Modulation of the Olfactory CNG Channel by Ptdlns(3,4,5)P<Subscript>3</Subscript>

Recent data suggest that the 3-phosphoinositides can modulate cyclic nucleotide signaling in rat olfactory receptor neurons (ORNs). Given the ability of diverse lipids to modulate ion channels, we asked whether phosphatidylinositol 3,4,5-trisphosphate (PIP₃) can regulate the olfactory cyclic nucleotide-gated (CNG) channel as a possible mechanism for this modulation. We show that applying PIP₃ to the intracellular side of inside-out patches from rat ORNs inhibits activation of the olfactory CNG channel by cAMP. The effect of PIP₃ is immediate and partially reversible, and reflects an increase in the EC₅₀ of cAMP, not a reduction in the single-channel current amplitude. The effect of PIP₃ is significantly stronger than that of PIP₂; other phospholipids tested have no appreciable effect on channel activity. PIP₃ similarly inhibits the recombinant heteromeric (A2/A4) and homomeric (A2) olfactory CNG channel expressed in HEK293 cells, suggesting that PIP₃ acts directly on the channel. These findings indicate that 3-phosphoinositides can be functionally important regulators of CNG channels.     

Flash photolysis of caged compounds in the cilia of olfactory sensory neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP or caged Ca. We use a flash lamp to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration.     

Odorant-induced responses recorded from olfactory receptor neurons using the suction pipette technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert & Zhao, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca(2+)-activated Cl(-) channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body. In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors. The suction pipette technique was later adapted to also record from ORNs. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.     

Cyclic nucleotide-gated ion channels in sensory transduction

Cyclic nucleotide-gated (CNG) channels, directly activated by the binding of cyclic nucleotides, were first discovered in retinal rods, cones and olfactory sensory neurons. In the visual and olfactory systems, CNG channels mediate sensory transduction by conducting cationic currents carried primarily by sodium and calcium ions. In olfactory transduction, calcium in combination with calmodulin exerts a negative feedback on CNG channels that is the main molecular mechanism responsible for fast adaptation in olfactory sensory neurons. Six mammalian CNG channel genes are known and some human visual disorders are caused by mutations in retinal rod or cone CNG genes.     

Mechanism of the excitatory Cl- response in mouse olfactory receptor neurons

In vertebrate olfactory receptor neurons (ORNs), the odorant-triggered receptor current flows through two distinct ion channels on the sensory cilia: Ca2+ influx through a cyclic nucleotide-gated (CNG) channel followed by Cl- efflux through a Ca2+-activated anion channel. The excitatory Cl- current amplifies the small CNG current and crucially depends on a high intracellular Cl- concentration. We show here that a (Na+)-(K+)-(2Cl-) cotransporter, NKCC1, is required for this Cl- current, in that ORNs deficient in Nkcc1 or incubated with an NKCC blocker (bumetanide) lack the Cl- current. Surprisingly, immunocytochemistry indicates that NKCC1 is located on the somata and dendrites of ORNs rather than the cilia, where transduction occurs. This topography is remarkably similar to the situation in secretory epithelial cells, where basolateral Cl- uptake and apical Cl- efflux facilitate transepithelial fluid movement. Thus, a single functional architecture serves two entirely different purposes, probably underscoring the epithelial origin of the ORNs.     

Regulation of cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels are found in several cell types, and are best studied in photoreceptors and olfactory sensory neurons. There, CNG channels are gated by the second messengers of the visual and olfactory signalling cascades, cGMP and cAMP respectively, and operate as transduction channels generating the stimulus-induced receptor potentials. In visual and olfactory sensory cells CNG channels conduct cationic currents. Calcium can contribute a large fraction of this current, and calcium influx serves a modulatory role in CNG-channel mediated signal transduction. There have been recent developments in our understanding of how the regulation of CNG channels contributes to the physiological properties of photoreceptors and olfactory sensory cells, and in particular on the role of calcium-mediated feedback.     

The electrochemical basis of odor transduction in vertebrate olfactory cilia

Most vertebrate olfactory receptor neurons share a common G-protein-coupled pathway for transducing the binding of odorant into depolarization. The depolarization involves 2 currents: an influx of cations (including Ca2+) through cyclic nucleotide-gated channels and a secondary efflux of Cl- through Ca2+-gated Cl- channels. The relation between stimulus strength and receptor current shows positive cooperativity that is attributed to the channel properties. This cooperativity amplifies the responses to sufficiently strong stimuli but reduces sensitivity and dynamic range. The odor response is transient, and prolonged or repeated stimulation causes adaptation and desensitization. At least 10 mechanisms may contribute to termination of the response; several of these result from an increase in intraciliary Ca2+. It is not known to what extent regulation of ionic concentrations in the cilium depends on the dendrite and soma. Although many of the major mechanisms have been identified, odor transduction is not well understood at a quantitative level.     

Molecular cloning and single-channel properties of the cyclic nucleotide-gated channel from catfish olfactory neurons.

We have cloned a functional cDNA encoding the cyclic nucleotide-gated channel selectively expressed in catfish olfactory sensory neurons. The cyclic nucleotide-gated channels share sequence and structural features with the family of voltage-gated ion channels. This homology is most evident in transmembrane region S4, the putative voltage sensor domain, and the H5 domain, thought to form the channel pore. We have characterized the single-channel properties of the cloned catfish channel and compared these properties with the channel in native catfish olfactory sensory neurons. The channel is activated equally well by cAMP and cGMP, shows only a slight voltage dependence of gating, and exhibits a pH- and voltage-dependent subconductance state similar to that observed for the voltage-gated L-type calcium channel.     

Odorant receptors activated by amino acids in sensory neurons of the channel catfish Ictalurus punctatus

Odorant receptors activated by amino acids were investigated with patch- clamp techniques in olfactory receptor neurons of the channel catfish, Ictalurus punctatus. The L-isomers of alanine, norvaline, arginine, and glutamate, known to act predominantly on different olfactory receptor sites, activated nondesensitizing inward currents with amplitudes of - 2.5 to -280 pA in olfactory neurons voltage-clamped at membrane potentials of -72 or -82 mV. Different amino acids were shown to induce responses in the same sensory neurons; however, the amplitude and the kinetics of the observed whole cell currents differed among the stimuli and may therefore reflect activation of different amino acid receptor types or combinations of receptor types in these cells. Amino acid- induced currents appeared to have diverse voltage dependence and could also be classified according to the amplitude of the spontaneous channel fluctuations underlying the macroscopic currents. A mean single- channel conductance (gamma) of 360 fS was estimated from small noise whole-cell currents evoked by arginine within the same olfactory neuron in which a mean gamma value of 23.6 pS was estimated from ''large noise'' response to norvaline. Quiescent olfactory neurons fired bursts of action potentials in response to either amino acid stimulation or application of 8-Br-cyclic GMP (100 microM), and voltage-gated channels underlying generation of action potentials were similar in these neurons. However, in whole-cell voltage-clamp, 8-Br-cyclic GMP evoked large rectangular current pulses, and single-channel conductances of 275, 220, and 110 pS were obtained from the discrete current levels. These results suggest that in addition to the cyclic nucleotide-gated transduction channels, olfactory neurons of the channel catfish possess a variety of odor receptors coupled to different types of transduction channels.     

The Ca-activated Cl channel and its control in rat olfactory receptor neurons

Odorants activate sensory transduction in olfactory receptor neurons (ORNs) via a cAMP-signaling cascade, which results in the opening of nonselective, cyclic nucleotide-gated (CNG) channels. The consequent Ca2+ influx through CNG channels activates Cl channels, which serve to amplify the transduction signal. We investigate here some general properties of this Ca-activated Cl channel in rat, as well as its functional interplay with the CNG channel, by using inside-out membrane patches excised from ORN dendritic knobs/cilia. At physiological concentrations of external divalent cations, the maximally activated Cl current was approximately 30 times as large as the CNG current. The Cl channels on an excised patch could be activated by Ca2+ flux through the CNG channels opened by cAMP. The magnitude of the Cl current depended on the strength of Ca buffering in the bath solution, suggesting that the CNG and Cl channels were probably not organized as constituents of a local transducisome complex. Likewise, Cl channels and the Na/Ca exchanger, which extrudes Ca2+, appear to be spatially segregated. Based on the theory of buffered Ca2+ diffusion, we determined the Ca2+ diffusion coefficient and calculated that the CNG and Cl channel densities on the membrane were approximately 8 and 62 micro m-2, respectively. These densities, together with the Ca2+ diffusion coefficient, demonstrate that a given Cl channel is activated by Ca2+ originating from multiple CNG channels, thus allowing low-noise amplification of the olfactory receptor current.     

Calcium Regulation of Cyclic Nucleotide Signaling in Lobster Olfactory Receptor Neurons

An elevated free Ca2+ concentration reduces odor-stimulated production of cyclic AMP (cAMP) in the outer dendritic membranes of lobster olfactory receptor neurons in vitro. This effect can occur within 50 ms of odor stimulation. The effect is concentration-dependent at submicromolar concentrations of free Ca2+. An elevated free Ca2+ concentration also reduces basal and forskolin-stimulated cAMP levels in a concentration-dependent manner, suggesting that Ca2+ is not targeting the activation of the odor receptor/G protein complex. The degradation of synthetic cAMP by phosphodiesterases is not enhanced by an increased free Ca2+ concentration, suggesting that Ca2+ acts by down-regulating the olfactory adenylyl cyclase. Western blot analysis of the lobster olfactory sensilla that contain the outer dendrites reveals a protein in the transduction zone with a molecular mass of approximately 138 kDa that is immunoreactive to an antiserum against adenylyl cyclase type III. Given earlier evidence that Ca2+ potentially enters the receptor cell through odor-activated inositol 1,4,5-trisphosphate-gated channels, our results suggest a possible route for cross talk between the cyclic nucleotide and the inositol phospholipid signaling pathways in lobster olfactory receptor neurons.     

Calcium,the two-faced messenger of olfactory transduction and adaptation

Exposure of olfactory receptor cells to odour stimulates the influx of Ca(2+) through cyclic nucleotide-gated channels into the small volume within the cilia, the site of olfactory transduction. The consequent rise in intraciliary Ca(2+) concentration has two opposing effects: activation of an unusual excitatory Cl(-) conductance, and negative feedback actions on various stages of the odour transduction mechanism. Recent studies are beginning to unravel how Ca(2+) performs this dual function, and how the spatial and temporal dynamics of Ca(2+) modulate the odour response. The feedback actions of Ca(2+) on different elements of the transduction cascade seem to occur on different timescales, and are therefore responsible for shaping different parts of the receptor current response to odour stimulation.     

Nitric Oxide Inhibition of the Rat Olfactory Cyclic Nucleotide-Gated Cation Channel

The effects of nitric oxide (NO) and other cysteine modifying agents were examined on cyclic nucleotide-gated (CNG) cation channels from rat olfactory receptor neurons. The NO compounds, S-nitroso-cysteine (SNC) and 3-morpholino-sydnonomine (SIN-1), did not activate the channels when applied for up to 10 min. The cysteine alkylating agent, N-ethylmaleimide (NEM), and the oxidising agent, dithionitrobensoate (DTNB), were also without agonist efficacy. Neither SNC nor DTNB altered the cAMP sensitivity of the channels. However, 2-min applications of SIN-1, SNC and DTNB inhibited the cAMP-gated current to approximately 50% of the control current level. This inhibition showed no spontaneous reversal for 5 min but was completely reversed by a 2-min exposure to DTT. The presence of cAMP protected the channels against NO-induced inhibition. These results indicate that inhibition is caused by S-nitrosylation of neighboring sulfhydryl groups leading to sulfhydryl bond formation. This reaction is favored in the closed channel state. Since recombinantly expressed rat olfactory α and β CNG channel homomers and α/β heteromers are activated and not inhibited by cysteine modification, the results of this study imply the existence of a novel subunit or tightly bound factor which dominates the effect of cysteine modification in the native channels. As CNG channels provide a pathway for calcum influx, the results may also have important implications for the physiological role of NO in mammalian olfactory receptor neurons. Received: 30 March 1998/Revised: 17 June 1998     

Presynaptic cyclic nucleotide-gated ion channels modulate neurotransmission in the mammalian olfactory bulb

Cyclic nucleotide-gated channels (CNGCs) on the dendritic cilia of olfactory receptor neurons (ORNs) are critical for sensory transduction in the olfactory system. Do CNGCs also play a role in the axons and/or nerve terminals of ORNs? We find that the cyclic nucleotides cAMP and cGMP can both facilitate and depress synaptic transmission between olfactory nerve fibers and their targets in olfactory bulb glomeruli. Cyclic nucleotides increase intracellular Ca(2+) in ORN terminals and enhance spontaneous transmitter release; at higher concentrations, cyclic nucleotides depress evoked transmission by altering olfactory nerve excitability. Cyclic nucleotides have no effect on transmission or nerve excitability, however, in mice lacking olfactory CNGCs. Taken together, our results identify a novel role for presynaptic CNGCs in modulating neurotransmission.     

Ca2+-dependent K+ channels from rat olfactory cilia characterized in planar lipid bilayers

Olfactory cilia contain cyclic nucleotide-gated and Ca2+-dependent Cl- conductances that underlie excitatory chemotransduction, and a Ca2+-dependent K+ (KCa) conductance, apparently involved in inhibitory transduction. Previous single-channel patch-clamp studies on olfactory cilia revealed four different KCas, with different conductances and kinetics. Here, we further characterized these channels in planar bilayers, where blockers could be properly tested. All four ciliary KCas were observed: The 16 pS channel, K0.5,Ca=40 microM and apamin-sensitive; the 30 and 50 pS channel, K0.5,Ca=59 microM, clotrimazole-sensitive and charybdotoxin-insensitive; the 60 pS channel, clotrimazole-sensitive and charybdotoxin-insensitive; and the 210 pS channel, K0.5,Ca=63 microM, blocked by charybdotoxin and iberiotoxin. The presence of the 16 and 210 pS channels was confirmed by immunoblotting.     

Distinct binding properties distinguish LQ-type calmodulin-binding domains in cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels operate as transduction channels in photoreceptors and olfactory receptor neurons. Direct binding of cGMP or cAMP opens these channels which conduct a mixture of monovalent cations and Ca(2+). Upon activation, CNG channels generate intracellular Ca(2+) signals that play pivotal roles in the transduction cascades of the visual and olfactory systems. Channel activity is controlled by negative feedback mechanisms that involve Ca(2+)-calmodulin, for which all CNG channels possess binding sites. Here we compare the binding properties of the two LQ-type calmodulin binding sites, both of which are thought to be involved in channel regulation. They reside on the isoforms CNGB1 and CNGA4. The CNGB1 subunit is present in rod photoreceptors and olfactory receptor neurons. The CNGA4 subunit is only expressed in olfactory receptor neurons, and there are conflicting results as to its role in calmodulin-mediated feedback inhibition. We examined the interaction of Ca(2+)-calmodulin with two recombinant proteins that encompass either of the two LQ sites. Comparing binding properties, we found that the LQ site of CNGB1 binds Ca(2+)-calmodulin at 10-fold lower Ca(2+) levels than the LQ site of CNGA4. Our data provide biochemical evidence against a contribution of CNGA4 to feedback inhibition. In accordance with previous work on photoreceptor CNG channels, our results indicate that feedback control is the exclusive role of the B-subunits in photoreceptors and olfactory receptor neurons.     

Cyclic nucleotide-activated channels in the frog olfactory receptor plasma membrane.

Patch clamp technique was used to record cyclic nucleotide-dependent current of the frog olfactory receptor cell plasma membrane. Data obtained indicate that the channels passing this current are permeable to Ca2+ or Mg2+ and moderately selective for monovalent cations according to the sequence Li+, Na+, K+ greater than Rb+ greater than Cs+ and are effectively blocked by 1-cis-diltiazem and 3',4'-dichlorobenzamil. The conductance of single cyclic nucleotide-gated channels in solutions with low Ca2+ and Mg2+ content is about 19 pS. The results demonstrate that cyclic nucleotide-activated channels of olfactory receptor cells are virtually identical to photoreceptor ones.     

Search for new cyclic AMP-binding proteins

Today, there is evidence that the cAMP-dependent kinases (PKA) are not the only intracellular receptors involved in intracellular cAMP signalling in eukaryotes. Other cAMP-binding proteins have been recently identified, including some cyclic nucleotide-gated channels and Epac (exchange protein directly activated by cAMP) proteins. All these proteins bind cAMP through conserved cyclic nucleotide monophosphate-binding domains. However, all putative cAMP-binding proteins having such domains, as revealed by computer analysis, do not necessarily bind cAMP, indicating that their presence is not a sufficient criteria to predict cAMP-binding property for a protein.     

Intracellular Ca2+ regulates the sensitivity of cyclic nucleotide-gated channels in olfactory receptor neurons.

In olfactory receptor neurons, odorants stimulate production of cAMP, which directly activates cyclic nucleotide-gated (CNG) channels. Olfactory adaptation is thought to result from a rise in intracellular Ca2+. To determine whether inhibition of CNG channels plays a role in adaptation, we have investigated the action of Ca2+ on these channels in inside-out "macro" patches from the dendrite and cilia of catfish olfactory neurons. Internal Ca2+, with a K1/2 of 3 microM, profoundly inhibits CNG channels by shifting the dose-response relationship to higher cAMP levels without altering the maximal response. The inhibition does not appear to result from a direct interaction of Ca2+ with the CNG channel. Thus, the inhibition washes out after excision of the inside-out patch, and Ca2+ does not inhibit the cloned catfish CNG channel expressed in Xenopus oocytes. Hence we propose that a regulatory Ca(2+)-binding protein, distinct from the CNG channel, controls the gain of signal transduction and contributes to olfactory adaptation by decreasing the sensitivity of the CNG channel to cAMP.     

Inositol-trisphosphate-dependent calcium currents precede cation currents in insect olfactory receptor neurons in vitro

Specialized olfactory receptor neurons in insects respond to species-specific sex pheromones with transient rises in inositol trisphosphate and by opening pheromone-dependent cation channels. These channels resemble cation channels which are directly or indirectly Ca²⁺-dependent. But there appear to be no internal Ca²⁺ stores in the outer dendrite where the olfactory transduction cascade is thought to start. Hence, it remains to be determined whether an influx of external Ca²⁺ precedes pheromone-dependent cation currents. Patch clamp measurements in cultured olfactory receptor neurons from Manduca sexta reveal that a transient inward current precedes pheromone-dependent cation currents. A transient inositol trisphosphate-dependent Ca²⁺ current, also preceding cation currents with the characteristics of pheromone-dependent cation currents, shares properties with the transient pheromone-dependent current. These results match the biochemical measurements with the electrophysiological data obtained in insect olfactory receptor neurons.Abbreviations ORNs Olfactory receptor neurons - IP₃ Inositol-1,4,5-trisphosphate - I_t Transient pheromone-dependent current - I_ir Transient IP₃-dependent current