Conformational rearrangements in the S6 domain and C-linker during gating in CNGA1 channels

Eur Biophys J. 2009 Sep;38(7):993-1002. doi: 10.1007/s00249-009-0491-4. Epub 2009 Jun 2.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cadmium / pharmacology
  • Cattle
  • Cross-Linking Reagents / pharmacology
  • Cyclic Nucleotide-Gated Cation Channels
  • Intracellular Space / metabolism
  • Ion Channel Gating* / drug effects
  • Ion Channels / antagonists & inhibitors
  • Ion Channels / chemistry*
  • Ion Channels / genetics
  • Ion Channels / metabolism*
  • Molecular Sequence Data
  • Movement
  • Mutation
  • Protein Structure, Tertiary / drug effects
  • Sulfhydryl Reagents / pharmacology

Substances

  • CNGA1 protein, bovine
  • Cross-Linking Reagents
  • Cyclic Nucleotide-Gated Cation Channels
  • Ion Channels
  • Sulfhydryl Reagents
  • Cadmium