Potentiation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ currents by the chemical solvent tetrahydrofuran

Abstract

The chemical solvent tetrahydrofuran (THF) increases short-circuit current (I_sc) in renal epithelia endogenously expressing the cystic fibrosis transmembrane conductance regulator (CFTR). To understand how THF increases I_sc, we employed the Ussing chamber and patch-clamp techniques to study cells expressing recombinant human CFTR. THF increased I_sc in Fischer rat thyroid (FRT) epithelia expressing wild-type CFTR with half-maximal effective concentration (K_D) of 134 mM. This THF-induced increase in I_sc was enhanced by forskolin (10 µM), inhibited by the PKA inhibitor H-89 (10 µM) and the thiazolidinone CFTR_inh-172 (10 µM) and attenuated greatly in FRT epithelia expressing the cystic fibrosis mutants F508del- and G551D-CFTR. By contrast, THF (100 mM) was without effect on untransfected FRT epithelia, while other solvents failed to increase I_sc in FRT epithelia expressing wild-type CFTR. In excised inside-out membrane patches, THF (100 mM) potentiated CFTR Cl⁻ channels open in the presence of ATP (1 mM) alone by increasing the frequency of channel openings without altering their duration. However, following the phosphorylation of CFTR by PKA (75 nM), THF (100 mM) did not potentiate channel activity. Similar results were obtained with the ▵R-S660A-CFTR Cl⁻ channel that is not regulated by PKA-dependent phosphorylation and using 2′deoxy-ATP, which gates wild-type CFTR more effectively than ATP. Our data suggest that THF acts directly on CFTR to potentiate channel gating, but that its efficacy is weak and dependent on the phosphorylation status of CFTR.

• ATP-binding cassette transporter
• chloride ion channel
• channel gating
• phosphorylation
• CFTR potentiators

Abbreviations
ABC transporter	=	ATP-binding cassette transporter
CF	=	cystic fibrosis
CFTR	=	cystic fibrosis transmembrane conductance regulator
2′-dATP	=	2′-deoxy-ATP
FRT cells	=	Fischer rat thyroid cells
H-89	=	N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2HCl
i	=	single-channel current amplitude
IBI	=	interburst interval
ICL	=	intracellular loop
Isc	=	short-circuit current
MBD	=	mean burst duration
MDCK cells	=	Madin-Darby canine kidney cells
MSD	=	membrane-spanning domain
N	=	number of active channels
NBD	=	nucleotide-binding domain
NMDG	=	N-methyl-D-glucamine
NPPB	=	5-nitro-2-(3-phenylpropylamino)-benzoic acid
Po	=	open-probability
RD	=	regulatory domain
Rt	=	transepithelial resistance
Tes	=	N-tris[Hydroxymethyl]methyl-2-aminoethanesulfonic acid
THF	=	tetrahydrofuran

Introduction

Tetrahydrofuran (THF) is an organic oxide, consisting of a four-carbon cyclic ether [1] (for the chemical structure of THF, see Figure 1A). It is an aprotic, electron donating solvent that dissolves a wide range of nonpolar and polar compounds [1]. As a result, THF is an important and widely used solvent in chemistry, the manufacture of dyes and adhesives.

Figure 1.  THF stimulates transepithelial ion transport in FRT epithelia expressing wild-type human CFTR. Representative I_sc recordings from FRT epithelia expressing wild-type human CFTR show the effects of (A) THF; (B) ethanol (EtOH); (C) methanol (MeOH); (D) DMSO (all tested at 0.8% v v⁻¹). During the periods denoted by the solid bars the indicated chemical solvents and forskolin (Fk; 10 µM) + genistein (Gen; 50 µM) were present in the solution bathing the apical membrane. Transepithelial voltage was clamped at 0 mV and there was a large Cl⁻ concentration gradient across the epithelium ([Cl⁻]_basolateral=149 mM; [Cl⁻]_apical=14.8 mM). The dotted lines indicate zero current. The chemical structure of each solvent is shown; (E) Effect of chemical solvents on I_sc. Column and error bars are means + SEM (solvents, n=4; Fk + Gen, n=7).

In previous work, we used THF as a vehicle to solubilise artificial anion transporters because it is the solvent of choice for biophysical studies of these molecules [2]. To our surprise, we found that THF, itself, stimulated weakly short-circuit current (I_sc; a measure of transepithelial ion transport) in type I Madin-Darby canine kidney (MDCK) epithelia [2]. However, the cellular mechanisms by which THF increased I_sc were not investigated in that study and we are unaware of reports in the literature of THF modulating the activity of ion channels and transporters.

We undertook the present study to identify how THF stimulates transepithelial ion transport. Type I MDCK epithelia endogenously express the ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) [3], a Cl⁻ channel with complex regulation that plays an essential role in fluid and electrolyte transport across epithelia [4]. CFTR is assembled from five domains: Two membrane-spanning domains (MSDs) that form an anion-selective pore, two nucleotide-binding domains (NBDs) that control channel gating and a unique regulatory domain (RD) phosphorylation of which is a prerequisite for channel activity [5], [6].

A large number of small molecules (termed CFTR potentiators) have been identified that interact directly with CFTR to enhance channel gating and hence, transepithelial ion transport [6]. We therefore hypothesised that THF might increase I_sc by enhancing CFTR-mediated transepithelial ion transport. To test this idea, we employed the Ussing chamber technique to study Fischer rat thyroid (FRT) epithelia expressing recombinant wild-type human CFTR. When we found that THF stimulates CFTR-mediated transepithelial ion transport, we employed the patch-clamp technique to investigate the effects of THF on the single-channel behaviour of CFTR. We discovered that THF acts directly on CFTR to potentiate channel gating, but that its efficacy is weak and dependent on the phosphorylation status of CFTR.

Materials and methods

Cells and cell culture

For this study, we used Fischer rat thyroid (FRT) epithelial cells expressing wild-type, F508del- and G551D- human CFTR [8] and mouse mammary epithelial (C127) cells stably expressing either wild-type human CFTR or the variant ▵R-S660A-CFTR [9]. FRT cells were supplied by Dr. L.J.V. Galietta (Istituto Giannina Gaslini, Genova, Italy) while C127 cells were gifts of Dr C.R. O'Riordan (Genzyme Corp., Framingham, MA, USA) and Prof. M.J. Welsh (University of Iowa, Iowa, USA). C127 and FRT cells were cultured as described previously [8], [10], with the exception that FRT cells expressing F508del-CFTR were not incubated at reduced temperatures to augment the cell surface expression of CFTR.

To grow FRT cells as polarized epithelia, trypsinized cells were seeded directly onto permeable filter supports (Millicell-PCF culture plate inserts 0.4 µm pore size, 12 mm diameter, Millipore Corp.; Fisher Scientific UK, Loughborough, UK) at a density of 4.5×10⁵ cells per 0.6 cm². Apical and basolateral media were replaced every 48 h and transepithelial resistance (R_t) measured using an epithelial voltohmeter (EVOM; World Precision Instruments, Stevenage, UK). R_t increased with time and five days after seeding (when R_t=3.36±0.08 kω cm²; n=79), we used FRT epithelia for experiments. For experiments using excised inside-out membrane patches, C127 cells were seeded onto glass coverslips and used within 48 h.

Short-circuit current measurements

FRT epithelia were mounted in modified Ussing chambers (Warner Instrument Corp., Dual Channel Chamber; Harvard Apparatus Ltd., Edenbridge, UK). The basolateral membrane of FRT epithelia was bathed in a solution containing (in mM): 140 NaCl, 5 KCl, 0.36 K₂HPO₄, 0.44 KH₂PO₄, 1.3 CaCl₂, 0.5 MgCl₂, 10 Hepes, and 4.2 NaHCO₃, pH 7.2 with Tris ([Cl⁻], 149 mM); osmolarity, 288±0.1 mOsm (n=4). The composition of the solution bathing the apical membrane of FRT epithelia was identical to that of the basolateral solution, with the exception that (in mM): 133.3 Na gluconate + 2.5 NaCl and 5 K gluconate replaced 140 NaCl and 5 KCl, respectively, to create a transepithelial Cl⁻ concentration gradient ([Cl⁻], 14.8 mM); osmolarity, 280±0.2 mOsm, (n=4). To compensate for the Ca^2 + buffering capacity of gluconate, we used 5.7 mM Ca^2 + in the apical solution. All solutions were maintained at 37°C and bubbled continuously with 5% CO₂.

We clamped transepithelial voltage (referenced to the basolateral solution) at 0 mV and recorded I_sc continuously using an epithelial voltage-clamp amplifier (Warner Instrument Corp., model EC-825, Harvard Apparatus Ltd.), digitising data as described previously [3]. The resistance of the filter and solutions, in the absence of cells, was subtracted from all measurements. Flow of current from the basolateral to the apical solution is shown as an upward deflection.

Patch-clamp experiments

CFTR Cl⁻ channels were recorded in excised inside-out membrane patches using an Axopatch 200A patch-clamp amplifier (Molecular Devices Corp., Union City, CA, USA) and pCLAMP data acquisition and analysis software (versions 6.03 and 9.0.0, Molecular Devices Corp.) as previously described [9], [10]. The established sign convention was used throughout; currents produced by positive charge moving from intra- to extracellular solutions (anions moving in the opposite direction) are shown as positive currents.

The pipette (extracellular) solution contained (mM): 140 N-methyl-D-glucamine (NMDG), 140 aspartic acid, 5 CaCl₂, 2 MgSO₄ and 10 N-tris[Hydroxymethyl]methyl-2-aminoethanesulfonic acid (Tes), adjusted to pH 7.3 with Tris ([Cl⁻], 10 mM; osmolarity, 279±0.5 mOsm; n=7). The bath (intracellular) solution contained (mM): 140 NMDG, 3 MgCl₂, 1 CsEGTA and 10 Tes, adjusted to pH 7.3 with HCl, ([Cl⁻], 147 mM; free [Ca^2 +],<10⁻⁸ M; osmolarity, 281±0.5 mOsm; n=4) and was maintained at 37°C; voltage was −50 mV.

After excision of inside-out membrane patches, CFTR Cl⁻ channels were activated by the addition of ATP (1 mM) and the catalytic subunit of PKA (75 nM) to the intracellular solution within 5 min of patch excision. To test the effects of THF, the solvent was added to the intracellular solution either before or after PKA addition. We recorded, filtered and digitised data as described previously [9]. For the purposes of illustration, data were filtered at 500 Hz and digitised at 1 kHz.

To investigate the effects of THF on fully phosphorylated CFTR Cl⁻ channels, we used membrane patches containing multiple active channels as previously described [11]. To measure single-channel current amplitude (i), Gaussian distributions were fit to current amplitude histograms. To study the effects of THF on CFTR channel gating prior to phosphorylation by PKA, we calculated burst duration and frequency of channel gating using membrane patches that contained only bursts of single level openings with no superimposed openings that were separated from one another by a minimum of 20 ms in the presence of ATP (1 mM) as described previously [10]. In membrane patches containing between 1 and 5 channels NP_o was calculated using the equation:1

where N is the number of channels, T_tot is the total time analysed and T₁ is the time that one or more channels are open, T₂ is the time two or more channels are open and so on. However, when membrane patches contained large numbers of active channels, NP_o was calculated by dividing I by i.

Reagents

PKA was purchased from Promega Ltd. (Southampton, UK). ATP (disodium salt), 2′-deoxyadenosine 5′-triphosphate disodium salt (2′-dATP), forskolin, Hepes, Tes, THF and vitamin C were obtained from the Sigma-Aldrich Company, Ltd. (Gillingham, UK). Genistein was purchased from LC Laboratories (Woburn, MA, USA), N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2HCl (H-89; Calbiochem) from Merck Biosciences, Ltd. (Nottingham, UK), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) from Semat Technical (UK) Ltd. (St Albans, UK) and CFTR_inh-172 from Asinex Ltd. (Moscow, Russia) and Calbiochem. All other chemicals were of reagent grade.

ATP, 2′-dATP and vitamin C were dissolved in distilled water, forskolin in methanol and all other reagents in DMSO. Stock solutions were stored at −20°C with the exception of those of (i) vitamin C, which was stored at ∼23°C and (ii) ATP and 2′-dATP, which were prepared directly before each experiment. Immediately before use, stock solutions were diluted to achieve final concentrations. Precautions against light-sensitive reactions were observed when using genistein and vitamin C.

Statistics

Results are expressed as means±SEM of n observations. To test for differences between two groups of data, we used paired and unpaired t tests, and for non-parametric data the Wilcoxon signed rank test (paired) and Mann-Whitney rank sum test (unpaired). To test for differences between multiple sets of data, we used a one-way analysis of variance (ANOVA) and the Dunn's and Holm Sidak method post tests where appropriate. Differences were considered statistically significant when p<0.05. Tests were performed using SigmaStat^TM (version 2.03, Jandel Scientific GmbH, Erkrath, Germany).

Results

THF causes a Cl⁻-dependent increase in I_sc

To investigate the effects of the chemical solvent THF on transepithelial ion transport, we grew FRT cells expressing wild-type human CFTR as polarised epithelia, mounted them in Ussing chambers and recorded I_sc, a measure of transepithelial ion transport. To study Cl⁻-dependent transepithelial ion transport, we imposed a large Cl⁻ concentration gradient ([Cl⁻]_basolateral=148 mM; [Cl⁻]_apical=14.8 mM) across epithelia without permeabilizing the basolateral membrane and clamped transepithelial voltage at 0 mV. As controls, we tested the effects of other chemical solvents including ethanol, methanol and DMSO. Figure 1A and 1E demonstrate that addition of THF (0.8% v v⁻¹; 100 mM) to the solution bathing the apical surface of FRT epithelia expressing wild-type CFTR increased I_sc. By contrast, ethanol, methanol and DMSO (all tested at 0.8% v v⁻¹) failed to increase I_sc (Figure 1B–1E). Following treatment of FRT epithelia with chemical solvents, addition to the apical solution of the cAMP agonist forskolin (10 µM) and the CFTR potentiator genistein (50 µM) generated a substantial I_sc, 5-fold greater than that achieved by THF (100 mM) (Figure 1).

Next we explored the Cl⁻-dependence of the THF-induced I_sc. When FRT epithelia expressing wild-type CFTR were bathed in symmetrical 148 mM Cl⁻ solutions, the I_sc elicited by THF (100 mM) decreased to 7% (4±1 µA cm⁻²; n=3) of that with the Cl⁻ gradient (Figure 2G; p=0.006). Similarly, the response to forskolin (10 µM) in the presence of symmetrical Cl⁻ concentrations was only 5% (12±3 µA cm⁻²; n=5) of that under gradient conditions (Figure 2G; p<0.001). In the presence of a Cl⁻-concentration gradient, addition of THF (100 mM) to the solution bathing the basolateral membrane elicited an I_sc of similar magnitude to that evoked when THF was added to the apical solution (n=3; data not shown). Finally, both THF (100 mM) and forskolin (10 µM) were without effect on I_sc in mock transfected FRT epithelia (Figure 2E, 2F). These experiments demonstrate that THF increases I_sc in FRT epithelia expressing recombinant CFTR and suggest that this I_sc is Cl⁻-dependent. They also argue that the increase in I_sc is not a consequence of the solvent acting non-specifically.

Figure 2.  THF stimulation of transepithelial ion transport is cAMP-dependent. Representative I_sc recordings from FRT epithelia expressing wild-type CFTR show the effects of (A) THF (100 mM); (B) forskolin (Fk; 10 µM); (C) forskolin (10 µM) and THF (100 mM); and (D) forskolin (10 µM) and genistein (Gen; 50 µM); (E, F) The effects of THF (100 mM) and forskolin (10 µM), respectively, on I_sc in mock transfected FRT epithelia (n=5). During the periods denoted by the solid bars the indicated agents were present in the solution bathing the apical membrane. (G, H) The change in I_sc evoked in wild-type, F508del- and G551D-CFTR FRT epithelia by different agents. Columns and error bars are means + SEM (n=6–23). The asterisks and cross denote values that are significantly different from those of THF alone and forskolin alone, respectively, (p<0.05). Note the change in ordinate scale between G and H. Other details as in Figure 1.

THF augments cAMP-stimulated I_sc

To characterise the I_sc elicited by THF, we compared the effects of THF (100 mM) and forskolin (10 µM) on transepithelial ion transport. Figure 2A demonstrates that the THF-induced I_sc was biphasic, with an initial transient peak followed by a sustained plateau 6-fold lower than the peak. By contrast, the I_sc response of FRT epithelia expressing wild-type CFTR to forskolin was characterised by a large initial peak followed by a high, sustained plateau (Figure 2B). Thus, the magnitude of the THF-elicited I_sc was only one quarter that of the forskolin-induced I_sc (Figure 2G).

Many agents have been identified that potentiate cAMP-stimulated I_sc mediated by the CFTR Cl⁻ channel [6]. To investigate whether THF potentiates cAMP-stimulated I_sc, we applied THF (100 mM) to FRT epithelia expressing wild-type CFTR after first stimulating I_sc with forskolin (10 µM). Figure 2A, 2C and 2G demonstrates that following the stimulation of I_sc by forskolin (10 µM), the I_sc evoked by THF (100 mM) was enhanced compared with that elicited by THF (100 mM) alone. As a result, the peak change in I_sc produced by forskolin (10 µM) and THF (100 mM) approached that generated by forskolin (10 µM) and genistein (50 µM; Figure 2D, 2G). We interpret these data to suggest that CFTR is involved in the THF-induced increase in I_sc.

To provide further evidence that THF increases I_sc by acting on CFTR, we tested its effects on the cystic fibrosis (CF) mutants F508del and G551D, both of which are located in NBD1 of CFTR and associated with a severe disease phenotype. F508del causes CF principally by disrupting the processing of CFTR protein and its delivery to the cell surface [12]. However, like G551D [13], F508del also perturbs severely CFTR channel gating [13]. Both mutations attenuate markedly cAMP-stimulated I_sc [14]. Figure 2H demonstrates that THF (100 mM), itself, had little or no effect on I_sc in FRT epithelia expressing ?F508del- and G551D-CFTR, whereas forskolin (10 µM) elicited a modest increase in I_sc in F508del- and G551D-expressing FRT epithelia. Moreover, THF (100 mM) failed to potentiate I_sc following treatment of F508del- and G551D-expressing FRT epithelia with forskolin (10 µM; Figure 2H). By contrast, genistein (50 µM) enhanced robustly cAMP-stimulated I_sc in FRT epithelia expressing F508del- and G551D-CFTR (Figure 2H). These data suggest that THF increases I_sc by potentiating the activity of CFTR. However, unlike genistein, THF is ineffective at restoring CFTR function to CF mutants.

Blockers of cAMP-stimulated transepithelial ion transport attenuate the THF-induced I_sc

As a final test to determine whether the CFTR Cl⁻ channel mediates the THF-induced I_sc, we examined the effects on the THF-induced I_sc of agents that inhibit CFTR by different mechanisms. We used the arylaminobenzoate NPPB (50 µM), the thiazolidinone CFTR_inh-172 (10 µM) and H-89 (10 µM), a membrane-permeant selective inhibitor of PKA. NPPB is an open-channel blocker of the CFTR Cl⁻ channel [15], CFTR_inh-172 likely acts by an allosteric mechanism [16], while H-89 impedes the phosphorylation of CFTR by PKA [17]. Because the THF-induced I_sc is transient, we added inhibitors to the apical solution bathing FRT epithelia expressing wild-type CFTR prior to the application of THF (100 mM). Figure 3 demonstrates that each of the agents tested markedly attenuated the I_sc elicited by THF (100 mM), diminishing the magnitude of both peak and steady-state I_sc. Taken together, our data argue that the THF-induced I_sc is mediated by the CFTR Cl⁻ channel.

Figure 3.  Inhibition of THF-stimulated I_sc by blockers of cAMP-stimulated transepithelial Cl⁻ transport. (A–D) Representative I_sc recordings show the effects of blockers of cAMP-stimulated transepithelial Cl⁻ transport on THF-stimulated I_sc in FRT epithelia expressing wild-type CFTR. During the periods indicated by the bars THF (100 mM) was present in the solution bathing the apical membrane. (A) Control; (B) NPPB (50 µM) added immediately prior to THF; (C) CFTR_inh-172 (10 µM) added 1 h prior to THF (D) H-89 (10 µM) added 2 h prior to THF; (E) The effects of inhibitors of transepithelial ion transport on THF-stimulated I_sc. Peak I_sc values measured in the presence of inhibitors are expressed as a percentage of the peak I_sc recorded in the presence of THF (100 mM) alone. Data are means + SEM (NPPB, n=11; CFTR_inh-172, n=5; H-89, n=8). The asterisks indicate values that are significantly different from the control value (p<0.05). Other details as in Figure 1.

THF causes a concentration-dependent increase in I_sc

To determine the relationship between THF concentration and I_sc, we added increasing concentrations of THF to the apical solution bathing FRT epithelia expressing wild-type CFTR in the absence of forskolin and measured peak I_sc. THF (50–400 mM) caused a concentration-dependent increase in I_sc, whereas THF (800 mM), the highest concentration tested, decreased I_sc and CFTR_inh-172 (10 µM) subsequently abolished I_sc (Figure 4A). Figure 4B demonstrates that the relationship between THF concentration and I_sc is fit by a Sigmoidal Hill equation (3 parameter) (R²=0.97) with I_{sc max} of 141 µA cm⁻², K_D of 134 mM and Hill coefficient of 2.4. Two conclusions can be drawn from these data: (i) THF weakly potentiates CFTR-mediated I_sc and (ii) THF might interact at multiple sites to stimulate I_sc.

Figure 4.  THF stimulation of transepithelial ion transport is concentration-dependent. (A) Representative I_sc recording from an FRT epithelium expressing wild-type CFTR shows the effects of THF (50–800 mM) on peak I_sc. During the periods indicated by the solid bars the indicated concentrations of THF and CFTR_inh-172 (10 µM) were present in the solution bathing the apical membrane. (B) Effect of THF concentration on I_sc in FRT epithelia expressing wild-type human CFTR. Data are means±SEM (n=8, except THF (800 mM), where n=6). The continuous line is the fit of the Hill equation to the data. Other details as in Figure 1.

THF alters the gating behaviour of the CFTR Cl⁻ channel

To understand how THF enhances CFTR-mediated transepithelial ion transport, we investigated the effects of THF on the single-channel activity of CFTR using the excised inside-out configuration of the patch-clamp technique. Excised membrane patches were clamped at −50 mV and a large Cl⁻ concentration gradient imposed across the membrane to magnify the size of CFTR Cl⁻ currents. To determine how the chemical solvent acts on CFTR, we added THF (100 mM) to the intracellular solution of freshly excised membrane patches with unknown numbers of channels present. In the absence of both ATP (1 mM) and PKA (75 nM), THF did not activate CFTR Cl⁻ channels in those patches that subsequently displayed channel activity in the presence of ATP and PKA (n=6; data not shown).

Because THF, by itself, increased I_sc in FRT epithelia expressing wild-type CFTR, we investigated whether the solvent might enhance the activity of CFTR prior to its phosphorylation with PKA. Figure 5A shows representative channel recordings of wild-type CFTR Cl⁻ channels in the absence and presence of THF (100 mM) in the solution bathing the intracellular face of the excised membrane patch; ATP (1 mM) was continuously present in the solution. Upon addition of the chemical solvent, there was a noticeable change in CFTR channel gating. THF (100 mM) increased the frequency of channel openings without appearing to alter their duration (Figure 5A).

Figure 5.  The effects of THF on the single-channel activity of wild-type human CFTR prior to PKA-dependent phosphorylation. (A) Representative recordings of CFTR Cl⁻ current in an excised inside-out membrane patch from a C127 cell expressing wild-type human CFTR. During the periods denoted by the bars ATP (1 mM), THF (100 mM) and PKA (75 nM) were present in the intracellular solution. Voltage was −50 mV and there was a large Cl⁻ concentration gradient across the membrane patch ([Cl⁻]_internal=147 mM; [Cl⁻]_external=10 mM). For the purpose of illustration, the time-course has been inverted so that upward deflections represent inward currents. The inserts show the sections of the record labelled 1 and 2 on an expanded time scale. The dotted lines indicate where channels are closed and downward deflections of the traces correspond to channel openings. (B, C, D and E) The effects of THF (100 mM) on percent time open, events per minute, single-channel current amplitude (i) and mean burst duration (MBD). Columns and error bars are means + SEM (percent time open, events per minute and MBD n=5; i, n=12). The asterisks indicate values that are significantly different from control values (p<0.05).

To begin to analyse the effects of THF (100 mM) on channel gating, we measured NP_o because the number of active channels in the membrane patch was unknown. Under control conditions (1 mM ATP) NP_o was 0.06±0.04 (n=9), whereas following the addition of THF (100 mM) NP_o increased to 0.13±0.08 (n=9), an amplification of 2.2-fold (p<0.05). However, following phosphorylation with PKA (75 nM), NP_o increased to 12.11±3.83 (n=9), an amplification of 94-fold (p<0.01). Next, we measured the frequency of channel gating in membrane patches where only a single CFTR Cl⁻ channel was active. (We did not calculate interburst interval (IBI) because the number of active channels in the membrane patch was unknown in the absence of PKA). THF (100 mM) increased markedly the percent time open and the frequency of channel gating (Figure 5B, 5C). However, THF was without effect on mean burst duration (MBD) (Figure 5E) and depressed slightly single-channel current amplitude (i; Figure 5D). Taken together, these results suggest that THF increases the activity of CFTR Cl⁻ channels that are not fully phosphorylated by PKA by increasing the frequency of channel opening.

To determine whether THF potentiates the activity of CFTR after its phosphorylation by PKA, we added THF to the intracellular solution following channel activation by PKA (75 nM) and ATP (1 mM). In both membrane patches containing large (≥6) or small numbers of active channels (<6), THF (100 mM) was without effect on CFTR Cl⁻ current, whereas THF (200 mM) depressed albeit not significantly current magnitude (Figure 6). We interpret these data to suggest that the effects of THF on CFTR channel gating are dependent on the phosphorylation status of CFTR.

Figure 6.  THF is without effect on CFTR following PKA-dependent phosphorylation in an excised membrane patch. (A) Time-course of CFTR Cl⁻ current in an excised inside-out membrane patch from a C127 cell expressing wild-type CFTR. During the periods indicated by the bars ATP (1 mM), PKA (75 nM) and THF (100 mM) were present in the intracellular solution. (B) NP_o of wild-type CFTR following phosphorylation with PKA in the absence and presence of THF (100–200 mM). NP_o values measured in the presence of THF are expressed as a percentage of control values recorded in the absence of the drug. Columns and error bars are means + SEM (n=6). Other details as in Figure 5.

Addition of THF (100 mM) to the intracellular solution increases osmolarity from 280±0.5 mOsm (n=5) to 396±7.4 mOsm (n=5). To exclude the possibility that a change in solution osmolarity accounts for the observed effects of THF, we mimicked the THF-induced change in osmolarity of the intracellular solution using D-mannitol (180 mM). Elevating the osmolarity of the intracellular solution did not enhance CFTR activity. Instead, high solution osmolarity caused small, but not statistically significant decreases in i and NP_o (i: control, −0.79±0.04 pA (n=3); D-mannitol (180 mM), −0.73±0.03 (n=3; p>0.05); NP_o: control, 0.47±0.3 (n=3); D-mannitol (180 mM), 0.27±0.15 (n=3; p>0.05). These results argue that the effects of THF on the CFTR Cl⁻ channel are not caused by a change in osmolarity of the intracellular solution.

THF potentiates the ▵R-S660A-CFTR Cl⁻ channel and wild-type CFTR gated by 2′-deoxy-ATP

The lack of effect of THF on the CFTR Cl⁻ channel after phosphorylation by PKA, suggests that the RD might be a target for the actions of THF. To test this hypothesis, we used the CFTR construct ▵R-S660A [8], performing the same experiments on this CFTR variant as those with wild-type CFTR. The CFTR mutant ▵R-S660A lacks much of the RD (residues 708-835) and disables the dibasic phosphorylation site at serine 660. This CFTR construct no longer requires PKA-dependent phosphorylation to open in the presence of intracellular ATP [9]. Supplementary Information (SI) Figure 7 (online version only) demonstrates that THF (100 mM) augments the activity of ▵R-S660A-CFTR Cl⁻ channels by increasing the frequency of channel openings. These data suggest that THF might potentiate CFTR channel gating by interacting with a site distinct from the RD.

SI Figure 1.  THF increases the opening rate of the ▵R-S660A-CFTR Cl⁻ channel. (A) Representative recordings show the single-channel activity of ▵R-S660A-CFTR in an excised inside-out membrane patch from a C127 cell. The upper and lower traces were recorded in the absence and presence of THF (100 mM), respectively, in the intracellular solution; ATP (1 mM) was continuously present in the intracellular solution. Voltage was −50 mV and there was a large Cl⁻ concentration gradient across the membrane patch ([Cl⁻]_internal=147 mM; [Cl⁻]_external=10 mM). The dotted lines indicate where channels are closed and downward deflections of the traces correspond to channel openings. (B, C, D and E) Percent time open, events per min, i and MBD of ▵R-S660A-CFTR in the absence and presence of THF (100 mM). For percent time open, events per minute and MBD columns and error bars are means + SD (n=2), for i columns and error bars are means + SEM (n=5). The asterisks indicate values that are significantly different from control values (p<0.05).

Previous work has demonstrated that many agents interact directly with the NBDs to enhance CFTR channel gating (for review, see [6]). We therefore speculated that THF might exert its effects on CFTR channel gating by modulating ATP-dependent channel gating [18]. To test this idea, we employed the hydrolysable ATP analogue 2′-deoxy ATP (2′-dATP), which interacts with the two ATP-binding sites at the NBD dimer interface to gate CFTR more effectively than ATP [10]. We reasoned that THF might be ineffective at potentiating wild-type CFTR Cl⁻ channels gated by 2′-dATP. However, SI Figure 8 (online version only) demonstrates that prior to channel activation by PKA, THF (100 mM) augmented strongly the single-channel activity of wild-type CFTR gated by 2′-dATP (1 mM). We interpret these results to suggest that THF potentiates CFTR channel gating by interacting with a site distinct from CFTR's two ATP-binding sites.

SI Figure 2.  THF enhances the single-channel activity of wild-type CFTR gated by 2′-dATP prior to PKA-dependent phosphorylation. (A) Representative recordings show the single-channel activity of CFTR in an excised inside-out membrane patch from a C127 cell. The upper and lower traces were recorded in the absence and presence of THF (100 mM), respectively, in the intracellular solution; 2′d-ATP (1 mM) was continuously present in the intracellular solution. One CFTR Cl⁻ channel was active under control conditions and two in the presence of THF (100 mM). (B, C, D and E) Percent time open, events per minute, i and MBD of wild-type CFTR in the absence and presence of THF (100 mM). Columns and error bars are means + SEM (n=7). The asterisks indicate values that are significantly different from control values (p<0.05). Other details as in SI Figure 1.

THF potentiates wild-type CFTR in the presence of the reducing agent vitamin C

We considered the possibility that the chemical properties of THF might provide insight into its mechanism of action. Because THF is an aprotic, electron donating solvent [1], it might act as a reducing agent. This characteristic is significant in two respects. First, the intracellular redox status influences CFTR channel gating [19]. Second, the reducing agent vitamin C, which shares some similarities in chemical structure to THF, has analogous effects on CFTR channel gating as those of the solvent [20]. These comparisons suggest that THF might potentiate CFTR channel gating by altering the redox status of CFTR. To explore this possibility, we investigate whether prior to channel activation by PKA, pre-treatment of CFTR Cl⁻ channels with vitamin C (100 µM) might prevent the potentiation of CFTR channel gating by THF. However, SI Figure 9 (0nline version only) demonstrates that THF (100 mM) enhanced robustly the single-channel activity of wild-type CFTR potentiated by vitamin C (100 µM). These results suggest that potentiation of CFTR channel gating by THF likely does not involve modulation of the redox status of CFTR.

SI Figure 3.  THF enhances CFTR channel gating in the presence of the reducing agent vitamin C. (A) Representative recordings show the single-channel activity of CFTR in an excised inside-out membrane patch from a C127 cell. The upper and lower traces were recorded in the absence and presence of THF (100 mM), respectively, in the intracellular solution; ATP (1 mM) and vitamin C (100 ?M) were continuously present in the intracellular solution. Two CFTR Cl⁻ channels were active under control conditions and three in the presence of THF (100 mM). (B, C, D and E) Percent time open, events per minute, i and MBD of wild-type CFTR in the absence and presence of THF (100 mM). Columns and error bars are means + SEM (n=4). The asterisks indicate values that are significantly different from control values (p<0.05). Other details as in SI Figure 1. In two experiments, we tested the effects of vitamin C (100 µM) on CFTR channel gating in the presence of ATP (1 mM), but absence of PKA (75 nM). Data in the absence and presence of vitamin C (100 µM) are as follows: (i) percent time open: control, 6.3±1.4%; vitamin C, 26.4±2.3%; (ii) events per min: control, 213.6±46.6 min⁻¹; vitamin C, 777.1±70.0 min⁻¹; (iii) i: control, −0.77±0.05 pA; vitamin C, −0.76±0.05 pA and (iv) MBD: control, 65.1±43 ms; vitamin C, 85.2±24.0 ms (all data are means±SD; n=2).

Discussion

In this study, we investigated the effects of the chemical solvent THF on CFTR-mediated transepithelial ion transport. Using I_sc measurements and single-channel recording, we demonstrated that THF interacts directly with CFTR to potentiate channel gating, but that its efficacy is weak and dependent on the phosphorylation status of CFTR.

Our data demonstrate that the potentiation of CFTR Cl⁻ currents by THF is enhanced by forskolin in FRT epithelia expressing wild-type human CFTR, but not by PKA in membrane patches excised from C127 cells expressing the same CFTR construct. One possible explanation for these conflicting results is the different cell types, which we used to study CFTR-mediated transepithelial ion transport and single-channel behaviour. However, a more likely explanation is the phosphorylation status of CFTR and hence, the level of channel activity. In excised membrane patches from C127 cells expressing wild-type CFTR bathed in a Cl⁻-rich intracellular solution containing ATP (1 mM), PKA (75 nM) robustly stimulates the CFTR Cl⁻ channel (P_o=0.52±0.04; n=18; [10]). However, in cell-attached recordings from the same cells, forskolin (20 µM) is less effective at stimulating CFTR (P_o=0.24±0.02; n=16; Z Xu & DN Sheppard, unpublished observation). Based on these data, we suggest that THF robustly enhances the activity of weakly phosphorylated CFTR Cl⁻ channels, but has little or no effect on the activity of strongly phosphorylated CFTR Cl⁻ channels. Support for this idea is provided by previous work on the best-studied CFTR potentiator genistein. The effect of genistein on channel activity depends on the phosphorylation status of CFTR: Genistein strongly enhances channel gating when CFTR is weakly phosphorylated by PKA, but has little or no effect on channel gating when CFTR is highly phosphorylated by PKA [12], [22].

A further difference between the effects of THF in FRT epithelia and excised membrane patches is the transient nature of the THF response in FRT epithelia compared with the solvent's sustained effects in excised membrane patches (present results and data not shown). In the presence of a Cl⁻ concentration gradient, there is unlikely to be a change in the driving force for Cl⁻ exit across the apical membrane as a result of changes in the activity of basolateral membrane K⁺ channels. Instead, the transient nature of the THF response in FRT epithelia likely reflects the deactivation of CFTR Cl⁻ channels in intact cells by protein kinases (e.g., AMP kinase; [22]) and phosphatases (e.g., PP2A and PP2C; [23]).

Many CFTR potentiators (e.g., genistein; [21]) enhance the gating behaviour of CFTR by (i) accelerating channel opening and (ii) slowing dramatically channel closure. Other CFTR potentiators (e.g., phloxine B; [24]) augment CFTR channel gating solely by retarding channel closure. By contrast, THF enhanced CFTR channel gating by increasing the rate of channel opening without altering the duration of bursts. This effect of THF is reminiscent of agents that modulate CFTR activity by altering the phosphorylation status of the RD. For example, phosphorylation of the RD by PKA and PKC enhances CFTR Cl⁻ currents by increasing the frequency of channel openings without altering their duration [25], [26]. These considerations suggest that the simplest interpretation of our data is that PKA-dependent phosphorylation and THF binding might elicit similar changes in the conformation of the CFTR Cl⁻ channel, albeit with markedly different efficacy. Thus, the effects of THF (the weaker agonist) are masked in fully phosphorylated channels by the overriding effects of phosphorylation on CFTR conformation.

Where might THF interact with CFTR to exert its effects? Our observation that THF potentiates the ▵R-S660A-CFTR Cl⁻ channel argues that the solvent interacts with a site distinct from the RD. Because the NBDs are the location of binding sites for a number of CFTR potentiators [6], THF might exert its effects by binding to the NBDs. Using the ATP-driven NBD dimerisation model of CFTR channel gating [18], we suggest that the interaction of THF with the NBDs accelerates channel opening by providing binding energy to drive NBD dimerisation. Because THF did not substitute for ATP in supporting channel gating, we consider it unlikely that THF interacts with site 1, where ATP binds tightly. Similarly, because THF was without effect on the duration of channel openings, it is unlikely to compete with ATP for binding to site 2, where ATP is hydrolysed rapidly. Consistent with this idea, THF potentiated CFTR Cl⁻ channels gated by 2′-dATP, which exerts its effects by acting at sites 1 and 2 [10]. Instead, the THF-binding site might be located at the NBD1:NBD2 interface based on the interaction of CFTR potentiators with a molecular model of the NBD dimer [27].

However, two further THF binding sites are worthy of consideration. First, structural models of CFTR based on the ABC transporter Sav1866 [28–30] suggest that the intracellular loops (ICLs) play a critical role in communication between the NBDs and MSDs. This raises the intriguing possibility that CFTR potentiators, such as THF, might exert their effects by altering the conformation of the ICLs. Second, THF might potentiate CFTR channel gating by influencing the conformation of the MSDs, which form the CFTR pore. THF might interact directly with the MSDs to alter the packing of the transmembrane segments. Alternatively, the solvent might influence the conformation of the MSDs indirectly by altering the mechanical properties of the lipid bilayer. In support of this latter mechanism, Hwang et al. [31] demonstrated that the best-studied CFTR potentiator genistein modulates the activity of gramicidin A channels in planar lipid bilayers by altering bilayer mechanics, while the data of Artigas et al. [32] suggest that 2,3-butanedione monoxime might affect CFTR function by a similar mechanism.

In conclusion, we demonstrated that THF potentiates CFTR-mediated transepithelial ion transport by accelerating channel opening. THF has the simplest chemical structure of any CFTR potentiator identified to date. Moreover, the chemical structure of a number of CFTR potentiators identified by high-throughput screening incorporate the structure of THF (e.g., CFTR_act-07; [14]). The effects of THF on CFTR channel gating suggest that it might interact directly with the NBDs to promote their dimerization. The data also highlight the importance of the phosphorylation status of CFTR for the action of CFTR potentiators.

Supplementary information

We undertook a series of experiments to understand better how the chemical solvent THF potentiates CFTR channel gating. Below, we present these data and discuss the results.

THF potentiates the ▵R-S660A-CFTR Cl⁻ channel and wild-type CFTR gated by 2′-deoxy-ATP

The lack of effect of THF on the CFTR Cl⁻ channel after phosphorylation by PKA, suggests that the R domain (RD) might be a target for the actions of THF. To test this hypothesis, we used the CFTR construct ▵R-S660A [33], performing the same experiments on this CFTR variant as those with wild-type CFTR. The CFTR mutant ▵R-S660A lacks much of the R domain (residues 708–835) and disables the dibasic phosphorylation site at serine 660. This CFTR construct no longer requires PKA-dependent phosphorylation to open in the presence of intracellular ATP [33].

Supplementary Information (SI) Figure 7A shows representative recordings of a single ▵R-S660A-CFTR Cl⁻ channel in the absence and presence of THF (100 mM). Visual inspection of these single-channel records suggests that THF (100 mM) increases markedly the frequency of channel openings. In membrane patches with only a single active ▵R-S660A-CFTR Cl⁻ channel, THF enhanced strongly the percent time open and the frequency of channel gating, was without effect on MBD and depressed slightly i (SI Figure 7). We interpret these results to suggest that THF potentiates CFTR channel gating by interacting with a site distinct from the RD.

Previous work has demonstrated that many agents interact directly with the NBDs to enhance CFTR channel gating (for review, see [34]). We therefore speculated that THF might exert its effects on CFTR channel gating by modulating ATP-driven NBD dimerisation and hence conformation changes in the MSDs, which open and close the channel pore [35]. To test this idea, we employed the hydrolysable ATP analogue 2′-deoxy ATP (2′-dATP), which interacts with the two ATP-binding sites at the NBD dimer interface to gate CFTR more effectively than ATP [36]. We reasoned that THF might be ineffective at potentiating wild-type CFTR Cl⁻ channels gated by 2’-dATP. However, SI Figure 8A demonstrates that THF (100 mM) augmented strongly the gating behaviour of wild-type CFTR prior to channel activation by PKA when ATP (1 mM) was substituted by 2′-dATP (1 mM) in the intracellular solution. Like its effects on wild-type CFTR gated by ATP (Figure 5), THF (100 mM) increased the percent time open and frequency of channel gating, but was without effect on MBD (SI Figure 8B, 8C and 8E). The only difference between ATP and 2′-dATP was that in the presence of 2′-dATP, THF (100 mM) did not decrease i (SI Figure 8D). We interpret these results to suggest that THF potentiates CFTR channel gating by interacting with a site distinct from CFTR's two ATP-binding sites.

THF potentiates wild-type CFTR in the presence of the reducing agent vitamin C

We considered the possibility that the chemical properties of THF might provide insight into its mechanism of action. Because THF is an aprotic, electron donating solvent [37], it might act as a reducing agent. Previous work has demonstrated that the intracellular redox status influences CFTR channel gating. For example, Harrington et al. [38] demonstrated that reducing agents accelerate and oxidising agents slow channel gating. Because the non-hydrolysable ATP analogue ATPγS attenuated the effects of the reducing agent β-mercaptoethanol, Harrington et al. [38] speculated that the NBDs are the site of action of redox reagents. Recently, Fischer et al. [39] demonstrated that the reducing agent vitamin C potentiates the CFTR Cl⁻ channel. The authors’ data are significant in two respects: First, the effects of vitamin C on CFTR channel gating are similar to those of THF. Second, the chemical structure of vitamin C contains a furan ring, the parent molecule of THF. These comparisons suggest that THF might potentiate CFTR channel gating by altering the redox status of CFTR.

To explore whether THF potentiates CFTR channel gating by altering the redox status of CFTR, we investigated whether pre-treatment of CFTR Cl⁻ channels with vitamin C might prevent the potentiation of CFTR channel gating by THF. For these studies, we selected a concentration of vitamin C (100 µM), three-fold greater than the vitamin C concentration required for half-maximal potentiation of CFTR-mediated transepithelial ion transport in Calu-3 epithelia [39]. Like THF (100 mM; Figure 5), addition of vitamin C (100 µM) to the intracellular solution in the presence of ATP (1 mM), but absence of PKA (75 nM) enhanced CFTR channel gating. Vitamin C (100 µM) increased percent time open and frequency of channel gating, but was without effect on MBD (see legend of SI Figure 9). However, unlike THF (100 mM; Figure 5D) vitamin C did not decrease i (SI Figure 9 legend).

SI Figure 9A demonstrates that THF (100 mM) potentiated robustly the gating behaviour of wild-type CFTR in the presence of ATP (1 mM) and vitamin C (100 µM), but absence of PKA (75 nM) in the intracellular solution. THF (100 mM) increased the percent time open and frequency of channel gating, but was without effect on i and MBD (SI Figure 9B–9E). We interpret these results to suggest that THF interacts with CFTR at a site distinct from that of vitamin C to potentiate CFTR channel gating. The data also suggest that potentiation of CFTR channel gating by THF likely does not involve modulation of the redox status of CFTR.

Acknowledgements

We thank Professor A.P. Davis, Dr. G. Magro and our departmental colleagues for valuable discussions and assistance, especially Dr. H. Li. We also thank Drs. L.J.V. Galietta (Istituto Giannina Gaslini), C.R. O'Riordan (Genzyme Corp.) and Professor M.J. Welsh (University of Iowa) for generous gifts of cells expressing wild-type and mutant CFTRs. This work was supported by the Biotechnology and Biological Sciences Research Council [grant no. BBS/B/11044] and the Cystic Fibrosis Trust. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.