Sequence of events underlying the allosteric transition of rod cyclic nucleotide-gated channels

Activation of cyclic nucleotide-gated (CNG) ion channels involves a conformational change in the channel protein referred to as the allosteric transition. The amino terminal region and the carboxyl terminal cyclic nucleotide-binding domain of CNG channels have been shown to be involved in the allosteric transition, but the sequence of molecular events occurring during the allosteric transition is unknown. We recorded single-channel currents from bovine rod CNG channels in which mutations had been introduced in the binding domain at position 604 and/or the rat olfactory CNG channel amino terminal region had been substituted for the bovine rod amino terminal region. Using a hidden Markov modeling approach, we analyzed the kinetics of these channels activated by saturating concentrations of cGMP, cIMP, and cAMP. We used thermodynamic mutant cycles to reveal an interaction during the allosteric transition between the purine ring of the cyclic nucleotides and the amino acid at position 604 in the binding site. We found that mutations at position 604 in the binding domain alter both the opening and closing rate constants for the allosteric transition, indicating that the interactions between the cyclic nucleotide and this amino acid are partially formed at the time of the transition state. In contrast, the amino terminal region affects primarily the closing rate constant for the allosteric transition, suggesting that the state-dependent stabilizing interactions between amino and carboxyl terminal regions are not formed at the time of the transition state for the allosteric transition. We propose that the sequence of events that occurs during the allosteric transition involves the formation of stabilizing interactions between the purine ring of the cyclic nucleotide and the amino acid at position 604 in the binding domain followed by the formation of stabilizing interdomain interactions.     

Three amino acids in the C-linker are major determinants of gating in cyclic nucleotide-gated channels.

The activation of cyclic nucleotide-gated (CNG) channels is a complex process comprising the initial ligand binding and a consecutive allosteric transition from a closed to an open configuration. The cone and olfactory CNG channels differ considerably in cyclic nucleotide affinity and efficacy. In each channel, the cyclic nucleotide-binding site is connected to the last transmembrane segment of the channel by a linker peptide (C-linker) of approximately 90 amino acids. Here we report that replacement of three amino acids in the cone C-linker by the corresponding amino acids of the olfactory channel (I439V, D481A and D494S) profoundly enhanced the cAMP efficacy and increased the affinities for cAMP and cGMP. Unlike the wild-type cone channel, the mutated channel exhibited similar single-channel kinetics for both cGMP and cAMP, explaining the increase in cAMP efficacy. We thus conclude that the identified amino acids are major determinants of channel gating.     

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.     

Mechanism of allosteric modulation of rod cyclic nucleotide-gated channels

The cyclic nucleotide-gated (CNG) channel of retinal rod photoreceptor cells is an allosteric protein whose activation is coupled to a conformational change in the ligand-binding site. The bovine rod CNG channel can be activated by a number of different agonists, including cGMP, cIMP, and cAMP. These agonists span three orders of magnitude in their equilibrium constants for the allosteric transition. We recorded single-channel currents at saturating cyclic nucleotide concentrations from the bovine rod CNG channel expressed in Xenopus oocytes as homomultimers of alpha subunits. The median open probability was 0.93 for cGMP, 0.47 for cIMP, and 0.01 for cAMP. The channels opened to a single conductance level of 26-30 pS at +80 mV. Using signal processing methods based on hidden Markov models, we determined that two closed and one open states are required to explain the gating at saturating ligand concentrations. We determined the maximum likelihood rate constants for two gating schemes containing two closed (denoted C) and one open (denoted O) states. For the C left and right arrow C left and right arrow O scheme, all rate constants were dependent on cyclic nucleotide. For the C left and right arrow O left and right arrow C scheme, the rate constants for only one of the transitions were cyclic nucleotide dependent. The opening rate constant was fastest for cGMP, intermediate for cIMP, and slowest for cAMP, while the closing rate constant was fastest for cAMP, intermediate for cIMP, and slowest for cGMP. We propose that interactions between the purine ring of the cyclic nucleotide and the binding domain are partially formed at the time of the transition state for the allosteric transition and serve to reduce the transition state energy and stabilize the activated conformation of the channel. When 1 microM Ni2+ was applied in addition to cyclic nucleotide, the open time increased markedly, and the closed time decreased slightly. The interactions between H420 and Ni2+ occur primarily after the transition state for the allosteric transition.     

Functional consequences of progressive cone dystrophy-associated mutations in the human cone photoreceptor cyclic nucleotide-gated channel CNGA3 subunit

Progressive cone dystrophies are a genetically heterogeneous group of disorders characterized by early deterioration of visual acuity and color vision, together with psychophysical and electrophysiological evidence of abnormal cone function and cone degeneration. Recently, three mutations in the gene encoding the CNGA3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels have been linked to progressive cone dystrophy in humans. To investigate the functional consequences of these mutations, we expressed mutant human CNGA3 subunits in Xenopus oocytes, alone or together with human CNGB3, and studied these channels using patch-clamp recording. Compared with wild-type channels, homomeric and heteromeric channels containing CNGA3-N471S or CNGA3-R563H subunits exhibited an increase in apparent affinity for cGMP and an increase in the relative agonist efficacy of cAMP compared with cGMP. In contrast, R277C subunits did not form functional homomeric or heteromeric channels. Cell surface expression levels, determined using confocal microscopy of green fluorescent protein-tagged subunits and patch-clamp recording, were significantly reduced for both R563H and R277C but unchanged for N471S. Overall, these results suggest that the plasma membrane localization and gating properties of cone CNG channels are altered by progressive cone dystrophy-associated mutations, providing evidence that supports the pathogenicity of these mutations. phosphodiesterase     

环核苷酸门控离子通道门控的分子机理

环核苷酸门控离子通道(CNG)最广泛地分布于神经细胞。近年来关于 CNG 通道门控的分子机制的研究取得了很大的进步。研究表明, CNG 通道的组成及组装影响通道的特性及门控。近年来有关 CNG 突变体的研究及半胱氨酸残基亲和性的分析表明, 环核苷酸首先结合到 CNG 通道 C 端的环核苷酸结合域(CNBD)上引起 CNBD 空间构像改变, 然后 4 个亚单元发生空间构像的协调改变, CNG 通道开放。本文详细讨论了 CNG 通道的门控机制、各亚单元之间的相互作用、组装的过程及其空间构想的变化, 为 CNG 通道的进一步研究, 尤其是离子通道疾病方面提供理论指导。     

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.     

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.     

A cysteine scan of the inner vestibule of cyclic nucleotide-gated channels reveals architecture and rearrangement of the pore

Cyclic nucleotide-gated (CNG) channels belong to the P-loop-containing family of ion channels that also includes KcsA, MthK, and Shaker channels. In this study, we investigated the structure and rearrangement of the CNGA1 channel pore using cysteine mutations and cysteine-specific modification. We constructed 16 mutant channels, each one containing a cysteine mutation at one of the positions between 384 and 399 in the S6 region of the pore. By measuring currents activated by saturating concentrations of the full agonist cGMP and the partial agonists cIMP and cAMP, we show that mutating S6 residues to cysteine caused both favorable and unfavorable changes in the free energy of channel opening. The time course of cysteine modification with 2-aminoethylmethane thiosulfonate hydrochloride (MTSEA) was complex. For many positions we observed decreases in current activated by cGMP and concomitant increases in current activated by cIMP and cAMP. A model where modification affected both gating and permeation successfully reproduced the complex time course of modification for most of the mutant channels. From the model fits to the time course of modification for each mutant channel, we quantified the following: (a) the bimolecular rate constant of modification in the open state, (b) the change in conductance, and (c) the change in the free energy of channel opening for modification of each cysteine. At many S6 cysteines, modification by MTSEA caused a decrease in conductance and a favorable change in the free energy of channel opening. Our results are interpreted within the structural framework of the known structures of KcsA and MthK. We conclude that: (a) MTSEA modification affects both gating and permeation, (b) the open configuration of the pore of CNGA1 channels is consistent with the structure of MthK, and (c) the modification of S6 residues disrupts the helical packing of the closed channel, making it easier for channels to open.     

Single-channel properties of ionic channels gated by cyclic nucleotides.

This paper presents an extensive analysis of single-channel properties of cyclic nucleotide gated (CNG) channels, obtained by injecting into Xenopus laevis oocytes the mRNA encoding for the alpha and beta subunits from bovine rods. When the alpha and beta subunits of the CNG channel are coexpressed, at least three types of channels with different properties are observed. One type of channel has well-resolved, multiple conductive levels at negative voltages, but not at positive voltages. The other two types of channel are characterized by flickering openings, but are distinguished because they have a low and a high conductance. The alpha subunit of CNG channels has a well-defined conductance of about 28 pS, but multiple conductive levels are observed in mutant channels E363D and T364M. The conductance of these open states is modulated by protons and the membrane voltage, and has an activation energy around 44 kJ/mol. The relative probability of occupying any of these open states is independent of the cGMP concentration, but depends on extracellular protons. The open probability in the presence of saturating cGMP was 0.78, 0.47, 0.5, and 0.007 in the w.t. and mutants E363D, T364M, and E363G, and its dependence on temperature indicates that the thermodynamics of the transition between the closed and open state is also affected by mutations in the pore region. These results suggest that CNG channels have different conductive levels, leading to the existence of multiple open states in homomeric channels and to the flickering behavior in heteromeric channels, and that the pore is an essential part of the gating of CNG channels.     

Concerted All-or-none Subunit Interactions Mediate Slow Deactivation of Human ether-à-go-go-related Gene K+ Channels

During the repolarization phase of a cardiac action potential, hERG1 K⁺ channels rapidly recover from an inactivated state then slowly deactivate to a closed state. The resulting resurgence of outward current terminates the plateau phase and is thus a key regulator of action potential duration of cardiomyocytes. The intracellular N-terminal domain of the hERG1 subunit is required for slow deactivation of the channel as its removal accelerates deactivation 10-fold. Here we investigate the stoichiometry of hERG1 channel deactivation by characterizing the kinetic properties of concatenated tetramers containing a variable number of wild-type and mutant subunits. Three mutations known to accelerate deactivation were investigated, including R56Q and R4A/R5A in the N terminus and F656I in the S6 transmembrane segment. In all cases, a single mutant subunit induced the same rapid deactivation of a concatenated channel as that observed for homotetrameric mutant channels. We conclude that slow deactivation gating of hERG1 channels involves a concerted, fully cooperative interaction between all four wild-type channel subunits.     

C-type inactivation of a voltage-gated K+ channel occurs by a cooperative mechanism.

The lymphocyte voltage-gated K+ channel, Kv1.3, inactivates by a C-type process. We have elucidated the molecular basis for this process using a kinetic analysis of wild-type and mutant (A413V) Kv1.3 homo- and heteromultimeric currents in a mammalian lymphoid expression system. The medians of the measured inactivation time constants for wild-type and A413V homotetrameric currents are 204 and 4 ms, respectively. Co-expression of these subunits produces heteromultimeric channels manifesting inactivation kinetics intermediate between those of wild-type and A413V homomultimers. We have considered several models in which each subunit acts either independently or cooperatively to produce the observed inactivation kinetics. The cooperative model gives excellent fits to the data for any heteromultimeric composition of subunits, clearly distinguishing it from the independent models. In the cooperative model, the difference in free energy between the open and inactivated states of the channel is invariant with subunit composition and equals approximately 1.5 kcal/mol. Each subunit contributes equally to the activation free energy for transitions between open and inactivated states, with an A413V subunit decreasing the free energy barrier for inactivation (and for recovery from inactivation) by approximately 0.6 kcal/mol. Our results are consistent with a physical model in which the outer mouth of the channel constricts during C-type inactivation (G. Yellen, D. Sodickson, T. Chen, and M.E. Jurman, 1994, Biophys. J. 66:1068-1075).     

Isolation of a single activating allosteric interaction in phosphofructokinase from Escherichia coli

Escherichia coli phosphofructokinase 1 (EcPFK) is a homotetramer with four active and four allosteric sites. Understanding of the structural basis of allosteric activation of EcPFK by MgADP is complicated by the multiplicity of binding sites. To isolate a single heterotropic allosteric interaction, hybrid tetramers were formed between wild-type and mutant EcPFK subunits in which the binding sites of the mutant subunits have decreased affinity for their respective ligands. The 1:3 (wild-type:mutant) hybrid that contained only one native active site and one native allosteric site was isolated. The affinity for the substrate fructose-6-phosphate (Fru-6-P) of a single wild-type active site is greatly decreased over that displayed by the wild-type tetramer due to the lack of homotropic activation. The free energy of activation by MgADP for this heterotropic interaction is -0.58 kcal/mol at 8.5 degrees C. This compares to -2.87 kcal/mol for a hybrid with no homotropic coupling but all four unique heterotropic interactions. Therefore, the isolated interaction contributes 20% of the total heterotropic coupling. By comparison, wild-type EcPFK exhibits a coupling free energy between Fru-6-P and MgADP of -1.56 kcal/mol under these conditions, indicating that the effects of MgADP are diminished by a homotropic activation equal to -1.3 kcal/mol. These data are not consistent with a concerted allosteric mechanism.     

Noncatalytic Inhibition of Cyclic Nucleotide–gated Channels by Tyrosine Kinase Induced by Genistein

Rod photoreceptor cyclic nucleotide–gated (CNG) channels are modulated by tyrosine phosphorylation. Rod CNG channels expressed in Xenopus oocytes are associated with constitutively active protein tyrosine kinases (PTKs) and protein tyrosine phosphatases that decrease and increase, respectively, the apparent affinity of the channels for cGMP. Here, we examine the effects of genistein, a competitive inhibitor of the ATP binding site, on PTKs. Like other PTK inhibitors (lavendustin A and erbstatin), cytoplasmic application of genistein prevents changes in the cGMP sensitivity that are attributable to tyrosine phosphorylation of the CNG channels. However, unlike these other inhibitors, genistein also slows the activation kinetics and reduces the maximal current through CNG channels at saturating cGMP. These effects occur in the absence of ATP, indicating that they do not involve inhibition of a phosphorylation event, but rather involve an allosteric effect of genistein on CNG channel gating. This could result from direct binding of genistein to the channel; however, the time course of inhibition is surprisingly slow (>30 s), raising the possibility that genistein exerts its effects indirectly. In support of this hypothesis, we find that ligands that selectively bind to PTKs without directly binding to the CNG channel can nonetheless decrease the effect of genistein. Thus, ATP and a nonhydrolyzable ATP derivative competitively inhibit the effect of genistein on the channel. Moreover, erbstatin, an inhibitor of PTKs, can noncompetitively inhibit the effect of genistein. Taken together, these results suggest that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel catalyzed by PTKs, genistein triggers a noncatalytic interaction between the PTK and the channel that allosterically inhibits gating.     

Stoichiometry and arrangement of subunits in rod cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) ion channels mediate the response to light in retinal rods. They are tetramers of two homologous subunits (alpha and beta), each of which is essential for the function of the channels in vivo. We have investigated the stoichiometry and arrangement of these two subunits to determine how they come together within an individual channel complex. We exploited the very specific geometric and spatial requirements for forming a high-affinity Ni2+-binding site to examine the number and relative positions of the subunits. We found that only an order of alpha/alpha/beta/beta could account qualitatively and quantitatively for the observed intersubunit coordination of Ni2+ in wild-type and mutant alpha/beta channels. Furthermore, our results suggest a structural dimerization among like subunits, at least at the level of the Ni2+-binding site.     

Shaker potassium channel gating. III: Evaluation of kinetic models for activation

Predictions of different classes of gating models involving identical conformational changes in each of four subunits were compared to the gating behavior of Shaker potassium channels without N-type inactivation. Each model was tested to see if it could simulate the voltage dependence of the steady state open probability, and the kinetics of the single-channel currents, macroscopic ionic currents and macroscopic gating currents using a single set of parameters. Activation schemes based upon four identical single-step activation processes were found to be incompatible with the experimental results, as were those involving a concerted, opening transition. A model where the opening of the channel requires two conformational changes in each of the four subunits can adequately account for the steady state and kinetic behavior of the channel. In this model, the gating in each subunit is independent except for a stabilization of the open state when all four subunits are activated, and an unstable closed conformation that the channel enters after opening. A small amount of negative cooperativity between the subunits must be added to account quantitatively for the dependence of the activation time course on holding voltage.     

Rod cyclic nucleotide-gated channels have a stoichiometry of three CNGA1 subunits and one CNGB1 subunit

Phototransduction relies on the precise balance of speed and sensitivity to achieve optimal performance. The cyclic nucleotide-gated (CNG) ion channels, with their Ca(2+) permeability, high sensitivity to changes in cytosolic cGMP, rapid gating kinetics, and Ca(2+)-calmodulin modulation, are beautifully optimized for their role in light detection. Many of these specializations come about from the heteromeric composition of the native channel, comprised of CNGA1 and CNGB1 subunits. However, the stoichiometry and arrangement of these subunits is unknown. Here we have used an approach based on fluorescence resonance energy transfer (FRET) to determine the composition of the intact functional channel in the surface membrane. We find, surprisingly, that the channel contains three CNGA1 subunits and only one CNGB1 subunit. These results have implications for CNG channel function in particular and assembly of membrane proteins in general.     

Isolation of a single carboxyl-carboxylate proton binding site in the pore of a cyclic nucleotide-gated channel.

The pore of the catfish olfactory cyclic nucleotide-gated (CNG) channel contains four conserved glutamate residues, one from each subunit, that form a high-affinity binding site for extracellular divalent cations. Previous work showed that these residues form two independent and equivalent high-pKa (approximately 7.6) proton binding sites, giving rise to three pH-dependent conductance states, and it was suggested that the sites were formed by pairing of the glutamates into two independent carboxyl-carboxylates. To test further this physical picture, wild-type CNG subunits were coexpressed in Xenopus oocytes with subunits lacking the critical glutamate residue, and single channel currents through hybrid CNG channels containing one to three wild-type (WT) subunits were recorded. One of these hybrid channels had two pH-dependent conductance states whose occupancy was controlled by a single high-pKa protonation site. Expression of dimers of concatenated CNG channel subunits confirmed that this hybrid contained two WT and two mutant subunits, supporting the idea that a single protonation site is made from two glutamates (dimer expression also implied the subunit makeup of the other hybrid channels). Thus, the proton binding sites in the WT channel occur as a result of the pairing of two glutamate residues. This conclusion places these residues in close proximity to one another in the pore and implies that at any instant in time detailed fourfold symmetry is disrupted.     

Structure and rearrangements in the carboxy-terminal region of SpIH channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels regulate the spontaneous firing activity and electrical excitability of many cardiac and neuronal cells. The modulation of HCN channel opening by the direct binding of cAMP underlies many physiological processes such as the autonomic regulation of the heart rate. Here we use a combination of X-ray crystallography and electrophysiology to study the allosteric mechanism for cAMP modulation of HCN channels. SpIH is an invertebrate HCN channel that is activated fully by cAMP, but only partially by cGMP. We exploited the partial agonist action of cGMP on SpIH to reveal the molecular mechanism for cGMP specificity of many cyclic nucleotide-regulated enzymes. Our results also elaborate a mechanism for the allosteric conformational change in the cyclic nucleotide-binding domain and a mechanism for partial agonist action. These mechanisms will likely extend to other cyclic nucleotide-regulated channels and enzymes as well.     

Structure of the SthK Carboxy-Terminal Region Reveals a Gating Mechanism for Cyclic Nucleotide-Modulated Ion Channels

Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the K_v1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels.