Importance of the αC-helix in the cyclic nucleotide binding domain for the stable channel regulation and function of cyclic nucleotide gated ion channels in Arabidopsis

Abstract

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 αC-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 °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 αC-helix of the CNBD is important for channel regulation, but not for basic function. Furthermore, another suppressor mutant, S136 that lacks the entire αC-helix due to a premature stop codon, lost channel function completely. Our data presented here indicate that the αC-helix is functionally important in plant CNGCs.

Multiple Calmodulin-binding Sites Positively and Negatively Regulate Arabidopsis CYCLIC NUCLEOTIDE-GATED CHANNEL12

“…Low temperature is known to enhance immune responses in plants (Hua, 2013), and shifting to lower temperature has been previously shown to enhance the PCD phenotype induced by CNGC11/12 mutants (Mosher et al, 2010;Chin et al, 2010). We observed a similar effect in our assays, wherein incubation at 16°C increased the PCD induced by both of our NT mutant CNGC12 constructs (Supplemental Figure 5).…”

“…The structure of the CNGC12 C-linker and CNBD regions was previously modeled (Chin et al, 2010;Abdel-Hamid et al, 2013) based on homology to the mammalian channels SpIH and HCN2 (PDB ID: 2PTM and PDB ID:IQ50, respectively) (Flynn et al, 2007;Zagotta et al, 2003). Such models have provided important insight into CNGC structure-function; however, they could not include the C-terminal region downstream of the CNBD due to lack of homology to mammalian templates (i.e., amino acids ;565 to 649 in At-CNGC12).…”

“…The R577C mutation was previously found to suppress CNGC11/12 PCD induction at normal temperatures, while PCD was restored at low temperature (Chin et al, 2010). While we again observed CNGC11/12 R577C -induced PCD at lower temperature, 11/12-IQ mut failed to induce PCD even at low temperature (Supplemental Figure 8), further indicating that this IQ site mutation leads to a full loss-of-function phenotype.…”

“…Because the chimeric CNGC11/12 is hypothesized to comprise a misregulated form of CNGC12 and constitutively activates PCD, it has proven to be an excellent tool to study channel structurefunction (Baxter et al, 2008;Chin et al, 2010;Abdel-Hamid et al, 2013). CNGC11/12 possesses the C-terminal half of CNGC12 and therefore shares the CNGC12 IQ and CT motifs.…”

“…However, in contrast to animal isoforms, a single CaMBD was mapped to a site that overlaps the C-terminal a-helix (aC) of the CNBD (Arazi et al, 2000;Köhler and Neuhaus, 2000). This aC CaMBD is characterized by a four-residue, Trp-Arg-Thr-Trp (WRTW) motif required for CaM binding (Arazi et al, 2000), which is widely conserved across the Arabidopsis CNGC family (Chin et al, 2010). Previous studies have hypothesized that the location of the CaMBD within the CNBD allows CaM to compete with cyclic nucleotide monophosphate as a ligand in the allosteric gating of channel conductance (Hua et al, 2003;Kaplan et al, 2007;Swarbreck et al, 2013).…”