Identification of a functionally essential amino acid for Arabidopsis cyclic nucleotide gated ion channels using the chimeric AtCNGC11/12 gene.
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.
Baxter
Joyce
J
Department of Cell and Systems Biology, University of Toronto, 25 Willcocks Street, Toronto, ON M5S 3B2, Canada.
Moeder
Wolfgang
W
Urquhart
William
W
Shahinas
Dea
D
Chin
Kimberley
K
Christendat
Dinesh
D
Kang
Hong-Gu
HG
Angelova
Magdalena
M
Kato
Naohiro
N
Yoshioka
Keiko
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics; D003433:Crosses, Genetic; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000737:chemistry* / Q000235:genetics; D017343:Genes, Plant; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050506:Mutant Chimeric Proteins / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D010641:Phenotype; D030821:Plants, Genetically Modified / Q000737:chemistry / Q000235:genetics; D010957:Plasmids; D017433:Protein Structure, Secondary; D018749:RNA, Plant / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014026:Nicotiana / Q000737:chemistry / Q000235:genetics
D000596:Amino Acids; D029681:Arabidopsis Proteins; C510425:CNGC11 protein, Arabidopsis; C510426:CNGC12 protein, Arabidopsis; D054815:Cyclic Nucleotide-Gated Cation Channels; D050506:Mutant Chimeric Proteins; D018749:RNA, Plant