Clustering of cyclic-nucleotide-gated channels in olfactory cilia

Olfactory cilia contain the known components of olfactory signal transduction, including a high density of cyclic-nucleotide-gated (CNG) channels. CNG channels play an important role in mediating odor detection. The channels are activated by cAMP, which is formed by a G-protein-coupled transduction cascade. Frog olfactory cilia are 25-200 microm in length, so the spatial distribution of CNG channels along the length should be important in determining the sensitivity of odor detection. We have recorded from excised cilia and modeled diffusion of cAMP into a cilium to determine the spatial distribution of the CNG channels along the ciliary length. The proximal segment, which in frog is the first 20% of the cilium, appears to express a small fraction of the CNG channels, whereas the distal segment contains the majority, mostly clustered in one region.     

Transmembrane currents in frog olfactory cilia

Summary We have measured transmembrane currents in intact single cilia from frog olfactory receptor neurons. A single cilium on a neuron was sucked into a patch pipette, and a high-resistance seal was formed near the base of the cilium. Action potentials could be induced by applying suction or a voltage ramp to the ciliary membrane. A transient current was seen in some cells on stimulation with odorants. After excision from the cell, most of the cilia showed increased conductance in a bath containing cAMP, indicating that the cytoplasmic face of the ciliary membrane was accessible to the bath. The estimated resistance of a single cilium was surprisingly low.     

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.     

Noise analysis of ion channels in non-space-clamped cables: estimates of channel parameters in olfactory cilia.

Ion channels in the cilia of olfactory neurons are part of the transduction machinery of olfaction. Odorant stimuli have been shown to induce a biphasic current response, consisting of a cAMP-activated current and a Ca(2+)-activated Cl- current. We have developed a noise analysis method to study ion channels in leaky cables, such as the olfactory cilium, under non-space-clamp conditions. We performed steady-state noise analysis on ligand-induced currents in excised cilia, voltage-clamped at input and internally perfused with cAMP or Ca2+. The cAMP-activated channels analyzed by this method gave results similar to those of single-channel recordings (gamma = 8.3 pS). Single-channel currents have not yet been recorded for the Ca(2+)-activated Cl- channels. Using our noise analysis method, we estimate a unit conductance, gamma = 0.8 pS, for these channels. The density of channels was found to be approximately 70 channels/micron2 for both channel species.     

Bovine olfactory cilia preparation: thiol-modulated odorant-sensitive adenylyl cyclase

We have characterized the adenylyl cyclase activity in a newly developed preparation of isolated olfactory cilia from the bovine chemosensory neuroepithelium. Like its counterparts from frog and rat, the ciliary enzyme was stimulated by guanine nucleotides, by forskolin, and by a variety of odorants in the presence of GTP. The main difference between the bovine olfactory cilia preparation and the frog and rat olfactory cilia preparation is that odorant stimulation of the bovine olfactory adenylyl cyclase is strongly inhibited by submillimolar concentrations of dithiothreitol. This inhibition is a consequence of a concomitant increase in the GTP-stimulated level and the decrease of the odorant stimulation of the enzyme. Nasal respiratory cilia have a much lower level of adenylyl cyclase activity and show no odorant stimulation. Owing to the large quantities of material available, the bovine olfactory cilia preparation is advantageous for studies of the proteins involved in chemosensory transduction.     

Gated conductances in native and reconstituted membranes from frog olfactory cilia.

Although cAMP is well established as a second messenger for olfactory transduction in vertebrates, the role of inositol 1,4,5-trisphosphate (IP3) in this process remains controversial. We addressed this issue by comparing currents evoked by cAMP and IP3 in native and reconstituted membranes from olfactory cilia. We detected only a cyclic nucleotide-gated conductance in the native membrane but both cyclic nucleotide-gated and IP3-gated conductances in the reconstituted membrane. The magnitudes of the cyclic nucleotide- and IP3-gated conductances were not correlated with each other in reconstituted membranes, suggesting that cyclic nucleotide- and IP3-gated channels originate in different cellular compartments.     

Odorant-induced kinetics of Ca<Superscript>2+</Superscript>, NADH,and oxidized flavoproteins in frog olfactory mucosa

A study was made of the odorant-induced changes in the fluorescence of the Ca²⁺-chlortetracycline-membrane complex, NADH, and oxidized flavoproteins in the frog olfactory epithelium. Cineole and vanillin induce faster changes than camphor and pentanol. The different kinetics of NADH and membrane calcium evoked by these odorants are attributed to the heterogeneity of the molecular mechanisms involved in olfactory signal transduction. By contrast, ammonia and β-mercaptoethanol permeate the olfactory cells and without second messengers inhibit the mitochondrial respiratory chain and suppress the motility of olfactory cilia.     

Spatial distribution of calcium-gated chloride channels in olfactory cilia

Background

In vertebrate olfactory receptor neurons, sensory cilia transduce odor stimuli into changes in neuronal membrane potential. The voltage changes are primarily caused by the sequential openings of two types of channel: a cyclic-nucleotide-gated (CNG) cationic channel and a calcium-gated chloride channel. In frog, the cilia are 25 to 200 µm in length, so the spatial distributions of the channels may be an important determinant of odor sensitivity.

Principal Findings

To determine the spatial distribution of the chloride channels, we recorded from single cilia as calcium was allowed to diffuse down the length of the cilium and activate the channels. A computational model of this experiment allowed an estimate of the spatial distribution of the chloride channels. On average, the channels were concentrated in a narrow band centered at a distance of 29% of the ciliary length, measured from the base of the cilium. This matches the location of the CNG channels determined previously. This non-uniform distribution of transduction proteins is consistent with similar findings in other cilia.

Conclusions

On average, the two types of olfactory transduction channel are concentrated in the same region of the cilium. This may contribute to the efficient detection of weak stimuli.     

Cyclic GMP diffusion coefficient in rod photoreceptor outer segments.

Cyclic GMP (cGMP) is the intracellular messenger that mediates phototransduction in retinal rods. As photoisomerizations of rhodopsin molecules are local events, the longitudinal diffusion of cGMP in the rod outer segment should be a contributing factor to the response of the cell to light. We have employed the truncated rod outer segment preparation from bullfrog (Rana catesbeiana) and tiger salamander (Ambystoma tigrinum) to measure the cGMP diffusion coefficient. In this preparation, the distal portion of a rod outer segment was drawn into a suction pipette for measuring membrane current, and the rest of the cell was then sheared off with a glass probe, allowing bath cGMP to diffuse into the outer segment and activate the cGMP-gated channels on the surface membrane. Addition and removal of bath cGMP were fast enough to produce effectively step changes in cGMP concentration at the open end of the outer segment. When cGMP hydrolysis is inhibited by isobutylmethylxanthine (IBMX), the equation for the diffusion of cGMP inside the truncated rod outer segment has a simple analytical solution, which we have used to analyze the rise and decay kinetics of the cGMP-elicited currents. From these measurements we have obtained a cGMP diffusion coefficient of approximately 70 x 10(-8) cm2 s-1 for bullfrog rods and approximately 60 x 10(-8) cm2 s-1 for tiger salamander rods. These values are six to seven times lower than the expected value in aqueous solution. The estimated diffusion coefficient is the same at high (20-1000 microM) and low (5-10 microM) concentrations of cGMP, suggesting no significant effect from buffering over these concentration ranges.     

The proteome of rat olfactory sensory cilia

Olfactory sensory neurons expose to the inhaled air chemosensory cilia which bind odorants and operate as transduction organelles. Odorant receptors in the ciliary membrane activate a transduction cascade which uses cAMP and Ca²⁺ for sensory signaling in the ciliary lumen. Although the canonical transduction pathway is well established, molecular components for more complex aspects of sensory transduction, like adaptation, regulation, and termination of the receptor response have not been systematically identified. Moreover, open questions in olfactory physiology include how the cilia exchange solutes with the surrounding mucus, assemble their highly polarized set of proteins, and cope with noxious substances in the ambient air. A specific ciliary proteome would promote research efforts in all of these fields. We have improved a method to detach cilia from rat olfactory sensory neurons and have isolated a preparation specifically enriched in ciliary membrane proteins. Using LC‐ESI‐MS/MS analysis, we identified 377 proteins which constitute the olfactory cilia proteome. These proteins represent a comprehensive data set for olfactory research since more than 80% can be attributed to the characteristic functions of olfactory sensory neurons and their cilia: signal processing, protein targeting, neurogenesis, solute transport, and cytoprotection. Organellar proteomics thus yielded decisive information about the diverse physiological functions of a sensory organelle.     

Proteomic analysis of a membrane preparation from rat olfactory sensory cilia

The cilia of mammalian olfactory receptor neurons (ORNs) represent the sensory interface that is exposed to the air within the nasal cavity. The cilia are the site where odorants bind to specific receptors and initiate olfactory transduction that leads to excitation of the neuron. This process involves a multitude of ciliary proteins that mediate chemoelectrical transduction, amplification, and adaptation of the primary sensory signal. Many of these proteins were initially identified by their enzymatic activities using a membrane protein preparation from olfactory cilia. This so-called "calcium-shock" preparation is a versatile tool for the exploration of protein expression, enzyme kinetics, regulatory mechanisms, and ciliary development. To support such studies, we present a first proteomic analysis of this membrane preparation. We subjected the cilia preparation to liquid chromatography-electrospray ionisation (LC-ESI-MS/MS) tandem mass spectrometry and identified 268 proteins, of which 49% are membrane proteins. A detailed analysis of their cellular and subcellular localization showed that the cilia preparation obtained by calcium shock not only is highly enriched in ORN proteins but also contains a significant amount of nonciliary material. Although our proteomic study does not identify the entire set of ciliary and nonciliary proteins, it provides the first estimate of the purity of the calcium-shock preparation and provides valuable biochemical information for further research.     

The participation of the cAMP intracellular signal system in the olfactory transduction of camphor and amyl alcohol]

In the frog isolated olfactory epithelium, the cAMP intracellular signal system was shown to take part in olfactory transduction of amyl alcohol but not camphor.     

Kinetics of Ca2+, NADH, and oxidized flavoproteins in the frog olfactory lining under the effect of odorants

The kinetics of fluorescence of Ca(2+) - chlortertacyclin-cell membrane complex as well as of NADH and oxidized flavoproteins in receptor cells of the frog olfactory lining under the effect of odorants has been studied. Changes in the fluorescence of the olfactory lining upon stimulation by cineole and vanillin occurred more rapidly than under the effect of camphor and amyl alcohol. Differences in the kinetics of reactions of NADH and the Ca(2+)-CTC-CM complex to different odorants are apparently due to heterogeneity of molecular mechanisms associated with the involvement of different intracellular signal systems in the transduction of these odorants in the olfactory lining. In contrast to them, ammonia and beta3-mercaptoethanol penetrate into olfactory cells and inhibit the mitochondrial respiratory chain without the participation of second messengers. At the same time, the motor activity of olfactory cilia is depressed.     

Perturbation approximation of solutions of a nonlinear inverse problem arising in olfaction experimentation

In this paper, a mathematical model of the diffusion of cAMP into olfactory cilia and the resulting electrical activity is presented. The model, which consists of two nonlinear differential equations, is studied using perturbation techniques. The unknowns in the problem are the concentration of cAMP, the membrane potential, and the quantity of most interest in this work: the distribution of CNG channels along the length of a cilium. Experimental measurements of the total current during this diffusion process provide an extra boundary condition which helps determine the unknown distribution function. A simple perturbation approximation is derived and used to solve this inverse problem and thus obtain estimates of the spatial distribution of CNG ion channels along the length of a cilium. A one-dimensional computer minimization and a special delay iteration are used with the perturbation formulas to obtain approximate channel distributions in the cases of simulated and experimental data.      

Ultrastructural aspects of olfactory transduction and perireceptor events

Ultrastructural/immunocytochemical studies with well defined antibodies suggest that distal segments of olfactory cilia are the main sites of early events in olfactory signal transduction. Such studies also begin to provide specifics of the cytoskeletal make-up of olfactory epithelial cells, but knowledge about relationships between cytoskeletal and transduction components is still incomplete. Probes to less well defined chemical entities, but that distinctly label olfactory cilia, supporting cell microvilli and microvilli of microvillous cells, may serve as markers for further studies on olfactory signaling. Ultrastructural/immunocytochemical studies also suggest that supporting cells help to balance the mucous environment of olfactory cilia.     

Mechanism of olfactory masking in the sensory cilia

Olfactory masking has been used to erase the unpleasant sensation in human cultures for a long period of history. Here, we show a positive correlation between the human masking and the odorant suppression of the transduction current through the cyclic nucleotide–gated (CNG) and Ca²⁺-activated Cl⁻ (Cl_(Ca)) channels. Channels in the olfactory cilia were activated with the cytoplasmic photolysis of caged compounds, and their sensitiveness to odorant suppression was measured with the whole cell patch clamp. When 16 different types of chemicals were applied to cells, cyclic AMP (cAMP)-induced responses (a mixture of CNG and Cl_(Ca) currents) were suppressed widely with these substances, but with different sensitivities. Using the same chemicals, in parallel, we measured human olfactory masking with 6-rate scoring tests and saw a correlation coefficient of 0.81 with the channel block. Ringer''s solution that was just preexposed to the odorant-containing air affected the cAMP-induced current of the single cell, suggesting that odorant suppression occurs after the evaporation and air/water partition of the odorant chemicals at the olfactory mucus. To investigate the contribution of Cl_(Ca), the current was exclusively activated by using the ultraviolet photolysis of caged Ca, DM-nitrophen. With chemical stimuli, it was confirmed that Cl_(Ca) channels were less sensitive to the odorant suppression. It is interpreted, however, that in the natural odorant response the Cl_(Ca) is affected by the reduction of Ca²⁺ influx through the CNG channels as a secondary effect. Because the signal transmission between CNG and Cl_(Ca) channels includes nonlinear signal-boosting process, CNG channel blockage leads to an amplified reduction in the net current. In addition, we mapped the distribution of the Cl_(Ca) channel in living olfactory single cilium using a submicron local [Ca²⁺]_i elevation with the laser photolysis. Cl_(Ca) channels are expressed broadly along the cilia. We conclude that odorants regulate CNG level to express masking, and Cl_(Ca) in the cilia carries out the signal amplification and reduction evenly spanning the entire cilia. The present findings may serve possible molecular architectures to design effective masking agents, targeting olfactory manipulation at the nano-scale ciliary membrane.     

Protein kinase C sensitizes olfactory adenylate cyclase

Effects of neurotransmitters on cAMP-mediated signal transduction in frog olfactory receptor cells (ORCs) were studied using in situ spike recordings and radioimmunoassays. Carbachol, applied to the mucosal side of olfactory epithelium, amplified the electrical response of ORCs to cAMP-generating odorants, but did not affect unstimulated cells. A similar augmentation of odorant response was observed in the presence of phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC). The electrical response to forskolin, an activator of adenylate cyclase (AC), was also enhanced by PDBu, and it was attenuated by the PKC inhibitor Goe 6983. Forskolin-induced accumulation of cAMP in olfactory tissue was potentiated by carbachol, serotonin, and PDBu to a similar extent. Potentiation was completely suppressed by the PKC inhibitors Goe 6983, staurosporine, and polymyxin B, suggesting that the sensitivity of olfactory AC to stimulation by odorants and forskolin was increased by PKC. Experiments with deciliated olfactory tissue indicated that sensitization of AC was restricted to sensory cilia of ORCs. To study the effects of cell Ca2+ on these mechanisms, the intracellular Ca2+ concentration of olfactory tissue was either increased by ionomycin or decreased by BAPTA/AM. Increasing cell Ca2+ had two effects on cAMP production: (a) the basal cAMP production was enhanced by a mechanism sensitive to inhibitors of calmodulin; and (b) similar to phorbol ester, cell Ca2+ caused sensitization of AC to stimulation by forskolin, an effect sensitive to Goe 6983. Decreasing cell Ca2+ below basal levels rendered AC unresponsive to stimulation by forskolin. These data suggest that a crosstalk mechanism is functional in frog ORCs, linking the sensitivity of AC to the activity of PKC. At increased activity of PKC, olfactory AC becomes more responsive to stimulation by odorants, forskolin, and cell Ca2+. Neurotransmitters appear to use this crosstalk mechanism to regulate olfactory sensitivity.     

A large contribution of a cyclic AMP-independent pathway to turtle olfactory transduction

Although multiple pathways are involved in the olfactory transduction mechanism, cAMP-dependent pathway has been considered to contribute mainly to the transduction. We examined the degree of contribution of cAMP-independent pathway to the turtle olfactory response by recording inward currents from isolated cells, nerve impulses from cilia and olfactory bulbar responses. The results obtained by the three recordings were essentially consistent with each other, but detail studies were carried out by recording the bulbar response to obtain quantitative data. Application of an odorant cocktail to the isolated olfactory neuron after injection of 1 mM cAMP from the patch pipette elicited a large inward current. Mean amplitude of inward currents evoked by the cocktail with 1 mM cAMP in the patch pipette was similar to that without cAMP in the pipette. Application of the cocktail after the response to 50 microM forskolin was adapted also induced a large inward current. Application of the odorant cocktail to the olfactory epithelium, after the response to 50 microM forskolin was adapted, brought about an appreciable increase in the impulse frequency. The bulbar response to forskolin alone reached a saturation level around 10 microM. After the response to 50 microM forskolin was adapted, 11 species of odorants were applied to the olfactory epithelium. The magnitudes of responses to the odorants after forskolin were 45-80% of those of the control responses. There was no essential difference in the degree of the suppression by forskolin between cAMP- and IP3- producing odorants classified in the rat, suggesting that certain part of the forskolin-suppressive component was brought about by nonspecific action of forskolin. Application of a membrane permeant cAMP analogue, cpt-cAMP elicited a large response, and 0.1 mM citralva after 3 mM cpt- cAMP elicited 51% of the control response which was close to the response to citralva after 50 microM forskolin. A membrane permeant cGMP analogue, db-cGMP elicited a small response and the response to 0.1 mM citralva was unaffected by db-cGMP. It was concluded that cAMP- independent (probably IP3-independent) pathway greatly contributes to the turtle olfactory transduction.     

The location of olfactory receptor sites. Inferences from latency measurements.

Excitatory responses recorded from vertebrate olfactory sensory neurons are characterized by long latencies compared with those from other sensory receptors. Explanations which assume free access of the stimuli to receptor molecules presumably located on the olfactory cilia necessarily imply an intrinsic delay in the transduction mechanism. In contrast, the possibility of restricted or delayed access due to diffusion of the stimulus to molecular receptors located on the dendritic know or proximal portions of the cilia suggests transduction processes having time courses similar to those in other sensory systems. We show that the threshold stimulus concentrations and the latency of the excitatory response of the salamander can be predicted primarily on the basis of a diffusional delay and that the receptor molecules are well below the surface of the mucus. Examination of response latencies for other species reported in the literature support the generality of diffusional delay. The predicted location of molecular receptor sites is largely insensitive to assumptions based on the mode of clearance of the stimuli. Additional access restrictions are discussed but are shown to generate qualitatively different latency functions than does diffusion, suggesting that they exert only minor influences on latency and threshold characteristics.     

The contrasting roles of primary cilia and cytonemes in Hh signaling

Hedgehog (Hh) is a paracrine signaling protein with major roles in development and disease. In vertebrates and invertebrates, Hh signal transduction is carried out almost entirely by evolutionarily conserved components, and in both, intercellular movement of Hh is mediated by cytonemes – specialized filopodia that serve as bridges that bring distant cells into contact. A significant difference is the role of the primary cilium, a slender, tubulin-based protuberance of many vertebrate cells. Although the primary cilium is essential for Hh signaling in cells that have one, most Drosophila cells lack a primary cilium. This perspective addresses the roles of primary cilia and cytonemes, and proposes that for Hh signaling, the role of primary cilia is to provide a specialized hydrophobic environment that hosts lipid-modified Hh and other components of Hh signal transduction after Hh has traveled from elsewhere in the cell. Implicit in this model is the idea that initial binding and uptake of Hh is independent of and segregated from the processes of signal transduction and activation.