Identification of a domain on the beta-subunit of the rod cGMP-gated cation channel that mediates inhibition by calcium-calmodulin

J Biol Chem. 1998 Apr 10;273(15):9148-57. doi: 10.1074/jbc.273.15.9148.

Abstract

The cGMP-gated cation channel mediating phototransduction in retinal rods has recently been shown to be inhibited by calcium-calmodulin, through direct binding of the latter to the beta-subunit of the heterotetrameric channel complex. Here, we report the characterization of this inhibition and the identification of a domain crucial for this modulation. Heterologous expression of the alpha- and beta-subunits of the human rod channel in HEK 293 cells produced a cGMP-gated current that was highly sensitive to calcium-calmodulin, with half-maximal inhibition at approximately 4 nM. In biochemical and electrophysiological experiments on deletion mutants of the beta-subunit, we have identified a region on its cytoplasmic N terminus that binds calmodulin and is necessary for the calmodulin-mediated inhibition of the channel. However, in gel shift assays and fluorescence emission experiments, peptides derived from this region indicated a low calmodulin affinity, with dissociation constants of approximately 3-10 microM. On the C terminus, a region was also found to bind calmodulin, but it was likewise of low affinity, and its deletion did not abolish the calmodulin-mediated inhibition. We suggest that although the identified region on the N terminus of the beta-subunit is crucial for the calmodulin effect, other regions are likely to be involved as well. In this respect, the rod channel appears to differ from the olfactory cyclic nucleotide-gated channel, which is also modulated by calcium-calmodulin.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Calcium / pharmacology*
  • Calmodulin / pharmacology*
  • Cattle
  • Cloning, Molecular
  • Cyclic GMP / metabolism*
  • Cyclic GMP / pharmacology
  • Cyclic Nucleotide-Gated Cation Channels
  • Diltiazem / pharmacology
  • Egtazic Acid / pharmacology
  • Humans
  • Ion Channels / antagonists & inhibitors*
  • Ion Channels / chemistry*
  • Macromolecular Substances
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Retinal Rod Photoreceptor Cells / metabolism*
  • Sequence Alignment
  • Sequence Homology, Amino Acid

Substances

  • Calmodulin
  • Cyclic Nucleotide-Gated Cation Channels
  • Ion Channels
  • Macromolecular Substances
  • Peptide Fragments
  • Recombinant Proteins
  • Egtazic Acid
  • Diltiazem
  • Cyclic GMP
  • Calcium