New perspectives in cyclic nucleotide-mediated functions in the CNS: the emerging role of cyclic nucleotide-gated (CNG) channels.

Pflugers Arch - Eur J Physiol (2014) 466:1241–1257 DOI 10.1007/s00424-013-1373-2

INVITED REVIEW

New perspectives in cyclic nucleotide-mediated functions in the CNS: the emerging role of cyclic nucleotide-gated (CNG) channels Maria Vittoria Podda & Claudio Grassi

Received: 31 August 2013 / Revised: 27 September 2013 / Accepted: 28 September 2013 / Published online: 19 October 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Cyclic nucleotides play fundamental roles in the central nervous system (CNS) under both physiological and pathological conditions. The impact of cAMP and cGMP signaling on neuronal and glial cell functions has been thoroughly characterized. Most of their effects have been related to cyclic nucleotide-dependent protein kinase activity. However, cyclic nucleotide-gated (CNG) channels, first described as key mediators of sensory transduction in retinal and olfactory receptors, have been receiving increasing attention as possible targets of cyclic nucleotides in the CNS. In the last 15 years, consistent evidence has emerged for their expression in neurons and astrocytes of the rodent brain. Far less is known, however, about the functional role of CNG channels in these cells, although several of their features, such as Ca2+ permeability and prolonged activation in the presence of cyclic nucleotides, make them ideal candidates for mediators of physiological functions in the CNS. Here, we review literature suggesting the involvement of CNG channels in a number of CNS cellular functions (e.g., regulation of membrane potential, neuronal excitability, and neurotransmitter release) as well as in more complex phenomena, like brain plasticity, adult neurogenesis, and pain sensitivity. The emerging picture is that functional and dysfunctional cyclic nucleotide signaling in the CNS has to be reconsidered including CNG channels among possible targets. However, concerted efforts and multidisciplinary approaches are still needed to get more in-depth knowledge in this field.

Keywords Cyclic nucleotide-gated channels . cAMP . cGMP . CNS . Ca2+ signaling M. V. Podda : C. Grassi (*) Institute of Human Physiology, Medical School, Università Cattolica, Largo Francesco Vito 1, 00168 Rome, Italy e-mail: [email protected]

Introduction Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are ubiquitous second messengers involved in a multitude of cellular functions. cAMP is synthetized from ATP and cGMP from GTP by the actions of adenylyl (AC) and guanylyl cyclases (GC), respectively [104, 177]. These enzymes are activated by a number of hormones, neurotransmitters, and growth factors, most, but not all of which, act through G protein-coupled receptor signaling (Fig. 1). Intracellular cAMP levels are increased by neurotransmitters (e.g., noradrenaline, dopamine, serotonin) and diverse neuromodulators, including pituitary adenylate cyclaseactivating polypeptide, vasoactive intestinal peptide, adenosine, and ATP, whereas elevated cGMP levels are primarily linked to signaling pathways activated by nitric oxide (NO) and natriuretic peptides. Cyclic nucleotide homeostasis and signaling is also critically regulated by cAMP- and cGMP-degrading enzymes named phosphodiesterases (PDEs) [5, 34]. A decade after the discovery of cAMP by Earl Sutherland, Edwin Krebs and Edmond Fischer identified one of its major targets: an enzyme capable of phosphorylating proteins known as cAMP-dependent protein kinase or protein kinase A (PKA) [35, 164, 170]. The impact of these discoveries on our understanding of the cellular mechanisms for second messenger signaling earned all three scientists a Nobel Prize in Physiology or Medicine (Sutherland in 1971, Fischer and Krebs in 1992). The first discovered target for cGMP was the cGMP-dependent protein kinase G (PKG) [92]. The cAMP and cGMP protein kinase families were intensively studied and well characterized. For many years, the members of these kinase families were considered the primary, if not the exclusive, effectors of cyclic nucleotides [28, 71, 91, 93, 159, 180, 182]. Years have passed since the discovery of PKs, and numerous other cAMP and cGMP targets have been described [160]. They include the bacterial cAMP receptor protein [41], cGMP-

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Pflugers Arch - Eur J Physiol (2014) 466:1241–1257

Fig. 1 cAMP and cGMP signaling pathways. cAMP is produced by adenylyl cyclase (AC ) stimulated by G protein-coupled receptors (GPCRs) coupled to stimulatory G protein (Gs). cAMP activates the following substrates: cyclic nucleotide-gated channels (CNGC) by binding to the cyclic nucleotide binding domain (CNBD); protein kinase A (PKA); the exchange protein activated by cAMP (Epac); hyperpolarization activated cyclic nucleotide-gated (HCN ) channels and inward

rectifier K+ channels (Kir); and phosphodiesterases (PDE) that hydrolyze cAMP to AMP. cGMP also binds to CNBD and activated CNGC. cGMP is produced either by membrane-bound particulate guanylyl cyclases (pGC), which are the receptors of natriuretic peptides (NPs), or by soluble GC (sGC) that are activated by the gaseous transmitter nitric oxide (NO). Other cGMP targets are protein kinase G (PKG), HCN channels, and PDE, the latter degrading cGMP to GMP

binding cyclic nucleotide PDEs [30, 43], cyclic nucleotidegated (CNG) channels [50, 117], hyperpolarization-activated cyclic nucleotide-gated (HCN) channels [37], extracellular cAMP receptors from slime mold [86, 154], the transcription factor CREB (cAMP response element-binding protein) [40, 161], and, more recently, Epac (exchange proteins activated by cAMP) [21, 42] and voltage-dependent channels (e.g., the inwardly rectifying K+ channels) that are inhibited by cAMP [74, 135]. Some of these effectors are activated downstream cyclic nucleotide-dependent PKs, but most are directly activated by cAMP or cGMP and mediate the PK-independent effects of cyclic nucleotides, which currently appear to be numerous and highly relevant to cell physiology. For example, Epac has been implicated as a mediator of PKA-independent effects of cAMP in a host of functions, including cell growth, adhesion, differentiation and division, exocytosis, inflammation, and neurotransmission [20, 188]. Among proteins directly activated by cyclic nucleotides, a critical role is played by CNG channels, a class of ion channels activated by cAMP and/or cGMP binding, which are responsible for visual and olfactory signal transduction in vertebrates. These channels are expressed in rod and cone photoreceptors, and olfactory receptor neurons (ORNs), where they are the sole final targets of cGMP and cAMP. As such, they

produce membrane depolarization in response to sensory stimuli [12, 27, 51, 81, 202]. CNG channels were first discovered in rod photoreceptors in 1985 [50, 190]. The contention that cyclic nucleotides acted by direct binding to a channel protein was initially greeted skeptically by the scientific community [110]. Since then, a growing body of electrophysiological, molecular, and pharmacological evidence has confirmed direct cGMP binding to and gating of these channels, and other members of the CNG channel family have been identified and linked to signal transduction in other sensory receptors, such as cone photoreceptors [32, 63] and ORNs [117]. CNG channels have been the focus of extensive research on the physiology and pathology of the visual and olfactory systems, which provided in-depth insight into the channel structural and functional properties. Less interest has been demonstrated in the possible roles of CNG channels in other areas, although several reports suggested that they are widely distributed and particularly abundant within the CNS. A role for cyclic nucleotides in the CNS has emerged in many aspects of neuronal and glial functions. The largest and most compelling body of evidence implicates cAMP and cGMP signaling in the processes of synaptic transmission and plasticity, neuromodulation, learning, memory, and neural development [14, 77, 100]. Cyclic nucleotides have also been shown to play critical roles as second messengers in glial cells.


For instance, increased levels of either cGMP or cAMP affect the morphology of astrocytes as well as their communication with neurons and blood vessel cells [7, 8, 19, 136]. The effects of cAMP and cGMP within the CNS have been mainly attributed to the modulation of protein kinase and phosphatase activities, and the possibility that CNG channels play a role in these effects has been often undervalued. This paper reviews the current state of art about the functional expression of CNG channels in the CNS, and the emerging picture points to CNG channels as major targets of cyclic nucleotides in the mammalian brain.

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in rod photoreceptors (3 CNGA1:1 CNGB1a) [186, 195, 197], cone photoreceptors (2 CNGA3:2 CNGB3) [124], and olfactory neurons (2 CNGA2:1 CNGA4:1 CNGB1b) [196] (Fig. 2b). The A and B subunits consist of six transmembrane segments (S1–S6), a reentrant pore (P) loop between S5 and S6, and intracellular N and C termini (Fig. 2c). The C-terminus comprises the cyclic nucleotide-binding domain (CNBD), the region connecting the CNBD to S6 in the pore region (C-linker) and the distal C-terminus. CNG channels, like the related voltage-gated K+ channels whose crystal structure has been recently solved [101], are tetramers with the four subunits arranged in a circular symmetric manner, which allows the S5 and S6 segments to form the ion conducting pore [37, 53, 142, 191].

CNG channel structure and properties Biophysical properties and ligand activation Structure CNG channels belong to the superfamily of voltage-gated ion channels, and they display particularly high homology with voltage-gated K+ channels [53, 70, 75]. From a functional point of view, they can be regarded as ligand-gated ion channels, although they differ from most other channels of this type in that the ligand binding occurs on the cytoplasmic side of the cell membrane. CNG channels are heteromeric complexes of subunits identified as A type and B type (formerly referred to as alpha and beta subunits, respectively). Four distinct A subunits (CNGA 1–4) and two B subunits (CNGB1 and CNGB3) have been identified in mammals [22] (Fig. 2a). The A and B subunits are often regarded as members of distinct CNG channel subfamilies. Splice variants further increase the diversity of mammalian CNG channels. CNG subunit homolog genes have also been identified in invertebrates such as Caenorhabditis elegans and Drosophila melanogaster [81, 187]. CNG channels are tetrameric proteins whose subunits are arranged around a central pore. The CNGA1 (formerly, CNG1, CNGα1, or RCNC1); CNGA2 (formerly, CNG2 or OCNC1); and CNGA3 (formerly, CNG3, CNGα2, or CCNC1) subunits are expressed in rod photoreceptors, ORNs, and cone photoreceptors, respectively. They are considered the principal subunits because in heterologous systems each forms a functional homomeric channel on its own. The A4 subunit (formerly referred to as CNG5, CNGB2, CNGα4, or OCNC2) and the two B subunits, CNGB1 (formerly CNG4, CNG) and CNGB3 (CNG6, CNGβ3) [70], are described as modulatory subunits: they do not form functional channels when expressed alone, but when co-expressed with CNGA subunits 1–3, they form functional heteromeric channels with properties that are distinct from those of the principal subunits alone. Naive CNG channels are believed to be expressed in various tissues as heteromeric complexes of A- and B-type subunits. In particular, recent biochemical and fluorescence resonance energy transfer (FRET) studies allowed defining the exact subunit compositions and stoichiometries of CNG channels expressed

The CNG channels characterized so far share some biophysical features. Under physiological conditions, they carry inward Na+ and Ca2+ currents. Being nonselective cation channels, they conduct the monovalent cations K+, Na+, Li+, Rb+, and Cs+ almost equally well [47, 81]. Because they are nonselective, the current-to-voltage relationship of CNG channels is almost linear and has a reversal potential close to 0 mV. Divalent cations can permeate the channel, but at high concentrations, they bind to specific sites within the channel pore and block further ion flow [46, 55, 148]. The CNG channel subtypes exhibit different Ca2+ permeability, which is higher in olfactory and cone-type channels than in the rod photoreceptor type [54, 55, 127, 129]. For instance, the relative ion permeability, P Ca/P Na, is more than three times larger in cone- than in rod-type CNG channels (21.7 and 6.5, respectively), and under physiological ionic conditions, the fraction of the dark current carried by Ca2+ is about twofold larger in cones than in rods [81]. In the absence of cyclic nucleotides, the open probability of CNG channels is extremely low (10−4–10−6). In CNGA1bearing channels, cGMP increases the open probability much more than cAMP. Indeed, CNG channels expressed in rods and cones sharply discriminate between cGMP and cAMP, the latter requiring about 50-fold higher concentrations to activate the channels [171]. Olfactory-type CNG channels are instead equally sensitive to physiological concentrations of cAMP and cGMP. Therefore, they are less selective but display a much higher sensitivity to both cyclic nucleotides than photoreceptor CNG channels [54, 81, 171, 184]. Activation of CNG channels requires two steps: ligand binding and channel gating. Functional domains involved in the activation process include (1) the channel gate located in the distal portion of the S6 segment; (2) the CNBD domain; and (3) the C-linker domain that is responsible for the allosteric coupling of cyclic nucleotide binding to the channel gate. Several models have been proposed to describe the link between ligand binding and channel gating. They were based

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Fig. 2 Structure of CNG channels. a Phylogenetic tree of mammalian CNG channel A and B subunits. b Tetrameric subunit assembly of CNG channels in rod, cone, and olfactory receptors (ORN). c Schematic representation of CNG channel subunit structure consisting of six transmembrane domains (S1–S6). Functionally relevant sites are indicated: the loop between S5 and S6 segments forming the ionconducting pore; the cyclic nucleotide-binding domain (CNBD); the C-linker connecting the CNBD to the pore; the regulatory site for Ca2+ calmodulin (CaM)

on data obtained from single-channel recordings from naive and mutated CNG channel proteins, FRET experiments, and information gained from the recently resolved structures of the C-linker and the CNBD of the closely related HCN channels [36, 37, 52, 99, 105, 116, 172, 198]. Unlike other ligand-gated channels, CNG channels do not desensitize or inactivate following prolonged exposure to cyclic nucleotides [50, 63, 81]. This feature, together with their cooperative activation, makes them suitable to serve as fast molecular switches faithfully tracking intracellular cAMP and cGMP concentrations. However, CNG channels are not static sensors of the cyclic nucleotide concentrations, and in keeping with the critical role they play in sensory transduction, their sensitivity to cyclic nucleotides is affected by several factors. Calcium ions and Ca2+-binding proteins, especially calmodulin, provide feedback channel inhibition playing a major role in the receptor adaptation to sensory stimuli, particularly to odor stimulation in ORNs [113, 178, 202]. Other factors affecting the functional state and the cyclic nucleotide sensitivity of CNG channels include channel phosphorylation, interactions with membrane proteins (e.g., protein tyrosine kinases), divalent cations, diacylglycerol, phospholipids, NO, and the recently documented insulin receptor activation [24, 45, 59, 79, 89, 184]. Methods and compounds used for studying CNG channel functions Our knowledge on the functional properties of CNG channels mainly arises from patch-clamp recordings on intact

photoreceptors, ORNs, or heterologous cell expression systems as well as on excised patches from these preparations. Laser scanning confocal microscopy allowed detecting for the first time spatially resolved Ca2+ signals through activated CNG channels in single olfactory cilia [97]. Functional studies on CNG channels have frequently relied on the use of cGMP and cAMP analogs which, compared to naive nucleotides, are membrane-permeant and, to a variable extent, PDE-resistant. Nevertheless, the pharmacology of CNG channels is still poorly understood, partly for the limited selectivity of available compounds. Some of the first-generation cyclic nucleotide analogs, such as the 8-bromo- and dibutyryl-substituted cAMP and cGMP [200], are still commonly used in CNG channel research. Second-generation compounds include hydrolysis-resistant phosphorothioate derivatives of cAMP and cGMP, Sp and Rp isomers, exerting opposite effects on different targets. For instance, Wei et al. [183] identified the first competitive antagonist of the photoreceptor CNG channel that is also an activator of PKG, Sp-8-Br-PET-cGMPS. Furthermore, a cGMP analog, Rp-8-Br-cGMPS, was shown to activate the rod CNG channels and inhibit PKG [184]. Sp-cAMPS is an agonist of the olfactory CNG channel, but a weak antagonist of the rod CNG channel. Rp-cGMPS is an antagonist of the olfactory CNG channel and an agonist of the rod CNG channel [90]. Recently identified molecules used to study CNG channels, which may be referred to as third-generation compounds, include photo- or bio-activatable cyclic nucleotide analogs which allow rapidly releasing the second messenger upon photolysis or endogenous enzyme action [15, 18, 61, 80, 81].


An exhaustive review of the cAMP and cGMP analogues commonly used to investigate cyclic nucleotide-dependent pathways has been provided by Schwede et al. [158]. More recently, Poppe et al. [137] reconsidered the selectivity of the most frequently used cyclic nucleotide analogs, suggesting that the majority of them activate multiple targets including endogenous PDEs. The authors’ conclusion was that results obtained with these agents should be considered with caution. A number of organic compounds have also been reported to block currents flowing through CNG channels, although many of them are not highly specific [81]. The most extensively studied and widely used compound is L -cis-diltiazem (LCD) which blocks the CNG channels of rods, cones, and ORNs in a voltage-dependent fashion [54, 62, 106, 141]. At present, the most potent blocking agent for CNG channels is pseudechetoxin, a peptide venom of the Australian king brown snake [25]. Besides pharmacological approaches, genetic studies based on CNG channel-deficient mouse models and human channelopathies have recently provided insights on the physiological and pathophysiological roles of these channels [11, 12]. In particular, knockout (KO) mouse models include (1) a CNGB1-deficient mouse model covering all potential CNGB1 splice variants, which exhibits impaired vision and olfaction [73]; (2) CNGA3-deficient mice displaying the principal hallmarks of human achromatopsia [12, 13]; (3) four different CNGA2-deficient mouse cell lines showing no detectable responses to odorants [6, 26, 193, 194]; (4) CNGA4deficient mice showing olfactory structure morphology comparable with that of wild-type animals, but impaired odor adaptation and lower CNG channel sensitivity to cAMP [82, 114]; and (5) CNGB1-deficient mice lacking both the retinal CNGB1a and the olfactory CNGB1b subunits and displaying decreased olfactory performance [109]. Although up to now mice lacking the CNGA1 subunit are not available, transgenic mice have been produced exhibiting ∼50 % reduction in CNGA1 transcript levels due to the overexpression of a CNGA1 antisense mRNA. These animals show some histological features reminiscent of retinitis pigmentosa [96]. The use of these animal models, together with pharmacological and functional approaches, provided fundamental information on CNG channels as well as on the physiology and pathophysiology of visual and olfactory systems. However, they may also allow getting insight on the role played by CNG channels in other body districts and, especially, in the CNS.

CNG channels in the CNS Molecular and functional evidence of CNG channel expression in the CNS Studies performed on visual and olfactory systems also provided evidence that CNG channel expression was not

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confined to sensory receptor neurons. Indeed, ON bipolar cells in the retina were shown to regulate membrane responses to synaptic input through CNG channels via a transduction cascade similar to that operating in photoreceptors [118, 162]. Subsequent studies reported molecular and electrophysiological data revealing the expression of a CNG channel in rat retinal ganglion cells exhibiting some properties consistent with those of the rod photoreceptor channel [1]. Furthermore, putative cGMP- and NO-activated conductances were detected in Müller glial cells of human and bovine retina [94], where the expression of CNGA1 was also confirmed by RT-PCR. Within the olfactory system, molecular findings indicate the expression of CNGA2 and CNGA3 in the mammalian olfactory bulb [84, 169]. Moreover, there are functional data suggesting a role for CNG channels in mediating synaptic transmission between olfactory nerve fibers and their targets in the olfactory bulb [115]. These findings prompted some investigators to explore CNG channel distribution within the mammalian brain. Several studies reported systematic analyses of the whole brain revealing an unexpected widespread distribution of CNG channels, especially CNGA1 and CNGA2 [44, 48, 81, 83, 150, 184]. The main results from these studies are summarized in Table 1. In 1995, el-Husseini et al. [48] first documented the expression of CNGA2 in the rat brain using RT-PCR, Northern blotting, and in situ hybridization. This study reported significant levels of CNGA2 in the pituitary gland, the olfactory bulb, and the cerebellum of adult rats and pups. Within the latter region, the strongest in situ hybridization signal was found in Purkinje cells that also expressed the channel protein when grown in culture. Two subsequent studies analyzed in greater detail the regional and cellular distribution of CNGA2 in the adult rat brain [84, 169]. The results confirmed previous observations of the widespread distribution of the channel subunit in morphologically identified neuronal cell populations of the olfactory bulb, cerebral cortex, hypothalamus, hippocampus (pyramidal neurons and dentate gyrus granule cells), cerebellum, and brainstem areas, including vestibular nuclei. The strongest signals were detected in the piriform cortex and cerebellar Purkinje cells. In their study investigating the tissue distribution of CNGA1, Ding et al. [44] also documented its expression in the cerebral cortex, hippocampus, and cerebellum of the rat brain. Based on these observations, more focused studies were performed on brain areas such as the hippocampus and cerebral cortex, where either high levels of CNG channel expression were found or the channels might play a particularly critical role. Leinders-Zufall et al. [98] provided the first evidence for functional CNG channels in the mammalian brain. Using whole-cell recordings, the authors demonstrated that cultured

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Table 1 Overview on the functional expression of CNG channels in the mammalian CNS Region

Subunit

Olfactory bulb Cortex Amygdala Hypothalamic neurons Anterior pituitary Hippocampus

CNGA2, A3, A4 CNGA1, A2, A3 CNGA3 CNGA1, A2, A4

Brainstem Cerebellum Spinal cord

Model

Rat, mouse, CNGA2-KO Rat Mouse, CNGA3-KO GnRH-secreting cell line (GT1) CNGA1 Rat CNGA1, A2, A3, A4 Rat, mouse, CNGA2-KO, CNGA3-KO CNGA1, A2 Rat CNGA1, A2, B1 Rat CNGA1, A2, A3, A4, Mouse, CNGA3-KO B1, B3

Technique

References

RT-PCR, Northern/RPA, Hyb, IF, E, Ca2+-I RT-PCR, Northern/RPA, HyB, WB, IF, E RT-PCR, WB, IF, Behav RT-PCR, E

[23, 48, 60, 84, 115] [23, 44, 48, 84, 131, 150, 169] [108] [31, 179]

RT-PCR RT-PCR, Northern/RPA, Hyb, WB, IF, E, Ca2+-I, Behav RT-PCR, WB, IF, E RT-PCR, Hyb, WB, IF, E, Ca2+-I RT-PCR, Hyb, WB, IF, E, Ca2+-I, Behav

[176] [23, 44, 83, 108, 122, 134, 169] [130, 132, 133] [23, 84, 102, 169] [66]

Data on CNG channel expression in the retina and olfactory epithelium are not included Behav behavioral analysis, Ca 2+ -I Ca2+ imaging, E electrophysiology, Hyb in situ hybridization, IF immunofluorescence, KO knockout, Northern Northern blot, RPA ribonuclease protection assay, RT-PCR reverse transcriptase polymerase chain reaction, WB Western immunoblot

rat embryonic hippocampal neurons displayed a cGMP-gated membrane conductance exhibiting properties consistent with CNG channels (i.e., divalent cation block and high Ca2+ permeability). In a subsequent study, Kingston et al. [83] demonstrated that CNGA1 and CNGA2 are expressed in most hippocampal pyramidal neurons and granule cells of the dentate gyrus. Another study documented CNGA2 and CNGA4 expression in hippocampal neurons of the adult rat brain using PCR, in situ hybridization, and immunofluorescence [23]. Interestingly, the CNGA4 was localized in the growth cones, axons, and dendrites of cultured embryonic neurons. By using whole-cell and single-channel recordings, the same authors demonstrated that cultured hippocampal neurons express functional CNG channels activated by cAMP and cGMP. The cGMP analog, 8-Br-cGMP, also elicited Ca2+ influx that was abolished by extracellular high Mg2+ concentration. CNG channel expression was also investigated in the visual cortex [150]. By using semiquantitative RT-PCR and in situ hybridization, the authors demonstrated that all three CNG channel functional subunits (A1, A2, and A3) are expressed in the developing visual cortex of the rat brain with distinct temporal and spatial patterns in sensorimotor and occipital regions. Although several studies documented particularly high expression levels of both CNGA1 and CNGA2 in the cerebellum, their function was only recently investigated [102]. This study showed that CNGA1 and CNGB1 (presumably the 1b isoform) are expressed in rat cerebellar granule cells both in situ and in vitro. The expression of mRNAs encoding these proteins is highest after 7 days in culture, when the channels are expressed at synapses and co-localize with the synaptic marker synapsin I. Electrophysiological recordings performed on cultured cerebellar granule cells showed currents activated by 8-Br-cGMP with time course, reversal potential, and

pharmacological sensitivity to LCD and divalent cations compatible with CNG channels. Furthermore, confocal Ca2+ imaging experiments showed that channel opening in response to increased intracellular cGMP levels resulted in Ca2+ entry into the cell that was prevented by LCD. In the last decade, our studies have provided novel evidence on the functional role played by CNG channels in three different cell types of the CNS: (1) neurons of the medial vestibular nucleus (MVN); (2) astrocytes; (3) hippocampal neural stem cells (NSCs) (Fig. 3). In particular, we found that neurons of the MVN express CNGA1 and CNGA2 proteins as revealed by nested PCR, immunofluorescence, and Western immunoblot assays [130, 132, 133]. More importantly, we demonstrated that cAMP and cGMP analogs modulate the membrane potential and intrinsic firing rates of these neurons via CNG channel activation (Fig. 3d). Indeed, in patch-clamp recordings carried out on brainstem slices containing the MVN, cyclic nucleotides induced PKA- and PKG-independent membrane depolarization associated with a significant decrease in the membrane input resistance. The effects of cGMP on membrane potential were almost completely abolished by the CNG channel blockers, Cd2+ and LCD. In voltage-clamp experiments, the cGMP analog, 8-Br-cGMP, induced non-inactivating inward currents with an estimated reversal potential near 0 mV. These currents were markedly inhibited by the reduction of extracellular Na+ and Ca2+ concentrations. Our immunofluorescence studies, initially focused on neurons, also revealed the expression of CNG channels in glial cells. In a subsequent study, we investigated this issue in more detail and found that CNGA2 expression is a common feature of astrocytes in the rodent brain [131] (Fig. 3b, c). Electrophysiological recordings performed on both cultured and in situ astrocytes showed cGMP-activated currents with


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Fig. 3 Immunofluorescence and electrophysiological evidence for the expression of CNGA1 and CNGA2 in neurons, astrocytes, and neural stem cells in the rodent brain. The schematic representation of the sagittal section of the rodent brain shows the areas from which data presented in the figure were obtained. CTx cortex, Hip hippocampus, VN vestibular nuclei complex. a CNGA2 immunolabeling (green) of cortical neurons (red) in culture (upper panel) and in brain slices (lower panel). b In situ cortical astrocytes (GFAP+ cells) expressing CNGA2 (upper panel) and showing cGMP-gated currents in voltage-clamp recordings (lower panel ). c Representative images of hippocampal astrocytes expressing CNGA2, as the majority of GFAP+ cells within the rat brain. d CNGA1 immunolabeling of neurons in the medial vestibular nucleus (lower

panel). The upper panel shows current-clamp recordings demonstrating that CNG channel activation by 8-Br-cGMP causes membrane depolarization responsible for the increased firing rate of medial vestibular nucleus neurons. e Representative images showing CNGA1 labeling in cultured hippocampal neural stem cells either undifferentiated (nestin+ cells, upper panel on the left) or differentiating toward neuronal phenotype (MAP2+ cells, lower panel on the left). The graph on the right shows typical current-to-voltage (IV) plot of the mean 8-Br-cGMP net currents elicited in NSCs differentiating to neurons (n =10) by the ramp protocol depicted in the lower part of the panel. Reversal potential close to 0 and the IV profile are compatible with CNG channel conductance. Cell nuclei (DAPI+) are labeled in blue. Scale bars, 25 μm

pharmacological and biophysical features suggestive of ion flow through CNG channels. More specifically, in cultured rat cortical astrocytes, we observed currents elicited by voltage ramps from −100 to +100 mV in the presence of the cGMP analogue, dB-cGMP, that were significantly reduced by LCD and Cd2+. The reversal potentials of the LCD- and Cd2+sensitive currents were more positive than that of K+, as expected for a mixed cation current such as that carried by CNG channels. Non-inactivating, voltage-independent currents were also elicited by extracellular application of the membrane-permeant cGMP analogue, 8-Br-cGMP. These effects were blocked by LCD and were mimicked by either natriuretic peptide receptor activation or inhibition of PDE

activity. Voltage-independent, LCD-sensitive currents were also elicited by 8-Br-cGMP in astrocytes of hippocampal and neocortical brain slices. As mentioned above, several studies documented the expression of CNGA1 and CNGA2 in hippocampal neurons. Our recent research focused on a specific cell subpopulation residing in the neurogenic niche of hippocampus (i.e., the subgranular zone of the dentate gyrus) that plays a critical role in adult neurogenesis, a process allowing the generation of new neurons throughout the entire life. By immunofluorescence and Western immunoblot assays, we demonstrated that CNGA1 and CNGA2 subunits are expressed in cultured hippocampal NSCs as well as in situ, in the subgranular zone of

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adult mice [134]. We also demonstrated that CNG channel activation plays a critical role in NSC differentiation toward the neuronal phenotype without significantly affecting the proliferation of undifferentiated neural progenitors. Consistently, we recorded 8-Br-cGMP-activated currents attributable to ion flow through CNG channels in neuron-like differentiating NSCs (Fig. 3e). CNG channel functions in the CNS Excitability Changes in resting membrane potential (RMP) due to cationic currents flowing through activated CNG channels provide visual and olfactory signal transduction [12]. However, accumulating evidence indicates that CNG channel-induced changes in RMP may represent a broad mechanism for modulating neuronal excitability in the CNS. Indeed, depolarizing voltagedependent and voltage-independent currents may trigger action potentials in silent neurons bringing the membrane potential closer to the threshold for firing. RMP changes have a greater impact on the output signals of spontaneously firing neurons in that they markedly increase the neuronal discharge rate and, consequently, the functional response of the connected neurons. In this context, our studies demonstrated that CNG channel activation by cAMP and cGMP increased the firing rate of spontaneously active MVN neurons in the rat brainstem [130, 132]. These effects were not mediated by PKG and PKA and were abolished by the CNG channel blocker, LCD. Involvement of CNG channels was fully supported by molecular evidence of CNGA1 and CNGA2 expression in these neurons as well as by pharmacological and biophysical characterization of CNG channel-like ion currents activated by cyclic nucleotides. We hypothesized that several physiological signals, including NO, may modulate MVN firing rate via CNG channel activation [133]. Similar modulation of neuronal excitability by CNG channels was demonstrated in other cell types showing intrinsic rhythmic activity such as neuroendocrine cells of the pineal gland and neurons of the mediobasal hypotalamus producing gonadotropin-releasing hormone (GnRH) [31, 152]. In particular, GnRH neurons show spontaneous bursts of action potentials and consequent oscillations in intracellular Ca2+ levels ([Ca2+]i), resulting in pulsatile release of GnRH. In an in vitro model of immortalized hypothalamic neurons, cAMP analogs and cAMP-producing agents increased the frequency of action potential-dependent intracellular Ca2+ oscillations, thus suggesting that CNG channel activation plays a critical role in hormone release by affecting RMP and cell excitability [31]. In keeping with these results, CNG channels have been proposed to contribute to plateau potentials, i.e., prolonged depolarizations induced by muscarinic receptor activation in CA1 pyramidal neurons [95].


Neurotransmission and synaptic plasticity The role of CNG channels in synaptic transmission was first described in the retina, where they mediate transmitter release induced by NO and cGMP at the cone bipolar cell synapses [147, 153]. In these cells, CNG channels have also been suggested to mediate the postsynaptic inhibitory response to glutamate [118, 162]. Through electrophysiological and Ca2+ imaging experiments, Murphy and Isaacson [115] showed that, in response to moderate elevations in cyclic nucleotides, presynaptic CNG channels increased Ca2+ entry, thereby enhancing spontaneous transmitter release in the mammalian olfactory bulb. However, in response to large elevations in cyclic nucleotides, CNG channel activation decreased evoked transmitter release, most likely by depolarizing the presynaptic terminal and thus inducing Na+ channel inactivation. The involvement of CNG channels was supported by the striking evidence that cAMP and cGMP modulation of synaptic transmission between olfactory nerve fibers and their targets in the olfactory bulb was absent in CNGA2-deficient mice. In addition to the retina and olfactory epithelium, CNG channels have been reported to increase the frequency of Ca2+ oscillations in immortalized hypothalamic neurons following activation by cAMP, noradrenaline or forskolin. As already pointed out, these effects were suggested to contribute to the regulation of spontaneous as well as neurotransmitter-evoked release of GnRH [31, 49]. According to Barnstable et al. [9], PKG rather than CNG channels mediates the inhibitory effects of cGMP on fast synaptic transmission in cortical neurons. However, it should be pointed out that most of their results were obtained from brain slices of adult rat visual cortex where the expression of CNG channels has been demonstrated to undergo developmental regulation with higher expression in the neonatal period [150]. As a result, reduced levels of CNG channel expression may have favored the prevalence of inhibitory PKG effects. In the mammalian CNS, CNG channels might be critically involved in the modulation of synaptic plasticity, a phenomenon consisting in functional and structural modification of neuronal connectivity, which is thought to underlie learning and memory [64, 68, 69]. Molecular data demonstrated that CNG channels are expressed in regions of the rodent brain where plasticity phenomena have been well documented, i.e., hippocampus and cerebellum. As mentioned in the previous section, functional data also supported CNG channel expression in the hippocampus. Additionally, Ca2+ permeability of CNG channels and their persistent activation by cyclic nucleotides are well suited for mediating some of the pre- and postsynaptic events underlying synaptic plasticity [23]. One of the best-characterized forms of synaptic plasticity, the N-methyl-D -aspartate (NMDA)-dependent long-term potentiation (LTP) at the hippocampal glutamatergic synapses, is


critically related to cellular cascades stimulated by increased intracellular cAMP concentrations [78]. Besides cAMP, an important role in the short- and long-term modulation of synaptic strength has also been attributed to cGMP, whose increased levels at presynaptic terminals have been linked to the retrograde transmitter NO [3, 4, 16, 120, 129, 157]. Interestingly, pharmacological and genetic manipulation of cAMP, cGMP, and NO levels resulted in modifications of LTP and memory [139]. In particular, Bohme and colleagues [16] demonstrated that inhibition of endogenous NO impaired spatial learning and olfactive memory. Mice lacking the NO synthetizing enzyme, nNOS, or rats treated with nNOS inhibitors showed impaired spatial and object recognition memory [29, 85, 88, 138, 201]. Moreover, NO donors and cGMP analogs rescued the spatial memory deterioration induced by NMDA receptor blockade [189]. Passive avoidance learning increased cGMP levels in the rat hippocampus, and administration of 8-Br-cGMP [10] or zaprinast, a selective cGMP PDE inhibitor [138], improved memory. Altogether, these data suggest that the positive effects of NO on behavior are mediated by cGMP. Although most of the cAMP and cGMP effects have been demonstrated to involve the activation of PKA and PKG, respectively (e.g., the well-known cAMP/PKA/CREB pathway in LTP), the involvement of CNG channels would, in our opinion, deserve further investigations. To prove the involvement of the cGMP/PKG pathway in hippocampal LTP induction, Zhuo et al. [199] used LY83583, as GC inhibitor, and Rp-8-Br-cGMPs, as PKG blocker. However, LY83583 also blocks olfactory CNG channels and Rp-8-Br-cGMPS acts as a weak channel agonist. Other compounds used as activators and inhibitors of protein kinases in hippocampus and cerebellum include Rp-CAMPS, Rp-8-pCPT-CAMPS, and SpCAMPS [149, 185], which also are CNG channel agonists and antagonists. Based on experimental evidence of CNGA2 functional expression in the hippocampus, Bradley et al. [23] proposed the following possible scenarios involving CNG channels in plasticity: (1) in CA1 neurons, they may be activated in the late phase of LTP by CaM-sensitive AC-dependent cAMP production and mediate Ca2+ influx at the postsynaptic level, which is critical for LTP maintenance; (2) in the NMDAindependent LTP at dentate granule cells—CA3 synapses, they could mediate the presynaptic effects of cAMP, facilitating Ca2+ influx at presynaptic terminals; (3) in the cerebellum, CNG channels might mediate cAMP effects on LTP and cGMP effects on LTD reportedly attributed to PKA and PKG, respectively. In the retinal ganglion cells, a link between CNG channel activation and NO/cGMP has been reported [1], leading to propose a model of how CNG channels might be involved in synaptic plasticity and LTP [9, 203]. NO produced at the postsynaptic level following NMDA receptor stimulation,

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acting as retrograde messenger, would diffuse to presynaptic terminals and stimulate sGC, thus leading to increased cGMP levels and consequent CNG channel activation. Ca2+ influx through activated CNG channels would promote increased neurotransmitter release either directly or through protein phosphorylation and modification of the cytoskeletal structure. Given the difficulty in isolating the contribution of CNG channels from that of other cyclic nucleotide targets, CNG-KO mice have been used to investigate the role of CNG channels in synaptic plasticity. Interestingly, the two studies performed so far seem to support such a role. In line with the above reported models, CNGA2-KO mice displayed attenuated LTP and posttetanic potentiation induced by theta-burst stimulation at CA3CA1 synapses [122]. However, an opposite role has been proposed for cone-type CNG channels. Indeed, CNGA3deficient mice showed increased hippocampal LTP [108]. Because CNGA3 proteins were localized at the presynaptic level, the authors suggested that cation influx through activated CNG channels may lead to the depolarization of presynaptic terminals and inactivation of voltage-gated Na+ channel, thus suppressing action potential propagation and, consequently, neurotransmitter release [115]. However, CNGA3 channels do not seem to impact hippocampal-dependent memory. Indeed, spatial memory and learning, assessed by water maze tests, were similar in KO and wild-type animals. This study also addressed the interesting issue of whether CNGA3 channels have a role in amygdala-dependent behaviors, given that CNGA3 subunits are also expressed in this structure. Interestingly, CNGA3-KO mice showed a markedly reduced freezing in the auditory-cued fear conditioning task. Besides supporting a role for CNG channels in hippocampal plasticity and revealing a novel function of CNGA3 channels in the amygdala-dependent consolidation of fear memory, this study highlighted the great contribution that KO mice models may offer to understanding of CNG channel functions in intact animals. Pain A series of experimental data have revealed that cGMP pathways activated by NO and natriuretic peptides play critical roles in nociception processing within the spinal cord, resulting in either pro- or anti-nociceptive effects [103, 155, 192]. Opposite effects have been attributed to the activation of different downstream targets of cGMP and/or to their expression in different subpopulations of neuronal or non-neuronal cells within the nociceptive system. Although a major role has been attributed to PKG, increasing evidence indicates that cGMP antinociceptive effects may be independent on this kinase [156, 173, 174], and CNG channels were proposed as alternative targets [66]. In particular, CNGA3 were reported to play an inhibitory role in hyperalgesia during inflammatory pain based on the following experimental evidences: (1) real-time RT-PCR revealed the expression of CNGA2 and CNGA3 subunits and

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the three modulatory subunits—CNGA4, CNGB1, and CNGB3—in the mouse lumbar spinal cord; (2) mRNA levels of CNGA3 increased in the spinal cord after hindpaw inflammation caused by intraplantar injection of zymosan, whereas the expression of other CNG channel subunits was unaffected; (3) in situ hybridization combined with immunohistochemistry revealed particularly high levels of CNGA3 expression in superficial dorsal horn where the signals co-localized with the marker of inhibitory neurons, VGAT, and upregulation of CNGA3 after zymosan-induced inflammation specifically occurred in this neuronal subpopulation; (4) in the dorsal root ganglion, CNGA3 was constitutively expressed and upregulated following paw inflammation in satellite glial cells, but not in neurons; (5) molecular findings were fully consistent with behavioral data showing that CNGA3-KO mice exhibited an increased nociceptive behavior in models of inflammatory pain, whereas their behavior was normal in models of acute or neuropathic pain. Moreover, CNGA3-KO mice developed an exaggerated pain hypersensitivity induced by intrathecal administration of cGMP analogs or NO donors. Based on these results, Heine and co-workers proposed that cGMP-mediated activation of CNGA3 in inhibitory interneurons results in GABA release that, in turn, inhibits inflammatory pain processing. They also speculated that the activation of CNG channels in satellite glial cells may exert a modulatory action in inflammatory pain processing. CNS development and neurogenesis Consistent evidence has implicated CNG channels in development and, in particular, in axonal outgrowth and guidance. These aspects of nervous system physiology have been thoroughly investigated given the relevance of axon regeneration in brain repair after CNS injuries. As a consequence, several extracellular guidance molecules and intracellular signaling pathways involved in these processes have been characterized [112, 119, 166–168]. In particular, growth cone turning induced by guidance molecules is finely regulated by intracellular Ca2+ and cyclic nucleotides, which may exert opposite effects (i.e., attraction or repulsion) depending on their concentrations [123]. For instance, 5–20 % [Ca2+]i increases in growth cones convert repulsion to attraction [65, 67, 72, 166, 181]. First evidence for the involvement of cAMP and cGMP in growth cone turning came from the original turning assay in which growth cones of chick dorsal root ganglion neurons were observed orienting to gradients of dB-cAMP and cGMP [58]. From then on, the role of either nucleotide and the interplay between them have been well characterized, especially in Xenopus neurons, a broadly used model for growth cone tuning assays. cAMP and cGMP may also play opposite effects on growth cone behavior [119]. Downstream targets of cyclic nucleotide action include different classes of Ca2+-conducting channels generating Ca2+


signals with different spatiotemporal domains [68, 76, 151]. CNG channels have been recently added to the list, including Cav1 channels and transient receptor potential canonical channels [119, 163]. In their elegant work on Xenopus neurons, Togashi et al. [175] convincingly demonstrated that the activation of CNG channels, most likely xCNGA1 type (70 % homology with mammalian CNGA1), is required for the repulsive growth cone guidance signaling of semaphorin 3A. By using combined electrophysiological, molecular, and calcium imaging approaches together with an in vitro model of CNG channel knockdown, these authors demonstrated that semaphorin 3A induces growth cone repulsion by triggering the elevation of cGMP levels that, in turn, increase [Ca2+]i via xCNGA1 channel activation. This study also showed that inactivation of xCNG channels converted the semaphorin 3A-induced repulsion to PKG-dependent attraction. This observation demonstrates that CNG channel- and PKG-dependent mechanisms coexist and suggests that their differential activation, resulting in different growth tuning behaviors, may depend on CNG channel expression levels, cGMP intracellular levels, and compartmentalization of cGMP and its targets. In C. elegans, the tax-2 and tax-4 gene-encoded proteins, similar to CNGA1 and CNGB, respectively, were associated with chemosensation and axon guidance [33, 87]. Although the majority of studies demonstrating the role of CNG channels in axonal guidance were performed in lower invertebrates, there is evidence that local growth cone signaling mechanisms are, at least partly, conserved among species and in different classes of neurons [123]. Data obtained in CNGA2-KO mice suggest that this channel protein is important for the correct pattern of convergence of axonal projection onto the glomeruli in the olfactory bulb [194], and 8-BrcGMP has been shown to trigger Ca2+ transients through putative CNG channels in rat ORN growth cones [77]. Studies performed on the rodent visual cerebral cortex [150], hippocampus [23], and cerebellum [102] strongly suggest a role of CNG channels in CNS development. In the visual cortex, which is regarded as a good model for studying development and plasticity, CNGA1, CNGA2, and CNGA3 subunit expressions appear to be developmentally regulated, with the highest mRNA levels in the early postnatal period. In particular, the mRNA levels of CNGA2 channels show a pronounced peak at postnatal day 7, which is the time point of maximal dendritic outgrowth in the developing visual cortex. This finding suggests that CNGA2 channels play a critical role in growth cone guidance because of their high Ca2+ permeability. The highest levels of CNGA1 were found instead at birth; thereinafter, channel expression declines to almost undetectable levels in adulthood. It is tempting to speculate that, given the selectivity of CNGA1 channels to cGMP, they may be downstream targets of cGMP produced by several extracellular signaling molecules including NO.


The developmental time course of CNGA1 and CNGA2 is also accompanied by specific patterns of regional expression: both channels are localized in layers II/III and V at P7, and before then, CNGA2 are expressed by putative newly differentiating neurons migrating from the germinal zone. Finally, the authors suggested that CNGA3-type channels have a role in plastic changes occurring after eye opening. A subsequent study demonstrated the specific localization of the olfactory-type CNGA4 subunit at the growth cones of embryonic hippocampal neurons [23]. A recent paper by Lopez-Jimenez et al. [102] demonstrated that CNGA1 and CNGB1 subunits are present and functional in cerebellar granule cells, where they mediate some of the previously documented effects of cGMP on synaptic bouton function and maturation. In particular, chronic exposure of cultured cerebellar neurons to the CNG channel blocker LCD reduced the proportion of synaptic boutons formed and impaired their maturation. The study also showed that CNG channels are expressed in cerebellar neuron growth cones at the very early developmental stages, and when they are pharmacologically inhibited, a reorganization of the actin cytoskeleton in the growth cone occurs, leading to an increased number of filopodia. Migration and neuronal differentiation do not occur only during embryonic or postnatal development, but they are also features of differentiating NSCs residing in specific regions of the adult mammalian brain, the subventricolar zone and the subgranular zone of the hippocampus, where adult neurogenesis takes place [111]. This process shares common regulatory mechanisms with CNS development, including the key role played by Ca2+ signals and cyclic nucleotides [38, 39, 128]. We recently demonstrated that mouse hippocampal NSCs express rod- and olfactory-type CNG channels [134]. They may represent a molecular substrate linking cyclic nucleotides to Ca2+ signals in these cells. In particular, experiments performed in an in vitro model of hippocampal NSCs indicate that the activation of CNG channels contribute to NSC differentiation toward the neuronal phenotype without affecting the proliferation of undifferentiated cells. We found that treatment of differentiating NSC with the CNG channel blocker LCD resulted in about 25–30 % decrease of neuronal yield, assessed by immunoreactivity for the neuronal marked MAP2. Furthermore, LCD prevented the increase in neuronal yield elicited by 8-Br-cGMP. Remarkably, our data also suggest that CNG channels play a role in fate determination of hippocampal NSCs by promoting their differentiation toward neuronal vs. glial phenotype. Indeed, the increases in MAP2+ cells induced by CNG channel activation were accompanied by a decrease in the number of cells acquiring the glial phenotype (GFAP+ cells), whereas the blockade of CNG channels increased the number of GFAP+ cells. These results are in line with the recently proposed role of NO/cGMP in promoting neuronal differentiation

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of NSCs during development [56] and point to CNG channels as possible candidates for mediating such effects. Glial function Our studies first documented the widespread expression and function of CNG channels in glial cells of the CNS [130, 131]. A previous report had suggested that cGMP and CNG channels regulate the functions of a glial cell type of the retina, named the Müller glia [94]. The authors of this study proposed a model in which local changes in K+ concentrations occurring as a result of retinal neuronal activity lead to NO release from amacrine cells, which increases cGMP levels and activates CNG channels in Müller glial cells. Calcium influx through these channels, along with the change in electrotonic driving force caused by the local changes in K+ levels, would open Ca2+-activated K+ channels, thus restoring K+ homeostasis. Strong CNGA3 immunoreactivity was also reported in the ensheating glia of the rat olfactory bulb [60]. Our demonstration of the widespread astrocytic expression of CNGA2 subunits in the rodent CNS suggests that these channels might be involved in glial cell function regulation by numerous physiological signals affecting intracellular levels of cyclic nucleotides. Astrocytes are the most numerous glial cells found in the brain and, probably, those having the greatest impact on neuronal functions. Besides providing trophic support to neurons, astrocytes are active regulators of several brain functions under both physiological and pathological conditions [165]. Bidirectional communication between astrocytes and neurons is critical for glial regulation of neuronal excitability and synaptic transmission [125, 126]. Indeed, astrocytes are responsive to numerous neurotransmitters, many of which increase [Ca2+]i, thus triggering the release of gliotransmitters (glutamate, ATP or D -serine) which, in turn, activate presynaptic and postsynaptic neuronal receptors. Astrocytes also produce and respond to various growth factors, cytokines, and chemokines, thereby contributing to the homeostatic regulation of blood flow, metabolic processes, and inflammatory responses in the brain [2, 57]. Given the potentially broad functional implication of astrocytic CNG channel activation, additional research seems warranted to define the function of these channels and the roles they play in glia-mediated modulation of neuronal, vascular, and metabolic functions in the CNS under physiological and pathological conditions. CNGA2-KO mice are invaluable experimental models for investigating such issues.

Conclusions and future perspectives Intracellular second messengers mediate many cell responses to extracellular signals. Cyclic nucleotides play such a role in a large number of physiological processes spanning from

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metabolism to memory through signal cascades including, but not limited to, protein kinases. In the visual and olfactory receptors, the primary targets of cGMP and cAMP are CNG channels that transduce changes in cyclic nucleotide concentration to membrane potential changes. For many years, the role of CNG channels had been confined to transduction processes in retinal and olfactory receptors. However, evolution rarely selects cell-specific molecules, and in keeping with this, CNG channels have been found in several tissues including the reproductive system [144]. With regard to the CNS, although a wealth of molecular studies have demonstrated CNG channel expression in the rodent brain, so far, less effort has been put in investigating their functional role. To gain further insight on this issue, new approaches are probably needed, such as a wider use of CNG channel KO mice and antisense or cell type-specific recombination methods. Pharmacological research has also to be developed to identify new channel agonists and antagonists with higher selectivity for the different CNG subtypes and no cross-reactivity with other cyclic nucleotide targets. Last but not least, we believe that research on CNG channels in “non-canonical districts” including, but not limited to, the CNS should be encouraged also overcoming a sort of skepticism in some members of the scientific community who believe that CNG channel action is necessarily confined to visual and olfactory systems. On the other hand, molecular and functional data provided so far on CNG channel expression in the CNS should prompt to take into account their contribution when dealing with physiological and pathophysiological aspects of brain functions involving cyclic nucleotides. For instance, the recently emerging concept of “compartmentalization” referred to cyclic nucleotide signaling [5] should be reformulated including CNG channels. Research in this field has advanced rapidly in recent years thanks to the use of sophisticated methods (e.g., two-photon excitation integrated to FRET, internal reflection microscopy, and quantum dot imaging) as well as of genetically engineered fluorescent probes based on cAMP/cGMPdependent protein kinases. These approaches, adapted to target CNG channels, will help in defining whether these channels are components of compartmentalized cAMP and cGMP signaling, thereby providing conclusive evidence for their roles in cell physiology [145]. Another important issue that deserves mentioning is the documented role of dysregulated cyclic nucleotide signaling in certain pathologies. In this regard, CNG channels may be the altered component of the signaling pathway or the downstream target of abnormal cyclic nucleotide levels. With regard to the CNS, pharmacological interventions based on inhibition of selective cAMP- and cGMP-degrading PDEs are under study for the treatment of depression, anxiety, and schizophrenia as well as neurodegenerative disorders [17,


107, 121, 139, 140, 143, 146]. CNG channels are possible targets of these treatments. In conclusion, it is becoming increasingly clear that CNG channels have a role in many CNS functions, though we are still far from getting a clear and complete picture. In our view, this is a goal that neuroscience research should pursue given the fundamental role played by cyclic nucleotides in health and disease and the biophysical properties of these channels, especially their Ca2+ permeability, which makes them new potential players in the crucial regulation of Ca2+ homeostasis and signaling in excitable and non-excitable cells. Acknowledgments This work was supported by grants from the Catholic University (D3.2 and D1 funds). Conflict of interest The authors declare that they have no conflicts of interest.

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Contribution of the cyclic nucleotide gated channel subunit, CNG-3, to olfactory plasticity in Caenorhabditis elegans.

The role of cyclic AMP in myogenesis.

The role of cyclic GMP in the regulation of cyclic AMP hydrolysis.

Role of Cyclic Nucleotide Gated Channels in Stress Management in Plants.

Alternative splicing governs cone cyclic nucleotide-gated (CNG) channel sensitivity to regulation by phosphoinositides.

Role of Dynamics in the Autoinhibition and Activation of the Hyperpolarization-activated Cyclic Nucleotide-modulated (HCN) Ion Channels.

The role of cyclic AMP in gonadal steroidogenesis.

The role of opportunistic migration in cyclic games.

The role of cyclic AMP in CRF-induced ACTH secretion.

Role of cyclic nucleotides in receptor mechanisms.

Cyclic nucleotide mapping of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.

Proton transfer unlocks inactivation in cyclic nucleotide-gated A1 channels.

Rapid single-molecule imaging in cyclic olefin copolymer channels.

Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channels in Epilepsy.

Capacitation and Ca(2+) influx in spermatozoa: role of CNG channels and protein kinase G.

Investigating cyclic nucleotide and cyclic dinucleotide binding to HCN channels by surface plasmon resonance.

Stimulus-secretion coupling: role of cyclic AMP, cyclic GMP and calcium in mediating enzyme (kallikrein) secretion in the submandibular gland.

Advanced Developments in Cyclic Polymers: Synthesis, Applications, and Perspectives.

Cyclic AMP in action of antidiuretic hormone: effects of exogenous cyclic AMP and its new analogue.

Emerging Roles for Glycogen in the CNS.

Glabridin-induced vasorelaxation: Evidence for a role of BKCa channels and cyclic GMP.

Cyclic nucleotides in the regulation of expression of differentiated functions in neuroblastoma cells.

Deciphering the function of the CNGB1b subunit in olfactory CNG channels.

The measurement of cyclic GMP and cyclic AMP phosphodiesterases.