Calcium/calmodulin modulation of olfactory and rod cyclic nucleotide-gated ion channels

Cyclic nucleotide-gated (CNG) ion channels mediate sensory transduction in olfactory sensory neurons and retinal photoreceptor cells. In these systems, internal calcium/calmodulin (Ca2+/CaM) inhibits CNG channels, thereby having a putative role in sensory adaptation. Functional differences in Ca2+/CaM-dependent inhibition depend on the different subunit composition of olfactory and rod CNG channels. Recent evidence shows that three subunit types (CNGA2, CNGA4, and CNGB1b) make up native olfactory CNG channels and account for the fast inhibition of native channels by Ca2+/CaM. In contrast, two subunit types (CNGA1 and CNGB1) appear sufficient to mirror the native properties of rod CNG channels, including the inhibition by Ca2+/CaM. Within CNG channel tetramers, specific subunit interactions also mediate Ca2+/CaM-dependent inhibition. In olfactory CNGA2 channels, Ca2+/CaM binds to an N-terminal region and disrupts an interaction between the N- and C-terminal regions, causing inhibition. Ca2+/CaM also binds the N-terminal region of CNGB1 subunits and disrupts an intersubunit, N- and C-terminal interaction between CNGB1 and CNGA1 subunits in rod channels. However, the precise N- and C-terminal regions that form these interactions in olfactory channels are different from those in rod channels. Here, we will review recent advances in understanding the subunit composition and the mechanisms and roles for Ca2+/CaM-dependent inhibition in olfactory and rod CNG channels.     

Mechanism of inhibition of cyclic nucleotide-gated ion channels by diacylglycerol

Cyclic nucleotide-gated (CNG) channels are critical components in the visual and olfactory signal transduction pathways, and they primarily gate in response to changes in the cytoplasmic concentration of cyclic nucleotides. We previously found that the ability of the native rod CNG channel to be opened by cGMP was markedly inhibited by analogues of diacylglycerol (DAG) without a phosphorylation reaction (Gordon, S.E., J. Downing-Park, B. Tam, and A.L. Zimmerman. 1995. Biophys. J. 69:409-417). Here, we have studied cloned bovine rod and rat olfactory CNG channels expressed in Xenopus oocytes, and have determined that they are differentially inhibited by DAG. At saturating [cGMP], DAG inhibition of homomultimeric (alpha subunit only) rod channels was similar to that of the native rod CNG channel, but DAG was much less effective at inhibiting the homomultimeric olfactory channel, producing only partial inhibition even at high [DAG]. However, at low open probability (P(o)), both channels were more sensitive to DAG, suggesting that DAG is a closed state inhibitor. The Hill coefficients for DAG inhibition were often greater than one, suggesting that more than one DAG molecule is required for effective inhibition of a channel. In single-channel recordings, DAG decreased the P(o) but not the single-channel conductance. Results with chimeras of rod and olfactory channels suggest that the differences in DAG inhibition correlate more with differences in the transmembrane segments and their attached loops than with differences in the amino and carboxyl termini. Our results are consistent with a model in which multiple DAG molecules stabilize the closed state(s) of a CNG channel by binding directly to the channel and/or by altering bilayer-channel interactions. We speculate that if DAG interacts directly with the channel, it may insert into a putative hydrophobic crevice among the transmembrane domains of each subunit or at the hydrophobic interface between the channel and the bilayer.     

Dynamics of Ca2+-calmodulin-dependent inhibition of rod cyclic nucleotide-gated channels measured by patch-clamp fluorometry

Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca(2+) ions. Rod CNG channels are directly inhibited by Ca(2+)-calmodulin (Ca(2+)/CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca(2+)/CaM-dependent modulation. The molecular basis for Ca(2+)/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca(2+)/CaM to a site in the NH(2)-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH(2)-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca(2+)/CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca(2+)/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH(2)- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca(2+)/CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH(2)- and COOH-terminal regions underlies Ca(2+)/CaM-dependent inhibition.     

Subunit interactions in the activation of cyclic nucleotide-gated ion channels.

Cyclic nucleotide-gated (CNG) ion channels of retinal photoreceptors and olfactory neurons are multimeric proteins of unknown stoichiometry. To investigate the subunit interactions that occur during CNG channel activation, we have used tandem cDNA constructs of the rod CNG channel to generate heteromultimeric channels composed of wild-type and mutant subunits. We introduced point mutations that affect channel activation: 1) D604M, which alters the relative ability of agonists to promote the allosteric conformational change(s) associated with channel opening, and 2) T560A, which primarily affects the initial binding affinity for cGMP, and to a lesser extent, the allosteric transition. At saturating concentrations of agonist, heteromultimeric channels were intermediate between wild-type and mutant homomultimers in agonist efficacy and apparent affinity for cGMP, cIMP, and cAMP, consistent with a model for the allosteric transition involving a concerted conformational change in all of the channel subunits. Results were also consistent with a model involving independent transitions in two or three, but not one or four, of the channel subunits. The behavior of the heterodimers implies that the channel stoichiometry is some multiple of 2 and is consistent with a tetrameric quaternary structure for the functional channel complex. Steady-state dose-response relations for homomultimeric and heteromultimeric channels were well fit by a Monod, Wyman, and Changeux model with a concerted allosteric opening transition stabilized by binding of agonist.     

An intersubunit interaction regulates trafficking of rod cyclic nucleotide-gated channels and is disrupted in an inherited form of blindness

A mutation in a cyclic nucleotide-gated channel (CNGA1) is associated with retinitis pigmentosa (RP), a common, inherited eye disease. Expression of mutant (CNGA1-RP) homomeric channels in Xenopus oocytes revealed no measurable differences compared to wild-type CNGA1 homomers. As native retinal rod CNG channels comprise CNGA1 and CNGB1 subunits, we coexpressed CNGA1-RP and CNGB1. Surprisingly, this subunit combination did not produce detectable channels at the membrane surface. We show that the mechanism underlying this defect involves an intersubunit interaction between CNGA1 and CNGB1 that was not formed between CNGA1-RP and CNGB1 subunits. In the absence of this interaction, a short N-terminal region in CNGB1 prevented membrane expression. Thus, disruption of a regulatory interaction by mutation in CNGA1 exposed a region of CNGB1 that disrupted surface expression of heteromeric CNGA1-RP/CNGB1 channels, accounting for this instance of RP.     

Subunit configuration of heteromeric cone cyclic nucleotide-gated channels

Cone photoreceptor cyclic nucleotide-gated (CNG) channels are thought to be tetrameric assemblies of CNGB3 (B3) and CNGA3 (A3) subunits. We have used functional and biochemical approaches to investigate the stoichiometry and arrangement of these subunits in recombinant channels. First, tandem dimers of linked subunits were used to constrain the order of CNGB3 and CNGA3 subunits; the properties of channels formed by B3/B3+A3/A3 dimers, or A3/B3+B3/A3 dimers, closely resembled those of channels arising from B3+A3 monomers. Functional markers in B3/B3 (or A3/A3) dimers confirmed that both B3 subunits (and both A3 subunits) gained membership into the pore-forming tetramer and that like subunits were positioned adjacent to each other. Second, chemical crosslinking and co-immunoprecipitation studies using epitope-tagged monomer subunits both demonstrated the presence of two CNGB3 subunits in cone channels. Together, these data support a preferred subunit arrangement for cone CNG channels (B3-B3-A3-A3) that is distinct from the 3A:1B configuration of rod channels.     

Interactions of cyclic nucleotide-gated channel subunits and protein tyrosine kinase probed with genistein

The cGMP sensitivity of cyclic nucleotide-gated (CNG) channels can be modulated by changes in phosphorylation catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases. Previously, we used genistein, a PTK inhibitor, to probe the interaction between PTKs and homomeric channels comprised of alpha subunits (RETalpha) of rod photoreceptor CNG channels expressed in Xenopus oocytes. We showed that in addition to inhibiting phosphorylation, genistein triggers a noncatalytic interaction between PTKs and homomeric RETalpha channels that allosterically inhibits channel gating. Here, we show that native CNG channels from rods, cones, and olfactory receptor neurons also exhibit noncatalytic inhibition induced by genistein, suggesting that in each of these sensory cells, CNG channels are part of a regulatory complex that contains PTKs. Native CNG channels are heteromers, containing beta as well as alpha subunits. To determine the contributions of alpha and beta subunits to genistein inhibition, we compared the effect of genistein on native, homomeric (RETalpha and OLFalpha), and heteromeric (RETalpha+beta, OLFalpha+beta, and OLFalpha+RETbeta) CNG channels. We found that genistein only inhibits channels that contain either the RETalpha or the OLFbeta subunits. This finding, along with other observations about the maximal effect of genistein and the Hill coefficient of genistein inhibition, suggests that the RETalpha and OLFbeta subunits contain binding sites for the PTK, whereas RETbeta and OLFalpha subunits do not.     

Purification and cloning of toxins from elapid venoms that target cyclic nucleotide-gated ion channels

In 1999, we purified pseudechetoxin (PsTx), the first peptide toxin known to block cyclic nucleotide-gated (CNG) ion channels, from the venom of Pseudechis australis [Brown, R. L., Haley, T. L., West, K. A., and Crabb, J. W. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 754-759]. Here we report the cloning of the cDNA encoding PsTx, as well as the discovery and cloning of pseudecin, a homologous toxin from the venom of Pseudechis porphyriacus. The mature proteins are 211 and 210 amino acids in length, and the amino acid sequences are 96.7% identical, differing in only seven residues. The purified toxins were applied to outside-out patches excised from Xenopus oocytes expressing CNG channels composed of the rod CNGA1 or olfactory CNGA2 channel subunits. Surprisingly, these patch-clamp studies revealed a 30-fold difference in affinity between PsTx and pseudecin for channels composed of CNGA2 subunits. The apparent K(i) of PsTx was 15 nM, while the affinity of pseudecin was 460 nM. The difference in affinities for the CNGA1 subunit from rod photoreceptors was less pronounced, but the affinity of PsTx was 70 nM, compared with 1000 nM for pseudecin. This difference in affinity may be instructive as we attempt to identify the regions of the toxins that contact CNG channels. As the only known protein blockers of CNG channels, these toxins promise to be valuable tools to study the structure of the external face of these channels.     

Functional activity of photoreceptor cyclic nucleotide-gated channels is dependent on the integrity of cholesterol- and sphingolipid-enriched membrane domains

Rod and cone photoreceptor cyclic nucleotide-gated (CNG) channels play pivotal roles in phototransduction. This work investigates the functional significance of photoreceptor CNG channel association with membrane microdomains enriched in raft lipids, cholesterol and sphingolipids. The primary subunits of cone and rod CNG channels, CNGA3 and CNGA1, respectively, were heterologously expressed in HEK 293 cells, and channel activity was determined by ratiometric measurement of [Ca (2+)] i in response to cyclic guanosine monophosphate (cGMP) stimulation. CNGA3 was found to be largely insoluble following Triton X-100 extraction and cofractionationed with biochemically isolated membrane domains enriched in caveolin-1. Cofractionation of both natively expressed CNGA3 and CNGB1 (the modulatory subunit of the rod CNG channel) with the low buoyant density, caveolin-1-enriched membranes was also confirmed in mouse retinas. The functional significance of this association was established by the observed negative effects of depletion of raft lipids on the channel activity. Treatment with the cholesterol depleting agent, methyl-beta-cyclodextrin (MCD), significantly inhibited CNGA3 and CNGA1 activation in response to cGMP stimulation. MCD treatment lowered cellular cholesterol levels by approximately 45% without altering fatty acid composition, suggesting that the inhibition of channel activity by MCD treatment is not due to perturbation of other membrane lipids. Treatment with the sphingolipid biosynthesis inhibitor myriocin resulted in impaired activation and cytosolic redistribution of CNGA3, suggesting that the integrity of the membrane domains is critical for the channel cellular processing and plasma membrane localization. This study demonstrates the association of photoreceptor CNG channels with membrane domains enriched in raft lipids and indicates, for the first time, that raft lipids modulate the plasma membrane localization and functional activity of photoreceptor CNG channels.     

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

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

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

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

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

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

Multimerization of the ligand binding domains of cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels comprise four subunits and are activated by the direct binding of cyclic nucleotide to an intracellular domain on each subunit. This ligand binding domain is thought to contain a beta roll followed by two alpha helices, designated the B and C helices. To examine the quaternary structure of CNG channels and how it changes during ion channel gating, we introduced single cysteines along the C helix of each subunit in an otherwise cysteineless channel. We found that cysteines on the C helices could form intersubunit disulfide bonds, even between diagonal subunits. Disulfide bond formation occurred primarily in closed channels and inhibited channel opening. These data suggest that the C helices from all four channel subunits are in close proximity in the closed state and move apart during channel opening.     

Accessibility of cysteines in the native bovine rod cGMP-gated channel

Cyclic nucleotide-gated channels of photoreceptors and olfactory sensory neurons are tetramers consisting of A and B subunits. Here, the accessibility of the cysteines of the bovine rod cyclic nucleotide-gated channel is examined as a function of ligand binding. N-Ethylmaleimide-modified cysteines of both subunits were identified by mass spectrometry after trypsin digestion. In the absence of ligand, the intracellular carboxy-terminal cysteines of both subunits were accessible to N-ethylmaleimide. Activation of the channel abolished the accessibility of Cys505 of the A subunit and Cys1104 of the B subunit, with both being conserved cysteines of the cyclic nucleotide-binding sites. The cysteine of the pore loop of the B subunit was also found to be modified by this reagent in the absence of ligand. The total number of accessible cysteines of each subunit was determined by mass shifting upon modification with polyethylene glycol maleimide. In the absence of cyclic nucleotides, this hydrophilic reagent only weakly labeled cysteines of the A subunit but readily labeled at least three cysteines of the B subunit. Ligand binding exposed two cysteines of the A subunit and one cysteine of the B subunit to chemical modification. Double-modification experiments suggest that some of these cysteines are in or close to membrane-spanning domains. However, these cysteines could not yet be identified. Together, the cysteine accessibility of the native rod cyclic nucleotide-gated channel varies markedly upon ligand binding, thus indicating major structural rearrangements, which are of functional importance for channel activation.     

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

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

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

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

Ciliary targeting of olfactory CNG channels requires the CNGB1b subunit and the kinesin-2 motor protein, KIF17

Nonmotile cilia on olfactory sensory neurons (OSNs) compartmentalize signaling molecules, including odorant receptors and cyclic nucleotide-gated (CNG) channels, allowing for efficient, spatially confined responses to sensory stimuli . Little is known about the mechanisms of the ciliary targeting of olfactory CNG channels, composed of three subunits: CNGA2, CNGA4, and CNGB1b . Recent reports suggest that subunit composition of the retinal CNG channel influences localization, leading to disease . However, the mechanistic role of subunits in properly targeting native olfactory CNG channels remains unclear. Here, we show that heteromeric assembly with CNGB1b, containing a critical carboxy-terminal motif (RVxP), is required for ciliary trafficking of olfactory CNG channels. Movement of proteins within the cilia is governed by intraflagellar transport (IFT), a process that facilitates bidirectional movement of cargo along microtubules. Work in C. elegans has established that heterotrimeric and homodimeric kinesin-2 family members play a critical role in anterograde transport . In mammalian systems, the heterotrimeric KIF3a/KIF3b/KAP-3 complex plays a clear role in IFT; however, no role has been established for KIF17, the mammalian homolog of OSM-3 . Here, we demonstrate that KIF17 is required for olfactory CNG channel targeting, providing novel insights into mechanisms of mammalian ciliary transport.     

Gene replacement therapy for retinal CNG channelopathies

Visual phototransduction relies on the function of cyclic nucleotide-gated channels in the rod and cone photoreceptor outer segment plasma membranes. The role of these ion channels is to translate light-triggered changes in the second messenger cyclic guanosine 3′–5′-monophosphate levels into an electrical signal that is further processed within the retinal network and then sent to higher visual centers. Rod and cone photoreceptors express distinct CNG channels. The rod photoreceptor CNG channel is composed of one CNGB1 and three CNGA1 subunits, whereas the cone channel is formed by one CNGB3 and three CNGA3 subunits. Mutations in any of these channel subunits result in severe and currently untreatable retinal degenerative diseases like retinitis pigmentosa or achromatopsia. In this review, we provide an overview of the human diseases and relevant animal models of CNG channelopathies. Furthermore, we summarize recent results from preclinical gene therapy studies using adeno-associated viral vectors and discuss the efficacy and translational potential of these gene therapeutic approaches.     

Odorant inhibition of the olfactory cyclic nucleotide-gated channel with a native molecular assembly

Human olfaction comprises the opposing actions of excitation and inhibition triggered by odorant molecules. In olfactory receptor neurons, odorant molecules not only trigger a G-protein-coupled signaling cascade but also generate various mechanisms to fine tune the odorant-induced current, including a low-selective odorant inhibition of the olfactory signal. This wide-range olfactory inhibition has been suggested to be at the level of ion channels, but definitive evidence is not available. Here, we report that the cyclic nucleotide-gated (CNG) cation channel, which is a key element that converts odorant stimuli into electrical signals, is inhibited by structurally unrelated odorants, consistent with the expression of wide-range olfactory inhibition. Interestingly, the inhibitory effect was small in the homo-oligomeric CNG channel composed only of the principal channel subunit, CNGA2, but became larger in channels consisting of multiple types of subunits. However, even in the channel containing all native subunits, the potency of the suppression on the cloned CNG channel appeared to be smaller than that previously shown in native olfactory neurons. Nonetheless, our results further showed that odorant suppressions are small in native neurons if the subsequent molecular steps mediated by Ca(2+) are removed. Thus, the present work also suggests that CNG channels switch on and off the olfactory signaling pathway, and that the on and off signals may both be amplified by the subsequent olfactory signaling steps.