Arnd Baumann

Primary structure and functional expression of a Drosophila cyclic nucleotide-gated channel present in eyes and antennae.

Cyclic nucleotide‐gated (CNG) ion channels serve as downstream targets of signalling pathways in vertebrate photoreceptors and olfactory sensory neurons. Whether CNG channels subserve similar functions in invertebrate photoreception and olfaction is unknown. We have cloned genomic DNA and cDNA encoding a cGMP‐gated channel from Drosophila. The gene contains at least seven exons. Heterologous expression of cloned cDNA in both Xenopus oocytes and HEK 293 cells gives rise to functional ion channels. The Drosophila CNG channel is approximately 50‐fold more sensitive to cGMP than to cAMP. The voltage dependence of blockage by divalent cations is different compared with the CNG channel of rod photoreceptors, and the Ca2+ permeability is much larger. The channel mRNA is expressed in antennae and the visual system of Drosophila. It is proposed that CNG channels are involved in transduction cascades of both invertebrate photoreceptors and olfactory sensillae.

Amtyr1: characterization of a gene from honeybee (Apis mellifera) brain encoding a functional tyramine receptor.

Abstract: Biogenic amine receptors are involved in the regulation and modulation of various physiological and behavioral processes in both vertebrates and invertebrates. We have cloned a member of this gene family from the CNS of the honeybee, Apis mellifera. The deduced amino acid sequence is homologous to tyramine receptors cloned from Locusta migratoria and Drosophila melanogaster as well as to an octopamine receptor cloned from Heliothis virescens. Functional properties of the honeybee receptor were studied in stably transfected human embryonic kidney 293 cells. Tyramine reduced forskolin‐induced cyclic AMP production in a dose‐dependent manner with an EC50 of ∼130 nM. A similar effect of tyramine was observed in membrane homogenates of honeybee brains. Octopamine also reduced cyclic AMP production in the transfected cell line but was both less potent (EC50 of ∼3 μM) and less efficacious than tyramine. Receptor‐encoding mRNA has a widespread distribution in the brain and subesophageal ganglion of the honeybee, suggesting that this tyramine receptor is involved in sensory signal processing as well as in higher‐order brain functions.

Characterization of a Dopamine D1 Receptor from Apis mellifera: Cloning, Functional Expression, Pharmacology, and mRNA Localization in the Brain

Abstract: The neurotransmitter dopamine is an important regulator of physiological and behavioral functions in both vertebrates and invertebrates. We have isolated a homologue of the vertebrate dopamine D1 receptor subfamily from the honeybee Apis mellifera. [3H]Lysergic acid diethylamide specifically binds to the heterologously expressed receptor with KD∼5 nM. Dopaminergic receptor ligands compete for this high‐affinity binding, with the following order of potency: R(+)‐lisuride > chlorpromazine = cis(Z)‐flupentixol > dopamine > S(+)‐butaclamol > R(+)‐SCH 23390 > haloperidol. Activation of the heterologously expressed receptor of Apis mellifera leads to cyclic AMP production. Receptor mRNA is expressed in perikarya of different brain neuropils, including those of mushroom body intrinsic neurons. These results suggest that this dopamine receptor is involved in signal processing of visual and olfactory information in the honeybee.

Molecular and functional characterization of an octopamine receptor from honeybee (Apis mellifera) brain

Biogenic amines and their receptors regulate and modulate many physiological and behavioural processes in animals. In vertebrates, octopamine is only found in trace amounts and its function as a true neurotransmitter is unclear. In protostomes, however, octopamine can act as neurotransmitter, neuromodulator and neurohormone. In the honeybee, octopamine acts as a neuromodulator and is involved in learning and memory formation. The identification of potential octopamine receptors is decisive for an understanding of the cellular pathways involved in mediating the effects of octopamine. Here we report the cloning and functional characterization of the first octopamine receptor from the honeybee, Apis mellifera. The gene was isolated from a brain‐specific cDNA library. It encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis. Signalling properties of the cloned receptor were studied in transiently transfected human embryonic kidney (HEK) 293 cells. Nanomolar to micromolar concentrations of octopamine induced oscillatory increases in the intracellular Ca2+ concentration. In contrast to octopamine, tyramine only elicited Ca2+ responses at micromolar concentrations. The gene is abundantly expressed in many somata of the honeybee brain, suggesting that this octopamine receptor is involved in the processing of sensory inputs, antennal motor outputs and higher‐order brain functions.

A family of octapamine receptors that specifically induce cyclic AMP production or Ca2+ release in Drosophila melanogaster

In invertebrates, the biogenic‐amine octopamine is an important physiological regulator. It controls and modulates neuronal development, circadian rhythm, locomotion, ‘fight or flight’ responses, as well as learning and memory. Octopamine mediates its effects by activation of different GTP‐binding protein (G protein)‐coupled receptor types, which induce either cAMP production or Ca2+ release. Here we describe the functional characterization of two genes from Drosophila melanogaster that encode three octopamine receptors. The first gene (Dmoa1) codes for two polypeptides that are generated by alternative splicing. When heterologously expressed, both receptors cause oscillatory increases of the intracellular Ca2+ concentration in response to applying nanomolar concentrations of octopamine. The second gene (Dmoa2) codes for a receptor that specifically activates adenylate cyclase and causes a rise of intracellular cAMP with an EC50 of ∼3 × 10−8 m octopamine. Tyramine, the precursor of octopamine biosynthesis, activates all three receptors at ≥ 100‐fold higher concentrations, whereas dopamine and serotonin are non‐effective. Developmental expression of Dmoa genes was assessed by RT–PCR. Overlapping but not identical expression patterns were observed for the individual transcripts. The genes characterized in this report encode unique receptors that display signature properties of native octopamine receptors.

Molecular determinants of a Ca2+-binding site in the pore of cyclic nucleotide-gated channels: S5/S6 segments control affinity of intrapore glutamates.

Cyclic nucleotide‐gated (CNG) channels play an important role in Ca2+ signaling in many cells. CNG channels from various tissues differ profoundly in their Ca2+ permeation properties. Using the voltage‐dependent Ca2+ blockage of monovalent current in wild‐type channels, chimeric constructs and point mutants, we have identified structural elements that determine the distinctively different interaction of Ca2+ with CNG channels from rod and cone photoreceptors and olfactory neurons. Segments S5 and S6 and the extracellular linkers flanking the pore region are the only structural elements that account for the differences between channels. Ca2+ blockage is strongly modulated by external pH. The different pH dependence of blockage suggests that the pKa of intrapore glutamates and their protonation pattern differ among channels. The results support the hypothesis that the S5–pore–S6 module, by providing a characteristic electrostatic environment, determines the protonation state of pore glutamates and thereby controls Ca2+ affinity and permeation in each channel type.

Postnatal Expression Pattern of HCN Channel Isoforms in Thalamic Neurons: Relationship to Maturation of Thalamocortical Oscillations

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels are the molecular substrate of the hyperpolarization-activated inward current (Ih). Because the developmental profile of HCN channels in the thalamus is not well understood, we combined electrophysiological, molecular, immunohistochemical, EEG recordings in vivo, and computer modeling techniques to examine HCN gene expression and Ih properties in rat thalamocortical relay (TC) neurons in the dorsal part of the lateral geniculate nucleus and the functional consequence of this maturation. Recordings of TC neurons revealed an approximate sixfold increase in Ih density between postnatal day 3 (P3) and P106, which was accompanied by significantly altered current kinetics, cAMP sensitivity, and steady-state activation properties. Quantification on tissue levels revealed a significant developmental decrease in cAMP. Consequently the block of basal adenylyl cyclase activity was accompanied by a hyperpolarizing shift of the Ih activation curve in young but not adult rats. Quantitative analyses of HCN channel isoforms revealed a steady increase of mRNA and protein expression levels of HCN1, HCN2, and HCN4 with reduced relative abundance of HCN4. Computer modeling in a simplified thalamic network indicated that the occurrence of rhythmic delta activity, which was present in the EEG at P12, differentially depended on Ih conductance and modulation by cAMP at different developmental states. These data indicate that the developmental increase in Ih density results from increased expression of three HCN channel isoforms and that isoform composition and intracellular cAMP levels interact in determining Ih properties to enable progressive maturation of rhythmic slow-wave sleep activity patterns.

Am5‐HT7: molecular and pharmacological characterization of the first serotonin receptor of the honeybee (Apis mellifera)

The biogenic amine serotonin (5‐HT) plays a key role in the regulation and modulation of many physiological and behavioural processes in both vertebrates and invertebrates. These functions are mediated through the binding of serotonin to its receptors, of which 13 subtypes have been characterized in vertebrates. We have isolated a cDNA from the honeybee Apis mellifera (Am5‐ht7) sharing high similarity to members of the 5‐HT7 receptor family. Expression of the Am5‐HT7 receptor in HEK293 cells results in an increase in basal cAMP levels, suggesting that Am5‐HT7 is expressed as a constitutively active receptor. Serotonin application to Am5‐ht7‐transfected cells elevates cyclic adenosine 3′,5′‐monophosphate (cAMP) levels in a dose‐dependent manner (EC50 = 1.1–1.8 nm). The Am5‐HT7 receptor is also activated by 5‐carboxamidotryptamine, whereas methiothepin acts as an inverse agonist. Receptor expression has been investigated by RT‐PCR, in situ hybridization, and western blotting experiments. Receptor mRNA is expressed in the perikarya of various brain neuropils, including intrinsic mushroom body neurons, and in peripheral organs. This study marks the first comprehensive characterization of a serotonin receptor in the honeybee and should facilitate further analysis of the role(s) of the receptor in mediating the various central and peripheral effects of 5‐HT.

Molecular, pharmacological, and signaling properties of octopamine receptors from honeybee (Apis mellifera) brain.

G protein‐coupled receptors are important regulators of cellular signaling processes. Within the large family of rhodopsin‐like receptors, those binding to biogenic amines form a discrete subgroup. Activation of biogenic amine receptors leads to transient changes of intracellular Ca2+‐([Ca2+]i) or 3′,5′‐cyclic adenosine monophosphate ([cAMP]i) concentrations. Both second messengers modulate cellular signaling processes and thereby contribute to long‐lasting behavioral effects in an organism. In vivo pharmacology has helped to reveal the functional effects of different biogenic amines in honeybees. The phenolamine octopamine is an important modulator of behavior. Binding of octopamine to its receptors causes elevation of [Ca2+]i or [cAMP]i. To date, only one honeybee octopamine receptor that induces Ca2+ signals has been molecularly and pharmacologically characterized. Here, we examined the pharmacological properties of four additional honeybee octopamine receptors. When heterologously expressed, all receptors induced cAMP production after binding to octopamine with EC50s in the nanomolar range. Receptor activity was most efficiently blocked by mianserin, a substance with antidepressant activity in vertebrates. The rank order of inhibitory potency for potential receptor antagonists was very similar on all four honeybee receptors with mianserin >> cyproheptadine > metoclopramide > chlorpromazine > phentolamine. The subroot of octopamine receptors activating adenylyl cyclases is the largest that has so far been characterized in arthropods, and it should now be possible to unravel the contribution of individual receptors to the physiology and behavior of honeybees.

Molecular and Pharmacological Characterization of Serotonin 5-HT2α and 5-HT7 Receptors in the Salivary Glands of the Blowfly Calliphora vicina

Secretion in blowfly (Calliphora vicina) salivary glands is stimulated by the biogenic amine serotonin (5-hydroxytryptamine, 5-HT), which activates both inositol 1,4,5-trisphosphate (InsP3)/Ca2+ and cyclic adenosine 3′,5′-monophosphate (cAMP) signalling pathways in the secretory cells. In order to characterize the signal-inducing 5-HT receptors, we cloned two cDNAs (Cv5-ht2α, Cv5-ht7) that share high similarity with mammalian 5-HT2 and 5-HT7 receptor genes, respectively. RT-PCR demonstrated that both receptors are expressed in the salivary glands and brain. Stimulation of Cv5-ht2α-transfected mammalian cells with 5-HT elevates cytosolic [Ca2+] in a dose-dependent manner (EC50 = 24 nM). In Cv5-ht7-transfected cells, 5-HT produces a dose-dependent increase in [cAMP]i (EC50 = 4 nM). We studied the pharmacological profile for both receptors. Substances that appear to act as specific ligands of either Cv5-HT2α or Cv5-HT7 in the heterologous expression system were also tested in intact blowfly salivary gland preparations. We observed that 5-methoxytryptamine (100 nM) activates only the Cv5-HT2α receptor, 5-carboxamidotryptamine (300 nM) activates only the Cv5-HT7 receptor, and clozapine (1 µM) antagonizes the effects of 5-HT via Cv5-HT7 in blowfly salivary glands, providing means for the selective activation of each of the two 5-HT receptor subtypes. This study represents the first comprehensive molecular and pharmacological characterization of two 5-HT receptors in the blowfly and permits the analysis of the physiological role of these receptors, even when co-expressed in cells, and of the modes of interaction between the Ca2+- and cAMP-signalling cascades.