A Suppressor Screen of the Chimeric AtCNGC11/12 Reveals Residues Important for Intersubunit Interactions of Cyclic Nucleotide-Gated Ion Channels

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.Cyclic nucleotide-gated ion channels (CNGCs) were first discovered in retinal photoreceptors and olfactory sensory neurons (Zagotta and Siegelbaum, 1996; Kaupp and Seifert, 2002). CNGCs play crucial roles for the signal transduction in these neurons that are excited by photons and odorants, respectively. In mammalian genomes, six CNGC genes have been found and named CNGA1 to CNGA4, CNGB1, and CNGB3 (Kaupp and Seifert, 2002). It has been reported that in mammalian cells, CNGCs function as heterotetramers that are composed of A and B subunits with cell-specific stoichiometry (Kaupp and Seifert, 2002; Cukkemane et al., 2011). For example, CNGCs in rod photoreceptors are composed of three A1 subunits and one B1a subunit, whereas in cone photoreceptors, they are believed to be composed of two A3 and two B3 subunits (Zhong et al., 2002; Peng et al., 2004). The structure of each subunit is similar to that of the voltage-gated K⁺-selective ion channel (Shaker) proteins, including a cytoplasmic N terminus, six membrane-spanning regions (S1–S6), a pore domain located between S5 and S6, and a cytoplasmic C terminus (Zagotta and Siegelbaum, 1996). However, CNGCs are only weakly voltage dependent and are opened by the direct binding of cyclic nucleotides (cAMP and cGMP), which are universally important secondary messengers that control diverse cellular responses (Fesenko et al., 1985). The cytoplasmic C terminus contains a cyclic nucleotide-binding domain (CNBD) and a C-linker region that connects the CNBD to the S6 domain. CNGC activity is also regulated by feedback inhibitory mechanisms involving the Ca²⁺ sensor protein, calmodulin (CaM). CaM-binding sites in animal CNGCs have been found in various regions of both the C- and N-terminal domains (Ungerer et al., 2011). It has been reported that the subunit composition has significant influence on the mode of CaM-mediated regulation (Kramer and Siegelbaum, 1992; Bradley et al., 2004; Song et al., 2008).On the other hand, plant CNGCs have only been investigated much more recently. The first plant CNGC, HvCBT1, was identified as a CaM-binding transporter protein in barley (Hordeum vulgare; Schuurink et al., 1998). Subsequently, several CNGCs were identified from Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum; Arazi et al., 1999; Köhler and Neuhaus, 1998; Köhler et al., 1999). Interestingly, the Arabidopsis genome sequencing project identified a large family comprising 20 members (AtCNGC1–AtCNGC20), indicating a significant expansion of Arabidopsis CNGCs that suggests a higher level of diversity and functional importance in plants (Mäser et al., 2001). To date, possible biological functions of Arabidopsis CNGCs in development, ion homeostasis, thermal sensing, as well as pathogen resistance have been reported (Kaplan et al., 2007; Chin et al., 2009; Dietrich et al., 2010; Moeder et al., 2011; Finka et al., 2012). With respect to structure, plant CNGCs are believed to have a similar architecture to their animal counterparts (Chin et al., 2009). However, only a handful of studies on the structure-function analysis of plant CNGCs have been published so far, and this field is still very much in its infancy (Hua et al., 2003; Bridges et al., 2005; Kaplan et al., 2007; Baxter et al., 2008; Chin et al., 2010).Previously, we have reported two functionally important residues in plant CNGCs (Baxter et al., 2008; Chin et al., 2010). These residues were discovered using a suppressor screen of the rare gain-of-function Arabidopsis mutant constitutive expresser of PR genes22 (cpr22; Yoshioka et al., 2006). The cpr22 mutant, which has a deletion between AtCNGC11 and AtCNGC12 resulting in a novel but functional chimeric CNGC (AtCNGC11/12), exhibits multiple resistance responses without pathogen infection in the hemizygous state and conditional lethality in the homozygous state (Yoshioka et al., 2001, 2006; Moeder et al., 2011). It has been reported that the cpr22 phenotype is attributable to the expression of AtCNGC11/12 and its channel activity (Yoshioka et al., 2006; Baxter et al., 2008), thereby making the suppressor screen an invaluable tool for identifying intragenic mutants to further elucidate the structure-function relationship of plant CNGCs (Baxter et al., 2008; Chin et al., 2010).In this study, we describe a total of 29 mutant alleles of AtCNGC11/12, including the three previously published alleles (Baxter et al., 2008; Chin at al., 2010), and compare their predicted three-dimensional structural positions with equivalent mutations of a human CNGC, CNGA3. In this analysis, two AtCNGC11/12 mutations emerged as counterparts of human mutations (Wissinger et al., 2001). Both the AtCNGC11/12 as well as the human CNGA3 mutations were computationally predicted to affect intersubunit interactions. This prediction was experimentally validated by size-exclusion chromatography (FPLC) as well as bimolecular fluorescence complementation (BiFC) in combination with site-direct mutagenesis using recombinant C-terminal peptides.