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Richard W. Mercier and Gerald A. Berkowitz, Department of Plant Science, University of Connecticut
The translocation of cations across biological membranes is an inherent feature associated with numerous physiological processes, including growth and development, signal transduction cascades, and cell homeostasis. Identifying the transport proteins involved in these processes and the roles they play is an intense area of research. Isolation and characterization of a moderately large family of putative cation channels, plant cyclic nucleotide gated cation channels (CNGC's), will invariably bring researchers closer to this goal. With the completion of whole genome sequencing projects, a significant challenge on the horizon will be to elucidate the structure activity relationship between the functional domains of cation channels and the ligands that interact with them. Primary sequence comparisons in tandem with electrophysiological characterizations and 3-dimensional structure analyses are leading scientists to a fundamental understanding as to how these channels facilitate ion conduction. Here we describe the proposed 3-dimensional structures of the pore domain and cyclic-nucleotide binding sites (CNBS's) for two CNGC's from Arabidopsis (AtCNGC1, AtCNGC2) (Leng et al., 1999; Köhler et al., 1999).
It is well known that adenosine and/or guanosine 3',5'-cyclic monophosphate (cNMP; cAMP or cGMP) are important secondary messenger signaling molecules in eukaryotic and prokaryotic cells. They are typically involved in the transduction of a signal into a specific cellular response. CNGC's were first identified in animals where they are involved in visual, gustatory, and olfactory signal transduction as well as other physiological processes. Although plant CNGC's show a relatively low overall sequence identity with animal CNGC's, they do share significant homology at their pore regions and CNBS's. In general, CNGC's possess similar primary sequence identities with the Shaker family of channels and consequently have similar structural motifs (Zagotta and Siegelbaum 1996). The functional channel is most likely represented as a homo-tetramer, although hetero-tetrameric channels organized in situ should not be ruled out. Each subunit consists of six transmembrane domains (S1–6) containing a pore-forming region, with lower hydrophobicity, between the S5 and S6 region. Moreover, CNGC include a CNBS intracellularly located down stream from the pore. These channels are directly gated upon binding with cAMP or cGMP and are permeable to a variety of monovalent cations as well as to Ca2+. In addition to these characteristics, plant CNGC's include a putative Ca2+/calmodulin binding site (CaMBS) embedded within the CNBS (Leng et al., 1999; Köhler et al., 1999 and 2000). Ca2+/calmodulin (Ca2+/CaM) and cNMPs are essential components in a number of well characterized signaling pathways strengthening the argument that plant CNGC's play critical roles in Ca2+ signal transduction cascades.
The Arabidopsis genome-sequencing project has revealed the presence of 20 putative members within the plant CNGC family. Primary amino acid sequence identities range between 35 and 95 percent for the different peptides (Leng et al., 1999). Such variability suggests that these CNGC's may function in an array of different physiological processes, as is the case in animal systems. A direct analysis of the expression patterns associated with each homologue should provide significant clues as to their function. No less then four individual clades are identified in a phylogenetic relationship between the 20 homologs (Maser et al., 2001). The redundancy of this class of channels and their sequence divergence within the Arabidopsis genome suggests multiple roles for the homo or hetero-tetrameric proteins.
Putative 3-dimensional structural models were generated for AtCNGC1 and 2. In order to identify appropriate modeling templates, query sequences corresponding to the plant CNGC's structural domains were run through the Swiss-Model Blast Protein Modeling Server. This utility searches the ExNRL-3D database derived from the protein database (PDB). Upon identification of a positive structural "hit," PDB records were downloaded for subsequent analysis. The experimental sequences were sent back through the Swiss-Model Protein Modeling Server using the identified (crystallized) templates. Utilizing the Swiss-Model "First Approach" mode with a lower BLAST P (N) limit of 0.00001, positive structures was rendered and analyzed locally through the Swiss-PdbViewer version 3.5 (Glaxo Wellcome Experimental Research). Reproductions of the modeled structures were either rendered by the Persistence of Vision Ray Tracer (POV-Ray) software, or loaded directly into Microsoft PowerPoint as bitmap files and annotated (Guex and Peitsch, 1997, Peitsch 1995 and 1996).
The pore region of K+-selective channels contains the highly conserved primary sequence motif (TxGYGD) termed the selectivity filter (Ketchum and Slayman, 1996)(Figure 1B).
Na+ exclusion is dictated by chemical interactions between amino acids from the selectivity filter and pore and inner helices (from adjacent subunits) that influence selectivity for the larger dehydrated K+ ion over the smaller Na+ ion by creating a rigid optimal geometry. In addition, the amide-carbonyl dipoles of the pore helices, slanting in towards the pore axis from the outside (partial negative charge carbonyl end in) stabilize the cations as they pass through the pore. A proposed 3-dimensional quaternary structure of the homo-tetrameric channel corresponding to AtCNGC2 is presented in Figure 1A.
In a 1998 publication Doyle et al. demonstrated the essential requirement of the glycine, tyrosine, glycine (GYG) residues for K+ selectivity. The pore region of AtCNGC2 was threaded through the crystallized K+ channel KcsA (Doyle et al., 1998). The putative 3-dimensional structure shares a number of features with KcsA, specifically with respect to the inner helix (S6 for plant CNGC's) and pore helix orientation (Figure 2A left).
Interestingly the most significant characteristic, which differs between AtCNGC2 and KcsA, is the core amino acids of the selectivity filter. The classical GYG motif identified in almost all K+ selective channels is absent in AtCNGC2. The aligned region consists of an alanine, asparagine, and aspartate (AND) motif in AtCNGC2. The orientation of the backbone carbonyls and side chain residues vary in the proposed model. H-bonding interactions between the Y residues with adjacent tryptophane (W) residues in the KcsA homo-tetramer (Doyle et al., 1998) leading to channel stability and ultimately contributing to K+ selectivity do not exist in AtCNGC2 (Figure 2B). How then do we fully account for K+ selectivity in the plant channel? Apparently, an undiscovered method for Na+ exclusion exists for AtCNGC2.
The pore domain corresponding to AtCNGC1 was modeled in a similar fashion as described above (Figure 2A right). Its structural architecture is remarkably similar to AtCNCC2. AtCNGC1 has conducted K+ ions, however, no electrophysiological data has been generated demonstrating it to be a Na+ conducting pathway.
The CNBS's corresponding to AtCNGC1 and 2, respectively, have also been modeled and 3-dimensional structures rendered based on the crystallized structures of the catabolite gene activator protein (CAP) (for AtCNGC1; McKay et al., 1982) and the regulatory subunit of cAMP dependent protein kinase A (RIα) (for AtCNGC2; Su et al., 1995). Several critical regions are preserved in the models of the plant CNBS's, specifically the formation of β-barrels, and the orientation of conserved residues associated with ligand binding (Figure 3A and B).
As stated above, plant CNGC's harbor a CaMBS closely associated with the CNBS (Köhler et al., 2000). Binding of Ca2+/CaM complexes to CNGC's probably reduces the affinity of the channel for cyclic nucleotides. Plant cells respond to a variety of stimuli via changes in their intracellular Ca2+ concentration ([Ca2+]i), which are perceived by CaM's and a family of structurally related Ca2+-binding proteins (Roberts and Harmon, 1992; Snedden and Fromm, 1998; Zielinski, 1998). CaM's in plants are a family of small (~16.8 kDa) protein isoforms. In Arabidopsis, for example, at least eight genes encode CaM (Gawienowski et al., 1993; Ling et al., 1991; Köhler and Neuhaus, 2000). These genes are expressed in unique but overlapping patterns. Increases in [Ca2+]i occur in response to a wide array of environmental stimuli (reviewed in Trewavas and Malho, 1998; Bush et al., 1993), including wounding and pathogen infection (Lamb and Dixon et al., 1999; Hammond-Kosack et al., 1996, Scheel, 1998). CaM transduces these signals by binding to and altering the activities of a variety of proteins, which in turn help to elicit the appropriate physiological response. The CNBS and juxtaposed CaMBS present in these plants CNGC's clearly provide molecular mechanisms for the regulation of ion conductance. Preliminary evidence shows that different CaM's display varying binding affinities to the plant CNGC's, and that the presence of channel specific CaM's has a negative effect on ion conductance (Köhler and Neuhaus, 1999). The manor in which cNMP and CaM interact and ultimately regulate channel conductance will invariably provide clues as to the controlling relationships between the various tetrameric proteins and their roles in plant cellular physiology.
Bush, D. S. (1993) Calcium regulation in plant cells and its role in signaling. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46: 95–122.
Doyle, D. A., Cabral, J. M., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) The structure of the potassium channel: Molecular basis of K conduction and selectivity. Science 280:69–76.
Gawienowski, M. C., Szymanski, D. Perera, I. Y., and Zielinski, R. E. (1993) Calmodulin isoforms in Arabidopsis encoded by multiple divergent mRNAs. Plant Mol. Biol. 22: 215–225.
Guex, N., and Peitsch, M. C. (1997) SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modelling. Electrophoresis 18:2714–2723.
Hammond-Kosack, K. E., and Jones, J. D. G. (1996) Resistance gene-dependent plant defense responses. Plant Cell 8: 1773–1791.
Ketchum, K. A., and Slayman, C. W. (1996) Isolation of an ion channel gene from Arabidopsis thaliana using the H5 signature sequence from voltage-dependent K+ channels. FEBS Lett. 378: 19–26.
Köhler, C., and Neuhaus, G. (2000) Characterisation of calmodulin binding to cyclic nucleotide-gated ion channels from Arabidopsis thaliana. FEBS Lett. 4710: 133–136.
Köhler, C., Merkle, T., and Neuhaus, G. (1999) Characterization of a novel gene family of putative cyclic nucleotide and calmodulin regulated ion channels in Arabidopsis. Plant J. 18: 97–104.
Lamb, C., and Dixon, R. A. (1999) Early events in the signal pathway for the oxidative burst in soybean cells exposed to avirulent pseudomonas syringae pv glycinea. Plant Physiol. 120: 1137–46.
Leng, Q., Mercier, R. W., Yao, W., and Berkowitz, G. A. (1999) Cloning and first functional characterization of a plant cyclic nucleotide-gated cation channel. Plant Physiol. 121:753–761.
Ling, V., Perera, I., and Zielinski, R. E. (1991) Primary structures of Arabidopsis calmodulin isoforms deduced from the sequences of cDNA clones. Plant Physiol. 96: 1196–1202.
Mäser P., Thomine S., Schroeder J., Ward J., Hirschi K., Sze H., Talke I. N., et al. (2001) Phylogenetic relationships within cation transporter families of Arabidopsis Plant Physiol. 126: 1646–1667.
McKay, D. B., Weber, I. T., and Steitz, T. A. (1982) Structure of catabolite gene activator protein at 2.9-A resolution. Incorporation of amino acid sequence and interactions with cyclic AMP. J Biol Chem. 257: 9518–24.
Peitsch, M. C. (1995) Protein modeling by E-mail. Bio/Technology 13: 658–660.
Peitsch, M. C. (1996) ProMod and Swiss-Model: Internet-based tools for automated comparative protein modeling. Biochem. Soc. Trans. 24:274–279.
Roberts, D. M., and Harmon, A. C. (1992) Calcium-modulated proteins: targets of intracellular calcium signals in higher plants. Annu Rev Plant Physiol Plant Mol Biol 42: 375–414.
Scheel, D. (1998) Resistance response physiology and signal transduction. Curr. Opinion. Plant Biol. 1: 305–310.
Snedden, W. A., and Fromm, H. (1998) Calmodulin, calmodulin-related proteins and plant responses to the environment. Trends Plant Sci. 3: 299–304.
Su, Y., Dostmann, W. R., Herberg, F. W., Durick, K., Xuong, N. H., Ten Eyck, L., Taylor, S. S., and Varughese, K. I. (1995) Regulatory subunit of protein kinase A: structure of deletion mutant with cAMP binding domains. Science 269: 807–13.
Trewavas, A. J. and Malho, R. (1998) Ca2+ signalling in plant cells: The big network! Curr. Opinion Plant Biol. 1:4 28–433.
Zagotta W. N., and Siegelbaum, S. A. (1996) Structure and function of cyclic nucleotide-gated channels. Annu Rev Neurosci 19: 235–263.
Zielinski, R. E. (1998) Calmodulin and calmodulin-binding proteins in plants. Annu.Rev.Plant Physiol.Plant Mol.Biol. 49: 697–725.
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