Importance of the alphaC-helix in the cyclic nucleotide binding domain for the stable channel regulation and function of cyclic nucleotide gated ion channels in Arabidopsis.
The involvement of cyclic nucleotide gated ion channels (CNGCs) in the signal transduction of animal light and odorant perception is well documented. Although plant CNGCs have recently been revealed to mediate multiple stress responses and developmental pathways, studies that aim to elucidate their structural and regulatory properties are still very much in their infancy. The structure-function relationship of plant CNGCs was investigated here by using the chimeric Arabidopsis AtCNGC11/12 gene that induces multiple defence responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22) for the identification of functionally essential residues. A genetic screen for mutants that suppress cpr22-conferred phenotypes identified over 20 novel mutant alleles in AtCNGC11/12. One of these mutants, suppressor S58 possesses a single amino acid substitution, arginine 557 to cysteine, in the alphaC-helix of the cyclic nucleotide-binding domain (CNBD). The suppressor S58 lost all cpr22 related phenotypes, such as spontaneous cell death formation under ambient temperature conditions. However, these phenotypes were recovered at 16 degrees C suggesting that the stability of channel function is affected by temperature. In silico modelling and site-directed mutagenesis analyses suggest that arginine 557 in the alphaC-helix of the CNBD is important for channel regulation, but not for basic function. Furthermore, another suppressor mutant, S136 that lacks the entire alphaC-helix due to a premature stop codon, lost channel function completely. Our data presented here indicate that the alphaC-helix is functionally important in plant CNGCs.
Chin
Kimberley
K
Department of Cell and Systems Biology, University of Toronto, 25 Willcocks Street, Toronto, ON, M5S 3B2 Canada.
Moeder
Wolfgang
W
Abdel-Hamid
Huda
H
Shahinas
Dea
D
Gupta
Deepali
D
Yoshioka
Keiko
K
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D029681:Arabidopsis Proteins; C510425:CNGC11 protein, Arabidopsis; C510426:CNGC12 protein, Arabidopsis; D054815:Cyclic Nucleotide-Gated Cation Channels