Mutations that influence the sensitivity of an organism to a volatile general anesthetic can be divided into two classes. In one, sensitivity to all other volatile agents is affected to a similar degree. Although this class may contain mutations of interest for understanding anesthesia, it is also likely to contain mutations that merely alter general health. In the second class, mutations confer larger effects for some volatile anesthetics than for others and thus display non-uniform effects on potency (abbreviated NEP). Members of this class are of special interest for studies of arousal and its pharmacological suppression because they not only avoid the pitfall of effects on global health, they imply the existence of drug targets that are preferentially affected by particular agents. We have now provided a systematic investigation of the relative frequency and diversity of NEP mutations. As a first step we isolated and characterized a set of P element insertion mutations that confer altered sensitivity of the fruit fly to the clinical anesthetic halothane. Then we tested the members of this collection for their effect on the sensitivity of flies to five other volatile agents. Not only do we find that most of the mutations show non-uniform effects, they share a characteristic arrangement of altered potencies (halothane >>desflurane ≥enflurane isoflurane methoxyflurane >sevoflurane). From this result, although we do not know how direct or indirect are the effects of the mutations, we infer the existence of a biologically relevant target for anesthetic action that has a distinct preference for halothane over other agents. Intriguingly, P elements with almost identical insertion points as those of several NEP mutations have been reported to alter the fly's response to cocaine and ethanol, suggesting that common genetic elements are involved in the response to all three drugs. Our current model is that the NEP loci are part of a gene network, central elements of which may represent critical targets for drug action. Accordingly, we are looking for interactions among these genes and we are testing their interaction with candidate genes. In early work from this lab, mutations that strongly influence sensitivity to halothane as well as several other anesthetics were mapped to a gene, narrow abdomen (na), which we showed encodes the ion-carrying subunit of a cation channel. Subsequently we found that na channel function depends on another gene, dunc-79, and that the two gene products participate in a complex. To identify additional proteins in this complex, in the past year we have explored immuno-purification followed by mass-spectroscopy. We first developed a novel protocol that efficiently solubilizes the channel while maintaining its contact with DUNC-79. We then used parallel immuno-precipitation of wild-type and mutant extracts so that the proteins identified by the NIDDK MS Core Facility could be sorted into specific and non-specific hits. To date 20 specific complex members have been found. The majority of these fall into two broad categories: cytoskeletal proteins and presynaptic components. At least one of the cytoskeletal partners, spectrin, has been confirmed by additional immune co-precipitation experiments. These results provide the first hints that this important channel is subject to complicated trafficking and targeting controls. Of many models to explain the strong effect of na mutations on anesthesia, the simplest is that the NA channel is a physiological target for volatile agents. To test this hypothesis, we attempted to reproduce the published claim that, upon expression in HEK293 cells, the mammalian ortholog of NA (NALCN) yields robust cation leak currents. As mentioned in last year's report, this result could not be reproduced. Since subsequent publications indicated that NALCN currents depend strongly on GPCR activation, using plasmids provided by the lab of Dejian Ren we have supplemented HEK293 transfections of NALCN so as to coexpress a mammalian tachykinin receptor and an accessory protein. Under these conditions, we do detect cation currents that are non-selective and non-voltage-gated. Our preliminary results suggest that this current is insensitive to halothane. Work is ongoing to test whether the recorded current is indeed carried by the NALCN channel and to establish the sensitivity of the current to other anesthetics. We are also using genetics to investigate the functional interaction of GPCRs with NA. Specifically, we acquired lines bearing mutations in Drosophila orthologs of tachykinin receptors and crossed them to lines bearing mutations in na. In at least one behavioral assay, the resulting double heterozygotes show a significant shift in their response to the anesthetic halothane. This promising lead will be followed up by genetic, anatomical, and functional studies. In addition to focused studies of general anesthesia, members of our laboratory are applying their expertise in Drosophila neurobiology via collaborative studies aimed at increased understanding of genes that are expressed in the nervous system and are conserved across the vertebrate/invertebrate boundary. In the past year, work has been done on three such genes. 1) Neuroligins, a family of cell adhesion molecules, are critical for synaptic maturation and have been implicated in autism spectrum disorder. Dr. Brian Mozer in the Nirenberg lab at NHLBI has generated mutants and transgenic flies for several of the Drosophila neuroligin genes. We analyzed synaptic function in mutant larvae and found that neuroligin2 (nrlg2) is required for normal neurotransmitter release. Analysis of the kinetics of synaptic currents indicates that the geometry of the synaptic cleft may be altered in nrlg2 mutants. Current work includes direct examination of the mutant synaptic morphology and determination of the tissue requirements for nrlg2 function. 2) BAG3 proteins are co-chaperones implicated in cell survival, proliferation and migration. In collaboration with Dr. Victoria Virador in the Kohn lab at NCI, we have found that the Drosophila homolog of BAG3, starvin (stv), is required in glia for viability and normal morphogenesis of the nervous system. Conversely glial overexpression of stv inhibits condensation of the central nervous system, a process that requires apoptosis. Tissue-specific knockdown with RNA interference (RNAi) produces a spectrum of phenotypes, one of which is altered retinal development, indicated by roughness of the adult eye. Experiments are ongoing to determine the spatial and temporal requirements for stv function in development of the eye and CNS, as well as the biochemical pathways involved. 3) TDP1 is an enzyme that is known to function in the repair of damaged DNA. Since dividing cells are most susceptible to serious consequences of such damage, one expects that TDP1 would rare or absent in post-mitotic neurons. Surprisingly, published studies report prominent expression of TDP1 in the brains of adult flies, mice, and humans. In collaboration with the lab of Dr. Yves Pommier at NCI, we are studying the role of TDP1 in the Drosophila nervous system. We acquired a mutant line in which TDP1 levels are severely reduced. Although the flies are viable and fertile, they have shortened lifespans and increased sensitivity to the lethal effects of DNA damaging agents. Interestingly, non-lethal doses of such agents interfere with eye development, suggesting a special sensitivity of developing neurons to DNA damage. Ongoing studies focus on rescue experiments, determining the effect of the mutation on adult behavior and brain structure, and defining the anatomical distribution of Tdp1 in the nervous system.
