IP3-Gated Channels and their Occurrence Relative to CNG Channels in the Soma and Dendritic Knob of Rat Olfactory Receptor Neurons

Olfactory receptor neurons respond to odorants with G protein-mediated increases in the concentrations of cyclic adenosine 3',5'-monophosphate (cAMP) and/or inositol-1,4,5-trisphosphate (IP3). This study provides evidence that both second messengers can directly activate distinct ion channels in excised inside-out patches from the dendritic knob and soma membrane of rat olfactory receptor neurons (ORNs). The IP3-gated channels in the dendritic knob and soma membranes could be classified into two types, with conductances of 40 +/- 7 pS (n = 5) and 14 +/- 3 pS (n = 4), with the former having longer open dwell times. Estimated values of the densities of both channels from the same inside-out membrane patches were very much smaller for IP3-gated than for CNG channels. For example, in the dendritic knob membrane there were about 1000 CNG channels x microm(-2) compared to about 85 IP3-gated channels x microm(-2). Furthermore, only about 36% of the dendritic knob patches responded to IP3, whereas 83% of the same patches responded to cAMP. In the soma, both channel densities were lower, with the CNG channel density again being larger ( approximately 57 channels x microm(-2)) than that of the IP3-gated channels ( approximately 13 channels x microm(-2)), with again a much smaller fraction of patches responding to IP3 than to cAMP. These results were consistent with other evidence suggesting that the cAMP-pathway dominates the IP3 pathway in mammalian olfactory transduction.     

Primary structure of cAMP-gated channel from bovine olfactory epithelium

The complete amino-acid sequence of the bovine olfactory epithelium adenosine 3',5'cyclic monophosphate (cAMP)-gated channel has been determined by cloning and sequencing its cDNA. It exhibits a high degree of sequence homology with the cGMP-gated channel of rod photoreceptors, suggesting that cyclic nucleotide-gated channels fall into a new family of genetically related proteins.     

Anomalous mole-fraction effects in recombinant and native cyclic nucleotide-gated channels in rat olfactory receptor neurons.

Anomalous mole-fraction effects (AMFE) were studied, using the inside-out configuration of the patchclamp technique, in both recombinant wild-type alpha-homomeric rat olfactory adenosine 3',5'-cyclic monophosphate (cAMP)-gated channels (rOCNC1) expressed in human embryonic kidney cells (HEK 293) and native cyclic nucleotide-gated (CNG) channels in acutely isolated rat olfactory receptor neurons. Single-channel and macroscopic currents were activated by 200 microM and 500 microM cAMP, respectively. Macroscopic currents, measured with mixtures of Na(+)-NH(4)(+) or Cs(+)-Li(+) in the cytoplasmic bathing solution, displayed AMFE in the rOCNC1 channels at both positive and negative membrane potentials. The rOCNC1 single-channel conductance showed a distinct minimum (or maximum) in an 80% Na(+)-20% NH(4)(+) mixture (or a 60% Cs(+)-40% Li(+) mixture), but only at positive membrane potentials. Macroscopic measurements in native olfactory CNG channels with mixtures of Na(+)-NH(4)(+) indicated similar AMFE. These results suggest that both native CNG channels and recombinant alpha-homomeric channels allow several ions to be present simultaneously within the channel pore. They also further validate the dominant role of the alpha-subunit in permeation through these channels, provide the first evidence to suggest that rOCNC1 channels have multi-ion properties and further justify the use of the rOCNC1 channel as an effective model for structure-function studies of ion permeation and selectivity in olfactory CNG channels.     

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.     

Mechanisms of modulation by internal protons of cyclic nucleotide-gated channels cloned from sensory receptor cells.

We have examined the modulation by internal protons of cyclic nucleotide-gated (CNG) channels cloned from bovine olfactory receptor cells and retinal rods. CNG channels were studied in excised inside-out membrane patches from Xenopus laevis oocytes previously injected with the mRNA encoding for the subunit 1 of olfactory or rod channels. Channels were activated by cGMP or cAMP, and currents as a function of cyclic nucleotide concentrations were measured as pHi varied between 7.6 and 5.0. Increasing internal proton concentrations caused a partial blockage of the single-channel current, consistent with protonation of a single acidic site with a pK1 of 4.5-4.7, both in rod and in olfactory CNG channels. Channel gating properties were also affected by internal protons. The open probability at low cyclic nucleotide concentrations was greatly increased by lowering pHi, and the increase was larger when channels were activated by cAMP than by cGMP. Therefore, internal protons affected both channel permeation and gating properties, causing a reduction in single-channel current and an increase in open probability. These effects are likely to be caused by different titratable groups on the channel.     

The styryl dye FM1-43 suppresses odorant responses in a subset of olfactory neurons by blocking cyclic nucleotide-gated (CNG) channels

Many olfactory receptor neurons use a cAMP-dependent transduction mechanism to transduce odorants into depolarizations. This signaling cascade is characterized by a sequence of two currents: a cation current through cyclic nucleotide-gated channels followed by a chloride current through calcium-activated chloride channels. To date, it is not possible to interfere with these generator channels under physiological conditions with potent and specific blockers. In this study we identified the styryl dye FM1-43 as a potent blocker of native olfactory cyclic nucleotide-gated channels. Furthermore, we characterized this substance to stain olfactory receptor neurons that are endowed with cAMP-dependent transduction. This allows optical differentiation and pharmacological interference with olfactory receptor neurons at the level of the signal transduction.     

Cyclic AMP diffusion coefficient in frog olfactory cilia

Cyclic AMP (cAMP) is one of the intracellular messengers that mediate odorant signal transduction in vertebrate olfactory cilia. Therefore, the diffusion coefficient of cAMP in olfactory cilia is an important factor in the transduction of the odorous signal. We have employed the excised cilium preparation from the grass frog (Rana pipiens) to measure the cAMP diffusion coefficient. In this preparation an olfactory cilium is drawn into a patch pipette and a gigaseal is formed at the base of the cilium. Subsequently the cilium is excised, allowing bath cAMP to diffuse into the cilium and activate the cyclic nucleotide-gated channels on the plasma membrane. In order to estimate the cAMP diffusion coefficient, we analyzed the kinetics of the currents elicited by step changes in the bath cAMP concentration in the absence of cAMP hydrolysis. Under such conditions, the kinetics of the cAMP-activated currents has a simple dependence on the diffusion coefficient. From the analysis we have obtained a cAMP diffusion coefficient of 2.7 +/- 0.2. 10(-6) cm2 s-1 for frog olfactory cilia. This value is similar to the expected value in aqueous solution, suggesting that there are no significant diffusional barriers inside olfactory cilia. At cAMP concentrations higher than 5 microM, diffusion slowed considerably, suggesting the presence of buffering by immobile cAMP binding sites. A plausible physiological function of such buffering sites would be to prolong the response of the cell to strong stimuli.     

Electrophysiological analysis of cloned cyclic nucleotide-gated ion channels

Electrophysiological studies were conducted on the cloned plant cyclic nucleotide-gated ion channels AtCNGC2 and AtCNGC1 from Arabidopsis, and NtCBP4 from tobacco (Nicotiana tobacum). The nucleotide coding sequences for these proteins were expressed in Xenopus laevis oocytes or HEK 293 cells. Channel characteristics were evaluated using voltage clamp analysis of currents in the presence of cAMP. AtCNGC2 was demonstrated to conduct K(+) and other monovalent cations, but exclude Na(+); this conductivity profile is unique for any ion channel not possessing the amino acid sequence found in the selectivity filter of K(+)-selective ion channels. Application of cAMP evoked currents in membrane patches of oocytes injected with AtCNGC2 cRNA. Direct activation of the channel by cyclic nucleotide, demonstrated by application of cyclic nucleotide to patches of membranes expressing such channels, is a hallmark characteristic of this ion channel family. Voltage clamp studies (two-electrode configuration) demonstrated that AtCNGC1 and NtCBP4 are also cyclic nucleotide-gated channels. Addition of a lipophilic analog of cAMP to the perfusion bath of oocytes injected with NtCBP4 and AtCNGC1 cRNAs induced inward rectified, noninactivating K(+) currents.     

Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice

Cyclic nucleotide-gated ion channels in olfactory sensory neurons (OSNs) are hypothesized to play a critical role in olfaction. However, it has not been demonstrated that the cAMP signaling is required for olfactory-based behavioral responses, and the contributions of specific adenylyl cyclases to olfaction have not been defined. Here, we report the presence of adenylyl cyclases 2, 3, and 4 in olfactory cilia. To evaluate the role of AC3 in olfactory responses, we disrupted the gene for AC3 in mice. Interestingly, electroolfactogram (EOG) responses stimulated by either cAMP- or inositol 1,4,5-triphosphate- (IP3-) inducing odorants were completely ablated in AC3 mutants, despite the presence of AC2 and AC4 in olfactory cilia. Furthermore, AC3 mutants failed several olfaction-based behavioral tests, indicating that AC3 and cAMP signaling are critical for olfactory-dependent behavior.     

Fusion of native or reconstituted membranes to liposomes, optimized for single channel recording.

We here describe a protocol for fusing vesicles into large structures suitable for patch clamp recording. The method may be used with native membrane vesicles or with liposomes containing reconstituted/purified ion channels. The resulting unilamellar membranes exhibit high channel surface abundance, yielding multiple channels in the average excised patch. The procedure has been used to record voltage-sensitive Na channels from three native membrane preparations (eel electroplax, rat skeletal muscle, squid optic nerve), and from reconstituted protein purified from eel electroplax. Channels treated with batrachotoxin (BTX) displayed characteristic activation voltage dependence, conductances, selectivity, and sensitivity to saxitoxin (STX).     

Cyclic AMP links amino acid chemoreceptors to ion channels in olfactory cilia

Amino acids, olfactory stimuli for the channel catfish (Ictaluruspunctatus), were tested for the ability to affect cyclic AMPmetabolism in isolated olfactory cilia. Ten stimuli, representativeof each receptor subtype and covering a wide range of electrophysiologicalpotency, elicited similar increases in guanine nucleotide-dependentcyclic AMP formation. Stimulus-dependent activation of adenylatecyclase was observed only when receptor occupancy approachedor exceeded 50%. Stimulus activation of adenylate cyclase wasadditive with that obtained with guanine nucleotide alone, wasindependent of receptor specificity, and poorly correlated withneural potency. Stimuli did not affect cyclic nucleotide phosphodiesteraseactivity in isolated cilia preparations. Both cyclic AMP andcyclic GMP reversibly increased membrane conductance in isolatedciliary membranes incorporated in artificial phospholipid bilayers.The cyclic nucleotide-gated conductance was activated by eithernucleotide with equal potency, did not require exogenous ATPor GTP, and was mediated by 44 pS non-selective cation channels.Taken together, these results suggest that, under appropriateconditions of receptor occupancy, cyclic AMP links amino acidchemoreceptor binding to membrane depolarization by directlygating cation channels in olfactory cilia.     

Three amino acids in the C-linker are major determinants of gating in cyclic nucleotide-gated channels.

The activation of cyclic nucleotide-gated (CNG) channels is a complex process comprising the initial ligand binding and a consecutive allosteric transition from a closed to an open configuration. The cone and olfactory CNG channels differ considerably in cyclic nucleotide affinity and efficacy. In each channel, the cyclic nucleotide-binding site is connected to the last transmembrane segment of the channel by a linker peptide (C-linker) of approximately 90 amino acids. Here we report that replacement of three amino acids in the cone C-linker by the corresponding amino acids of the olfactory channel (I439V, D481A and D494S) profoundly enhanced the cAMP efficacy and increased the affinities for cAMP and cGMP. Unlike the wild-type cone channel, the mutated channel exhibited similar single-channel kinetics for both cGMP and cAMP, explaining the increase in cAMP efficacy. We thus conclude that the identified amino acids are major determinants of channel gating.     

Isolation and functional characterization of an inositol trisphosphate receptor from brain

We have identified an IP3 receptor protein in brain membranes through the binding of radiolabelled IP3. Autoradiographic studies localize the receptor to various areas of the brain with highest densities in Purkinje cells of the cerebellum. IP3 binding is inhibited by physiologic intracellular concentrations of calcium. Purification of the IP3 receptor to homogeneity reveals it to be comprised of four identical subunits of 260 kD each. Antisera to the purified receptor protein have been employed for immunohistochemical studies which, at the electron microscopic level, localize the IP3 receptor to a subdivision of the rough endoplasmic reticulum occurring in synaptic areas and in close association with the nuclear membrane. The IP3 receptor protein is selectively phosphorylated by cyclic AMP (cAMP) dependent protein kinase. This phosphorylation decreases 10-fold the potency of IP3 in releasing calcium from brain membranes.     

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.     

Functional reconstitution of the olfactory membrane: incorporation of the olfactory adenylate cyclase in liposomes

Chemosensory cilia isolated from the olfactory epithelium of Rana catesbeiana were solubilized with Lubrol PX in the presence of supplementary lipid, forskolin, and sodium fluoride. Subsequent removal of the detergent by adsorption onto Biobeads SM2 results in the formation of proteoliposomes that display forskolin- and GTP gamma S-sensitive adenylate cyclase activity. Sucrose gradient centrifugation of liposomes formed in the presence of fluorescently labeled phosphatidylcholine demonstrates association between the olfactory adenylate cyclase and the exogenously added lipid. Forskolin stimulates the enzyme in reconstituted membranes with the same potency as in native membranes (EC50 = 1-2 microM). However, GTP gamma S is 350-fold more potent in native membranes (EC50 = 4.0 +/- 0.5 nM) than in reconstituted membranes (EC50 = 1.4 +/- 0.3 microM). These studies represent a first step toward the functional reconstitution and molecular dissection of the olfactory membrane.     

Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion

Cyclic AMP is a ubiquitous second messenger that coordinates diverse cellular functions. Current methods for measuring cAMP lack both temporal and spatial resolution, leading to the pervasive notion that, unlike Ca(2+), cAMP signals are simple and contain little information. Here we show the development of adenovirus-expressed cyclic nucleotide-gated channels as sensors for cAMP. Homomultimeric channels composed of the olfactory alpha subunit responded rapidly to jumps in cAMP concentration, and their cAMP sensitivity was measured to calibrate the sensor for intracellular measurements. We used these channels to detect cAMP, produced by either heterologously expressed or endogenous adenylyl cyclase, in both single cells and cell populations. After forskolin stimulation, the endogenous adenylyl cyclase in C6-2B glioma cells produced high concentrations of cAMP near the channels, yet the global cAMP concentration remained low. We found that rapid exchange of the bulk cytoplasm in whole-cell patch clamp experiments did not prevent the buildup of significant levels of cAMP near the channels in human embryonic kidney 293 (HEK-293) cells expressing an exogenous adenylyl cyclase. These results can be explained quantitatively by a cell compartment model in which cyclic nucleotide-gated channels colocalize with adenylyl cyclase in microdomains, and diffusion of cAMP between these domains and the bulk cytosol is significantly hindered. In agreement with the model, we measured a slow rate of cAMP diffusion from the whole-cell patch pipette to the channels (90% exchange in 194 s, compared with 22-56 s for substances that monitor exchange with the cytosol). Without a microdomain and restricted diffusional access to the cytosol, we are unable to account for all of the results. It is worth noting that in models of unrestricted diffusion, even in extreme proximity to adenylyl cyclase, cAMP does not reach high enough concentrations to substantially activate PKA or cyclic nucleotide-gated channels, unless the entire cell fills with cAMP. Thus, the microdomains should facilitate rapid and efficient activation of both PKA and cyclic nucleotide-gated channels, and allow for local feedback control of adenylyl cyclase. Localized cAMP signals should also facilitate the differential regulation of cellular targets.     

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.     

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.     

Characterization of inositol-1,4,5-trisphosphate-gated channels in the plasma membrane of rat olfactory neurons

It is generally accepted that inositol-1,4,5-trisphosphate (InsP3) plays a role in olfactory transduction. However, the precise mode of action of InsP3 remains controversial. We have characterized the conductances activated by the addition of 10 microM InsP3 to excised patches of soma plasma membrane from rat olfactory neurons. InsP3 induced current fluctuations in 25 of 121 inside-out patches. These conductances could be classified into two groups according to the polarity of the current at a holding potential of +40 to +60 mV (with Ringer's in the pipette and pseudointracellular solution in the bath). Conductances mediating outward currents could be further divided into large- (64 +/- 4 pS, n = 4) and small- (16 +/- 1.7 pS, n = 11) conductance channels. Both small- and large-conductance channels were nonspecific cation channels. The large-conductance channel displayed bursting behavior at +40 mV, with flickering increasing at negative holding potentials to the point where single-channel currents were no longer discernible. The small-conductance channel did not display flickering behavior. The conductance mediating inward currents at +40 to +60 mV reversed at +73 +/- 4 mV (n = 4). The current traces displayed considerable fluctuations, and single-channel currents could not be discerned. The current fluctuations returned to baseline after removal of InsP3. The power density spectrum for the excess noise generated by InsP3 followed a 1/f dependence consistent with conductance fluctuations in the channel mediating this current, although other mechanisms are not excluded. These experiments demonstrate the presence of plasma membrane InsP3-gated channels of different ionic specificity in olfactory receptor cells.     

Characterization of a phosphoinositide-mediated odor transduction pathway reveals plasma membrane localization of an inositol 1,4, 5-trisphosphate receptor in lobster olfactory receptor neurons

The role of phosphoinositide signaling in olfactory transduction is still being resolved. Compelling functional evidence for the transduction of odor signals via phosphoinositide pathways in olfactory transduction comes from invertebrate olfactory systems, in particular lobster olfactory receptor neurons. We now provide molecular evidence for two components of the phosphoinositide signaling pathway in lobster olfactory receptor neurons, a G protein alpha subunit of the G(q) family and an inositol 1,4, 5-trisphosphate-gated channel or an inositol 1,4,5-trisphosphate (IP(3)) receptor. Both proteins localize to the site of olfactory transduction, the outer dendrite of the olfactory receptor neurons. Furthermore, the IP(3) receptor localizes to membranes in the ciliary transduction compartment of these cells at both the light microscopic and electron microscopic levels. Given the absence of intracellular organelles in the sub-micron diameter olfactory cilia, this finding indicates that the IP(3) receptor is associated with the plasma membrane and provides the first definitive evidence for plasma membrane localization of an IP(3)R in neurons. The association of the IP(3) receptor with the plasma membrane may be a novel mechanism for regulating intracellular cations in restricted cellular compartments of neurons.