Dibasic protein kinase A sites regulate bursting rate and nucleotide sensitivity of the cystic fibrosis transmembrane conductance regulator chloride channel.

1. The relationship between phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and its gating by nucleotides was examined using the patch clamp technique by comparing strongly phosphorylated wild-type (WT) channels with weakly phosphorylated mutant channels lacking four (4SA) or all ten (10SA) dibasic consensus sequences for phosphorylation by protein kinase A (PKA). 2. The open probability (Po) of strongly phosphorylated WT channels in excised patches was about twice that of 4SA and 10SA channels, after correcting for the number of functional channels per patch by addition of adenylylimidodiphosphate (AMP-PNP). The mean burst durations of WT and mutant channels were similar, and therefore the elevated Po of WT was due to its higher bursting rate. 3. The ATP dependence of the 10SA mutant was shifted to higher nucleotide concentrations compared with WT channels. The relationship between Po and [ATP] was noticeably sigmoid for 10SA channels (Hill coefficient, 1.8), consistent with positive co-operativity between two sites. Increasing ATP concentration to 10 mM caused the Po of both WT and 10SA channels to decline. 4. Wild-type and mutant CFTR channels became locked in open bursts when exposed to mixtures of ATP and the non-hydrolysable analogue AMP-PNP. The rate at which the low phosphorylation mutants became locked open was about half that of WT channels, consistent with Po being the principal determinant of locking rate in WT and mutant channels. 5. We conclude that phosphorylation at 'weak' PKA sites is sufficient to sustain the interactions between the ATP binding domains that mediate locking by AMP-PNP. Phosphorylation of the strong dibasic PKA sites controls the bursting rate and Po of WT channels by increasing the apparent affinity of CFTR for ATP.

The CFTR chloride channel: nucleotide interactions and temperature-dependent gating.

The gating cycle of CFTR (Cystic Fibrosis Transmembrane conductance Regulator) chloride channels requires ATP hydrolysis and can be interrupted by exposure to the nonhydrolyzable nucleotide AMP-PNP. To further characterize nucleotide interactions and channel gating, we have studied the effects of AMP-PNP, protein kinase C (PKC) phosphorylation, and temperature on gating kinetics. The rate of channel locking increased from 1.05 x 10(-3) sec-1 to 58.7 x 10(-3) sec-1 when AMP-PNP concentration was raised from 0.5 to 5 mM in the presence of 1 mM MgATP and 180 nM protein kinase A catalytic subunit (PKA). Although rapid locking precluded estimation of Po or opening rate immediately after the addition of AMP-PNP to wild-type channels, analysis of locking rates in the presence of high AMP-PNP concentrations revealed two components. The appearance of a distinct, slow component at high [AMP-PNP] is evidence for AMP-PNP interactions at a second site, where competition with ATP would reduce Po and thereby delay locking. All channels exhibited locking when they were strongly phosphorylated by PKA, but not when exposed to PKC alone. AMP-PNP increased Po at temperatures above 30 degrees C but did not cause locking, evidence that the stabilizing interactions between domains, which have been proposed to maintain CFTR in the open burst state, are relatively weak. The temperature dependence of normal CFTR gating by ATP was strongly asymmetric, with the opening rate being much more temperature sensitive (Q10 = 9.6) than the closing rate (Q10 = 3.6). These results are consistent with a cyclic model for gating of phosphorylated CFTR.

Halide permeation in wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels.

Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22 degrees C or 8.7 pS at 37 degrees C. The current-voltage relationship became linear when patches were excised into symmetrical, -tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22 degrees C to 10.9 pS at 37 degrees C. The conductance at 22 degrees C was approximately 1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl activity was hyperbolic and well fitted by a Michaelis-Menten-type function having a of approximately 38 mM and maximum conductance of 10 pS at 22 degrees C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P/P = 0.003-0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P(1.8) > P(1. 3) > P(1.0) > P(0.17), consistent with a "weak field strength" selectivity site. The same sequence was obtained for external halides, although inward F flow was not observed. Iodide currents were protocol dependent and became blocked after 1-2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I/Cl permeability ratio (P/P< 0.4). The switch to low I permeability was enhanced at potentials that favored Cl entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl and I ions may influence I permeation and be responsible for the wide range of P/P ratios that have been reported for the CFTR channel. The low P/P ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a "weak field strength" anion selectivity sequence.

Permeability of wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels to polyatomic anions.

Permeability of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to polyatomic anions of known dimensions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. Biionic reversal potentials measured with external polyatomic anions gave the permeability ratio (P/P) sequence NO > Cl > HCO > formate > acetate. The same selectivity sequence but somewhat higher permeability ratios were obtained when anions were tested from the cytoplasmic side. Pyruvate, propanoate, methane sulfonate, ethane sulfonate, and gluconate were not measurably permeant (P/P < 0.06) from either side of the membrane. The relationship between permeability ratios from the outside and ionic diameters suggests a minimum functional pore diameter of approximately 5.3 A. Permeability ratios also followed a lyotropic sequence, suggesting that permeability is dependent on ionic hydration energies. Site-directed mutagenesis of two adjacent threonines in TM6 to smaller, less polar alanines led to a significant (24%) increase in single channel conductance and elevated permeability to several large anions, suggesting that these residues do not strongly bind permeating anions, but may contribute to the narrowest part of the pore.

Multi-Ion mechanism for ion permeation and block in the cystic fibrosis transmembrane conductance regulator chloride channel.

The mechanism of Cl ion permeation through single cystic fibrosis transmembrane conductance regulator (CFTR) channels was studied using the channel-blocking ion gluconate. High concentrations of intracellular gluconate ions cause a rapid, voltage-dependent block of CFTR Cl channels by binding to a site approximately 40% of the way through the transmembrane electric field. The affinity of gluconate block was influenced by both intracellular and extracellular Cl concentration. Increasing extracellular Cl concentration reduced intracellular gluconate affinity, suggesting that a repulsive interaction occurs between Cl and gluconate ions within the channel pore, an effect that would require the pore to be capable of holding more than one ion simultaneously. This effect of extracellular Cl is not shared by extracellular gluconate ions, suggesting that gluconate is unable to enter the pore from the outside. Increasing the intracellular Cl concentration also reduced the affinity of intracellular gluconate block, consistent with competition between intracellular Cl and gluconate ions for a common binding site in the pore. Based on this evidence that CFTR is a multi-ion pore, we have analyzed Cl permeation and gluconate block using discrete-state models with multiple occupancy. Both two- and three-site models were able to reproduce all of the experimental data with similar accuracy, including the dependence of blocker affinity on external Cl (but not gluconate) ions and the dependence of channel conductance on Cl concentration. The three-site model was also able to predict block by internal and external thiocyanate (SCN) ions and anomalous mole fraction behavior seen in Cl/SCN mixtures.

Regulation of the CFTR chloride channel from humans and sharks.

The cystic fibrosis transmembrane conductance regulator (CFTR) in an ATP-dependent channel which mediates cAMP-stimulated chloride secretion by epithelia, particularly those of the pancreas, airways, and intestine. CFTR homologues have been found in all higher vertebrates examined to date and also in some lower vertebrates, although only the human, shark, and Xenopus genes have been heterologously expressed and shown to generate protein kinase A-activated Cl channels. Once phosphorylated, CFTR channels require hydrolyzable nucleotides to be active, but they can be locked in an open burst state when exposed to mixtures of ATP and its hydrolysis-resistant analogue AMP-PNP. This locking requires low-level phosphorylation at unidentified sites that are not among the ten "strong" (dibasic) PKA consensus sequences on CFTR. Mutagenesis of the dibasic PKA sites, which reduces in vitro phosphorylation by > 98%, reduces open probability (Po) by about 50% whilst having no effect on burst duration. Thus, incremental phosphorylation of these sites under normal conditions does not increase Po by slowing down ATP hydrolysis and stabilizing the open burst state, although locking does strictly require low-level phosphorylation at one or more cryptic sites. In addition to serving as a Cl channel, there is compelling evidence that CFTR inhibits the amiloride-sensitive, epithelial sodium channel (ENaC). The mechanism of coupling is not known but most likely involves physical interactions between the channels, perhaps mediated by an intermediate protein that impinges on other transport proteins. CFTR does not function as a conductive channel for ATP; however, extracellular ATP does regulate epithelial channels through activation of P2U purinergic receptors and, after being hydrolyzed extracellularly, through activation of adenosine receptors which elevate cAMP.

CFTR channels expressed in CHO cells do not have detectable ATP conductance.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated, ATP-dependent chloride channel which may have additional functions. Recent reports that CFTR mediates substantial electrodiffusion of ATP from epithelial cells have led to the proposal that CFTR regulates other ion channels through an autocrine mechanism involving ATP. The aim of this study was to determine the ATP conductance of wild-type CFTR channels stably expressed in Chinese hamster ovary cells using patch clamp techniques. In the cell-attached configuration with 100 mM Mg middle dot ATP or Tris middle dot ATP solution in the pipette and 140 mM NaCl in the bath, exposing cells to forskolin caused the activation of a low-conductance channel having kinetics resembling those of CFTR. Single channel currents were negative at the resting membrane potential (Vm), consistent with net diffusion of Cl from the cell into the pipette. The transitions decreased in amplitude, but did not reverse direction, as Vm was clamped at increasingly positive potentials to enhance the driving force for inward ATP flow (>+80 mV). In excised patches, single channel currents did not reverse under essentially biionic conditions (Clin/ATPout or ATPin/Clout), although PKA-activated currents were clearly visible in the same patches at voltages where they would be carried by chloride ions. Moreover, with NaCl solution in the bath and a mixture of ATP and Cl in the pipette, the single channel I/V curve reversed at the predicted equilibrium potential for chloride. CFTR channel currents disappeared when patches were exposed to symmetrical ATP solutions and were restored by reexposure to Cl solution. Finally, in the whole-cell configuration with NaCl in the bath and 100 mM MgATP or TrisATP in the pipette, cAMP-stimulated cells had time-independent, outwardly rectifying currents consistent with CFTR selectivity for external Cl over internal ATP. Whole-cell currents reversed near Vm = -55 mV under these conditions, however the whole cell resistance measured at -100 mV was comparable to that of the gigaohm seal between the plasma membrane and glass pipette (7 Gomega). We conclude that CFTR does not mediate detectable electrodiffusion of ATP.

Failure of the cystic fibrosis transmembrane conductance regulator to conduct ATP.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel regulated by protein kinase A and adenosine triphosphate (ATP). Loss of CFTR-mediated chloride ion conductance from the apical plasma membrane of epithelial cells is a primary physiological lesion in cystic fibrosis. CFTR has also been suggested to function an an ATP channel, although the size of the ATP anion is much larger than the estimated size of the CFTR pore. ATP was not conducted through CFTR in intact organs, polarized human lung cell lines, stably transfected mammalian cell lines, or planar lipid bilayers reconstituted with CFTR protein. These findings suggest that ATP permeation through the CFTR is unlikely to contribute to the normal function of CFTR or to the pathogenesis of cystic fibrosis.

cAMP-dependent protein kinase-mediated phosphorylation of cystic fibrosis transmembrane conductance regulator residue Ser-753 and its role in channel activation.

Hormonal regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is largely mediated via cAMP-dependent protein kinase (PKA). CFTR contains 10 dibasic consensus sites for potential PKA phosphorylation ((R/K) (R/K)X(S*/T*)). Previous studies (Chang, X.-B., Tabcharani, J. A., Hou, Y.-X., Jensen, T. J., Kartner, N., Alon, N., Hanrahan, J. W., and Riordan, J.R (1993) J. Biol. Chem. 268, 11304-11311) showed that approximately 25% of the CFTR wild-type response to PKA activation remained upon inhibition of most detectable phosphorylation by in vitro mutagenesis of all 10 dibasic consensus sites (10SA CFTR). To identify potential additional sites responsible for the residual activity, large amounts of this mutant CFTR were phosphorylated with PKA using high specific activity [gamma-32P]ATP. Cyanogen bromide cleavage indicated that a large portion of the observed PKA phosphorylation occurred within a 5.8-kDa fragment of the R domain between residues 722-773. Removal of serines at potential PKA sites in this fragment showed that Ser-753 accounted for all of the gamma-32P labeling of the 5.8-kDa peptide. Replacement of Ser-753 with alanine reduced the level of residual CFTR activity by a further 40%, indicating that phosphorylation at this previously unidentified site contributes to the activation of 10SA CFTR.

Basolateral K channel activated by carbachol in the epithelial cell line T84.

Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (Isc), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T84 cells. Carbachol had little effect on Isc when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent Isc response to calcium ionophores. Carbachol (100 microM) transiently elevated intracellular free calcium ([Ca2+]i) by approximately 3-fold in confluent cells cultured on glass coverslips with a time course resembling the Isc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (-54 mV) with pipette solution containing 150 mM KCl (37 degrees C). This rectification persisted when patches were bathed in symmetrical 150 mM KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na >> Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mM barium. It is referred to as the KBIC channel based on its most distinctive properties (Ba-insensitive, inwardly rectifying, Ca-activated). Like single KBIC channels, the carbachol-stimulated Isc was relatively insensitive to several blockers on the basolateral side and was unaffected by barium. These comparisons between the properties of the macroscopic current and single channels suggest that the KBIC channel mediates basolateral membrane K conductance in T84 cell monolayers during stimulation by cholinergic secretagogues.

Regulation of an inwardly rectifying K channel in the T84 epithelial cell line by calcium, nucleotides and kinases.

Agonists that elevate calcium in T84 cells stimulate chloride secretion by activating KBIC, an inwardly rectifying K channel in the basolateral membrane. We have studied the regulation of this channel by calcium, nucleotides and phosphorylation using patch clamp and short-circuit current (ISC) techniques. Open probability (Po) was independent of voltage but declined spontaneously with time after excision. Rundown was slower if patches were excised into a bath solution containing ATP (10 microM-5 mM), ATP (0.1 mM)+protein kinase A (PKA; 180 nM), or isobutylmethylxanthine (IBMX; 1 mM). Analysis of event durations suggested that the channel has at least two open and two closed states, and that rundown under control conditions is mainly due to prolongation of the long closed time. Channel activity was restimulated after rundown by exposure to ATP, the poorly hydrolyzable ATP analogue AMP-PNP, or ADP. Activity was further enhanced when PKA was added in the presence of MgATP, but only if free calcium concentration was elevated (400 nM). Nucleotide stimulation and inward rectification were both observed in nominally Mg-free solutions. cAMP modulation of basolateral potassium conductance in situ was confirmed by measuring currents generated by a transepithelial K gradient after permeabilization of the apical membrane using alpha-toxin. Finally, protein kinase C (PKC) inhibited single KBIC channels when it was added directly to excised patches. These results suggest that nonhydrolytic binding of nucleotides and phosphorylation by PKA and PKC modulate the responsiveness of the inwardly rectifying K channel to Ca-mediated secretagogues.

Multi-ion pore behaviour in the CFTR chloride channel.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a non-rectifying, low-conductance channel regulated by ATP and phosphorylation, which mediates apical chloride conductance in secretory epithelia and malfunctions in cystic fibrosis (CF). Mutations at Lys 335 and Arg 347 in the sixth predicted transmembrane helix of CFTR alter its halide selectivity in whole-cell studies and its single channel conductance, but the physical basis of these alterations is unknown and permeation in CFTR is poorly understood. Here we present evidence that wild-type CFTR can contain more than one anion simultaneously. The conductance of CFTR passes through a minimum when channels are bathed in mixtures of two permeant anions. This anomalous mole fraction effect can be abolished by replacing Arg 347 with an aspartate and can be toggled on or off by varying the pH after the same residue is replaced with a histidine. Thus the CFTR channel should provide a convenient model in which to study multi-ion pore behaviour and conduction. The loss of multiple occupancy may explain how naturally occurring CF mutations at this site cause disease.

Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites.

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in transepithelial ion transport by acting as a tightly regulated apical chloride channel. Regulation is achieved by the concerted action of ATP at conserved nucleotide binding folds and serine phosphorylation at multiple sites by protein kinases A (PKA) and C (PKC). A previous investigation concluded that activation by PKA is critically dependent on phosphorylation at four of the nine predicted PKA sites in the R domain (S660A, S737A, S795A, S813A), because a "Quad" mutant lacking these sites could not be activated. We show in the present work that not only can this mutant be phosphorylated and activated, but a mutant in which all 10 predicted PKA sites have been altered still retains significant PKA-activated function. Potentiation of the PKA response by PKC is also preserved in this mutant. Thus CFTR may be regulated by cryptic PKA sites which also mediate interactions between different kinases. Such hierarchical phosphorylation of CFTR by obvious and cryptic PKA sites could provide a metered response to secretagogues.

On the activation of outwardly rectifying anion channels in excised patches.

Previous studies have shown that outwardly rectifying anion channels can be activated in excised patches by exposure to protein kinases or large depolarizing voltage pulses and by raising the bath temperature to 37 degrees C. These maneuvers presumably induce some conformational change in the channel or a regulatory molecule. However, the mechanisms underlying stimulation have not been defined. We have tested several procedures known to influence the structure and solubility of proteins for their effects on activation of anion channels in excised patches. Spontaneous and voltage-induced activation of outward rectifiers was enhanced by increasing the ionic strength or the pH of the bath solution. These maneuvers had no effect on the activity of calcium-activated cation nonselective channels. Activation of outward rectifiers depended on the anion used and followed the (inverse) Hofmeister series consistent with a salting-in process. Divalent cations also enhanced activation with relative potencies Ba greater than Ca greater than Mg but at a much lower concentration (4 meq/l). Exposing patches from T84 cells to purified catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent kinase had no effect on the outward rectifier but did activate the low-conductance Cl channel. The results indicate that the outward rectifier is labile and raise the possibility that nonspecific physical mechanisms may contribute to its activation in excised patches by kinases and other stimuli.

Phosphorylation-regulated Cl- channel in CHO cells stably expressing the cystic fibrosis gene.

A cyclic AMP-stimulated chloride conductance appears when the cystic fibrosis gene is expressed in non-epithelial cells by infection with recombinant viruses. Cyclic AMP-stimulated conductance in this system is mediated by the same ohmic, low-conductance Cl- channel as in human secretory epithelia, but control of this channel by phosphorylation has not been directly demonstrated. Here we report the appearance of the low-conductance Cl- channel in Chinese hamster ovary cells after stable transfection with the cystic fibrosis gene. The channel is regulated on-cell by membrane-permeant analogues of cAMP and off-cell by protein kinases A and C and by alkaline phosphatase. These results are further evidence that the cystic fibrosis transmembrane regulator is a Cl- channel which can be activated by specific phosphorylation events and inactivated by dephosphorylation; they reveal an unsuspected synergism between converging kinase regulatory pathways.

Low-conductance chloride channel activated by cAMP in the epithelial cell line T84.

We have studied the modulation and pharmacological properties of two anion channels in T84 cells by recording single channel and transepithelial currents. One channel had an outwardly rectifying current-voltage I/V curve, was rarely active in cell-attached patches, and was unaffected by cAMP. The other channel had lower conductance (8.7 pS at 37 degrees C) and a more ohmic I/V relationship. Exposure to cAMP increased the probability of observing low-conductance channel activity in cell-attached patches greater than 6-fold. Extracellular DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) or [IAA-94 (an indanyloxyacetic acid) inhibited the outward rectifier but did not affect the low-conductance channel or cAMP-stimulated transepithelial current. These results suggest the low-conductance Cl channel may contribute to apical membrane conductance during cAMP-stimulated secretion.

Inhibition of an outwardly rectifying anion channel by HEPES and related buffers.

The effect of pH buffers and related compounds on the conductance of an outwardly rectifying anion channel has been studied using the patch-clamp technique. Single-channel current-voltage relationships were determined in solutions buffered by trace amounts of bicarbonate and in solutions containing N-substituted taurines (HEPES, MES, BES, TES) and glycines (glycylglycine, bicine and tricine), Tris and bis-Tris at millimolar concentrations. HEPES (pK alpha = 7.55) reduced the conductance of the channel when present on either side of the membrane. Significant inhibition was observed with 0.6 mM HEPES on the cytoplasmic side (HEPESi) and this effect increased with [HEPESi] so that conductance at the reversal potential was diminished approximately 25% with 10 mM HEPEsi and approximately 70% at very high [HEPESi]. HEPESi block was relieved by applying positive voltage but positive currents were not consistent with a Woodhull-type blocking scheme in that calculated dissociation constants and electrical distances depended on HEPES concentration. Results obtained by varying total HEPESi concentration at constant [HEPES-] and vice versa suggest both the anionic and zwitterionic (protonated) forms of HEPES inhibit. Structure-activity studies with related compounds indicate the sulfonate group and heterocyclic aliphatic groups are both required for inhibition from the cytoplasmic side. TES (pK alpha = 7.54), substituted glycine buffers (pK alpha = 8.1-8.4) and bis-Tris (pK alpha = 6.46) had no measurable effect on conductance and appear suitable for use with this channel.

Transformed sweat gland and nasal epithelial cell lines from control and cystic fibrosis individuals.

We undertook to extend the in vitro lifespan of epithelial cell cultures useful for the study of the cellular defect underlying cystic fibrosis (CF). Primary cultures from sweat glands of four CF and four non-CF and from nasal polyps of one non-CF and two CF individuals were transformed using a chimaeric virus, Ad5/SV40 1613 ori-. The extended lifespans ranged from 20 to more than 250 population doublings beyond that of the primary cultures. Despite some degree of aneuploidy (as assayed by total cellular DNA content) all samples tested retained at least one copy of the region of chromosome 7 containing the CF gene (as assayed by probing with flanking DNA markers). Epithelial characteristics, including an epithelioid morphology, tight junctions and desmosomes, apical microvilli, keratin networks, and dome formation were positive in the majority of cells examined, although variably expressed. All cells tested demonstrated outwardly rectifying chloride channels by patch clamp, with some from non-CF cells responsive to the catalytic subunit of cyclic AMP-dependent protein kinase. The cells were used for DNA transfection assays with selectable marker genes in appropriate vectors, in order to develop methodology for assaying the function of the CF gene product and the effects of mutations.