Olfactory CNG channel desensitization by Ca2+/CaM via the B1b subunit affects response termination but not sensitivity to recurring stimulation

Ca2+/calmodulin-mediated negative feedback is a prototypical regulatory mechanism for Ca2+-permeable ion channels. In olfactory sensory neurons (OSNs), such regulation on the cyclic nucleotide-gated (CNG) channel is considered a major mechanism of OSN adaptation. To determine the role of Ca2+/calmodulin desensitization of the olfactory CNG channel, we introduced a mutation in the channel subunit CNGB1b in mice that rendered the channel resistant to fast desensitization by Ca2+/calmodulin. Contrary to expectations, mutant OSNs showed normal receptor current adaptation to repeated stimulation. Rather, they displayed slower response termination and, consequently, reduced ability to transmit olfactory information to the olfactory bulb. They also displayed reduced response decline during sustained odorant exposure. These results suggest that Ca2+/calmodulin-mediated CNG channel fast desensitization is less important in regulating the sensitivity to recurring stimulation than previously thought and instead functions primarily to terminate OSN responses.     

Pulse stimulation with odors or IBMX/forskolin potentiates responses in isolated olfactory neurons

Many odor responses are mediated by the adenosine 3',5'-cyclic monophosphate (cAMP) pathway in which the cAMP-gated current is amplified by Ca2+-dependent Cl- current. In olfactory neurons, prolonged exposure to odors decreases the odor response and is an adaptive effect. Several studies suggest that odor adaptation is linked to elevated intracellular Ca2+. In the present study, using the perforated configuration of the patch clamp technique, we found that repetitive odor stimulation elicits a potentiation of the subsequent responses in olfactory neurons. This potentiation is mimicked by stimulating the cAMP pathway and does not appear to be related to phosphorylation of ion channels since protein kinase inhibitors could not block it. Our data suggest that local increases in [Ca2+]i via activation of the cAMP pathway mediate the pulse-elicited potentiation. In the first odor application, entry of Ca2+ through cyclic nucleotide-gated channels appears to be buffered. Repetitive stimulation allows local increases in [Ca2+]i, recruiting more Ca2+-dependent Cl- channels with each subsequent odor pulse.     

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.     

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.     

Mechanisms of Regulation of Olfactory Transduction and Adaptation in the Olfactory Cilium

Olfactory adaptation is a fundamental process for the functioning of the olfactory system, but the underlying mechanisms regulating its occurrence in intact olfactory sensory neurons (OSNs) are not fully understood. In this work, we have combined stochastic computational modeling and a systematic pharmacological study of different signaling pathways to investigate their impact during short-term adaptation (STA). We used odorant stimulation and electroolfactogram (EOG) recordings of the olfactory epithelium treated with pharmacological blockers to study the molecular mechanisms regulating the occurrence of adaptation in OSNs. EOG responses to paired-pulses of odorants showed that inhibition of phosphodiesterases (PDEs) and phosphatases enhanced the levels of STA in the olfactory epithelium, and this effect was mimicked by blocking vesicle exocytosis and reduced by blocking cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and vesicle endocytosis. These results suggest that G-coupled receptors (GPCRs) cycling is involved with the occurrence of STA. To gain insights on the dynamical aspects of this process, we developed a stochastic computational model. The model consists of the olfactory transduction currents mediated by the cyclic nucleotide gated (CNG) channels and calcium ion (Ca²⁺)-activated chloride (CAC) channels, and the dynamics of their respective ligands, cAMP and Ca²⁺, and it simulates the EOG results obtained under different experimental conditions through changes in the amplitude and duration of cAMP and Ca²⁺ response, two second messengers implicated with STA occurrence. The model reproduced the experimental data for each pharmacological treatment and provided a mechanistic explanation for the action of GPCR cycling in the levels of second messengers modulating the levels of STA. All together, these experimental and theoretical results indicate the existence of a mechanism of regulation of STA by signaling pathways that control GPCR cycling and tune the levels of second messengers in OSNs, and not only by CNG channel desensitization as previously thought.     

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.     

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.     

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.     

Ca2+-activated K+ currents regulate odor adaptation by modulating spike encoding of olfactory receptor cells

The olfactory system is thought to accomplish odor adaptation through the ciliary transduction machinery in olfactory receptor cells (ORCs). However, ORCs that have lost their cilia can exhibit spike frequency accommodation in which the action potential frequency decreases with time despite a steady depolarizing stimulus. This raises the possibility that somatic ionic channels in ORCs might serve for odor adaptation at the level of spike encoding, because spiking responses in ORCs encode the odor information. Here I investigate the adaptational mechanism at the somatic membrane using conventional and dynamic patch-clamp recording techniques, which enable the ciliary mechanism to be bypassed. A conditioning stimulus with an odorant-induced current markedly shifted the response range of action potentials induced by the same test stimulus to higher concentrations of the odorant, indicating odor adaptation. This effect was inhibited by charybdotoxin and iberiotoxin, Ca2+-activated K+ channel blockers, suggesting that somatic Ca2+-activated K+ currents regulate odor adaptation by modulating spike encoding. I conclude that not only the ciliary machinery but also the somatic membrane currents are crucial to odor adaptation.     

Responses of Xenopus laevis water nose to water-soluble and volatile odorants.

Using the whole-cell mode of the patch-clamp technique, we recorded action potentials, voltage-activated cationic currents, and inward currents in response to water-soluble and volatile odorants from receptor neurons in the lateral diverticulum (water nose) of the olfactory sensory epithelium of Xenopus laevis. The resting membrane potential was -46.5 +/- 1.2 mV (mean +/- SEM, n = 68), and a current injection of 1-3 pA induced overshooting action potentials. Under voltage-clamp conditions, a voltage-dependent Na+ inward current, a sustained outward K+ current, and a Ca2+-activated K+ current were identified. Application of an amino acid cocktail induced inward currents in 32 of 238 olfactory neurons in the lateral diverticulum under voltage-clamp conditions. Application of volatile odorant cocktails also induced current responses in 23 of 238 olfactory neurons. These results suggest that the olfactory neurons respond to both water-soluble and volatile odorants. The application of alanine or arginine induced inward currents in a dose-dependent manner. More than 50% of the single olfactory neurons responded to multiple types of amino acids, including acidic, neutral, and basic amino acids applied at 100 microM or 1 mM. These results suggest that olfactory neurons in the lateral diverticulum have receptors for amino acids and volatile odorants.     

Ca2+-Activated Cl? Channels of the ClCa Family Express in the Cilia of a Subset of Rat Olfactory Sensory Neurons

The Ca²⁺-activated Cl⁻ channel is considered a key constituent of odor transduction. Odorant binding to a specific receptor in the cilia of olfactory sensory neurons (OSNs) triggers a cAMP cascade that mediates the opening of a cationic cyclic nucleotide-gated channel (CNG), allowing Ca²⁺ influx. Ca²⁺ ions activate Cl⁻ channels, generating a significant Cl⁻ efflux, with a large contribution to the receptor potential. The Anoctamin 2 channel (ANO2) is a major constituent of the Cl⁻ conductance, but its knock-out has no impairment of behavior and only slightly reduces field potential odorant responses of the olfactory epithelium. Likely, an additional Ca²⁺-activated Cl⁻ channel of unknown molecular identity is also involved. In addition to ANO2, we detected two members of the ClCa family of Ca²⁺-activated Cl⁻ channels in the rat olfactory epithelium, ClCa4l and ClCa2. These channels, also expressed in the central nervous system, may correspond to odorant transduction channels. Whole Sprague Dawley olfactory epithelium nested RT-PCR and single OSNs established that the mRNAs of both channels are expressed in OSNs. Real time RT-PCR and full length sequencing of amplified ClCa expressed in rat olfactory epithelium indicated that ClCa4l is the most abundant. Immunoblotting with an antibody recognizing both channels revealed immunoreactivity in the ciliary membrane. Immunochemistry of olfactory epithelium and OSNs confirmed their ciliary presence in a subset of olfactory sensory neurons. The evidence suggests that ClCa4l and ClCa2 might play a role in odorant transduction in rat olfactory cilia.     

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.     

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.     

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.     

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.     

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.     

Two bestrophins cloned from Xenopus laevis oocytes express Ca(2+)-activated Cl(-) currents

Ca2+-activated Cl- channels play important diverse roles from fast block to polyspermy to olfactory transduction, but their molecular identity has not been firmly established. By searching sequence databases with the M2 pore domain of ligand-gated anion channels, we identified potential Ca2+-activated Cl- channels, which included members of the bestrophin family. We cloned two bestrophins from Xenopus oocytes, which express high levels of Ca2+-activated Cl- channels. The Xenopus bestrophins were expressed in a variety of tissues. We predict that bestrophin has six transmembrane domains with the conserved RFP domain playing an integral part in ionic selectivity. When Xenopus bestrophins were heterologously expressed in human embryonic kidney-293 cells, large Ca2+-activated Cl- currents were observed. The currents are voltage- and time-independent, do not rectify, have a Kd for Ca2+ of approximately 210 nm, and exhibit a permeability ratio of I- > Br- > Cl- > aspartate. The W93C and G299E mutations produce non-functional channels that exert a dominant negative effect on wild type channels. We conclude that bestrophins are the first molecularly identified Cl- channels that are dependent on intracellular Ca2+ in a physiological range.     

Regulation of intracellular cyclic GMP levels in olfactory sensory neurons

Cyclic AMP is the primary second messenger mediating odorant signal transduction in mammals. A number of studies indicate that cyclic GMP is also involved in a variety of other olfactory signal transduction processes, including adaptation, neuronal development, and long-term cellular responses in the setting of odorant stimulation. However, the mechanisms that control the production and degradation of cGMP in olfactory sensory neurons (OSNs) remain unclear. Here, we investigate these mechanisms using primary cultures of OSNs. We demonstrate that odorants increase cGMP levels in intact OSNs in vitro. Different from the rapid and transient cAMP responses to odorants, the cGMP elevation is both delayed and sustained. Inhibition of soluble guanylyl cyclase and heme oxygenase blocks these odorant-induced cGMP increases, whereas inhibition of cGMP PDEs (phosphodiesterases) increases this response. cGMP PDE activity is increased by odorant stimulation, and is sensitive to both ambient calcium and cAMP concentrations. Calcium stimulates cGMP PDE activity, whereas cAMP and protein kinase A appears to inhibit it. These data demonstrate a mechanism by which odorant stimulation may regulate cGMP levels through the modulation of cAMP and calcium level in OSNs. Such interactions between odorants and second messenger systems may be important to the integration of immediate and long-term responses in the setting odorant stimulation.     

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.