The role of cystic fibrosis transmembrane conductance regulator phenylalanine 508 side chain in ion channel gating.
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.
Cui
Liying
L
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, 27599, USA.
Aleksandrov
Luba
L
Hou
Yue-Xian
YX
Gentzsch
Martina
M
Chen
Jey-Hsin
JH
Riordan
John R
JR
Aleksandrov
Andrei A
AA
D000200:Action Potentials / Q000502:physiology; D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D018118:Chloride Channels / Q000502:physiology; D006224:Cricetinae; D003545:Cysteine / Q000032:analysis; D003550:Cystic Fibrosis / Q000209:etiology / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006868:Hydrolysis; D015640:Ion Channel Gating / Q000502:physiology*; D008856:Microscopy, Fluorescence; D009154:Mutation; D010649:Phenylalanine / Q000032:analysis*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D018118:Chloride Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D010649:Phenylalanine; D000255:Adenosine Triphosphate; D003545:Cysteine