Effects of permeating ions and cGMP on gating and conductance of rod-type cyclic nucleotide-gated (CNGA1) channels

Cyclic nucleotide-gated (CNG) channels are tetrameric non-specific cation channels. They mediate the receptor potentials in photoreceptors and cells of the olfactory epithelium and they are activated by the binding of cyclic nucleotides such as cGMP and cAMP. Previous studies in homotetrameric CNGA1 channels, activated with covalently bound cGMP, presented evidence that partially liganded channels cause partial channel opening ( Ruiz & Karpen, 1997, 1999 ). Here, homotetrameric CNGA1 channels were expressed in Xenopus oocytes. Conductance and gating of these channels were studied as a function of the concentration of freely diffusible cGMP and with different permeating ions. At saturating cGMP the current levels distributed around a single mean in a Gaussian fashion and the open times were long. At low cGMP, however, the current levels were heterogeneous: they were smaller than those at saturating cGMP, equal, or larger. The open times were short. Ions generating the larger single-channel currents (Na⁺ > K⁺ > Rb⁺) concomitantly increased the heterogeneity of current levels and decreased the open probability and open times. The results suggest that the activation of CNGA1 channels by cGMP and ions staying longer in the pore is associated with less extensive and less frequent conformational fluctuations of the channel pore.     

Cyclic nucleotide-gated ion channels

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.     

Calmodulin permanently associates with rat olfactory CNG channels under native conditions

An important mechanism by which vertebrate olfactory sensory neurons rapidly adapt to odorants is feedback modulation of the Ca(2+)-permeable cyclic nucleotide-gated (CNG) transduction channels. Extensive heterologous studies of homomeric CNGA2 channels have led to a molecular model of channel modulation based on the binding of calcium-calmodulin to a site on the cytoplasmic amino terminus of CNGA2. Native rat olfactory CNG channels, however, are heteromeric complexes of three homologous but distinct subunits. Notably, in heteromeric channels, we found no role for CNGA2 in feedback modulation. Instead, an IQ-type calmodulin-binding site on CNGB1b and a similar but previously unidentified site on CNGA4 are necessary and sufficient. These sites seem to confer binding of Ca(2+)-free calmodulin (apocalmodulin), which is then poised to trigger inhibition of native channels in the presence of Ca(2+).     

Phosphodiesterase 3 and 5 and cyclic nucleotide-gated ion channel expression in rat trigeminovascular system

Activation of the trigeminovascular pain signalling system appears involved in migraine pathophysiology. However, the molecular mechanisms are only partially known. Stimulation of cAMP and cGMP production as well as inhibition of their breakdown induce migraine-like headache. Additionally, migraine may be associated with mutations in ion channels. The aim of the present study was to describe the expression of phosphodiesterase 3 (PDE3) and 5 (PDE5) and cyclic nucleotide-gated ion channels (CNG) in cerebral arteries, meninges, and the trigeminal ganglion. mRNA for PDE and CNG was determined in the rat middle cerebral artery, basilar artery, trigeminal ganglion, and dura mater using real-time PCR. PDE and CNG proteins were identified using Western blot. For comparison, rat aorta and mesenteric artery were analysed. PDE3A, PDE3B, and PDE5A mRNA were detected in all tissues examined except for PDE3A mRNA in dura mater and the trigeminal ganglion. PDE5A and PDE3A protein expression was present in both cerebral and peripheral arteries, whereas PDE3B protein was present only in the cerebral arteries. The CNGA4 and B1 subunit mRNAs were detected in cerebral arteries and CNGA2 also in the mesenteric artery. CNGA2 and A3 proteins were found in cerebral arteries and dura and CNGA1, CNGA2 and CNGA3 in the trigeminal ganglion. In conclusion, PDE3A, PDE3B, PDE5A, and five CNG subunits were expressed in several components of the trigeminovascular system of the rat. This suggests that modulation of cAMP and cGMP levels by PDE and activation of CNG may play a role in trigeminovascular pain signalling leading to migraine headache.     

Subunit contributions to phosphorylation-dependent modulation of bovine rod cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels in rod photoreceptors transduce a decrease in cGMP into hyperpolarization during the light response. Insulin-like growth factor-1 (IGF-1) increases light responses by increasing the cGMP sensitivity of CNG channels, an event mediated by a protein tyrosine phosphatase. Native rod CNG channels are heteromultimers, composed of three CNGA1 subunits and one CNGB1 subunit. Previous studies on heterologously expressed rod CNG channels show that a specific tyrosine in the CNGA1 subunit (Y498) is required for modulation by protein tyrosine phosphatases, protein tyrosine kinases and IGF-1. Here we show that the CNGB1 subunit contains a specific tyrosine (Y1097) that is important for modulation of heteromeric channels by tyrosine phosphorylation. Direct biochemical measurements demonstrate ³²P-labelling of CNGA1_Y498 and CNGB1_Y1097. Replacement of either Y498 of CNGA1 or Y1097 of CNGB1 with phenylalanine reduces modulation, and removal of both tyrosines eliminates modulation. Unlike CNGA1, CNGB1 does not exhibit activity dependence of modulation by tyrosine phosphorylation. Hence both CNGA1 and CNGB1 subunits contribute to phosphorylation-dependent modulation of rod CNG channels, but the phosphorylation states of the two subunits are regulated in different ways.     

A novel cyclic nucleotide-gated ion channel enriched in synaptic terminals of isotocin neurons in zebrafish brain and pituitary

Cyclic nucleotide-gated (CNG) channels are nonselective cation channels opened by binding of intracellular cyclic GMP or cyclic AMP. CNG channels mediate sensory transduction in the rods and cones of the retina and in olfactory sensory neurons, but in addition, CNG channels are also expressed elsewhere in the CNS, where their physiological roles have not yet been well defined. Besides the CNG channel subtypes that mediate vision and olfaction, zebrafish has an additional subtype, CNGA5, which is expressed almost exclusively in the brain. We have generated CNGA5-specific monoclonal antibodies, which we use here to show that immunoreactivity for CNGA5 channels is highly enriched in synaptic terminals of a discrete set of neurons that project to a subregion of the pituitary, as well as diffusely in the brain and spinal cord. Double labeling with a variety of antibodies against pituitary hormones revealed that CNGA5 is located in the terminals of neuroendocrine cells that secrete the nonapeptide hormone/transmitter isotocin in the neurohypophysis, brain, and spinal cord. Furthermore, we show that CNGA5 channels expressed in Xenopus oocytes are highly permeable to Ca²⁺, which suggests that the channels are capable of modulating isotocin release in the zebrafish brain and pituitary. Isotocin is the teleost homolog of the mammalian hormone oxytocin, and like oxytocin, it regulates reproductive and social behavior. Therefore, the high calcium permeability of CNGA5 channels and their strategic location in isotocin-secreting synaptic terminals suggest that activation of CNGA5 channels in response to cyclic nucleotide signaling may have wide-ranging neuroendocrine and behavioral effects.     

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

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.     

Odorant responses of dual polarity are mediated by cAMP in mouse olfactory sensory neurons

Some olfactory sensory neurons (OSNs) respond to odors with hyperpolarization. Although transduction for excitatory responses is mediated by opening of a cyclic nucleotide-gated (CNG) channel, there is controversy on the mechanism underlying inhibitory responses. We find that mouse OSNs respond to odorants by either depolarizing or hyperpolarizing responses in loose-patch measurements. In the perforated-patch configuration, OSNs not only responded with a current consistent with CNG channel-mediated excitation but also displayed enhancement of outward currents, consistent with inhibitory responses. Increasing cAMP levels pharmacologically elicited excitatory or inhibitory responses in different OSNs. In addition, OSNs from mice defective for the CNGA2 subunit of the CNG channel displayed neither excitatory nor inhibitory responses. Thus CNG channels mediate inhibitory olfactory responses.     

Distribution of the A3 subunit of the cyclic nucleotide-gated ion channels in the main olfactory bulb of the rat

Previous data suggest that cyclic GMP (cGMP) signaling can play key roles in the circuitry of the olfactory bulb (OB). Therefore, the expression of cGMP-selective subunits of the cyclic nucleotide-gated ion channels (CNGs) can be expected in this brain region. In the present study, we demonstrate a widespread expression of the cGMP-selective A3 subunit of the cyclic nucleotide-gated ion channels (CNGA3) in the rat OB. CNGA3 appears in principal cells, including mitral cells and internal, medium and external tufted cells. Moreover, it appears in two populations of interneurons, including a subset of periglomerular cells and a group of deep short-axon cells. In addition to neurons, CNGA3-immunoreactivity is found in the ensheathing glia of the olfactory nerve. Finally, an abundant population of CNGA3-containing cells with fusiform morphology and radial processes is found in the inframitral layers. These cells express doublecortin and have a morphology similar to that of the undifferentiated cells that leave the rostral migratory stream and migrate radially through the layers of the OB. Altogether, our results suggest that CNGA3 can play important and different roles in the OB. Channels composed of this subunit can be involved in the processing of the olfactory information taking place in the bulbar circuitry. Moreover, they can be involved in the function of the ensheathing glia and in the radial migration of immature cells through the bulbar layers.     

Mutations in CNGA3 impair trafficking or function of cone cyclic nucleotide-gated channels, resulting in achromatopsia

CNGA3 encodes the A-subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel, which is a crucial component of the phototransduction cascade in cone outer segments. Mutations in the CNGA3 gene have been associated with complete and incomplete forms of achromatopsia (ACHR), a congenital, autosomal recessively inherited retinal disorder characterized by lack of color discrimination, reduced visual acuity, nystagmus, and photophobia. Here we report the identification of three novel CNGA3 missense mutations in ACHR patients: c.682G>A (p.E228 K), c.1315C>T (p.R439W), and c.1405G>A (p.A469 T), and the detailed functional analyses of these new as well as five previously reported mutations (R283Q, T291R, F547L, G557R, and E590 K), in conjunction with clinical data of patients carrying these mutations, to establish genotype-phenotype correlations. The functional characterization of mutant CNGA3 channels was performed with calcium imaging and patch clamp recordings in a heterologous HEK293 cell expression system. Results were corroborated by immunostaining and colocalization experiments of the channel protein with the plasma membrane. Several mutations evoked pronounced alterations of the apparent cGMP sensitivity of mutant channels. These functional defects were fully or partially compensated by coexpressing the mutant CNGA3 subunit with the wild-type CNGB3 subunit for channels with the mutations R439W, A469 T, F547L, and E590 K. We could show that several mutant channels with agonist dose-response relationships similar to the wild-type exhibited severely impaired membrane targeting. In addition, this study presents the positive effect of reduced cell culture temperature on surface expression and functional performance of mutant CNG channels with protein folding or trafficking defects.     

Functional role of cyclic nucleotide-gated channels in rat medial vestibular nucleus neurons

Although cyclic nucleotide-gated (CNG) channels are expressed in numerous brain areas, little information is available on their functions in CNS neurons. The aim of the present study was to define the distribution of CNG channels in the rat medial vestibular nucleus (MVN) and their possible involvement in regulating MVN neuron (MVNn) excitability. The majority of MVNn expressed both CNG1 and CNG2 A subunits. In whole-cell current-clamp experiments carried out on brainstem slices containing the MVNn, the membrane-permeant analogues of cyclic nucleotides, 8-Br-cGMP and 8-Br-cAMP (1 m m ), induced membrane depolarizations (8.9 ± 0.8 and 9.2 ± 1.0 mV, respectively) that were protein kinase independent. The cGMP-induced depolarization was 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 l - cis -diltiazem, but they were unaffected by blockade of hyperpolarization-activated cyclic nucleotide-gated channels. In voltage-clamp experiments, 8-Br-cGMP induced non-inactivating inward currents (−22.2 ± 3.9 pA) with an estimated reversal potential near 0 mV, which were markedly inhibited by reduction of extracellular Na⁺ and Ca²⁺ concentrations. Membrane depolarization induced by CNG channel activation increased the firing rate of MVNn without changing the action potential shape. Collectively, these findings provide novel evidence that CNG channels affect membrane potential and excitability of MVNn. Such action should have a significant impact on the function of these neurons in sensory–motor integration processes. More generally, it might represent a broad mechanism for regulating the excitability of different CNS neurons.     

Cyclic AMP cascade mediates the inhibitory odor response of isolated toad olfactory receptor neurons

Odor stimulation may excite or inhibit olfactory receptor neurons (ORNs). It is well established that the excitatory response involves a cyclic AMP (cAMP) transduction mechanism that activates a nonselective cationic cyclic nucleotide-gated (CNG) conductance, accompanied by the activation of a Ca2+-dependent Cl(-) conductance, both causing a depolarizing receptor potential. In contrast, odor inhibition is attributed to a hyperpolarizing receptor potential. It has been proposed that a Ca2+-dependent K+ (K(Ca)) conductance plays a key role in odor inhibition, both in toad and rat isolated olfactory neurons. The mechanism underlying odor inhibition has remained elusive. We assessed its study using various pharmacological agents and caged compounds for cAMP, Ca2+, and inositol 1,4,5-triphosphate (InsP3) on isolated toad ORNs. The odor-triggered K(Ca) current was reduced on exposing the cell either to the CNG channel blocker LY83583 (20 microM) or to the adenylyl cyclase inhibitor SQ22536 (100 microM). Photorelease of caged Ca2+ activated a Cl- current sensitive to niflumic acid (10 microM) and a K+ current blockable by charybdotoxin (20 nM) and iberiotoxin (20 nM). In contrast, photoreleased Ca2+ had no effect on cells missing their cilia, indicating that these conductances are confined to the cilia. Photorelease of cAMP induced a charybdotoxin-sensitive K+ current in intact ORNs. Photorelease of InsP3 did not increase the membrane conductance of olfactory neurons, arguing against a direct role of InsP3 in chemotransduction. We conclude that a cAMP cascade mediates the activation of the ciliary Ca2+-dependent K+ current and that the Ca2+ ions that activate the inhibitory current enter the cilia through CNG channels.     

Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive, lung liquid absorption

Impairment of lung liquid absorption can lead to severe respiratory symptoms, such as those observed in pulmonary oedema. In the adult lung, liquid absorption is driven by cation transport through two pathways: a well-established amiloride-sensitive Na(+) channel (ENaC) and, more controversially, an amiloride-insensitive channel that may belong to the cyclic nucleotide-gated (CNG) channel family. Here, we show robust CNGA1 (but not CNGA2 or CNGA3) channel expression principally in rat alveolar type I cells; CNGA3 was expressed in ciliated airway epithelial cells. Using a rat in situ lung liquid clearance assay, CNG channel activation with 1?mM 8Br-cGMP resulted in an approximate 1.8-fold stimulation of lung liquid absorption. There was no stimulation by 8Br-cGMP when applied in the presence of either 100?μM L: -cis-diltiazem or 100?nM pseudechetoxin (PsTx), a specific inhibitor of CNGA1 channels. Channel specificity of PsTx and amiloride was confirmed by patch clamp experiments showing that CNGA1 channels in HEK 293 cells were not inhibited by 100?μM amiloride and that recombinant αβγ-ENaC were not inhibited by 100?nM PsTx. Importantly, 8Br-cGMP stimulated lung liquid absorption in situ, even in the presence of 50?μM amiloride. Furthermore, neither L: -cis-diltiazem nor PsTx affected the β(2)-adrenoceptor agonist-stimulated lung liquid absorption, but, as expected, amiloride completely ablated it. Thus, transport through alveolar CNGA1 channels, located in type I cells, underlies the amiloride-insensitive component of lung liquid reabsorption. Furthermore, our in situ data highlight the potential of CNGA1 as a novel therapeutic target for the treatment of diseases characterised by lung liquid overload.     

Electrophysiological characteristics of rat gustatory cyclic nucleotide--gated channel expressed in Xenopus oocytes

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(+) approximately 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(i)s) of intracellular Mg(2+) were determined as 360 +/- 40 microM at 70 mV and 8.2 +/- 1.5 mM at -70 mV with z delta value of 1.04, while K(i)s of extracellular Mg(2+) were as 1.1 +/- 0.3 mM at 70 mV and 20.0 +/- 0.1 microM at -70 mV with z delta of 0.94. Although 100 microM 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.     

Enrichment of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate in cholinergic axon terminals of the Torpedo electric organ

On fractionation of intact electric organ, cAMP and cGMP are enriched in the synaptosome fraction by factors of 14 and 6 respectively. For all regions of the electromotor neuron the absolute contents of nucleotides are in the order ATP greater than cAMP greater than cGMP. However, the molar ratios of cAMP/cGMP vary between tissues. Specific contents of both cyclic nucleotides are lowest in the nerve. For cAMP, specific contents in the axon terminal and in the perikaryal region are similar. As compared to this, cGMP is considerably enriched in the presynaptic nerve ending. This points to a physiological role of cGMP in presynaptic function.     

Cyclic AMP inhibits secretion from electroporated human neutrophils.

It has long been known that intracellular cAMP inhibits and cGMP enhances intact neutrophil function. However, these effects are modest and require relatively high concentrations of the cyclic nucleotides. We decided to re-examine the effects of cyclic nucleotides on Ca2(+)-induced secretion by electroporated cells. This system allowed us to bypass normal cell surface receptor-ligand interactions as well as to directly expose the intracellular space to native cyclic nucleotides. We found that concentrations of cAMP as low as 3 microM inhibited Ca2(+)-induced secretion; 30-300 microM cAMP was maximally inhibitory. cAMP was actually slightly more potent than dibutyryl cAMP, a membrane-permeant derivative. In contrast, cGMP was only slightly stimulatory at 3 microM and modestly inhibitory at 300 microM; dibutyryl cGMP was ineffective. A more detailed investigation of the effects of cAMP showed that inhibition was only obtained in the presence of Mg2+. Half-maximal inhibition by cAMP occurred at 10-30 microM. Inhibition by cAMP was achieved by shifting the Ca2+ dose-response curve for secretion to the right; this was observed for the release of both specific granules (vitamin B12 binding protein) and azurophil granules (B-glucuronidase). We previously showed that ATP could enhance Ca2(+)-induced secretion in the presence of Mg2+, apparently by interacting with a cell surface purine receptor. However, increasing concentrations of ATP could not overcome inhibition by cAMP; this suggested that cAMP acted at some site other than the purine receptor. Inhibition by cAMP was also less apparent in the presence of the protein kinase C agonist phorbol myristate acetate (PMA), suggesting that the cyclic nucleotide did not produce systemic desensitization of the neutrophils. In summary, these results demonstrate that low, physiologically relevant concentrations of cAMP can modulate neutrophil responsiveness.     

Comparative analysis between cyclic GMP and cyclic AMP signalling in human sperm

The principal involvement of cyclic nucleotides in regulating sperm functions is well established, but the factors controlling their generation and actions have not yet been entirely resolved. In particular, specific roles for cyclic (c)GMP in mammalian sperm are poorly understood. In this study, we have characterized comparatively the cAMP and cGMP signalling systems in ejaculated human sperm. Mean concentrations of cGMP (0.1 micromol/l) were found to be 100-fold lower than those of cAMP in non-stimulated cells, and adenylyl cyclase (AC) activities predominate by far guanylyl cyclase (GC) activities in both particulate and soluble protein fractions. By different experimental approaches (photoaffinity labelling, cyclase assays, immunoblotting), we provide evidence for the presence (guanylyl cyclase-A, soluble guanylyl cyclase, regulatory and catalytic subunits of cAMP-dependent protein kinase) or absence (guanylyl cyclase-B, natriuretic peptide clearance receptor, neuronal nitric oxide synthase, cGMP-dependent protein kinase I) of different factors involved in either cAMP or cGMP pathways. Functional studies showed that cGMP, at high concentrations, can enhance sperm protein tyrosine phosphorylation but not serine phosphorylation of glycogen synthase kinase. This study reveals that human sperm are characterized by an exceptional predominance of cAMP signalling and indicates potential roles for cGMP.     

Odorants suppress a voltage-activated K+ conductance in rat olfactory neurons.

Stimulation of olfactory receptor neurons (ORNs) with odors elicits an increase in the concentration of cAMP leading to opening of cyclic nucleotide-gated (CNG) channels and subsequent depolarization. Although opening of CNG channels is thought to be the main mechanism mediating signal transduction, modulation of other ion conductances by odorants has been postulated. To determine whether K+ conductances are modulated by odorants in mammalian ORNs, we examined the response of rat ORNs to odors by recording membrane current under perforated-patch conditions. We find that rat ORNs display two predominant types of responses. Thirty percent of the cells responded to odorants with activation of a CNG conductance. In contrast, in 55% of the ORNs, stimulation with odorants inhibited a voltage-activated K+ conductance (IKo). In terms of pharmacology, ion permeation, outward rectification, and time course for inactivation, IKo resembled a delayed rectifier K+ conductance. The effect of odorants on IKo was specific (only certain odorants inhibited IKo in each ORN) and concentration dependent, and there was a significant latency between arrival of odorants to the cell and the onset of suppression. These results indicate that indirect suppression of a K+ conductance (IKo) by odorants plays a role in signal transduction in mammalian ORNs.     

Dissecting the pathogenic mechanisms of mutations in the pore region of the human cone photoreceptor cyclic nucleotide‐gated channel

The CNGA3 gene encodes the A3 subunit of the cone photoreceptor cyclic nucleotide‐gated (CNG) channel, an essential component of the phototransduction cascade. Certain mutations in CNGA3 cause autosomal recessive achromatopsia, a retinal disorder characterized by severely reduced visual acuity, lack of color discrimination, photophobia, and nystagmus. We identified three novel mutations in the pore‐forming region of CNGA3 (L363P, G367V, and E376K) in patients diagnosed with achromatopsia. We assessed the expression and function of channels with these three new and two previously described mutations (S341P and P372S) in a heterologous HEK293 cell expression system using Western blot, subcellular localization on the basis of immunocytochemistry, calcium imaging, and patch clamp recordings. In this first comparative functional analysis of disease‐associated mutations in the pore of a CNG channel, we found impaired surface expression of S341P, L363P, and P372S mutants and reduced macroscopic currents for channels with the mutations S341P, G367V, and E376K. Calcium imaging and patch clamp experiments after incubation at 37°C revealed nonfunctional homo‐ and heteromeric channels in all five mutants, but incubation at 27°C combined with coexpression of the B3 subunit restored residual function of channels with the mutations S341P, G367V, and E376K. Hum Mutat 31:830–839, 2010. © 2010 Wiley‐Liss, Inc.     

Cyclic nucleotide-activated currents in cultured olfactory receptor neurons of the hawkmoth Manduca sexta

Moth pheromones cause rises in intracellular Ca(2+) concentrations that activate Ca(2+)-dependent cation channels in antennal olfactory receptor neurons. In addition, mechanisms of adaptation and sensitization depend on changes in cyclic nucleotide concentrations. Here, cyclic nucleotide-activated currents in cultured olfactory receptor neurons of the moth Manduca sexta are described, which share properties with currents through vertebrate cyclic nucleotide-gated channels. The cyclic nucleotide-activated currents of M. sexta carried Ca(2+) and monovalent cations. They were directly activated by cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), modulated by Ca(2+)/calmodulin, and inhibited by lanthanum. M. sexta cyclic nucleotide-activated currents developed in an all-or-none manner, which suggests that the underlying channels are coupled and act coordinately. At least one cAMP- and two cGMP-activated nonselective cation currents could be distinguished. Compared with the cAMP-activated current, both cGMP-activated currents appeared to conduct more Ca(2+) and showed a stronger down-regulation by Ca(2+)/calmodulin-dependent negative feedback. Furthermore, both cGMP-activated currents differed in their Ca(2+)-dependent inhibition. Thus M. sexta olfactory receptor neurons, like vertebrate sensory neurons, appear to express nonselective cyclic nucleotide-activated cation channels with different subunit compositions. Besides the nonselective cyclic nucleotide-activated cation currents, olfactory receptor neurons express a cAMP-dependent current. This current resembled a protein kinase-modulated low-voltage-activated Ca(2+) current.