Identification and experimental verification of a novel family of bacterial cyclic nucleotide-gated (bCNG) ion channels

Studies of bacterial ion channels have provided significant insights into the structure-function relationships of mechanosensitive and voltage-gated ion channels. However, to date, very few bacterial channels that respond to small molecules have been identified, cloned, and characterized. Here, we use bioinformatics to identify a novel family of bacterial cyclic nucleotide-gated (bCNG) ion channels containing a channel domain related by sequence homology to the mechanosensitive channel of small conductance (MscS). In this initial report, we clone selected members of this channel family, use electrophysiological measurements to verify their ability to directly gate in response to cyclic nucleotides, and use osmotic downshock to demonstrate their lack of mechanosensitivity. In addition to providing insight into bacterial physiology, these channels will provide researchers with a useful model system to investigate the role of ligand-gated ion channels (LGICs) in the signaling processes of higher organisms. The identification of these channels provides a foundation for structural and functional studies of LGICs that would be difficult to perform on mammalian channels. Moreover, the discovery of bCNG channels implies that bacteria have cyclic nucleotide-gated and cyclic nucleotide-modulated ion channels, which are analogous to the ion channels involved in eukaryotic secondary messenger signaling pathways.     

Construction of a cyclic nucleotide-gated KcsA K+ channel

The ability of an ion channel to open in response to a defined stimulus is central to its function. In ligand-gated channels, pore opening is conferred through transduction of a conformational change in a gating domain to the helices of the pore. Here, we present the construction of a designed cyclic nucleotide-gated (CNG) channel, named KcsA-CNG, by addition of a prokaryotic cyclic nucleotide-binding domain to a KcsA-derived K+ channel. This channel is functional in lipid bilayers at physiological pH and has the combined properties of both of its parent-derived components. It conducts K+ and is blocked by the K+ channel inhibitors Na+ and agitoxin-2. Channel open times are increased by about two orders of magnitude compared to wild-type KcsA. The average number of open channels increases by approximately 50% upon addition of cAMP. Although the absolute open probabilities are somewhat variable from one channel to the next, the property of cyclic nucleotide sensitivity is very reproducible. An apparent Kd value of approximately 90 nM was estimated. The successful construction of a cyclic nucleotide-gated KcsA K+ channel suggests that it should be possible to produce channels that will respond to novel ligands.     

Resonance assignment of the cyclic nucleotide binding domain from a cyclic nucleotide-gated K+ channel in complex with cAMP

In order to determine the structure of the 15 kDa cyclic nucleotide binding domain of a cyclic nucleotide-activated K⁺ channel from Mesorhizobium loti and its interaction with cAMP, nearly complete ¹H, ¹³C, and ¹⁵N chemical shifts were assigned.     

Influence of permeant ions on gating in cyclic nucleotide-gated channels

Cyclic nucleotide-gated channels are key components in the transduction of visual and olfactory signals where their role is to respond to changes in the intracellular concentration of cyclic nucleotides. Although these channels poorly select between physiologically relevant monovalent cations, the gating by cyclic nucleotide is different in the presence of Na(+) or K(+) ions. This property was investigated using rod cyclic nucleotide-gated channels formed by expressing the subunit 1 (or alpha) in HEK293 cells. In the presence of K(+) as the permeant ion, the affinity for cGMP is higher than the affinity measured in the presence of Na(+). At the single channel level, subsaturating concentrations of cGMP show that the main effect of the permeant K(+) ions is to prolong the time channels remain open without major changes in the shut time distribution. In addition, the maximal open probability was higher when K(+) was the permeant ion (0.99 for K(+) vs. 0.95 for Na(+)) due to an increase in the apparent mean open time. Similarly, in the presence of saturating concentrations of cAMP, known to bind but unable to efficiently open the channel, permeant K(+) ions also prolong the time channels visit the open state. Together, these results suggest that permeant ions alter the stability of the open conformation by influencing of the O-->C transition.     

All-trans-retinal is a closed-state inhibitor of rod cyclic nucleotide-gated ion channels

Rod vision begins when 11-cis-retinal absorbs a photon and isomerizes to all-trans-retinal (ATR) within the photopigment, rhodopsin. Photoactivated rhodopsin triggers an enzyme cascade that lowers the concentration of cGMP, thereby closing cyclic nucleotide-gated (CNG) ion channels. After isomerization, ATR dissociates from rhodopsin, and after a bright light, this release is expected to produce a large surge of ATR near the CNG channels. Using excised patches from Xenopus oocytes, we recently showed that ATR shuts down cloned rod CNG channels, and that this inhibition occurs in the nanomolar range (aqueous concentration) at near-physiological concentrations of cGMP. Here we further characterize the ATR effect and present mechanistic information. ATR was found to decrease the apparent cGMP affinity, as well as the maximum current at saturating cGMP. When ATR was applied to outside-out patches, inhibition was much slower and less effective than when it was applied to inside-out patches, suggesting that ATR requires access to the intracellular surface of the channel or membrane. The apparent ATR affinity and maximal inhibition of heteromeric (CNGA1/CNGB1) channels was similar to that of homomeric (CNGA1) channels. Single-channel and multichannel data suggest that channel inhibition by ATR is reversible. Inhibition by ATR was not voltage dependent, and the form of its dose-response relation suggested multiple ATR molecules interacting per channel. Modeling of the data obtained with cAMP and cGMP suggests that ATR acts by interfering with the allosteric opening transition of the channel and that it prefers closed, unliganded channels. It remains to be determined whether ATR acts directly on the channel protein or instead alters channel-bilayer interactions.     

Molecular mechanisms of cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels are highly specialized to carry out their unique role in cell signaling. Significant progress has been made in the last several years determining the molecular mechanisms for these specializations. The activation of the channels begins with the binding of cyclic nucleotide to a domain in the carboxyl terminal region. This binding, in turn, produces an induced fit of the protein that involves a movement of the C-helix portion of the binding domain. The induced fit of the binding domain is coupled to an allosteric conformational change that opens the channel pore. The pore is formed primarily from the sequence between the S5 and S6 segments. A single glutamic acid in the pore represents the binding site for multiple monovalent cations, the blocking site for external divalent cations, and the site for the effect of protons on permeation.     

A cyclic nucleotide-gated channel in the brain regulates olfactory modulation through neuropeptide F in fruit fly Drosophila melanogaster

Olfactory sensing and its modulation are important for the insects in recognizing diverse odors from the environment and in making correct decisions to survive. Identifying new genes involved in olfactory modulation and unveiling their mechanisms may lead us to understand decision making processes in the central nervous system. Here, we report a novel olfactory function of the cyclic nucleotide-gated (CNG) channel CG42260 in modulating ab3A olfactory sensory neurons, which specifically respond to food-derived odors in fruit fly Drosophila melanogaster. We found that two independent CG42260 mutants show reduced responses in the ab3A neurons. Unlike mammalian CNGs, CG42260 is not expressed in the odorant sensory neurons but broadly in the central nervous system including neuropeptide-producing cells. By using molecular genetic tools, we identified CG42260 expression in one pair of neuropeptide F (NPF) positive L1-l cells known to modulate food odor responsiveness. Knockdown of CG42260 in the NPF neurons reduced production of NPF in Ll-1 cells, which in turn, led to reduction of neuronal responses of the ab3A neurons. Our findings show the novel biological function of CG42260 in modulating olfactory responses to food odor through NPF.     

Efficient coupling of ligand binding to channel opening by the binding domain of a modulatory (beta) subunit of the olfactory cyclic nucleotide-gated channel.

CNG channels in vivo are heteromers of homologous alpha and beta subunits that each contain a six-transmembrane segment domain and a COOH-terminal cytoplasmic cyclic nucleotide binding domain (BD). In heterologous expression systems, heteromeric alphabeta channels activate with greater sensitivity to ligand than do homomeric alpha channels; however, ligand-gating of channels containing only beta subunit BDs has never been studied because beta subunits cannot form functional homomeric CNG channels. To characterize directly the contribution of the beta subunit BD to ligand-gating, we constructed a chimeric subunit, X-beta, whose BD sequence was that of the beta subunit CNG5 from rat, but whose sequence outside the BD was derived from alpha subunits. For comparison, we constructed another chimera, X-alpha, whose sequence outside the BD was identical to that of X-beta, but whose BD sequence was that of the alpha subunit CNG2 from catfish. When expressed in Xenopus oocytes, X-beta and X-alpha each formed functional homomeric channels activated by both cAMP and cGMP. This is the first demonstration that the beta subunit BD can couple ligand binding to activation in the absence of alpha subunit BD residues. Notably, both agonists activate X-beta more effectively than X-alpha (higher opening efficacy and lower K(1/2)). The BD is believed to comprise two functionally distinct subdomains: (1) the roll subdomain (beta-roll and flanking A- and B-helices) and (2) the C-helix subdomain. Opening efficacy was previously believed to be controlled primarily by the C-helix, but when we made additional chimeras by exchanging the subdomains between X-beta and X-alpha, we found that both subdomains contain significant determinants of efficacy and agonist selectivity. In particular, only channels containing the roll subdomain of the beta subunit had high efficacy. Thermodynamic linkage analysis shows that interaction between the two subdomains accounts for a significant portion of their contribution to activation energetics.     

Phylogeny and evolution of plant cyclic nucleotide-gated ion channel (CNGC) gene family and functional analyses of tomato CNGCs

Cyclic nucleotide-gated ion channels (CNGCs) are calcium-permeable channels that are involved in various biological functions. Nevertheless, phylogeny and function of plant CNGCs are not well understood. In this study, 333 CNGC genes from 15 plant species were identified using comprehensive bioinformatics approaches. Extensive bioinformatics analyses demonstrated that CNGCs of Group IVa were distinct to those of other groups in gene structure and amino acid sequence of cyclic nucleotide-binding domain. A CNGC-specific motif that recognizes all identified plant CNGCs was generated. Phylogenetic analysis indicated that CNGC proteins of flowering plant species formed five groups. However, CNGCs of the non-vascular plant Physcomitrella patens clustered only in two groups (IVa and IVb), while those of the vascular non-flowering plant Selaginella moellendorffii gathered in four (IVa, IVb, I and II). These data suggest that Group IV CNGCs are most ancient and Group III CNGCs are most recently evolved in flowering plants. Furthermore, silencing analyses revealed that a set of CNGC genes might be involved in disease resistance and abiotic stress responses in tomato and function of SlCNGCs does not correlate with the group that they are belonging to. Our results indicate that Group IVa CNGCs are structurally but not functionally unique among plant CNGCs.     

The chimeric cyclic nucleotide-gated ion channel ATCNGC11/12 constitutively induces programmed cell death in a Ca2+ dependent manner

The hypersensitive response (HR) involves programmed cell death (PCD) in response to pathogen infection. To investigate the pathogen resistance signaling pathway, we previously identified the Arabidopsis mutant cpr22, which displays constitutive activation of multiple defense responses including HR like cell death. The cpr22 mutation has been identified as a 3 kb deletion that fuses two cyclic nucleotide-gated ion channel (CNGC)-encoding genes, ATCNGC11 and ATCNGC12, to generate a novel chimeric gene, ATCNGC11/12. In this study, we conducted a characterization of cell death induced by transient expression of ATCNGC11/12 in Nicotiana benthamiana. Electron microscopic analysis of this cell death showed similar characteristics to PCD, such as plasma membrane shrinkage and vesicle formation. The hallmark of animal PCD, fragmentation of nuclear DNA, was also observed in ATCNGC11/12-induced cell death. The development of cell death was significantly suppressed by caspase-1 inhibitors, suggesting the involvement of caspases in this process. Recently, vacuolar processing enzyme (VPE) was isolated as the first plant caspase-like protein, which is involved in HR development. In VPE-silenced plants development of cell death induced by ATCNGC11/12 was much slower and weaker compared to control plants, suggesting the involvement of VPE as a caspase in ATCNGC11/12-induced cell death. Complementation analysis using a Ca²⁺ uptake deficient yeast mutant demonstrated that the ATCNGC11/12 channel is permeable to Ca²⁺. Additionally, calcium channel blockers such as GdCl₃ inhibited ATCNGC11/12-induced HR formation, whereas potassium channel blockers did not. Taken together, these results indicate that the cell death that develops in the cpr22 mutant is indeed PCD and that the chimeric channel, ATCNGC11/12, is at the point of, or up-stream of the calcium signal necessary for the development of HR.     

Expression of the hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 during mouse heart development

HCN4 is a hyperpolarization-activated nucleotide-gated cation channel involved in the generation of the I(f) current that drives cardiac pacemaker activity. Previous studies have demonstrated that HCN4 is highly expressed in a restricted manner in adult sinoatrial (SA) node [Eur. J. Biochem. 268 (2001) 1646]. However, its developmental expression pattern is unknown. We have examined expression of HCN4 mRNA during mouse heart development. HCN4 mRNA was first detected in the cardiac crescent at embryonic day (ED) 7.5. At ED 8 it was symmetrically located in the most caudal portion of the heart tube, the sinus venosus where pacemaker activity has previously been reported [Am. J. Physiol. 212 (1967) 407]. With further development, HCN4 expression became asymmetrically distributed, occupying the dorsal wall of the right atria, and was progressively restricted to the junction of the right atrial appendage and the superior vena cava. The site of HCN4 expression in late embryonic heart coincided with the location of the SA node in postnatal and adult heart [Cardiovasc. Res. 52 (2001) 51]. Our results suggest that HCN4 may be a unique marker of the developing SA node.     

The conduction system and expressions of hyperpolarization-activated cyclic nucleotide-gated cation channel 4 and connexin43 expressions in the hearts of fetal day 13 mice

We investigated the development of the sinus node of the heart conduction system by localizing hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4) and connexin43 (Cx43) in the hearts of fetal day 13 mice. Horizontal serial sections of day 13 whole fetuses were stained by hematoxylin and eosin and immunofluorescence to identify myocardial cells that express HCN4, hyperpolarization-activated cyclic nucleotide-gated cation channel 2 (HCN2) and Cx43. Expression levels of HCN4 and Cx43 were determined by quantitative RT-PCR in both fetal day 13 and adult mice. We found that both Cx43 and HCN4 expressions were located on the cell membranes in the hearts of fetal day 13 mice, but Cx43 was distributed throughout the myocardial cells. HCN4 expression was concentrated mainly in the left dorsal epicardium of the right atrium where Cx43 expression was low or absent. Quantitative RT-PCR demonstrated that HCN4 expression was significantly higher and HCN2 expression was significantly lower in fetal day 13 mice than in adults. We found no statistically significant difference in Cx43 expression between fetal day 13 mice and adults. HCN4 stained myocardial cells in the left dorsal epicardium of the right atrium are the origin of the sinus node and the remainder of the heart conduction system.     

Barley necrotic locus nec1 encodes the cyclic nucleotide-gated ion channel 4 homologous to the Arabidopsis HLM1

Barley homolog of the Arabidopsis necrotic (disease lesion mimic) mutant HLM1 that encodes the cyclic nucleotide-gated ion channel 4 was cloned. Barley gene was mapped genetically to the known necrotic locus nec1 and subsequent sequence analysis identified mutations in five available nec1 alleles confirming barley homolog of Arabidopsis HLM1 as the NEC1 gene. Two fast neutron (FN) induced mutants had extensive deletions in the gene, while two previously described nec1 alleles had either a STOP codon in exon 1 or a MITE insertion in intron 2 which caused alternative splicing, frame shift and production of a predicted non-functional protein. The MITE insertion was consistent with the reported spontaneous origin of the nec1 Parkland allele. The third FN mutant had a point mutation in the coding sequence which resulted in an amino acid change in the conserved predicted cyclic nucleotide-gated ion channel pore region. The expression of two pathogenesis-related genes, HvPR-1a and β-1,3-glucanase, was elevated in two FN necrotic lines. Ten other members of the barley cyclic nucleotide-gated ion channel gene family were identified and their position on barley linkage map is reported. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

A new hyperpolarization-activated, cyclic nucleotide-gated channel from sea urchin sperm flagella

A sea urchin sperm flagellar hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel is known (SpHCN1) that is modulated by cAMP. Here, we describe a second flagellar HCN channel (SpHCN2) cloned from the same sea urchin species. SpHCN2 is 638 amino acids compared to 767 for SpHCN1. SpHCN2 has all the domains of an HCN channel, including six transmembrane segments (S1-S6), the ion pore, and the cyclic nucleotide-binding domain. The two full-length proteins are 33% identical and 51% similar. The six transmembrane segments vary from 46-79% identity. S4, which is the voltage sensor, is 79% identical between the two proteins. The ion selectivity filter sequence is GYG in the ion pore of SpHCN1 and GFG in SpHCN2. By sequence, SpHCN2 is 73.5kDa, but it migrates on SDS-PAGE at 64kDa. Western immunoblots show localization to flagella, which is confirmed by immunofluorescence. A neighbor-joining tree shows that SpHCN2 is basal to all known HCN channels. SpHCN2 might be the simplest pacemaker channel yet discovered.     

A heat-activated calcium-permeable channel--Arabidopsis cyclic nucleotide-gated ion channel 6--is involved in heat shock responses

An increased concentration of cytosolic calcium ions (Ca²⁺) is an early response by plant cells to heat shock. However, the molecular mechanism underlying the heat‐induced initial Ca²⁺ response in plants is unclear. In this study, we identified and characterized a heat‐activated Ca²⁺‐permeable channel in the plasma membrane of Arabidopsis thaliana root protoplasts using reverse genetic analysis and the whole‐cell patch‐clamp technique. The results indicated that A. thaliana cyclic nucleotide‐gated ion channel 6 (CNGC6) mediates heat‐induced Ca²⁺ influx and facilitates expression of heat shock protein (HSP) genes and the acquisition of thermotolerance. GUS and GFP reporter assays showed that CNGC6 expression is ubiquitous in A. thaliana, and the protein is localized to the plasma membrane of cells. Furthermore, it was found that the level of cytosolic cAMP was increased by a mild heat shock, that CNGC6 was activated by cytosolic cAMP, and that exogenous cAMP promoted the expression of HSP genes. The results reveal the role of cAMP in transduction of heat shock signals in plants. The correlation of an increased level of cytosolic cAMP in a heat‐shocked plant with activation of the Ca²⁺ channels and downstream expression of HSP genes sheds some light on how plants transduce a heat stimulus into a signal cascade that leads to a heat shock response.     

Extracellular proton sensing of the rat gustatory cyclic nucleotide-gated channel

Elevations of the intracellular levels of cyclic nucleotides appear to cause the cation influx through gustatory cyclic nucleotide-gated (CNGgust) channels expressed in taste cells. Although changes in the oral pH may directly regulate the activity of the CNGgust channel, the mechanism of pH-dependent control of the channel is not understood. In the present study, we combined the whole-cell patch-clamp recording and the site-directed mutagenesis to investigate the effect of extracellular pH on the ion permeation through CNGgust channels expressed in HEK293 cells. Extracellular acidification strongly inhibited ion permeation through open CNGgust channels. Mutation of Glu(289) remarkably attenuated the pH-dependence of the channel, suggesting that Glu(289) in the pore-forming region is a major proton acceptor site. However, the mutant E289A-CNGgust channel possesses the other residual protonation/deprotonation site. The channel activity, tightly regulated by pH(o) and [cNMP](i), suggests the involvement of its pH(o)-dependent ion permeation in taste signal transduction events.