Covalent modification of the nucleotide binding domains of cystic fibrosis transmembrane conductance regulator.
The cytosolic nucleotide binding domains of cystic fibrosis transmembrane conductance regulator (NBD1 and NBD2) mediate ATP-dependent opening and closing of the Cl- channel pore. To learn more about NBD structure and function, we introduced a cysteine residue into the Walker A motif or the LSGGQ motif of each NBD and examined modification by N-ethylmaleimide (NEM). Covalent modification of either Walker A motif partially inhibited cystic fibrosis transmembrane conductance regulator channel activity, decreasing the open state probability by prolonging the long closed duration. An increase in cytosolic ATP concentration slowed the rate of modification. The data suggest that both NBDs interact with ATP to influence channel opening and that inhibition by NEM modification was in part due to decreased ATP binding. When cysteine was placed in the NBD2 Walker A motif, it was modified more rapidly than when it was placed in NBD1, suggesting that the NBDs are not structurally or functionally identical. Modification of a cysteine inserted in the LSGGQ motif of either NBD1 or NBD2 also inhibited channel activity. The rate of modification was comparable with that of a thiol in free solution, suggesting that the LSGGQ motif resides in a surface-exposed position in both NBDs.
Cotten
J F
JF
Howard Hughes Medical Institute and Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Welsh
M J
MJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000502:physiology; D016384:Consensus Sequence; D003545:Cysteine; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000502:physiology*; D005033:Ethylmaleimide / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D008564:Membrane Potentials / Q000502:physiology; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C505032:CFTR protein, human; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D003545:Cysteine; D005033:Ethylmaleimide