Journal articles on the topic 'Cyclic nucleotide-gated channel'
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1
Kaupp, U. Benjamin, and Reinhard Seifert. "Cyclic Nucleotide-Gated Ion Channels." Physiological Reviews 82, no. 3 (January 7, 2002): 769–824. http://dx.doi.org/10.1152/physrev.00008.2002.
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Cyclic nucleotide-gated (CNG) channels are nonselective cation channels first identified in retinal photoreceptors and olfactory sensory neurons (OSNs). They are opened by the direct binding of cyclic nucleotides, cAMP and cGMP. Although their activity shows very little voltage dependence, CNG channels belong to the superfamily of voltage-gated ion channels. Like their cousins the voltage-gated K+ channels, CNG channels form heterotetrameric complexes consisting of two or three different types of subunits. Six different genes encoding CNG channels, four A subunits (A1 to A4) and two B subunits (B1 and B3), give rise to three different channels in rod and cone photoreceptors and in OSNs. Important functional features of these channels, i.e., ligand sensitivity and selectivity, ion permeation, and gating, are determined by the subunit composition of the respective channel complex. The function of CNG channels has been firmly established in retinal photoreceptors and in OSNs. Studies on their presence in other sensory and nonsensory cells have produced mixed results, and their purported roles in neuronal pathfinding or synaptic plasticity are not as well understood as their role in sensory neurons. Similarly, the function of invertebrate homologs found in Caenorhabditis elegans, Drosophila,and Limulus is largely unknown, except for two subunits of C. elegans that play a role in chemosensation. CNG channels are nonselective cation channels that do not discriminate well between alkali ions and even pass divalent cations, in particular Ca2+. Ca2+ entry through CNG channels is important for both excitation and adaptation of sensory cells. CNG channel activity is modulated by Ca2+/calmodulin and by phosphorylation. Other factors may also be involved in channel regulation. Mutations in CNG channel genes give rise to retinal degeneration and color blindness. In particular, mutations in the A and B subunits of the CNG channel expressed in human cones cause various forms of complete and incomplete achromatopsia.
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James, Zachary M., and William N. Zagotta. "Structural insights into the mechanisms of CNBD channel function." Journal of General Physiology 150, no. 2 (December 12, 2017): 225–44. http://dx.doi.org/10.1085/jgp.201711898.
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Cyclic nucleotide-binding domain (CNBD) channels are a family of ion channels in the voltage-gated K+ channel superfamily that play crucial roles in many physiological processes. CNBD channels are structurally similar but functionally very diverse. This family includes three subfamilies: (1) the cyclic nucleotide-gated (CNG) channels, which are cation-nonselective, voltage-independent, and cyclic nucleotide-gated; (2) the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are weakly K+ selective, hyperpolarization-activated, and cyclic nucleotide-gated; and (3) the ether-à-go-go-type (KCNH) channels, which are strongly K+ selective, depolarization-activated, and cyclic nucleotide-independent. Recently, several high-resolution structures have been reported for intact CNBD channels, providing a structural framework to better understand their diverse function. In this review, we compare and contrast the recent structures and discuss how they inform our understanding of ion selectivity, voltage-dependent gating, and cyclic nucleotide–dependent gating within this channel family.
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Becchetti, Andrea, Katia Gamel, and Vincent Torre. "Cyclic Nucleotide–Gated Channels." Journal of General Physiology 114, no. 3 (September 1, 1999): 377–92. http://dx.doi.org/10.1085/jgp.114.3.377.
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In voltage- and cyclic nucleotide–gated ion channels, the amino-acid loop that connects the S5 and S6 transmembrane domains, is a major component of the channel pore. It determines ion selectivity and participates in gating. In the α subunit of cyclic nucleotide–gated channels from bovine rod, the pore loop is formed by the residues R345–S371, here called R1-S27. These 24 residues were mutated one by one into a cysteine. Mutant channels were expressed in Xenopus laevis oocytes and currents were recorded from excised membrane patches. The accessibility of the substituted cysteines from both sides of the plasma membrane was tested with the thiol-specific reagents 2-aminoethyl methanethiosulfonate (MTSEA) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). Residues V4C, T20C, and P22C were accessible to MTSET only from the external side of the plasma membrane, and to MTSEA from both sides of the plasma membrane. The effect of MTSEA applied to the inner side of T20C and P22C was prevented by adding 10 mM cysteine to the external side of the plasma membrane. W9C was accessible to MTSET from the internal side only. L7C residue was accessible to internal MTSET, but the inhibition was partial, ∼50% when the MTS compound was applied in the absence of cGMP and 25% when it was applied in the presence of cGMP, suggesting that this residue is not located inside the pore lumen and that it changes its position during gating. Currents from T15C and T16C mutants were rapidly potentiated by intracellular MTSET. In T16C, a slower partial inhibition took place after the initial potentiation. Current from I17C progressively decayed in inside-out patches. The rundown was accelerated by inwardly applied MTSET. The accessibility results of MTSET indicate a well-defined topology of the channel pore in which residues between L7 and I17 are inwardly accessible, residue G18 and E19 form the narrowest section of the pore, and T20, P21, P22 and V4 are outwardly accessible.
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Santy, Lorraine C., and Guido Guidotti. "Expression of a single gene produces both forms of skeletal muscle cyclic nucleotide-gated channels." American Journal of Physiology-Endocrinology and Metabolism 273, no. 6 (December 1, 1997): E1140—E1148. http://dx.doi.org/10.1152/ajpendo.1997.273.6.e1140.
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Cyclic nucleotide-gated cation channels in skeletal muscle are responsible for insulin-activated sodium entry into this tissue (J. E. M. McGeoch and G. Guidotti. J. Biol. Chem. 267: 832–841, 1992). These channels have previously been isolated from rabbit skeletal muscle by 8-bromoguanosine 3′,5′-cyclic monophosphate (8-BrcGMP) affinity chromatography, which separates them into two populations differing in nucleotide affinity [L. C. Santy and G. Guidotti. Am. J. Physiol. 271 ( Endocrinol. Metab. 34): E1051-E1060, 1996]. In this study, a polymerase chain reaction approach was used to identify skeletal muscle cyclic nucleotide-gated channel cDNAs. Rabbit skeletal muscle expresses the same cyclic nucleotide-gated channel as rabbit aorta (M. Biel, W. Altenhofen, R. Hullin, J. Ludwig, M. Freichel, V. Flockerzi, N. Dascal, U. B. Kaupp, and F. Hofmann. FEBS Lett. 329: 134–138, 1993). The entire cDNA for this gene was cloned from rabbit skeletal muscle and an antiserum to this protein produced. Expression of this cDNA produces a 63-kDa protein with cyclic nucleotide-gated channel activity. A similarly sized immunoreactive protein is present in sarcolemma. Purification of the expressed channels reveals that this single gene produces both native skeletal muscle channel populations.
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Bej, Aritra, and James B. Ames. "Retinal Cyclic Nucleotide-Gated Channel Regulation by Calmodulin." International Journal of Molecular Sciences 23, no. 22 (November 16, 2022): 14143. http://dx.doi.org/10.3390/ijms232214143.
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Retinal cyclic nucleotide-gated (CNG) ion channels bind to intracellular cGMP and mediate visual phototransduction in photoreceptor rod and cone cells. Retinal rod CNG channels form hetero-tetramers comprised of three CNGA1 and one CNGB1 protein subunits. Cone CNG channels are similar tetramers consisting of three CNGA3 and one CNGB3 subunits. Calmodulin (CaM) binds to two distinct sites (CaM1: residues 565–587 and CaM2: residues 1120–1147) within the cytosolic domains of rod CNGB1. The binding of Ca2+-bound CaM to CNGB1 promotes the Ca2+-induced desensitization of CNG channels in retinal rods that may be important for photoreceptor light adaptation. Mutations that affect Ca2+-dependent CNG channel function are responsible for inherited forms of blindness. In this review, we propose structural models of the rod CNG channel bound to CaM that suggest how CaM might cause channel desensitization and how dysregulation of the channel may lead to retinal disease.
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Fodor, Anthony A., Sharona E. Gordon, and William N. Zagotta. "Mechanism of Tetracaine Block of Cyclic Nucleotide-gated Channels." Journal of General Physiology 109, no. 1 (January 1, 1997): 3–14. http://dx.doi.org/10.1085/jgp.109.1.3.
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Local anesthetics are a diverse group of ion channel blockers that can be used to probe conformational changes in the pore. We examined the effects of the local anesthetic tetracaine on rod and olfactory cyclic nucleotide-gated channels expressed from subunit 1 in Xenopus oocytes. We found that 40 μM tetracaine effectively blocked the bovine rod channel but not the rat olfactory channel at saturating concentrations of cGMP. By testing chimeric channels containing regions of sequence from both rod and olfactory channels, we found that determinants of apparent affinity for tetracaine at saturating cGMP did not map to any one region of the channel sequence. Rather, the differences in apparent affinity could be explained by differences between the chimeras in the free energy of the opening allosteric transition. If a channel construct (such as the rod channel) spent appreciable time in the closed state at saturating cGMP, then it had a high apparent affinity for tetracaine. If, on the other hand, a channel construct (such as the olfactory channel) spent little time in the closed state at saturating cGMP, then it had a low apparent affinity for tetracaine. Furthermore, tetracaine became more effective at low concentrations of cGMP and at saturating concentrations of cAMP, conditions which permit the channels to spend more time in the closed configuration. These results were well fit by a model in which tetracaine binds more tightly to the closed channel than to the open channel. Dose-response curves for tetracaine in the presence of saturating cGMP are well fit with a Michaelis-Menten binding scheme Indicating that a single tetracaine molecule is sufficient to produce block. In addition, tetracaine block is voltage dependent with an effective zδ of +0.56. These data are consistent with a pore-block hypothesis. The finding that tetracaine is a state-dependent pore blocker suggests that the inner mouth of the pore of cyclic nucleotide-gated channels undergoes a conformational change during channel opening.
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James, Zachary M., Andrew J. Borst, Yoni Haitin, Brandon Frenz, Frank DiMaio, William N. Zagotta, and David Veesler. "CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel." Proceedings of the National Academy of Sciences 114, no. 17 (April 10, 2017): 4430–35. http://dx.doi.org/10.1073/pnas.1700248114.
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Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxyl-terminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryoEM to determine the structure of the intact LliK CNG channel isolated from Leptospira licerasiae—which shares sequence similarity to eukaryotic CNG and HCN channels—in the presence of a saturating concentration of cAMP. A short S4–S5 linker connects nearby voltage-sensing and pore domains to produce a non–domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies.
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Santy, L. C., and G. Guidotti. "Reconstitution and characterization of two forms of cyclic nucleotide-gated channels from skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 271, no. 6 (December 1, 1996): E1051—E1060. http://dx.doi.org/10.1152/ajpendo.1996.271.6.e1051.
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A cyclic nucleotide-gated channel present in skeletal muscle plasma membrane has previously been identified as being responsible for insulin-activated sodium entry into muscle cells (J. E. M. McGeoch and G. Guidotti. J. Biol. Chem. 267:832-841, 1992). We have isolated this channel activity to further study and characterize it. The channel was solubilized from rabbit skeletal muscle sarcolemma and functionally reconstituted into phospholipid vesicles, as assayed by patch-clamp analysis of the reconstituted proteins. Channel activity was isolated by 8-bromo-guanosine 3',5'-cyclic monophosphate affinity chromatography, producing two distinct peaks of cyclic nucleotide-gated channel activity. These two types of channel activity differ in guanosine 3',5'-cyclic monophosphate affinity and in the ability to be opened by adenosine 3',5'-cyclic monophosphate. The cyclic nucleotide-gated channel from rod outer segments also forms two peaks of activity when purified in this manner. The presence of two forms of channel activity could have implications for the mechanism of insulin-activated sodium entry.
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Paoletti, Pierre, Edgar C. Young, and Steven A. Siegelbaum. "C-Linker of Cyclic Nucleotide–gated Channels Controls Coupling of Ligand Binding to Channel Gating." Journal of General Physiology 113, no. 1 (January 1, 1999): 17–34. http://dx.doi.org/10.1085/jgp.113.1.17.
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Cyclic nucleotide–gated channels are composed of a core transmembrane domain, structurally homologous to the voltage-gated K+ channels, and a cytoplasmic ligand-binding domain. These two modules are joined by ∼90 conserved amino acids, the C-linker, whose precise role in the mechanism of channel activation by cyclic nucleotides is poorly understood. We examined cyclic nucleotide–gated channels from bovine photoreceptors and Caenorhabditis elegans sensory neurons that show marked differences in cyclic nucleotide efficacy and sensitivity. By constructing chimeras from these two channels, we identified a region of 30 amino acids in the C-linker (the L2 region) as an important determinant of activation properties. An increase in both the efficacy of gating and apparent affinity for cGMP and cAMP can be conferred onto the photoreceptor channel by the replacement of its L2 region with that of the C. elegans channel. Three residues within this region largely account for this effect. Despite the profound effect of the C-linker region on ligand gating, the identity of the C-linker does not affect the spontaneous, ligand-independent open probability. Based on a cyclic allosteric model of activation, we propose that the C-linker couples the opening reaction in the transmembrane core region to the enhancement of the affinity of the open channel for agonist, which underlies ligand gating.
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Gerhardt, Maximilian J., Siegfried G. Priglinger, Martin Biel, and Stylianos Michalakis. "Biology, Pathobiology and Gene Therapy of CNG Channel-Related Retinopathies." Biomedicines 11, no. 2 (January 19, 2023): 269. http://dx.doi.org/10.3390/biomedicines11020269.
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The visual process begins with the absorption of photons by photopigments of cone and rod photoreceptors in the retina. In this process, the signal is first amplified by a cyclic guanosine monophosphate (cGMP)-based signaling cascade and then converted into an electrical signal by cyclic nucleotide-gated (CNG) channels. CNG channels are purely ligand-gated channels whose activity can be controlled by cGMP, which induces a depolarizing Na+/Ca2+ current upon binding to the channel. Structurally, CNG channels belong to the superfamily of pore-loop cation channels and share structural similarities with hyperpolarization-activated cyclic nucleotide (HCN) and voltage-gated potassium (KCN) channels. Cone and rod photoreceptors express distinct CNG channels encoded by homologous genes. Mutations in the genes encoding the rod CNG channel (CNGA1 and CNGB1) result in retinitis-pigmentosa-type blindness. Mutations in the genes encoding the cone CNG channel (CNGA3 and CNGB3) lead to achromatopsia. Here, we review the molecular properties of CNG channels and describe their physiological and pathophysiological roles in the retina. Moreover, we summarize recent activities in the field of gene therapy aimed at developing the first gene therapies for CNG channelopathies.
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Jarratt-Barnham, Edwin, Limin Wang, Youzheng Ning, and Julia M. Davies. "The Complex Story of Plant Cyclic Nucleotide-Gated Channels." International Journal of Molecular Sciences 22, no. 2 (January 16, 2021): 874. http://dx.doi.org/10.3390/ijms22020874.
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Plant cyclic nucleotide-gated channels (CNGCs) are tetrameric cation channels which may be activated by the cyclic nucleotides (cNMPs) adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP). The genome of Arabidopsis thaliana encodes 20 CNGC subunits associated with aspects of development, stress response and immunity. Recently, it has been demonstrated that CNGC subunits form heterotetrameric complexes which behave differently from the homotetramers produced by their constituent subunits. These findings have widespread implications for future signalling research and may help explain how specificity can be achieved by CNGCs that are known to act in disparate pathways. Regulation of complex formation may involve cyclic nucleotide-gated channel-like proteins.
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Crary, Jennifer I., Dylan M. Dean, Wang Nguitragool, Peri T. Kurshan, and Anita L. Zimmerman. "Mechanism of Inhibition of Cyclic Nucleotide–Gated Ion Channels by Diacylglycerol." Journal of General Physiology 116, no. 6 (December 1, 2000): 755–68. http://dx.doi.org/10.1085/jgp.116.6.755.
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Cyclic nucleotide–gated (CNG) channels are critical components in the visual and olfactory signal transduction pathways, and they primarily gate in response to changes in the cytoplasmic concentration of cyclic nucleotides. We previously found that the ability of the native rod CNG channel to be opened by cGMP was markedly inhibited by analogues of diacylglycerol (DAG) without a phosphorylation reaction (Gordon, S.E., J. Downing-Park, B. Tam, and A.L. Zimmerman. 1995. Biophys. J. 69:409–417). Here, we have studied cloned bovine rod and rat olfactory CNG channels expressed in Xenopus oocytes, and have determined that they are differentially inhibited by DAG. At saturating [cGMP], DAG inhibition of homomultimeric (α subunit only) rod channels was similar to that of the native rod CNG channel, but DAG was much less effective at inhibiting the homomultimeric olfactory channel, producing only partial inhibition even at high [DAG]. However, at low open probability (Po), both channels were more sensitive to DAG, suggesting that DAG is a closed state inhibitor. The Hill coefficients for DAG inhibition were often greater than one, suggesting that more than one DAG molecule is required for effective inhibition of a channel. In single-channel recordings, DAG decreased the Po but not the single-channel conductance. Results with chimeras of rod and olfactory channels suggest that the differences in DAG inhibition correlate more with differences in the transmembrane segments and their attached loops than with differences in the amino and carboxyl termini. Our results are consistent with a model in which multiple DAG molecules stabilize the closed state(s) of a CNG channel by binding directly to the channel and/or by altering bilayer–channel interactions. We speculate that if DAG interacts directly with the channel, it may insert into a putative hydrophobic crevice among the transmembrane domains of each subunit or at the hydrophobic interface between the channel and the bilayer.
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Tang, Chih-Yung, and Diane M. Papazian. "Transfer of Voltage Independence from a Rat Olfactory Channel to the Drosophila Ether-à-go-go K+ Channel." Journal of General Physiology 109, no. 3 (March 1, 1997): 301–11. http://dx.doi.org/10.1085/jgp.109.3.301.
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The S4 segment is an important part of the voltage sensor in voltage-gated ion channels. Cyclic nucleotide-gated channels, which are members of the superfamily of voltage-gated channels, have little inherent sensitivity to voltage despite the presence of an S4 segment. We made chimeras between a voltage-independent rat olfactory channel (rolf) and the voltage-dependent ether-à-go-go K+ channel (eag) to determine the basis of their divergent gating properties. We found that the rolf S4 segment can support a voltage-dependent mechanism of activation in eag, suggesting that rolf has a potentially functional voltage sensor that is silent during gating. In addition, we found that the S3-S4 loop of rolf increases the relative stability of the open conformation of eag, effectively converting eag into a voltage-independent channel. A single charged residue in the loop makes a significant contribution to the relative stabilization of the open state in eag. Our data suggest that cyclic nucleotide-gated channels such as rolf contain a voltage sensor which, in the physiological voltage range, is stabilized in an activated conformation that is permissive for pore opening.
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Voss, Logan J., Sebastian Karalus, Vilhelm Englund, and James W. Sleigh. "Ketamine Action in the In Vitro Cortical Slice Is Mitigated by Potassium Channel Blockade." Anesthesiology 128, no. 6 (June 1, 2018): 1167–74. http://dx.doi.org/10.1097/aln.0000000000002147.
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Abstract Background Ketamine is a general anesthetic thought to act by antagonizing N-methyl-d-aspartate receptors. However, ketamine acts on multiple channels, many of which are potential targets―including hyperpolarization-activated cyclic nucleotide-gated and potassium channels. In this study we tested the hypothesis that potassium leak channels contribute to the anesthetic action of ketamine. Methods Adult mouse cortical slices (400 µm) were exposed to no-magnesium artificial cerebrospinal fluid to generate seizure-like event activity. The reduction in seizure-like event frequency after exposure to ketamine (n = 14) was quantified as a signature of anesthetic effect. Pharmacologic manipulation of hyperpolarization-activated cyclic nucleotide-gated and potassium channels using ZD7288 (n = 11), cesium chloride (n = 10), barium chloride (n = 10), low-potassium (1.5 mM) artificial cerebrospinal fluid (n = 10), and urethane (n = 7) were investigated. Results Ketamine reduced the frequency of seizure-like events (mean [SD], –62 [22]%, P < 0.0001). Selective hyperpolarization-activated cyclic nucleotide-gated channel block with ZD7288 did not significantly alter the potency of ketamine to inhibit seizure-like event activity. The inhibition of seizure-like event frequency by ketamine was fully antagonized by the potassium channel blockers cesium chloride and barium chloride (8 [26]% and 39 [58%] increase, respectively, P < 0.0001, compared to ketamine control) and was facilitated by the potassium leak channel opener urethane (–93 [8]%, P = 0.002 compared to ketamine control) and low potassium artificial cerebrospinal fluid (–86 [11]%, P = 0.004 compared to ketamine control). Conclusions The results of this study show that mechanisms additional to hyperpolarization-activated cyclic nucleotide-gated channel block are likely to explain the anesthetic action of ketamine and suggest facilitatory action at two-pore potassium leak channels.
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Zufall, F., and S. Firestein. "Divalent cations block the cyclic nucleotide-gated channel of olfactory receptor neurons." Journal of Neurophysiology 69, no. 5 (May 1, 1993): 1758–68. http://dx.doi.org/10.1152/jn.1993.69.5.1758.
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1. The effects of external divalent cations on odor-dependent, cyclic AMP-activated single-channel currents from olfactory receptor neurons of the tiger salamander (Ambystoma tigrinum) were studied in inside-out membrane patches taken from dendritic regions of freshly isolated sensory cells. 2. Channels were reversibly activated by 100 microM cyclic AMP. In the absence of divalent cations, the channel had a linear current-voltage relation giving a conductance of 45 pS. With increasing concentrations of either Ca2+ or Mg2+ in the external solution, the channel displayed a rapid flickering behavior. At higher concentrations of divalent cations, the transitions were too rapid to be fully resolved and appeared as a reduction in mean unitary single-channel current amplitude. 3. This effect was voltage dependent, and on analysis was shown to be due to an open channel block by divalent ions. In the case of Mg2+, the block increased steadily with hyperpolarization. In contrast, for Ca2+ the block first increased with hyperpolarization and then decreased with further hyperpolarization beyond -70 mV, providing evidence for Ca2+ permeation of this channel. 4. This block is similar to that seen in voltage-gated calcium channels. Additionally, the cyclic nucleotide-gated channel shows some pharmacological similarities with L-type calcium channels, including a novel block of the cyclic nucleotide channel by nifedipine (50 microM). 5. Our results indicate that the sensory generator current simultaneously depends on the presence of the second messenger and on the membrane potential of the olfactory neuron.
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Middendorf, Thomas R., Richard W. Aldrich, and Denis A. Baylor. "Modification of Cyclic Nucleotide–Gated Ion Channels by Ultraviolet Light." Journal of General Physiology 116, no. 2 (July 31, 2000): 227–52. http://dx.doi.org/10.1085/jgp.116.2.227.
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We irradiated cyclic nucleotide–gated ion channels in situ with ultraviolet light to probe the role of aromatic residues in ion channel function. UV light reduced the current through excised membrane patches from Xenopus oocytes expressing the α subunit of bovine retinal cyclic nucleotide–gated channels irreversibly, a result consistent with permanent covalent modification of channel amino acids by UV light. The magnitude of the current reduction depended only on the total photon dose delivered to the patches, and not on the intensity of the exciting light, indicating that the functionally important photochemical modification(s) occurred from an excited state reached by a one-photon absorption process. The wavelength dependence of the channels' UV light sensitivity (the action spectrum) was quantitatively consistent with the absorption spectrum of tryptophan, with a small component at long wavelengths, possibly due to cystine absorption. This spectral analysis suggests that UV light reduced the currents at most wavelengths studied by modifying one or more “target” tryptophans in the channels. Comparison of the channels' action spectrum to the absorption spectrum of tryptophan in various solvents suggests that the UV light targets are in a water-like chemical environment. Experiments on mutant channels indicated that the UV light sensitivity of wild-type channels was not conferred exclusively by any one of the 10 tryptophan residues in a subunit. The similarity in the dose dependences of channel current reduction and tryptophan photolysis in solution suggests that photochemical modification of a small number of tryptophan targets in the channels is sufficient to decrease the currents.
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Li, Minghui, Xiaoyuan Zhou, Shu Wang, Ioannis Michailidis, Ye Gong, Deyuan Su, Huan Li, Xueming Li, and Jian Yang. "Structure of a eukaryotic cyclic-nucleotide-gated channel." Nature 542, no. 7639 (January 18, 2017): 60–65. http://dx.doi.org/10.1038/nature20819.
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Johnson, J. P., and William N. Zagotta. "Rotational movement during cyclic nucleotide-gated channel opening." Nature 412, no. 6850 (August 2001): 917–21. http://dx.doi.org/10.1038/35091089.
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Goulding, Evan H., Gareth R. Tibbs, and Steven A. Siegelbaum. "Molecular mechanism of cyclic-nucleotide-gated channel activation." Nature 372, no. 6504 (November 1994): 369–74. http://dx.doi.org/10.1038/372369a0.
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Lam, Yee Ling, Weizhong Zeng, Mehabaw Getahun Derebe, and Youxing Jiang. "Structural implications of weak Ca2+ block in Drosophila cyclic nucleotide–gated channels." Journal of General Physiology 146, no. 3 (August 17, 2015): 255–63. http://dx.doi.org/10.1085/jgp.201511431.
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Calcium permeability and the concomitant calcium block of monovalent ion current (“Ca2+ block”) are properties of cyclic nucleotide–gated (CNG) channel fundamental to visual and olfactory signal transduction. Although most CNG channels bear a conserved glutamate residue crucial for Ca2+ block, the degree of block displayed by different CNG channels varies greatly. For instance, the Drosophila melanogaster CNG channel shows only weak Ca2+ block despite the presence of this glutamate. We previously constructed a series of chimeric channels in which we replaced the selectivity filter of the bacterial nonselective cation channel NaK with a set of CNG channel filter sequences and determined that the resulting NaK2CNG chimeras displayed the ion selectivity and Ca2+ block properties of the parent CNG channels. Here, we used the same strategy to determine the structural basis of the weak Ca2+ block observed in the Drosophila CNG channel. The selectivity filter of the Drosophila CNG channel is similar to that of most other CNG channels except that it has a threonine at residue 318 instead of a proline. We constructed a NaK chimera, which we called NaK2CNG-Dm, which contained the Drosophila selectivity filter sequence. The high resolution structure of NaK2CNG-Dm revealed a filter structure different from those of NaK and all other previously investigated NaK2CNG chimeric channels. Consistent with this structural difference, functional studies of the NaK2CNG-Dm chimeric channel demonstrated a loss of Ca2+ block compared with other NaK2CNG chimeras. Moreover, mutating the corresponding threonine (T318) to proline in Drosophila CNG channels increased Ca2+ block by 16 times. These results imply that a simple replacement of a threonine for a proline in Drosophila CNG channels has likely given rise to a distinct selectivity filter conformation that results in weak Ca2+ block.
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Sunderman, Elizabeth R., and William N. Zagotta. "Mechanism of Allosteric Modulation of Rod Cyclic Nucleotide–gated Channels." Journal of General Physiology 113, no. 5 (May 1, 1999): 601–20. http://dx.doi.org/10.1085/jgp.113.5.601.
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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 α 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 ↔ C ↔ O scheme, all rate constants were dependent on cyclic nucleotide. For the C ↔ O ↔ 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 μM 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.
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Schwiebert, E. M., E. D. Potter, T. H. Hwang, J. S. Woo, C. Ding, W. Qiu, W. B. Guggino, M. A. Levine, and S. E. Guggino. "cGMP stimulates sodium and chloride currents in rat tracheal airway epithelia." American Journal of Physiology-Cell Physiology 272, no. 3 (March 1, 1997): C911—C922. http://dx.doi.org/10.1152/ajpcell.1997.272.3.c911.
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To test the hypothesis that guanosine 3',5'-cyclic monophosphate (cGMP) regulates ion transport in airway epithelial cells, we measured short-circuit current (I(sc)) and (22)Na+ fluxes in primary cultured rat tracheal epithelial cells. In Cl- -containing Ringer solution, I(sc) was increased by approximately 17 microA/cm2 after application of 1 mM 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), whereas, in Cl- -free solutions, the Na+ -mediated component was approximately 5 microA/cm2, suggesting a cGMP stimulation of Cl-secretory current and a smaller Na+ absorptive current. Inward and net mucosal-to-serosal (22)Na+ flux was doubled in the presence of 2 mM 8-BrcGMP. To determine whether nucleotide-gated channels play a role in this transepithelial Na+ absorption, blockers of nucleotide-gated cation channels were used to inhibit I(sc). The cGMP-stimulated Na+-mediated I(sc) was blocked by as little as 500 nM dichlorobenzamil or 50 microM L-cis-diltiazem, which are known blockers for cyclic nucleotide-gated cation channels. These agents also blocked the basal (non-cGMP-stimulated) current when measured in the presence of 10 microM amiloride, which blocks current through 5-pS amiloride-sensitive Na+ channels. To document whether the distribution of nucleotide-gated nonselective cation channels was consistent with a role in airway epithelial transport, in situ hybridization was performed. In situ hybridization of mRNA encoding for nucleotide-gated cation channels was found in epithelial cell layers of rat trachea, bronchi, bronchioles, and alveolar cells but not in smooth muscle layers or tracheal cartilage. Reverse transcriptase-polymerase chain reaction, restriction enzyme analysis, and sequencing of the cDNA transcribed from mRNA of whole lung and tracheal epithelial cells indicate that a channel highly homologous to the retinal nucleotide-gated nonselective cation channel (CNG1) is present. Thus these data, along with evidence supporting the existence of signal transduction pathways elevating intracellular levels of cGMP, indicate that cGMP regulates transepithelial ion transport in lung epithelial tissues.
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Qiu, W., B. Lee, M. Lancaster, W. Xu, S. Leung, and S. E. Guggino. "Cyclic nucleotide-gated cation channels mediate sodium and calcium influx in rat colon." American Journal of Physiology-Cell Physiology 278, no. 2 (February 1, 2000): C336—C343. http://dx.doi.org/10.1152/ajpcell.2000.278.2.c336.
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We found mRNA for the three isoforms of the cyclic nucleotide-gated nonselective cation channel expressed in the mucosal layer of the rat intestine from the duodenum to the colon and in intestinal epithelial cell lines in culture. Because these channels are permeable to sodium and calcium and are stimulated by cGMP or cAMP, we measured 8-bromo-cGMP-stimulated sodium-mediated short-circuit current ( I sc) in proximal and distal colon and unidirectional45Ca2+fluxes in proximal colon to determine whether these channels could mediate transepithelial sodium and calcium absorption across the colon. Sodium-mediated I sc, stimulated by 8-bromo-cGMP, were inhibited by dichlorobenzamil and l-cis-diltiazem, blockers of cyclic nucleotide-gated cation channels, suggesting that these ion channels can mediate transepithelial sodium absorption. Sodium-mediated I sc and net transepithelial45Ca2+absorption were stimulated by heat-stable toxin from Escherichia coli that increases cGMP. Addition of l-cis-diltiazem inhibited the enhanced transepithelial absorption of both ions. These results suggest that cyclic nucleotide-gated cation channels simultaneously increase net sodium and calcium absorption in the colon of the rat.
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ORANAB, S., A. GHAFFAR, A. AHMAD, MFK PASHA, B. MUNIR, S. ARIF, S. ISHAQ, et al. "GENOME-WIDE ANALYSIS OF CYCLIC NUCLEOTIDE-GATED ION CHANNELS (CNGCS) OF ARABIDOPSIS THALIANA UNDER ABIOTIC STRESSES." SABRAO Journal of Breeding and Genetics 55, no. 1 (February 28, 2023): 38–49. http://dx.doi.org/10.54910/sabrao2023.55.1.4.
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Cyclic nucleotide-gated ion channels (CNGCs) in plants play a significant role in abiotic and biotic stress tolerance. This study analyzed 20 CNGCs of Arabidopsis thaliana for their potential role under different stresses. According to phylogenetic analysis, the abiotic stress-tolerating gene CNGC19 in A. thaliana showed as closely related to Hordeum vulgare cyclic nucleotide-gated ion channel 19 (HvCNGC19) in barley and Oryza sativa cyclic nucleotide-gated ion channels 4 and 11 (OsCNGC4 and OsCNGC11) of rice. All CNGCs of A. thaliana contains an ion transport domain. HvCNGC19, OsCNGC4, OsCNGC11, and AtCNGC19 contained the same motif 24, which depicted that they might be expressed similarly to AtCNGC19 under salt stress. CNGCs expression signals under abiotic stress showed high expression of AtCNGC19 and AtCNGC20 under salt stress in roots and AtCNGC2 and AtCNGC4 in shoots, yet very low in roots under approximately all stresses. The findings provide widespread implications for future cell signaling research and characterization of CNGCs for their roles under different stresses.
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Schmidpeter, Philipp A. M., Xiaolong Gao, Vikrant Uphadyay, Jan Rheinberger, and Crina M. Nimigean. "Ligand binding and activation properties of the purified bacterial cyclic nucleotide–gated channel SthK." Journal of General Physiology 150, no. 6 (May 11, 2018): 821–34. http://dx.doi.org/10.1085/jgp.201812023.
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Cyclic nucleotide–modulated ion channels play several essential physiological roles. They are involved in signal transduction in photoreceptors and olfactory sensory neurons as well as pacemaking activity in the heart and brain. Investigations of the molecular mechanism of their actions, including structural and electrophysiological characterization, are restricted by the availability of stable, purified protein obtained from accessible systems. Here, we establish that SthK, a cyclic nucleotide–gated (CNG) channel from Spirochaeta thermophila, is an excellent model for investigating the gating of eukaryotic CNG channels at the molecular level. The channel has high sequence similarity with its eukaryotic counterparts and was previously reported to be activated by cyclic nucleotides in patch-clamp experiments with Xenopus laevis oocytes. We optimized protein expression and purification to obtain large quantities of pure, homogeneous, and active recombinant SthK protein from Escherichia coli. A negative-stain electron microscopy (EM) single-particle analysis indicated that this channel is a promising candidate for structural studies with cryo-EM. Using radioactivity and fluorescence flux assays, as well as single-channel recordings in lipid bilayers, we show that the protein is partially activated by micromolar concentrations of cyclic adenosine monophosphate (cAMP) and that channel activity is increased by depolarization. Unlike previous studies, we find that cyclic guanosine monophosphate (cGMP) is also able to activate SthK, but with much lower efficiency than cAMP. The distinct sensitivities to different ligands resemble eukaryotic CNG and hyperpolarization-activated and cyclic nucleotide–modulated channels. Using a fluorescence binding assay, we show that cGMP and cAMP bind to SthK with similar apparent affinities, suggesting that the large difference in channel activation by cAMP or cGMP is caused by the efficacy with which each ligand promotes the conformational changes toward the open state. We conclude that the functional characteristics of SthK reported here will permit future studies to analyze ligand gating and discrimination in CNG channels.
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Holmgren, Miguel. "Influence of Permeant Ions on Gating in Cyclic Nucleotide–gated Channels." Journal of General Physiology 121, no. 1 (December 30, 2002): 61–72. http://dx.doi.org/10.1085/jgp.20028722.
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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 α) 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.
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Henry, Diane, Stephanie Burke, Emiko Shishido, and Gary Matthews. "Retinal Bipolar Neurons Express the Cyclic Nucleotide-Gated Channel of Cone Photoreceptors." Journal of Neurophysiology 89, no. 2 (February 1, 2003): 754–61. http://dx.doi.org/10.1152/jn.00771.2002.
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Cyclic nucleotide-gated (CNG) channels link intracellular cyclic nucleotides to changes in membrane ionic conductance in a variety of physiological contexts. In the retina, in addition to their central role in phototransduction, CNG channels may be involved in nitric oxide signaling in bipolar neurons or in the hyperpolarizing synaptic response to glutamate inon-type (depolarizing) bipolar cells. Despite their potential physiological significance, however, expression of CNG channels has not yet been demonstrated in bipolar cells. To identify CNG channel subtypes in retinal bipolar neurons, we used single-cell molecular biological techniques in morphologically distinctiveon bipolar cells from goldfish retina. Both single-cell in situ hybridization and single-cell RT-PCR demonstrated inon bipolar cells the presence of mRNA for the CNG channel subtype that is also found in cone photoreceptors. Other bipolar cells, which likely represent off cells, did not express the cone CNG channel. Thus the CNG channel of cone photoreceptors is expressed in on bipolar cells, where it may be involved in physiological responses to nitric oxide, or in the sign-inverting glutamatergic synapse that gives rise to the on visual pathway.
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Yao, X. Q. "Cyclic nucleotide-gated channels: an old channel family with new function?" International Journal of Cardiology 125 (February 2008): S25. http://dx.doi.org/10.1016/s0167-5273(08)70168-2.
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Evans, Eric G. B., Jacob L. W. Morgan, Frank DiMaio, William N. Zagotta, and Stefan Stoll. "Allosteric conformational change of a cyclic nucleotide-gated ion channel revealed by DEER spectroscopy." Proceedings of the National Academy of Sciences 117, no. 20 (May 1, 2020): 10839–47. http://dx.doi.org/10.1073/pnas.1916375117.
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Cyclic nucleotide-gated (CNG) ion channels are essential components of mammalian visual and olfactory signal transduction. CNG channels open upon direct binding of cyclic nucleotides (cAMP and/or cGMP), but the allosteric mechanism by which this occurs is incompletely understood. Here, we employed double electron-electron resonance (DEER) spectroscopy to measure intersubunit distance distributions in SthK, a bacterial CNG channel from Spirochaeta thermophila. Spin labels were introduced into the SthK C-linker, a domain that is essential for coupling cyclic nucleotide binding to channel opening. DEER revealed an agonist-dependent conformational change in which residues of the B′-helix displayed outward movement with respect to the symmetry axis of the channel in the presence of the full agonist cAMP, but not with the partial agonist cGMP. This conformational rearrangement was observed both in detergent-solubilized SthK and in channels reconstituted into lipid nanodiscs. In addition to outward movement of the B′-helix, DEER-constrained Rosetta structural models suggest that channel activation involves upward translation of the cytoplasmic domain and formation of state-dependent interactions between the C-linker and the transmembrane domain. Our results demonstrate a previously unrecognized structural transition in a CNG channel and suggest key interactions that may be responsible for allosteric gating in these channels.
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Kramer, Richard H., and Elena Molokanova. "Modulation of cyclic-nucleotide-gated channels and regulation of vertebrate phototransduction." Journal of Experimental Biology 204, no. 17 (September 1, 2001): 2921–31. http://dx.doi.org/10.1242/jeb.204.17.2921.
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SUMMARY Cyclic-nucleotide-gated (CNG) channels are crucial for sensory transduction in the photoreceptors (rods and cones) of the vertebrate retina. Light triggers a decrease in the cytoplasmic concentration of cyclic GMP in the outer segments of these cells, leading to closure of CNG channels and hyperpolarization of the membrane potential. Hence, CNG channels translate a chemical change in cyclic nucleotide concentration into an electrical signal that can spread through the photoreceptor cell and be transmitted to the rest of the visual system. The sensitivity of phototransduction can be altered by exposing the cells to light, through adaptation processes intrinsic to photoreceptors. Intracellular Ca2+ is a major signal in light adaptation and, in conjunction with Ca2+-binding proteins, one of its targets for modulation is the CNG channel itself. However, other intracellular signals may be involved in the fine-tuning of light sensitivity in response to cues internal to organisms. Several intracellular signals are candidates for mediating changes in cyclic GMP sensitivity including transition metals, such as Ni2+ and Zn2+, and lipid metabolites, such as diacylglycerol. Moreover, CNG channels are associated with protein kinases and phosphatases that catalyze changes in phosphorylation state and allosterically modulate channel activity. Recent studies suggest that the effects of circadian rhythms and retinal transmitters on CNG channels may be mediated by such changes in phosphorylation. The goal of this paper is to review the molecular mechanisms underlying modulation of CNG channels and to relate these forms of modulation to the regulation of light sensitivity.
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Lee, Han Mi, Young Sun Park, Wonjae Kim, and Chul-Seung Park. "Electrophysiological Characteristics of Rat Gustatory Cyclic Nucleotide–Gated Channel Expressed in Xenopus Oocytes." Journal of Neurophysiology 85, no. 6 (June 1, 2001): 2335–49. http://dx.doi.org/10.1152/jn.2001.85.6.2335.
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The complementary DNA encoding gustatory cyclic nucleotide–gated ion channel (or gustCNG channel) cloned from rat tongue epithelial tissue was expressed in Xenopus oocytes, and its electrophysiological characteristics were investigated using tight-seal patch-clamp recordings of single and macroscopic channel currents. Both cGMP and cAMP directly activated gustCNG channels but with markedly different affinities. No desensitization or inactivation of gustCNG channel currents was observed even in the prolonged application of the cyclic nucleotides. Single-channel conductance of gustCNG channel was estimated as 28 pS in 130 mM of symmetric Na+. Single-channel current recordings revealed fast open-close transitions and longer lasting closure states. The distribution of both open and closed events could be well fitted with two exponential components and intracellular cGMP increased the open probability ( P o) of gustCNG channels mainly by increasing the slower opening rate. Under bi-ionic conditions, the selectivity order of gustCNG channel among divalent cations was determined as Na+ ∼ K+ > Rb+ > Li+ > Cs+ with the permeability ratio of 1:0.95:0.74:0.63:0.49. Magnesium ion blocked Na+ currents through gustCNG channels from both intracellular and extracellular sides in voltage-dependent manners. The inhibition constants ( K is) of intracellular Mg2+ were determined as 360 ± 40 μM at 70 mV and 8.2 ± 1.5 mM at −70 mV with zδ value of 1.04, while K is of extracellular Mg2+ were as 1.1 ± 0.3 mM at 70 mV and 20.0 ± 0.1 μM at −70 mV with zδ of 0.94. Although 100 μM l- cis-diltiazem blocked significant portions of outward Na+ currents through both bovine rod and rat olfactory CNG channels, the gustCNG channel currents were minimally affected by the same concentration of the drug.
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Liu, Chang, Changan Xie, Khade Grant, Zhuocheng Su, Weihua Gao, Qinglian Liu, and Lei Zhou. "Patch-clamp fluorometry–based channel counting to determine HCN channel conductance." Journal of General Physiology 148, no. 1 (June 27, 2016): 65–76. http://dx.doi.org/10.1085/jgp.201511559.
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Counting ion channels on cell membranes is of fundamental importance for the study of channel biophysics. Channel counting has thus far been tackled by classical approaches, such as radioactive labeling of ion channels with blockers, gating current measurements, and nonstationary noise analysis. Here, we develop a counting method based on patch-clamp fluorometry (PCF), which enables simultaneous electrical and optical recordings, and apply it to EGFP-tagged, hyperpolarization-activated and cyclic nucleotide–regulated (HCN) channels. We use a well-characterized and homologous cyclic nucleotide–gated (CNG) channel to establish the relationship between macroscopic fluorescence intensity and the total number of channels. Subsequently, based on our estimate of the total number of HCN channels, we determine the single-channel conductance of HCN1 and HCN2 to be 0.46 and 1.71 pS, respectively. Such a small conductance would present a technical challenge for traditional electrophysiology. This PCF-based technique provides an alternative method for counting particles on cell membranes, which could be applied to biophysical studies of other membrane proteins.
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Shiba, Kogiku, and Kazuo Inaba. "The Roles of Two CNG Channels in the Regulation of Ascidian Sperm Chemotaxis." International Journal of Molecular Sciences 23, no. 3 (January 31, 2022): 1648. http://dx.doi.org/10.3390/ijms23031648.
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Spermatozoa sense and respond to their environmental signals to ensure fertilization success. Reception and transduction of signals are reflected rapidly in sperm flagellar waveforms and swimming behavior. In the ascidian Ciona intestinalis (type A; also called C. robusta), an egg-derived sulfated steroid called SAAF (sperm activating and attracting factor), induces both sperm motility activation and chemotaxis. Two types of CNG (cyclic nucleotide-gated) channels, Ci-tetra KCNG (tetrameric, cyclic nucleotide-gated, K+-selective) and Ci-HCN (hyperpolarization-activated and cyclic nucleotide-gated), are highly expressed in Ciona testis from the comprehensive gene expression analysis. To elucidate the sperm signaling pathway to regulate flagellar motility, we focus on the role of CNG channels. In this study, the immunochemical analysis revealed that both CNG channels are expressed in Ciona sperm and localized to sperm flagella. Sperm motility analysis and Ca2+ imaging during chemotaxis showed that CNG channel inhibition affected the changes in flagellar waveforms and Ca2+ efflux needed for the chemotactic turn. These results suggest that CNG channels in Ciona sperm play a vital role in regulating sperm motility and intracellular Ca2+ regulation during chemotaxis.
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Li, Mo, Lige Tonggu, Lan Tang, and Liguo Wang. "Effects of N-glycosylation on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels." Biochemical Journal 466, no. 1 (February 6, 2015): 77–84. http://dx.doi.org/10.1042/bj20140692.
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The results suggest that N-glycosylation is not required for the opening of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and some but not all of the four subunits of the HCN channel need to be glycosylated for trafficking to cell membrane.
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Molokanova, Elena, Alexei Savchenko, and Richard H. Kramer. "Noncatalytic Inhibition of Cyclic Nucleotide–gated Channels by Tyrosine Kinase Induced by Genistein." Journal of General Physiology 113, no. 1 (January 1, 1999): 45–56. http://dx.doi.org/10.1085/jgp.113.1.45.
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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.
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Sunderman, Elizabeth R., and William N. Zagotta. "Sequence of Events Underlying the Allosteric Transition of Rod Cyclic Nucleotide–gated Channels." Journal of General Physiology 113, no. 5 (May 1, 1999): 621–40. http://dx.doi.org/10.1085/jgp.113.5.621.
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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.
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Liston, Conor. "Targeting pacemaker channels in depression." Science Translational Medicine 11, no. 477 (January 30, 2019): eaaw5318. http://dx.doi.org/10.1126/scitranslmed.aaw5318.
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Wang, Zhengchao, Yongqing Jiang, Lizhi Lu, Ruihua Huang, Qingchao Hou, and Fangxiong Shi. "Molecular Mechanisms of Cyclic Nucleotide-Gated Ion Channel Gating." Journal of Genetics and Genomics 34, no. 6 (June 2007): 477–85. http://dx.doi.org/10.1016/s1673-8527(07)60052-6.
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Shah, Mala M. "Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Currents in Neurons." Cold Spring Harbor Protocols 2016, no. 7 (July 2016): pdb.top087346. http://dx.doi.org/10.1101/pdb.top087346.
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Ohndorf, Uta-Maria, and Roderick MacKinnon. "Construction of a Cyclic Nucleotide-gated KcsA K+ Channel." Journal of Molecular Biology 350, no. 5 (July 2005): 857–65. http://dx.doi.org/10.1016/j.jmb.2005.05.050.
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Firestein, Stuart, and Frank Zufall. "The cyclic nucleotide gated channel of olfactory receptor neurons." Seminars in Cell Biology 5, no. 1 (February 1994): 39–46. http://dx.doi.org/10.1006/scel.1994.1006.
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Galindo, Blanca Estela, José Luis de la Vega-Beltrán, Pedro Labarca, Victor D. Vacquier, and Alberto Darszon. "Sp-tetraKCNG: A novel cyclic nucleotide gated K+ channel." Biochemical and Biophysical Research Communications 354, no. 3 (March 2007): 668–75. http://dx.doi.org/10.1016/j.bbrc.2007.01.035.
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Gao, Xiaolong, Philipp A. M. Schmidpeter, and Crina M. Nimigean. "Functional Characterization of the Cyclic Nucleotide-Gated Channel SthK." Biophysical Journal 114, no. 3 (February 2018): 129a. http://dx.doi.org/10.1016/j.bpj.2017.11.730.
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Schmidpeter, Philipp A. M., Jan Rheinberger, Di Wu, Haiping Tang, Carol V. Robinson, and Crina M. Nimigean. "Lipid-Modulation of SthK, a Cyclic Nucleotide-Gated Channel." Biophysical Journal 116, no. 3 (February 2019): 54a. http://dx.doi.org/10.1016/j.bpj.2018.11.338.
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Fodor, Anthony A., Kevin D. Black, and William N. Zagotta. "Tetracaine Reports a Conformational Change in the Pore of Cyclic Nucleotide–gated Channels." Journal of General Physiology 110, no. 5 (November 1, 1997): 591–600. http://dx.doi.org/10.1085/jgp.110.5.591.
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Local anesthetics are a diverse group of clinically useful compounds that act as pore blockers of both voltage- and cyclic nucleotide–gated (CNG) ion channels. We used the local anesthetic tetracaine to probe the nature of the conformational change that occurs in the pore of CNG channels during the opening allosteric transition. When applied to the intracellular side of wild-type rod CNG channels expressed in Xenopus oocytes from the α subunit, the local anesthetic tetracaine exhibits state-dependent block, binding with much higher affinity to closed states than to open states. Here we show that neutralization of a glutamic acid in the conserved P region (E363G) eliminated this state dependence of tetracaine block. Tetracaine blocked E363G channels with the same effectiveness at high concentrations of cGMP, when the channel spent more time open, and at low concentrations of cGMP, when the channel spent more time closed. In addition, Ni2+, which promotes the opening allosteric transition, decreased the effectiveness of tetracaine block of wild-type but not E363G channels. Similar results were obtained in a chimeric CNG channel that exhibits a more favorable opening allosteric transition. These results suggest that E363 is accessible to internal tetracaine in the closed but not the open configuration of the pore and that the conformational change that accompanies channel opening includes a change in the conformation or accessibility of E363.
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Molokanova, Elena, and Richard H. Kramer. "Mechanism of Inhibition of Cyclic Nucleotide–Gated Channel by Protein Tyrosine Kinase Probed with Genistein." Journal of General Physiology 117, no. 3 (February 26, 2001): 219–34. http://dx.doi.org/10.1085/jgp.117.3.219.
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Rod cyclic nucleotide–gated (CNG) channels are modulated by changes in tyrosine phosphorylation catalyzed by protein tyrosine kinases (PTKs) and phosphatases (PTPs). We used genistein, a PTK inhibitor, to probe the interaction between the channel and PTKs. Previously, we found that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel α-subunit (RETα), genistein triggers a noncatalytic inhibitory interaction between the PTK and the channel. These studies suggest that PTKs affects RETα channels in two ways: (1) by catalyzing phosphorylation of the channel protein, and (2) by allosterically regulating channel activation. Here, we study the mechanism of noncatalytic inhibition. We find that noncatalytic inhibition follows the same activity dependence pattern as catalytic modulation (phosphorylation): the efficacy and apparent affinity of genistein inhibition are much higher for closed than for fully activated channels. Association rates with the genistein–PTK complex were similar for closed and fully activated channels and independent of genistein concentration. Dissociation rates were 100 times slower for closed channels, which is consistent with a much higher affinity for genistein–PTK. Genistein–PTK affects channel gating, but not single channel conductance or the number of active channels. By analyzing single channel gating during genistein–PTK dissociation, we determined the maximal open probability for normal and genistein–PTK-bound channels. genistein–PTK decreases open probability by increasing the free energy required for opening, making opening dramatically less favorable. Ni2+, which potentiates RETα channel gating, partially relieves genistein inhibition, possibly by disrupting the association between the genistein–PTK and the channel. Studies on chimeric channels containing portions of RETα, which exhibits genistein inhibition, and the rat olfactory CNG channel α-subunit, which does not, reveals that a domain containing S6 and flanking regions is the crucial for genistein inhibition and may constitute the genistein–PTK binding site. Thus, genistein–PTK stabilizes the closed state of the channel by interacting with portions of the channel that participate in gating.
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Middendorf, Thomas R., and Richard W. Aldrich. "Effects of Ultraviolet Modification on the Gating Energetics of Cyclic Nucleotide–Gated Channels." Journal of General Physiology 116, no. 2 (July 31, 2000): 253–82. http://dx.doi.org/10.1085/jgp.116.2.253.
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Middendorf et al. (Middendorf, T.R., R.W. Aldrich, and D.A. Baylor. 2000. J. Gen. Physiol. 116:227–252) showed that ultraviolet light decreases the current through cloned cyclic nucleotide–gated channels from bovine retina activated by high concentrations of cGMP. Here we probe the mechanism of the current reduction. The channels' open probability before irradiation, Po(0), determined the sign of the change in current amplitude that occurred upon irradiation. UV always decreased the current through channels with high initial open probabilities [Po(0) > 0.3]. Manipulations that promoted channel opening antagonized the current reduction by UV. In contrast, UV always increased the current through channels with low initial open probabilities [Po(0) ≤ 0.02], and the magnitude of the current increase varied inversely with Po(0). The dual effects of UV on channel currents and the correlation of both effects with Po(0) suggest that the channels contain two distinct classes of UV target residues whose photochemical modification exerts opposing effects on channel gating. We present a simple model based on this idea that accounts quantitatively for the UV effects on the currents and provides estimates for the photochemical quantum yields and free energy costs of modifying the UV targets. Simulations indicate that UV modification may be used to produce and quantify large changes in channel gating energetics in regimes where the associated changes in open probability are not measurable by existing techniques.
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Wong, Ching-On, and Xiaoqiang Yao. "Cyclic nucleotide-gated channels: a familiar channel family with a new function?" Future Cardiology 4, no. 5 (September 2008): 505–15. http://dx.doi.org/10.2217/14796678.4.5.505.
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Vandorpe, D. H., F. Ciampolillo, R. B. Green, and B. A. Stanton. "Cyclic nucleotide-gated cation channels mediate sodium absorption by IMCD (mIMCD-K2) cells." American Journal of Physiology-Cell Physiology 272, no. 3 (March 1, 1997): C901—C910. http://dx.doi.org/10.1152/ajpcell.1997.272.3.c901.
Full textAbstract:
The inner medullary collecting duct cell line, mIMCD-K2, absorbs Na+ by an amiloride-sensitive, electrogenic mechanism. The goal of the present study was to characterize the amiloride-sensitive, Na+ -conducting channels responsible for electrogenic Na+ absorption. To this end, we measured Na+ currents in single cells with the patch-clamp technique and Na+ currents across monolayers mounted in Ussing-type chambers. In whole cell patch-clamp experiments, amiloride-sensitive, inward Na+ currents were mediated by nonselective cation channels. In single-channel patch-clamp experiments, amiloride- and guanosine 3',5'-cyclic monophosphate (cGMP)-sensitive, 20-pS nonselective cation channels (i.e., CNG channels) were identified in the apical membrane. CNG channels were inhibited by amiloride, diltiazem, ethylisopropylamiloride (EIPA), and 8-bromo-cGMP and were permeable to Ca2+ and Mg2+. Epithelial Na+ channels were never observed in whole cell or single-channel recordings. Na+ absorption across confluent monolayers was inhibited with a rank order potency of benzamil > amiloride > phenamil >> EIPA > diltiazem. Our data are most consistent with the view that CNG channels mediate electrogenic Na+ absorption across mIMCD-K2 cells.
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Thompson, Stuart H. "Cyclic GMP–gated Channels in a Sympathetic Neuron Cell Line." Journal of General Physiology 110, no. 2 (August 1, 1997): 155–64. http://dx.doi.org/10.1085/jgp.110.2.155.
Full textAbstract:
The stimulation of IP3 production by muscarinic agonists causes both intracellular Ca2+ release and activation of a voltage-independent cation current in differentiated N1E-115 cells, a neuroblastoma cell line derived from mouse sympathetic ganglia. Earlier work showed that the membrane current requires an increase in 3′,5′-cyclic guanosine monophosphate (cGMP) produced through the NO-synthase/guanylyl cyclase cascade and suggested that the cells may express cyclic nucleotide–gated ion channels. This was tested using patch clamp methods. The membrane permeable cGMP analogue, 8-br-cGMP, activates Na+ permeable channels in cell attached patches. Single channel currents were recorded in excised patches bathed in symmetrical Na+ solutions. cGMP-dependent single channel activity consists of prolonged bursts of rapid openings and closings that continue without desensitization. The rate of occurrence of bursts as well as the burst length increase with cGMP concentration. The unitary conductance in symmetrical 160 mM Na+ is 47 pS and is independent of voltage in the range −50 to +50 mV. There is no apparent effect of voltage on opening probability. The dose response curve relating cGMP concentration to channel opening probability is fit by the Hill equation assuming an apparent KD of 10 μm and a Hill coefficient of 2. In contrast, cAMP failed to activate the channel at concentrations as high as 100 μm. Cyclic nucleotide gated (CNG) channels in N1E-115 cells share a number of properties with CNG channels in sensory receptors. Their presence in neuronal cells provides a mechanism by which activation of the NO/cGMP pathway by G-protein–coupled neurotransmitter receptors can directly modify Ca2+ influx and electrical excitability. In N1E-115 cells, Ca2+ entry by this pathway is necessary to refill the IP3-sensitive intracellular Ca2+ pool during repeated stimulation and CNG channels may play a similar role in other neurons.