title: A suppressor screen of the chimeric AtCNGC11/12 reveals residues important for intersubunit interactions of cyclic nucleotide-gated ion channels.
abstract: To investigate the structure-function relationship of plant cyclic nucleotide-gated ion channels (CNGCs), we identified a total of 29 mutant alleles of the chimeric AtCNGC11/12 gene that induces multiple defense responses in the Arabidopsis (Arabidopsis thaliana) mutant, constitutive expresser of PR genes22 (cpr22). Based on computational modeling, two new alleles, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), were identified as counterparts of human CNGA3 (a human CNGC) mutants. Both mutants lost all cpr22-mediated phenotypes. Transient expression in Nicotiana benthamiana as well as functional complementation in yeast (Saccharomyces cerevisiae) showed that both AtCNGC11/12:G459R and AtCNGC11/12:R381H have alterations in their channel function. Site-directed mutagenesis coupled with fast-protein liquid chromatography using recombinantly expressed C-terminal peptides indicated that both mutations significantly influence subunit stoichiometry to form multimeric channels. This observation was confirmed by bimolecular fluorescence complementation in planta. Taken together, we have identified two residues that are likely important for subunit interaction for plant CNGCs and likely for animal CNGCs as well.
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada M5S 3B2.
authors.forename: Huda
authors.initials: H
authors.lastname: Abdel-Hamid
authors.forename: Kimberley
authors.initials: K
authors.lastname: Chin
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Moeder
authors.forename: Dea
authors.initials: D
authors.lastname: Shahinas
authors.forename: Deepali
authors.initials: D
authors.lastname: Gupta
authors.forename: Keiko
authors.initials: K
authors.lastname: Yoshioka
mesh_terms: D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001120:Arginine; D001483:Base Sequence; D049108:Cell Enlargement; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016153:Genes, Suppressor; D005816:Genetic Complementation Test; D005998:Glycine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D029681:Arabidopsis Proteins; C518238:CNGA3 protein, human; C510425:CNGC11 protein, Arabidopsis; C510426:CNGC12 protein, Arabidopsis; D054815:Cyclic Nucleotide-Gated Cation Channels; D011994:Recombinant Proteins; D001120:Arginine; D005998:Glycine