A Short Segment of the R Domain of Cystic Fibrosis Transmembrane Conductance Regulator Contains Channel Stimulatory and Inhibitory Activities That Are Separable by Sequence Modification*

The regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) contains consensus phosphorylation sites for cAMP-dependent protein kinase (PKA) that are the basis for physiological regulation of the CFTR chloride channel. A short peptide segment in the R domain with a net negative charge of B9 (amino acids 817–838, NEG2) and predicted helical tendency is shown to play a critical role in CFTR chloride channel function. Deletion of NEG2 from CFTR completely eliminates the PKA dependence of channel activity. Exogenous NEG2 peptide interacts with CFTR to exert both stimulatory and inhibitory effects on the channel function. The NEG2 peptide with sequence scrambled to remove helical tendencies also inhibits channel function, but does not stimulate. Similar results are found for a NEG2 peptide whose helical structure is disrupted by a proline residue. When six of the negatively charged carboxylic acid residues are replaced by their cognate amides, reducing net negative charge to B3, but increasing helical propensity as assessed by circular dichroism, the peptide stimulates CFTR channel function,

The regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) contains consensus phosphorylation sites for cAMP-dependent protein kinase (PKA) that are the basis for physiological regulation of the CFTR chloride channel. A short peptide segment in the R domain with a net negative charge of B9 (amino acids 817-838, NEG2) and predicted helical tendency is shown to play a critical role in CFTR chloride channel function. Deletion of NEG2 from CFTR completely eliminates the PKA dependence of channel activity. Exogenous NEG2 peptide interacts with CFTR to exert both stimulatory and inhibitory effects on the channel function. The NEG2 peptide with sequence scrambled to remove helical tendencies also inhibits channel function, but does not stimulate. Similar results are found for a NEG2 peptide whose helical structure is disrupted by a proline residue. When six of the negatively charged carboxylic acid residues are replaced by their cognate amides, reducing net negative charge to B3, but increasing helical propensity as assessed by circular dichroism, the peptide stimulates CFTR channel function, but does not inhibit. We speculate that the NEG2 region interacts with other cytosolic domains of CFTR to control opening and closing transitions of the chloride channel.
Defects in CFTR, 1 a chloride channel located in the apical membrane of epithelial cells, are associated with the common genetic disease, cystic fibrosis (1)(2)(3). CFTR is a 1480-amino acid protein that is a member of the ATP binding cassette transporter family (4). The general structure of these membrane proteins includes two membrane spanning domains, each consisting of six transmembrane segments, and two nucleotide binding folds (NBF1 and NBF2). Most members of the ATP binding cassette family use the free energy of ATP hydrolysis to actively transport substrates across the membrane (5). However, unlike the other members of this family, CFTR contains a unique regulatory (R) domain, and encodes a cAMP-regulated chloride channel (6 -8).
The R domain of CFTR contains several consensus PKA phosphorylation sites (9 -11) that are the basis for physiological regulation of this chloride channel. CFTR channel opening requires phosphorylation of serine residues in the R domain, and ATP binding and hydrolysis at the nucleotide binding folds (7,12,13). Phosphorylation adds negative charges to the R domain, and introduces global conformational changes reflected by a reduction in the ␣-helical content of the R domain protein (14). Thus, electrostatic and/or allosteric changes mediated by phosphorylation are likely responsible for interactions between the R domain and other CFTR domains that regulate channel function (15,16).
Rich et al. (17) showed that deletion of amino acids 708 -835 from the R domain (⌬R-CFTR), which removes most of the PKA consensus sites, allows the CFTR chloride channel to open without phosphorylation. The open probability of ⌬R-CFTR is one-third that of the wild type (wt) CFTR channel and does not increase upon PKA phosphorylation, although other biophysical properties of the channel (i.e. conductance and anion selectivity) are similar to wt-CFTR (18,19). These data suggest that deletion of the R domain removes both inhibitory and stimulatory effects conferred by the R domain on CFTR chloride channel function. In support of this suggestion, addition of exogenous unphosphorylated R domain protein (amino acids 588 -858) to wt-CFTR blocks the chloride channel (20,21), and the block is relieved if the R domain becomes phosphorylated, indicating that the unphosphorylated R domain is inhibitory. Conversely, exogenous phosphorylated R domain protein (amino acids 588 -855 or 645-834) stimulates the ⌬R-CFTR channel, suggesting that the phosphorylated R domain is stimulatory (18,19). Therefore, it appears that the phosphorylation state of the R domain determines whether it functions to stimulate or inhibit chloride channel activity.
In this work we identify a stretch of negatively charged amino acids at the carboxyl terminus of the R domain (817-838, NEG2), with a net charge of Ϫ9, which appears to be involved in both the stimulatory and inhibitory functions of the R domain on chloride channel activity. Furthermore, by modifying the sequence of this peptide, we were able to separate its stimulatory and inhibitory functions.

EXPERIMENTAL PROCEDURES
Subcloning of CFTR Gene-The wt-CFTR cDNA was cloned into an Epstein-Barr virus-based episomal eukaryotic expression vector, pCEP4 (Invitrogen, San Diego, CA), between the NheI and XhoI restriction sites (18, 20 -22). The ⌬NEG1 and ⌬NEG2 deletion mutants were created using the pALTER mutagenesis system and shuttled from pALTER into pCEP4 by substituting the corresponding fragment in pCEP4 wt-CFTR with the mutant fragment between the BstZ171 and XhoI restriction sites. The ⌬NEG1-CFTR cDNA has 27 bases deleted (amino acids 725-733). The ⌬NEG2-CFTR cDNA has 66 bases deleted (amino acids 817-838). Deletions were confirmed by sequencing across the junction site.
Expression of CFTR in HEK 293 Cells-A human embryonic kidney cell line (293-EBNA HEK; Invitrogen) was used for transfection and expression of the CFTR proteins (18, 20 -22). The HEK-293 cell line contains a pCMV-EBNA vector, which constitutively expresses the Epstein-Barr virus nuclear antigen-1 (EBNA-1) gene product and increases the transfection efficiency of Epstein-Barr virus-based vectors. Cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% L-glutamine. Geneticin (G418, 250 g/ml) was added to the cell culture medium to maintain selection of the cells containing the pCMV-EBNA vector. LipofectAMINE reagent (Invitrogen) in Opti-MEM media (serum-free) was used to transfect the HEK-293 cells with pCEP4(wt), pCEP4(⌬NEG1), or pCEP4(⌬NEG2). After 5 h, serum was added to the media (10% final serum concentration). Twenty-four hours after transfection, the transfection media was replaced with fresh media. The cells were harvested 2 days after transfection and microsomal membrane vesicles were prepared for single channel measurements in the lipid bilayer reconstitution system (18, 20 -22).
In Vitro Phosphorylation of CFTR Proteins-CFTR proteins isolated in membrane vesicles were bound to protein G-agarose using a mouse monoclonal anti-human CFTR antibody (mAb 24 -1, Genzyme). The protein G-agarose was washed and [␥-32 P]ATP (10 Ci) and protein kinase A (ϳ10 units/50 l) was added. Samples were incubated at 30°C for 1 h during phosphorylation. Excess [␥-32 P]ATP was removed, and SDS-PAGE sample buffer (200 mM Tris-Cl, pH 6.7, 9% SDS, 6% ␤-mercaptoethanol, 15% glycerol, and 0.01% bromphenol blue) was added to denature CFTR and release it from the protein G-agarose. The samples were subjected to electrophoresis on a 5% SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and exposed to film. The phosphorylated proteins from 12.5-l vesicles were loaded in each lane. Densitometry was performed and normalized to CFTR protein band intensity recorded from Western blots performed as previously described (18, 20 -22) on 10-l vesicles for wt-CFTR and the ⌬NEG2-CFTR samples, and 20-l vesicles containing ⌬NEG1-CFTR, using the Genzyme 24-1 monoclonal antibody developed with ECL.
Peptide Studies-Twenty-two residue amino acid peptides corresponding to the NEG2, NEG2i, s-NEG2, p-NEG2, and h-NEG2i sequences were custom synthesized (Quality Controlled Biochemicals, Hopkinton, MA) (see Table I). The NH 2 -and COOH-terminal residues of each peptide were acetylated and amidated, respectively. The peptides were determined to be Ͼ95% pure by high performance liquid chromatography mass spectral analysis. Molecular modeling was performed with Insight II software (MSI Inc., San Diego, CA) using standard amino acid side chain conformations. Secondary structure predictions were made using the secondary structure consensus prediction feature of the Network Protein Sequence analysis site at npsapbil.icp.fr (23,24). Circular dichroism (CD) spectra were obtained on a Jasco J-1810 Spectra Polarimeter (Jasco, Easton, MD) equipped with Jasco Spectra  Manager (version 1.51.00) data acquisition software. Spectra were obtained at 4°C from 1-mm path length quartz cells. CD spectra were the average of eight scans collected at 0.1-nm intervals from 260 to 190 nm using standard instrument settings. Peptide concentrations ranged between 15 and 49 M, at pH ϳ 6.7. Peptide stock solution concentrations were obtained by quantitative amino acid analysis performed by the Protein/Peptide Core Facility of the Massachusetts General Hospital.
Reconstitution of CFTR Channels in Lipid Bilayer Membranes-Electrophysiological analysis of single channel activity was performed as previously described (17,21). Briefly, lipid bilayer membranes were formed across an aperture of ϳ200 m diameter with a mixture of phosphatidylethanolamine:phosphatidylserine:cholesterol in a ratio of 5:5:1. The lipids were dissolved in decane at a concentration of 33 mg/ml. The recording solutions contained (in mM): cis (intracellular), 200 CsCl, 1 MgCl 2 , 2 ATP, and 10 HEPES-Tris (pH 7.4); and trans (extracellular), 50 CsCl, 10 HEPES-Tris (pH 7.4). Vesicles (1-4 l) containing wt-, ⌬NEG1-, or ⌬NEG2-CFTR were added to the cis solution. Unless otherwise noted, the PKA catalytic subunit was present at a concentration of 50 units/ml in the cis solution. Single channel currents were recorded with an Axopatch 200A patch clamp unit (Axon Instruments). The currents were sampled at 1-2.5 ms/point. Single channel data analyses were performed with pClamp and TIPS software.

RESULTS
Activity of CFTR Channels Lacking the NEG1 or NEG2 Sequence-Examination of the primary amino acid sequence of CFTR revealed two regions with high proportion of negatively charged residues in the R domain, amino acids 725-733 (NEG1) and amino acids 817-838 (NEG2) (Fig. 1A). The NEG2 sequence is highly conserved across species (Table I). To investigate the roles of NEG1 and NEG2 in CFTR function, these FIG. 3. Structure of wt NEG2 peptide and CD spectroscopy of NEG2 peptide analogues. A, space filling model of the predicted ␣-helical structure of the NEG2 peptide. The negatively charged Glu and Asp residues are colored purple, the hydrophoblic residues Leu, Ile, and Phe are orange, and Lys, light blue. The remaining residues are gray. B-E, circular dichroism spectra of NEG2i (pink), h-NEG2i (blue), s-NEG2 (light blue), p-NEG2 (red), and NEG2 (green) at 0, 33, 50, and 66% TFE in water at pH 6.7. F and G, mean residue molar ellipticity plots at 193 and 222 nm for the peptides as a function of TFE. Percent helicity was estimated from the band intensities using the maximum helical values of Chen et al. (32). regions were deleted from CFTR. The resulting ⌬NEG1and ⌬NEG2-CFTR proteins were transiently expressed in HEK 293 cells. Membrane vesicles containing CFTR proteins were isolated and subjected to SDS-PAGE. Like wt-CFTR, both ⌬NEG1and ⌬NEG2-CFTR are present both in the core glycosylated (band B) and the fully glycosylated form (band C) (Fig.   1B). The PKA dependence of the ⌬NEG1-CFTR channel is similar to wt-CFTR (Fig. 1C). No channel activity is observed in the absence of PKA, and the open probability (P o ) of the ⌬NEG1-CFTR channel in the presence of PKA and ATP is similar to wt-CFTR. In contrast, the ⌬NEG2-CFTR channel opens without PKA (Fig. 1C, right). The "constitutive" activity of the ⌬NEG2-CFTR channel is unlikely to be due to the endogenous phosphorylation of the ⌬NEG2-CFTR protein, since protein phosphatase 2A, which decreases activity of the wt-CFTR opened by PKA and ATP (18), has no effect on the ⌬NEG2-CFTR channel (n ϭ 4). Moreover, addition of PKA up to 200 units/ml, four times the concentration required to fully activate wt-CFTR (18), does not increase the open probability of the ⌬NEG2-CFTR channel (Fig. 2). Although the conductance properties of ⌬NEG2-CFTR are similar to those of wild type CFTR (25), its open probability is much less and cannot be increased with PKA (wt-CFTR P o ϭ 0.254 Ϯ 0.024, n ϭ 11; ⌬NEG2-CFTR, P o ϭ 0.061 Ϯ 0.015 without PKA and 0.053 Ϯ 0.016, with PKA, n ϭ 8).
The failure of the ⌬NEG2-CFTR channel to respond to PKA does not result from inability of the channel to be phosphorylated, an in vitro assay using [␥-32 P]ATP shows comparable phosphorylation of wt-CFTR and ⌬NEG2-CFTR (Fig. 1B). Densitometry readings for this gel were: wt-CFTR, Band C, 1063 units, Band B, 818 units; ⌬NEG2-CFTR, Band C, 1000 units, Band B, 1145 units. However, since expression of ⌬NEG2-CFTR in vesicles was slightly less than wt-CFTR, on Western blot, values for phosphorylated protein normalized to the amount of CFTR present were slightly higher for the ⌬NEG2-CFTR. Densitometry units of radioactive phosphate per densitometry unit of ECL-labeled CFTR on Western blot for identical amounts of vesicles were as follows: for wt-CFTR, Band C, 1.3, Band B, 1.16; for ⌬NEG2-CFTR, Band C, 1.58, Band B, 2.38. In a second experiment, the values were: wt-CFTR, Band C, 1.15, Band B, 0.99; for ⌬NEG2-CFTR, Band C, 1.62, Band B, 2.08. Thus, ⌬NEG2-CFTR was at least as well phosphorylated as the wt-CFTR under these conditions. The apparent molecular weight of the phosphorylated ⌬NEG-2 CFTR protein is less than that of the wild type protein or the ⌬NEG1-CFTR (apparent M r 169,000 for wild type-CFTR Band C versus 160,000 for the ⌬NEG1-CFTR Band C versus M r 155,000 for the ⌬NEG2-CFTR Band C). The apparent reduction in molecular weight with deleting these negatively charged sequences is greater than can be accounted for by the mass of the amino acids alone. This difference probably results from the deletion of substantial negative charges along with the sequence, since disproportion of acidic amino acids, especially when concentrated in a short sequence, is well known as a cause of anomalously high molecular weight determinations in SDS-PAGE (26 -28).
The ⌬NEG2-CFTR channel still contains all 10 consensus  PKA phosphorylation sites. Riordan and co-workers (29,30) showed that even wt-CFTR with all 10 consensus PKA sites mutated increases its P o in response to PKA, presumably by phosphorylation of a weak consensus sequence at Ser 753 . Elimination of PKA responsiveness is therefore an unusual property of the ⌬NEG2-CFTR channel. Since the ⌬NEG2-CFTR channel exhibited reduced P o , it is possible that removal of the NEG2 sequence also alters the stimulatory function of the R domain on the CFTR channel. To test this possibility, the following experiments were performed.
Structural Properties of the NEG2 Sequence-NEG2 is predicted to have high ␣-helical content by several secondary structure prediction algorithms (23,24). Molecular modeling of the NEG2 sequence as an ␣-helix reveals a long negatively charged stripe circling the helix, paralleled by two hydrophobic patches (Fig. 3A). To distinguish the effect of bulk negative charge from that of a specific structural motif due to this putative helical conformation, several sequence variant peptides were designed with different predicted helical propensities (Table II). To retain the negative charge but disrupt the helix, we synthesized a scrambled NEG2 peptide (s-NEG2) containing the same amino acids but designed to disrupt the ␣-helical content; and in a second variant, we inserted a proline residue into the sequence to disrupt the helical structure (p-NEG2). To reduce the negative charge, but retain the helical structure, four aspartic acid residues were replaced with asparagines, and two glutamic acid residues were replaced with glutamines, as well as substituting serine for the native cysteine residue (Cys 832 ) to avoid disulfide cross-linking of the peptide and replacing the native methionine (Met 837 ) with norleucine to avoid oxidation. The resulting peptide was designated h-NEG2i for helical NEG2-inert. The h-NEG2i peptide has net negative charge of Ϫ3, but is predicted to have strong helical propensity (Table II). An attempt to replace all of the aspartic acid and glutamic acid residues with their cognate amides resulted in an insoluble peptide. In addition, we obtained an inert NEG2 variant, NEG2i, with the cysteine to serine and methionine to norleucine substitutions, to assure that the structural changes we observed in h-NEG2i were not the result of these substitutions.
Circular dichroism spectra obtained in aqueous solution of increasing concentrations of trifluoroethanol (TFE, 0 -66%) were used to assess the helical propensities of these peptides (Fig. 3, panels B-E). There was little or no helical tendency of the p-NEG2 or s-NEG2 peptides in the presence or absence of TFE as demonstrated by the lack of characteristic positive and negative ␣-helical bands at 193 and 222 nm, respectively (Fig.  3, panels F and G). Instead the large negative bands at 200 nm suggest predominantly random coil structures for these peptides (31). The NEG2 and NEG2i peptides demonstrate relatively little helicity in aqueous solution, however, with increasing TFE concentration, the helical content steadily increases as shown by the increase in the characteristic helical bands at 193 and 222 nm. Interestingly, h-NEG2i has clear helical tendency in the absence of TFE, which can be further enhanced by TFE addition (Fig. 3, F and G). Mean residue molar ellipticity plots at 193 and 222 nm for these peptides as a function of TFE concentration are given in Fig. 3, F and G, as well as estimates of percent helicity (32). These plots clearly indicate that h-NEG2i is significantly more helical than NEG2 or NEG2i and that s-NEG2 and p-NEG2 almost completely lack helical structures. The different helical propensities between NEG2 and s-NEG2 were also confirmed by two-dimensional NOESY proton NMR spectroscopy.
Effect of Exogenous NEG2 and Its Congeners on CFTR Channel Activity-To test whether the NEG2 region is responsible for both stimulatory and inhibitory interactions between the R domain and other domains of CFTR, the wild type NEG2 peptide was added to the cis-intracellular side of a single CFTR channel captured in the planar lipid bilayer (Fig. 4). The diary plot of P o as a function of time shows the activity of a single wt-CFTR channel during the course of the experiment (Fig. 4). After peptide addition (at arrows), there are periods of intense stimulation that last 4 -8 min, often followed by return to the basal level of activity observed before peptide addition. In the example shown, activity reverted to baseline levels 4 -8 min after addition of NEG2 peptide at concentrations 2.2 and 4.4 M, but at 8.8 M, stimulation persisted until a still higher concentration was added, at which time the channel closed. At higher concentrations, NEG2 produces an almost complete inhibition of the channel, where only a flickery 3 pS conductance is observed. During stimulation, the open probability more than doubled and more transitions were observed between the open and closed states (Fig. 4). In 10 of 12 experiments in which a peptide concentration Ն0.44 M was achieved, a stimulatory response was observed. For the two experiments for which it was not, one channel was inhibited upon initial peptide addition at a concentration of 4.4 M and no stimulation was observed, and another channel did not display stimulation. Statistically significant increases in channel open probability are documented for NEG2 concentrations of 4.4 and 8.8 M, and increased activity, with p ϭ 0.09, was observed at 2.2 M NEG2. Profound inhibition, comparable with that shown in the example, was observed in five channels exposed to NEG2 peptide at concentrations Ն4.  (33). Therefore, to better identify the closed times between bursts, a delimiter of t c ϭ 40 ms was set at the nadir between the fast and intermediate closed times (arrow) to generate the closed-burst duration histograms (C). The solid lines represent the fit according to a double exponential equation: y ϭ P 2 *exp[t-a 2 -exp(t-a 2 )] ϩ P 3 *exp[t-a 3exp(t-a 3 )], where a 2 ϭ log t c2 , a 3 ϭ log t c3 , P 2 ϭ probability of the intermediate closed component, and P 3 ϭ probability of the long closed component. The best fit parameters are P 2 ϭ 0.811, t c2 ϭ 459 ms, P 3 ϭ 0.189, t c3 ϭ 2494 ms (control); P 2 ϭ 0.957, t c2 ϭ 105 ms, P 3 ϭ 0.043, t c3 ϭ 1652 ms (peptide-stimulated). Changes in P o and t c2 of the wt-CFTR channel in paired experiments, before and after addition of 4.4 M wt-NEG2 peptide is shown in D. the NEG2 peptide was added to the wt-CFTR channel, we speculated that there might be competition from the endogenous NEG2 sequence for the binding sites, and the endogenous sequence might have the advantage of presentation and proximity. We therefore tested the ⌬NEG-2-CFTR channel for its ability to undergo stimulation or inhibition by the exogenous NEG2 peptide. The NEG2 peptide at concentrations 2.2 to 4.4 M significantly stimulates the ⌬NEG2-CFTR channel activity, but at higher concentrations, ranging from 2.2 to 13.2 M, the channel is markedly inhibited (Fig. 5). The NEG2-peptideinduced increase in ⌬NEG2-CFTR channel activity results almost entirely from an increase in channel openings, from 2.14 Ϯ 0.42 openings/s to 3.94 Ϯ 1.04 openings/s after application of 2.2 M NEG2, whereas the reduction of channel activity at higher NEG2 peptide concentrations is due to a combination of decrease in channel open lifetime and decrease in channel open events. Inhibition of channel activity occurred in seven of eight experiments in the absence of PKA, and in all three experiments performed in its presence.
To elucidate the mechanism responsible for the increase in P o at the lower peptide concentrations, the gating kinetics of wt-CFTR without peptide and during stimulation by NEG2 peptide were analyzed. The open time distributions of the wt-CFTR did not change during peptide stimulation, as both control (without NEG2 peptide) and peptide-stimulated channels had an open lifetime of ϳ120 ms (Fig. 6A). Thus, the increase in P o is not due to a change in the closing rate of the channel. However, the closed time distribution for the stimulated channel is clearly shifted to the left compared with the control channel (Fig. 6, B and C). There are three components to the closed state, a fast (t c1 ), an intermediate (t c2 ), and a long (t c3 ) closed component. Following peptide stimulation, the intermediate closed time was reduced significantly, from 459 to 105 ms (p Ͻ 0.005). The long closed time was also shortened, but because of the variability in the measurements (and their paucity in the stimulated condition), this did not achieve statistical significance. However, there were significantly fewer episodes in the long closed time category in the presence of the NEG2 peptide. Thus, the channel opening rate has increased in the presence of the NEG2 peptide, and this appears to account for the increase in P o of the channel (Fig. 6D). This mechanism is similar to what we, and others, observe for phosphorylated R domain protein (amino acids 588 -855 or 645-834) when it stimulates the wt-CFTR channel (18,19).
The s-NEG2 peptide has no stimulatory effect on the wt-CFTR channel, but exhibits inhibition on the wt-CFTR channel (Fig. 7A). In paired experiments, the wt-CFTR channel has an average P o of 0.266 Ϯ 0.034 (n ϭ 6) under control conditions of 2 mM ATP and 50 units/ml PKA in the intracellular solution.
With the addition of as little as 0.44 M s-NEG2 peptide, the P o was reduced by 70%, and with 2.2 M s-NEG2, the inhibition was 80% (Fig. 7A). The NEG2 peptide with a proline residue inserted in the sequence to disrupt the helical conformation, p-NEG2, also produced significant channel inhibition (60% at 11 M) (Fig. 7B). Inhibition of wt-CFTR has also been reported with the NEG1 peptide (which carries a total negative charge of Ϫ6) by other investigators (33). Thus, it is likely that charge interactions contribute to the inhibitory effects of the exogenous peptides on the CFTR channel.
To further dissect the stimulatory and inhibitory effects of the NEG2 sequence, we synthesized another peptide, h-NEG2i, in which negative charge was reduced from Ϫ9 to Ϫ3 (Table I). As shown in Fig. 3C, the h-NEG2i peptide exhibits higher propensity of ␣-helical structure in aqueous solution. At the single channel level, h-NEG2i gave persistent concentrationdependent stimulation of the wt-CFTR without inhibitory activity (Fig. 8).  Table II. DISCUSSION The R domain of CFTR contains two negatively charged regions, amino acids 725-733 (NEG1) and amino acids 817-838 (NEG2) which reside in close proximity to two PKA phosphorylation sites, Ser 737 and Ser 813 , that are used in vivo (9). One amino acid substitution, noted in a patient with CF, is reported in the CF Mutation Consortium data base in the NEG1 region (E725K). Three mutations are reported in the NEG2 region (E822K, E826K, and D836Y), two of which were obtained from patients with cystic fibrosis (E822K and D836Y). Single channel studies of E822K and E826K indicate that both mutations result in reduced P o compared with wt-CFTR (34). Moreover, Cotten and Welsh (35) showed that N-ethylmaleimide modification of a cysteine residue in the NEG2 region (C832) produced irreversible stimulation of PKA-phosphorylated CFTR channel activity. The NEG2 region is highly conserved among species. Taken together, these data indicate the importance of this portion of the R domain for the regulation of the CFTR channel. Our data demonstrate that the NEG2 region of CFTR can both stimulate and inhibit chloride channel function. When this region is deleted from CFTR, the resultant channel opens without PKA, indicating loss of inhibitory function, but the P o never achieves that of wt-CFTR, and does not increase when phosphorylated with PKA, indicating a loss of stimulatory function. In support of a dual action for the NEG2 sequence, addition of the NEG2 sequence as a synthetic peptide to the intracellular side of the CFTR channel results in stimulation of channel openings at lower concentrations, but inhibition of channel activity at higher concentrations.
Stimulatory and inhibitory activities can be separated by sequence modifications. Inhibition alone is evident with peptides designed to retain the negative charge but disrupt the helical tendencies. Conversely, stimulation occurs with a peptide designed to retain and enhance the helical structure of the sequence, but to reduce the negative charge. Molecular modeling reveals an amphipathic feature of the NEG2 sequence, i.e. the negatively charged residues mostly line up as a barber-pole stripe on the ␣-helix. The surrounding residues are mostly hydrophobic. We speculate that the NEG2 sequence, presented in different ways, could interact with CFTR at different sites to either stimulate or inhibit channel openings. Which function the NEG2 sequence performs might be determined by how it is presented in the context of the intact molecule, which could be entrained, at least in part, by the phosphorylation state of the R domain. In this model, phosphorylated R domain favors presentation of the hydrophobic stripe of the helical conformation of NEG2 to the stimulatory site, whereas the unphosphorylated R domain favors access of the negative charge in NEG2 to an inhibitory site. Since the mechanism of increasing channel activity appears to be, both for NEG2 and for h-NEG2i, mainly by increasing the number of channel openings, we speculate that the stimulatory activity might result from peptide binding increasing either binding or hydrolysis of ATP, probably at the first nucleotide-binding domain, which is often assigned the role of channel opening (36).
When the exogenous NEG2 peptide is added to the intracellular side of the wt-CFTR channel, it could interact with either the stimulatory site or the inhibitory site, and it may compete with the endogenous sequence for access to these sites. The degree and duration of stimulation or inhibition by exogenous peptide will depend on the on and off rates at the stimulatory site and the inhibitory site, the effective concentration of the relevant structural form of the peptide, and competition from endogenous sequences (either the NEG2 sequence itself, or sequences at other sites) at those two sites. When the channel is closed, as it is most of the time even in the phosphorylated state (since the P o is only about 30%), the endogenous site for inhibition may often be occupied by the endogenous NEG2 sequence, and thus binding of the exogenous peptide is favored at the stimulatory site. However, as stimulation increases, the inhibitory site may become more available, and eventually binding occurs at this site, and the channel is inhibited.
Naren and co-workers (37) reported that a sequence in the NH 2 terminus of CFTR interacts with sequences in the proximal end of the R domain to increase CFTR channel activity. This work, taken together with the work reported here, is consistent with our earlier studies of segments of the R domain which inhibit channel function (21). Two segments, one containing amino acids 588 -805 and the other, amino acids 672-855, proved inhibitory. The first segment may inhibit channel activity by interacting with the stimulatory NH 2 -terminal sequence in CFTR, preventing it from interacting with its target in the R domain, thereby preventing channel stimulation. The second inhibitory segment contains the NEG2 sequence, and probably inhibits channel openings by presenting NEG2 to its inhibitory site. It is intriguing that the binding portion of the NH 2 terminus, which stimulates channel activity, appears to be a helical segment of the protein with one negatively charged face, and its activity is abrogated by reducing the negative charges in this segment (38). This observation reinforces the concept that negatively charged sequences may exert profound regulatory influence on channel activity.
A recent study by Baldursson et al. (39) found that deletion of amino acids 760 -783 in the R domain resulted in CFTR channel activity in the absence of PKA phosphorylation; however, deletion of amino acids 784 to 835, which includes most of the NEG2 sequence, did not allow the channel to open without phosphorylation. It may be that the two deletion mutants delete the NEG2 sequence in whole or in part, ours (⌬NEG2(817-838)-CFTR) and that of Baldursson et al. (39) (⌬(784 -835)-CFTR), assume different conformations compared with the native CFTR molecule and thus may behave differently in channel function. Baldursson's (39) construct deletes 33 more amino acids (amino acids 783-816, including a putative PKCphosphorylation site (Ser 790 )) than our ⌬NEG2-CFTR construct. Since PKC phosphorylation has an essential role in PKA phosphorylation-dependent regulation of the CFTR channel (40), removal of this site may alter channel regulation. The potential role of PKC phosphorylation on the ⌬(784 -835)-CFTR and ⌬(817-838)-CFTR remains to be studied. It is also possible that the R domain contains more than one inhibitory site, or both identified segments of R domain sequences (760 -783 and 817-838) could act together to inhibit the unphosphorylated CFTR channel, and the lack of either one disrupts the inhibitory function of the R domain. King and Sorscher (41) also identify the COOH-terminal portion of the R domain (amino acids 723-836, which includes both the putative inhibitory sequences) as crucial in conferring PKA regulation on the channel.
Future studies to identify the sites of NEG2 interaction within the CFTR molecule should provide new insights into the regulation of the CFTR channel. Particularly, understanding the stimulatory interaction of NEG2 may facilitate the design of therapeutics to stimulate the CFTR opening to treat patients whose mutant forms of CFTR reach the cell surface.