Expression of olfactory-type cyclic nucleotide-gated channels in rat cortical astrocytes

Cyclic nucleotide-gated (CNG) channels are nonselective cation channels activated by cyclic AMP (cAMP) or cyclic GMP (cGMP). They were originally identified in retinal and olfactory receptors, but evidence has also emerged for their expression in several mammalian brain areas. Because cGMP and cAMP control important aspects of glial cell physiology, we wondered whether CNG channels are expressed in astrocytes, the most functionally relevant glial cells in the CNS. Immunoblot and immunofluorescence experiments demonstrated expression of the CNG channel olfactory-type A subunit, CNGA2, in cultured rat cortical astrocytes. In patch-clamp experiments, currents elicited in these cells by voltage ramps from -100 to +100 mV in the presence of the cGMP analogue, dB-cGMP, were significantly reduced by the CNG channel blockers, L-cis-diltiazem (LCD) and Cd(2+) . The reversal potentials of the LCD- and Cd(2+) -sensitive currents were more positive than that of K(+) , as expected for a mixed cation current. Noninactivating, 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 natriuretic peptide receptor activation and inhibition of phosphodiesterase activity. Voltage-independent, LCD-sensitive currents were also elicited by 8-Br-cGMP in astrocytes of hippocampal and neocortical brain slices. Immunohistochemistry confirmed a broad distribution of CNG channels in astrocytes of the rat forebrain, midbrain, and hindbrain. These findings suggest that CNG channels are downstream targets of cyclic nucleotides in astrocytes, and they may be involved in the glial-mediated regulation of CNS functions under physiological and pathological conditions.     

CNG-modulin: a novel Ca-dependent modulator of ligand sensitivity in cone photoreceptor cGMP-gated ion channels

The transduction current in several different types of sensory neurons arises from the activity of cyclic nucleotide-gated (CNG) ion channels. The channels in these sensory neurons vary in structure and function, yet each one demonstrates calcium-dependent modulation of ligand sensitivity mediated by the interaction of the channel with a soluble modulator protein. In cone photoreceptors, the molecular identity of the modulator protein was previously unknown. We report the discovery and characterization of CNG-modulin, a novel 301 aa protein that interacts with the N terminus of the β subunit of the cGMP-gated channel and modulates the cGMP sensitivity of the channels in cone photoreceptors of striped bass (Morone saxatilis). Immunohistochemistry and single-cell PCR demonstrate that CNG-modulin is expressed in cone but not rod photoreceptors. Adding purified recombinant CNG-modulin to cone membrane patches containing the native CNG channels shifts the midpoint of cGMP dependence from ～91 μM in the absence of Ca(2+) to ～332 μM in the presence of 20 μM Ca(2+). At a fixed cGMP concentration, the midpoint of the Ca(2+) dependence is ～857 nM Ca(2+). These restored physiological features are statistically indistinguishable from the effects of the endogenous modulator. CNG-modulin binds Ca(2+) with a concentration dependence that matches the calcium dependence of channel modulation. We conclude that CNG-modulin is the authentic Ca(2+)-dependent modulator of cone CNG channel ligand sensitivity. CNG-modulin is expressed in other tissues, such as brain, olfactory epithelium, and the inner ear, and may modulate the function of ion channels in those tissues as well.     

Co-expression of wild-type and mutant olfactory cyclic nucleotide-gated channels: restoration of the native sensitivity to Ca(2+) and Mg(2+) blockage

In the pore of homomeric cyclic nucleotide-gated (CNG) channels, Ca(2+) and Mg(2+) bind to a set of glutamate residues, which in the bovine olfactory CNG channel are located at position 340. However, native CNG channels from olfactory sensory neurons are composed by the assembly of three different types of subunits, each having a different residue -- glutamate, aspartate or glycine -- at the position corresponding to the binding site for external Ca(2+) and Mg(2+). We co-expressed the wild-type principal alpha subunit with its mutants E340G and E340D in different combinations in Xenopus laevis oocytes, and measured Ca(2+) and Mg(2+) blockage in excised outside-out membrane patches. The comparison between our results and data from native olfactory CNG channels indicates that the presence of all three residues -- glutamate, aspartate and glycine -- in the different subunits, is necessary to restore the sensitivity to external Ca(2+) and Mg(2+) measured in native channels.     

Functional reconstitution of a heteromeric cyclic nucleotide-gated channel of Caenorhabditis elegans in cultured cells

The tax-4 and tax-2 genes of Caenorhabditis elegans are essential for normal olfaction, gustation, and thermosensation, suggesting that they have a role in sensory transduction. The predicted products of these genes are similar to the cyclic nucleotide-gated (CNG) channel subunits used in vertebrate vision and olfaction: TAX-4 is highly related to those alpha subunits, while TAX-2 is most closely related to the beta subunits of the rod phototransduction channels. TAX-4 has previously been shown to form a highly sensitive cGMP-gated channel when expressed in human HEK293 cells. Here we show that TAX-4 and TAX-2 form a heteromeric channel when expressed in HEK293 cells, but TAX-2 does not form a channel on its own. Since these genes are expressed in the same neurons, most of the native channels in C. elegans are likely to be hetero-oligomers of TAX-4 and TAX-2 subunits, with TAX-4 as the alpha subunit and TAX-2 acting as a modifying beta subunit. The heteromeric TAX-4/TAX-2 channel is 25-fold less sensitive to cGMP than the TAX-4 channel, but it remains highly selective for cGMP over cAMP. The heteromeric channel and the TAX-4 homomeric channel differ in their blockage by divalent cations and in their single channel properties. These results suggest that cGMP is used as the second messenger during sensory signal transduction in C. elegans, and that distinct roles for alpha and beta subunits of CNG channels are conserved in vertebrate and invertebrate signal transduction.     

Localization of mRNAs encoding alpha and beta subunits of soluble guanylyl cyclase in the brain of rainbow trout: comparison with the distribution of neuronal nitric oxide synthase

Detailed distribution of mRNAs encoding alpha and beta subunits of soluble guanylyl cyclase (sGC) was examined in the brain of rainbow trout by in situ hybridization. In addition, distribution of nitric oxide synthase (NOS) was mapped in adjacent parallel sections by neuronal NOS (nNOS) immunocytochemistry and NADPH-diaphorase (NADPHd) histochemistry. Following application of digoxigenin-labeled riboprobes for sGC alpha and beta subunit mRNAs, we found comparatively intense hybridization signals in the telencephalon, preoptic area, thalamus, hypothalamus, pretectum and tegmentum. Both nNOS immunocytochemistry and NADPHd histochemistry showed extensive distribution of nitroxergic neurons in various brain areas, although various degrees of dissociation of nNOS immunoreactivity (ir) and NADPHd staining were detected. In comparison with sGC subunit mRNAs, nNOS signals were more widely distributed in many neurons, including parvocellular neurons in the preoptic area, nucleus anterior tuberis in the hypothalamus, periventricular neurons in the optic tectum, most of the rhombencephalic neurons and pituitary cells. However, wide overlaps of sGC mRNA-containing neurons and nNOS-positive neurons were observed in the olfactory bulb, telencephalon, preoptic area, thalamus, hypothalamus, pretectum, optic tectum, tegmentum and cerebellum. The widespread overlapping in sGC subunit mRNAs and nNOS distribution suggests a role for sGC in various neuronal functions, such as processing of olfactory and visual signals and neuroendocrine function, possibly via NO/cGMP signaling in the brain of rainbow trout.     

The native rat olfactory cyclic nucleotide-gated channel is composed of three distinct subunits.

Cyclic nucleotide-gated (CNG) channels play central roles in visual and olfactory signal transduction. In the retina, rod photoreceptors express the subunits CNCalpha1 and CNCbeta1a. In cone photoreceptors, only CNCalpha2 expression has been demonstrated so far. Rat olfactory sensory neurons (OSNs) express two homologous subunits, here designated CNCalpha3 and CNCalpha4. This paper describes the characterization of CNCbeta1b, a third subunit expressed in OSNs and establishes it as a component of the native channel. CNCbeta1b is an alternate splice form of the rod photoreceptor CNCbeta1a subunit. Analysis of mRNA and protein expression together suggest co-expression of all three subunits in sensory cilia of OSNs. From single-channel analyses of native rat olfactory channels and of channels expressed heterologously from all possible combinations of the CNCalpha3, -alpha4, and -beta1b subunits, we conclude that the native CNG channel in OSNs is composed of all three subunits. Thus, CNG channels in both rod photoreceptors and olfactory sensory neurons result from coassembly of specific alpha subunits with various forms of an alternatively spliced beta subunit.     

Analysis of single cyclic nucleotide-gated channels in olfactory receptor cells.

In the accompanying article (Firestein et al., 1991b), we have demonstrated that odor- and cyclic nucleotide-sensitive channels exist at a low density in the dendritic membranes of isolated salamander olfactory receptor neurons. Here, we analyze the cyclic nucleotide sensitivity of these channels using the inside-out patch recording technique. Both cAMP and cGMP, at micromolar concentrations, are capable of inducing channel openings. The biophysical parameters of channel activity are nearly the same in response to either ligand. The unitary conductance is about 45 pS, the reversal potential of single-channel currents is +5 mV, and the I/V relation is linear over the range -80 to +80 mV. The channel activity shows no obvious voltage dependence in divalent cation-free symmetrical solutions. The channel shows no desensitization, even to agonist exposures lasting 15 sec. Mean open time is about 1.5 msec; the closed time distribution is best fit by two exponentials with a fast time constant in the submillisecond range (ca. 0.15 msec) and a slower time constant in the millisecond range (ca. 1.5 msec). The only clear difference in the activity of the two ligands is in their affinity constants. The K1/2 for cAMP is 20 microM; that for cGMP is 4 microM. In both cases, the Hill coefficient is greater than 2, suggesting that channel opening requires the cooperative action of three ligand molecules.     

Cyclic Nucleotide-gated Channels in Identified Human Olfactory Receptor Neurons

Patch-clamp recordings revealed the presence of a non-desensitizing cyclic nucleotide-gated channel on human olfactory receptor neurons and a fast-desensitizing non-specific cation channel activated by nucleotides on human supporting cells. Cyclic nucleotide-gated channels on olfactory receptor neurons showed selective channel activation by cAMP (K_1/2= 5 μM) and cGMP (K_1/2= 2 μM), a unitary conductance of ∼20 pS, a reversal potential of single-channel currents close to 0 mV, a linear current-voltage relationship over the range of −80 to 80 mV and a strong extracellular but a weaker intracellular blocking effect of Ca²⁺. The channel activity outlasted the cyclic nucleotide pulses for hundreds of milliseconds when higher agonist concentrations (>50 μM cAMP) were applied. The duration of the response was longer than in cyclic nucleotide-gated channels from other species studied so far. The plateau duration and the decay remained constant for pulses with a length of 50−150 ms, whereas pulses shorter than 50 ms successively reduced the time required by shortening the plateau phase. A larger difference for the K_1/2 values of cAMP (K_1/2= 22 μM) and cGMP (K_1/2= 2.5 μM) were found for a small group (n = 3) of cyclic nucleotide-gated channels, pointing to the selective expression of the a-subunit in a small subgroup of olfactory receptor neurons.     

Functional reconstitution of a heteromeric cyclic nucleotide-gated channel of Caenorhabditis elegans in cultured cells

The tax-4 and tax-2 genes of Caenorhabditis elegans are essential for normal olfaction, gustation, and thermosensation, suggesting that they have a role in sensory transduction. The predicted products of these genes are similar to the cyclic nucleotide-gated (CNG) channel subunits used in vertebrate vision and olfaction: TAX-4 is highly related to those α subunits, while TAX-2 is most closely related to the β subunits of the rod phototransduction channels. TAX-4 has previously been shown to form a highly sensitive cGMP-gated channel when expressed in human HEK293 cells. Here we show that TAX-4 and TAX-2 form a heteromeric channel when expressed in HEK293 cells, but TAX-2 does not form a channel on its own. Since these genes are expressed in the same neurons, most of the native channels in C. elegans are likely to be hetero-oligomers of TAX-4 and TAX-2 subunits, with TAX-4 as the α subunit and TAX-2 acting as a modifying β subunit. The heteromeric TAX-4/TAX-2 channel is 25-fold less sensitive to cGMP than the TAX-4 channel, but it remains highly selective for cGMP over cAMP. The heteromeric channel and the TAX-4 homomeric channel differ in their blockage by divalent cations and in their single channel properties. These results suggest that cGMP is used as the second messenger during sensory signal transduction in C. elegans, and that distinct roles for α and β subunits of CNG channels are conserved in vertebrate and invertebrate signal transduction.     

Functional analysis of human CNGA3 mutations associated with colour blindness suggests impaired surface expression of channel mutants A3(R427C) and A3(R563C)

Mutations in the CNGA3 gene have been associated with complete and incomplete forms of total colour blindness (achromatopsia), a disorder characterized by reduced visual acuity, lack of colour discrimination, photophobia and nystagmus. CNGA3 encodes the A-subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel, an essential component of the phototransduction cascade. Here we report the identification of three new CNGA3 mutations in patients with achromatopsia. To assess the pathogenicity of these newly identified and four previously reported mutations, mutant CNGA3 channels were heterologously expressed in a human embryonic kidney cell line (HEK293 cells) and functionally analysed using calcium imaging. Channels with the mutations R427C and R563C showed a response in imaging experiments and were subsequently characterized in-depth with the patch-clamp technique. The mutant channels were analysed as homooligomers and also as heterooligomers with the wild-type B-subunit present in native channels. Overall, cyclic guanosine monophosphate (cGMP) maximum currents of mutant channels were profoundly reduced in homo- and heteromers. Treatment with the chemical chaperone glycerol effectively increased macroscopic currents, presumably by enhancing surface expression of mutant channels as confirmed by immunocytochemistry. These results suggest decreased channel density in the cell membrane due to impaired folding or trafficking of the channel protein as the main pathogenic effect of the mutations R427C and R563C. Moreover, A3(R427C) homomers showed distinctly increased cGMP and cyclic adenosine monophosphate (cAMP) sensitivities as well as cAMP fractional currents that were raised to over 90% of cGMP maximum currents. Co-expression of A3(R427C) with the B3 subunit compensated for most of these aberrant properties, apart from the reduced cGMP maximum currents.     

Direct suppression by odorants of cyclic nucleotide-gated currents in the newt photoreceptors

Odorants are known to suppress the cyclic nucleotide-gated (CNG) current in olfactory receptor cells. It is unclear, however, whether odorants suppress the olfactory CNG current directly or whether they suppress the current by decreasing the second messenger (cAMP) through the activation of phosphodiesterase. We found that odorants also suppress CNG currents in photoreceptor cells. Under voltage clamp, an odorant puff immediately suppressed the currents induced by the intracellular cGMP in isolated newt rods and cones. Odorants also suppressed the currents induced by another cGMP analog (8-p-chlorophenylthio-cGMP, which strongly resists hydrolysis by phosphodiesterase), suggesting that the second messenger metabolism via phosphodiesterase is not involved in the suppression by odorants. This suggests that odorants suppress the CNG currents directly rather than via the second messenger system in photoreceptors, and also likely in olfactory receptor cells.     

Granule neurons generated during development extend divergent axon collaterals to hippocampal area CA3

cAMP is a ubiquitous second messenger, which acts mainly through specific protein kinases that consist of two regulatory and two catalytic subunits. An unsolved problem in cAMP physiology is how it can regulate so many cellular functions through this simple enzymatic cascade. A tentative explanation is related to the different biochemical properties of the four regulatory subunit isoforms (RI alpha and RI beta, RII alpha and RII beta) and to their differential cell and tissue distribution. For example, detergent insoluble aggregates of RI alpha are present in some cholinergic neurons of the adult rat brain. Rat brains, from the embryonic stage to old age, were examined for the presence of highly concentrated clusters of RI alpha. They are present only in some neurons of restricted brain areas, for a limited time span. During development, labeled neurons appear in different brain areas after neuron migration, at a stage of advanced functional maturation. They have their greatest expression after birth but before sexual maturation, and then they slowly decline, persisting only in a few brain areas throughout life. The first appearance, time course, and eventual disappearance is different in the different brain areas: RI alpha clusters appear in brainstem, hypothalamus, and accessory olfactory bulb at a late embryonic stage; in the main olfactory bulb, hippocampus, and medial thalamic nuclei shortly after birth; and in the cortex as late as in the third and fourth postnatal week. During the rat's lifespan, the distribution of these peculiar RI alpha clusters undergo changes that may contribute to shape neuronal responses differentially to agents modifying cAMP levels.     

The cyclic nucleotide-gated channels of vertebrate photoreceptors and olfactory epithelium.

Cation channels that are directly gated by guanosine 3', 5'-cyclic monophosphate (cGMP) control the flow of ions across the surface membrane of vertebrate rod and cone photoreceptor cells. A similar channel, gated by adenosine 3',5'-cyclic monophosphate (cAMP), exists in vertebrate olfactory sensory neurons. The channel polypeptide of rod photoreceptors has been identified and the amino acid sequence of the channel polypeptide in rod and olfactory cells has been determined by cloning cDNA. Although the cyclic nucleotide-gated channels functionally belong to the class of ligand-gated channels, they share some structural features with voltage-gated channels.     

Phosphorylation of voltage-gated ion channels in rat olfactory receptor neurons

In olfactory receptor neurons (ORNs), ligand-odorant receptor interactions cause G protein-mediated activation of adenylate cyclase and a subsequent increase in concentration of the intracellular messenger cAMP. Odorant-evoked elevation in cAMP is thought to directly activate a cation-selective cyclic nucleotide-gated channel, which causes external Ca2+ influx, leading to membrane depolarization and the generation of action potentials. Our data show that in freshly dissociated rat ORNs, odorant-induced elevation in cAMP also activates cAMP-dependent protein kinase (PKA), which is then able to phosphorylate various protein targets in the olfactory signal transduction pathway, specifically voltage-gated sodium and calcium channels. The presence of PKI (PKA inhibitor peptide) blocked the modulatory action of cAMP on voltage-gated ion channels. By modulating the input/output properties of the sensory neurons, this mechanism could take part in the complex adaptation process in odorant perception. In addition, we found modulation of voltage-gated sodium and calcium channel currents by 5-hydroxytryptamine and the dopamine D1 receptor agonist SKF 38393. These findings suggest that in situ ORNs might also be a target for efferent modulation.     

Ethanol withdrawal is accompanied by downregulation of calcium channel alpha 1B subunit in rat inferior colliculus neurons

Ethanol withdrawal enhances the current density of calcium (Ca(2+)) channels in inferior colliculus (IC) neurons. The present report shows that ethanol withdrawal markedly enhanced the susceptibility to seizures as it decreased significantly the protein levels of alpha(1B) subunit associated with N-type Ca(2+) channel in IC neurons of animals not tested for seizures. Thus, remodeling of N-type Ca(2+) channels may play an important role in neuronal hyperexcitability that leads to ethanol withdrawal seizures.     

Grueneberg ganglion olfactory subsystem employs a cGMP signaling pathway

The mammalian olfactory sense employs several olfactory subsystems situated at characteristic locations in the nasal cavity to detect and report on different classes of odors. These olfactory subsystems use different neuronal signal transduction pathways, receptor expression repertoires, and axonal projection targets. The Grueneberg ganglion (GG) is a newly appreciated olfactory subsystem with receptor neurons located just inside of the nostrils that project axons to a unique domain of interconnected glomeruli in the caudal olfactory bulb. It is not well understood how the GG relates to other olfactory subsystems in contributing to the olfactory sense. Furthermore, the range of chemoreceptors and the signal transduction cascade utilized by the GG have remained mysterious. To resolve these unknowns, we explored the molecular relationship between the GG and the GC‐D neurons, another olfactory subsystem that innervates similarly interconnected glomeruli in the same bulbar region. We found that mouse GG neurons express the cGMP‐associated signaling proteins phosphodiesterase 2a, cGMP‐dependent kinase II, and cyclic nucleotide gated channel subunit A3 coupled to a chemoreceptor repertoire of cilia‐localized particulate guanylyl cyclases (pGC‐G and pGC‐A). The primary cGMP signaling pathway of the GG is shared with the GC‐D neurons, unifying their target glomeruli as a unique center of olfactory cGMP signal transduction. However, the distinct chemoreceptor repertoire in the GG suggests that the GG is an independent olfactory subsystem. This subsystem is well suited to detect a unique set of odors and to mediate behaviors that remained intact in previous olfactory perturbations. J. Comp. Neurol. 516:36–48, 2009. © 2009 Wiley‐Liss, Inc.