title: Multiple Calmodulin-Binding Sites Positively and Negatively Regulate Arabidopsis CYCLIC NUCLEOTIDE-GATED CHANNEL12.
abstract: Ca(2+) signaling is critical to plant immunity; however, the channels involved are poorly characterized. Cyclic nucleotide-gated channels (CNGCs) are nonspecific, Ca(2+)-permeable cation channels. Plant CNGCs are hypothesized to be negatively regulated by the Ca(2+) sensor calmodulin (CaM), and previous work has focused on a C-terminal CaM-binding domain (CaMBD) overlapping with the cyclic nucleotide binding domain of plant CNGCs. However, we show that the Arabidopsis thaliana isoform CNGC12 possesses multiple CaMBDs at cytosolic N and C termini, which is reminiscent of animal CNGCs and unlike any plant channel studied to date. Biophysical characterizations of these sites suggest that apoCaM interacts with a conserved isoleucine-glutamine (IQ) motif in the C terminus of the channel, while Ca(2+)/CaM binds additional N- and C-terminal motifs with different affinities. Expression of CNGC12 with a nonfunctional N-terminal CaMBD constitutively induced programmed cell death, providing in planta evidence of allosteric CNGC regulation by CaM. Furthermore, we determined that CaM binding to the IQ motif was required for channel function, indicating that CaM can both positively and negatively regulate CNGC12. These data indicate a complex mode of plant CNGC regulation by CaM, in contrast to the previously proposed competitive ligand model, and suggest exciting parallels between plant and animal channels.
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3B2, Canada.
authors.forename: Thomas A
authors.identifier: 0000-0003-2897-1485
authors.initials: TA
authors.lastname: DeFalco
authors.affiliation: Department of Medical Biophysics, Campbell Family Cancer Research Institute/Princess Margaret Cancer Centre, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Marshall
authors.affiliation: Protein Function Discovery Facility, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Kim
authors.initials: K
authors.lastname: Munro
authors.affiliation: Department of Biology, Texas State University, San Marcos, Texas 78666.
authors.forename: Hong-Gu
authors.identifier: 0000-0002-5842-8945
authors.initials: HG
authors.lastname: Kang
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3B2, Canada.
authors.forename: Wolfgang
authors.identifier: 0000-0003-3889-6183
authors.initials: W
authors.lastname: Moeder
authors.affiliation: Department of Medical Biophysics, Campbell Family Cancer Research Institute/Princess Margaret Cancer Centre, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
authors.forename: Mitsuhiko
authors.initials: M
authors.lastname: Ikura
authors.affiliation: Department of Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Wayne A
authors.identifier: 0000-0001-9564-3057
authors.initials: WA
authors.lastname: Snedden
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3B2, Canada Center for the Analysis of Genome Evolution and Function (CAGEF), University of Toronto, Toronto, Ontario M5S 3B2, Canada keiko.yoshioka@utoronto.ca.
authors.forename: Keiko
authors.identifier: 0000-0002-3797-4277
authors.initials: K
authors.lastname: Yoshioka
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000235:genetics / Q000378:metabolism*; D057865:Plant Immunity / Q000235:genetics / Q000502:physiology; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology
chemical_list: D029681:Arabidopsis Proteins; C510426:CNGC12 protein, Arabidopsis; D002147:Calmodulin; D054815:Cyclic Nucleotide-Gated Cation Channels; D002118:Calcium