Characterisation of calmodulin binding to cyclic nucleotide-gated ion channels from Arabidopsis thaliana

The recently identified cyclic nucleotide-gated ion channels (AtCNGCs) from Arabidopsis thaliana have the ability to bind calmodulin. Using two different methods, we mapped the binding site of AtCNGC1 to the last predicted alpha helix of the cyclic nucleotide binding domain. This is in contrast to CNGCs from animals, where the calmodulin binding site is located in the N-terminus, implying that different mechanisms for CNGC modulation have evolved in animals and plants. Furthermore, we demonstrate that AtCNGC1 and AtCNGC2 have different calmodulin binding affinities and we provide evidence for target specificities among calmodulin isoforms.     

The cyclic nucleotide-gated calmodulin-binding channel AtCNGC10 localizes to the plasma membrane and influences numerous growth responses and starch accumulation in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Cyclic nucleotide gated channels (CNGCs) that are regulated by calmodulin (CaM) have been shown to play essential roles in signal transduction, metabolism, and growth in animals. By contrast, very little is known about the subcellular location and the function of these channels in plants. Here we report on the effects of antisense suppression of the expression of AtCNGC10, a putative K⁺ channel, and the immunolocalization of the protein using an AtCNGC10-specific antiserum. In Arabidopsis thaliana leaves, AtCNGC10 was localized to the plasma membrane of mesophyll and parenchyma cells. Antisense AtCNGC10 plants had 40% of the AtCNGC10 mRNA levels and virtually undetectable protein levels relative to wild type plants. Antisense expression of AtCNGC10 did not affect the mRNA levels of AtCNGC13, the most closely related CNGC family member in the genome. Relative to wild type Columbia, antisense AtCNGC10 plants flowered 10 days earlier, and had a 25% reduction in leaf surface area, thickness and palisade parenchyma cell length. Their roots responded more slowly to gravitropic changes and the chloroplasts accumulated more starch. We propose that AtCNGC10, through interactions with CaM and cGMP, modulates cellular K⁺ balance across the plasma membrane, and that perturbations of this K⁺ gradient affect numerous growth and developmental processes.     

Identification of a functionally essential amino acid for Arabidopsis cyclic nucleotide gated ion channels using the chimeric AtCNGC11/12 gene

We used the chimeric Arabidopsis cyclic nucleotide-gated ion channel AtCNGC11/12 to conduct a structure-function study of plant cyclic nucleotide-gated ion channels (CNGCs). AtCNGC11/12 induces multiple pathogen resistance responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22). A genetic screen for mutants that suppress cpr22-conferred phenotypes identified an intragenic mutant, #73, which has a glutamate to lysine substitution (E519K) at the beginning of the eighth beta-sheet of the cyclic nucleotide-binding domain in AtCNGC11/12. The #73 mutant is morphologically identical to wild-type plants and has lost cpr22-related phenotypes including spontaneous cell death and enhanced pathogen resistance. Heterologous expression analysis using a K(+)-uptake-deficient yeast mutant revealed that this Glu519 is important for AtCNGC11/12 channel function, proving that the occurrence of cpr22 phenotypes requires active channel function of AtCNGC11/12. Additionally, Glu519 was also found to be important for the function of the wild-type channel AtCNGC12. Computational structural modeling and in vitro cAMP-binding assays suggest that Glu519 is a key residue for the structural stability of AtCNGCs and contributes to the interaction of the cyclic nucleotide-binding domain and the C-linker domain, rather than the binding of cAMP. Furthermore, a mutation in the alpha-subunit of the human cone receptor CNGA3 that causes total color blindness aligned well to the position of Glu519 in AtCNGC11/12. This suggests that AtCNGC11/12 suppressors could be a useful tool for discovering important residues not only for plant CNGCs but also for CNGCs in general.     

Electrophysiological analysis of cloned cyclic nucleotide-gated ion channels

Electrophysiological studies were conducted on the cloned plant cyclic nucleotide-gated ion channels AtCNGC2 and AtCNGC1 from Arabidopsis, and NtCBP4 from tobacco (Nicotiana tobacum). The nucleotide coding sequences for these proteins were expressed in Xenopus laevis oocytes or HEK 293 cells. Channel characteristics were evaluated using voltage clamp analysis of currents in the presence of cAMP. AtCNGC2 was demonstrated to conduct K(+) and other monovalent cations, but exclude Na(+); this conductivity profile is unique for any ion channel not possessing the amino acid sequence found in the selectivity filter of K(+)-selective ion channels. Application of cAMP evoked currents in membrane patches of oocytes injected with AtCNGC2 cRNA. Direct activation of the channel by cyclic nucleotide, demonstrated by application of cyclic nucleotide to patches of membranes expressing such channels, is a hallmark characteristic of this ion channel family. Voltage clamp studies (two-electrode configuration) demonstrated that AtCNGC1 and NtCBP4 are also cyclic nucleotide-gated channels. Addition of a lipophilic analog of cAMP to the perfusion bath of oocytes injected with NtCBP4 and AtCNGC1 cRNAs induced inward rectified, noninactivating K(+) currents.     

Functional interaction of calmodulin with a plant cyclic nucleotide gated cation channel

A family of plant ligand gated nonselective cation channels (cngcs) can be activated by direct, and reversible binding of cyclic nucleotide. These proteins have a cytoplasm-localized cyclic nucleotide binding domain (CNBD) at the carboxy-terminus of the polypeptide. A portion of the cngc CNBD also acts as a calmodulin (CaM) binding domain (CaMBD). The objective of this work is to further characterize interaction of cyclic nucleotide and CaM in gating plant cngc currents. The three-dimensional structure of an Arabidopsis thaliana cngc (Atcngc2) CNBD was modeled, indicating cAMP binding to the Atcngc2 CNBD in a pocket formed by a β barrel structure appressing a shortened (relative to animal cngc CNBDs) αC helix. The Atcngc2 CaMBD was expressed as a fusion peptide linking blue and green fluorescent proteins, and used to quantify CaM (A. thaliana CaM isoform 4) binding. CaM bound the fusion protein in a Ca²⁺–dependent manner with a K_d of 7.6 nM and a Ca²⁺ binding K_d of 200 nM. Functional characterization (voltage clamp analysis) of Atcngc2 was undertaken by expression in human embryonic kidney cells. CaM reversed cAMP activation of Atcngc2 currents. This functional interaction was dependent on free cytosolic Ca²⁺. Increasing cytosolic Ca²⁺ was found to inhibit cAMP activation of the channel in the absence of added CaM. We conclude that the physical interaction of Ca²⁺/CaM with plant cngcs blocks cyclic nucleotide activation of these channels. Thus, the cytosolic secondary messengers CaM, cAMP, and Ca²⁺ can act in an integrated fashion to gate currents through these plant ion channels.     

Cloning and first functional characterization of a plant cyclic nucleotide-gated cation channel

Cyclic nucleotide-gated (cng) non-selective cation channels have been cloned from a number of animal systems. These channels are characterized by direct gating upon cAMP or cGMP binding to the intracellular portion of the channel protein, which leads to an increase in channel conductance. Animal cng channels are involved in signal transduction systems; they translate stimulus-induced changes in cytosolic cyclic nucleotide into altered cell membrane potential and/or cation flux as part of a signal cascade pathway. Putative plant homologs of animal cng channels have been identified. However, functional characterization (i.e. demonstration of cyclic-nucleotide-dependent ion currents) of a plant cng channel has not yet been accomplished. We report the cloning and first functional characterization of a plant member of this family of ion channels. The Arabidopsis cDNA AtCNGC2 encodes a polypeptide with deduced homology to the alpha-subunit of animal channels, and facilitates cyclic nucleotide-dependent cation currents upon expression in a number of heterologous systems. AtCNGC2 expression in a yeast mutant lacking a low-affinity K(+) uptake system complements growth inhibition only when lipophilic cyclic nucleotides are present in the culture medium. Voltage clamp analysis indicates that Xenopus laevis oocytes injected with AtCNGC2 cRNA demonstrate cyclic-nucleotide-dependent, inward-rectifying K(+) currents. Human embryonic kidney cells (HEK293) transfected with AtCNGC2 cDNA demonstrate increased permeability to Ca(2+) only in the presence of lipophilic cyclic nucleotides. The evidence presented here supports the functional classification of AtCNGC2 as a cyclic-nucleotide-gated cation channel, and presents the first direct evidence (to our knowledge) identifying a plant member of this ion channel family.     

Five-group distribution of the Shaker-like K+ channel family in higher plants

Abstract In higher plants, potassium channels of the Shaker family have been shown to play crucial roles in the uptake of K⁺ from the soil solution and subsequent transport of this ion at the cell, tissue, and organ levels. In the model plant Arabidopsis thaliana, this family is composed of nine members, which are the best characterized among plant channels at the protein, gene, and functional property levels. Plant Shaker channels share a common structure: a hydrophobic core composed of six transmembrane segments, a long cytoplasmic C-terminal region harboring a putative cyclic nucleotide binding domain, and a K_HA domain. Many channels also contain an ankyrin domain between the putative cyclic nucleotide binding domain and the K_HA domain. The analysis of 44 Shaker channels from plants revealed a five-group classification. The members of each group share high sequence and structure similarities. This grouping also correlates with the diversification of the functional properties of the proteins, as members of an individual group have roughly the same electrophysiological characteristics. Analysis of the intron positions showed that the gene structures are also quite well conserved within the five groups. A correlation linking the evolution of the sequences and the positioning of the introns was established. Finally, a moss sequence provided additional clues about the hypothetical structure of an ancestor of the present channels and suggested that the diversification of plant Shaker channels happened before the separation of monocots and dicots and after the separation of bryophytes and tracheophytes.     

High throughput chemical screening supports the involvement of Ca2+ in cyclic nucleotide-gated ion channel-mediated programmed cell death in Arabidopsis

Recently, we reported the role of Arabidopsis cyclic nucleotide-gated ion channel (AtCNGC) 11 and 12 in Ca²⁺-dependent physiological responses. AtCNGC11 and 12 have been reported to be involved in plant immunity, but whether these channels play additional physiological roles was not clear before. Using single and double knockout mutants, we have found that these channels play significant roles in Ca²⁺ signaling, which mediates several physiological processes, such as gravitropic bending and senescence. Here, we conducted a high throughput, non-biased chemical screen using the gain-of-function mutant of AtCNGC11 and 12, cpr22. Our data presented here indicates that Ca²⁺ but not K⁺ channel blockers suppress AtCNGC11/12-induced lethality. Our data further suggest that AtCNGC11 and 12 are involved in Ca²⁺-dependent, but not K⁺-dependent physiological responses in planta.     

CNGCs: prime targets of plant cyclic nucleotide signalling?

Cyclic nucleotide-gated channels (CNGCs) are a recently identified family of plant ion channels. They show a high degree of similarity to Shaker-type voltage-gated channels and contain a C-terminal cyclic nucleotide-binding domain with an overlapping calmodulin-binding domain. Heterologously expressed plant CNGCs show activation by cyclic nucleotides and permeability to monovalent and divalent cations. In plants, downstream effectors of cyclic nucleotide signals have so far remained obscure, and CNGCs might be their prime targets. The unique position of CNGCs as ligand-gated Ca(2+)-permeable channels suggests that they function at key sites where cyclic nucleotide and Ca(2+) signalling pathways interact. Such processes include plant defence responses, and two recently characterized Arabidopsis mutants in CNGC genes indeed show altered pathogen responses.     

The cyclic nucleotide-gated channels AtCNGC11 and 12 are involved in multiple Ca²⁺-dependent physiological responses and act in a synergistic manner

Arabidopsis cyclic nucleotide-gated ion channels (AtCNGCs) form a large family consisting of 20 members. These channels have so far been reported to be involved in a diverse range of physiological phenomena. For example, AtCNGC18 was reported to play an important role in pollen tube growth, while AtCNGC2, 4, 11, and 12 were implicated in mediating pathogen defence. To identify additional functions for AtCNGC11 and 12, various physiological aspects were analysed using both AtCNGC11 and 12 single knockout mutants as well as a double mutant. Although AtCNGC11 and 12 can function as K(+) and Ca(2+) channels in yeast, it was found that the loss of AtCNGC11 and 12 in Arabidopsis caused increased sensitivity to Ca(2+) but not K(+), indicating a specific function for these genes in Ca(2+) signalling in planta. However, they did not show an alteration in Ca(2+) accumulation, suggesting that AtCNGC11 and 12 are not involved in general Ca(2+) homeostasis but rather in the endogenous movement of Ca(2+) and/or Ca(2+) signalling. Furthermore, these channels synergistically contribute to the generation of a Ca(2+) signal that leads to gravitropic bending. Finally, AtCNGC11 and 12 gene expression was induced during dark-induced senescence and AtCNGC11 and 12 knockout mutants displayed enhanced chlorophyll loss, which was even more pronounced in the double mutant, also indicating synergistic roles in senescence. The findings indicate that (i) some CNGC family members have multiple physiological functions and (ii) some plant CNGCs share the same biological function and work in a synergistic manner.     

Characterization of plant phenotypes associated with loss-of-function of AtCNGC1, a plant cyclic nucleotide gated cation channel.

Of the 57 cation channel genes in the Arabidopsis genome, over a third encode cyclic nucleotide gated cation channels (CNGCs). CNGCs are ion channels regulated by cytosolic signaling molecules (cyclic nucleotides, calmodulin, and Ca(2+)), and which conduct Ca(2+) as well as K(+) and in some cases Na(+). Little is currently known about the role CNGCs may play in plant growth and development. Here, we examined the hypothesis that an Arabidopsis thaliana genotype containing a null mutation in one of the CGNC genes (AtCNGC1) would display cation uptake-related growth phenotype differences from wild type (WT) plants. We determined that AtCNGC1 protein is primarily expressed in the roots of Arabidopsis seedlings. Seedlings lacking this protein had slightly (6-22%) lower shoot Ca(2+) than WT plants. Primary roots of Atcngc1 mutant seedlings grew faster than roots of WT plants, and had larger angles of gravicurvature and less nitric oxide generation upon gravistimulation. We conclude that channels formed (at least in part) by AtCNGC1 contribute (along with other channels) to Ca(2+) uptake into plants, and that Ca(2+) uptake into roots through AtCNGC1 affects some aspects of growth in the primary root of Arabidopsis seedlings.     

Expression of plant cyclic nucleotide-gated cation channels in yeast

The functional properties of inwardly conducting plant cyclic nucleotide-gated cation channels (CNGCs) have not been thoroughly characterized due in part to the recalcitrance of their functional expression in heterologous systems. Here, K+ uptake-deficient mutants of yeast (trk1,2) and Escherichia coli (LB650), as well as the Ca2+-uptake yeast mutant mid1,cch1, were used for functional characterization of Arabidopsis thaliana CNGCs, with the aim of identifying some of the cultural and physiological conditions that impact on plant CNGC function in heterologous systems. Use of the Ca2+-uptake yeast mutant provided the first evidence consistent with Ca2+ conduction by the A. thaliana CNGC AtCNGC1. Expression of AtCNGC1 in LB650 demonstrated that mutants of Escherichia coli (which has no endogenous calmodulin) can also be used to study functional properties of CNGCs. Expression of AtCNGC2 and AtCNGC4 enhanced growth of trk1,2 in the presence of hygromycin; AtCNGC1 has less of an effect. Deletion of the AtCNGC1 calmodulin-binding domain enhanced growth of trk1,2 at low external K+ but not of LB650, suggesting that yeast calmodulin may bind to, and down-regulate this plant channel. In vitro binding studies confirmed this physical interaction. Northern analysis, green fluorescent protein:AtCNGC1 fusion protein expression, as well as an antibody raised against a portion of AtCNGC1, were used to monitor expression of AtCNGC1 and deletion constructs of the channel in the heterologous systems. In the presence of the activating ligand cAMP, expression of the AtCNGC1 channel with the calmodulin-binding domain deleted increased intracellular [K+] of trk1,2. Trk1,2 is hypersensitive to the toxic cations spermine, tetramethylamine, and NH4+. These compounds, as well as amiloride, inhibited trk1,2 growth and thereby improved the efficacy of this yeast mutant as a heterologous expression system for CNGCs. In addition to characterizing mutants of yeast and E. coli as assay systems for plant CNGCs, work presented in this report demonstrates, for the first time, that a plant CNGC can retain ion channel function despite (partial) deletion of its calmodulin-binding domain and that yeast calmodulin can bind to and possibly down-regulate a plant CNGC.     

Calmodulin binding to Arabidopsis cyclic nucleotide gated ion channels

Recently we have reported that the αC-helix in the cyclic nucleotide binding domain (CNBD) is required for channel regulation and function of cyclic nucleotide gated ion channels (CNGCs) in Arabidopsis. A mutation at arginine 557 to cysteine (R557C) in the αC-helix of the CNBD caused an alteration in channel regulation. Protein sequence alignments revealed that R557 is located in a region that is important for calmodulin (CaM) binding. It has been hypothesized that CaM negatively regulates plant CNGCs similar to their counter parts in animals. However, only a handful of studies has been published so far and we still do not have much information about the regulation of CNGCs by CaM. Here, we conducted in silico binding prediction of CaM and Arabidopsis CNGC12 (AtCNGC12) to further study the role of R557. Our analysis revealed that R557 forms salt bridges with both D79 and E83 in AtCaM1. Interestingly, a mutation of R557 to C causes the loss of these salt bridges. Our data further suggests that this alteration in CaM binding causes the observed altered channel regulation and that R557 plays an important role in CaM binding.Key words: calmodulin, CaM, cyclic nucleotide gated ion channels, CNGC, environmental effect, defense responses, temperatureWe recently reported about the role of the αC-helix in the cyclic nucleotide binding domain (CNBD) for channel regulation and function of cyclic nucleotide gated ion channels (CNGCs) in Arabidopsis.¹ CNGCs were first discovered in vertebrate retinal photoreceptors and olfactory sensory neurons.^2,3 They are composed of a cytoplasmic N-terminus, six membrane spanning regions (S1–S6), a pore domain located between S5 and S6 and a cytoplasmic C-terminus and share similarities with the voltage-gated outward rectifying K⁺-selective ion channel (Shaker) proteins.² However, CNGCs are ligand gated and opened by the direct binding of cyclic nucleotides monophosphates (cNMPs), such as cAMP and cGMP (cNMPs).⁴ The cytoplasmic C-terminus contains a CNBD that is connected to the pore domain by a C-linker region.⁵ The function and regulation of CNGCs has been extensively studied in retinal photoreceptor and olfactory sensory neurons and it has been reported that their channel activity is moderated by Ca²⁺/calmodulin (CaM).⁶ A variety of Ca²⁺ permeable channels are known to be regulated by Ca²⁺/CaM. In many cases, CaM downregulates their activity, thereby creating negative feedback regulation for Ca²⁺ entry.⁷ CNGCs are also considered to follow this mode of regulation. Some animal CNGCs possess a CaM binding domain in the cytoplasmic N-terminus. For example, it has been reported that CaM binds to a short segment in the N-terminal region of the A2 subunit of CNGCs of olfactory sensory neurons in a Ca²⁺ dependent manner.^8,9The first plant CNGC, HvCBT1 (Hordeum vulgare calmodulin (CaM)-binding transporter), was identified as a CaM-binding protein in barley.¹⁰ Interestingly, in contrast to animal CNGCs, the CaM binding domain in HvCBT1 was shown to be located at the cytoplasmic C-terminal region.¹⁰ Subsequently, several CNGCs were identified from Arabidopsis and Nicotiana tabacum^11,12 and some of these CNGCs have also been shown to possess the CaM binding domain in the C-terminal region that partially overlaps with the CNBD.^11–14 This difference in the location of the CaM binding domain between animal and plant CNGCs implies that different mechanisms for CNGC regulation may have evolved in animals and plants.The Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22), which contains a novel chimeric CNGC, AtCNGC11/12, shows environmentally sensitive constitutive defense responses, such as heightened salicylic acid accumulation and hypersensitive response-like spontaneous programmed cell death.^15,16 It has been reported that the expression of AtCNGC11/12 and its channel activity is attributable for the cpr22 phenotype.^17,18 A genetic screen for mutants that suppress cpr22-conferred phenotypes identified over 20 novel mutant alleles in AtCNGC11/12.^1,18 These intragenic mutants are excellent tools to study the structure-function relationship of plant CNGCs. One of these mutants, suppressor S58, possesses a single amino acid substitution, arginine 557 to cysteine (R557C), in the αC-helix of the 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.¹ Interestingly, this temperature sensitivity was also observed in the original mutant, cpr22.¹⁹ All salicylic acid-dependent phenotypes of cpr22 are enhanced under low temperature and low humidity conditions. Furthermore, this type of environmental sensitivity has been reported not only for cpr22, but also for various other pathogen resistance mutants as well as for defense responses in wild type plants.¹⁶ Therefore, it is possible that the basis of the temperature sensitivity observed in S58 may be related to a general environmental sensitivity in defense responses.Characterization of S58 and functional complementation using heterologous expression analyses suggested that R557 in the αC-helix of the CNBD is important for channel regulation, but not for basic channel function.¹ To further investigate this, we aimed to elucidate the molecular mechanism by which R557C (S58 mutation) alters channel regulation to determine the functional role of R557. Since R557 is located in the CNBD, it is possible that R557C alters cNMP binding resulting in disruption of channel opening. In animal systems, it has been reported that cNMPs bind within the pocket formed by the αC-helix and the β-barrel that is composed of the eight β sheets in the CNBD.^20,21 The αC-helix was suggested to function as the lid of this pocket that stabilizes cNMP binding by forming hydrophobic interactions with the bound cNMP.²¹ However, it is unlikely that R557 interacts directly with the bound cNMP because of its hydrophilic nature.Interestingly, a 19–20 amino acid sequence of the αC-helix was suggested to function as CaM binding domain in AtCNGC1 and AtCNGC2 by Köhler and Neuhaus using yeast two hybrid analysis.¹⁴ Arazi et al.¹³ biochemically demonstrated that a 23 amino acid sequence that overlaps with this 19–20 amino acids is the CaM binding domain in the tobacco CNGC, NtCBP4. Furthermore, they reported that the 4 additional amino acids (W R T/S W) which are located just outside of the 19–20 amino acid sequence are crucial for efficient binding to CaM. Sequence alignment revealed that R557 is located in this crucial sequence (W R T/S W).¹ Thus, it can be hypothesized that the R557C mutation causes a modification in the binding affinity to CaM resulting in an alteration in channel regulation. Therefore, we generated in silico models of CaM binding with the αC-helix of the CNBD in AtCNGC12 (identical to AtCNGC11/12) and AtCNGC12:R557C (S58). We first modeled Arabidopsis CaM1 (AtCaM1) based on the crystal structure of a potato CaM, PCM6 (PDB# 1RFJ).²² There are seven different CaM genes in Arabidopsis that encode two sets of identical isoforms (CaM1 and CaM4; CaM2, CaM3 and CaM5) and two additional distinct isoforms (CaM6 and CaM7).^23,24 All of them share high similarity with PCM6. Using the AtCaM1 model, we modeled possible interactions between AtCaM1 and the αC-helix of the CNBD in AtCNGC12 or AtCNGC12:R557C. As shown in Figure 1 (center part), a hydrophobic pocket of CaM that is necessary for binding with target proteins²⁵ was seen in AtCaM1 creating a binding pocket for the αC-helix of the CNBD in AtCNGC12. In this model, R557 creates salt bridges with both D79 and E83 of AtCaM1 (indicated by a box) and these salt bridges appear to play a role for binding to CaM. This type of salt bridges have been reported to be crucial for CaM binding with several different target proteins.²⁶ Interestingly, these salt bridges are no longer seen in AtCNGC12:R557C (Fig. 1, right part, indicated by a box); indicating that the mutation may cause an affinity change in CaM binding. Such an affinity change will likely cause an alteration in channel regulation. Since CaM is likely a negative regulator of CNGCs, this alteration in CaM affinity does not provide a simple mechanism for the R557C mutation. However, the change in CaM binding affinity may cause complex regulatory changes in CNGCs. Thus, we are currently conducting various biochemical analyses to validate this hypothesis.Open in a separate windowFigure 1Computational structural modeling of CaM binding with AtCNGC12 and AtCNGC12:R557C. Modeling of the tertiary structure of AtCaM1, and the αC-helix of AtCNGC12 and AtCNGC12:R557 was conducted using the crystallized structures of the potato CaM, PCM 6 (PDB# 1RFJ)²² and the cytoplasmic C-terminus of the invertebrate CNGC, SpIH (Flynn et al. 2007, PDB# 2PTM),²⁷ respectively, as templates. The protein fold recognition server (Phyre)²⁸ was used to model these proteins. The binding modeling was performed using an algorithm for molecular docking (PatchDock).²⁹ All the images were generated using PyMOL.³⁰ CaM is colored in cyan and the αC-helix is shown in magenta. Left part: overall binding model between AtCaM1 and AtCNGC12, Center part: close up of the boxed area of the left part in AtCNGC12, Right part: the same area in AtCNGC12:R557C. M73, M52 and M37 of AtCaM1 create a hydrophobic pocket together with F562 and I564 of AtCNGC12, which is a typical binding configuration between CaM and target proteins. R557 creates salt bridges with both D79 and E83 (center part). These salt bridges are no longer seen between AtCaM1 and AtCNGC12:R557C (right part).Although plants CNGCs have only recently been revealed to mediate multiple stress responses and also play important roles in some developmental pathways, studies that aim to elucidate their structural and regulatory properties are still very much in their infancy. Our current study will certainly contribute to a better understanding of the structure-function relationship and regulation of plant CNGCs.     

Resonance assignments and secondary structure of calmodulin in complex with its target sequence in rat olfactory cyclic nucleotide-gated ion channel

Calmodulin (CaM), the primary receptor for intracellular Ca²⁺, regulates a large number of key enzymes and controls a wide spectrum of important biological responses. Olfactory cyclic nucleotide-gated ion channels (OLF channels) mediate olfactory transduction in olfactory receptor neurons. The opening of OLF leads to a rise in cytosolic concentration of Ca²⁺, upon binding to Ca²⁺, CaM disrupts the open conformation by binding to the CaM-binding domain in the N-terminal region and triggers the close mechanism. In order to unravel the regulatory role of CaM from structural point of view, NMR techniques were used to characterize the structure of CaM in association with the CaM binding domain of rat OLF channel (OLFp, 28 residues). Our data indicated that two distinct CaM/OLFp complexes existed simultaneously with stable structures that were not inter-exchangeable within the NMR time scale. Here, we report the full backbone and side chain resonance assignments of these two complexes of CaM/OLFp.     

The cyclic nucleotide‐gated channel AtCNGC10 transports Ca2+ and Mg2+ in Arabidopsis

The suppression of the cyclic nucleotide‐gated channel (CNGC) AtCNGC10 alters K⁺ transport in Arabidopsis plants. Other CNGCs have been shown to transport Ca²⁺, K⁺, Li⁺, Cs⁺ and Rb⁺ across the plasma membrane when expressed in heterologous systems; however, the ability of the AtCNGC10 channel to transport nutrients other than K⁺ in plants has not been previously tested. The ion fluxes along different zones of the seedling roots, as estimated by the non‐invasive ion‐specific microelectrode technique, were significantly different in two AtCNGC10 antisense lines (A2 and A3) in comparison to the wild type (WT). Most notably, the influxes of H⁺, Ca²⁺ and Mg²⁺ in the meristem and distal elongation zones of the antisense A2 and A3 lines were significantly lower than in the WT. The lower Ca²⁺ influx from the external media corresponded to a lower intracellular Ca²⁺ activity, which was estimated by fluorescence lifetime imaging measurements (FLIM). On the other hand, the intracellular pH values in the meristem zone of the roots of A2 and A3 seedlings were significantly lower (more acidic) than that of the WT, which might indicate a feedback block of H⁺ influx into meristematic cells caused by low intracellular pH. Under the control conditions, mature plants from the A2 and A3 lines contained significantly higher K⁺ and lower Ca²⁺ and Mg²⁺ content in the shoots, indicating disturbed long‐distance ion transport of these cations, possibly because of changes in xylem loading/retrieval and/or phloem loading. Exposing the plants in the flowering stage to various K⁺, Ca²⁺ and Mg²⁺ concentrations in the solution led to altered K⁺, Ca²⁺ and Mg²⁺ content in the shoots of A2 and A3 plants in comparison with the WT, suggesting a primary role of AtCNGC10 in Ca²⁺ (and probably Mg²⁺) transport in plants, which in turn regulates K⁺ transporters' activities.     

External Cd2+ and protons activate the hyperpolarization-gated K+ channel KAT1 at the voltage sensor

The functionally diverse cyclic nucleotide binding domain (CNBD) superfamily of cation channels contains both depolarization-gated (e.g., metazoan EAG family K⁺ channels) and hyperpolarization-gated channels (e.g., metazoan HCN pacemaker cation channels and the plant K⁺ channel KAT1). In both types of CNBD channels, the S4 transmembrane helix of the voltage sensor domain (VSD) moves outward in response to depolarization. This movement opens depolarization-gated channels and closes hyperpolarization-gated channels. External divalent cations and protons prevent or slow movement of S4 by binding to a cluster of acidic charges on the S2 and S3 transmembrane domains of the VSD and therefore inhibit activation of EAG family channels. However, a similar divalent ion/proton binding pocket has not been described for hyperpolarization-gated CNBD family channels. We examined the effects of external Cd²⁺ and protons on Arabidopsis thaliana KAT1 expressed in Xenopus oocytes and found that these ions strongly potentiate voltage activation. Cd²⁺ at 300 µM depolarizes the V₅₀ of KAT1 by 150 mV, while acidification from pH 7.0 to 4.0 depolarizes the V₅₀ by 49 mV. Regulation of KAT1 by Cd²⁺ is state dependent and consistent with Cd²⁺ binding to an S4-down state of the VSD. Neutralization of a conserved acidic charge in the S2 helix in KAT1 (D95N) eliminates Cd²⁺ and pH sensitivity. Conversely, introduction of acidic residues into KAT1 at additional S2 and S3 cluster positions that are charged in EAG family channels (N99D and Q149E in KAT1) decreases Cd²⁺ sensitivity and increases proton potentiation. These results suggest that KAT1, and presumably other hyperpolarization-gated plant CNBD channels, can open from an S4-down VSD conformation homologous to the divalent/proton-inhibited conformation of EAG family K⁺ channels.     

Calcineurin B‐like protein CBL10 directly interacts with AKT1 and modulates K+ homeostasis in Arabidopsis

Potassium transporters and channels play crucial roles in K⁺ uptake and translocation in plant cells. These roles are essential for plant growth and development. AKT1 is an important K⁺ channel in Arabidopsis roots that is involved in K⁺ uptake. It is known that AKT1 is activated by a protein kinase CIPK23 interacting with two calcineurin B‐like proteins CBL1/CBL9. The present study showed that another calcineurin B‐like protein (CBL10) may also regulate AKT1 activity. The CBL10‐over‐expressing lines showed a phenotype as sensitive as that of the akt1 mutant under low‐K⁺ conditions. In addition, the K⁺ content of both CBL10‐over‐expressing lines and akt1 mutant plants were significantly reduced compared with wild‐type plants. Moreover, CBL10 directly interacted with AKT1, as verified in yeast two‐hybrid, BiFC and co‐immunoprecipitation experiments. The results of electrophysiological analysis in both Xenopus oocytes and Arabidopsis root cell protoplasts demonstrated that CBL10 impairs AKT1‐mediated inward K⁺ currents. Furthermore, the results from the yeast two‐hybrid competition assay indicated that CBL10 may compete with CIPK23 for binding to AKT1 and negatively modulate AKT1 activity. The present study revealed a CBL‐interacting protein kinase‐independent regulatory mechanism of calcineurin B‐like proteins in which CBL10 directly regulates AKT1 activity and affects ion homeostasis in plant cells.     

Solution structure of the Mesorhizobium loti K1 channel cyclic nucleotide‐binding domain in complex with cAMP

Cyclic nucleotide‐sensitive ion channels, known as HCN and CNG channels, are crucial in neuronal excitability and signal transduction of sensory cells. HCN and CNG channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide‐binding domain (CNBD). However, the mechanism by which the binding of cyclic nucleotides opens these channels is not well understood. Here, we report the solution structure of the isolated CNBD of a cyclic nucleotide‐sensitive K⁺ channel from Mesorhizobium loti. The protein consists of a wide anti‐parallel β‐roll topped by a helical bundle comprising five α‐helices and a short 3₁₀‐helix. In contrast to the dimeric arrangement (‘dimer‐of‐dimers’) in the crystal structure, the solution structure clearly shows a monomeric fold. The monomeric structure of the CNBD supports the hypothesis that the CNBDs transmit the binding signal to the channel pore independently of each other.