COMMUNICATION
A Single Conductance Pore for Chloride Ions Formed by Two Cystic Fibrosis Transmembrane Conductance Regulator Molecules*

Bryan Zerhusen, Jiying Zhao, Junxia XieDagger , Pamela B. DavisDagger , and Jianjie Ma§

From the Departments of Physiology and Biophysics and Dagger  Pediatrics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)- and ATP-regulated chloride channel, whose gating process involves intra- or intermolecular interactions among the cytosolic domains of the CFTR protein. Tandem linkage of two CFTR molecules produces a functional chloride channel with properties that are similar to those of the native CFTR channel, including trafficking to the plasma membrane, ATP- and PKA-dependent gating, and a unitary conductance of 8 picosiemens (pS). A heterodimer, consisting of a wild type and a mutant CFTR, also forms an 8-pS chloride channel with mixed gating properties of the wild type and mutant CFTR channels. The data suggest that two CFTR molecules interact together to form a single conductance pore for chloride ions.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
REFERENCES

CFTR1 is a multi-functional protein, which provides the pore of a linear conductance chloride channel (1-5) and also functions to regulate other membrane proteins (6, 7). Mutations in CFTR leading to defective regulation or transport of chloride ions across the apical surface of epithelial cells are the primary cause of the genetic disease of cystic fibrosis (8-10). Comprehensive genotype-phenotype studies have indicated possible contribution of protein-protein interactions to the severity of the disease (11), but little is known on the stoichiometry of CFTR as a chloride channel.

The native CFTR chloride channel is activated by PKA-phosphorylation of serine residues in its regulatory or R domain and then gated by binding and hydrolysis of ATP by the nucleotide binding folds (12). The actual pore of the chloride channel is presumably formed by portions of the two membrane spanning domains of CFTR, each consisting of six transmembrane segments (13), with an ohmic conductance of ~8 pS in 200 mM KCl solution (14, 15). An early study by Rich et al. (16) showed that deletion of amino acids 708-835 from the R domain (Delta R) renders the CFTR channel PKA independent. The open probability of Delta R-CFTR is approximately one-third that of the wt channel and does not increase upon PKA phosphorylation (17, 18). Based on these different gating properties of the wt and Delta R channels, we set out to test the intermolecular interactions of CFTR by constructing tandem cDNAs between the wt-CFTR and the Delta R-CFTR. Our rationale is as follows. If a monomer of CFTR is sufficient to form an 8-pS chloride channel, the dimeric CFTR molecules would form either a 16-pS chloride channel or two 8-pS chloride channels that may gate independently or together. On the other hand, if two CFTR molecules are required to function as a chloride channel, we expected the tandem construct to form a single 8-pS chloride channel, provided that the linker sequence does not affect the CFTR channel function. Furthermore, we predicted that the wt-Delta R (or Delta R-wt) channel should exhibit mixed properties of the wt and Delta R channels.

    EXPERIMENTAL PROCEDURES

Subcloning of CFTR cDNAs-- The wild type and Delta R (708-835) CFTR cDNAs were cloned into the NheI/XhoI sites of the pCEP4 expression vector (14, 17). The tandem constructs, wt-wt, wt-Delta R, Delta R-wt, and Delta R-Delta R, were generated in three steps. First, site-directed mutageneses were used to remove the stop codon and to introduce a BssHII restriction site at the 3' end of the CFTR cDNA, to create C-BssH. Second, similar approach was taken to remove the Kozak sequence and to introduce a BssHII site at the 5' end of the CFTR cDNA, to yield N-BssH. Third, the entire CFTR cDNA from N-BssH was released from the pCEP4 vector through digestion with BssHII and XhoI and ligated into the BssHII/XhoI sites of the C-BssH, to create the wt-wt dimer. This represents a direct head-to-tail linkage of two CFTR cDNAs. A double stranded oligonucleotide containing the recognition sequence for thrombin (underlined), Arg-Ala-Ala-Ser-Leu-Val-Pro-Arg-Gly-Ser-Gly-Gly-Gly-Gly, was ligated to the BssHII site of wt-wt, to yield the wt-e-wt construct.

Expression of CFTR in HEK 293 Cells-- The human embryonic kidney (HEK 293) cells were used for expression of CFTR proteins. The different CFTR cDNAs were introduced into the HEK 293 cells using the LipofectAMINE reagent. Two days after transfection, the cells were used for Western blot assay, SPQ measurement, or isolation of membrane vesicles followed by reconstitution studies in the lipid bilayer membranes, as described previously (14, 15).

SPQ Assay of Chloride Transport-- The CFTR-mediated chloride transport was measured by SPQ assay with HEK 293 cells expressing the wt, Delta R, wt-wt, Delta R-Delta R, and wt-Delta R CFTR proteins, following the procedure described previously (14). Basically, the cells were loaded with SPQ dye (Molecular Probes) using hypotonic shock, and chloride flux across the plasma membrane was measured upon stimulation with 10 µM forskolin.

Lipid Bilayer Reconstitution of CFTR Channel-- The procedure for single channel measurements of CFTR using the lipid bilayer reconstitution technique has been described elsewhere (17). Briefly, microsomal membrane vesicles were isolated from HEK 293 cells transiently expressing either the wt-, Delta R-, wt-wt, wt-Delta R, Delta R-wt, or Delta R-Delta R proteins and added to the cis (intracellular) solution containing 200 mM KCl, 2 mM Mg-ATP, 10 mM HEPES-Tris (pH 7.4). The trans solution contained 50 mM KCl, 10 mM HEPES-Tris (pH 7.4). To study the PKA-dependent regulation of the CFTR channel, 100 units/ml of the catalytic subunit of PKA was added to the cis solution. Single channel currents were recorded using an Axopatch 200A patch clamp unit (Axon Instruments). Data acquisition and pulse generation were performed with a 486 Computer and a 1200 Digidata A/D-D/A converter. The currents were sampled at 1-2.5 ms/point and filtered at 100 Hz. Single channel analysis were performed with the pClamp7 software.

    RESULTS AND DISCUSSION

Fig. 1A shows a Western blot of CFTR expressed in HEK 293 cells. Both fully glycosylated (~170 kDa) and core glycosylated (~140 kDa) proteins can be detected in cells transfected with the wt-CFTR cDNA (lane 1). The corresponding bands for the Delta R-CFTR run at apparent molecular masses of ~150 and ~120 kDa (lane 2), reflecting the deletion of 128 amino acids from the R domain (amino acids 708-835). The wt-wt protein has molecular masses of ~340 and ~280 kDa (lane 4), as expected for a dimer of the wt-CFTR. Similarly, the wt-Delta R (lane 5), Delta R-wt (lane 6), and Delta R-Delta R (lane 7) proteins can all be expressed in HEK 293 cells, with the expected size as dimers. To be able to manipulate the oligomerization state of the CFTR proteins, we engineered an enzymatic digestion site for thrombin in the linker sequence of the wt-wt dimer. This construct is named wt-e-wt (Fig. 1A, lane 10). Digestion of wt-e-wt with thrombin resulted in reduction of the apparent size from dimer to monomers (lane 9), whereas thrombin had no effect on the wt monomer (lane 8) or the wt-wt dimer (lane 11).


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Fig. 1.   Expression of CFTR in HEK 293 cells. A, Western blot of CFTR expressed in HEK 293 cells. The pCEP4 plasmids containing the different CFTR constructs were introduced into the HEK 293 cells using the LipofectAMINE reagent. 24-48 h after transfection, the cells were harvested and the whole cell lysate was used in the Western blot. The proteins were separated on a 3-12% polyacrylamide gel, and probed with a monoclonal antibody against the COOH terminus of the CFTR protein (mAb24-1, Genzyme). Lane 1, wt; lane 2, Delta R; lane 3, untransfected HEK cells; lane 4, wt-wt; lane 5, wt-Delta R; lane 6, Delta R-wt; lane 7, Delta R-Delta R; lane 10, wt-e-wt. 10 units of thrombin (purchased from Sigma) cleaved the wt-e-wt dimer into monomers (lane 9) in a reaction run at 37 °C for 10 min with a buffer containing 200 mM KCl, 2 mM Mg-ATP, 10 mM HEPES-Tris (pH 7.4). The wt CFTR has no internal cleavage site for thrombin, as revealed by the lack of effect of thrombin on the wt monomer (lane 8) and wt-wt dimer (lane 11). B, SPQ assay of CFTR-mediated chloride transport. HEK 293 cells loaded with SPQ were preincubated with nitrate solution to allow depletion of intracellular chloride ions (1). The perfusion solution is then replaced by a buffer containing chloride to quench fluorescence as chloride ions enter the cell (2). When fluorescence reaches a minimum, nitrate solution is replaced to restore fluorescence (3). This sequence is repeated with chloride and nitrate buffers containing 10 µM foskolin (4). At the completion of the experiment, the cells are perfused with KSCN and valinomycin to quench the fluorescence and dissipate the membrane potential (5). Each trace represents a single cell responding over the course of the experiment.

SPQ assays indicate that the wt-wt dimer, similar to the wt monomer, supports chloride transport in HEK 293 cells upon stimulation with forskolin (Fig. 1B). Those cells expressing the Delta R and Delta R-Delta R proteins exhibit basal chloride transport activities without stimulation with forskolin, which is consistent with the studies of Rich et al. (16). Interestingly, the cells expressing wt-Delta R have basal chloride transport activity in the absence of forskolin, which became significantly higher upon stimulation by forskolin (relative changes in fluorescence per minute, 0.095 ± 0.010, -forskolin; 0.203 ± 0.007, +forskolin, n = 65). These results indicate that CFTR dimers can traffic properly to the plasma membrane of HEK 293 cells.

To study the single channel functions of the CFTR dimers, microsomal membrane vesicles containing the wt-wt or Delta R-Delta R proteins are incorporated into the lipid bilayer membrane. Fig. 2A shows representative current traces from the wt, wt-wt, Delta R, and Delta R-Delta R channels, and their corresponding current-voltage relationships are plotted in Fig. 2B. As can be seen, all four constructs give rise to chloride channels with unitary conductances of ~8 pS. In 9 out of 13 experiments with wt-wt, and 8 out of 12 experiments with Delta R-Delta R, we only observed openings of a single channel (not two channels) in the bilayer membrane. Similar to the wt channel, opening of the wt-wt channel absolutely requires the presence of both ATP and PKA in the cytosolic solution; and similar to Delta R, opening of the Delta R-Delta R channel is independent of PKA phosphorylation. Interestingly, the activity of the wt-wt channel appears to be significantly lower than that of the wt channel (Fig. 2C). Studies from other laboratories have shown that the amino- and carboxyl-terminal tails of CFTR contribute to the overall function of the CFTR channel (19, 20). We speculate that the head-to-tail connection in the dimeric construct probably constrains the movement of the amino- and carboxyl-terminal portions of CFTR, reducing activity of the wt-wt channel (see also Fig. 4).


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Fig. 2.   Bilayer reconstitution of single CFTR channels. A, representative current traces from a wt, wt-wt, Delta R, and Delta R-Delta R CFTR channel were obtained at a test potential of -100 mV. The recording solution contained: cis (intracellular), 200 mM KCl, 2 mM Mg-ATP, 10 mM HEPES-Tris (pH 7.4); and trans(extracellular), 50 mM KCl, 10 mM HEPES-Tris (pH 7.4). For the wt and wt-wt channel, 100 units/ml of the catalytic subunit of PKA (Promega) was present in the cis solution, and for the Delta R and Delta R-Delta R channel, no PKA was present in the cis solution. Downward deflections represent the movement of chloride ions from cis intracellular to trans extracellular solutions. B, current-voltage relationships for the wt-wt, Delta R-Delta R, and wt-Delta R channels. The wt-wt channel had a slope conductance of G = 8.1 ± 0.8 pS, with a reversal potential of Vrev = 15.6 ± 8.4 mV (open circle ). G = 8.6 ± 0.6 pS, Vrev = 9.5 ± 6.0 mV for the wt-Delta R channel (), and G = 7.1 ± 0.1 pS, Vrev = 15.4 ± 7.3 mV for the Delta R-Delta R channel (black-down-triangle ). The solid line represents the best fit to data from all three channels, with G = 8.1 ± 0.5 pS and Vrev = 12.0 ± 4.7 mV. C, average open probability of the CFTR channels were calculated at a test potential of -100 mV. Data are presented as mean ± S.E. (n = 6-16).

Thus, it appears that the dimeric constructs of CFTR form functional chloride channels with conduction properties that are indistinguishable from the monomers of CFTR, i.e. all of them have single channel conductance of ~8 pS. The dimeric constructs could in principle have two separate pores with conductance of 8 pS for chloride ions, and because of some physical constraint due to the linker sequence, opening of one pore could prevent opening of the other pore, which would result in the overall appearance of a single CFTR channel. The other possibility is that the 8-pS channel normally recorded in single channel measurements (2, 4, 5, 14, 17) actually represents dimeric complexes of CFTR that naturally assemble in the cell surface membrane. Data from the following sets of experiments support the latter hypothesis.

The heterodimers of CFTR, wt-Delta R and Delta R-wt, also form functional chloride channels with unitary conductance of 8 pS, which display mixed gating properties of the wt and Delta R channels (Fig. 3). First, opening of the wt-Delta R and Delta R-wt channels exhibit bursting kinetics, but unlike either the wt or Delta R channels, these bursting patterns are interrupted by fast closing transitions (compare traces of Fig. 3, A and B, with Fig. 2A). The wt channel has an average open lifetime of tau o = 96.0 ± 9.3 ms, and the Delta R channel has a tau o = 54.2 ± 6.5 ms (17), whereas the wt-Delta R channel has a tau o = 24.4 ± 5.8 ms (n = 9), and the Delta R-wt channel has a tau o = 32.4 ± 3.4 ms (n = 7). Second, open probability of the wt-Delta R and Delta R-wt channels display a clear PKA dependence (Fig. 3C). The channels exhibit constitutive activity in the absence of PKA, which becomes significantly higher upon PKA phosphorylation (Fig. 2C). In contrast, open probabilities of the Delta R and Delta R-Delta R channels are completely independent of PKA phosphorylation (Fig. 3D).


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Fig. 3.   PKA dependence of the wt-Delta R and Delta R-wt channels. Selected single channel currents through the wt-Delta R (A) and Delta R-wt (B) channels at -100 mV test potential, in the absence of PKA (-PKA) and presence of PKA (+PKA) in the cis solution. Notice the fast gating kinetics of the wt-Delta R and Delta R-wt channels compared with the wt and Delta R channels (see Fig. 2A). Diary plot of channel open probability (Po) as a function of time with the wt-Delta R channel (C) and the Delta R-Delta R channel (D). Po was calculated as the fractional time occupied by the open state during the 5-s test pulse to -100 mV. The plot showed that PKA phosphorylation increased activity of the wt-Delta R channel, but it did not affect open probability of the Delta R-Delta R channel. 3 mM diphenyl carboxylate added to the extracellular solution completely inhibited activities of both wt-Delta R and Delta R-Delta R channels. The plot shown in C is representative of four other experiments with wt-Delta R and three other experiments with Delta R-wt and that in D is representative of four other experiments with the Delta R-Delta R channel.

Fig. 4 shows the effect of thrombin on the wt-e-wt channel. Compared with the wt-wt and wt-Delta R constructs, the wt-e-wt dimer contains 14 extra amino acids in the linker sequence corresponding to the thrombin cleavage site (underlined) (R-A-A-S-L-V-P-R-G-S-G-G-G-G). As shown in Fig. 4A, the wt-e-wt channel opens predominantly to a single 8-pS conductance state, with an average Po of 0.186 ± 0.045 (n = 11) at -100 mV. 3-5 min following the addition of 10 units/ml of thrombin to the cytosolic solution, the activity of the wt-e-wt channel increases approximately 4-fold, but the apparent number of channels in the bilayer membrane remains unchanged. During the course of the experiment, simultaneous opening of two channels were never observed, even though open probability of the thrombin-treated wt-e-wt channel was as high as p = 0.723 (Fig. 4B). Thrombin would separate the halves of the CFTR dimer and presumably remove constraints on the movement of the amino- and carboxyl-terminal tails of CFTR. If such constraints limit channel openings, this may explain why the wt-wt channel has a lower open probability than the wt channel (Fig. 2C).


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Fig. 4.   Effect of thrombin on the wt-e-wt CFTR channel. A, selected current traces at -100 mV from a single wt-e-wt channel in the presence of 2 mM ATP and 100 units/ml of PKA (control), and 3 min after the addition of 10 units of thrombin to the intracellular solution (+thrombin). Treatment of the wt-e-wt channel with thrombin resulted in significant increase of Po from 0.186 ± 0.045 (n = 11, control) to 0.456 ± 0.064 (n = 7, +thrombin), without affecting the distribution of single channel conductance states, i.e. the number of channels in the bilayer remained unchanged. B, time-dependent effect of thrombin on the wt-e-wt channel. Following stable incorporation of a single wt-e-wt channel in the bilayer membrane, 10 units of thrombin was first added to the cis solution. The second addition of thrombin was 40 units.

Taken together, we have shown that the dimeric constructs of CFTR can be expressed in the plasma membrane of HEK 293 cells, and these CFTR dimers form functional chloride channels with unitary conductance of 8 pS, similar to the native CFTR channel (2, 4). The gating properties of the wt-wt and Delta R-Delta R channels are similar to the wt and Delta R channels, whereas the wt-Delta R and Delta R-wt channels exhibit mixed properties of the wt and Delta R channels. The fact that both wt-Delta R and Delta R-wt channels exhibit similar PKA dependence and similar gating kinetics (Fig. 3, A and B) suggests that both halves of the CFTR dimer are properly expressed and inserted in the membrane of HEK 293 cells. The tandem linkage of CFTR apparently reduces the overall activity of the chloride channel due to physical constraints introduced at the junction of the two CFTR molecules, but does not affect the conduction property of the chloride channel, since cleavage of wt-e-wt with thrombin did not change the conductance state of the channel. Our data suggest that two CFTR molecules are required for the chloride channel to open to the 8-pS conductance state.

Marshall et al. (21) used co-immunoprecipitation to search for protein-protein interactions between different mutant forms of CFTR and concluded that CFTR exists predominantly in a monomeric state. It may be that the intermolecular interactions between the CFTR molecules are weak and do not survive strong detergent solubilization (i.e. SDS) or that the CFTR dimers only represent a small percentage of the total CFTR proteins that could not be detected by the co-immunoprecipitation procedure. A recent study by Eskandari et al. (22) established structural evidence for a dimeric complex with the CFTR proteins. These investigators used freeze fracture electron microscopy to investigate the oligomeric assembly of membrane proteins expressed in Xenopus oocytes, and they concluded that the intramembrane structure of CFTR was consistent with a dimeric assembly of 12-transmembrane helix of the CFTR monomers.

Besides being of fundamental importance in understanding the mechanism by which the CFTR channel works, the concept that CFTR functions as a dimer may have implication for persons who are compound heterozygotes for CFTR mutations. Some mutant pairs may be able to complement each other thereby increasing the overall CFTR function, but other mutant pairs may not. This may explain some of the phenotypic variations in patients with CFTR mutations, especially those with some residual function. It will be important to know at what stage in the biosynthetic pathway of CFTR the dimers are formed, i.e. before leaving the endoplasmic reticulum or after trafficking to the apical membrane. More specifically, which portions of the CFTR molecule are involved in the contact interaction or constitute the binding site(s) for accessory proteins that could interact with CFTR?

    ACKNOWLEDGEMENTS

We thank Drs. M. L. Drumm, J. W. Hanrahan, and D. C. Gadsby for helpful discussions.

    FOOTNOTES

* This work was supported by Grants RO1-DK51770 (to J. M.) and RO1-DK27561 (to P. B. D.) from the National Institutes of Health and by an Established Investigatorship from the American Heart Association (to J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 216-368-2684; Fax: 216-368-1693; E-mail: jxm63{at}po.cwru.edu.

    ABBREVIATIONS

The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; PKA, cAMP-dependent protein kinase; S, siemens; wt, wild type; SPQ, 6-methoxy-N-(3 sulfopropyl)quinolinium.

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