Johannes Reisert

ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification

For vertebrate olfactory signal transduction, a calcium-activated chloride conductance serves as a major amplification step. However, the molecular identity of the olfactory calcium-activated chloride channel (CaCC) is unknown. Here we report a proteomic screen for cilial membrane proteins of mouse olfactory sensory neurons (OSNs) that identified all the known olfactory transduction components as well as Anoctamin 2 (ANO2). Ano2 transcripts were expressed specifically in OSNs in the olfactory epithelium, and ANO2::EGFP fusion protein localized to the OSN cilia when expressed in vivo using an adenoviral vector. Patch-clamp analysis revealed that ANO2, when expressed in HEK-293 cells, forms a CaCC and exhibits channel properties closely resembling the native olfactory CaCC. Considering these findings together, we propose that ANO2 constitutes the olfactory calcium-activated chloride channel.

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 ∼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 ∼8 and 62 μm−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.

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.

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.

Loss of CNGB1 protein leads to olfactory dysfunction and subciliary cyclic nucleotide-gated channel trapping.

Olfactory receptor neurons (ORNs) employ a cyclic nucleotide-gated (CNG) channel to generate a receptor current in response to an odorant-induced rise in cAMP. This channel contains three types of subunits, the principal CNGA2 subunit and two modulatory subunits (CNGA4 and CNGB1b). Here, we have analyzed the functional relevance of CNGB1 for olfaction by gene targeting in mice. Electro-olfactogram responses of CNGB1-deficient (CNGB1-/-) mice displayed a reduced maximal amplitude and decelerated onset and recovery kinetics compared with wild-type mice. In a behavioral test, CNGB1-/- mice exhibited a profoundly decreased olfactory performance. Electrophysiological recordings revealed that ORNs of CNGB1-/- mice weakly expressed a CNG current with decreased cAMP sensitivity, very rapid flicker-gating behavior and no fast modulation by Ca2+-calmodulin. Co-immunoprecipitation confirmed the presence of a CNGA2/CNGA4 channel in the olfactory epithelium of CNGB1-/- mice. This CNGA2/CNGA4 channel was targeted to the plasma membrane of olfactory knobs, but failed to be trafficked into olfactory cilia. Interestingly, we observed a similar trafficking defect in mice deficient for the CNGA4 subunit. In conclusion, these results demonstrate that CNGB1 has a dual function in vivo. First, it endows the olfactory CNG channel with a variety of biophysical properties tailored to the specific requirements of olfactory transduction. Second, together with the CNGA4 subunit, CNGB1 is needed for ciliary targeting of the olfactory CNG channel.

Olfactory marker protein modulates the cAMP kinetics of the odour‐induced response in cilia of mouse olfactory receptor neurons

Olfactory marker protein (OMP), a phylogenetically conserved protein, is highly, and almost exclusively, expressed in vertebrate olfactory receptor neurons (ORNs). Although OMP is widely used as a marker for ORNs, its function has remained largely elusive. Here we used suction‐pipette recordings from isolated ORNs of OMP−/− mice to investigate its role in olfactory transduction. Vertebrate olfactory transduction is initiated when odourants bind to receptor proteins to activate an adenylyl cyclase via a G protein‐coupled signalling pathway. This leads to an increase in cAMP and the opening of a cyclic nucleotide‐gated (CNG), non‐selective cation channel which depolarizes the cells. Ca2+ influx through the CNG channel in turn activates a Ca2+‐activated Cl− channel, causing a Cl− efflux and further depolarization. In the absence of OMP, the time‐to‐transient‐peak of the response, the latency to first spike, and the response termination were slowed 2‐ to 8‐fold, indicating its role in regulating olfactory response kinetics and termination. This phenotype persisted in OMP−/− ORNs even in low external Ca2+ solution chosen to prevent Cl− channel activation, suggesting OMP acts upstream of Cl− channel activation. Furthermore, the response kinetics in cilia are virtually indistinguishable between OMP−/− and wild‐type ORNs when intracellular cAMP level was elevated by the phospho‐diesterase inhibitor, IBMX, suggesting OMP acts upstream of cAMP production. Together, our results suggest a role for OMP in regulating the kinetics and termination of olfactory responses, implicating a novel mechanism for fast and robust response termination to ensure the temporal resolution of the odour stimulus. These observations also help explain the deficits in odour detection threshold and odour quality discrimination seen in the OMP−/− mice.

Origin of basal activity in mammalian olfactory receptor neurons.

Mammalian odorant receptors form a large, diverse group of G protein–coupled receptors that determine the sensitivity and response profile of olfactory receptor neurons. But little is known if odorant receptors control basal and also stimulus-induced cellular properties of olfactory receptor neurons other than ligand specificity. This study demonstrates that different odorant receptors have varying degrees of basal activity, which drives concomitant receptor current fluctuations and basal action potential firing. This basal activity can be suppressed by odorants functioning as inverse agonists. Furthermore, odorant-stimulated olfactory receptor neurons expressing different odorant receptors can have strikingly different response patterns in the later phases of prolonged stimulation. Thus, the influence of odorant receptor choice on response characteristics is much more complex than previously thought, which has important consequences on odor coding and odor information transfer to the brain.

Signaling by olfactory receptor neurons near threshold

An important contributing factor for the high sensitivity of sensory systems is the exquisite sensitivity of the sensory receptor cells. We report here the signaling threshold of the olfactory receptor neuron (ORN). We first obtained a best estimate of the size of the physiological electrical response successfully triggered by a single odorant-binding event on a frog ORN, which was ∼0.034 pA and had an associated transduction domain spanning only a tiny fraction of the length of an ORN cilium. We also estimated the receptor-current threshold for an ORN to fire action potentials in response to an odorant pulse, which was ∼1.2 pA. Thus, it takes about 35 odorant-binding events successfully triggering transduction during a brief odorant pulse in order for an ORN to signal to the brain.

Perspectives on: Information and coding in mammalian sensory physiology: Response kinetics of olfactory receptor neurons and the implications in olfactory coding

Olfaction begins with the detection of odorants by olfactory receptor neurons (ORNs) in the nasal cavity. Olfactory transduction is mediated by a G protein–coupled transduction cascade culminating in the opening of the two olfactory transduction ion channels, the olfactory CNG channel and the Ca2+-activated Cl− channel anoctamin 2 (Ano2), and ultimately action potential (AP) generation. The mechanisms that activate olfactory transduction have been understood quite well over the last two decades. Mechanisms of response adaptation, however, have actually become much less clear, with mechanisms previously thought to be important now suggested to play less significant roles, raising the question of which transduction components are the target of adaptational feedback. Because ORNs are often stimulated rhythmically by the inhalation of odorants, fast response termination should be a prerequisite to adequately resolve the temporal aspect of the stimulus. Recent progress suggests that mechanisms that regulate ciliary Ca2+ transients dictate kinetics of transduction termination. Ultimately, the question to answer is how ORNs code for “natural” stimuli in the behaving animal.