Essay 6.1 Potassium Channels

Richard W. Mercier and Gerald A. Berkowitz, Department of Plant Science, University of Connecticut

September, 2006

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).

Figure 1   Proposed 3-dimensional quaternary structure of the pore of AtCNGC2 (panel A). This model was generated utilizing the PDB record 1BL8 as a template (KcsA crystallized homotetramer). The model is presented in ribbon format and colored according to the individual peptide chains. A multiple amino acid sequence alignment of the pore domains from AtCNGC1 and 2, animal CNGC's and K+ selective channels is shown in panel B. The region depicted includes the pore helix, pore selectivity filter (boxed in panel B) and inner helix (relative to KcsA) or S6 membrane spanning domain. An invariant tryptophan (W) is highlighted in blue. Similar residues are highlighted in yellow. The crystal structure of KcsA provides an array of structural information regarding K+ pore architecture leading to ion selectivity. As is demonstrated for the KcsA subunits, AtCNGC2 consist of 2 membrane-spanning helices S5 (not shown) and S6 (homologous to the inner and outer helices in KcsA, respectively) connected by the pore domain. In the homotetramer the four inner helix pairs have an "inverted teepee" configuration. The short selectivity filter is oriented in the outer portion of the 'teepee.' In a similar fashion with KcsA, the amide-carbonyl dipoles of the pore helices from AtCNGC2 are slanted in towards the pore axis from the outside orienting a partial negative charge inward towards the center of the pore. However, the chemical interactions between amino acids from the selectivity filter and pore and inner helices (from adjacent subunits) in KcsA which appear to influence selectivity for the larger dehydrated K+ ion over the smaller Na+ ion by creating a rigid optimal geometry are not observed in AtCNGC2 (also see Figure 2). GenBank accession numbers corresponding to the various peptides are as follows: AtCNGC2 from Arabidopsis thaliana, AF067798; AtCNGC1, Y16327; BOLF a bovine olfactory CNG channel, X55010; BRET a bovine retinal CNG channel, X51604; KAT1 from A. thaliana, U25088; KcsA from Streptomyces lividans Z37969.

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).

Figure 2   Proposed 3-dimensional structures of the pore region and S6 domain of AtCNGC1 and 2. Panel A ribbon diagrams were generated with the PDB record 1BL8a (subunit of KcsA). In A (left panel) the inner helix of KcsA (green; as with corresponding pore helix) and the S6 membrane-spanning domain of AtCNGC2 (blue; as with corresponding pore helix) are superimposed. The selectivity filter (yellow for AtCNGC2 and red for KcsA) includes annotations in single letter code; backbone and sidechains are in CPK. A similar model with AtCNGC1 and KcsA superimposed is presented in A (right panel). In panel B, a 3-dimensional structural model of the four pore helices and selectivity filter for AtCNGC2, in comparison to KcsA, is shown as a cross section looking down into the pore. Coloring is according to subunits; backbone and side chains are in CPK, oxygen atoms are red, nitrogen blue. Invariant Ws are shown, and H-bonding (green lines) between the Y residues and adjacent Ws are presented in the KcsA image. The structural architecture of the KcsA pore confirmed a number of hypotheses previously set forth regarding ion permeability and selectivity. Several of these characteristics are identified in the AtCNGC2 model. As a K+ ion passes through the channel it is completely stripped of its hydration shell. The axis of the K+ selectivity filter is lined by backbone carbonyl oxygen atoms, which stabilize and coordinate the dehydrated K+ ion. The inner helix (S6 in AtCNGC2) acts as the inner portion of the pore. Its predominately hydrophobic amino acid composition creates an inert surface (or "low resistance pathway") to the diffusing ion over a significant portion of the channel length. However, the core amino acids of the selectivity filter are different between AtCNGC2 and KcsA. The highly conserved GYG motif identified in K+ selective channels is an AND triplet in AtCNGC2. The orientation of the backbone carbonyls and sidechain residues vary in the proposed model, optional orientations may be possible (the above models were generated offsite by the Swiss-MODEL Protein Modeling Server and have not been modified locally). H-bonding between the Y and W residues of the 4 subunits in KcsA does not occur in AtCNGC2. However, the N or D residues of the selectivity filter in AtCNGC2 could conceivably H-bond with pore helix Ws (conserved residue) within the same subunit.

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).

Figure 3   Proposed 3-dimensional structure of the CNBS's corresponding to AtCNGC1 and 2. The PDB record 1RGS (RIα crystallized structure; panel A, right) was used to generate the structural prediction for AtCNGC2 (panel A left) and the PDB record 3GAP (CAP crystallized structure; B, right) was used to generate the structural prediction for AtCNGC1 (B left). The 3- dimensional structures are presented as ribbon diagrams and colored according to secondary structure succession. Denoted amino acid residues as well as the hetero-ligand are in CPK, oxygen atoms are red, nitrogen blue and phosphorous orange. A structural sequence alignment of RIα, CAP and the two plant CNBS's is presented in panel C. Identical amino acids are indicated with asterisks, similar residues with a period. Secondary structures corresponding to the 3-dimensional models are annotated, helices are boxed, and β sheets are overlined for RIα and underlined for CAP. The classic structure of a CNBS includes an N terminal α helix (αA) preceding eight antiparallel β sheets forming a β barrel (β1-8) followed by two C terminal α helices (αB and C). The orientation of the cAMP (CMP1 in panel b; syn conformation for RIα and AtCNGC2, anti conformation for CAP and AtCNGC1) relative to the β barrel is shown. Amino acid residues which interact with the ligand by H-bonding are denoted with arrows in (C) and are presented in the crystallized structures (dashed lines); conserved residues (glutamate (E) 75, serine (S) 93 and tyrosine (T) 94 in AtCNGC2; aspartate (D) 73, serine (S) 91 and serine (S) 92 in AtCNGC2) which may interact in a similar fashion with cNMP are presented in the proposed models.

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.


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