Analyse der Struktur, Funktion und Evolution der Astacin-Protein-Familie

Astacin (E.C. 3.4.24.21) aus dem Magen des Flusskrebses Astacus astacus ist der Prototyp einer Familie von Zink-Endopeptidasen, deren Mitglieder bei Vertebraten, Invertebraten und Bakterien gefunden werden. Bei dem Nematoden Caenorhabditis elegans konnte die überraschend große Zahl von 40 Astacin-homologen Genen identifiziert werden. Eine Transkriptom-Analyse ergab, dass mit Ausnahme eines Pseudogens alle anderen Astacine tatsächlich exprimiert werden. GFP-Reporter-Proteine zeigten für zwei Astacine eine Verdauungsfunktion und eine entwicklungsrelevante Rolle. Bei einer Proteom-Analyse durch 2D-Elektrophorese konnten neue Proteine der C. elegans Proteinkarte zugeordnet werden. Bei dem Modellorganismus Hydractinia echinata konnten zwei neue Astacin-homologe Gene charakterisiert werden, die als HEA-1 und HEA-2 bezeichnet wurden. In situ Hybridisierungen deuten eine Rolle bei der Kopfbildung und bei der Differenzierung von Tentakelzellen an. Durch BLAST- und FASTA-Suchen in den Datenbanken konnten insgesamt 106 verschiedene Astacin-homologe Proteine gefunden werden. Die phylogenetische Analyse und die Analyse der Domänenstruktur ließen ein allgemeines Evolutions-Prinzip erkennen, wonach die katalytische Kette des Astacins nach Anhängung von regulatorischen Einheiten als ein Modul in unterschiedlicher Umgebung und mit neuen Funktionen eingesetzt werden kann. Weiterhin erlaubte die Stammbaum-Analyse eine Einteilung der Astacine in mehrere phylogenetische Gruppen. Als Modellprotein für die Untersuchung des Aktivierungsmechansimus von Pro-Astacinen wurde das Flusskrebs-Astacin verwendet. Die immunhistochemischen, proteinbiochemischen und die massenspektrometrischen Untersuchungen von Substrat-Peptiden zeigten, dass Pro-Astacin in Astacus astacus autokatalytisch im proximalen Bereich des Hepatopankreas vor Übertritt in den Magen aktiviert wird. Darüber hinaus konnte auch gezeigt werden, dass die Mehrzahl der Pro-Astacine ebenfalls autokatalytisch aktiviert werden kann

Calmodulin Contributes to Gating Control in Olfactory Calcium-activated Chloride Channels

In sensory neurons of the peripheral nervous system, receptor potentials can be amplified by depolarizing Cl currents. In mammalian olfactory sensory neurons (OSNs), this anion-based signal amplification results from the sequential activation of two distinct types of transduction channels: cAMP-gated Ca channels and Ca-activated Cl channels. The Cl current increases the initial receptor current about 10-fold and leads to the excitation of the neuron. Here we examine the activation mechanism of the Ca-dependent Cl channel. We focus on calmodulin, which is known to mediate Ca effects on various ion channels. We show that the cell line Odora, which is derived from OSN precursor cells in the rat olfactory epithelium, expresses Ca-activated Cl channels. Single-channel conductance, ion selectivity, voltage dependence, sensitivity to niflumic acid, and Ca sensitivity match between Odora channels and OSN channels. Transfection of Odora cells with CaM mutants reduces the Ca sensitivity of the Cl channels. This result points to the participation of calmodulin in the gating process of Ca-ativated Cl channels, and helps to understand how signal amplification works in the olfactory sensory cilia. Calmodulin was previously shown to mediate feedback inhibition of cAMP-synthesis and of the cAMP-gated Ca channels in OSNs. Our results suggest that calmodulin may also be instrumental in the generation of the excitatory Cl current. It appears to play a pivotal role in the peripheral signal processing of olfactory sensory information. Moreover, recent results from other peripheral neurons, as well as from smooth muscle cells, indicate that the calmodulin-controlled, anion-based signal amplification operates in various cell types where it converts Ca signals into membrane depolarization

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

<p>Abstract</p> <p>Background</p> <p>Chloride 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.</p> <p>Results</p> <p>We developed an <it>in vitro </it>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 PGE₂for 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.</p> <p>Conclusion</p> <p>Our 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.</p

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

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, Ca2+-permeable channels and Ca2+-activated chloride channels. The cAMP-gated channels open at micromolar concentrations of their ligand and are subject to a Ca2+-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 Ca2+-dependent feedback inhibition. Each subunit could be assigned to one of these functions that, together, define the gating logic of the olfactory transduction channel

An evolutionary conserved role of wnt signaling in stem cell fate decision

AbstractWnt/Frizzled/ß-catenin-based signaling systems play diverse roles in metazoan development, being involved not only in the establishment of body axes in embryogenesis but also in regulating stem cell fate in mammalian post-embryonic development. We have studied the role the canonical Wnt cascade plays in stem cell fate determination in Hydractinia, a member of the ancient metazoan phylum Cnidaria, by analyzing two key molecules in this pathway, frizzled and ß-catenin, and blocking GSK-3. Generally, frizzled was expressed in cells able to divide but absent in post-mitotic, terminally differentiated cells such as nerve cells and nematocytes. Transcripts of frizzled were identified in all embryonic stages beginning with maternal transcripts in the oocyte. Following gastrulation and in the planula larva, frizzled expression concentrated in the central endodermal mass from which the first interstitial stem cells and their derivatives arise. In post-metamorphic development, high levels of frizzled transcripts were detected in interstitial stem cells. Activating downstream events of the Wnt-cascade in the post-metamorphic life phase by blocking GSK-3 with paullones induced recruitment of nematocytes and nerve cells from the pool of interstitial stem cells. Terminal differentiation was preceded by an initial burst of proliferation of frizzled-positive i-cells. In activated i-cells, ß-catenin appeared in the cytoplasm, later in the nucleus. It was subsequently again observed in the cytoplasm and eventually faded out during terminal differentiation. Our results suggest an ancient role of Wnt signaling in stem cell fate determination

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