©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Intracellular Loop between Transmembrane Segments IV and V of Cystic Fibrosis Transmembrane Conductance Regulator Is Involved in Regulation of Chloride Channel Conductance State (*)

(Received for publication, August 1, 1995; and in revised form, September 21, 1995)

Junxia Xie (1) Mitchell L. Drumm (2) Jianjie Ma (1) Pamela B. Davis (1) (2)(§)

From the  (1)Departments of Physiology and Biophysics and (2)Pediatrics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cystic fibrosis transmembrane conductance regulator (CFTR) contains two membrane-spanning domains; each consists of six transmembrane segments joined by three extracellular and two intracellular loops of different length. To examine the role of intracellular loops in CFTR channel function, we studied a deletion mutant of CFTR (Delta19 CFTR) in which 19 amino acids were removed from the intracellular loop joining transmembrane segments IV and V. This mutant protein was expressed in a human embryonic kidney cell line (293 HEK). Fully mature glycosylated CFTR (170 kDa) was immunoprecipitated from cells transfected with wild-type CFTR cDNA, while cells transfected with the mutant gene expressed only a core-glycosylated form (140 kDa). The chloride efflux rate (measured by 6-methoxyl-N-(3-sulfopropyl) quinolinium SPQ fluorescence) from cells expressing wild-type CFTR increased 600% in response to forskolin. In contrast, Delta19 CFTR-expressing cells had no significant response to forskolin. Western blotting performed on subcellular membrane fractions showed that Delta19 CFTR was located in the same fractions as DeltaF508 CFTR, a processing mutant of CFTR. These results suggest that Delta19 CFTR is located in the intracellular membranes, without reaching the cell surface. Upon reconstitution into lipid bilayer membranes, Delta19 CFTR formed a functional Cl channel with gating properties nearly identical to those of the wild-type CFTR channel. However, Delta19 CFTR channels exhibited frequent transitions to a 6-picosiemens subconductance state, whereas wild-type CFTR channels rarely exist in this subconductance state. These data suggest that the intracellular loop is involved in stabilizing the full conductance state of the CFTR Cl channel.


INTRODUCTION

The cystic fibrosis transmembrane conductance regulator (CFTR) (^1)consists of five distinct regions, with two putative membrane-spanning domains, two nucleotide-binding folds, and a regulatory domain(1) . CFTR forms a Cl channel of linear conductance(2, 3) , which is regulated by cAMP-dependent protein kinase phosphorylation (2, 4, 5, 6) at multiple sites in the regulatory domain and by binding and hydrolysis of ATP by the nucleotide-binding folds (7, 8, 9, 10) . The structure and function of these five domains of CFTR have been extensively studied(11, 12, 13, 14) . In contrast, little is known about the role of intracellular loops and their contribution to the function of the CFTR Cl channel.

The intracellular loops of other channel proteins appear to participate in channel function. For instance, mutations in the second intracellular loop of the Shaker K channel affect channel inactivation(15) . Deletion of a portion of the putative cytosolic loops between two transmembrane repeats of the Na channel slows the rate of channel inactivation(16) . To investigate the role of the intracellular loops of CFTR in Cl channel function, we studied a deletion mutant of CFTR (Delta19 CFTR) in which 19 amino acids were removed from the intracellular loop joining transmembrane segments IV and V in the first membrane-spanning domain. This is the largest and most hydrophilic loop in the first membrane-spanning domain, as one-third of the residues in this loop are charged. These features make this loop a candidate for electrostatic or allosteric interactions with other cytosolic domains of CFTR (nucleotide-binding folds, the regulatory domain, or other intracellular loops) and cellular proteins to contribute to channel function.


EXPERIMENTAL PROCEDURES

Subcloning of CFTR Gene

The wild-type and DeltaF508 CFTR cDNAs (17) were subcloned into an Epstein-Barr virus-based episomal eukaryotic expression vector, pCEP4 (Invitrogen, San Diego, CA), between the NheI and XhoI restriction sites. The mutant gene, which lacks 57 nucleotides between nucleotides 930 and 987, was shuttled from pBluescript into pCEP4 by substituting the corresponding fragment in pCEP4(WT) (where WT is wild type) with the mutant one between the KpnI and AflII restriction sites. The mutant clone (pCEP4(Delta19)) was confirmed by restriction enzyme digestion and DNA sequencing of the shuttled fragment from nucleotides 874 to 1040.

Cell Culture

A human embryonic kidney cell line (293-EBNA, Invitrogen) was used for transfection and expression of wild-type and mutant CFTR proteins. This cell line contains a pCMV-EBNA vector, which constitutively expresses the Epstein-Barr virus EBNA-1 gene product and increases the transfection efficiency of Epstein-Barr virus-based vectors. The cell line exhibited high transfection efficiency (up to 80%) with Lipofectin reagent (Life Technologies, Inc.) following the manufacturer's instructions. The cells were maintained in Dulbecco's modified Eagle's medium (Biofluids, Inc., Rockville, MD) containing 10% fetal bovine serum (Life Technologies, Inc.) and 1% glutamine. Geneticin (G418 sulfate, 250 µg/ml; Life Technologies, Inc.) was added to cell culture medium for the continuous selection of cells containing the pCMV-EBNA vector. The parent cell line was grown to confluence in a 37 °C incubator with 5% CO(2) and passed 1:5 2 days before the gene transfer. pCEP4(WT), pCEP4(DeltaF508), or pCEP4(Delta19) was then introduced into the cells, using Lipofectin reagent. Two days after transfection, the cells were passed and selected for hygromycin resistance in medium containing hygromycin B (Boehringer, Mannheim, Germany) at 260 µg/ml. After 3 weeks in selection, the cells were used for immunoprecipitation and Western blot assay, SPQ assay, isolation of microsomal vesicles, and reconstitution studies in lipid bilayers.

Immunoprecipitation/Western Blot Assay of CFTR

293 HEK cells transfected with pCEP4(WT), pCEP4(/delta]F508), or pCEP4(Delta19) were cultured to confluence in 162-cm^2 flasks (Costar, Cambridge, MA). Cells were washed three times with ice-cold phosphate-buffered saline (137 mM NaCl, 10 mM Na(2)HPO(4), 2.7 mM KCl, 1.8 mM KH(2)PO(4), 5 µM diisopropyl fluorophosphate, pH 7.4), lysed with 1 ml of ice-cold radioimmune precipitation assay solution (150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) in the presence of protease inhibitors (10 µg/ml aprotinin, 10 µg/ml pepstatin A, and 5 µM diisopropyl fluorophosphate), and spun at 48,000 times g for 1 h at 4 °C. A mouse anti-human CFTR monoclonal antibody that is specific for the regulatory domain of CFTR (monoclonal antibody 13-1, 2 µg; Genzyme Corp., Cambridge, MA) was added to 350 µl of the above cell lysis supernatant solution and incubated on ice for 90 min. Antibody complexes were then precipitated with 20 µl of protein G-agarose beads (Boehringer) by incubation at 4 °C for 30 min on a rocker. The beads were washed with radioimmune precipitation assay solution three times, and the bound proteins were solubilized with 20 µl of gel sample buffer (200 mM Tris-Cl, pH 6.7, 9% SDS, 6% beta-mercaptoethanol, 15% glycerol, 0.01% bromphenol blue) and loaded onto a 5% SDS-polyacrylamide gel. The proteins were then transferred to a polyvinylidene difluoride membrane (Bio-Rad) and probed with monoclonal antibody 13-1 (0.8 µg/ml). A secondary peroxidase-conjugated affinity-purified goat antibody to mouse IgG (Organon Teknika Corp., West Chester, PA) was added to allow visualization of the CFTR-antibody complex. The proteins were detected by chemiluminescence according to the manufacturer's recommendations (ECL kit, Amersham Corp.).

SPQ Assay of Cl Transport

Cells were transferred to glass coverslips, cultured for 2-3 days, and then loaded with SPQ fluorescent dye by hypotonic loading as described previously(18) . Briefly, cells were incubated at room temperature for 4 min with a 1:1 mixture of Cl buffer (126 mM NaCl, 5 mM KCl, 1.5 mM CaCl(2), 1.0 mM MgCl(2), 20 mM Hepes, 0.1% bovine serum albumin, 0.1% D-glucose, pH 7.2) and distilled H(2)O containing 5 mM SPQ. The glass coverslip was mounted in a chamber on a stage heated to 37 °C, which was arranged for continuous flow of warmed buffer solutions and studied one-by-one using an upright Zeiss epifluorescence microscope. Fluorescence was excited at 355 nm (Omega Optical Inc., Brattleboro, VT) by a 75-watt xenon lamp. Excitation light was reflected by a 400-nm fused silica dichroic mirror (Omega Optical Inc.) and illuminated the cells through a 20times objective (numerical aperature of 0.75, working distance of 0.66 mm; Nikon Inc., Garden City, NY). Emitted light was filtered by a 450-nm lens and detected by a microchannel plate image intensifier (Model KS-1381) in series with a CCD-200 solid-state video camera (Video Scope International, Ltd., Washington, D. C.). The images were quantified with a Model MVP-AT frame grabber board (Matrox, Quebec, Canada) mounted internally in a host 386 computer and processed with Image-1 Fl software (Universal Imaging Corp., West Chester, PA). Every image taken (0.26 s) represents the average of 16 images. Successive images were recorded at a rate of 1 every 5 s. The ``blank field'' obtained by closing the camera shutter was used as background image and subtracted prior to acquisition of the images. SPQ fluorescence was calibrated by measuring the fluorescence in the absence of chloride (maximum fluorescence) and in the presence of a solution that quenched all the SPQ fluorescence (150 mM KSCN, 10 µM valinomycin). Photobleaching was corrected by subtracting a straight line that connects the two points at the beginning of the experiment and before KSCN + valinomycin application. Results are expressed as rates (relative fluorescence units/minute). The rates were calculated by fitting a single exponential equation to the initial portion of each single curve after correction for photobleaching. Cells that exhibited >15% photobleaching or dye leakage were not used for analysis.

Subcellular Fractionation of Membrane Vesicles

The protocol used was a modified version of that of Gunderson and Kopito (19) (see Fig. 5A). Briefly, 12 75-cm^2 flasks of 293 HEK cells transfected with the pCEP4(WT), pCEP4(DeltaF508), or pCEP4(Delta19) vector were harvested by scraping from the flask bottom following three washes with ice-cold phosphate-buffered saline. The cell pellet (600 times g, 5 min, 4 °C) was then resuspended by incubation for 10 min in ice-cold hypotonic lysis buffer (10 mM Hepes, pH 7.2, 1 mM EDTA, 5 µM diisopropyl fluorophosphate, 10 µg/ml pepstatin A, 10 µg/ml aprotinin, 9.6 mg/ml benzamidine) prior to lysis by 10 strokes in tight-fitting Dounce homogenizer, followed by 15 strokes after addition of an equal volume of sucrose buffer (500 mM sucrose, 1 mM EDTA, 10 mM Hepes, pH 7.2). Microsomes were collected by centrifugation of the post-mitochondrial supernatant (6000 times g, 20 min, 4 °C) at 100,000 times g for 45 min and resuspended with either 600 µl of isotonic buffer (250 mM sucrose, 1 mM EDTA, 10 mM Hepes, pH 7.2) or prephosphorylation buffer (250 mM sucrose, 10 mM Hepes, pH 7.2, 5 mM ATP, 5 mM MgCl(2), 100 units/ml cAMP-dependent protein kinase catalytic subunit). Further separation of plasma and intracellular membranes was carried out in a discontinuous sucrose gradient (10,000 times g, 6 h, 4 °C) in a swinging bucket rotor (SW 28). Each fraction (I-V) was collected at the interface of the two sucrose gradients. Subsequently, an abbreviated fractionation scheme was used that separated DeltaF508 CFTR from the wild type and ``intracellular membrane'' from ``plasma membrane.'' In this experiment, cells were washed and lysed in buffer as before, except that diisopropyl fluorophosphate was not included. The post-mitochondrial supernatant was spun at 12,000 times g for 30 min to give intracellular membrane (Fig. 5C, IM), and the supernatant was spun at 100,000 times g for 45 min to give plasma membrane (PM).


Figure 5: Subcellular fractionation and Western blotting of CFTR. A, a discontinuous sucrose gradient with five densities (28, 33, 36, 38.7, and 43.7%) was used to separate the plasma membrane from intracellular membranes. B, shown is the top of the gradient (lane I), 28/33% (lane II), 33/36% (lane III), 36/38.7% (lane IV), 38/43.7% (lane V), and pellet (lane VI). 50 µg of total protein from each fraction were subjected to 5% SDS-polyacrylamide gel electrophoresis; the CFTR protein was detected as described under ``Experimental Procedures.'' Protein markers are labeled in the middle. C, plasma (PM) and intracellular membrane (IM) fractions of DeltaF508 CFTR- and Delta19 CFTR-expressing cells grown at 37 °C were run on a 5% SDS-polyacrylamide gel and detected as described for B. Protein markers are labeled on the left. Arrowhead a, wild-type (WT) CFTR protein (170 kDa); arrowhead b, DeltaF508 CFTR protein (140 kDa); arrowhead c, Delta19 CFTR protein (140 kDa).



Detection of CFTR by Western Blotting

The membrane fractions collected from the above procedure were washed and resuspended in isotonic buffer. 50 µg of protein from each fraction were denatured with 6 times gel sample buffer (300 mM Tris-Cl, pH 6.8, 600 mM dithiothreitol, 12% SDS, 0.6% bromphenol blue) and loaded onto a 5% SDS-polyacrylamide gel. The proteins were then transferred to a polyvinylidene difluoride membrane and detected as described under ``Immunoprecipitation/Western Blot Assay of CFTR.''

Reconstitution of CFTR Cl Channel

Lipid bilayer membranes were formed across an aperture of 200-µm diameter with a lipid mixture of phosphatidylethanolamine/phosphatidylserine/cholesterol at a ratio of 6:6:1; the lipids are dissolved in decane at a concentration of 40 mg/ml(20) . The recording solutions contained the following: cis-side solution (intracellular), 200 mM KCl, 2 mM MgATP, and 10 mM Hepes/Tris, pH 7.4; and trans-side solution (extracellular), 50 mM KCl, 10 mM Hepes/Tris, pH 7.4. Microsomal vesicles (3-6 µl) containing the wild-type or Delta19 CFTR protein were added to the cis-side solution. 50 units/ml cAMP-dependent protein kinase catalytic subunit (Promega, Madison, WI) was always present in the cis-side solution. Single channel currents were recorded with an Axopatch 200A patch clamp unit (Axon Instruments, Inc., Foster City, CA). Data acquisition and pulse generation were performed with a 486 computer and a 1200 Digidata A/D-D/A convertor (Axon Instruments, Inc.). The currents were sampled at 1-2.5 ms/point and filtered at 100 Hz. Single channel data analyses were performed with pClamp software (Axon Instruments, Inc.).


RESULTS

Subcloning of CFTR Mutant Gene into Eukaryotic Expression Vector

The CFTR mutant studied in this paper was a spontaneous deletion mutation in the CFTR cDNA in which 57 nucleotides corresponding to 19 amino acids (MIENIQSVKAYCWEEAMEK, residues 266-284) were deleted. These residues are located in the intracellular loop joining transmembrane segments IV and V in the first membrane-spanning domain (Fig. 1). The mutant gene was shuttled from pBluescript into pCEP4 for eukaryotic cell expression. The pCEP4 vector contains the cytomegalovirus promoter, which drives the transcription of the cloned gene.


Figure 1: Predicted topology of CFTR. The predicted membrane topology of CFTR is shown, with the deleted 19 amino acids indicated. L, loop; TM, transmembrane segment; NBF, nucleotide-binding fold; R, regulatory.



Expression of Wild-type and Mutant CFTR Proteins

The CFTR protein was expressed in the eukaryotic cells by transfecting pCEP4(WT), pCEP4(DeltaF508), or pCEP4(Delta19) into 293 HEK cells using the Lipofectin reagent(21) . To confirm CFTR expression, the immunoprecipitation/Western blot assay was performed on the transfected cells. Cells transfected with pCEP4(WT) expressed a readily detectable amount of a fully glycosylated form of the CFTR protein with a molecular mass of 170 kDa, while the untransfected cells did not (Fig. 2). Cells transfected with either pCEP4(DeltaF508) or pCEP4(Delta19) showed a dominant band at 140 kDa (Fig. 2), which probably corresponds to the core-glycosylated form of CFTR. Consistent with other studies(22, 23) , DeltaF508 CFTR, which is incompletely glycosylated at 37 °C, displayed increased glycosylation when the culture temperature was lowered to 26 °C (Fig. 2, lane 5). However, incubation of pCEP4(Delta19)-transfected cells at 26 °C for 2 days did not increase the amount of Delta19 CFTR protein in the fully glycosylated form (Fig. 2, lane 7). The amount of 140-kDa protein associated with both DeltaF508 CFTR and Delta19 CFTR increased significantly following the lower temperature incubation (Fig. 2, lanes 5 and 7), probably due to the slower degradation of the abnormal protein in the intracellular organelles.


Figure 2: Immunoprecipitation and Western blotting of CFTR. 293 HEK cells transfected with pCEP4(WT), pCEP4(DeltaF508), or pCEP4(Delta19) were immunoprecipitated and blotted as described under ``Experimental Procedures.'' A similar number of cells were lysed for each cell line. The proteins were visualized by exposing the blot to a Kodak X-Omat AR film for 3 min. Lane 1, untransfected 293 HEK cells; lanes 2 and 3, wild-type CFTR-expressing cells; lanes 4 and 5, DeltaF508 CFTR-expressing cells; lanes 6 and 7, Delta19 CFTR-expressing cells. Lanes labeled 26 °C contained cells cultured to 8090% confluence at 37 °C (5% CO(2)), followed by incubation at 26 °C (6% CO(2)) for 2 days.



SPQ Assay of Cl Transport

The immunoprecipitation/Western blot assay detected no fully glycosylated Delta19 CFTR protein. However, other studies have shown that some CFTR mutants produce the fully glycosylated form in quantities too small to be detected by the above assay, but can nevertheless result in functional channels at the cell surface(23, 24) . To test the surface function of Delta19 CFTR, the SPQ assay was performed on pCEP4(Delta19)-transfected cells using pCEP4(WT)-transfected cells as a positive control and untransfected cells as a negative control. Representative traces of chloride movement for five to six cells of each cell line in the absence and presence of 10 µM forskolin are depicted in Fig. 3. A decrease in SPQ fluorescence indicates Cl influx, and an increase in fluorescence indicates Cl efflux. Forskolin failed to elicit an increase in the rate of Cl efflux in untransfected 293 HEK cells (Fig. 3A). Wild-type CFTR-transfected cells, on the other hand, exhibited a significant increase in the rate of Cl efflux upon forskolin stimulation (Fig. 3B). In cells transfected with Delta19 CFTR, stimulation of the rate of Cl efflux by forskolin was not statistically different from that of untransfected cells (Fig. 3C). Data from multiple experiments are summarized in Fig. 4. Incubation of the Delta19 CFTR-expressing cells at 26 °C for 15-48 h did not increase the rate of Cl efflux following forskolin stimulation (Fig. 4). This result is consistent with the immunoprecipitation/Western blot studies (Fig. 2), which indicate that little or no Delta19 CFTR reaches the Golgi apparatus for final glycosylation and presumably does not transport to the plasma membrane.


Figure 3: SPQ assay of Cl transport. Cells loaded with SPQ were preincubated with nitrate solution (126 mM NaNO(3), 5 mM KNO(3), 1.5 mM Ca(NO(3))(2), 1.0 mM Mg(NO(3))(2), 20 mM Hepes, 0.1% bovine serine albumin, 0.1% D-glucose, pH 7.2) on ice for 15 min to allow the depletion of intracellular chloride. At the beginning of the experiment, the cells were first perfused with nitrate solution until the fluorescence was stable (1 min). Perfusion solution was then switched to Cl-containing solution. SPQ fluorescence was quenched as Cl entered the cell. When fluorescence reached its steady state, perfusion was switched to nitrate solution until the fluorescence came back to its base line. This cycle was repeated with 10 µM forskolin (FSK) added to the Cl and nitrate solution. At the conclusion of the experiment, cells were perfused with KSCN (150 mM) plus valinomycin (VAL; 10 µM) solution to quench all the fluorescence. Each trace represents the fluorescence of a single cell from the beginning to the end. A, untransfected 293 HEK cells; B, wild-type (WT) CFTR-transfected cells; C, Delta19 CFTR-transfected cells. Fluorescence of five to six cells was plotted for each of the above three cases.




Figure 4: Forskolin stimulation of the rate of Cl efflux. The Cl efflux rate was calculated as described under ``Experimental Procedures'' and is plotted as the relative fluorescence units/minute (A). Open bars indicate the rate of basal Cl efflux; hatched bars indicate the rate of Cl efflux upon forskolin (FSK; 10 µM) stimulation. The percentage of forskolin-stimulated Cl efflux over basal Cl efflux is also plotted (B). The untransfected cells (Control) have a forskolin-stimulated efflux rate of 157 ± 7% of the base line (198 cells at 37 °C, n = 7). The wild-type (WT) CFTR-transfected cells have a forskolin-stimulated efflux rate of 618 ± 37% of the base line (297 cells at 37 °C, n = 8). The Delta19 CFTR-expressing cells have a forskolin-stimulated efflux rate of 178 ± 15% of the base line (170 cells at 37 °C, n = 10), which is not statistically different from the control. Data labeled with 26 °C (wild-type CFTR: 453 ± 144% forskolin stimulation, 40 cells, n = 3; Delta19 CFTR: 90 ± 10% forskolin stimulation, 65 cells, n = 4) were obtained from cells incubated at 26 °C for 15-48 h before the experiment.



Localization of Delta19 CFTR Protein

Subcellular membrane fractionation was performed with 293 HEK cells transfected with CFTR cDNA to localize the Delta19 CFTR protein. Intracellular membranes were separated from the plasma membrane using a discontinuous sucrose density gradient (Fig. 5A). Wild-type CFTR was used as a marker for the plasma membrane, and DeltaF508 CFTR, a known CFTR processing mutant, as a marker for the intracellular membranes. Western blotting showed that the wild-type CFTR protein (170 kDa) was detected mainly in fractions II and III, while the DeltaF508 CFTR protein (140 kDa) was detected mainly in fraction IV of the sucrose density gradient (Fig. 5B). Intracellular membrane fractions isolated from Delta19 CFTR-expressing cells contained a 140-kDa CFTR protein, which colocalized with the DeltaF508 CFTR protein (Fig. 5C). Together with the immunoprecipitation/Western blot and SPQ assays, these data suggest that Delta19 CFTR is a processing mutant.

Functional Characterization of Wild-type and Delta19 CFTR Cl Channels

To further study the function of Delta19 CFTR, microsomal vesicles, which contain mixtures of plasma membrane, endoplasmic reticulum, and Golgi membranes, were incorporated into planar lipid bilayers. Its function was compared with that of wild-type CFTR, incorporated into planar lipid bilayers from plasma membrane vesicles. Consistent with previous studies(19) , wild-type CFTR showed a cAMP-dependent protein kinase phosphorylation-dependent Cl channel with linear conductance of 8.2 ± 0.6 pS (Fig. 6). The extrapolated reversal potential was +22.2 ± 1.4 mV in a KCl gradient of 200 mM (cis) and 50 mM (trans), consistent with a chloride-selective channel. Without cAMP-dependent protein kinase, this 8-pS Cl channel was never observed in vesicles isolated from 293 HEK cells with (n > 30) or without (n > 20) transfection with CFTR cDNA.


Figure 6: Reconstitution of CFTR Cl channel in lipid bilayer membranes. Represented traces are selected single channel currents at the given test potential from a wild-type CFTR channel (A) and a Delta19 CFTR channel (B). Both wild-type and Delta19 CFTR channels exhibited a linear current-voltage relationship with a slope conductance of 8.2 ± 0.6 pS and an extrapolated reversal potential of +22.2 ± 1.4 mV. , wild-type CFTR (n = 76); circle, Delta19 CFTR (n = 14).



The reconstituted CFTR channels had slow kinetics of gating. The open lifetime histogram contained at least two exponentials, with mean open lifetimes of = 23.6 ms and = 111.9 ms (Fig. 7A). The relative occurrence of was y/(y + y) = 0.38. An average open probability (p(O)) of 0.318 ± 0.028 was measured at -80 mV. In separate studies, we found that the wild-type CFTR channel contained two distinct subconductance states with conductances of 6 pS (O2) and 2.7 pS, in addition to the full conductance state (8 pS, O1). (^2)The occurrence of O2, however, was low for wild-type CFTR and accounted for <10% of the open events (see Fig. 10A).


Figure 7: Open time histograms at -80 mV. Open events were calculated at -80 mV with wild-type (WT; A) and Delta19 (B) CFTR channels. The histograms were constructed with a total of 4040 open events obtained in three separate experiments for wild-type CFTR and of 4843 open events obtained in four separate experiments for Delta19 CFTR. The solid lines represent the best fit according to the following equation: y = y(1)/ exp(-t/) + y(2)/ exp(-t/), where = 23.6 ms, = 111.9 ms, and y(1)/(y(1) + y(2)) = 0.38 (A) and = 30.6 ms, = 115.8 ms, and y(1)/(y(1) + y(2)) = 0.48 (B).




Figure 10: Open current histogram at -80 mV. The current amplitudes of the individual open events were calculated with the 50% threshold detection method using the pClamp software. The currents were sorted at a resolution of 0.0025 pA/bin. The histogram in A contained three separate experiments with the wild-type (WT) CFTR channel, and that in B contained four separate experiments with the Delta19 CFTR channel. The solid lines represent the best fit with the sum of two gaussian distribution functions: y = W(1)/(&cjs3484;(2)(1)) exp(-(x - µ(1))^2/(1)^2) + W(2)/(&cjs3484;(2)(2)) exp(-(x - µ(2))^2/(2)^2), where W(1) = 5.04, µ(1) = -0.63 pA, (1) = 0.07 pA, W(2) = 0.49, µ(2) = -0.45 pA, and (2) = 0.06 pA (A) and W(1) = 8.32, µ(1) = -0.66 pA, (1) = 0.06 pA, W(2) = 1.97, µ(2) = -0.47 pA, and (2) = 0.07 pA (B).



When Delta19 CFTR was incorporated into the lipid bilayer membrane, functional Cl channel activity was identified. The full conductance state (O1) of the Delta19 CFTR Cl channel was identical to that of the wild-type CFTR channel (Fig. 6C and Fig. 8). Similar to the wild-type CFTR channel, the Delta19 CFTR channel had an average open probability of 0.308 ± 0.043 and mean open lifetimes of = 30.6 ms and = 115.8 ms at -80 mV (Fig. 7B). The relative occurrence of was y/(y + y) = 0.48. Thus, gating of the Delta19 CFTR channel is not significantly different from that of the wild-type CFTR channel at a time resolution of 100-Hz cutoff frequency in the bilayer system.


Figure 8: Subconductance states of Delta19 CFTR. Consecutive traces for wild-type (WT) CFTR and Delta19 CFTR were measured at a test potential of -80 mV. In both cases, the bilayer contained three active CFTR channels. O1 corresponds to the full open state (8 pS), and O2 corresponds to the subconductance state of the channel (6 pS).



A significant difference between Delta19 CFTR and wild-type CFTR was found in the distribution of channel subconductance states. Unlike the wild-type CFTR channel, the Delta19 CFTR channel displayed a prominent subconductance state (O2; Fig. 8). Fig. 9shows the amplitude histogram of two separate experiments with the wild-type and Delta19 CFTR channels. The histograms were different from each other, particularly in the first open level. The frequent occurrence of subconductance state resulted in an asymmetric distribution of the first open level of the Delta19 CFTR channel (indicated by O1 and O2). Similar phenomena were observed in 13 other experiments with Delta19 CFTR channels. There was a relative paucity of instances of two channels in these recordings, especially for the Delta19 mutant, in records in which two channels are clearly present (e.g.Fig. 8and Fig. 9). The reasons for this are unclear, and detailed analysis is beyond the scope of this paper.


Figure 9: Amplitude histogram of CFTR channel. Each histogram was constructed with 16 episodes of data shown in Fig. 8. All acquisition points were included. The three open levels of wild-type (WT) CFTR could be fitted by gaussian distributions (first open level amplitude of -0.68 pA, second of -1.41 pA, and third of -2.11 pA). The first open level of the Delta19 CFTR channels could be fitted by two gaussian distribution functions with amplitudes of -0.66 pA (O1) and -0.45 pA (O2).



Fig. 10shows the amplitude histograms of open channel current pooled from multiple experiments in which a single CFTR channel was incorporated into the bilayer membrane. Clearly, the Delta19 CFTR channel contained more openings to the O2 state than the wild-type CFTR channel (Fig. 10, compare A with B). The histograms could be fitted with the sum of two gaussian distribution functions, with mean currents of -0.65 pA (O1) and -0.46 pA (O2), at -80 mV. The relative occurrence of the O2 state was estimated at 9% for the wild-type CFTR channel and at 23% for the Delta19 CFTR channel.


DISCUSSION

In this study, we examined the function of a CFTR deletion mutant, Delta19 CFTR. This deletion mutation results in incomplete glycosylation and intracellular retention of CFTR based on the following observations. (i) An immunoprecipitation/Western blot assay identified a core-glycosylated form of Delta19 CFTR (140 kDa), different from the fully mature glycosylated protein (170 kDa); (ii) subcellular fractionation showed the Delta19 CFTR protein localized predominantly in the intracellular membranes; and (iii) an SPQ assay of cells expressing Delta19 CFTR showed no forskolin-stimulated Cl transport. However, this processing mutant maintained functional Cl channel activity, presumably in the intracellular organelles, when reconstituted into lipid bilayer membranes. The most notable difference between wild-type and Delta19 CFTR channels lies in the distribution of conductance states. The deletion mutation caused frequent occurrence of a subconductance state within the Cl channel.

The most common disease-causing mutation of CFTR is the deletion of a single phenylalanine residue at position 508 (DeltaF508 CFTR). Most of the DeltaF508 CFTR protein is retained in the endoplasmic reticulum and fails to reach its intended site of action in the plasma membrane. With regard to processing at 37 °C, Delta19 CFTR appears to be similar to DeltaF508 CFTR. However, DeltaF508 CFTR appears in the fully mature glycosylated form and can traffic to the plasma membrane following incubation at lower temperature (26-30 °C) (Fig. 2)(22, 23) . Unlike DeltaF508 CFTR, the misprocessing of Delta19 CFTR is temperature-insensitive: the protein remained in the core-glycosylated state in intracellular organelles even at 26 °C. The DeltaF508 CFTR proteins, once at the plasma membrane, give rise to functional Cl channels similar to those of wild-type CFTR in terms of open probability (26) and conductance state(24, 26) .

A cardiac-specific isoform of CFTR lacks 30 amino acids in the intracellular loop between transmembrane segments II and III. It is caused by alternative splicing of exon 5 of the CFTR mRNA(27) . Cardiac CFTR functions as a cAMP-dependent protein kinase-regulated Cl channel with conduction properties similar to those of epithelial CFTR(9, 28) . However, when expressed in HeLa cells, human CFTR lacking exon 5 failed to generate a cAMP-mediated chloride transport by the SPQ assay, apparently due to defective intracellular processing(29) . The strategy applied here to the study of CFTR channel function can be used to study even misprocessed or mislocalized channels and therefore ought to be applicable to the study of the effects of deleting exon 5 on CFTR channel function as well.

The 19 amino acids deleted in Delta19 CFTR span a major part of the intracellular loop joining transmembrane segments IV and V. This segment is highly hydrophilic, with about one-third of the residues being charged (two lysines and four glutamates). Several factors could account for the subconductance state associated with the Delta19 CFTR channel.

One possibility is that retention of the Delta19 CFTR protein in intracellular membranes alters the function of the Cl channel. Maturation of CFTR from the endoplasmic reticulum through the Golgi apparatus to the plasma membrane involves several processing and post-translational modifications (e.g. folding and glycosylation). The endoplasmic reticulum membrane contains other proteins, including various types of chaperons, that could interact with CFTR and alter the function of the Cl channel. However, the gating kinetics and open probability of the Delta19 CFTR channel in the intracellular membrane are similar to those of the wild-type CFTR channel in the plasma membrane, so the deletion of these 19 amino acids did not alter the essential function of the CFTR Cl channel. Moreover, the wild-type CFTR channel occasionally enters the O2 subconductance state characteristic of the Delta19 CFTR channel. These observations suggest that the subconductance state associated with the Delta19 CFTR Cl channel is probably an intrinsic property of the CFTR protein, but the deletion affects CFTR in such a way as to favor the O2 conductance state.

A second possibility is that this loop interacts with other domains (nucleotide-binding folds, the regulatory domain, and other intracellular loops) in CFTR or with other cellular proteins that are involved in the conductance state transition. Deletion of part of this loop might then affect the channel structure allosterically by changing these inter- or intramolecular interactions and thus induce a quasi-stable open configuration in addition to the full open state. Understanding the distribution of subconductance states associated with the single Cl channel should provide valuable information about structure-function relationships in the CFTR Cl channel.

The approach reported here allows functional characterization of CFTR in any intracellular membranes besides the endoplasmic reticulum membrane(25) . By preparing vesicles from intracellular membranes separated from the plasma membrane, in principle, one can study any processing mutant in the lipid bilayer system. Further experiments are necessary to test the role of specific amino acids of the intracellular loops (e.g. positively and negatively charged and hydrophobic amino acids) in the regulation of the conductance state of the CFTR Cl channel.


FOOTNOTES

*
This work was supported by a research development program grant from the Cystic Fibrosis Foundation and by Grants P30 DK27651 and RO1HL/DK49003 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Depts. of Physiology and Biophysics and Pediatrics, Case Western Reserve University School of Medicine, BRB, 2109 Adelbert Rd., Cleveland, OH 44106. Tel.: 216-368-4370; Fax: 216-368-4223.

(^1)
The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; pS, picosiemens; SPQ, 6-methoxyl-N-(3-sulfopropyl) quinolinium.

(^2)
T. Tao, J. Xie, M. L. Drumm, J. Zhao, P. B. Davis, and J. Ma, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. A. Perez for help in the SPQ assay and graphics, Drs. T. Tao and J. Zhao for help in membrane preparations, and Y. Hervey for help in the immunoprecipitation assay.


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