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• W1974398081 abstract "Volatile anesthetics like halothane and enflurane are of interest to clinicians and neuroscientists because of their ability to preferentially disrupt higher functions that make up the conscious state. All volatiles were once thought to act identically; if so, they should be affected equally by genetic variants. However, mutations in two distinct genes, one in Caenorhabditis and one in Drosophila, have been reported to produce much larger effects on the response to halothane than enflurane [1Morgan P.G. Sedensky M. Meneely P.M. Multiple sites of action of volatile anesthetics in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 1990; 87: 2965-2969Crossref PubMed Scopus (93) Google Scholar, 2Campbell D.B. Nash H.A. Use of Drosophila mutants to distinguish among volatile general anesthetics.Proc. Natl. Acad. Sci. USA. 1994; 91: 2135-2139Crossref PubMed Scopus (67) Google Scholar]. To see whether this anesthesia signature is adventitious or fundamental, we have identified orthologs of each gene and determined the mutant phenotype within each species. The fly gene, narrow abdomen (na), encodes a putative ion channel whose sequence places it in a unique family; the nematode gene, unc-79, is identified here as encoding a large cytosolic protein that lacks obvious motifs. In Caenorhabditis, mutations that inactivate both of the na orthologs produce an Unc-79 phenotype; in Drosophila, mutations that inactivate the unc-79 ortholog produce an na phenotype. In each organism, studies of double mutants place the genes in the same pathway, and biochemical studies show that proteins of the UNC-79 family control NA protein levels by a posttranscriptional mechanism. Thus, the anesthetic signature reflects an evolutionarily conserved role for the na orthologs, implying its intimate involvement in drug action. Volatile anesthetics like halothane and enflurane are of interest to clinicians and neuroscientists because of their ability to preferentially disrupt higher functions that make up the conscious state. All volatiles were once thought to act identically; if so, they should be affected equally by genetic variants. However, mutations in two distinct genes, one in Caenorhabditis and one in Drosophila, have been reported to produce much larger effects on the response to halothane than enflurane [1Morgan P.G. Sedensky M. Meneely P.M. Multiple sites of action of volatile anesthetics in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 1990; 87: 2965-2969Crossref PubMed Scopus (93) Google Scholar, 2Campbell D.B. Nash H.A. Use of Drosophila mutants to distinguish among volatile general anesthetics.Proc. Natl. Acad. Sci. USA. 1994; 91: 2135-2139Crossref PubMed Scopus (67) Google Scholar]. To see whether this anesthesia signature is adventitious or fundamental, we have identified orthologs of each gene and determined the mutant phenotype within each species. The fly gene, narrow abdomen (na), encodes a putative ion channel whose sequence places it in a unique family; the nematode gene, unc-79, is identified here as encoding a large cytosolic protein that lacks obvious motifs. In Caenorhabditis, mutations that inactivate both of the na orthologs produce an Unc-79 phenotype; in Drosophila, mutations that inactivate the unc-79 ortholog produce an na phenotype. In each organism, studies of double mutants place the genes in the same pathway, and biochemical studies show that proteins of the UNC-79 family control NA protein levels by a posttranscriptional mechanism. Thus, the anesthetic signature reflects an evolutionarily conserved role for the na orthologs, implying its intimate involvement in drug action. Volatile anesthetics have long had a prominent place in the practice of medicine, but there remains much uncertainty about the mechanism of action of these drugs [3Eckenhoff R.G. Promiscuous ligands and attractive cavities: How do the inhaled anesthetics work?.Mol. Interv. 2002; 1: 258-268Google Scholar, 4Hemmings Jr., H.C. Akabas M.H. Goldstein P.A. Trudell J.R. Orser B.A. Harrison N.L. Emerging molecular mechanisms of general anesthetic action.Trends Pharmacol. Sci. 2005; 26: 503-510Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar]. Most current studies examine the way volatile anesthetics affect biochemical and physiological processes [5Urban B.W. Bleckwenn M. Concepts and correlations relevant to general anaesthesia.Br. J. Anaesth. 2002; 89: 3-16Crossref PubMed Scopus (64) Google Scholar, 6Antognini J.F. Carstens E.E. Raines D.E. Neural Mechanisms of Anesthesia. Humana Press, Totowa, N.J.2003Google Scholar]. To provide a different perspective, we have used a pharmacogenetic approach with the invertebrates Caenorhabditis elegans and Drosophila melanogaster, organisms that respond to volatiles in ways that are reminiscent of mammalian responses. Among the mutations identified in our studies, a few showed larger effects with one anesthetic than with another; this phenotype is of particular interest because it undermines the classical notion [7Koblin D.D. Mechanisms of Action.in: Miller R. Anesthesia. Volume 1. Churchill Livingstone, San Francisco1994: 67-99Google Scholar, 8Urban B.W. Current assessment of targets and theories of anaesthesia.Br. J. Anaesth. 2002; 89: 167-183Crossref PubMed Scopus (87) Google Scholar] that all volatile agents work via the same mechanism and at the same targets. The earliest report of agent-specific effects involved unc-79 mutants [9Morgan P.G. Sedensky M.M. Meneely P.M. Cascorbi H.F. The effect of two genes on anesthetic response in the nematode Caenorhabditis elegans.Anesthesiology. 1988; 69: 246-251Crossref PubMed Scopus (37) Google Scholar, 10Sedensky M. Meneely P.M. Genetic analysis of halothane sensitivity in Caenorhabditis elegans.Science. 1987; 236: 952-954Crossref PubMed Scopus (64) Google Scholar]. Three alleles of the C. elegans gene conferred strikingly increased sensitivity to the immobilizing action of some volatile anesthetics, such as halothane, but left sensitivity to other volatiles, such as enflurane, unaffected or even slightly lowered [1Morgan P.G. Sedensky M. Meneely P.M. Multiple sites of action of volatile anesthetics in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 1990; 87: 2965-2969Crossref PubMed Scopus (93) Google Scholar, 9Morgan P.G. Sedensky M.M. Meneely P.M. Cascorbi H.F. The effect of two genes on anesthetic response in the nematode Caenorhabditis elegans.Anesthesiology. 1988; 69: 246-251Crossref PubMed Scopus (37) Google Scholar]. We tested seven other alleles (see the Supplemental Data available online) and found that each conferred a similar phenotype. Remarkably, mutations that were subsequently mapped to the narrow abdomen (na) gene of D. melanogaster produce a closely related pattern of altered sensitivity, at least in assays of one anesthetic endpoint [2Campbell D.B. Nash H.A. Use of Drosophila mutants to distinguish among volatile general anesthetics.Proc. Natl. Acad. Sci. USA. 1994; 91: 2135-2139Crossref PubMed Scopus (67) Google Scholar]. In addition to the common pattern of sensitivity, the mutants share a nonanesthetic phenotype in that they each display a pattern of locomotion characterized by periods of quiescence—“fainting” in unc-79 [9Morgan P.G. Sedensky M.M. Meneely P.M. Cascorbi H.F. The effect of two genes on anesthetic response in the nematode Caenorhabditis elegans.Anesthesiology. 1988; 69: 246-251Crossref PubMed Scopus (37) Google Scholar, 10Sedensky M. Meneely P.M. Genetic analysis of halothane sensitivity in Caenorhabditis elegans.Science. 1987; 236: 952-954Crossref PubMed Scopus (64) Google Scholar, 11Rajaram S. Spangler T.L. Sedensky M.M. Morgan P.G. A stomatin and a degenerin interact to control anesthetic sensitivity in Caenorhabditis elegans.Genetics. 1999; 153: 1673-1682Crossref PubMed Google Scholar] and “hesitant walking” in na mutants [12Krishnan K.S. Nash H.A. A genetic study of the anesthetic response: Mutants of Drosophila melanogaster altered in sensitivity to halothane.Proc. Natl. Acad. Sci. USA. 1990; 87: 8632-8636Crossref PubMed Scopus (72) Google Scholar, 13Guan Z. Scott R.L. Nash H.A. A new assay for the genetic study of general anesthesia in Drosophila melanogaster: Use in analysis of mutations in the 12E region.J. Neurogenet. 2000; 14: 25-42Crossref PubMed Scopus (26) Google Scholar]. To explore whether the similar phenotypes imply a significant connection between the genes, one needs to look at their effects in the same organism. Here, we use molecular analysis of unc-79 and na to acquire orthologous mutations and determine whether the agent-specific phenotype reflects a conserved role for these genes in anesthesia. The na gene of Drosophila has been shown to encode a polypeptide whose predicted topology resembles that of voltage-gated sodium and calcium channels [14Nash H.A. Scott R.L. Lear B.C. Allada R. An unusual cation channel mediates photic control of locomotion in Drosophila.Curr. Biol. 2002; 12: 2152-2158Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar]. Indeed, the aligned sequences of na and its invertebrate and vertebrate orthologs [15Lee J.H. Cribbs L.L. Perez-Reyes E. Cloning of a novel four repeat protein related to voltage-gated sodium and calcium channels.FEBS Lett. 1999; 445: 231-236Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar] clearly form a branch of this superfamily, designated the α1U branch [16Littleton J.T. Ganetzky B. Ion channels and synaptic organization: analysis of the Drosophila genome.Neuron. 2000; 26: 35-43Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar], whose members share distinct intracellular domains and pore signatures. Although failure of heterologous expression has so far precluded a definitive demonstration of α1U channel activity, recent evidence for the importance of a presumptive pore-lining residue of NA [17Lear B.C. Lin J.M. Keath J.R. McGill J.J. Raman I.M. Allada R. The ion channel narrow abdomen is critical for neural output of the Drosophila circadian pacemaker.Neuron. 2005; 48: 965-976Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar] supports its assignment as an ion channel. The α1U family was first discerned in C. elegans [15Lee J.H. Cribbs L.L. Perez-Reyes E. Cloning of a novel four repeat protein related to voltage-gated sodium and calcium channels.FEBS Lett. 1999; 445: 231-236Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar], where it is represented by two genes, nca-1 and nca-2. When deletion alleles of the putative-channel genes (Figure S1) are combined, the resulting nca-2(gk5);nca-1(gk9) double mutant is virtually identical to an unc-79 mutant in sensitivity to various anesthetics (Figure 1A) and in displaying a “fainting” pattern of locomotion (Movie S1). In fact, all double-null-allele combinations of nca-2;nca-1 are similar to unc-79 in anesthetic sensitivity (Figure 1B) and move in a fainting manner. In contrast, animals with mutations in nca-1 or nca-2 individually resemble the wild-type strain (N2) in both locomotion (not shown) and anesthetic sensitivity (Figure 1B). Because the mutations tested, nca-1(gk9) and nca-2(gk5), are null alleles (Supplemental Experimental Procedures), the paralogs appear to function redundantly for the endpoints we have examined. This is confirmed by the ability of either an nca-1 or an nca-2 transgene to fully rescue both the fainting locomotion and the anesthetic hypersensitivity of the double mutant (Table S1). The nca genes not only yield a similar phenotype to unc-79 but, as evidenced by genetic interaction studies, also appear to have a strong functional relationship with it. Four mutations have been shown to suppress the anesthetic phenotype of unc-79 [1Morgan P.G. Sedensky M. Meneely P.M. Multiple sites of action of volatile anesthetics in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 1990; 87: 2965-2969Crossref PubMed Scopus (93) Google Scholar], and each of them suppresses the nca-2;nca-1 double mutant (Figure 1C). Moreover, triple mutants with either unc-79(ec1) or unc-79(e1068) together with nca-2(gk-5) and nca-1(gk9) have a fainting phenotype and anesthetic-sensitivity profile that is identical to that of the single unc-79 mutant (not shown). In a parallel way, we wished to ask whether loss of UNC-79 function in Drosophila produced a phenotype similar to that showed by na mutants. The first, and most difficult, step was to molecularly identify the C. elegans gene associated with the unc-79 mutations. After recombination mapping, the unc-79 gene was localized by cosmid rescue of the mutant phenotype (Table S1). The gene occupies almost all of a 19 kb fragment that contains what had been predicted to be four separate genes (Figure 2A). That unc-79 comprises all four predicted ORFs is demonstrated by both the location of sequence changes in various alleles and the existence of cDNAs that span them (Figure 2B). Although the predicted sequence for the gene product provides no hint as to the function of UNC-79, database searches show that flies, mice, and humans each contain a single close ortholog. The Drosophila ortholog is annotated as CG5237 [18Drysdale R.A. Crosby M.A. FlyBase: Genes and gene models.Nucleic Acids Res. 2005; 33: D390-D395Crossref PubMed Scopus (273) Google Scholar], and from a public repository [19Thibault S.T. Singer M.A. Miyazaki W.Y. Milash B. Dompe N.A. Singh C.M. Buchholz R. Demsky M. Fawcett R. Francis-Lang H.L. et al.A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac.Nat. Genet. 2004; 36: 283-287Crossref PubMed Scopus (627) Google Scholar] we obtained a strain bearing a transposon that disrupts the gene (hereafter called dunc79). When assayed for the ability of anesthetics to interfere with reactive climbing [13Guan Z. Scott R.L. Nash H.A. A new assay for the genetic study of general anesthesia in Drosophila melanogaster: Use in analysis of mutations in the 12E region.J. Neurogenet. 2000; 14: 25-42Crossref PubMed Scopus (26) Google Scholar], the dunc79 mutant has a halothane sensitivity exactly like that of an na mutant, and an na;dunc79 double-mutant strain has a halothane sensitivity no different than that of either single mutant (Figure 3A). In addition, like na mutant flies, dunc79 flies elute more slowly than control flies during inebriometer tests of postural control [2Campbell D.B. Nash H.A. Use of Drosophila mutants to distinguish among volatile general anesthetics.Proc. Natl. Acad. Sci. USA. 1994; 91: 2135-2139Crossref PubMed Scopus (67) Google Scholar] in response to halothane but not to enflurane (Figure 3B). Flies bearing mutations in na or dunc79 also share nonanesthetic phenotypes, including altered circadian locomotor patterns ([14Nash H.A. Scott R.L. Lear B.C. Allada R. An unusual cation channel mediates photic control of locomotion in Drosophila.Curr. Biol. 2002; 12: 2152-2158Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar], Bridget Lear, personal communication), cylindrically shaped abdomen (not shown), and hesitant walking mode (Movie S2). In addition, both na and dunc79 mutants display an oscillation that can be recorded electroretinographically (unpublished data) or visualized as periodic twitching in restrained animals [14Nash H.A. Scott R.L. Lear B.C. Allada R. An unusual cation channel mediates photic control of locomotion in Drosophila.Curr. Biol. 2002; 12: 2152-2158Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar].Figure 3The Effect of a dunc79 Mutation on Anesthetic Sensitivity in D. melanogasterShow full caption(A) Distribution tests of reactive climbing. Halothane concentration-response curves are fitted as above with a slope constant of 8. As reported before for different alleles in a different strain background [13Guan Z. Scott R.L. Nash H.A. A new assay for the genetic study of general anesthesia in Drosophila melanogaster: Use in analysis of mutations in the 12E region.J. Neurogenet. 2000; 14: 25-42Crossref PubMed Scopus (26) Google Scholar, 21Campbell J.L. Nash H.A. Volatile general anesthetics reveal a neurobiological role for the white and brown genes of Drosophila melanogaster.J. Neurobiol. 2001; 49: 339-349Crossref PubMed Scopus (68) Google Scholar], the curve for an na mutant (e04385) is greatly left-shifted compared to an isogenic control strain. In the same genetic background [19Thibault S.T. Singer M.A. Miyazaki W.Y. Milash B. Dompe N.A. Singh C.M. Buchholz R. Demsky M. Fawcett R. Francis-Lang H.L. et al.A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac.Nat. Genet. 2004; 36: 283-287Crossref PubMed Scopus (627) Google Scholar], a dunc79 mutation (f03453) confers similar hypersensitivity to halothane, and the na;dunc79 double mutant is no more sensitive than either single mutant.(B) Inebriometer tests of postural control. Parallel tests were run simultaneously for the control strain and a single mutant; each such pair is presented as solid and dashed lines of the same color (black for na, red for dunc79). The step plots show the time-dependent increase in the cumulative fraction of flies of each genotype that tumble out of the inebriometer column. Note that the mutants elute much more slowly than the wild-type when exposed to 0.5% halothane (top) but elute very similarly to the wild-type control when exposed to 0.7% enflurane (bottom).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Distribution tests of reactive climbing. Halothane concentration-response curves are fitted as above with a slope constant of 8. As reported before for different alleles in a different strain background [13Guan Z. Scott R.L. Nash H.A. A new assay for the genetic study of general anesthesia in Drosophila melanogaster: Use in analysis of mutations in the 12E region.J. Neurogenet. 2000; 14: 25-42Crossref PubMed Scopus (26) Google Scholar, 21Campbell J.L. Nash H.A. Volatile general anesthetics reveal a neurobiological role for the white and brown genes of Drosophila melanogaster.J. Neurobiol. 2001; 49: 339-349Crossref PubMed Scopus (68) Google Scholar], the curve for an na mutant (e04385) is greatly left-shifted compared to an isogenic control strain. In the same genetic background [19Thibault S.T. Singer M.A. Miyazaki W.Y. Milash B. Dompe N.A. Singh C.M. Buchholz R. Demsky M. Fawcett R. Francis-Lang H.L. et al.A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac.Nat. Genet. 2004; 36: 283-287Crossref PubMed Scopus (627) Google Scholar], a dunc79 mutation (f03453) confers similar hypersensitivity to halothane, and the na;dunc79 double mutant is no more sensitive than either single mutant. (B) Inebriometer tests of postural control. Parallel tests were run simultaneously for the control strain and a single mutant; each such pair is presented as solid and dashed lines of the same color (black for na, red for dunc79). The step plots show the time-dependent increase in the cumulative fraction of flies of each genotype that tumble out of the inebriometer column. Note that the mutants elute much more slowly than the wild-type when exposed to 0.5% halothane (top) but elute very similarly to the wild-type control when exposed to 0.7% enflurane (bottom). Given the similar phenotype conferred by their inactivation within each organism (Table 1) plus the effect of mutant combinations described above, the genes we have studied appear to act in a common pathway. The simplest model for their relationship would thus be for one to control the expression of the other. Indeed, western blots and immunohistochemistry (Figure 4) reveal that levels of the putative channel are reduced to background in the absence of UNC-79/DUNC79 function. Given the sensitivity of these assays, we cannot rule out that low levels of protein are present; such residual expression in flies might explain why some aspects of the dunc79 mutant phenotype (e.g., Figure 3B) are weaker than those of na mutants. Nevertheless, the expression defect is at least 10-fold and is specific, because western blots revealed no change in level of other fly and nematode membrane proteins (not shown). Interestingly, in both organisms the UNC-79 ortholog appears to have little or no effect on transcription of the genes encoding the putative channel. Probing northern blots for the nca-1 and nca-2 transcripts revealed no effect of unc-79(e1291) on message size and amount (Figure S2A). Similarly, RT-PCR revealed that disruption of dunc79 caused no perturbation in structure or gross level of na transcripts (Figure S2B). This implies that UNC-79/DUNC79 normally serves in the posttranscriptional processing of the putative channel, e.g., by affecting translation, protein modification, trafficking, protection against degradation, etc. The same may be true for the nematode unc-80 gene, known to generate a mutant phenotype that precisely mimics the Unc-79 phenotype [9Morgan P.G. Sedensky M.M. Meneely P.M. Cascorbi H.F. The effect of two genes on anesthetic response in the nematode Caenorhabditis elegans.Anesthesiology. 1988; 69: 246-251Crossref PubMed Scopus (37) Google Scholar, 10Sedensky M. Meneely P.M. Genetic analysis of halothane sensitivity in Caenorhabditis elegans.Science. 1987; 236: 952-954Crossref PubMed Scopus (64) Google Scholar], because the e1272 mutant also lacks detectable NCA1/2 protein despite the presence of normal transcripts (not shown).Table 1Effects of unc79/dunc79 and nca/na MutationsC. elegansAssay/conditionunc-79nca-2;nca-1Locomotion/airfainterfainterImmobility/halothanevery hypersensitivevery hypersensitiveImmobility/enfluranenear wild-typenear wild-typeD. melanogasterAssay/conditiondunc79naLocomotion/airhesitanthesitantInebriometer/halothaneslowvery slowInebriometer/enfluranenear wild-typenear wild-type Open table in a new tab How directly do the UNC-79 orthologs control the expression of the putative channel? One way to explore this issue is to see whether the corresponding gene products are present in the same tissue at the same time. Toward this end, we examined GFP expression in C. elegans from constructs in which this reporter was linked to the promoter regions of unc-79, nca-1, or nca-2. Although each reporter shows conspicuous (but not exclusive) expression in the nervous system, the pattern of unc-79 does not coincide with that of nca-1 or nca-2 or with their sum (Figure S3). This disagreement might merely reflect infidelity of a reporter pattern, but, taken at face value, it hints that the regulation is indirect, e.g., that UNC-79 might be used nonautonomously. An intriguing feature of the genes studied in this paper is that inactivating them produces agent-specific effects on anesthesia. There are several implications of this phenomenon. First, differences between mutant effects on halothane and enflurane sensitivity make it unlikely that the affected genes are merely needed for vigorous neuromuscular function; if they were, inactivation would render either organism hypersusceptible to both agents. Further evidence against such a trivial model comes from the observation [20van Swinderen B. A succession of anesthetic endpoints in the Drosophila brain.J. Neurobiol. 2006; 66: 1195-1211Crossref PubMed Scopus (23) Google Scholar] that an na mutation increases the potency with which a volatile anesthetic alters local field potentials recorded directly from fly brains. To this background, as summarized in Table 1, our current work establishes that agent-specific effects of the unc-79/dunc79 and na/nca genes are conserved between organisms. This evolutionary conservation is particularly incisive, arguing against many scenarios for indirect effects. According to such models, the putative channel is not in neurons that are affected directly by anesthetics but only influences sensitivity because neurons that depend on it for optimal performance are in communication with such target neurons. However, because the neuronal circuitry of the nematode is strikingly different from that of the fruit fly, it is hard to imagine that in each organism there are halothane-sensitive and enflurane-insensitive neurons that just by chance are connected to neurons containing the putative channel. Thus, indirect models for the effect of na and its orthologs on anesthesia are disfavored. It must be noted that, at least in fruit flies, some anesthetic endpoints do not show the same agent-specific effects described in Table 1. For example, in the distribution test na and dunc79 mutants are hypersensitive to both halothane ([13Guan Z. Scott R.L. Nash H.A. A new assay for the genetic study of general anesthesia in Drosophila melanogaster: Use in analysis of mutations in the 12E region.J. Neurogenet. 2000; 14: 25-42Crossref PubMed Scopus (26) Google Scholar, 21Campbell J.L. Nash H.A. Volatile general anesthetics reveal a neurobiological role for the white and brown genes of Drosophila melanogaster.J. Neurobiol. 2001; 49: 339-349Crossref PubMed Scopus (68) Google Scholar] and above) and enflurane ([21Campbell J.L. Nash H.A. Volatile general anesthetics reveal a neurobiological role for the white and brown genes of Drosophila melanogaster.J. Neurobiol. 2001; 49: 339-349Crossref PubMed Scopus (68) Google Scholar] and data not shown). Because these effects lack a distinctive signature, it is hard to rule out indirect models for their generation. Nevertheless, for the circuits that subserve endpoints with agent-specific dependence on NCA or NA function, our observations suggest that neurons within them directly depend on these gene products for resisting the effects of halothane. In this population, the putative channel could be a molecular target of halothane (but not enflurane) or could serve to stabilize neuronal performance against the deleterious effects of halothane on some other component in that cell (one that is insensitive to enflurane). In considering these models, one must keep in mind that volatiles are likely to have more than one molecular target, each of which may be necessary but not sufficient to produce the desired endpoint [22Nash H.A. In vivo genetics of anesthetic action.Br. J. Anaesth. 2002; 89: 143-155Crossref PubMed Scopus (30) Google Scholar]. Thus, although the putative channel may play a critical role in setting anesthesia sensitivity, it is unlikely to be the only factor. Nevertheless, our observations provide a strong clue that, at least for some endpoints, the relationship between the α1U family and halothane action is intimate. In summary, this work has established that there is a remarkable parallelism between two sets of genes in two distantly related organisms. One set of genes, unc-79/dunc79, acts as a posttranscriptional regulator of the other set, nca/na, which encodes a putative ion channel. Moreover, there is a strong parallelism in the phenotypes of animals carrying mutations in these genes, with subtle effects on locomotion and strong effects on sensitivity to certain volatile anesthetics found in both organisms. The conserved nature of the agent-specific effects implies that the channel is present in anesthetic-sensitive neurons and has important effects on the degree to which these neurons resist the effects of volatile agents. Because both sets of genes are found in vertebrates and have been shown to be expressed neuronally, there is every reason to believe that they will strongly influence the clinical effects of volatile anesthetics. P.G.M. and M.M.S. were supported in part by National Institutes of Health (NIH) grant #GM45402. J.A.H. was supported by NIH grant #GM45402 and by NIH training grant T32GM008613. J.A.H, M.M.S., and P.G.M. are indebted to the technical assistance of Julie Seifker, Qiao-yun Jiang, and Judy Preston and to insightful discussions with Erik Jorgensen and Mei Zhen. Work in the Michael Smith Laboratories was supported by a grant from the Canadian Institutes of Health Research (CIHR) of Canada and a Canada Research Chair in Genomics-Neurobiology to T.P.S. and the Natural Sciences and Engineering Research Council of Canada and the Killam Trusts to K.S.H. Work in the Laboratory of Molecular Biology was supported by the Intramural Research Program of the National Institute of Mental Health. We are grateful to Dr. B. van Swinderen for his suggestions for improving this manuscript. R.L.S and H.A.N thank Joy Qun Gu for help with the anesthesia assays; these authors also acknowledge George Dold and David Ide of the Research Services Branch of the National Institute of Mental Health/National Institute of Neurological Disorders and Stroke (NIMH/NINDS) for design and construction of the hardware and software used in determining inebriometer elution profiles. Download .pdf (.53 MB) Help with pdf files Document S1. Experimental Procedures, Three Figures, and One Table Download .mov (14.4 MB) Help with mov files Movie S1. Fainting Locomotion in nca-2;nca-1 and unc-79 MutantsLoss-of-function mutations in unc-79(e1291) and nca-2(gk5);nca-1(gk9) move in a hesitating motion when stimulated by touch. Although mutants with this phenotype were classically described as “fainters,” the term is descriptive only of locomotion and does not imply loss of consciousness. All alleles of unc-79 animals and nca-2;nca-1 animals have similar fainting locomotion. In contrast, the wild-type nematode, N2, moves continuously after stimulation. When unperturbed, animals with loss-of-function mutations in unc-79(e1291) and nca-2(gk5);nca-1(gk9) display normal sinusoidal motion but are less active than their wild-type counterparts, N2. Download .mov (.5 MB) Help with mov files Movie S2. Hesitant Walking in na and dunc79 MutantsFlies with a transposon insert into the na gene, a randomly chosen gene (CG11674), or the dunc79 gene were loaded into centrifuge tubes and acclimated briefly. After being tapped to the bottom of the tube, like wild-type flies, the control flies (center tube) climb vigorously. In contrast, although the na and dunc79 mutants (flanking tubes) promptly right themselves and start walking upward, they often pause and thus lag behind the controls. The following sequences have been deposited in GenBank with the corresponding accession number: the C. elegans unc-79 cDNA sequence, DQ858354 ; the D. melanogaster dunc79 cDNA sequence, DQ923614 ; the alternatively spliced C. elegans nca-1 cDNA sequences, DQ917240 and AY555271 ; and the alternatively spliced C. elegans nca-21 cDNA sequences, DQ917241 and AY555272 ." @default.
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• W1974398081 date "2007-04-01" @default.
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• W1974398081 title "A Putative Cation Channel and Its Novel Regulator: Cross-Species Conservation of Effects on General Anesthesia" @default.
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