Identification of a functionally essential amino acid for Arabidopsis cyclic nucleotide gated ion channels using the chimeric AtCNGC11/12 gene

Plant J. 2008 Nov;56(3):457-69. doi: 10.1111/j.1365-313X.2008.03619.x. Epub 2008 Aug 14.

Abstract

We used the chimeric Arabidopsis cyclic nucleotide-gated ion channel AtCNGC11/12 to conduct a structure-function study of plant cyclic nucleotide-gated ion channels (CNGCs). AtCNGC11/12 induces multiple pathogen resistance responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22). A genetic screen for mutants that suppress cpr22-conferred phenotypes identified an intragenic mutant, #73, which has a glutamate to lysine substitution (E519K) at the beginning of the eighth beta-sheet of the cyclic nucleotide-binding domain in AtCNGC11/12. The #73 mutant is morphologically identical to wild-type plants and has lost cpr22-related phenotypes including spontaneous cell death and enhanced pathogen resistance. Heterologous expression analysis using a K(+)-uptake-deficient yeast mutant revealed that this Glu519 is important for AtCNGC11/12 channel function, proving that the occurrence of cpr22 phenotypes requires active channel function of AtCNGC11/12. Additionally, Glu519 was also found to be important for the function of the wild-type channel AtCNGC12. Computational structural modeling and in vitro cAMP-binding assays suggest that Glu519 is a key residue for the structural stability of AtCNGCs and contributes to the interaction of the cyclic nucleotide-binding domain and the C-linker domain, rather than the binding of cAMP. Furthermore, a mutation in the alpha-subunit of the human cone receptor CNGA3 that causes total color blindness aligned well to the position of Glu519 in AtCNGC11/12. This suggests that AtCNGC11/12 suppressors could be a useful tool for discovering important residues not only for plant CNGCs but also for CNGCs in general.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / chemistry*
  • Amino Acids / genetics
  • Arabidopsis / chemistry
  • Arabidopsis / genetics*
  • Arabidopsis Proteins / chemistry*
  • Arabidopsis Proteins / genetics
  • Crosses, Genetic
  • Cyclic Nucleotide-Gated Cation Channels / chemistry*
  • Cyclic Nucleotide-Gated Cation Channels / genetics
  • Genes, Plant
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis
  • Mutant Chimeric Proteins / chemistry
  • Mutant Chimeric Proteins / genetics
  • Mutation
  • Phenotype
  • Plants, Genetically Modified / chemistry
  • Plants, Genetically Modified / genetics
  • Plasmids
  • Protein Structure, Secondary
  • RNA, Plant / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / genetics
  • Sequence Alignment
  • Structure-Activity Relationship
  • Tobacco / chemistry
  • Tobacco / genetics

Substances

  • Amino Acids
  • Arabidopsis Proteins
  • CNGC11 protein, Arabidopsis
  • CNGC12 protein, Arabidopsis
  • Cyclic Nucleotide-Gated Cation Channels
  • Mutant Chimeric Proteins
  • RNA, Plant