Channelrhodopsin-2 Oligomerization in Cell Membrane Revealed by Photo-Activated Localization Microscopy.

Microbial rhodopsins are retinal membrane proteins that found a broad application in optogenetics. The oligomeric state of rhodopsins is important for their functionality and stability. Of particular interest is the oligomeric state in the cellular native membrane environment. Fluorescence microscopy provides powerful tools to determine the oligomeric state of membrane proteins directly in cells. Among these methods is quantitative photoactivated localization microscopy (qPALM) allowing the investigation of molecular organization at the level of single protein clusters. Here, we apply qPALM to investigate the oligomeric state of the first and most used optogenetic tool Channelrhodopsin-2 (ChR2) in the plasma membrane of eukaryotic cells. ChR2 appeared predominantly as a dimer in the cell membrane and did not form higher oligomers. The disulfide bonds between Cys34 and Cys36 of adjacent ChR2 monomers were not required for dimer formation and mutations disrupting these bonds resulted in only partial monomerization of ChR2. The monomeric fraction increased when the total concentration of mutant ChR2 in the membrane was low. The dissociation constant was estimated for this partially monomerized mutant ChR2 as 2.2±0.9 proteins/µm2 . Our findings are important for understanding the mechanistic basis of ChR2 activity as well as for improving existing and developing future optogenetic tools.

Examination of Intracellular GPCR-Mediated Signaling with High Temporal Resolution.

The GTP-binding protein-coupled receptors (GPCRs) play important roles in physiology and neuronal signaling. More than a thousand genes, excluding the olfactory receptors, have been identified that encode these integral membrane proteins. Their pharmacological and functional properties make them fascinating targets for drug development, since various disease states can be treated and overcome by pharmacologically addressing these receptors and/or their downstream interacting partners. The activation of the GPCRs typically causes transient changes in the intracellular second messenger concentrations as well as in membrane conductance. In contrast to ion channel-mediated electrical signaling which results in spontaneous cellular responses, the GPCR-mediated metabotropic signals operate at a different time scale. Here we have studied the kinetics of two common GPCR-induced signaling pathways: (a) Ca2+ release from intracellular stores and (b) cyclic adenosine monophosphate (cAMP) production. The latter was monitored via the activation of cyclic nucleotide-gated (CNG) ion channels causing Ca2+ influx into the cell. Genetically modified and stably transfected cell lines were established and used in stopped-flow experiments to uncover the individual steps of the reaction cascades. Using two homologous biogenic amine receptors, either coupling to Go/q or Gs proteins, allowed us to determine the time between receptor activation and signal output. With ~350 ms, the release of Ca2+ from intracellular stores was much faster than cAMP-mediated Ca2+ entry through CNG channels (~6 s). The measurements with caged compounds suggest that this difference is due to turnover numbers of the GPCR downstream effectors rather than the different reaction cascades, per se.

The Functional Characterization of GCaMP3.0 Variants Specifically Targeted to Subcellular Domains.

Calcium (Ca2+) ions play a pivotal role in physiology and cellular signaling. The intracellular Ca2+ concentration ([Ca2+]i) is about three orders of magnitude lower than the extracellular concentration, resulting in a steep transmembrane concentration gradient. Thus, the spatial and the temporal dynamics of [Ca2+]i are ideally suited to modulate Ca2+-mediated cellular responses to external signals. A variety of highly sophisticated methods have been developed to gain insight into cellular Ca2+ dynamics. In addition to electrophysiological measurements and the application of synthetic dyes that change their fluorescent properties upon interaction with Ca2+, the introduction and the ongoing development of genetically encoded Ca2+ indicators (GECI) opened a new era to study Ca2+-driven processes in living cells and organisms. Here, we have focused on one well-established GECI, i.e., GCaMP3.0. We have systematically modified the protein with sequence motifs, allowing localization of the sensor in the nucleus, in the mitochondrial matrix, at the mitochondrial outer membrane, and at the plasma membrane. The individual variants and a cytosolic version of GCaMP3.0 were overexpressed and purified from E. coli cells to study their biophysical properties in solution. All versions were examined to monitor Ca2+ signaling in stably transfected cell lines and in primary cortical neurons transduced with recombinant Adeno-associated viruses (rAAV). In this comparative study, we provide evidence for a robust approach to reliably trace Ca2+ signals at the (sub)-cellular level with pronounced temporal resolution.

PaOctß2R: Identification and Functional Characterization of an Octopamine Receptor Activating Adenylyl Cyclase Activity in the American Cockroach Periplaneta americana.

Biogenic amines constitute an important group of neuroactive substances that control and modulate various neural circuits. These small organic compounds engage members of the guanine nucleotide-binding protein coupled receptor (GPCR) superfamily to evoke specific cellular responses. In addition to dopamine- and 5-hydroxytryptamine (serotonin) receptors, arthropods express receptors that are activated exclusively by tyramine and octopamine. These phenolamines functionally substitute the noradrenergic system of vertebrates Octopamine receptors that are the focus of this study are classified as either α- or ß-adrenergic-like. Knowledge on these receptors is scarce for the American cockroach (Periplaneta americana). So far, only an α-adrenergic-like octopamine receptor that primarily causes Ca2+ release from intracellular stores has been studied from the cockroach (PaOctα1R). Here we succeeded in cloning a gene from cockroach brain tissue that encodes a ß-adrenergic-like receptor and leads to cAMP production upon activation. Notably, the receptor is 100-fold more selective for octopamine than for tyramine. A series of synthetic antagonists selectively block receptor activity with epinastine being the most potent. Bioinformatics allowed us to identify a total of 19 receptor sequences that build the framework of the biogenic amine receptor clade in the American cockroach. Phylogenetic analyses using these sequences and receptor sequences from model organisms showed that the newly cloned gene is an ß2-adrenergic-like octopamine receptor. The functional characterization of PaOctß2R and the bioinformatics data uncovered that the monoaminergic receptor family in the hemimetabolic P. americana is similarly complex as in holometabolic model insects like Drosophila melanogaster and the honeybee, Apis mellifera. Thus, investigating these receptors in detail may contribute to a better understanding of monoaminergic signaling in insect behavior and physiology.

Loss of HCN2 in Dorsal Hippocampus of Young Adult Mice Induces Specific Apoptosis of the CA1 Pyramidal Neuron Layer.

Neurons inevitably rely on a proper repertoire and distribution of membrane-bound ion-conducting channels. Among these proteins, the family of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels possesses unique properties giving rise to the corresponding Ih-current that contributes to various aspects of neural signaling. In mammals, four genes (hcn1-4) encode subunits of HCN channels. These subunits can assemble as hetero- or homotetrameric ion-conducting channels. In order to elaborate on the specific role of the HCN2 subunit in shaping electrical properties of neurons, we applied an Adeno-associated virus (AAV)-mediated, RNAi-based knock-down strategy of hcn2 gene expression both in vitro and in vivo. Electrophysiological measurements showed that HCN2 subunit knock-down resulted in specific yet anticipated changes in Ih-current properties in primary hippocampal neurons and, in addition, corroborated that the HCN2 subunit participates in postsynaptic signal integration. To further address the role of the HCN2 subunit in vivo, we injected recombinant (r)AAVs into the dorsal hippocampus of young adult male mice. Behavioral and biochemical analyses were conducted to assess the contribution of HCN2-containing channels in shaping hippocampal network properties. Surprisingly, knock-down of hcn2 expression resulted in a severe degeneration of the CA1 pyramidal cell layer, which did not occur in mice injected with control rAAV constructs. This finding might pinpoint to a vital and yet unknown contribution of HCN2 channels in establishing or maintaining the proper function of CA1 pyramidal neurons of the dorsal hippocampus.

Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides.

BACKGROUND: Approximately 40% of prescribed drugs exert their activity via GTP-binding protein-coupled receptors (GPCRs). Once activated, these receptors cause transient changes in the concentration of second messengers, e.g., cyclic adenosine 3',5'-monophosphate (cAMP). Specific and efficacious genetically encoded biosensors have been developed to monitor cAMP fluctuations with high spatial and temporal resolution in living cells or tissue. A well characterized biosensor for cAMP is the Förster resonance energy transfer (FRET)-based Epac1-camps protein. Pharmacological characterization of newly developed ligands acting at GPCRs often includes numerical quantification of the second messenger amount that was produced. RESULTS: To quantify cellular cAMP concentrations, we bacterially over-expressed and purified Epac1-camps and applied the purified protein in a cell-free detection assay for cAMP in a multi-well format. We found that the biosensor can detect as little as 0.15 pmol of cAMP, and that the sensitivity is not impaired by non-physiological salt concentrations or pH values. Notably, the assay tolerated desiccation and storage of the protein without affecting Epac1-camps cyclic nucleotide sensitivity. CONCLUSIONS: We found that determination cAMP in lysates obtained from cell assays or tissue samples by purified Epac1-camps is a robust, fast, and sensitive assay suitable for routine and high throughput analyses.

AmOctα2R: Functional Characterization of a Honeybee Octopamine Receptor Inhibiting Adenylyl Cyclase Activity.

The catecholamines norepinephrine and epinephrine are important regulators of vertebrate physiology. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they use the phenolamines tyramine and octopamine for similar physiological functions. These biogenic amines activate specific members of the large protein family of G protein-coupled receptors (GPCRs). Based on molecular and pharmacological data, insect octopamine receptors were classified as either α- or ß-adrenergic-like octopamine receptors. Currently, one α- and four ß-receptors have been molecularly and pharmacologically characterized in the honeybee. Recently, an α2-adrenergic-like octopamine receptor was identified in Drosophila melanogaster (DmOctα2R). This receptor is activated by octopamine and other biogenic amines and causes a decrease in intracellular cAMP ([cAMP]i). Here, we show that the orthologous receptor of the honeybee (AmOctα2R), phylogenetically groups in a clade closely related to human α2-adrenergic receptors. When heterologously expressed in an eukaryotic cell line, AmOctα2R causes a decrease in [cAMP]i. The receptor displays a pronounced preference for octopamine over tyramine. In contrast to DmOctα2R, the honeybee receptor is not activated by serotonin. Its activity can be blocked efficiently by 5-carboxamidotryptamine and phentolamine. The functional characterization of AmOctα2R now adds a sixth member to this subfamily of monoaminergic receptors in the honeybee and is an important step towards understanding the actions of octopamine in honeybee behavior and physiology.

Dorsal BNST α2A-Adrenergic Receptors Produce HCN-Dependent Excitatory Actions That Initiate Anxiogenic Behaviors.

Stress is a precipitating agent in neuropsychiatric disease and initiates relapse to drug-seeking behavior in addicted patients. Targeting the stress system in protracted abstinence from drugs of abuse with anxiolytics may be an effective treatment modality for substance use disorders. α2A-adrenergic receptors (α2A-ARs) in extended amygdala structures play key roles in dampening stress responses. Contrary to early thinking, α2A-ARs are expressed at non-noradrenergic sites in the brain. These non-noradrenergic α2A-ARs play important roles in stress responses, but their cellular mechanisms of action are unclear. In humans, the α2A-AR agonist guanfacine reduces overall craving and uncouples craving from stress, yet minimally affects relapse, potentially due to competing actions in the brain. Here, we show that heteroceptor α2A-ARs postsynaptically enhance dorsal bed nucleus of the stria terminalis (dBNST) neuronal activity in mice of both sexes. This effect is mediated by hyperpolarization-activated cyclic nucleotide-gated cation channels because inhibition of these channels is necessary and sufficient for excitatory actions. Finally, this excitatory action is mimicked by clozapine-N-oxide activation of the Gi-coupled DREADD hM4Di in dBNST neurons and its activation elicits anxiety-like behavior in the elevated plus maze. Together, these data provide a framework for elucidating cell-specific actions of GPCR signaling and provide a potential mechanism whereby competing anxiogenic and anxiolytic actions of guanfacine may affect its clinical utility in the treatment of addiction.SIGNIFICANCE STATEMENT Stress affects the development of neuropsychiatric disorders including anxiety and addiction. Guanfacine is an α2A-adrenergic receptor (α2A-AR) agonist with actions in the bed nucleus of the stria terminalis (BNST) that produces antidepressant actions and uncouples stress from reward-related behaviors. Here, we show that guanfacine increases dorsal BNST neuronal activity through actions at postsynaptic α2A-ARs via a mechanism that involves hyperpolarization-activated cyclic nucleotide gated cation channels. This action is mimicked by activation of the designer receptor hM4Di expressed in the BNST, which also induces anxiety-like behaviors. Together, these data suggest that postsynaptic α2A-ARs in BNST have excitatory actions on BNST neurons and that these actions can be phenocopied by the so-called "inhibitory" DREADDs, suggesting that care must be taken regarding interpretation of data obtained with these tools.

Caspase-3 and GFAP as early markers for apoptosis and astrogliosis in shRNA-induced hippocampal cytotoxicity.

Genetic manipulation of cells and tissue by RNA interference has significantly contributed to the functional characterization of individual proteins and their role in physiological processes. Despite its versatility, RNA interference can have detrimental side effects, including reduced cell viability. We applied recombinant adeno-associated viruses by stereotaxic injection into the murine hippocampus to express different short hairpin RNA (shRNA) constructs along with eGFP. Tissue responses were assessed immunohistochemically for up to 8 weeks post-infection. Strong hippocampal degeneration and tissue atrophy was observed, most likely induced by high shRNA expression. The effect was entirely absent in mice injected with vectors driving only expression of eGFP. Active caspase-3 (Casp-3) and glial fibrillary acidic protein (GFAP) were identified as molecular markers and early indicators of adverse tissue responses. Our findings also demonstrate that detrimental effects of high shRNA expression in hippocampal tissue can be monitored even before the onset of tissue degeneration.

PeaTAR1B: Characterization of a Second Type 1 Tyramine Receptor of the American Cockroach, Periplaneta americana.

The catecholamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. In insects; these neuroactive substances are functionally replaced by the phenolamines octopamine and tyramine. Phenolamines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Type 1 tyramine receptors are better activated by tyramine than by octopamine. In contrast; type 2 tyramine receptors are almost exclusively activated by tyramine. Functionally; activation of type 1 tyramine receptors leads to a decrease in the intracellular concentration of cAMP ([cAMP]i) whereas type 2 tyramine receptors can mediate Ca2+ signals or both Ca2+ signals and effects on [cAMP]i. Here; we report that the American cockroach (Periplaneta americana) expresses a second type 1 tyramine receptor (PeaTAR1B) in addition to PeaTAR1A (previously called PeaTYR1). When heterologously expressed in flpTM cells; activation of PeaTAR1B by tyramine leads to a concentration-dependent decrease in [cAMP]i. Its activity can be blocked by a series of established antagonists. The functional characterization of two type 1 tyramine receptors from P. americana; PeaTAR1A and PeaTAR1B; which respond to tyramine by changing cAMP levels; is a major step towards understanding the actions of tyramine in cockroach physiology and behavior; particularly in comparison to the effects of octopamine.

Transiently increasing cAMP levels selectively in hippocampal excitatory neurons during sleep deprivation prevents memory deficits caused by sleep loss.

The hippocampus is particularly sensitive to sleep loss. Although previous work has indicated that sleep deprivation impairs hippocampal cAMP signaling, it remains to be determined whether the cognitive deficits associated with sleep deprivation are caused by attenuated cAMP signaling in the hippocampus. Further, it is unclear which cell types are responsible for the memory impairments associated with sleep deprivation. Transgenic approaches lack the spatial resolution to manipulate specific signaling pathways selectively in the hippocampus, while pharmacological strategies are limited in terms of cell-type specificity. Therefore, we used a pharmacogenetic approach based on a virus-mediated expression of a Gαs-coupled Drosophila octopamine receptor selectively in mouse hippocampal excitatory neurons in vivo. With this approach, a systemic injection with the receptor ligand octopamine leads to increased cAMP levels in this specific set of hippocampal neurons. We assessed whether transiently increasing cAMP levels during sleep deprivation prevents memory consolidation deficits associated with sleep loss in an object-location task. Five hours of total sleep deprivation directly following training impaired the formation of object-location memories. Transiently increasing cAMP levels in hippocampal neurons during the course of sleep deprivation prevented these memory consolidation deficits. These findings demonstrate that attenuated cAMP signaling in hippocampal excitatory neurons is a critical component underlying the memory deficits in hippocampus-dependent learning tasks associated with sleep deprivation.

Distinct expression patterns of HCN channels in HL-1 cardiomyocytes.

BACKGROUND: Cardiac rhythmic activity is initiated in functionally specialized areas of the heart. Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are fundamental for these processes of cardiac physiology. RESULTS: Here we investigated transcript and protein expression patterns of HCN channels in HL-1 cardiomyocytes using a combination of quantitative PCR analysis and immunocytochemistry. Gene expression profiles of hcn1, hcn2 and hcn4 were acutely affected during HL-1 cell propagation. In addition, distinct expression patterns were uncovered for HCN1, HCN2 and HCN4 proteins. CONCLUSIONS: Our results suggest that HCN channel isoforms might be involved in the concerted differentiation of HL-1 cells and may indirectly affect the occurrence of contractile HL-1 cell activity. We expect that these findings will promote studies on other molecular markers that contribute to cardiac physiology.

Molecular and functional profiling of histamine receptor-mediated calcium ion signals in different cell lines.

Calcium ions (Ca(2+)) play a pivotal role in cellular physiology. Often Ca(2+)-dependent processes are studied in commonly available cell lines. To induce Ca(2+) signals on demand, cells may need to be equipped with additional proteins. A prominent group of membrane proteins evoking Ca(2+) signals are G-protein coupled receptors (GPCRs). These proteins register external signals such as photons, odorants, and neurotransmitters and convey ligand recognition into cellular responses, one of which is Ca(2+) signaling. To avoid receptor cross-talk or cross-activation with introduced proteins, the repertoire of cell-endogenous receptors must be known. Here we examined the presence of histamine receptors in six cell lines frequently used as hosts to study cellular signaling processes. In a concentration-dependent manner, histamine caused a rise in intracellular Ca(2+) in HeLa, HEK 293, and COS-1 cells. The concentration for half-maximal activation (EC50) was in the low micromolar range. In individual cells, transient Ca(2+) signals and Ca(2+) oscillations were uncovered. The results show that (i) HeLa, HEK 293, and COS-1 cells express sufficient amounts of endogenous receptors to study cellular Ca(2+) signaling processes directly and (ii) these cell lines are suitable for calibrating Ca(2+) biosensors in situ based on histamine receptor evoked responses.

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 Ca²âº-([Ca²âº](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 [Ca²âº](i) or [cAMP](i). To date, only one honeybee octopamine receptor that induces Ca²âº 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 EC50(s) 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.

Characterization of an invertebrate-type dopamine receptor of the American cockroach, Periplaneta americana.

We have isolated a cDNA coding for a putative invertebrate-type dopamine receptor (Peadop2) from P. americana brain by using a PCR-based strategy. The mRNA is present in samples from brain and salivary glands. We analyzed the distribution of the PeaDOP2 receptor protein with specific affinity-purified polyclonal antibodies. On Western blots, PeaDOP2 was detected in protein samples from brain, subesophageal ganglion, thoracic ganglia, and salivary glands. In immunocytochemical experiments, we detected PeaDOP2 in neurons with their somata being located at the anterior edge of the medulla bilaterally innervating the optic lobes and projecting to the ventro-lateral protocerebrum. In order to determine the functional and pharmacological properties of the cloned receptor, we generated a cell line constitutively expressing PeaDOP2. Activation of PeaDOP2-expressing cells with dopamine induced an increase in intracellular cAMP. In contrast, a C-terminally truncated splice variant of this receptor did not exhibit any functional property by itself. The molecular and pharmacological characterization of the first dopamine receptor from P. americana provides the basis for forthcoming studies focusing on the significance of the dopaminergic system in cockroach behavior and physiology.

Baculovirus Actin Rearrangement-Inducing Factor 1 Can Remodel the Mammalian Actin Cytoskeleton.

The actin rearrangement-inducing factor 1 (Arif-1) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an early viral protein that manipulates the actin cytoskeleton of host insect cells. Arif-1 is conserved among alphabaculoviruses and is responsible for the accumulation of F-actin at the plasma membrane during the early phase of infection. However, the molecular mechanism underlying Arif-1-induced cortical actin accumulation is still open. Recent studies have demonstrated the formation of invadosome-like structures induced by Arif-1, suggesting a function in systemic virus spread. Here, we addressed whether Arif-1 is able to manipulate the actin cytoskeleton of mammalian cells comparably to insect cells. Strikingly, transient overexpression of Arif-1 in B16-F1 mouse melanoma cells revealed pronounced F-actin remodeling. Actin assembly was increased, and intense membrane ruffling occurred at the expense of substrate-associated lamellipodia. Deletion mutagenesis studies of Arif-1 confirmed that the C-terminal cytoplasmic region was not sufficient to induce F-actin remodeling, supporting that the transmembrane region for Arif-1 function is also required in mammalian cells. The similarities between Arif-1-induced actin remodeling in insect and mammalian cells indicate that Arif-1 function relies on conserved cellular interaction partners and signal transduction pathways, thus providing an experimental tool to elucidate the underlying mechanism. IMPORTANCE Virus-induced changes of the host cell cytoskeleton play a pivotal role in the pathogenesis of viral infections. The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is known for intervening with the regulation of the host actin cytoskeleton in a wide manner throughout the infection cycle. The actin rearrangement-inducing factor 1 (Arif-1) is a viral protein that causes actin rearrangement during the early phase of AcMNPV infection. Here, we performed overexpression studies of Arif-1 in mammalian cells to establish an experimental tool that allows elucidation of the mechanism underlying the Arif-1-induced remodeling of actin dynamics in a well-characterized and genetically accessible system.

Coumarin-based octopamine phototriggers and their effects on an insect octopamine receptor.

We have developed and characterized efficient caged compounds of the neurotransmitter octopamine. For derivatization, we introduced [6-bromo-8-(diethylaminomethyl)-7-hydroxycoumarin-4-yl]methoxycarbonyl (DBHCMOC) and {6-bromo-7-hydroxy-8-[(piperazin-1-yl)methyl]coumarin-4-yl}methoxycarbonyl (PBHCMOC) moieties as novel photo-removable protecting groups. The caged compounds were functionally inactive when applied to heterologously expressed octopamine receptors (AmOctα1R). Upon irradiation with UV-visible or IR light, bioactive octopamine was released and evoked Ca2+ signals in AmOctα1R-expressing cells. The pronounced water solubility of compounds 2-4 in particular holds great promise for these substances as excellent phototriggers of this important neurotransmitter.

A single amino acid residue controls Ca2+ signaling by an octopamine receptor from Drosophila melanogaster.

Rhythmic activity of cells and cellular networks plays an important role in physiology. In the nervous system oscillations of electrical activity and/or second messenger concentrations are important to synchronize neuronal activity. At the molecular level, rhythmic activity can be initiated by different routes. We have recently shown that an octopamine-activated G-protein-coupled receptor (GPCR; DmOctα1Rb, CG3856) from Drosophila initiates Ca(2+) oscillations. Here, we have unraveled the molecular basis of cellular Ca(2+) signaling controlled by the DmOctα1Rb receptor using a combination of pharmacological intervention, site-directed mutagenesis, and functional cellular Ca(2+) imaging on heterologously expressed receptors. Phosphorylation of a single amino acid residue in the third intracellular loop of the GPCR by PKC is necessary and sufficient to desensitize the receptor. From its desensitized state, DmOctα1Rb is resensitized by dephosphorylation, and a new Ca(2+) signal occurs on octopamine stimulation. Our findings show that transient changes of the receptor's surface profile have a strong effect on its physiological signaling properties. We expect that the detailed knowledge of DmOctα1Rb-dependent signal transduction fosters the identification of specific drugs that can be used for GPCR-mediated pest control, since octopamine serves important physiological and behavioral functions in arthropods.

Characterization of the 5-HT1A receptor of the honeybee (Apis mellifera) and involvement of serotonin in phototactic behavior.

Serotonin plays a key role in modulating various physiological and behavioral processes in both protostomes and deuterostomes. The vast majority of serotonin receptors belong to the superfamily of G-protein-coupled receptors. We report the cloning of a cDNA from the honeybee (Am5-ht1A) sharing high similarity with members of the 5-HT(1) receptor class. Activation of Am5-HT(1A) by serotonin inhibited the production of cAMP in a dose-dependent manner (EC(50) = 16.9 nM). Am5-HT(1A) was highly expressed in brain regions known to be involved in visual information processing. Using in vivo pharmacology, we could demonstrate that Am5-HT(1A) receptor ligands had a strong impact on the phototactic behavior of individual bees. The data presented here mark the first comprehensive study-from gene to behavior-of a 5-HT(1A) receptor in the honeybee, paving the way for the eventual elucidation of additional roles of this receptor subtype in the physiology and behavior of this social insect.

AAV-Mediated CRISPRi and RNAi Based Gene Silencing in Mouse Hippocampal Neurons.

Uncovering the physiological role of individual proteins that are part of the intricate process of cellular signaling is often a complex and challenging task. A straightforward strategy of studying a protein's function is by manipulating the expression rate of its gene. In recent years, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-based technology was established as a powerful gene-editing tool for generating sequence specific changes in proliferating cells. However, obtaining homogeneous populations of transgenic post-mitotic neurons by CRISPR/Cas9 turned out to be challenging. These constraints can be partially overcome by CRISPR interference (CRISPRi), which mediates the inhibition of gene expression by competing with the transcription machinery for promoter binding and, thus, transcription initiation. Notably, CRISPR/Cas is only one of several described approaches for the manipulation of gene expression. Here, we targeted neurons with recombinant Adeno-associated viruses to induce either CRISPRi or RNA interference (RNAi), a well-established method for impairing de novo protein biosynthesis by using cellular regulatory mechanisms that induce the degradation of pre-existing mRNA. We specifically targeted hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, which are widely expressed in neuronal tissues and play essential physiological roles in maintaining biophysical characteristics in neurons. Both of the strategies reduced the expression levels of three HCN isoforms (HCN1, 2, and 4) with high specificity. Furthermore, detailed analysis revealed that the knock-down of just a single HCN isoform (HCN4) in hippocampal neurons did not affect basic electrical parameters of transduced neurons, whereas substantial changes emerged in HCN-current specific properties.