The Arabidopsis thaliana genome encodes twenty putative cyclic nucleotide-gated channel (CNGC) genes. Studies on A. thaliana CNGCs so far have revealed their ability to selectively transport cations that play a role in various stress responses and development, however, the regulation of plant CNGCs is not yet fully understood. Thus, in this study I have attempted to analyze the structure-function relationship of AtCNGCs, mainly by using suppressor mutants of the rare gain-of function mutant, cpr22. The A. thaliana mutant cpr22 resulted from an approximately 3kb deletion that fused the 5’ half and the 3’ half of two CNGC-encoding genes, AtCNGC11 and AtCNGC12, respectively. The expression of this chimeric CNGC, the AtCNGC11/12 gene confers easily detectable characteristics such as stunted morphology with curly leaves and hypersensitive response-like spontaneous lesion formation. Through a suppressor screen, twenty nine new alleles were identified in AtCNGC11/12. Since the cytosolic C-terminal region contains important regulatory domains, such as a cyclic-nucleotide binding domain, eleven cytosolic C-terminal mutants, S17, S35, S81, S83, S84, S100, S135, S136, S137, S140 and S144, were analyzed. A detailed analysis of two mutants, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), suggested that G459 and R381 are important for basic channel function rather than channel regulation. Site-directed mutagenesis and fast protein liquid chromatography (FPLC) showed that these two amino acids influence both intra- and inter-subunit interactions that are involved in stabilizing the tertiary structure of the channel. In addition, calmodulin binding domain(s) (CaMBD) and cyclic nucleotide binding domain(s) (CNBD) of some of AtCNGCs were studied using computational modeling and biophysical analyses. The data indicated that AtCNGC12 has two CaMBDs in both N- and C- cytosolic termini, whereas AtCNGC11 has only one CaMBD located in the N-terminal region of the channel. In addition, a thermal shift assay suggested that AtCNGC12 has higher affinity to bind cAMP over cGMP. Taken together, the current study contributes to identify key residues for channel function and provides new insights into CaMBD and CNBD in plant CNGCs.

The Arabidopsis thaliana genome encodes twenty putative cyclic nucleotide-gated channel (CNGC) genes. Studies on A. thaliana CNGCs so far have revealed their ability to selectively transport cations that play a role in various stress responses and development, however, the regulation of plant CNGCs is not yet fully understood. Thus, in this study I have attempted to analyze the structure-function relationship of AtCNGCs, mainly by using suppressor mutants of the rare gain-of function mutant, cpr22. The A. thaliana mutant cpr22 resulted from an approximately 3kb deletion that fused the 5’ half and the 3’ half of two CNGC-encoding genes, AtCNGC11 and AtCNGC12, respectively. The expression of this chimeric CNGC, the AtCNGC11/12 gene confers easily detectable characteristics such as stunted morphology with curly leaves and hypersensitive response-like spontaneous lesion formation. Through a suppressor screen, twenty nine new alleles were identified in AtCNGC11/12. Since the cytosolic C-terminal region contains important regulatory domains, such as a cyclic-nucleotide binding domain, eleven cytosolic C-terminal mutants, S17, S35, S81, S83, S84, S100, S135, S136, S137, S140 and S144, were analyzed. A detailed analysis of two mutants, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), suggested that G459 and R381 are important for basic channel function rather than channel regulation. Site-directed mutagenesis and fast protein liquid chromatography (FPLC) showed that these two amino acids influence both intra- and inter-subunit interactions that are involved in stabilizing the tertiary structure of the channel. In addition, calmodulin binding domain(s) (CaMBD) and cyclic nucleotide binding domain(s) (CNBD) of some of AtCNGCs were studied using computational modeling and biophysical analyses. The data indicated that AtCNGC12 has two CaMBDs in both N- and C- cytosolic termini, whereas AtCNGC11 has only one CaMBD located in the N-terminal region of the channel. In addition, a thermal shift assay suggested that AtCNGC12 has higher affinity to bind cAMP over cGMP. Taken together, the current study contributes to identify key residues for channel function and provides new insights into CaMBD and CNBD in plant CNGCs.