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.

Signaling with Ions: The Keystone for Apical Cell Growth and Morphogenesis in Pollen Tubes

“…Lastly, AtCNGC16 only plays a role under stress conditions (Tunc-Ozdemir et al, 2013b). In animals, CNGCs have calmodulin (CaM)-binding domains with regulatory functions, a mechanism recently confirmed for AtCNGC12 in plants (DeFalco et al, 2016). In tobacco, the Glutamate receptor agonist D-Ser induces a Ca 2+ current while the antagonist CNQX inhibits the Ca 2+ conductivity of PT protoplasts (Michard et al, 2011).…”

“…More recently, a function was identified for AtCML24 in PT growth, acting on the interface between the actin cytoskeleton and Ca 2+ . Given the relevance of AtCNGCs in PT growth, the recent description of CaM regulation of AtCNGC12 (DeFalco et al, 2016) opens the possibility that such a protein also may also be involved as a decoder of Ca 2+ signals in PTs, as previously suggested by injection experiments (Moutinho et al, 1998;Rato et al, 2004).…”

Calcium transport across plant membranes: mechanisms and functions

“…Using CNGC12 as a model, DeFalco et al . () found that this channel has three CaMBSs, allowing [Ca 2+ ] cyt. to be maintained at the basal concentration ( c .…”

“…According to DeFalco et al . () CNGC 12 has three Ca MBS s – one in the N‐terminus ( NT domain), and two in the C‐terminus ( CT and IQ domains). The NT and CT domains did not bind free CaM but they do show high affinity for CaM–Ca 2+ complexes ( K d of 7 and 34 nM, respectively).…”

“…Recent data reveal that plant CNGC subunits possess more CaMBS than initially shown (Fischer et al ., , ; DeFalco et al ., ,b). CNGC20 and potentially some other CNGCs do not show CNBD overlapping with CaMBSs.…”