation of mutant forms of C R cannot overcome G protein inhibition. Recent observations demonstrating that CFTR Cl- channels with the AF508 mutation can be activated by cAMP are consistent with our proposal (47, 48). The present study suggests that inhibition of G proteins may be useful in circumventing the cellular defect in Clsecretion in CF airway epithelia. The observation that PITX vaccine improved pulmonary function in a patient with CF supports such a speculation (49). A successful strategy to block the inhibitory action of G proteins in CF airway epithelia could be directed at interfering with any step in the receptor-G protein-effector pathway. Because inhibition of G protein activity is required to restore cAMP activation of Cl- channels in CF cells, it is likely that G proteins are tonically active in CF airway epithelial cells. Accordingly, identification of the apical membrane receptor coupled to the inhibitory G protein, the agonist tonically activating the receptor and, ultimately, a receptor antagonist would be useful in inactivating the G protein. Because the receptor-G protein complex is located in the apical membrane, the antagonist could be delivered by aerosol therapy and used to prevent the endogenous autocoid from activating the receptor. One possible autocoid is adenosine, which has been shown to act as a feedback inhibitor of cAMP-activated Clsecretion in shark rectal gland (50). Other potential strategies to block G protein inactivation of CFTR Cl- channels include desensitization of the receptor coupled to the G protein, inhibition of autocoid release, inhibition of a putative second messenger activated by the inhibitory G protein, and antisense oligodeoxynucleotide therapy to inhibit Gai gene expression. It is unlikely that PTX treatment would be a useful approach. Clearly, further studies are necessary to elucidate the mechanisms of G protein regulation of Clchannels in normal and CF airway epithelial cells and to determine whether modulation of the inhibitory G protein

Medical Sciences: Schwiebert et al. signaling pathway is a viable approach to correct defective regulation of C1- secretion by cAMP. In conclusion, the present study demonstrates that heterotrimeric G proteins inhibit CFTR Cl- channels in normal and CF airway epithelial cells and suggests that modulation of the inhibitory G protein signaling pathway may be a useful strategy to correct defective regulation of Cl- secretion by cAMP. We thank Drs. Louis Ercolani, Henry Bourne, and William Boyle for advice and stimulating discussions; Drs. William Boyle, Worth Parker, and Walter Noll for providing cells from CF patients, and Ms. Katherine Karlson and Michele LaBotz for their expert assistance. This research was supported by the National Institutes of Health (DK-34533 to B.A.S. and DK-39619 to D.C.G.), Basic Research Support Grant at Dartmouth Medical School, and the Cystic Fibrosis Foundation (to B.A.S.). 1. Widdicombe, J. H. & Wine, J. J. (1991) Trends Biochem. Sci. 16, 474-477. 2. Rich, D. P., Anderson, M. P., Gregory, R. J., Cheng, S. H., Paul, S., Jefferson, D. M., McCann, J. D., Klinger, K. W., Smith, A. E. & Welsh, M. J. (1990) Nature (London) 347, 358-363. 3. Drumm, M. L., Pope, H. A., Cliff, W. H., Rommens, J. M., Marvin, S. A., Tsui, L.-C., Collins, F. S., Frizzell, R. A. & Wilson, J. M. (1990) Cell 62, 1227-1233. 4. Kartner, N., Hanrahan, J. W., Jensen, T. J., Naismith, A. L., Sun, S., Ackerley, C. A., Reyes, E. F., Tsui, L.-C., Rommens, J. M., Bear, C. E. & Riordan, J. R. (1991) Cell 64, 681-691. 5. Lemna, W. K., Feldman, G. L., Kerem, B.-S., Fernbach, S. D., Zevkovich, E. P., O'Brien, W. E., Riordan, J. R., Collins, F. S., Tsui, L.-C. & Beaudet, A. L. (1990) Proc. Natl. Acad. Sci. USA 322, 291-2%. 6. Kerem, B.-S., Rommens, J. M., Buchanan, J. A., Markiewicz, D., Cox, T. K., Chakravarti, A., Buchwald, M. & Tsui, L.-C. (1989) Science 245, 1073-1080. 7. Rich, D. P., Anderson, M. P., Gregory, R. J., Cheng, S. H., Paul, S., Jefferson, D. M., McCann, J. D., Klinger, K. W., Smith, A. E. & Welsh, M. J. (1990) Nature (London) 347, 358-363. 8. Gregory, R. J., Cheng, S. H., Rich, D. P., Marshall, J., Paul, S., Hehir, K., Ostedgaard, L., Klinger, K. W., Welsh, M. J. & Smith, A. E. (1990) Nature (London) 347, 382-386. 9. Bear, C. E., Li, C., Kartner, N., Bridges, R. J., Jensen, T. J., Ramjeesingh, M. & Riordan, J. R. (1992) Cell 68, 809-818. 10. Cliff, W. H. & Frizzell, R. A. (1990) Proc. Natl. Acad. Sci. USA 87, 4956-4960. 11. Anderson, M. P., Rich, D. P., Gregory, R. J., Smith, A. E. & Welsh, M. J. (1991) Science 251, 679-682. 12. Anderson, M. P. & Welsh, M. J. (1991) Proc. Natl. Acad. Sci. USA 88, 6003-6007. 13. Tabcharani, J. A., Chang, X.-B., Riordan, J. R. & Hanrahan, J. W. (1991) Nature (London) 352, 628-631. 14. Berger, H. A., Anderson, M. P., Gregory, R. J., Thompson, S., Howard, P. W., Maurer, R. A., Mulligan, R., Smith, A. E. & Welsh, M. J. (1991) J. Clin. Invest. 88, 1422-1431. 15. Cheng, S. H., Gregory, R. J., Marshall, J., Paul, S., Souza, D. W., White, G. A., O'Riordan, C. R. & Smith, A. E. (1990) Cell 63, 827-834. 16. Barasch, J., Kiss, B., Prince, A., Saiman, L., Gruenert, D. & Al-Awqati, Q. (1991) Nature (London) 352, 70-73. 17. Schwiebert, E. M., Light, D. B., Fejes-Toth, G., Naray-FejesToth, A. & Stanton, B. A. (1990) J. Biol. Chem. 265, 77257728. 18. Tilly, B. C., Kansen, M., Van Gageldonk, P. G. M., Van den

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GTP-binding proteins inhibit cAMP activation of chloride channels in cystic fibrosis airway epithelial cells.

Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl- secretion by airway epithelial cells. In CF, cAMP doe...
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