Olfactory stimulation Inhibits Nociceptive Signal Processing at the Input Stage of the Central Trigeminal System.

The spinal trigeminal nucleus caudalis (SpVc) in the mammalian brainstem serves a pivotal function in pain processing. As the main relay center for nociceptive signals, SpVc conducts pain-related signals from various regions of the head toward higher levels of central processing such as the thalamus. SpVc also receives modulatory signals from other brain areas, which can alleviate the perception of headache. We studied the impact of olfactory co-stimulation on pain-related behavior and SpVc neural activity in mice. Using the TRPA1 agonist allyl isothiocyanate (AITC) as noxious stimulus, we quantified the aversive response and the perceived pain intensity by evaluating explorative running and the mouse grimace scale, respectively. We found that the floral odorants phenylethyl alcohol (PEA) and lavender oil mitigated the aversive response to AITC. Consistent with this finding, a newly developed, automated quantification of c-Fos expression in SpVc revealed that co-stimulation with PEA or lavender profoundly reduced network activity in the presence of AITC. These results demonstrated a substantial analgesic potential of odor stimulation in the trigeminal system and provide an explanation for the palliative effect of odors in the treatment of headache.

Anoctamin 2-chloride channels reduce simple spike activity and mediate inhibition at elevated calcium concentration in cerebellar Purkinje cells.

Modulation of neuronal excitability is a prominent way of shaping the activity of neuronal networks. Recent studies highlight the role of calcium-activated chloride currents in this context, as they can both increase or decrease excitability. The calcium-activated chloride channel Anoctamin 2 (ANO2 alias TMEM16B) has been described in several regions of the mouse brain, including the olivo-cerebellar system. In inferior olivary neurons, ANO2 was proposed to increase excitability by facilitating the generation of high-threshold calcium spikes. An expression of ANO2 in cerebellar Purkinje cells was suggested, but its role in these neurons remains unclear. In the present study, we confirmed the expression of Ano2 mRNA in Purkinje cells and performed electrophysiological recordings to examine the influence of ANO2-chloride channels on the excitability of Purkinje cells by comparing wildtype mice to mice lacking ANO2. Recordings were performed in acute cerebellar slices of adult mice, which provided the possibility to study the role of ANO2 within the cerebellar cortex. Purkinje cells were uncoupled from climbing fiber input to assess specifically the effect of ANO2 channels on Purkinje cell activity. We identified an attenuating effect of ANO2-mediated chloride currents on the instantaneous simple spike activity both during strong current injections and during current injections close to the simple spike threshold. Moreover, we report a reduction of inhibitory currents from GABAergic interneurons upon depolarization, lasting for several seconds. Together with the role of ANO2-chloride channels in inferior olivary neurons, our data extend the evidence for a role of chloride-dependent modulation in the olivo-cerebellar system that might be important for proper cerebellum-dependent motor coordination and learning.

Photoactivation of olfactory sensory neurons does not affect action potential conduction in individual trigeminal sensory axons innervating the rodent nasal cavity.

Olfactory and trigeminal chemosensory systems reside in parallel within the mammalian nose. Psychophysical studies in people indicate that these two systems interact at a perceptual level. Trigeminal sensations of pungency mask odour perception, while olfactory stimuli can influence trigeminal signal processing tasks such as odour localization. While imaging studies indicate overlap in limbic and cortical somatosensory areas activated by nasal trigeminal and olfactory stimuli, there is also potential cross-talk at the level of the olfactory epithelium, the olfactory bulb and trigeminal brainstem. Here we explored the influence of olfactory and trigeminal signaling in the nasal cavity. A forced choice water consumption paradigm was used to ascertain whether trigeminal and olfactory stimuli could influence behaviour in mice. Mice avoided water sources surrounded by both volatile TRPV1 (cyclohexanone) and TRPA1 (allyl isothiocyanate) irritants and the aversion to cyclohexanone was mitigated when combined with a pure odorant (rose fragrance, phenylethyl alcohol, PEA). To determine whether olfactory-trigeminal interactions within the nose could potentially account for this behavioural effect we recorded from single trigeminal sensory axons innervating the nasal respiratory and olfactory epithelium using an isolated in vitro preparation. To circumvent non-specific effects of chemical stimuli, optical stimulation was used to excite olfactory sensory neurons in mice expressing channel-rhodopsin (ChR2) under the olfactory marker protein (OMP) promoter. Photoactivation of olfactory sensory neurons produced no modulation of axonal action potential conduction in individual trigeminal axons. Similarly, no evidence was found for collateral branching of trigeminal axon that might serve as a conduit for cross-talk between the olfactory and respiratory epithelium and olfactory dura mater. Using direct assessment of action potential activity in trigeminal axons we observed neither paracrine nor axon reflex mediated cross-talk between olfactory and trigeminal sensory systems in the rodent nasal cavity. Our current results suggest that olfactory sensory neurons exert minimal influence on trigeminal signals within the nasal cavity.

Expression and function of Anoctamin 1/TMEM16A calcium-activated chloride channels in airways of in vivo mouse models for cystic fibrosis research.

Physiological processes of vital importance are often safeguarded by compensatory systems that substitute for primary processes in case these are damaged by gene mutation. Ca2+-dependent Cl- secretion in airway epithelial cells may provide such a compensatory mechanism for impaired Cl- secretion via cystic fibrosis transmembrane conductance regulator (CFTR) channels in cystic fibrosis (CF). Anoctamin 1 (ANO1) Ca2+-gated Cl- channels are known to contribute to calcium-dependent Cl- secretion in tracheal and bronchial epithelia. In the present study, two mouse models of CF were examined to assess a potential protective function of Ca2+-dependent Cl- secretion, a CFTR deletion model (cftr-/-), and a CF pathology model that overexpresses the epithelial Na+ channel ß-subunit (ßENaC), which is encoded by the Scnn1b gene, specifically in airway epithelia (Scnn1b-Tg). The expression levels of ANO1 were examined by mRNA and protein content, and the channel protein distribution between ciliated and non-ciliated epithelial cells was analyzed. Moreover, Ussing chamber experiments were conducted to compare Ca2+-dependent Cl- secretion between wild-type animals and the two mouse models. Our results demonstrate that CFTR and ANO1 channels were co-expressed with ENaC in non-ciliated cells of mouse tracheal and bronchial epithelia. Ciliated cells did not express these proteins. Despite co-localization of CFTR and ANO1 in the same cell type, cells in cftr-/- mice displayed no altered expression of ANO1. Similarly, ANO1 expression was unaffected by ßENaC overexpression in the Scnn1b-Tg line. These results suggest that the CF-related environment in the two mouse models did not induce ANO1 overexpression as a compensatory system.

Tracking of unfamiliar odors is facilitated by signal amplification through anoctamin 2 chloride channels in mouse olfactory receptor neurons.

Many animals follow odor trails to find food, nesting sites, or mates, and they require only faint olfactory cues to do so. The performance of a tracking dog, for instance, poses the question on how the animal is able to distinguish a target odor from the complex chemical background around the trail. Current concepts of odor perception suggest that animals memorize each odor as an olfactory object, a percept that enables fast recognition of the odor and the interpretation of its valence. An open question still is how this learning process operates efficiently at the low odor concentrations that typically prevail when animals inspect an odor trail. To understand olfactory processing under these conditions, we studied the role of an amplification mechanism that boosts signal transduction at low stimulus intensities, a process mediated by calcium-gated anoctamin 2 chloride channels. Genetically altered Ano2-/- mice, which lack these channels, display an impaired cue-tracking behavior at low odor concentrations when challenged with an unfamiliar, but not with a familiar, odor. Moreover, recordings from the olfactory epithelium revealed that odor coding lacks sensitivity and temporal resolution in anoctamin 2-deficient mice. Our results demonstrate that the detection of an unfamiliar, weak odor, as well as its memorization as an olfactory object, require signal amplification in olfactory receptor neurons. This process may contribute to the phenomenal tracking abilities of animals that follow odor trails.

Cellular distribution and function of ion channels involved in transport processes in rat tracheal epithelium.

Transport of water and electrolytes in airway epithelia involves chloride-selective ion channels, which are controlled either by cytosolic Ca2+ or by cAMP The contributions of the two pathways to chloride transport differ among vertebrate species. Because rats are becoming more important as animal model for cystic fibrosis, we have examined how Ca2+- dependent and cAMP- dependent Cl- secretion is organized in the rat tracheal epithelium. We examined the expression of the Ca2+-gated Cl- channel anoctamin 1 (ANO1), the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, the epithelial Na+ channel ENaC, and the water channel aquaporin 5 (AQP5) in rat tracheal epithelium. The contribution of ANO1 channels to nucleotide-stimulated Cl- secretion was determined using the channel blocker Ani9 in short-circuit current recordings obtained from primary cultures of rat tracheal epithelial cells in Ussing chambers. We found that ANO1, CFTR and AQP5 proteins were expressed in nonciliated cells of the tracheal epithelium, whereas ENaC was expressed in ciliated cells. Among nonciliated cells, ANO1 occurred together with CFTR and Muc5b and, in addition, in a different cell type without CFTR and Muc5b. Bioelectrical studies with the ANO1-blocker Ani9 indicated that ANO1 mediated the secretory response to the nucleotide uridine-5'-triphosphate. Our data demonstrate that, in rat tracheal epithelium, Cl- secretion and Na+ absorption are routed through different cell types, and that ANO1 channels form the molecular basis of Ca2+-dependent Cl- secretion in this tissue. These characteristic features of Cl--dependent secretion reveal similarities and distinct differences to secretory processes in human airways.

Impaired Motor Coordination and Learning in Mice Lacking Anoctamin 2 Calcium-Gated Chloride Channels.

Neurons communicate through excitatory and inhibitory synapses. Both lines of communication are adjustable and allow the fine tuning of signal exchange required for learning processes in neural networks. Several distinct modes of plasticity modulate glutamatergic and GABAergic synapses in Purkinje cells of the cerebellar cortex to promote motor control and learning. In the present paper, we present evidence for a role of short-term ionic plasticity in the cerebellar circuit activity. This type of plasticity results from altered chloride driving forces at the synapses that molecular layer interneurons form on Purkinje cell dendrites. Previous studies have provided evidence for transiently diminished chloride gradients at these GABAergic synapses following climbing fiber activity. Electrical stimulation of climbing fibers in acute slices caused a decline of inhibitory postsynaptic currents recorded from Purkinje cells. Dendritic calcium-gated chloride channels of the type anoctamin 2 (ANO2) were proposed to mediate this short-term modulation of inhibition, but the significance of this process for motor control has not been established yet. Here, we report results of behavioral studies obtained from Ano2 -/- mice, a mouse line that was previously shown to lack this particular mode of ionic plasticity. The animals display motor coordination deficits that constitute a condition of mild ataxia. Moreover, motor learning is severely impaired in Ano2 -/- mice, suggesting cerebellar dysfunction. This reduced motor performance of Ano2 -/- mice highlights the significance of inhibitory control for cerebellar function and introduces calcium-dependent short-term ionic plasticity as an efficient control mechanism for neural inhibition.

Possible role of calcitonin gene-related peptide in trigeminal modulation of glomerular microcircuits of the rodent olfactory bulb.

Chemosensation in the mammalian nose comprises detection of odorants, irritants and pheromones. While the traditional view assigned one distinct sub-system to each stimulus type, recent research has produced a more complex picture. Odorants are not only detected by olfactory sensory neurons but also by the trigeminal system. Irritants, in turn, may have a distinct odor, and some pheromones are detected by the olfactory epithelium. Moreover, it is well established that irritants change odor perception and vice versa. A wealth of psychophysical evidence on olfactory-trigeminal interactions in humans contrasts with a paucity of structural insight. In particular, it is unclear whether the two systems communicate just by sharing stimuli, or whether neuronal connections mediate cross-modal signaling. One connection could exist in the olfactory bulb that performs the primary processing of olfactory signals and receives trigeminal innervation. In the present study, neuroanatomical tracing of the mouse ethmoid system illustrates how peptidergic fibers enter the glomerular layer of the olfactory bulb, where local microcircuits process and filter the afferent signal. Biochemical assays reveal release of calcitonin gene-related peptide from olfactory bulb slices and attenuation of cAMP signaling by the neuropeptide. In the non-stimulated tissue, the neuropeptide specifically inhibited the basal activity of calbindin-expressing periglomerular interneurons, but did not affect the basal activity of neurons expressing calretinin, parvalbumin, or tyrosine hydroxylase, nor the activity of astrocytes. This study represents a first step towards understanding trigeminal neuromodulation of olfactory-bulb microcircuits and provides a working hypothesis for trigeminal inhibition of olfactory signal processing. This article is protected by copyright. All rights reserved.

Protein O-Mannosylation in the Murine Brain: Occurrence of Mono-O-Mannosyl Glycans and Identification of New Substrates.

Protein O-mannosylation is a post-translational modification essential for correct development of mammals. In humans, deficient O-mannosylation results in severe congenital muscular dystrophies often associated with impaired brain and eye development. Although various O-mannosylated proteins have been identified in the recent years, the distribution of O-mannosyl glycans in the mammalian brain and target proteins are still not well defined. In the present study, rabbit monoclonal antibodies directed against the O-mannosylated peptide YAT(α1-Man)AV were generated. Detailed characterization of clone RKU-1-3-5 revealed that this monoclonal antibody recognizes O-linked mannose also in different peptide and protein contexts. Using this tool, we observed that mono-O-mannosyl glycans occur ubiquitously throughout the murine brain but are especially enriched at inhibitory GABAergic neurons and at the perineural nets. Using a mass spectrometry-based approach, we further identified glycoproteins from the murine brain that bear single O-mannose residues. Among the candidates identified are members of the cadherin and plexin superfamilies and the perineural net protein neurocan. In addition, we identified neurexin 3, a cell adhesion protein involved in synaptic plasticity, and inter-alpha-trypsin inhibitor 5, a protease inhibitor important in stabilizing the extracellular matrix, as new O-mannosylated glycoproteins.

Properties of an optogenetic model for olfactory stimulation.

KEY POINTS: In olfactory research it is difficult to deliver stimuli with defined intensity and duration to olfactory sensory neurons. Expression of channelrhodopsin 2 (ChR2) in olfactory sensory neurons provides a means to activate these neurons with light flashes. Appropriate mouse models are available. The present study explores the suitability of an established olfactory marker protein (OMP)/ChR2-yellow fluorescent protein (YFP) mouse model for ex vivo experimentation. Expression of ChR2 in sensory neurons of the main olfactory epithelium, the septal organ and vomeronasal organ is characterized. Expression pattern of ChR2 in olfactory receptor neurons and the properties of light responses indicate that light stimulation does not impact on signal transduction in the chemosensory cilia. Light-induced electro-olfactograms are characterized with light flashes of different intensities, durations and frequencies. The impact of light-induced afferent stimulation on the olfactory bulb is examined with respect to response amplitude, polarity and low-pass filtering. ABSTRACT: For the examination of sensory processing, it is helpful to deliver stimuli in precisely defined temporal and spatial patterns with accurate control of stimulus intensity. This is challenging in experiments with the mammalian olfactory system because airborne odorants have to be transported into the intricate sensory structures of the nose and must dissolve in mucus to be detected by sensory neurons. Defined and reproducible activity can be generated in olfactory sensory neurons that express the light-gated ion channel channelrhodopsin 2 (ChR2). The neurons can be stimulated by light flashes in a controlled fashion by this optogenetic approach. Here we examined the application of an olfactory marker protein (OMP)/ChR2-yellow fluorescent protein (YFP) model for ex vivo exploration of the olfactory epithelium and the olfactory bulb of the mouse. We studied the expression patterns of ChR2 in the main olfactory system, the vomeronasal system, and the septal organ, and we found that ChR2 is absent from the sensory cilia of olfactory sensory neurons. In the olfactory epithelium, we characterized light-induced electro-olfactograms with respect to peripheral encoding of stimulus intensity, stimulus duration and stimulus frequency. In acute slices of the olfactory bulb, we identified specific aspects of the ChR2-induced input signal, concerning its dynamic range, its low-pass filter property and its response to prolonged stimulation. Our study describes the performance of the OMP/ChR2-YFP model for ex vivo experimentation on the peripheral olfactory system and documents its versatility and its limitations for olfactory research.

Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex.

Calcium-activated chloride channels of the anoctamin (alias TMEM16) protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca2+-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum.

Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels.

Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca(2+)/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca(2+)/calmodulin, one at submicromolar Ca(2+) concentrations and one in the micromolar Ca(2+) range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca(2+)/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca(2+) signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca(2+) regulation in anoctamin Cl(-) channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types.

Targeted expression of anoctamin calcium-activated chloride channels in rod photoreceptor terminals of the rodent retina.

PURPOSE: In the vertebrate retina, calcium-activated chloride channels are expressed in photoreceptor synaptic terminals. These channels are involved in the control of transmitter release in the dark. The search for their molecular identity has recently lead to the localization of the protein anoctamin 2 (also TMEM16B) in the outer plexiform layer of the rodent retina. Since both rod and cone photoreceptors have their terminals in this layer, it was not clear which of these express anoctamin 2. Here, we examine rod spherules and cone pedicles for expression of anoctamin 2. METHODS: Expression of anoctamin genes was studied in the rat eye using RT-PCR. Immunohistochemical experiments were used to localize anoctamins and chloride transporters with their regulatory kinases. Photoreceptor synaptic proteins, as well as the lectins Peanut agglutinin and Griffonia simplicifolia agglutinin, were used to distinguish retinal structures. RESULTS: Anoctamin 1, 2, and 10 were found to be expressed in the eye. Anoctamin 2 was expressed as a splice variant that includes exon 15 of the genomic structure. The protein is exclusively expressed in rod terminals and is not present in cone pedicles. Expression is not clustered at the ribbon complex, but spread across the presynaptic membrane where it colocalizes with the plasma membrane calcium pump. The electroneutral chloride transporter NKCC1 is expressed in photoreceptor terminals, together with its regulatory kinases SPAK and OSR1. CONCLUSIONS: Rod photoreceptor terminals possess the molecular machinery for chloride accumulation and for the generation of calcium-dependent chloride currents conducted through anoctamin 2 channels. We discuss this finding in the framework of the established hypothesis that calcium-activated chloride channels are part of a feedback inhibition mechanism that limits transmitter release in the dark.

Neuropeptide receptors provide a signalling pathway for trigeminal modulation of olfactory transduction.

The mammalian olfactory epithelium contains olfactory receptor neurons and trigeminal sensory endings. The former mediate odor detection, the latter the detection of irritants. The two apparently parallel chemosensory systems are in reality interdependent in various well-documented ways. Psychophysical studies have shown that virtually all odorants can act as irritants, and that most irritants have an odor. Thus, the sensory perception of odorants and irritants is based on simultaneous input from the two systems. Moreover, functional interactions between the olfactory system and the trigeminal system exist on both peripheral and central levels. Here we examine the impact of trigeminal stimulation on the odor response of olfactory receptor neurons. Using an odorant with low trigeminal potency (phenylethyl alcohol) and a non-odorous irritant (CO(2) ), we have explored this interaction in psychophysical experiments with human subjects and in electroolfactogram (EOG) recordings from rats. We have demonstrated that simultaneous activation of the trigeminal system attenuates the perception of odor intensity and distorts the EOG response. On the molecular level, we have identified a route for this cross-modal interaction. The neuropeptide calcitonin-gene related peptide (CGRP), which is released from trigeminal sensory fibres upon irritant stimulation, inhibits the odor response of olfactory receptor neurons. CGRP receptors expressed by these neurons mediate this neuromodulatory effect. This study demonstrates a site of trigeminal-olfactory interaction in the periphery. It reveals a pathway for trigeminal impact on olfactory signal processing that influences odor perception.

Expression patterns of anoctamin 1 and anoctamin 2 chloride channels in the mammalian nose.

Calcium-activated chloride channels are expressed in chemosensory neurons of the nose and contribute to secretory processes and sensory signal transduction. These channels are thought to be members of the family of anoctamins (alternative name: TMEM16 proteins), which are opened by micromolar concentrations of intracellular Ca(2+). Two family members,ANO 1 (TMEM16A) and ANO 2 (TMEM16B), are expressed in the various sensory and respiratory tissues of the nose.We have examined the tissue specificity and sub-cellular localization of these channels in the nasal respiratory epithelium and in the five chemosensory organs of the nose: the main olfactory epithelium, the septal organ of Masera, the vomeronasal organ, the Grueneberg ganglion and the trigeminal system. We have found that the two channels show mutually exclusive expression patterns. ANO 1 is present in the apical membranes of various secretory epithelia in which it is co-localized with the water channel aquaporin 5. It has also been detected in acinar cells and duct cells of subepithelial glands and in the supporting cells of sensory epithelia. In contrast, ANO 2 expression is restricted to chemosensory neurons in which it has been detected in microvillar and ciliary surface structures. The different expression patterns of ANO 1 and ANO 2 have been observed in the olfactory, vomeronasal and respiratory epithelia. No expression has been detected in the Grueneberg ganglion or trigeminal sensory fibers. On the basis of this differential expression, we derive the main functional features of ANO 1 and ANO 2 chloride channels in the nose and suggest their significance for nasal physiology.

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.

Molecular components of signal amplification in olfactory sensory cilia.

The mammalian olfactory system detects an unlimited variety of odorants with a limited set of odorant receptors. To cope with the complexity of the odor world, each odorant receptor must detect many different odorants. The demand for low odor selectivity creates problems for the transduction process: the initial transduction step, the synthesis of the second messenger cAMP, operates with low efficiency, mainly because odorants bind only briefly to their receptors. Sensory cilia of olfactory receptor neurons have developed an unusual solution to this problem. They accumulate chloride ions at rest and discharge a chloride current upon odor detection. This chloride current amplifies the receptor potential and promotes electrical excitation. We have studied this amplification process by examining identity, subcellular localization, and regulation of its molecular components. We found that the Na(+)/K(+)/2Cl(-) cotransporter NKCC1 is expressed in the ciliary membrane, where it mediates chloride accumulation into the ciliary lumen. Gene silencing experiments revealed that the activity of this transporter depends on the kinases SPAK and OSR1, which are enriched in the cilia together with their own activating kinases, WNK1 and WNK4. A second Cl(-) transporter, the Cl(-)/HCO(3)(-) exchanger SLC4A1, is expressed in the cilia and may support Cl(-) accumulation. The calcium-dependent chloride channel TMEM16B (ANO2) provides a ciliary pathway for the excitatory chloride current. These findings describe a specific set of ciliary proteins involved in anion-based signal amplification. They provide a molecular concept for the unique strategy that allows olfactory sensory neurons to operate as efficient transducers of weak sensory stimuli.

Characterization of the astacin family of metalloproteases in C. elegans.

BACKGROUND: Astacins are a large family of zinc metalloproteases found in bacteria and animals. They have diverse roles ranging from digestion of food to processing of extracellular matrix components. The C. elegans genome contains an unusually large number of astacins, of which the majority have not been functionally characterized yet. RESULTS: We analyzed the expression pattern of previously uncharacterized members of the astacin family to try and obtain clues to potential functions. Prominent sites of expression for many members of this family are the hypodermis, the alimentary system and several specialized cells including sensory sheath and sockets cells, which are located at openings in the body wall. We isolated mutants affecting representative members of the various subfamilies. Mutants in nas-5, nas-21 and nas-39 (the BMP-1/Tolloid homologue) are viable and have no apparent phenotypic defects. Mutants in nas-6 and nas-6; nas-7 double mutants are slow growing and have defects in the grinder of the pharynx, a cuticular structure important for food processing. CONCLUSIONS: Expression data and phenotypic characterization of selected family members suggest a diversity of functions for members of the astacin family in nematodes. In part this might be due to extracellular structures unique to nematodes.

Activation and desensitization of the olfactory cAMP-gated transduction channel: identification of functional modules.

Olfactory receptor neurons respond to odor stimulation with a receptor potential that results from the successive activation of cyclic AMP (cAMP)-gated, Ca(2+)-permeable channels and Ca(2+)-activated chloride channels. The cAMP-gated channels open at micromolar concentrations of their ligand and are subject to a Ca(2+)-dependent feedback inhibition by calmodulin. Attempts to understand the operation of these channels have been hampered by the fact that the channel protein is composed of three different subunits, CNGA2, CNGA4, and CNGB1b. Here, we explore the individual role that each subunit plays in the gating process. Using site-directed mutagenesis and patch clamp analysis, we identify three functional modules that govern channel operation: a module that opens the channel, a module that stabilizes the open state at low cAMP concentrations, and a module that mediates rapid Ca(2+)-dependent feedback inhibition. Each subunit could be assigned to one of these functions that, together, define the gating logic of the olfactory transduction channel.

Bestrophin 2: an anion channel associated with neurogenesis in chemosensory systems.

The chemosensory neuroepithelia of the vertebrate olfactory system share a life-long ability to regenerate. Novel neurons proliferate from basal stem cells that continuously replace old or damaged sensory neurons. The sensory neurons of the mouse and rat olfactory system specifically express bestrophin 2, a member of the bestrophin family of calcium-activated chloride channels. This channel was recently proposed to operate as a transduction channel in olfactory sensory cilia. We raised a polyclonal antibody against bestrophin 2 and characterized the expression pattern of this protein in the mouse main olfactory epithelium, septal organ of Masera, and vomeronasal organ. Comparison with the maturation markers growth-associated protein 43 and olfactory marker protein revealed that bestrophin 2 was expressed in developing sensory neurons of all chemosensory neuroepithelia, but was restricted to proximal cilia in mature sensory neurons. Our results suggest that bestrophin 2 plays a critical role during differentiation and growth of axons and cilia. In mature olfactory receptor neurons, it appears to support growth and function of sensory cilia.