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Disease-associated Mutations in the Fourth Cytoplasmic Loop of Cystic Fibrosis Transmembrane Conductance Regulator Compromise Biosynthetic Processing and Chloride Channel Activity

1996; Elsevier BV; Volume: 271; Issue: 25 Linguagem: Inglês

10.1074/jbc.271.25.15139

1083-351X

Fabian S. Seibert, Paul Linsdell, Tip W. Loo, John W. Hanrahan, David M. Clarke, John R. Riordan,

Legume Nitrogen Fixing Symbiosis

A cluster of 18 point mutations in exon 17b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been detected in patients with cystic fibrosis. These mutations cause single amino acid substitutions in the most C-terminal cytoplasmic loop (CL4, residues 1035-1102) of the CFTR chloride channel. Heterologous expression of the mutants showed that 12 produced only core-glycosylated CFTR, which was retained in the endoplasmic reticulum; the other six mutants matured and reached the cell surface. In some cases substitution of one member of pairs of adjacent residues resulted in misprocessing, whereas the other did not. Thus, the secondary structure of CL4 may contribute crucially to the proper folding of the entire CFTR molecule. Cyclic AMP-stimulated iodide efflux was not detected from cells expressing the misprocessed variants but was from the other six, indicating that their mutations cause relatively subtle channel defects. Consistent with this, these latter mutations generally are present in patients who are pancreatic-sufficient, while the processing mutants are mostly from patients who are pancreatic-insufficient. Single-channel patch-clamp analysis demonstrated that the processed mutants had the same ohmic conductance as wild-type CFTR, but a lower open probability, generally due to an increase in channel mean closed time and a reduction in mean open time. This suggests that mutations in CL4 do not affect pore properties of CFTR, but disrupt the mechanism of channel gating. A cluster of 18 point mutations in exon 17b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been detected in patients with cystic fibrosis. These mutations cause single amino acid substitutions in the most C-terminal cytoplasmic loop (CL4, residues 1035-1102) of the CFTR chloride channel. Heterologous expression of the mutants showed that 12 produced only core-glycosylated CFTR, which was retained in the endoplasmic reticulum; the other six mutants matured and reached the cell surface. In some cases substitution of one member of pairs of adjacent residues resulted in misprocessing, whereas the other did not. Thus, the secondary structure of CL4 may contribute crucially to the proper folding of the entire CFTR molecule. Cyclic AMP-stimulated iodide efflux was not detected from cells expressing the misprocessed variants but was from the other six, indicating that their mutations cause relatively subtle channel defects. Consistent with this, these latter mutations generally are present in patients who are pancreatic-sufficient, while the processing mutants are mostly from patients who are pancreatic-insufficient. Single-channel patch-clamp analysis demonstrated that the processed mutants had the same ohmic conductance as wild-type CFTR, but a lower open probability, generally due to an increase in channel mean closed time and a reduction in mean open time. This suggests that mutations in CL4 do not affect pore properties of CFTR, but disrupt the mechanism of channel gating. INTRODUCTIONThe cystic fibrosis transmembrane conductance regulator (CFTR) 1The abbreviations used are: CFTRcystic fibrosis transmembrane conductance regulatorCFcystic fibrosisAMP-PNP5′-adenylylimidodiphosphateCHOChinese hamster ovaryCLcytoplasmic loopERendoplasmic reticulumNBFnucleotide binding foldPAGEpolyacrylamide gel electrophoresisTMtransmembrane helixTES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. is a chloride channel located in the apical membrane of many epithelia, where it plays a key role in the regulation of salt and water homeostasis (Welsh et al., 34Welsh M.J. Anderson M.P. Rich D.P. Berger H.A. Denning G.M. Ostedgaard L.S. Sheppard D.N. Cheng S.H. Gregory R.J. Smith A.E. Neuron. 1992; 8: 821-829Google Scholar). CFTR was predicted to form a transmembrane pore with regulatory segments protruding into the cell cytoplasm (Riordan et al., 27Riordan J.R. Rommens J.M. Kerem B. 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: 1066-1073Google Scholar); several features of this model have been confirmed experimentally (Hanrahan et al., 17Hanrahan J.W. Tabcharani J.A. Becq F. Mathews C.J. Augustinas O. Jensen T.J. Chang X.-B. Riordan J.R. Soc. Gen. Physiol. Ser. 1995; 50: 125-137Google Scholar). The channel consists of two structurally similar halves, each containing six transmembrane segments (TMs) and a nucleotide binding fold (NBF) with which ATP interacts. The two halves are connected by the R-domain, rich in consensus sequences for phosphorylation by several kinases, including cyclic AMP-dependent protein kinase and protein kinase C. On the cytoplasmically exposed side of the protein the TMs are linked to each other by cytoplasmic loops (CLs), which vary between 55 and 65 amino acids in length. There is evidence that the concerted action of ATP binding and ATP hydrolysis at the NBFs (Anderson et al., 2Anderson M.P. Berger H.A. Rich D.P. Gregory R.J. Smith A.E. Welsh M.J. Cell. 1991; 67: 775-784Google Scholar; Hwang et al., 20Hwang T.-C. Baukrowtiz G. Nagel G. Horie A.C. Gadsby D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4698-4702Google Scholar) and of phosphorylation at the R-domain (Cheng et al., 8Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.E. Cell. 1991; 66: 1027-1036Google Scholar; Chang et al., 5Chang X.B. Tabcharani J.A. Hou Y.X. Jensen T.J. Kartner N. Alon N. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1993; 268: 11304-11311Google Scholar) are necessary to allow channel activation, however, little information is available regarding the importance of the CLs in CFTR function. Only recently, a report by Xie et al. (36Xie J. Drumm M.L. Ma J. Davis P.B. J. Biol. Chem. 1995; 270: 28084-28091Google Scholar), which studied the effect of a 19-amino acid deletion in CL2 (residues 242–307), suggested that this region is involved in stabilizing the full conductance state of the CFTR chloride channel.The CLs are highly conserved between CFTRs expressed in different species (Diamond et al., 9Diamond G. Scanlin T.F. Zasloff M.A. Bevins C.L. J. Biol. Chem. 1991; 266: 22761-22769Google Scholar) and moderately conserved in comparison to the CLs of other ABC transporters (Manavalan et al., 23Manavalan P. Smith A.E. McPherson J.M. J. Protein Chem. 1993; 12: 279-290Google Scholar). Their possible significance is highlighted by the fact that 20% of all identified CF-associated mutations lie within CLs. Thus far in CF patients, 18 different point mutations have been described in exon 17b, which approximately corresponds to CL4 (amino acids 1035-1142); however, as yet the mechanistic impact of alterations in this segment of CFTR on its function and biosynthetic processing has not been investigated. The detailed analysis of amino acid substitutions in other CFTR domains has shown that the CF phenotype usually is attributable to either a lack of the channel at the cell surface or the production of a channel with impaired function (Welsh and Smith, 33Welsh M.J. Smith A.E. Cell. 1993; 73: 1251-1254Google Scholar). The failure of many mutant forms of CFTR, including the most frequent ΔF508 variant, to be biosynthetically processed beyond the core-glycosylated form present in the endoplasmic reticulum (ER), has become a hallmark of CFTR (Cheng et al., 7Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Google Scholar) and the focus of intense research. The aim of the present study was to examine the effects of mutations in CL4 on processing, intracellular transport, and chloride channel activity of CFTR.DISCUSSIONCurrent models of CFTR suggest that the molecule utilizes three major cytoplasmic domains (NBF-1, NBF-2, and the R-domain) in the regulation of its chloride channel activity (Hanrahan et al., 18Hanrahan J.W. Mathews C.J. Grygorczyk R. Tabcharani J.A. Grzelczak Z. Chang X.-B. Riordan J.R. J. Exp. Zool. 1996; 275 (in press)Google Scholar). An additional set of cytoplasmic domains is constituted by four fairly large CLs, connecting the TMs on the cytoplasmic side of the protein; little is known about their function. It would not be surprising if these loops also were involved in regulation of channel activity due to their position close to the putative pore-forming TM segments and their potential proximity to the major regulatory domains. Such a general view is consistent with the recent findings of Xie et al. (36Xie J. Drumm M.L. Ma J. Davis P.B. J. Biol. Chem. 1995; 270: 28084-28091Google Scholar) who concluded from studies of a 19-residue deletion variant (amino acids 266–284) that CL2 is required to stabilize the full conductance state of the CFTR chloride channel. For the investigation presented here, a different strategy was applied to study CLs. Eighteen CF-associated point mutations in CL4 were reconstructed to determine which cause CF, because the channel cannot reach the plasma membrane, and which cause disease due to defective functioning at the cell surface.Previous studies have shown that many mutations in CFTR, including the most frequent Δ508 deletion, prevent biosynthetic maturation (Gregory et al., 15Gregory R.J. Rich D.P. Cheng S.H. Souza D.W. Paul S. Manavalan P. Anderson M.P. Welsh M.I. Smith A.E. Mol. Cell. Biol. 1991; 11: 3886-3893Google Scholar). We find that this is also true of several mutations in CL4, in that 12 of the 18 resulted in misprocessing of the protein and its retention in the ER. Thus, for patients that carry these mutations, the CF phenotype most likely is a result of lack of channel expression at the cell surface. In the case of previously studied CFTR mutations known to result in ER retention, including ΔF508, it is believed that local misfolding prevents the attainment of the global cytoplasmic conformation of CFTR that is required for maturation and ER to Golgi transport. CL4 would be expected to contribute to this overall tertiary structure of the cytoplasmic aspect of the molecule on the ER surface. Disturbance of the local secondary structure of CL4 due to point mutations may prevent it from adequately making this contribution. It is not obvious why amino acid substitutions at 9 of the 14 different positions may compromise secondary structure, whereas those at the remaining 5 positions appear not to (see Table I). However, all of the substitutions resulting in misprocessing involve either charge changes or the introduction of a proline. Alterations at all of the positions in the C-terminal half of CL4 result in misprocessing, but only half of those in the N-terminal portion do so. Mutations at adjacent residues in that portion may either prevent maturation or not, viz. 1060 and 1061; 1066 and 1067; 1070 and 1071. Different substitutions at the same residue always produced the same effect, i.e. R1066C, R1066H, and R1066L, as well as M1101K and M1101R all inhibited maturation, whereas R1070W and R1070Q were both normally processed. The impacts of the mutations on CFTR maturation are compared with the reported pancreatic status of the patients in which they were detected in Table I. For 8 of 11 mutations resulting in misprocessing and failure of maturation, the patients were pancreatic-insufficient and for two of three mutations allowing processing the patients were pancreatic-sufficient. This is consistent with the possibility that biosynthetic arrest of CFTR in the ER may cause severe disease, whereas some point mutations, compatible with transport of the protein to the cell surface, may result in less severe disease.Table I.Pancreatic status of patients containing CL4 mutationsMutationBiosynthetic processingPancreatic statusRef.F1052V+—aNot reported. PS/PI for R1070Q because of five patients analyzed, one was PS, one PI and three unspecified.Mercier et al., 25Mercier B. Lissens W. Novelli G. Kalaydjieva L. DeArce M. Kapranov N. Klain N. Lenoir G. Chauveau P. Lenaerts C. Rault G. Cashman S. Sangiuolo F. Audrezet M.P. Dallapiccola B. Guillermit H. Bonduelle M. Liebaers I. Quere I. Verlingue C. Ferec C. Genomics. 1993; 16: 296-297Google ScholarH1054D−PSFerec et al., 12Ferec C. Verlingue C. Guillermit H. Quere J. Raguenes O. Felgelson J. Audrezet M.-P. Moullier P. Mercier B. Hum. Mol. Genet. 1993; 2: 1557-1560Google ScholarK1060T+PSCasals et al., 4Casals T. Bassas L. Ruiz-Romero J. Chillon M. Gimenez J. Ramos M.D. Tapia G. Narvacz H. Nunes V. Estivill X. Hum. Genet. 1995; 95: 205-211Google ScholarG1061R−PIMercier et al., 25Mercier B. Lissens W. Novelli G. Kalaydjieva L. DeArce M. Kapranov N. Klain N. Lenoir G. Chauveau P. Lenaerts C. Rault G. Cashman S. Sangiuolo F. Audrezet M.P. Dallapiccola B. Guillermit H. Bonduelle M. Liebaers I. Quere I. Verlingue C. Ferec C. Genomics. 1993; 16: 296-297Google ScholarL1065P−PIGhanem et al., 13Ghanem N. Costes B. Girodon E. Martin J. Fanen P. Goossens M. Genomics. 1994; 21: 434-436Google ScholarR1066C−—aNot reported. PS/PI for R1070Q because of five patients analyzed, one was PS, one PI and three unspecified.Fanan et al., 10Fanan P. Ghanem N. Vidaud M. Besmond C. Martin J. Costes B. Plassa F. Goossens M. Genomics. 1992; 13: 770-776Google ScholarR1066H−PIFerec et al., 11Ferec C. Audrezet M.-P. Mercier B. Guillermit H. Moullier P. Quiere I. Verlingue C. Nat. Genet. 1992; 1: 188-191Google ScholarR1066L−PIMercier et al., 25Mercier B. Lissens W. Novelli G. Kalaydjieva L. DeArce M. Kapranov N. Klain N. Lenoir G. Chauveau P. Lenaerts C. Rault G. Cashman S. Sangiuolo F. Audrezet M.P. Dallapiccola B. Guillermit H. Bonduelle M. Liebaers I. Quere I. Verlingue C. Ferec C. Genomics. 1993; 16: 296-297Google ScholarA1067T+PIFerec et al., 11Ferec C. Audrezet M.-P. Mercier B. Guillermit H. Moullier P. Quiere I. Verlingue C. Nat. Genet. 1992; 1: 188-191Google ScholarG1069R+—aNot reported. PS/PI for R1070Q because of five patients analyzed, one was PS, one PI and three unspecified.Savov et al., 28Savov A. Mercier B. Kalaydjieva I. Ferec C. Hum. Mol. Genet. 1994; 3: 57-60Google ScholarR1070W+PSCasals et al., 4Casals T. Bassas L. Ruiz-Romero J. Chillon M. Gimenez J. Ramos M.D. Tapia G. Narvacz H. Nunes V. Estivill X. Hum. Genet. 1995; 95: 205-211Google ScholarR1070Q+PS/PIMercier et al., 25Mercier B. Lissens W. Novelli G. Kalaydjieva L. DeArce M. Kapranov N. Klain N. Lenoir G. Chauveau P. Lenaerts C. Rault G. Cashman S. Sangiuolo F. Audrezet M.P. Dallapiccola B. Guillermit H. Bonduelle M. Liebaers I. Quere I. Verlingue C. Ferec C. Genomics. 1993; 16: 296-297Google ScholarQ1071P−PIGhanem et al., 13Ghanem N. Costes B. Girodon E. Martin J. Fanen P. Goossens M. Genomics. 1994; 21: 434-436Google ScholarL1077P−PSBozon et al., 3Bozon D. Zielenski J. Rininsland F. Tsui L.-C. Hum. Mutat. 1994; 3: 330-332Google ScholarH1085R−PSMercier et al., 25Mercier B. Lissens W. Novelli G. Kalaydjieva L. DeArce M. Kapranov N. Klain N. Lenoir G. Chauveau P. Lenaerts C. Rault G. Cashman S. Sangiuolo F. Audrezet M.P. Dallapiccola B. Guillermit H. Bonduelle M. Liebaers I. Quere I. Verlingue C. Ferec C. Genomics. 1993; 16: 296-297Google ScholarW1098R−PIZielenski et al., 38Zielenski J. Markiewicz D. Chen H.S. Schappert K. Seller A. Durie P. Corey M. Tsui L.-C. Hum. Mutat. 1995; 5: 43-47Google ScholarM1101K−PIZielenski et al., 37Zielenski J. Fujiwara T.M. Markiewicz D. Paradis A.J. Anaeleto A.I. Richards B. Schwartz R.H. Klinger K.W. Tsui L.-C. Morgan K. Am. J. Hum. Genet. 1993; 52: 609-615Google ScholarM1101R−PIMercier et al., 25Mercier B. Lissens W. Novelli G. Kalaydjieva L. DeArce M. Kapranov N. Klain N. Lenoir G. Chauveau P. Lenaerts C. Rault G. Cashman S. Sangiuolo F. Audrezet M.P. Dallapiccola B. Guillermit H. Bonduelle M. Liebaers I. Quere I. Verlingue C. Ferec C. Genomics. 1993; 16: 296-297Google Scholara Not reported. PS/PI for R1070Q because of five patients analyzed, one was PS, one PI and three unspecified. Open table in a new tab Those amino acid substitutions that do not interfere with maturation have relatively minor effects on channel activity. Bulk measurements of cyclic AMP-stimulated iodide efflux from entire populations of cells expressing these variants showed that they did not differ drastically from wild type. Patch-clamp analysis demonstrated that the CFTR channel in all six processed mutants had similar current-voltage relationships and conductances as the wild-type channel, indicating that once the channel was open, chloride permeation was not altered. This suggests that despite their close proximity to the putative pore entrance, these residues do not contribute to interactions between the channel and the permeant anion. However, all of these mutant channels had decreased open probability. Their residual partial activity correlates well with a less severe phenotype in that two out of three of the fully processed were found in pancreatic-sufficient patients. The significance of the altered open probabilities is highlighted by the fact that each of the single amino acid substitutions in CL4 reduced the open probability of the channel, whereas deletion of 19 residues in CL2 had no effect on the open probability (Xie et al., 36Xie J. Drumm M.L. Ma J. Davis P.B. J. Biol. Chem. 1995; 270: 28084-28091Google Scholar). Generally, the decreased open probability of the CL4 mutants resulted from both a decrease in channel open time and an increase in closed time. An understanding of how residues Phe1052, Lys1060, Ala1067, Gly1069, and Arg1070 in the N-terminal half of CL4 affect channel opening and closing must await more information about the structure of the molecule. However, it seems reasonable to postulate that this segment may interact with one or more of the large regulatory cytoplasmic domains, possibly providing a link between these domains and the pore-forming parts of CFTR.Comparison of CFTR sequences among species shows that 13 of the 14 residues investigated in this study are 100% conserved (Diamond et al., 9Diamond G. Scanlin T.F. Zasloff M.A. Bevins C.L. J. Biol. Chem. 1991; 266: 22761-22769Google Scholar), consistent with the hypothesis that these amino acids are important to the proper folding and function of CFTR. Only Gly1069 is replaced by different residues in other species. Interestingly in murine CFTR, Gly1069 is replaced by Arg, a human mutation that we found to leave biosynthetic processing unaffected but to reduce channel open probability. Alignment of CFTR with a number of other traffic ATPases (Manavalan et al., 23Manavalan P. Smith A.E. McPherson J.M. J. Protein Chem. 1993; 12: 279-290Google Scholar) indicates that Arg1066 is the most conserved of the 14 residues studied in CL4. All three of the mutations reported at this position result in failure of maturation. The conservation of this residue may thus be a result of its importance for the proper biosynthetic processing of these proteins.In summary, it was found that 12 of the mutations identified in CL4 result in the CF phenotype due to misprocessing of CFTR, thus showing that point mutations in CL4 can perturb the overall structure of the molecule sufficiently to mark it for retention in the ER. Six mutations allowed the protein to mature, and in these, the CF phenotype is likely a consequence of the decreased channel open probability detected. The decrease in open probability could generally be attributed to an increase in the channel mean closed time and a decrease in the mean open time. This result together with the unaltered conductance suggests that mutations in CL4 do not affect pore properties of CFTR, but rather the mechanism of channel gating. Finally, these data provide the first functional evidence that this set of mutations probably does cause disease. INTRODUCTIONThe cystic fibrosis transmembrane conductance regulator (CFTR) 1The abbreviations used are: CFTRcystic fibrosis transmembrane conductance regulatorCFcystic fibrosisAMP-PNP5′-adenylylimidodiphosphateCHOChinese hamster ovaryCLcytoplasmic loopERendoplasmic reticulumNBFnucleotide binding foldPAGEpolyacrylamide gel electrophoresisTMtransmembrane helixTES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. is a chloride channel located in the apical membrane of many epithelia, where it plays a key role in the regulation of salt and water homeostasis (Welsh et al., 34Welsh M.J. Anderson M.P. Rich D.P. Berger H.A. Denning G.M. Ostedgaard L.S. Sheppard D.N. Cheng S.H. Gregory R.J. Smith A.E. Neuron. 1992; 8: 821-829Google Scholar). CFTR was predicted to form a transmembrane pore with regulatory segments protruding into the cell cytoplasm (Riordan et al., 27Riordan J.R. Rommens J.M. Kerem B. 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: 1066-1073Google Scholar); several features of this model have been confirmed experimentally (Hanrahan et al., 17Hanrahan J.W. Tabcharani J.A. Becq F. Mathews C.J. Augustinas O. Jensen T.J. Chang X.-B. Riordan J.R. Soc. Gen. Physiol. Ser. 1995; 50: 125-137Google Scholar). The channel consists of two structurally similar halves, each containing six transmembrane segments (TMs) and a nucleotide binding fold (NBF) with which ATP interacts. The two halves are connected by the R-domain, rich in consensus sequences for phosphorylation by several kinases, including cyclic AMP-dependent protein kinase and protein kinase C. On the cytoplasmically exposed side of the protein the TMs are linked to each other by cytoplasmic loops (CLs), which vary between 55 and 65 amino acids in length. There is evidence that the concerted action of ATP binding and ATP hydrolysis at the NBFs (Anderson et al., 2Anderson M.P. Berger H.A. Rich D.P. Gregory R.J. Smith A.E. Welsh M.J. Cell. 1991; 67: 775-784Google Scholar; Hwang et al., 20Hwang T.-C. Baukrowtiz G. Nagel G. Horie A.C. Gadsby D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4698-4702Google Scholar) and of phosphorylation at the R-domain (Cheng et al., 8Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.E. Cell. 1991; 66: 1027-1036Google Scholar; Chang et al., 5Chang X.B. Tabcharani J.A. Hou Y.X. Jensen T.J. Kartner N. Alon N. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1993; 268: 11304-11311Google Scholar) are necessary to allow channel activation, however, little information is available regarding the importance of the CLs in CFTR function. Only recently, a report by Xie et al. (36Xie J. Drumm M.L. Ma J. Davis P.B. J. Biol. Chem. 1995; 270: 28084-28091Google Scholar), which studied the effect of a 19-amino acid deletion in CL2 (residues 242–307), suggested that this region is involved in stabilizing the full conductance state of the CFTR chloride channel.The CLs are highly conserved between CFTRs expressed in different species (Diamond et al., 9Diamond G. Scanlin T.F. Zasloff M.A. Bevins C.L. J. Biol. Chem. 1991; 266: 22761-22769Google Scholar) and moderately conserved in comparison to the CLs of other ABC transporters (Manavalan et al., 23Manavalan P. Smith A.E. McPherson J.M. J. Protein Chem. 1993; 12: 279-290Google Scholar). Their possible significance is highlighted by the fact that 20% of all identified CF-associated mutations lie within CLs. Thus far in CF patients, 18 different point mutations have been described in exon 17b, which approximately corresponds to CL4 (amino acids 1035-1142); however, as yet the mechanistic impact of alterations in this segment of CFTR on its function and biosynthetic processing has not been investigated. The detailed analysis of amino acid substitutions in other CFTR domains has shown that the CF phenotype usually is attributable to either a lack of the channel at the cell surface or the production of a channel with impaired function (Welsh and Smith, 33Welsh M.J. Smith A.E. Cell. 1993; 73: 1251-1254Google Scholar). The failure of many mutant forms of CFTR, including the most frequent ΔF508 variant, to be biosynthetically processed beyond the core-glycosylated form present in the endoplasmic reticulum (ER), has become a hallmark of CFTR (Cheng et al., 7Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Google Scholar) and the focus of intense research. The aim of the present study was to examine the effects of mutations in CL4 on processing, intracellular transport, and chloride channel activity of CFTR.