A Single Conductance Pore for Chloride Ions Formed by Two Cystic Fibrosis Transmembrane Conductance Regulator Molecules
1999; Elsevier BV; Volume: 274; Issue: 12 Linguagem: Inglês
10.1074/jbc.274.12.7627
ISSN1083-351X
AutoresBryan Zerhusen, Jiying Zhao, Junxia Xie, Pamela B. Davis, Jianjie Ma,
Tópico(s)Congenital Ear and Nasal Anomalies
ResumoThe 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. 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. cystic fibrosis transmembrane conductance regulator cAMP-dependent protein kinase siemens wild type 6-methoxy-N-(3 sulfopropyl)quinolinium CFTR1 is a multi-functional protein, which provides the pore of a linear conductance chloride channel (1Riordan J.R. Rommens J.M. Kerem B.-S. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.-L. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L.-C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5907) Google Scholar, 2Bear C.E. Li C. Kartner N. Bridges R.J. Jensen T.J. Ramjeesingh M. Riordan J.R. Cell. 1992; 68: 809-818Abstract Full Text PDF PubMed Scopus (778) Google Scholar, 3Smit L.S. Wilkinson D.J. Mansoura M.K. Collins F.S. Dawson D.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9963-9967Crossref PubMed Scopus (87) Google Scholar, 4Tabcharani J.A. Rommens J.M. Hou Y.X. Chang X.B. Tsui L.C. Riordan J.R. Hanrahan J.W. Nature. 1993; 366: 79-82Crossref PubMed Scopus (206) Google Scholar, 5Gunderson K.L. Kopito R.R. J. Biol. Chem. 1994; 269: 19349-19353Abstract Full Text PDF PubMed Google Scholar) and also functions to regulate other membrane proteins (6Egan M. Flotte T. Afione S. Solow R. Zeitlin P.L. Carter B.J. Guggino W.B. Nature. 1992; 358: 581-584Crossref PubMed Scopus (377) Google Scholar, 7Stutts M.J. Canessa C.M. Olsen J.C. Hamrick M. Cohn J.A. Rossier B.C. Boucher R.C. Science. 1995; 269: 847-850Crossref PubMed Scopus (955) Google Scholar). 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 (8Quinton P.M. Am. J. Physiol. 1986; 251: C649-C652Crossref PubMed Google Scholar, 9Welsh M.J. Smith A.E. Cell. 1993; 73: 1251-1254Abstract Full Text PDF PubMed Scopus (1224) Google Scholar, 10Tsui L.C. Am. J. Respir. Crit. Care Med. 1995; 151: S47-S53Crossref PubMed Google Scholar). Comprehensive genotype-phenotype studies have indicated possible contribution of protein-protein interactions to the severity of the disease (11Zielenski J. Tsui L.C. Annu. Rev. Genet. 1995; 29: 777-807Crossref PubMed Scopus (517) Google Scholar), 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 (12Gadsby D.C. Nairn A.C. Trends Biochem. Sci. 1994; 19: 513-518Abstract Full Text PDF PubMed Scopus (82) Google Scholar). 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 (13Cheung M. Akabas M.H. J. Gen. Physiol. 1997; 109: 289-299Crossref PubMed Scopus (85) Google Scholar), with an ohmic conductance of ∼8 pS in 200 mm KCl solution (14Xie J. Drumm M.L. Ma J. Davis P.B. J. Biol. Chem. 1995; 270: 28084-28096Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 15Ma J. Tasch J. Tao T. Zhao J. Xie J. Drumm M.L. Davis P.B. J. Biol. Chem. 1995; 271: 7351-7356Abstract Full Text Full Text PDF Scopus (42) Google Scholar). An early study by Richet al. (16Rich D.P. Gregory R.J. Anderson M.P. Manavalan P. Smith A.E. Welsh M.J. Science. 1991; 253: 205-207Crossref PubMed Scopus (193) Google Scholar) showed that deletion of amino acids 708–835 from the R domain (ΔR) renders the CFTR channel PKA independent. The open probability of ΔR-CFTR is approximately one-third that of the wt channel and does not increase upon PKA phosphorylation (17Ma J. Zhao J. Drumm M.L. Xie J. Davis P.B. J. Biol. Chem. 1997; 273: 28133-28141Abstract Full Text Full Text PDF Scopus (69) Google Scholar, 18Winter M.C. Welsh M.J. Nature. 1997; 398: 294-296Crossref Scopus (124) Google Scholar). Based on these different gating properties of the wt and ΔR channels, we set out to test the intermolecular interactions of CFTR by constructing tandem cDNAs between the wt-CFTR and the ΔR-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-ΔR (or ΔR-wt) channel should exhibit mixed properties of the wt and ΔR channels. The wild type and ΔR (708–835) CFTR cDNAs were cloned into theNheI/XhoI sites of the pCEP4 expression vector (14Xie J. Drumm M.L. Ma J. Davis P.B. J. Biol. Chem. 1995; 270: 28084-28096Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 17Ma J. Zhao J. Drumm M.L. Xie J. Davis P.B. J. Biol. Chem. 1997; 273: 28133-28141Abstract Full Text Full Text PDF Scopus (69) Google Scholar). The tandem constructs, wt-wt, wt-ΔR, ΔR-wt, and ΔR-ΔR, were generated in three steps. First, site-directed mutageneses were used to remove the stop codon and to introduce aBssHII 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. 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 (14Xie J. Drumm M.L. Ma J. Davis P.B. J. Biol. Chem. 1995; 270: 28084-28096Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 15Ma J. Tasch J. Tao T. Zhao J. Xie J. Drumm M.L. Davis P.B. J. Biol. Chem. 1995; 271: 7351-7356Abstract Full Text Full Text PDF Scopus (42) Google Scholar). The CFTR-mediated chloride transport was measured by SPQ assay with HEK 293 cells expressing the wt, ΔR, wt-wt, ΔR-ΔR, and wt-ΔR CFTR proteins, following the procedure described previously (14Xie J. Drumm M.L. Ma J. Davis P.B. J. Biol. Chem. 1995; 270: 28084-28096Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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. The procedure for single channel measurements of CFTR using the lipid bilayer reconstitution technique has been described elsewhere (17Ma J. Zhao J. Drumm M.L. Xie J. Davis P.B. J. Biol. Chem. 1997; 273: 28133-28141Abstract Full Text Full Text PDF Scopus (69) Google Scholar). Briefly, microsomal membrane vesicles were isolated from HEK 293 cells transiently expressing either the wt-, ΔR-, wt-wt, wt-ΔR, ΔR-wt, or ΔR-ΔR 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 thecis 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. Fig. 1 A 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 ΔR-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-ΔR (lane 5), ΔR-wt (lane 6), and ΔR-ΔR (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.1 A, 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). 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. 1 B). Those cells expressing the ΔR and ΔR-ΔR proteins exhibit basal chloride transport activities without stimulation with forskolin, which is consistent with the studies of Rich et al. (16Rich D.P. Gregory R.J. Anderson M.P. Manavalan P. Smith A.E. Welsh M.J. Science. 1991; 253: 205-207Crossref PubMed Scopus (193) Google Scholar). Interestingly, the cells expressing wt-ΔR 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 ΔR-ΔR proteins are incorporated into the lipid bilayer membrane. Fig.2 A shows representative current traces from the wt, wt-wt, ΔR, and ΔR-ΔR channels, and their corresponding current-voltage relationships are plotted in Fig.2 B. 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 ΔR-ΔR, 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 ΔR, opening of the ΔR-ΔR 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. 2 C). Studies from other laboratories have shown that the amino- and carboxyl-terminal tails of CFTR contribute to the overall function of the CFTR channel (19Loo M.A. et al.Pediatr. Pulmonol. Suppl. 1997; 14: 212Google Scholar, 20Naren A.P. et al.Pediatr. Pulmonol. Suppl. 1998; 17: 94Google Scholar). 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).Figure 4Effect 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 P o 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.View Large Image Figure ViewerDownload (PPT) 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 (2Bear C.E. Li C. Kartner N. Bridges R.J. Jensen T.J. Ramjeesingh M. Riordan J.R. Cell. 1992; 68: 809-818Abstract Full Text PDF PubMed Scopus (778) Google Scholar, 4Tabcharani J.A. Rommens J.M. Hou Y.X. Chang X.B. Tsui L.C. Riordan J.R. Hanrahan J.W. Nature. 1993; 366: 79-82Crossref PubMed Scopus (206) Google Scholar, 5Gunderson K.L. Kopito R.R. J. Biol. Chem. 1994; 269: 19349-19353Abstract Full Text PDF PubMed Google Scholar, 14Xie J. Drumm M.L. Ma J. Davis P.B. J. Biol. Chem. 1995; 270: 28084-28096Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 17Ma J. Zhao J. Drumm M.L. Xie J. Davis P.B. J. Biol. Chem. 1997; 273: 28133-28141Abstract Full Text Full Text PDF Scopus (69) Google Scholar) 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-ΔR and ΔR-wt, also form functional chloride channels with unitary conductance of 8 pS, which display mixed gating properties of the wt and ΔR channels (Fig.3). First, opening of the wt-ΔR and ΔR-wt channels exhibit bursting kinetics, but unlike either the wt or ΔR channels, these bursting patterns are interrupted by fast closing transitions (compare traces of Fig. 3, A and B,with Fig. 2 A). The wt channel has an average open lifetime of τo = 96.0 ± 9.3 ms, and the ΔR channel has a τo = 54.2 ± 6.5 ms (17Ma J. Zhao J. Drumm M.L. Xie J. Davis P.B. J. Biol. Chem. 1997; 273: 28133-28141Abstract Full Text Full Text PDF Scopus (69) Google Scholar), whereas the wt-ΔR channel has a τo = 24.4 ± 5.8 ms (n= 9), and the ΔR-wt channel has a τo = 32.4 ± 3.4 ms (n = 7). Second, open probability of the wt-ΔR and ΔR-wt channels display a clear PKA dependence (Fig. 3 C). The channels exhibit constitutive activity in the absence of PKA, which becomes significantly higher upon PKA phosphorylation (Fig.2 C). In contrast, open probabilities of the ΔR and ΔR-ΔR channels are completely independent of PKA phosphorylation (Fig. 3 D). Fig. 4 shows the effect of thrombin on the wt-e-wt channel. Compared with the wt-wt and wt-ΔR 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.4 A, the wt-e-wt channel opens predominantly to a single 8-pS conductance state, with an average P o 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 asp = 0.723 (Fig. 4 B). 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.2 C). 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 (2Bear C.E. Li C. Kartner N. Bridges R.J. Jensen T.J. Ramjeesingh M. Riordan J.R. Cell. 1992; 68: 809-818Abstract Full Text PDF PubMed Scopus (778) Google Scholar, 4Tabcharani J.A. Rommens J.M. Hou Y.X. Chang X.B. Tsui L.C. Riordan J.R. Hanrahan J.W. Nature. 1993; 366: 79-82Crossref PubMed Scopus (206) Google Scholar). The gating properties of the wt-wt and ΔR-ΔR channels are similar to the wt and ΔR channels, whereas the wt-ΔR and ΔR-wt channels exhibit mixed properties of the wt and ΔR channels. The fact that both wt-ΔR and ΔR-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. (21Marshall J. Fang S. Ostedgaard L.S. O'Riordan C.R. Ferrara D. Amara J.F. Hoppe H. Scheule R.K. Welsh M.J. Smith A.E. Cheng S.H. J. Biol. Chem. 1994; 269: 2987-2995Abstract Full Text PDF PubMed Google Scholar) 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. (22Eskandari S. Wright E.M. Kreman M. Starace D.M. Zampighi G.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11235-11240Crossref PubMed Scopus (161) Google Scholar) 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? We thank Drs. M. L. Drumm, J. W. Hanrahan, and D. C. Gadsby for helpful discussions.
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