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Molecular and Genomic |
Departments of
1 Biochemistry and Biophysics
2 Cell and Developmental Biology
3 Biomedical Engineering, University of North Carolina, Chapel Hill, NC 27599, USA
4 Mayo Clinic Scottsdale, Scottsdale, AZ 85259, USA
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel employing the ABC transporter structural motif. Deletion of a single residue (Phe508) in the first nucleotide-binding domain (NBD1), which occurs in most patients with cystic fibrosis, impairs both maturation and function of the protein. However, substitution of the Phe508 with small uncharged amino acids, including cysteine, is permissive for maturation. To explore the possible role of the phenylalanine aromatic side chain in channel gating we introduced a cysteine at this position in cysless CFTR, enabling its selective chemical modification by sulfhydryl reagents. Both cysless and wild-type CFTR ion channels have identical mean open times when activated by different nucleotide ligands. Moreover, both channels could be locked in an open state by introducing an ATPase inhibiting mutation (E1371S). However, the introduction of a single cysteine (F508C) prevented the cysless E1371S channel from maintaining the permanently open state, allowing closing to occur. Chemical modification of cysless E1371S/F508C by sulfhydryl reagents was used to probe the role of the side chain in ion channel function. Specifically, benzyl-methanethiosulphonate modification of this variant restored the gating behaviour to that of cysless E1371S containing the wild-type phenylalanine at position 508. This provides the first direct evidence that a specific interaction of the Phe508 aromatic side chain plays a role in determining the residency time in the closed state. Thus, despite the fact that this aromatic side chain is not essential for CFTR folding, it is important in the ion channel function.
(Received 18 October 2005;
accepted after revision 14 February 2006;
first published online 16 February 2006)
Corresponding author A. A. Aleksandrov: University of North Carolina, 5009 Thurston Bowles Building, CB no. 7248, Chapel Hill, NC 27599, USA. Email: aleksand{at}email.unc.edu
This article has been cited by other articles:
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  L. He, A. A. Aleksandrov, A. W. R. Serohijos, T. Hegedus, L. A. Aleksandrov, L. Cui, N. V. Dokholyan, and J. R. Riordan Multiple Membrane-Cytoplasmic Domain Contacts in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mediate Regulation of Channel Gating J. Biol. Chem., September 26, 2008; 283(39): 26383 - 26390. [Abstract] [Full Text] [PDF]
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 A. W. R. Serohijos, T. Hegedus, A. A. Aleksandrov, L. He, L. Cui, N. V. Dokholyan, and J. R. Riordan Phenylalanine-508 mediates a cytoplasmic-membrane domain contact in the CFTR 3D structure crucial to assembly and channel function PNAS, March 4, 2008; 105(9): 3256 - 3261. [Abstract] [Full Text] [PDF]
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