Functional roles for redox genes in ethanol sensitivity in Drosophila.

Whilst the effects and associated costs of excessive alcohol consumption in the human population are obvious at one level, the roles played by genetic factors at the molecular level are still unclear. Drosophila melanogaster has an alcohol response comparable to humans and is used as a genetic model to study the functional roles of genes regulated in response to ethanol. In the current study, the biological processes associated with behavioural responses to acute alcohol exposure in Drosophila have been analysed using whole genome expression profiling. Ethanol response genes differentially expressed (a) at a single time point (2 h) and (b) in a time series (0-4 h) were identified using microarrays. In addition, a subset of differentially expressed genes was validated using behavioural sedation and recovery assays. The study shows that genes involved in redox processes, neuron development, and specific signalling and metabolic pathways (including glutathione metabolism) form part of the response to ethanol in Drosophila. Biological processes for the regulation of oxidative stress are the common functional denominator of many of the ethanol response genes identified. These upregulated genes work to rescue cells from oxidative stress and its consequences such as protein misfolding, apoptosis and ageing. In the current study, an enrichment of Drosophila genes linked to ageing is observed for the first time. The functional genomics data revealed by such studies can be used to predict transcription networks of ethanol response genes, but the future lies in mapping these networks to the human population, with the ultimate aim of identifying genetic factors for alcohol use disorders.

Molecular mechanism of rectification at identified electrical synapses in the Drosophila giant fiber system.

Electrical synapses are neuronal gap junctions that mediate fast transmission in many neural circuits. The structural proteins of gap junctions are the products of two multigene families. Connexins are unique to chordates; innexins/pannexins encode gap-junction proteins in prechordates and chordates. A concentric array of six protein subunits constitutes a hemichannel; electrical synapses result from the docking of hemichannels in pre- and postsynaptic neurons. Some electrical synapses are bidirectional; others are rectifying junctions that preferentially transmit depolarizing current anterogradely. The phenomenon of rectification was first described five decades ago, but the molecular mechanism has not been elucidated. Here, we demonstrate that putative rectifying electrical synapses in the Drosophila Giant Fiber System are assembled from two products of the innexin gene shaking-B. Shaking-B(Neural+16) is required presynaptically in the Giant Fiber to couple this cell to its postsynaptic targets that express Shaking-B(Lethal). When expressed in vitro in neighboring cells, Shaking-B(Neural+16) and Shaking-B(Lethal) form heterotypic channels that are asymmetrically gated by voltage and exhibit classical rectification. These data provide the most definitive evidence to date that rectification is achieved by differential regulation of the pre- and postsynaptic elements of structurally asymmetric junctions.

Molecular basis of gap junctional communication in the CNS of the leech Hirudo medicinalis.

Gap junctions are intercellular channels that allow the passage of ions and small molecules between cells. In the nervous system, gap junctions mediate electrical coupling between neurons. Despite sharing a common topology and similar physiology, two unrelated gap junction protein families exist in the animal kingdom. Vertebrate gap junctions are formed by members of the connexin family, whereas invertebrate gap junctions are composed of innexin proteins. Here we report the cloning of two innexins from the leech Hirudo medicinalis. These innexins show a differential expression in the leech CNS: Hm-inx1 is expressed by every neuron in the CNS but not in glia, whereas Hm-inx2 is expressed in glia but not neurons. Heterologous expression in the paired Xenopus oocyte system demonstrated that both innexins are able to form functional homotypic gap junctions. Hm-inx1 forms channels that are not strongly gated. In contrast, Hm-inx2 forms channels that are highly voltage-dependent; these channels demonstrate properties resembling those of a double rectifier. In addition, Hm-inx1 and Hm-inx2 are able to cooperate to form heterotypic gap junctions in Xenopus oocytes. The behavior of these channels is primarily that predicted from the properties of the constituent hemichannels but also demonstrates evidence of an interaction between the two. This work represents the first demonstration of a functional gap junction protein from a Lophotrochozoan animal and supports the hypothesis that connexin-based communication is restricted to the deuterostome clade.

Cloning and expression of a leech complexin.

Release of neurotransmitter from presynaptic nerve terminals is mediated by SNARE proteins, which are located on the vesicle and plasma membranes. These proteins form a SNARE complex thought to mediate membrane fusion. Complexin is a soluble protein essential for transmitter release, which has been postulated to bind to and stabilise the SNARE complex. We have cloned a complexin homologue, Hm-cpx1, from the leech, Hirudo medicinalis. This protein is expressed in only a subset of neurons in the leech CNS, including the Retzius and P neurons. It is 33% identical to rat complexin I, and 44% identical to squid complexin. Sequence conservation is particularly high in the predicted SNARE binding domain.

Gap junctions in Drosophila: developmental expression of the entire innexin gene family.

Invertebrate gap junctions are composed of proteins called innexins and eight innexin encoding loci have been identified in the now complete genome sequence of Drosophila melanogaster. The intercellular channels formed by these proteins are multimeric and previous studies have shown that, in a heterologous expression system, homo- and hetero-oligomeric channels can form, each combination possessing different gating characteristics. Here we demonstrate that the innexins exhibit complex overlapping expression patterns during oogenesis, embryogenesis, imaginal wing disc development and central nervous system development and show that only certain combinations of innexin oligomerization are possible in vivo. This work forms an essential basis for future studies of innexin interactions in Drosophila and outlines the potential extent of gap-junction involvement in development.