Frank Möhrlen

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−/HCO3− 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.

The proteome of rat olfactory sensory cilia.

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

Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons

BackgroundChloride currents in peripheral nociceptive neurons have been implicated in the generation of afferent nociceptive signals, as Cl- accumulation in sensory endings establishes the driving force for depolarizing, and even excitatory, Cl- currents. The intracellular Cl- concentration can, however, vary considerably between individual DRG neurons. This raises the question, whether the contribution of Cl- currents to signal generation differs between individual afferent neurons, and whether the specific Cl- levels in these neurons are subject to modulation. Based on the hypothesis that modulation of the peripheral Cl- homeostasis is involved in the generation of inflammatory hyperalgesia, we examined the effects of inflammatory mediators on intracellular Cl- concentrations and on the expression levels of Cl- transporters in rat DRG neurons.ResultsWe developed an in vitro assay for testing how inflammatory mediators influence Cl- concentration and the expression of Cl- transporters. Intact DRGs were treated with 100 ng/ml NGF, 1.8 μM ATP, 0.9 μM bradykinin, and 1.4 μM PGE2 for 1–3 hours. Two-photon fluorescence lifetime imaging with the Cl--sensitive dye MQAE revealed an increase of the intracellular Cl- concentration within 2 hours of treatment. This effect coincided with enhanced phosphorylation of the Na+-K+-2Cl- cotransporter NKCC1, suggesting that an increased activity of that transporter caused the early rise of intracellular Cl- levels. Immunohistochemistry of NKCC1 and KCC2, the main neuronal Cl- importer and exporter, respectively, exposed an inverse regulation by the inflammatory mediators. While the NKCC1 immunosignal increased, that of KCC2 declined after 3 hours of treatment. In contrast, the mRNA levels of the two transporters did not change markedly during this time. These data demonstrate a fundamental transition in Cl- homeostasis toward a state of augmented Cl- accumulation, which is induced by a 1–3 hour treatment with inflammatory mediators.ConclusionOur findings indicate that inflammatory mediators impact on Cl- homeostasis in DRG neurons. Inflammatory mediators raise intracellular Cl- levels and, hence, the driving force for depolarizing Cl- efflux. These findings corroborate current concepts for the role of Cl- regulation in the generation of inflammatory hyperalgesia and allodynia. As the intracellular Cl- concentration rises in DRG neurons, afferent signals can be boosted by excitatory Cl- currents in the presynaptic terminals. Moreover, excitatory Cl- currents in peripheral sensory endings may also contribute to the generation or modulation of afferent signals, especially in inflamed tissue.

Characterization of the proteolytic enzymes in the midgut of the European Cockchafer, Melolontha melolontha (Coleoptera: Scarabaeidae)

In previous studies we showed that the resistance of the European Cockchafer, Melolontha melolontha, towards the Scarab specific Cry8C toxin of Bacillus thuringiensis japonensis strain Buibui is due to the complexity of proteinases in the midgut of the pest insect. In this study these proteinases were identified and characterized using a combination of synthetic substrates and specific inhibitors in zymograms, activity blots, and photometric/fluorometric assays. In the midgut juice three trypsin-like and three elastase-like serine proteinases are predominantly present. In addition, two metalloendoproteinases were detected. At least one of them is most likely to belong to the astacin family, proteinases which normally do not play a role in general protein digestion outside the decapod crustacean. Furthermore, a free aminopeptidase as well as a membrane-associated aminopeptidase, isolated from the brush boarder membrane vesicles (BBMV) of the midgut epithelium, were characterized.

Differential maturation of chloride homeostasis in primary afferent neurons of the somatosensory system.

Recent research into the generation of hyperalgesia has revealed that both the excitability of peripheral nociceptors and the transmission of their afferent signals in the spinal cord are subject to modulation by Cl− currents. The underlying Cl− homeostasis of nociceptive neurons, in particular its postnatal maturation, is, however, poorly understood. Here we measure the intracellular Cl− concentration, [Cl−]i, of somatosensory neurons in intact dorsal root ganglia of mice. Using two‐photon fluorescence‐lifetime imaging microscopy, we determined [Cl−]i in newborn and adult animals. We found that the somatosensory neurons undergo a transition of Cl− homeostasis during the first three postnatal weeks that leads to a decline of [Cl−]i in most neurons. Immunohistochemistry showed that a major fraction of neurons in the dorsal root ganglia express the cation–chloride co‐transporters NKCC1 and KCC2, indicating that the molecular equipment for Cl− accumulation and extrusion is present. RT‐PCR analysis showed that the transcription pattern of electroneutral Cl− co‐transporters does not change during the maturation process.

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 Ca2+/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 Ca2+/calmodulin, one at submicromolar Ca2+ concentrations and one in the micromolar Ca2+ 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 Ca2+/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca2+ 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 Ca2+ regulation in anoctamin Cl− channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types.

Caged Capsaicins: New Tools for the Examination of TRPV1 Channels in Somatosensory Neurons

The vanilloid capsaicin, N‐(4‐hydroxy‐3‐methoxybenzyl)‐8‐methylnon‐6‐enamide, is the pungent ingredient of chili peppers and is used in pain research as an activating ligand of heat‐sensitive transduction channels in nociceptive neurons. Here we describe the synthesis and application of two capsaicin derivatives modified at the hydroxy function of the vanillyl motif: α‐carboxy‐4,5‐dimethoxy‐2‐nitrobenzyl‐caged (CDMNB‐caged) capsaicin and {7‐[bis(carboxymethyl)amino]coumarin‐4‐yl}methoxycarbonyl‐caged (BCMACMOC‐caged) capsaicin. These compounds show dramatically reduced pungency, but release active capsaicin upon irradiation with UV light. CDMNB‐caged capsaicin can be used to perform concentration‐jump experiments, while BCMACMOC‐caged capsaicin is membrane‐impermeant and can be applied selectively to the intracellular or extracellular sides of a plasma membrane. Both compounds can serve as valuable research tools in pain physiology.

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 Ca2+. 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.

Calcium-signaling networks in olfactory receptor neurons.

The olfactory neuroepithelium represents a unique interface between the brain and the external environment. Olfactory function comprises a distinct set of molecular tasks: sensory signal transduction, cytoprotection and adult neurogenesis. A multitude of biochemical studies has revealed the central role of Ca(2+) signaling in the function of olfactory receptor neurons (ORNs). We set out to establish Ca(2+)-dependent signaling networks in ORN cilia by proteomic analysis. We subjected a ciliary membrane preparation to Ca(2+)/calmodulin-affinity chromatography using mild detergent conditions in order to maintain functional protein complexes involved in olfactory Ca(2+) signaling. Thus, calmodulin serves as a valuable tool to gain access to novel Ca(2+)-regulated protein complexes. Tandem mass spectrometry (nanoscale liquid-chromatography-electrospray injection) identified 123 distinct proteins. Ninety-seven proteins (79%) could be assigned to specific olfactory functions, including 32 to sensory signal transduction and 40 to cytoprotection. We point out novel perspectives for research on the Ca(2+)-signaling networks in the olfactory system of the rat.

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

BackgroundAstacins 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.ResultsWe 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.ConclusionsExpression 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.