INTRODUCTION

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CYSTIC FIBROSIS is a genetic disease most prominently characterized by the absence of a adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl conductance (i.e., the cystic fibrosis transmembrane conductance regulator; CFTR) in the apical membranes of ion-transporting epithelia (38). Disease-associated mutations in the gene encoding CFTR manifest themselves in one of four ways: lack of protein production, defective protein processing, defective regulation, or defective ion conduction (38). Some mutant proteins, including the most common disease-causing mutation (F508), are affected with both errant processing and regulation (5, 7, 11). However, it remains unclear whether altered channel gating behavior or ion conduction properties are related to the changes in protein processing that occur for some mutant forms of CFTR. Also, the relationship of channel gating to acute regulation of CFTR trafficking to or from the plasma membrane is unresolved. Electrophysiological techniques are available to evaluate the ion conductive capacity of mutant CFTR channels that reside in the plasma membrane. However, methods for evaluating protein processing and regulated trafficking and recycling at the cell surface have, until recently, relied on antibodies directed against intracellular CFTR domains and are thus unable to clearly resolve protein residing in the cell membrane from that in subcellular compartments (9, 18, 29). We recently reported development of a construct that, when expressed exogenously, contains the antigenic eight-amino acid FLAG epitope in the fourth extracellular loop of CFTR (M2-901/CFTR; see Ref. 15). We were able to detect plasma membrane expression of M2-901/CFTR with the commercially available M2 antibody. CFTR mutants previously reported to be fully glycosylated (as demonstrated by the presence of the C-band; see Ref. 7) during cellular processing were identified in the plasma membrane by the M2 antibody when doubly mutated to also include the FLAG epitope in the fourth extracellular loop. Furthermore, mutant forms of CFTR known to have defects in processing (F508, N1303K) were not localized in the plasma membrane. Thus constructs of wild-type (wt) and mutant CFTRs containing the FLAG epitope in this extracellular domain are useful in evaluating protein trafficking, processing, and cAMP-dependent membrane recycling of CFTR.

Our initial studies demonstrated that the expression of M2-901/CFTR conferred cAMP-sensitive anion permeability on mammalian cells as assessed by the halide-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Furthermore, injection of M2-901/CFTR cRNA into Xenopus oocytes produced a cAMP-sensitive Cl conductance. However, more detailed physiological and pharmacological evaluations, along with analysis of biophysical characteristics, are required to fully validate this protein construct as a useful tool for the variety of research approaches currently employed to understand the pathogenesis of cystic fibrosis. Therefore, we evaluated the channel characteristics of M2-901/CFTR in a variety of contexts to determine if the conduction or regulatory properties were altered by insertion of the FLAG epitope at this site. Our results indicate that M2-901/CFTR is biophysically identical to wtCFTR and thus is a useful tool to evaluate protein processing and trafficking.

	METHODS

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M2-901/CFTR Expression

Transient expression in HeLa cells. Transient expression of M2-901/CFTR in HeLa cells was carried out as previously described (15). Briefly, HeLa cells seeded on coverslips in 35-mm dishes were infected with vaccinia virus (vTF7-3, multiplicity of infection = 8) expressing the T7 polymerase. After adsorption, the cells were washed and transfected with 10 µg/dish of plasmid DNA (M2-901/CFTR subcloned into pTM1) using serum-free media and lipofectin (10 µg/ml; GIBCO-BRL, Grand Island, NY). Five hours posttransfection, the cells were placed in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). M2-901/CFTR channel activity was evaluated 1-5 h later.

Transient expression in HEK-293 cells. M2-901/CFTR cDNA was cloned into pCMV (Clontech, Palo Alto, CA) in place of the -galactosidase gene and thus the pCMV/M2-901/CFTR construct was obtained. HEK-293 cells were seeded on plastic coverslips coated with human placental collagen (collagen type VI; Sigma Chemical, St. Louis, MO) in 35-mm dishes at density of 40-50%. On the following day, cells were transfected with 5 µg/dish plasmid DNA (pCMV/M2-901/CFTR) by the calcium phosphate coprecipitation method as previously described (14). Cells were maintained in standard incubation conditions, and M2-901/CFTR channel activity was evaluated 2 days later.

Xenopus laevis oocyte expression. Oocyte isolation, in vitro cRNA transcription, and cRNA injection were performed as described previously with minor modifications (15, 34). Briefly, Xenopus laevis were obtained from Xenopus 1 (Ann Arbor, MI). After surgical isolation, oocytes were separated from follicular cells by incubation in nominally Ca²⁺-free Barth's solution, 10 mg/ml collagenase (GIBCO), and 1 mg/ml trypsin inhibitor (Sigma Chemical) on a low-speed rocker at room temperature for 60-90 min. The oocytes were rinsed five times and were incubated in K₂HPO₄ (100 mM; pH 6.5) with bovine serum albumin (0.1% wt/vol; Sigma Chemical) for 1 h followed by overnight recovery in modified Barth's solution at 18-20°C to remove follicular cells. Oocytes were injected with 2.5 ng of either wt or M2-901/CFTR cRNA in 50 nl of water or with water alone. Oocytes were maintained in modified Barth's solution at 18-20°C until current recordings were made 2-3 days later.

Stable expression in Madin-Darby canine kidney and C127 cells. The vector pMEP4 containing the cDNA for M2-901/CFTR subcloned behind the metallothionein promoter was used to stably transfect Madin-Darby canine kidney (MDCK) type II and C127 cells. Transfections were performed as previously described (13). Briefly, MDCK and C127 cells were grown in DMEM-F-12 supplemented with 10% FBS or DMEM supplemented with 10% FBS, respectively, for 24 h. The cells were trypsinized, and 2 × 10⁶ cells were suspended in 1 ml of media. Calcium phosphate-precipitated DNA (0.5 ml containing 15 µg) was added to cells in a 10-cm dish at room temperature and was incubated for 30 min. Medium containing 100 µg/ml chloroquine was added, and the cells were incubated at 37°C for 6 h. Cells were treated with 10% glycerol (wt/vol) for 3 min at room temperature, washed, and incubated in media at 37°C until reaching confluency. Cells were split 1:10 into 10-cm dishes and were incubated in media containing 900 µg/ml of hygromycin B. After 1 wk, cloning rings were placed over individual cells. Clonal cell lines were propagated, and M2-901/CFTR expression was induced 48-72 h before experiments using 50 µM ZnSO₄. Clonal cell lines were screened for cAMP-sensitive Cl conductance using the SPQ fluorescence assay as previously described (15). For electrophysiological evaluation, positive clones were seeded on glass coverslips, and M2-901/CFTR expression was induced with either zinc or 5 mM sodium butyrate. Channel activity was evaluated after 21 h of induction.

Voltage-Clamp Recording and Analysis

Data were acquired from excised membrane patches and were analyzed as described previously (25, 27, 37) with minor modifications. Before solution composition was changed at the cytosolic face of the membrane, CFTR channel activity was recorded for a control period in excess of 80 s to ensure steady-state channel activity. Unless otherwise noted, the 0.75-ml bath was refreshed at a rate of four bath volumes per minute and was maintained at 34-37°C throughout all experiments. The pipette solution contained (in mM) 140 NMDG-Cl, 1 CaCl₂, 2 MgCl₂, and 10 1,3-bis[tris(hydroxymethyl)-methylamino]propane-HCl (BTP), pH 7.35. The standard bathing solution contained (in mM) 150 NaCl, 2 MgCl₂, 10 NaF, 0.5 EGTA, 0.26 CaCl₂, and 10 BTP, pH 7.35. Free Ca²⁺ concentration was calculated to be 100 nM (6). F was included as a nonspecific inhibitor of phosphatases that might be active on patch excision and can lead to channel inactivation (31). We have previously reported that the inclusion of F in the cytosolic bath does not affect nucleotide dependence of CFTR channel gating kinetics (24, 27). Some experiments (anion substitution experiments; see below) were performed in the absence of F, and channel gating in control conditions (i.e., normal Cl with no F) was indistinguishable from experiments in which F was present. Anion permselectivity was determined by replacing Cl in the bathing solution with either or gluconate. When replaced Cl in the bath, the solution contained (in mM) 150 NaNO₃, 2 MgSO₄, 0.5 EGTA, 0.26 Ca(NO₃)₂, and 10 BTP, pH 7.35. The gluconate-containing bath included (in mM) 11 NaCl, 139 sodium gluconate, 2 MgCl₂, 0.5 EGTA, 0.26 CaCl₂, and 10 BTP, pH 7.35. ATP and ADP were made as stock solutions (200 mM) in 200 mM BTP, and the pH was adjusted to 7.2. Aliquots of each stock solution were frozen at 20°C until use. Fresh stock solutions (100 mM) of glibenclamide or LY-295501 were prepared in dimethyl sulfoxide on the day of the experiment.

Single-channel amplitude (i), mean current for the duration of an observation period (macroscopic current; I), and number of channels (N) present in the patch were determined as previously described (27), and these parameters were used to calculate channel open probability (P_o) from the relationship P_o = I/Ni. Only patches containing fewer than eight channels were subjected to statistical analysis for concentration-dependent effects on P_o (37). For determination of concentration-dependent changes in I (i.e., I in the presence of various concentrations of ATP expressed as a percent of I in the presence of 300 µM ATP; I/I₃₀₀), determination of P_o, and fluctuation analysis, recordings 85-170 s in length were analyzed for each control or experimental condition with Bio-Patch software (version 3.21; Molecular Kinetics, Pullman, WA) as previously described (27). User-defined equations for linear regression and a simple Michaelis-Menten function were fitted to the data sets for I-V relationships and concentration dependencies of P_o and I, respectively, using SigmaPlot (version 4.14; Jandel Scientific, San Rafael, CA). Values are presented as means and SE unless otherwise noted.

Chemical Sources

	RESULTS

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M2-901/CFTR-Mediated Conductance Is Stimulated by cAMP-Dependent Agonists

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Stimulation of whole cell Cl current and increased membrane capacitance by cAMP-mediated agonists in Xenopus oocytes injected with wild-type (wt) cystic fibrosis transmembrane conductor (CFTR) cRNA or with FLAG epitope inserted in fourth extracellular loop of CFTR cRNA (M2-901/CFTR). Oocytes were impaled, and currents were recorded using the double-electrode voltage-clamp technique. Resting membrane current at 60 mV was 27 ± 10 nA (n = 9) and 35 ± 13 nA (n = 7) in wtCFTR- and M2-901/CFTR-injected oocytes, respectively. CFTR-mediated changes in Cl currents were stimulated by exposing the oocytes to a cocktail containing 1 mM 3-isobutyl-1-methylxanthine (IBMX) and 10 µM forskolin. Maximal currents were 1,178 ± 108 and 1,147 ± 156 nA for wtCFTR- and M2-901/CFTR-injected oocytes, respectively. Membrane capacitance, an indicator of cell surface area, was determined as previously described (34) before and after stimulation. Effects of stimulation on M2-901/CFTR cRNA-injected oocytes were not different from those observed in wtCFTR cRNA-injected oocytes. No effects were observed in water-injected oocytes.

C127 cells stably expressing M2-901/CFTR had low Cl conductance in control conditions, as assessed by the whole cell patch-clamp technique (Fig. 2, A and C). Whole cell conductance was relatively small and showed slight outward rectification, and the currents reversed at potentials significantly negative (E_rev = 39 mV) relative to the expected reversal potential for Cl (E_Cl = 3 mV). Upon stimulation by forskolin (10 µM) and CPT-cAMP (300 µM), agents known to activate PKA, whole cell conductance increased sixfold and became linear, and the E_rev shifted toward E_Cl (E_rev = 20 mV; Fig. 2, B and C). After agonist washout, whole cell conductance returned to basal levels (n = 2; data not shown). Effects of stimulants were not observed in either nontransfected or mock-transfected cells. These results are as expected for the activation of CFTR-mediated Cl conductance by PKA.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Stimulation of whole cell Cl conductance by cAMP-dependent protein kinase (PKA)-mediated agonists in an M2-901/CFTR-transfected C127 cell. A: overlay of cell current records obtained from the current-voltage (I-V) pulse protocol (see METHODS) in basal conditions. B: overlay of cell current records obtained from the I-V pulse protocol in the same cell after the addition of forskolin (10 µM) and 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP; 300 µM) to the bathing solution. C: I-V relationships determined by averaging current values during the final 300 ms of each voltage pulse in A () and B (). Em, membrane potential. Lines represent the best fit of the data to a simple linear regression. Slope conductances were 2.7 and 16.6 mS and reversal potentials (Erev) were 39 and 20 mV for basal (A) and stimulated (B) conditions, respectively. See METHODS for complete description of conditions and protocol.

M2-901/CFTR Channel Activity Is Regulated by Phosphorylation and Nucleotides

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Phosphorylation and nucleotide-dependent regulation of channel gating in an M2-901/CFTR-transfected HeLa cell. Shown are excerpts from a continuous current record of an excised membrane patch containing >20 M2-901/CFTR channels. A: in the presence of 300 µM ATP, a maximum of two simultaneously open M2-901/CFTR channels was observed. PKA (500 units) was pipetted into the bath (arrow), resulting in the immediate activation of >20 M2-901/CFTR Cl channels (static bath). B: bath perfusion was initiated with a solution containing no ATP or PKA. M2-901/CFTR channels closed in a stepwise fashion over a 3-min period until a single channel remained "locked" open (perfused bath). C: perfusion solution was changed to include 300 µM ATP (arrow). Introduction of ATP resulted in the immediate reactivation of >12 M2-901/CFTR channels (perfused bath). D: ADP (200 µM final concentration) was pipetted into the bath (arrow) in the continued presence of 300 µM ATP and caused an immediate reduction in M2-901/CFTR channel open probability (Po; static bath). All other bath constituents were constant throughout the entire recording period. Data were acquired at a sampling rate of 2 kHz with an analog 8-pole Bessel filter (800-Hz cutoff). For clarity of presentation, data were plotted at 100 Hz.

Figure 4 shows the nucleotide concentration dependency of M2-901/CFTR gating behavior. Figure 4, A and D, provides excerpts from the continuous current record of an HEK-293 cell membrane patch containing at least 17 M2-901/CFTR Cl channels in the presence of 300 and 30 µM ATP, respectively. Figure 4, B and C, shows the amplitude histogram and power density spectra, respectively, for the current trace in Fig. 4A, whereas Fig. 4, E and F, provides parallel information for the current trace in Fig. 4D. The 10-fold reduction in ATP concentration reduced I to 35% of the control value (I/I₃₀₀ = 3.7/10.6 pA), whereas i was unchanged (0.91 ± 0.02 vs. 0.91 ± 0.04 pA). Fluctuation analysis of these data resulted in the construction of power density spectra that contained two Lorentzian components as previously described (27). The corner frequencies (f_c) of the lower-frequency component was reduced from 1.63 to 1.04 Hz when ATP concentration was reduced, indicating a decrease in the nucleotide-dependent opening rate of 3.7 s¹. The higher-frequency component accounted for <1% of the total power and was affected little by the change in nucleotide concentration (86 and 77 Hz at 300 and 30 µM ATP, respectively). These results are consistent with earlier observations that we reported for wtCFTR (37); decreased concentrations of ATP lead to a decrease in P_o, which is caused by a decrease in the opening rate of M2-901/CFTR.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Nucleotide-dependent gating behavior of M2-901/CFTR Cl channels in an excised membrane patch from an HEK-293 cell containing >17 channels. A and D: excerpts of a continuous current record in the presence of 300 and 30 µM ATP, as indicated. B and E: amplitude histograms constructed from 125 s of continuous current records in the presence of 300 and 30 µM ATP, respectively. Multi-Gaussian fits to the data revealed single-channel amplitudes (i) of 0.91 ± 0.02 and 0.91 ± 0.04 pA, respectively (not shown). C and F: power density spectra (PDS) constructed by averaging 30 nonoverlapping windows of 8,192 data points in the presence of 300 and 30 µM ATP, respectively. In each case, data were well fitted by 2 Lorentzian components. In the presence of 300 µM ATP (C) corner frequencies (fc) were 1.63 and 86 Hz with respective associated plateau powers of 1.0 × 1024 and 1.2 × 1027 A2 · s. In the presence of 30 µM ATP (F) fc values were 1.04 and 77 Hz with associated plateau powers of 1.1 × 1024 and 6.0 × 1028 A2 · s, respectively. Methods are as indicated in the text. Data were acquired at a sampling rate of 2 kHz with an analog 8-pole Bessel filter (800-Hz cutoff). For clarity of presentation, data in A and D were plotted at 100 Hz.

The comparison of fluctuation results derived from recordings of M2-901/CFTR and wtCFTR obtained in the same conditions reveals no difference in gating properties. Nucleotide-dependent gating frequencies of M2-901/CFTR (i.e., the sum of the nucleotide-dependent opening rate and the closing rate from this state; 2f_c) were similar to those that we previously reported for wtCFTR (Table 1). Furthermore, gating frequencies were consistently reduced by the addition of ADP (e.g., Fig. 3) as expected from our previous observations (27). Results presented in Figs. 3 and 4 and in Table 1 demonstrate that, at the single channel level, the regulation of M2-901/CFTR gating behavior is unchanged from that of wtCFTR.

Previously, we reported that wtCFTR opening rate and thus P_o were increased by ATP over the concentration range of 1 to 1,000 µM (K_s = 24 ± 8 µM; see Ref. 37) and that ADP was a competitive inhibitor of ATP-dependent channel opening (K_i = 16 ± 9 µM; see Ref. 27). In the present study, most membrane patches contained more than seven M2-901/CFTR channels (e.g., Figs. 3 and 4) such that P_o could not be reliably determined. However, data presented in Fig. 4 and in a previous report (24) show that patch current can be normalized to a single set of conditions and related to channel P_o (i.e., I/I₃₀₀). Data presented in Fig. 5 demonstrate that normalized current (I/I₃₀₀) carried by M2-901/CFTR in 14 excised membrane patches increases as a saturating function of ATP concentration. Although this is a limited data set with three or fewer experimental observations at four of the six concentrations evaluated, all observations and the derived results are consistent with the ATP dependency reported for wtCFTR (37). That the concentration dependence of relative current (I/I₃₀₀) is reflective of P_o is demonstrated by the virtually identical ATP dependence of P_o in a subset of these observations from patches containing fewer than eight channels. Observations of either normalized Cl current carried by M2-901/CFTR or P_o were well fitted by a simple Michaelis-Menten function for activation with K_s of 76 ± 10 and 66 ± 36 µM, respectively. These values are slightly larger than that previously reported for wtCFTR (37), but, because of the small sample size, are not different. Thus all aspects of nucleotide- and phosphorylation-dependent ion channel regulation of M2-901/CFTR are indistinguishable from that of wtCFTR.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   ATP dependence of normalized M2-901/CFTR current and Po in excised membrane patches. Data are derived from observations on 14 excised membrane patches in which mean current (I) was determined in the presence of 300 µM ATP and at least one other ATP concentration. Relative current levels (; left axis) are expressed as the fraction of current observed at 300 µM ATP. Curve on right indicates the best fit of a simple Michaelis-Menten function to these data. The derived Ks (76 ± 10 µM) indicates the concentration of ATP predicted to support one-half of the predicted maximal current (I/I300 = 1.27 ± 0.18). The number of observations comprising each data point and used in the construction of the fitted line are as indicated in line at the top. Po, determined as indicated in the text, from a subset of these patches (; right axis) are presented for comparison. Curve on left indicates the best fit of a simple Michaelis-Menten function to these observations. The derived Ks (66 ± 36 µM) indicates the concentration of ATP predicted to support a Po of one-half the maximum value (0.61 ± 0.08). The number of observations comprising each data point and used in the construction of the fitted line are as indicated on the second line at top.

M2-901/CFTR Conductance and Selectivity Are Identical to wtCFTR

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Voltage dependency of M2-901/CFTR single channel amplitude (i) in the presence of intracellular Cl, , or gluconate. In nominally symmetrical Cl concentrations (), linear regression to the data revealed a slope conductance of 11.4 pS over the voltage range indicated. Erev, 3.8 mV, was near the expected value of 2.4 mV. Substitution of for Cl in the cytoplasmic solution () resulted in a rightward shift of the I-V relationship. Linear regression to these data revealed an increased slope conductance of 13.4 pS. After correction for junction potentials, Erev was 18.0 mV. The 14.2-mV rightward shift in Erev is indicative of a permeability ratio (PNO3/PCl) = 1.66. When Cl was substituted by gluconate in the cytoplasmic solution (), virtually all inward currents were blocked, whereas outward currents were increased as expected for an increase in the electrochemical driving force for Cl. Data points represent means and SE as determined by multi-Gaussian functions fitted to the amplitude histograms. Error bars are presented only for those sets of observations in which the error was larger than the symbol. Complete ionic compositions of pipette (extracellular) and bath (intracellular) are as indicated in the text.

Sulfonylurea Sensitivity of M2-901/CFTR

View larger version (47K): [in this window] [in a new window]   Fig. 7.   Glibenclamide-induced reduction in M2-901/CFTR channel Po and associated changes in gating characteristics. Data in A and B are excerpts from a continuous current record and the associated amplitude histograms from an excised membrane patch containing 7 CFTR Cl channels held at 80 mV. A: control conditions [channel amplitude (i) = 1.07 pA and mean current (I) = 3.43 pA]. B: in the presence of 100 µM glibenclamide, i = 1.03 pA and I = 0.35 pA. C: power density spectrum (PDS) and associated multi-Lorentzian fits of current records from A (control) and B (glibenclamide). Each spectrum was fitted to two Lorentzian components. In control conditions, fc values were seen at 3.3 and 130 Hz. The associated plateau powers were 2.1 × 1025 and 4.2 × 1028 A2 · s, respectively. In the presence of glibenclamide, fc values were 2.8 and 34.5 Hz with associated plateau powers of 2.4 × 1026 and 4.2 × 1027 A2 · s, respectively. Effects of glibenclamide on Po and PDS reversed with washout (not shown). Current records were sampled at 2 kHz and filtered at 800 Hz for analysis and the construction of amplitude histograms. For clarity, current records were plotted at 100 Hz. Dashed line indicates the current level when all channels were closed. Other conditions were as stated in METHODS.

	DISCUSSION

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The data show that positioning of the eight-amino acid FLAG epitope in the M2-901/CFTR does not affect the regulatory properties, biophysical characteristics, or pharmacological modulation of CFTR channel activity. The implications from these studies are twofold. First, the M2-901/CFTR is able to tolerate the addition of this epitope, which increases its length by 20% and triples the number of charged residues, without altering the electrophysiological parameters associated with anion conduction or channel gating. Second, the demonstration that M2-901/CFTR functions normally validates the use of this tagged construct as a tool for further studies characterizing the protein associations, processing, trafficking, and other regulated functions of CFTR.

In the present study, we chose the best-understood, most completely characterized, and most widely accepted aspects of CFTR function as benchmarks to evaluate M2-901/CFTR. The aspects evaluated included physiologically relevant nucleotide and phosphorylation dependence, kinetic parameters, pore conductance and selectivity, and pharmacology. Additionally, we showed that CFTR-dependent changes in membrane capacitance, an indicator of regulated membrane insertion, were retained by this construct. Because the context of expression (i.e., expression vector, expression level, expression method, cell system) might affect channel function, we evaluated this protein's activities and regulatory properties in transient and stable mammalian and nonmammalian expression systems.

Results show that M2-901/CFTR is regulated physiologically by the same compounds previously shown to regulate wtCFTR by others and ourselves. Most notably, the kinase and nucleotide dependence of channel activity is identical to that reported by a variety of laboratories (3, 22, 31, 35) and is identical to our previous quantitative characterization of wtCFTR channel kinetics (27, 37).

CFTR is a selective anion permeation pathway, and the candidate amino acids comprising at least a portion of the pore and/or the selectivity filter have been identified in various putative membrane-spanning segments (1, 8, 19). Numerous anions have been shown to permeate the ion conductive pathway, with the permeabilites for halides falling in a relatively narrow range (1, 29, 35). Organic anions such as SCN (24, 33) or (32) have also been shown to readily permeate the channel, whereas glutamate and gluconate are virtually impermeant (17). Loops separating the transmembrane segments have also been shown to affect anion conductance. Indeed, reduction in the length of the second intracellular loop resulted in the stabilization of an alternative conductance state (39). Because the fourth extracellular loop is relatively short and the flanking membrane-spanning regions might comprise or contribute to the ion pore, one might expect that changes in the length or charge density of this loop would affect ion selectivity or conductance. Insertion of the FLAG epitope increases the length of this loop by 20%, increases the number of charged residues from 4 to 11, and changes the net charge from +2 to 1. However, our results showed that the extracellular positioning of the FLAG epitope did not alter either the conductance or selectivity of the ion pore for the three anions that were evaluated (Cl, , and gluconate) or the shape of the I-V relationship. Therefore, this extracellular portion of the protein likely resides away from critical structural components of the permeation pathway that determine conduction and selectivity. The accumulation or depletion of ions at that site due to surface charge effects does not influence permeation, presumably because this locus is not near the limiting pore diameter (i.e., selectivity filter). These observations are consistent with earlier reports suggesting that the selectivity filter is near the cytoplasmic end of the sixth transmembrane segment (8) and that the second membrane-spanning domain of CFTR is not required for anion permeation (20, 28).

Block of Cl conductance by sulfonylureas is widely employed as a diagnostic indication of CFTR participation in anion transport. Sheppard and Welsh (30) first reported that tolbutamide and glibenclamide reduced CFTR-mediated currents in whole cell membrane patches. Additional work has demonstrated that this inhibition is due to a direct interaction with the open state of CFTR (25, 36). Because glibenclamide potently interacts with the ATP-sensitive potassium channel (2) and has been shown to affect other Cl channels (23, 25), there has been some question regarding the diagnostic use of this compound. Therefore, we also employed a diarylsulfonylurea (LY-295501) that blocks wtCFTR but that does not affect the ATP-sensitive K⁺ channel (26). Our data indicate that the most potent direct inhibitors of wtCFTR currently available, glibenclamide and LY-295501, modulate the activity of M2-901/CFTR over the same concentration range and by an identical mechanism. It was previously shown that glibenclamide interacts exclusively with the open state of CFTR to transiently interrupt ion permeation (25). Fluctuation analysis indicates an identical mechanism of interaction for these compounds with M2-901/CFTR. Overall, these results strengthen the conclusion that the pharmacological profile of M2-901/CFTR is indistinguishable from that of wtCFTR.

Extensive work has been completed to identify, produce, and understand the effects of mutations throughout the CFTR gene. Although >700 putative disease-associated mutations and numerous polymorphisms have been identified, to date, only splicing and truncation mutations have been reported for the fourth extracellular loop, which includes amino acids Leu-881 through Ser-911 (10). This loop sequence includes two N-linked glycosylation sites that have been simultaneously disrupted without an apparent effect on permeation characteristics (15, 21). The location selected for the FLAG epitope may result in a hemiglycosylated form of the protein because it interrupts the consensus Asn-Xoa-Ser/Thr sequence that encodes N-linked glycosylation at position Asn-900. However, biophysical properties associated with ion conduction remained intact. Because maximum currents obtained with whole cell recordings in oocytes or M2-901/CFTR-expressing cells did not differ from those of wtCFTR, this suggests that either Asn-900 is glycosylated or that a hemiglycosylated protein has similar residency in the plasma membrane. Previous studies have indicated that a deglycosylated CFTR (N894/900Q) yields 25-50% of the maximal current associated with stimulation of wtCFTR (15). Taken together, these observations suggest that a fully deglycosylated protein results in a decreased number of channels expressed, whereas a protein retaining a single glycosylation site at position Asn-894 is sufficient for normal membrane residency time.

The FLAG epitope has been successfully employed to monitor protein activity, association, and trafficking in a variety of systems. Perhaps most important to note is that positioning the FLAG sequence in extracellular loops of the epithelial sodium channel subunits did not interrupt trafficking to the cell membrane or ion permeation (12). Likewise, when the substance P receptor was similarly tagged in an extracellular loop, internalization by endothelial cells, and thus membrane recycling, was not apparently affected (4). Positioning of the FLAG epitope cytoplasmically in other proteins allowed for monitoring of protein subunit association, expression, and processing without affecting protein function (either receptor binding or second messenger production), recycling, or the expression of endogenous proteins. To this list we now add that an epithelial anion channel, CFTR, can be successfully tagged without loss of biological function.

Previous studies have demonstrated that positioning the FLAG epitope in the M2-901/CFTR allows for observations of membrane expression of a variety of clinically relevant mutant CFTR constructs (15, 16). Because the electrophysiology and pharmacology of M2-901/CFTR are indistinguishable from endogenously or exogenously expressed wtCFTR and because of the unique extracellular location of its FLAG epitope, M2-901/CFTR will be a tool for the ongoing study of protein associations, trafficking, and functions of CFTR.