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Mutations at Arginine 352 Alter the Pore Architecture of CFTR

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Abstract

Arginine 352 (R352) in the sixth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) previously was reported to form an anion/cation selectivity filter and to provide positive charge in the intracellular vestibule. However, mutations at this site have nonspecific effects, such as inducing susceptibility of endogenous cysteines to chemical modification. We hypothesized that R352 stabilizes channel structure and that charge-destroying mutations at this site disrupt pore architecture, with multiple consequences. We tested the effects of mutations at R352 on conductance, anion selectivity and block by the sulfonylurea drug glipizide, using recordings of wild-type and mutant channels. Charge-altering mutations at R352 destabilized the open state and altered both selectivity and block. In contrast, R352K-CFTR was similar to wild-type. Full conductance state amplitude was similar to that of wild-type CFTR in all mutants except R352E, suggesting that R352 does not itself form an anion coordination site. In an attempt to identify an acidic residue that may interact with R352, we found that permeation properties were similarly affected by charge-reversing mutations at D993. Wild-type-like properties were rescued in R352E/D993R-CFTR, suggesting that R352 and D993 in the wild-type channel may interact to stabilize pore architecture. Finally, R352A-CFTR was sensitive to modification by externally applied MTSEA+, while wild-type and R352E/D993R-CFTR were not. These data suggest that R352 plays an important structural role in CFTR, perhaps reflecting its involvement in forming a salt bridge with residue D993.

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References

  • Abbott DW, Boraston AB (2007) Specific recognition of saturated and 4,5-unsaturated hexuronate sugars by a periplasmic binding protein involved in pectin catabolism. J Mol Biol 369:759–770

    Article  PubMed  CAS  Google Scholar 

  • Anderson MP, Gregory RJ, Thompson S, Souza DW, Paul S, Mulligan RC, Smith AE, Welsh MJ (1991) Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science 253:202–205

    Article  PubMed  CAS  Google Scholar 

  • Audrézet M-P, Mercier B, Guillermit H, Quere I, Verlingue C, Rault G, Ferec C (1993) Identification of 12 novel mutations in the CFTR gene. Hum Mol Genet 2:51–54

    Article  PubMed  Google Scholar 

  • Baukrowitz T, Hwang T-C, Nairn AC, Gadsby DC (1994) Coupling of CFTR Cl– channel gating to an ATP hydrolysis cycle. Neuron 12:473–482

    Article  PubMed  CAS  Google Scholar 

  • Brancolini V, Cremonesi L, Belloni E, Pappalardo E, Bordoni R, Seia M, Rady M, Russo MP, Romeo G, Devoto M (1995) Search for mutations in pancreatic sufficient cystic fibrosis Italian patients: detection of 90% of molecular defects and identification of three novel mutations. Hum Genet 96:312–318

    Article  PubMed  CAS  Google Scholar 

  • Braun V, Herrmann C (2007) Docking of the periplasmic FecB binding protein to the FecCD transmembrane proteins in the ferric citrate transport system of Escherichia coli. J Bacteriol 189:6913–6918

    Article  PubMed  CAS  Google Scholar 

  • Chen JM, Cutler C, Jacques C, Boefu G, Denamur E, Lecointre G, Mercier B, Cramb G, Férec C (2001) A combined analysis of the cystic fibrosis transmembrane conductance regulator: implications for structure and disease models. Mol Biol Evol 18:1771–1788

    PubMed  CAS  Google Scholar 

  • Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, White GA, O’Riordan CR, Smith AE (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63:827–834

    Article  PubMed  CAS  Google Scholar 

  • Cheng SH, Rich DP, Marshall J, Gregory RJ, Welsh MW, Smith AE (1991) Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel. Cell 66:1027–1036

    Article  PubMed  CAS  Google Scholar 

  • Cheung M, Akabas MH (1997) Locating the anion-selectivity filter of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. J Gen Physiol 109:289–299

    Article  PubMed  CAS  Google Scholar 

  • Claydon TW, Makary SY, Dibb KM, Boyett MR (2003) The selectivity filter may act as the agonist-activated gate in the G protein-activated Kir3.1/Kir3.4 K+ channel. J Biol Chem 278:50654–50663

    Article  PubMed  CAS  Google Scholar 

  • Cotten JF, Welsh MJ (1999) Cystic fibrosis-associated mutations at arginine 347 alter the pore architecture for CFTR: evidence for disruption of a salt bridge. J Biol Chem 274:5429–5435

    Article  PubMed  CAS  Google Scholar 

  • Craven KB, Zagotta WN (2004) Salt bridges and gating in the COOH-terminal region of HCN2 and CNGA1 channels. J Gen Physiol 124:663–677

    Article  PubMed  CAS  Google Scholar 

  • Creighton TE (1993) Proteins: structure and molecular properties. WH Freeman, New York

    Google Scholar 

  • Cremonesi L, Ferrari M, Belloni E, Magnani C, Seia M, Ronchetto P, Rady M, Russo MP, Romeo G, Devoto M (1992) Four new mutations of the CFTR gene (541delC, R347H, R352Q, E585X) detected by DGGE analysis in Italian CF patients, associated with different clinical phenotypes. Hum Mutat 1:314–319

    Article  PubMed  CAS  Google Scholar 

  • Dean M, Homon Y, Chimini G (2001) The human ATP-binding cassette (ABC) transporter superfamily. J Lipid Res 42:1007–1017

    PubMed  CAS  Google Scholar 

  • Devidas S, Guggino WB (1997) CFTR: domains, structure, and function. J Bioenerg Biomemb 29:443–451

    Article  CAS  Google Scholar 

  • Feldmann D, Couderc R, Audrézet M-P, Ferec C, Bienvenu T, Desgeorges M, Claustres M, Mittre H, Blayau M, Bozon D, Malinge MC, Monnier M, Bonnefont JP, Iron A, Bieth E, Dumur V, Clavel C, Cazeneuve C, Girodon E (2003) CFTR genotypes in patients with normal or borderline sweat chloride levels. Hum Mutat 22:340

    Article  PubMed  CAS  Google Scholar 

  • Gadsby DC, Vergani P, Csanády L (2006) The ABC protein turned chloride channel whose failure causes cystic fibrosis. Nature 440:477–483

    Article  PubMed  CAS  Google Scholar 

  • Ge N, Muise C, Gong X, Linsdell P (2004) Direct comparison of the functional roles played by different transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore. J Biol Chem 279:55283–55289

    Article  PubMed  CAS  Google Scholar 

  • Glaser F, Steinberg DM, Vakser IA, Ben-Tal N (2001) Residue frequencies and pairing preferences at protein–protein interfaces. Proteins 43:89–102

    Article  PubMed  CAS  Google Scholar 

  • Guggino WB, Stanton BA (2006) New insights into cystic fibrosis: molecular switches that regulate CFTR. Nat Rev Mol Cell Biol 7:426–436

    Article  PubMed  CAS  Google Scholar 

  • Guinamard R, Akabas MH (1999) Arg352 is a major determinant of charge selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel. Biochemistry 38:5528–5537

    Article  PubMed  CAS  Google Scholar 

  • Hwang T-C, Nagel G, Nairn AC, Gadsby DC (1994) Regulation of the gating of cystic fibrosis transmembrane conductance regulator Cl channels by phosphorylation and ATP hydrolysis. Proc Natl Acad Sci USA 91:4698–4702

    Article  PubMed  CAS  Google Scholar 

  • King SC, Hansen CL, Wilson TH (1991) The interaction between aspartic acid 237 and lysine 358 in the lactose carrier of Escherichia coli. Biochim Biophys Acta 1062:177–186

    Article  PubMed  CAS  Google Scholar 

  • Kitaoka S, Wada K, Hasegawa Y, Minami Y, Fukuyama K, Takahashi Y (2006) Crystal structure of Escherichia coli SufC, an ABC-type ATPase component of the SUF iron–sulfur cluster assembly machinery. FEBS Lett 580:137–143

    Article  PubMed  CAS  Google Scholar 

  • Linsdell P (2001) Thiocyanate as a probe of the cystic fibrosis transmembrane conductance regulator chloride channel pore. Can J Physiol Pharmacol 79:573–579

    Article  PubMed  CAS  Google Scholar 

  • Linsdell P (2005) Location of a common inhibitor binding site in the cytoplasmic vestibule of the cystic fibrosis transmembrane conductance regulator chloride channel pore. J Biol Chem 280:8945–8950

    Article  PubMed  CAS  Google Scholar 

  • Linsdell P (2006) Mechanism of chloride permeation in the cystic fibrosis transmembrane conductance regulator chloride channel. Exp Physiol 91:123–129

    Article  PubMed  CAS  Google Scholar 

  • Linsdell P, Hanrahan JW (1996a) Disulphonic stilbene block of cystic fibrosis transmembrane conductance regulator Cl− channels expressed in a mammalian cell line, and its regulation by a critical pore residue. J Physiol 496:687–693

    PubMed  CAS  Google Scholar 

  • Linsdell P, Hanrahan JW (1996b) Flickery block of single CFTR chloride channels by intracellular anions and osmolytes. Am J Physiol 271:C628–C634

    PubMed  CAS  Google Scholar 

  • Linsdell P, Tabcharani JA, Rommens JM, Hou Y-X, Chang X-B, Tsui L-C, Riordan JR, Hanrahan JW (1997) Permeability of wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels to polyatomic anions. J Gen Physiol 110:355–364

    Article  PubMed  CAS  Google Scholar 

  • Linsdell P, Evagelidis A, Hanrahan JW (2000) Molecular determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel pore. Biophys J 78:2973–2982

    PubMed  CAS  Google Scholar 

  • Liu NZ, Delcour AH (1998) The spontaneous gating activity of OmpC porin is affected by mutations of a putative hydrogen bond network or of a salt bridge between the L3 loop and the barrel. Protein Eng 11:797–802

    Article  PubMed  CAS  Google Scholar 

  • Liu X, Zhang Z-R, Fuller MD, Billingsley J, McCarty NA, Dawson DC (2004) CFTR: a cysteine at position 338 in TM6 senses a positive electrostatic potential in the pore. Biophys J 87:3826–3841

    Article  PubMed  CAS  Google Scholar 

  • McCarty NA (2000) Permeation through the CFTR chloride channel. J Exp Biol 203:1947–1962

    PubMed  CAS  Google Scholar 

  • McCarty NA, Zhang Z-R (2001) Identification of a region of strong discrimination in the pore of CFTR. Am J Physiol 281:L852–L867

    CAS  Google Scholar 

  • McDonough S, Davidson N, Lester HA, McCarty NA (1994) Novel pore-lining residues in CFTR that govern permeation and open-channel block. Neuron 13:623–634

    Article  PubMed  CAS  Google Scholar 

  • Perutz MF (1978) Electrostatic effects in proteins. Science 201:1187–1191

    Article  PubMed  CAS  Google Scholar 

  • Qin F, Auerbach A, Sachs F (1996) Estimating single-channel kinetic parameters from idealized patch clamp data containing missed events. Biophys J 70:264–280

    Article  PubMed  CAS  Google Scholar 

  • Ramjeesingh M, Li C, Kogan I, Wang Y, Huan L-J, Bear CE (2001) A monomer is the minimum function unit required for channel and ATPase activity of the cystic fibrosis transmembrane conductance regulator. Biochemistry 40:10700–10706

    Article  PubMed  CAS  Google Scholar 

  • Ramjeesingh M, Ugwu F, Li C, Dhani S, Huan L-J, Wang Y, Bear CE (2003) Stable dimeric assembly of the second membrane-spanning domain of CFTR (cystic fibrosis transmembrane conductance regulator) reconstitutes a chloride-selective pore. Biochemistry 375:633–641

    Article  CAS  Google Scholar 

  • Riordan JR, Rommens JM, Kerem B-S, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou J-L, Drumm ML, Iannuzzi MC, Collins FS, Tsui L-C (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066–1072

    Article  PubMed  CAS  Google Scholar 

  • Schultz BD, DeRoos ADG, Venglarik CJ, Singh AK, Frizzell RA, Bridges RJ (1996) Glibenclamide blockade of CFTR chloride channels. Am J Physiol 271:L192–L200

    PubMed  CAS  Google Scholar 

  • Sharp KA, Honig B (1990) Electrostatic interactions in macromolecules: theory and applications. Annu Rev Biophys Biophys Chem 19:301–332

    Article  PubMed  CAS  Google Scholar 

  • Sheppard DN, Robinson KA (1997) Mechanism of glibenclamide inhibition of cystic fibrosis transmembrane conductance regulator Cl− channels expressed in a murine cell line. J Physiol 503:333–345

    Article  PubMed  CAS  Google Scholar 

  • Sheppard DN, Welsh MJ (1992) Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents. J Gen Physiol 100:573–592

    Article  PubMed  CAS  Google Scholar 

  • Sheppard DN, Ostedgaard LS, Rich DP, Welsh MJ (1994) The amino-terminal portion of CFTR forms a regulated Cl− channel. Cell 76:1091–1098

    Article  PubMed  CAS  Google Scholar 

  • Sheppard DN, Travis SM, Ishihara H, Welsh MJ (1996) Contributions of proline residues in the membrane-spanning domains of the cystic fibrosis transmembrane conductance regulator to chloride channel function. J Biol Chem 271:14995–15001

    Article  PubMed  CAS  Google Scholar 

  • Smith SS, Steinle ED, Meyerhoff ME, Dawson DC (1999) Cystic fibrosis transmembrane conductance regulator: physical basis for lyotropic anion selectivity patterns. J Gen Physiol 114:799–817

    Article  PubMed  CAS  Google Scholar 

  • Smith SS, Liu X, Zhan Z-R, Sun F, Kriewall TE, McCarty NA, Dawson DC (2001) CFTR: covalent and noncovalent modification suggests a role for fixed charges in anion conduction. J Gen Physiol 118:407–431

    Article  PubMed  CAS  Google Scholar 

  • St. Aubin CN, Linsdell P (2006) Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel. J Gen Physiol 128:535–545

    Article  CAS  Google Scholar 

  • Stockner T, Vogel HJ, Tieleman DP (2005) A salt-bridge motif involved in ligand binding and large-scale domain motions of the maltose-binding protein. Biophys J 89:3362–3371

    Article  PubMed  CAS  Google Scholar 

  • Tabcharani JA, Rommens JM, Hou Y-X, Chang X-B, Tsui L-C, Riordan JR, Hanrahan JW (1993) Multi-ion pore behavior in the CFTR chloride channel. Nature 366:79–82

    Article  PubMed  CAS  Google Scholar 

  • Tabcharani JA, Linsdell P, Hanrahan JW (1997) Halide permeation in wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels. J Gen Physiol 110:341–351

    Article  PubMed  CAS  Google Scholar 

  • Tector M, Hartl FU (1999) An unstable transmembrane segment in the cystic fibrosis transmembrane conductance regulator. EMBO J 18:6290–6298

    Article  PubMed  CAS  Google Scholar 

  • Tsui L-C, Zielenski J, O’Brien A (2007) Cystic Fibrosis Mutation Database, http://www.genet.sickkids.on.ca/cftr/Home.html

  • Yang J, Yu M, Jan YN, Jan LY (1997) Stabilization of ion selectivity filter by pore loop pairs in an inwardly rectifying potassium channel. Proc Natl Acad Sci USA 94:1568–1572

    Article  PubMed  CAS  Google Scholar 

  • Yue H, Devidas S, Guggino WB (2000) The two halves of CFTR form a dual-pore ion channel. J Biol Chem 275:10030–10034

    Article  PubMed  CAS  Google Scholar 

  • Zaitseva J, Oswald C, Jumpertz T, Jenewein S, Wiedenmann A, Holland IB, Schmitt L (2006) A structural analysis of asymmetry required for catalytic activity of an ABC-ATPase domain dimer. EMBO J 25:3432–3443

    Article  PubMed  CAS  Google Scholar 

  • Zhang Z-R, McDonough SI, McCarty NA (2000) Interaction between permeation and gating in a putative pore-domain mutant in CFTR. Biophys J 79:298–313

    PubMed  CAS  Google Scholar 

  • Zhang Z-R, Zeltwanger S, Smith SS, Dawson DC, McCarty NA (2001) Voltage-sensitive gating induced by a mutation in the fifth transmembrane domain of CFTR. Am J Physiol 282:L135–L145

    Google Scholar 

  • Zhang Z-R, Cui G, Zeltwanger S, McCarty NA (2004a) Time-dependent interactions of glibenclamide with CFTR: kinetically complex block of macroscopic currents. J Membr Biol 201:139–155

    Article  PubMed  CAS  Google Scholar 

  • Zhang Z-R, Zeltwanger S, McCarty NA (2004b) Steady-state interactions of glibenclamide with CFTR: evidence for multiple sites. J Membr Biol 199:15–28

    Article  PubMed  CAS  Google Scholar 

  • Zhang Z-R, Cui G, Liu X, Song B, Dawson DC, McCarty NA (2005a) Determination of the functional unit of the cystic fibrosis transmembrane conductance regulator chloride channel: one polypeptide forms one pore. J Biol Chem 280:458–468

    PubMed  CAS  Google Scholar 

  • Zhang Z-R, Song B, McCarty NA (2005b) State-dependent chemical reactivity of an engineered cysteine reveals conformational changes in the outer vestibule of CFTR. J Biol Chem 280:41997–42003

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We thank Chris Thompson for comments. This work was supported by the National Institute for Diabetes, Digestive and Kidney Diseases (DK056481 to N. A. M.) and the Cystic Fibrosis Foundation (MCCART07P0). During the performance of these studies, N. A. M. was an Established Investigator of the American Heart Association.

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  1. School of Biology, Georgia Institute of Technology, Atlanta, GA, 30332-0230, USA

    Guiying Cui, Zhi-Ren Zhang, Andrew R. W. O’Brien, Binlin Song & Nael A. McCarty

  2. Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Emory University School of Medicine, 2015 Uppergate Drive, Atlanta, GA, 30322, USA

    Guiying Cui & Nael A. McCarty

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  1. Guiying Cui
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Correspondence to Nael A. McCarty.

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Cui, G., Zhang, ZR., O’Brien, A.R.W. et al. Mutations at Arginine 352 Alter the Pore Architecture of CFTR. J Membrane Biol 222, 91–106 (2008). https://doi.org/10.1007/s00232-008-9105-9

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