Conformational rearrangements in the S6 domain and C-linker during gating in CNGA1 channels.
This work completes previous findings and, using cysteine scanning mutagenesis (CSM) and biochemical methods, provides detailed analysis of conformational changes of the S6 domain and C-linker during gating of CNGA1 channels. Specific residues between Phe375 and Val424 were mutated to a cysteine in the CNGA1 and CNGA1(cys-free) background and the effect of intracellular Cd(2+) or cross-linkers of different length in the open and closed state was studied. In the closed state, Cd(2+) ions inhibited mutant channels A406C and Q409C and the longer cross-linker reagent M-4-M inhibited mutant channels A406C(cys-free) and Q409C(cys-free). Cd(2+) ions inhibited mutant channels D413C and Y418C in the open state, both constructed in a CNGA1 and CNGA1(cys-free) background. Our results suggest that, in the closed state, residues from Phe375 to approximately Ala406 form a helical bundle with a three-dimensional (3D) structure similar to those of the KcsA; furthermore, in the open state, residues from Ser399 to Gln409 in homologous subunits move far apart, as expected from the gating in K(+) channels; in contrast, residues from Asp413 to Tyr418 in homologous subunits become closer in the open state.
Nair
Anil V
AV
International School for Advanced Studies, via Beirut 2-4, 34014, Trieste, Italy.
Nguyen
Chuong H H
CH
Mazzolini
Monica
M
D000595:Amino Acid Sequence; D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D002417:Cattle; D003432:Cross-Linking Reagents / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels; D042541:Intracellular Space / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009068:Movement; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000187:drug effects; D013439:Sulfhydryl Reagents / Q000494:pharmacology
C475741:CNGA1 protein, bovine; D003432:Cross-Linking Reagents; D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D013439:Sulfhydryl Reagents; D002104:Cadmium