In vivo genetics of anaesthetic action

Abstract

Over the past decade the use of genetics as a means to investigate volatile anaesthetic action has increased in scale, growing from a few preliminary trials into a substantial field that is confidently addressing basic questions. Although genetic studies can be performed at a variety of levels of biological organization, the present review focuses on the branch of genetics in which variants are identified in intact organisms. The principal impetus for the growing interest in such in vivo genetics of anaesthetic action is a set of characteristics of volatile agents, described in every textbook of anaesthesia, that has made them difficult to analyse. (i)Volatiles are not very potent, requiring near-millimolar concentrations in blood and producing much higher levels in lipid-rich tissues.(ii)At clinical concentrations, these agents produce a staggering array of effects on a wide variety of cellular and biochemical processes.(iii)There are no specific antagonists that reverse their effects.(iv)The clinical state they produce is highly complex and may well be an amalgam of several distinct effects on separate functions.(v)This state lacks a characteristic signature that can be readily identified in studies with isolated tissues and/or experimental organisms. Collectively, these characteristics have encumbered biochemical and physiological studies and thus made it attractive to consider additional strategies. Since anaesthesia is defined by observations on patients and experiments with whole animals, in vivo genetics seems particularly relevant. As detailed below, this approach has its own limitations but these are likely to be different from those of other approaches to anaesthetic action. Thus, genetic studies can be viewed as a promising complement to pharmacological, physiological, and biochemical studies. Another reason for the growth of genetic studies of anaesthetic action has been the avalanche of new genetic techniques and new information about genes. Most notable amongst these are the publication of nearly complete genome sequences for several experimental organisms, including metazoans like nematodes and fruit flies.69Rubin GM Yandell MD Wortman JR et al.Comparative genomics of the eukaryotes.Science. 2000; 287: 2204-2215Crossref PubMed Scopus (1368) Google Scholar This vastly simplifies the chore of making the transition from characterizing a mutated gene at the chromosomal level to determining the molecular nature of its gene product. Although the sequence has not yet been assembled, work is ongoing on the genome of the mouse.66Rogers J Bradley A The mouse genome sequence: status and prospects.Genomics. 2001; 77: 117-118Crossref PubMed Scopus (9) Google Scholar The mouse holds a special place in the realm of genetic studies since it has been the principal focus of techniques for gene targeting, the purposeful replacement of a gene with a manipulated copy. Such manipulations have recently become more and more sophisticated, advancing from simple knock-outs to constructs in which gene function is altered at specific times during the life and/or in specific tissues of the mouse.45Lewandoski M Conditional control of gene expression in the mouse.Nat Rev Genet. 2001; 2: 743-755Crossref PubMed Scopus (625) Google Scholar Although gene replacement is only rarely achieved in Drosophila,24Gloor GB Gene-targeting in Drosophila validated.Trends Genet. 2001; 17: 549-551Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar emerging techniques offer similarly specific alteration of gene function.92White B Osterwalder T Keshishian H Molecular genetic approaches to the targeted suppression of neuronal activity.Curr Biol. 2001; 11: R1041-R1053Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar The tool kit for experimental genetic studies has thus become much more diverse and powerful. And, the recent publication of a draft sequence of the human genome4Baltimore D Our genome unveiled.Nature. 2001; 409: 814-816Crossref PubMed Scopus (255) Google Scholar greatly simplifies the task of evaluating the clinical significance of a gene identified in a model organism. The use of genetics to investigate anaesthetic action was last summarized over five years ago.76Simpson VJ Johnson TE Genetic models in the study of anesthetic drug action.Int Rev Neurobiol. 1996; 39: 223-241Crossref PubMed Google Scholar In this review, I begin by outlining the many choices from which an investigator must select in designing a genetic study. Each such selection carries with it opportunities and limitations and I will attempt to highlight which of these are most critical for the study of anaesthetic action. The second part of this review presents examples from recent and ongoing work from this field illustrating the power of the genetic approach. The final section discusses an important issue that remains underdeveloped. As for any experiment, the design of a genetic project involves many decisions. Although some projects may demand the development of novel approaches, most will come down to choices between established alternatives. Each of these will in large measure determine what one can hope to find, but it must be emphasized that the alternatives are not exclusive: similar information can be obtained in more than one way. Nevertheless, it is vital to fully consider the implications of each choice. Perhaps the most fundamental of the strategic alternatives is that of forward versus reverse genetics. Reverse genetics focuses on a particular gene, usually chosen because its product is suspected to be important for the process under study. Typically, mutations are deliberately made in a cloned copy of the gene, the construct is introduced into the germline of the organism, and the effect on the process is examined. Reverse genetics is thus a way to ask directed questions about the involvement of a gene in a pharmacological effect. Its power is exemplified by the demonstration that the sedative action of benzodiazepines is lost in mice in which a specific mutation has been engineered into the α1 subunit of the GABAA receptor.51McKernan RM Rosahl TW Reynolds DS et al.Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype.Nat Neurosci. 2000; 3: 587-592Crossref PubMed Scopus (833) Google Scholar 70Rudolph U Crestani F Benke D et al.Benzodiazepine actions mediated by specific gamma-aminobutyric acid(A) receptor subtypes.Nature. 1999; 401: 796-800Crossref PubMed Scopus (1046) Google Scholar In contrast to reverse genetics, forward genetics refers to study of untargeted mutations, i.e. those that are randomly generated and chosen for study simply because they yield a particular phenotype. Thus, forward genetics can uncover an important role for a gene previously unsuspected to be involved. As stated by Hartwell and colleagues,31Hartwell LH Szankasi P Roberts CJ Murray AW Friend SH Integrating genetic approaches into the discovery of anticancer drugs.Science. 1997; 278: 1064-1068Crossref PubMed Scopus (608) Google Scholar the ability of those who use forward genetics to identify new players in a process is because, ‘…we approach biology with humility – we allow the organism to tell us which are the important functions.’ It is this basic philosophical distinction that, despite the experimental difficulties described in subsequent sections of this review, makes it attractive to consider a forward genetic approach to anaesthetic action, a subject where it is still possible to imagine there are novel mechanisms at work. If one chooses the forward genetic approach, one must also decide between undertaking a mutagenesis, generating new gene variants, or exploiting polymorphisms, variants that occur naturally in animal populations.85Tully T Discovery of genes involved with learning and memory: an experimental synthesis of Hirschian and Benzerian perspectives.Proc Natl Acad Sci USA. 1996; 93: 13460-13467Crossref PubMed Scopus (92) Google Scholar Although in both cases one must select the variants of interest on the basis of their phenotype, there are significant differences in the two kinds of undertaking. Mutagenesis has the advantage of starting with a single parental strain, which is often reared so as to be genetically homogeneous. A new mutation generated in such an inbred or isogenic strain can thus be studied against a very uniform background. In contrast, relevant polymorphisms are detected by selecting individuals from an outbred (genetically heterogenous) population or by selecting individuals derived from a cross between two inbred strains. As a result, unless there is a single polymorphism of major effect, it may be hard to distinguish the effect of a variation in an individual gene. This is because each animal inheriting the variation has a distinct (and presumably random) combination of other genetic variants. A disadvantage of mutagenesis is that, even with the help of environmental mutagens, the rate at which new mutations can be generated in any particular locus is very low. Thus, a mutagenesis approach requires screening large numbers (typically tens of thousands) of individuals to find a few with mutations that alter the phenotype of interest. Many fewer animals are required to detect relevant polymorphisms because the mutations have already accrued in the starting stocks. The choice of experimental organism is no less important than those discussed above. Moreover, it illustrates dramatically the degree to which the choices are interconnected. For example, as described above, a forward mutagenesis approach implies the ability to test large numbers of offspring. This limits the choice of experimental animal to a few possibilities. (i)Bacteria and fungi. Single-celled microbes are the most facile subjects for a genetic study. Their generation time is usually less than an hour, they can be raised by the billion, their complement of genes is small (2000–6000), and several microbial genomes have been completely sequenced. Previous work has shown that at least one microbe is affected by volatile anaesthetics and that genes which perturb its sensitivity to anaesthetics can be identified.93Wolfe D Reiner T Keeley JL Pizzini M Keil RL Ubiquitin metabolism affects cellular response to volatile anesthetics in yeast.Mol Cell Biol. 1999; 19: 8254-8262Crossref PubMed Scopus (22) Google Scholar(ii)Caenorhabditis elegans. This nematode comprises 959 somatic cells, about 300 of which are neurones. Despite its simplicity, the adult organism displays a coordinated locomotor pattern and its movements reveal the capacity for thermotaxis and chemotaxis.6Bargmann CI Mori I Chemotaxis and thermotaxis.in: Riddle DL Blumenthal T Meyer BJ Priess JR C. elegans II. Cold Spring Harbor Laboratory Press, Plainview, NY1997Google Scholar Since its introduction to the research community in the 1960s, Caenorhabditis has been a favourite of geneticists, many of whom have studied genes that affect neural function and behaviour.12Chalfie M Jorgensen EM C. elegans neuroscience: genetics to genome.Trends Genet. 1998; 14: 506-512Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Its small genome (∼97 Mbp) has now been completely sequenced69Rubin GM Yandell MD Wortman JR et al.Comparative genomics of the eukaryotes.Science. 2000; 287: 2204-2215Crossref PubMed Scopus (1368) Google Scholar and many of its ∼19,000 genes are easily identifiable as homologues of vertebrate genes that encode components of the nervous system.5Bargmann CI Neurobiology of the Caenorhabditis elegans genome.Science. 1998; 282: 2028-2033Crossref PubMed Scopus (722) Google Scholar For more than 15 years, C. elegans has been the subject of genetic studies of anaesthesia.57Morgan PG Cascorbi HF Effect of anesthetics and a convulsant on normal and mutant Caenorhabditis elegans.Anesthesiology. 1985; 62: 738-744Crossref PubMed Scopus (57) Google Scholar(iii)Drosophila melanogaster. The nervous system of the fruit fly contains more than 100,000 neurones and mediates an impressive behavioural repertory, including land-based and aerial navigation20Ferrus A Canal I The behaving brain of a fly.Trends Neurosci. 1994; 17: 479-486Abstract Full Text PDF PubMed Scopus (8) Google Scholar and elaborate courtship rituals.30Hall JC The mating of a fly.Science. 1994; 264: 1702-1714Crossref PubMed Scopus (533) Google Scholar Flies also display circadian cycles of locomotor activity that have many of the characteristics of sleep cycles in vertebrates39Kilduff TS What rest in flies can tell us about sleep in mammals.Neuron. 2000; 26: 295-298Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar and flies have the capacity to remember salient events.67Roman G Davis RL Molecular biology and anatomy of Drosophila olfactory associative learning.Bioessays. 2001; 23: 571-581Crossref PubMed Scopus (116) Google Scholar The genome of Drosophila is of a similar size and complexity as that of the nematode and it too has been sequenced.69Rubin GM Yandell MD Wortman JR et al.Comparative genomics of the eukaryotes.Science. 2000; 287: 2204-2215Crossref PubMed Scopus (1368) Google Scholar Classical fly genetics provides ample tools for analysing mutants26Greenspan RJ Fly Pushing. The Theory and Practice of Drosophila Genetics. Cold Spring Harbor Laboratory Press, Plainview, NY1997Google Scholar and a host of newer methods permit tissue and temporal control of gene action.92White B Osterwalder T Keshishian H Molecular genetic approaches to the targeted suppression of neuronal activity.Curr Biol. 2001; 11: R1041-R1053Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar For more than 70 yr researchers have used diethyl ether to immobilize flies and more than 20 yr ago differences between strains of flies were noted in sensitivity to ether.23Gamo S Ogaki M Nakashima-Tanaka E Strain differences in minimum anesthetic concentrations in Drosophila melanogaster.Anesthesiology. 1981; 54: 289-293Crossref PubMed Scopus (43) Google Scholar In more recent times, single gene analyses of anaesthetic action in Drosophila using forward genetics have become prominent.(iv)Danio rerio. The zebrafish is a vertebrate whose life cycle and ease of culture make it amenable to forward genetic studies.78Solnica-Krezel L Schier AF Driever W Efficient recovery of ENU-induced mutations from the zebrafish germline.Genetics. 1994; 136: 1401-1420Crossref PubMed Google Scholar Although most of the mutagenesis efforts to date have been devoted to developmental biology, there is an increasing focus on the function of the zebrafish nervous system, which has the brain anatomy and physiology of a typical vertebrate.14Darland T Dowling JE Behavioral screening for cocaine sensitivity in mutagenized zebrafish.Proc Natl Acad Sci USA. 2001; 98: 11691-11696Crossref PubMed Scopus (306) Google Scholar To the knowledge of this reviewer, there are no published studies of anaesthetic action in this organism but the opportunities look inviting. Although genetics of anaesthesia in non-mammalian model organisms has much to offer, anaesthesia is defined by a clinical state in human patients. Accordingly, as much as possible one wants to examine the impact on anaesthesia of genetic variations in mammals. Two species dominate such efforts. (i)Mus musculus. Anaesthetics have long been known to alter the behavioural state in the mouse and genetic polymorphisms that are present in populations of mice can result in lines with significantly altered anaesthetic sensitivity.41Koblin DD Deady JE Eger 2nd., EI Potencies of inhaled anesthetics and alcohol in mice selectively bred for resistance and susceptibility to nitrous oxide anesthesia.Anesthesiology. 1982; 56: 18-24Crossref PubMed Scopus (30) Google Scholar 79Sonner JM Gong D Eger 2nd., EI Naturally occurring variability in anesthetic potency among inbred mouse strains.Anesth Analg. 2000; 91: 720-726Crossref PubMed Scopus (114) Google Scholar However, until recently single gene studies were lacking. It is possible to mount a forward genetic scheme with this organism,3Balling R ENU Mutagenesis: analyzing gene function in mice.Annu Rev Genomics Hum Genet. 2001; 2: 463-492Crossref PubMed Scopus (106) Google Scholar but such an effort requires enormous resources. In contrast, as acknowledged in the recent Lasker Award,25Goldstein JL Laskers for 2001: knockout mice and test-tube babies.Nat Med. 2001; 7: 1079-1080Crossref PubMed Scopus (18) Google Scholar the development of gene targeting strategies in the mouse has revolutionized and simplified mammalian genetics. Although it is still a major undertaking, engineering the genome of a mouse so as to alter the structure and/or expression of a single gene can now be accomplished predictably. Accordingly, there has been a wave of studies that have used reverse genetics to evaluate candidate genes in anaesthetic action.(ii)Homo sapiens. As opposed to the case with experimental animals, studying genetic effects in humans depends entirely on naturally occurring polymorphisms. There are hundreds of single gene variants that are known to cause alterations in human health.52McKusick VA Mendelian Inheritance in Man. Catalogs of Human Genes and Genetic Disorders. 12th edn. Johns Hopkins University Press, Baltimore, MD1998Google Scholar One expects that many of these will alter the affected individual's response to anaesthetics, and some may do so in ways that are informative of anaesthetic action. Yet, with the exception of malignant hyperthermia,34Hopkins PM Malignant hyperthermia: advances in clinical management and diagnosis.Br J Anaesth. 2000; 85: 118-128Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar to the knowledge of this reviewer the number of published studies that examine the relationship between familial traits and anaesthesia is vanishingly small. In part, this is due to the substantial non-genetic variance in human populations'due to factors like age, sex, debilitation, etc.'in the potency of anaesthetics. Against this background it may be hard to discern a genetic component. However, it also seems true that this issue has not had the full attention of anaesthesiologists. As discussed below (in the section on the gas-1 gene), this is likely to change soon. At the Sixth International Conference on Molecular and Basic Mechanisms of Anaesthesia held in Bonn in June 2001, among the most welcome features were the repeated and clearly enunciated reminders of the need to define anaesthetic endpoints. In patients, it has been long understood that the anaesthetized state has many components'absence of movement in response to noxious stimuli, absence of visceral response to pain, muscle relaxation, amnesia for perioperative events, etc. How each of these relates to each of the others, relates to monitors of neural activity, and relates to the molecular actions of anaesthetics is largely unknown. The situation becomes even less clear in the case of the experimental animals used in genetic research. Here, in addition to the problems just listed, there is the added problem of relating anaesthetic effects in the model organism to those in humans. The problem of relating endpoints for anaesthetic effects is most obvious for microbes, where the readily available endpoint is inhibition of growth. Most researchers believe that the clinically relevant effects of anaesthetics are those that are peculiar to the nervous system, especially those that concern synaptic transmission. For this, growth of a microbe may be a very poor model. (Microbes can enjoy cell–cell communication, e.g. during formation of mating pairs; perhaps anaesthetic influences on this process would be fruitful.) Compared to microbes, the invertebrate models have the advantage of containing a nervous system that incorporates representatives of virtually all of the components found in neurones of vertebrates. And they have motor programmes that are easy to observe on a very short time scale and thus offer a variety of convenient endpoints that are informative of the state of the neuromuscular system. For example, in nematodes anaesthetics cause alterations in spontaneous movement that range from lack of coordination13Crowder CM Shebester LD Schedl T Behavioral effects of volatile anesthetics in Caenorhabditis elegans.Anesthesiology. 1996; 85: 901-912Crossref PubMed Scopus (54) Google Scholar to complete immobility.57Morgan PG Cascorbi HF Effect of anesthetics and a convulsant on normal and mutant Caenorhabditis elegans.Anesthesiology. 1985; 62: 738-744Crossref PubMed Scopus (57) Google Scholar Similarly, anaesthetics can render fruit flies unable to climb,28Guan Z Scott RL Nash HA 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 unable to stand,42Krishnan KS Nash HA 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 or unable to respond to an irritating stimulus.9Campbell DB Nash HA Use of Drosophila mutants to distinguish among volatile general anesthetics.Proc Natl Acad Sci USA. 1994; 91: 2135-2139Crossref PubMed Scopus (69) Google Scholar 23Gamo S Ogaki M Nakashima-Tanaka E Strain differences in minimum anesthetic concentrations in Drosophila melanogaster.Anesthesiology. 1981; 54: 289-293Crossref PubMed Scopus (43) Google Scholar However, a linear relationship between any of these endpoints and those in a mouse, e.g. the response to a tail pinch, is virtually impossible. Even with a mouse as the experimental organism, it is hard to know how an endpoint like the loss of righting reflex relates to the hypnotic actions of anaesthetics in the clinic. But at least the loss of movement in response to a tail pinch seems like a straightforward model for a patient's loss of movement in response to surgical incision. Moreover, the recent demonstration that anaesthetics disrupt fear conditioning in rats80Sonner JM Li J Eger 2nd., EI Desflurane and the nonimmobilizer 1,2-dichlorohexafluorocyclobutane suppress learning by a mechanism independent of the level of unconditioned stimulation.Anesth Analg. 1998; 87: 200-205PubMed Google Scholar extends the hope of modeling in the mouse the phenomenon of perioperative amnesia in humans. One criterion that is frequently used to judge the relevance to human anaesthesia of anaesthetic endpoints in an experimental organism is that they can be observed at concentrations used in the clinic. On this basis, endpoints like immobility of nematodes and inhibition of yeast growth (which are observed, respectively, at isoflurane concentrations of ∼6 and ∼12%57Morgan PG Cascorbi HF Effect of anesthetics and a convulsant on normal and mutant Caenorhabditis elegans.Anesthesiology. 1985; 62: 738-744Crossref PubMed Scopus (57) Google Scholar 93Wolfe D Reiner T Keeley JL Pizzini M Keil RL Ubiquitin metabolism affects cellular response to volatile anesthetics in yeast.Mol Cell Biol. 1999; 19: 8254-8262Crossref PubMed Scopus (22) Google Scholar) may be less relevant. However, to this reviewer, such a conclusion seems short-sighted. Volatile agents almost certainly cause their effects in humans by binding to molecular targets. Let us assume that the resulting alteration in function of one such target makes a major contribution to a particular anaesthetic endpoint. Without having to specify the identity of this target in humans, one can easily imagine that it is present in your favourite organism (YFO) and that it is similarly responsible for producing an anaesthetic endpoint. Despite such conservation, there are at least two ways in which anaesthetic potency in YFO could be much different from that in human subjects. First, there might be substantial differences between organisms in the degree to which target function must be altered so as to produce the assayed endpoint.86Urban BW Friederich P Anesthetic mechanisms in-vitro and in general anesthesia.Toxicol Lett. 1998; 100-101: 9-16Crossref PubMed Scopus (30) Google Scholar In other words, humans and YFO might have different safety factors. Second, there might be substantial differences in the potency with which anaesthetics disrupt target function in the two organisms. Ample precedent exists for this possibility from studies of receptor pharmacology, where the same (homologous) receptor from a vertebrate and an invertebrate can have radically different IC50 values for a given specific antagonist.7Blenau W Baumann A Molecular and pharmacological propertoes of insect biogenic amine receptors: lessons from Drosophila melanogaster and Apis mellifera.Arch Insect Biochem Physiol. 2001; 48: 13-38Crossref PubMed Scopus (310) Google Scholar In either case, it would be wrong to eliminate the anaesthetic effect on a particular behaviour in YFO from consideration just because it required higher concentrations of volatiles than does clinical anaesthesia. In the end, the value of an endpoint will be determined by the quality of information provided to the neuroscience/anaesthesia community from genetic studies that use the endpoint. In recent years, such studies have passed through the exploratory phase and have begun to focus on individual genes. In the next section of this review, I discuss some of the leading characters implicated by this approach. Each of the principal organisms for experimental genetic study'worms, flies and mice'has now been subjected to substantial scrutiny for mutations that affect the response to volatile anaesthetics. From such studies a small set of genes has been identified, typically because they yield large effects and/or because they are known to encode plausible candidates. As yet, the same genes have not been studied in more than one system, so it is impossible to make cross-species comparisons. Nevertheless, these genes provide a window on to the methods used, the difficulties encountered, and the promise of the enterprise. The following sections give details for selected examples from this set. All of these come from studies of random or targeted mutagenesis; genetic studies of naturally occurring polymorphisms have yet to be refined to the level at which individual genes are identified. And although the example of malignant hyperthermia makes it clear that genetic variation in muscle can influence the course of anaesthesia,34Hopkins PM Malignant hyperthermia: advances in clinical management and diagnosis.Br J Anaesth. 2000; 85: 118-128Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar because of the unique capacity of volatile anaesthetics to perturb higher brain function, I focus on those genes known to be involved in neurones. Syntaxin is well-established as an integral part of the apparatus responsible for the release of the contents of synaptic vesicles into the synaptic cleft.46Lewis JL Dong M Earles CA Chapman ER The transmembrane domain of syntaxin 1A is critical for cytoplasmic domain protein–protein interactions.J Biol Chem. 2001; 276: 15458-15465Crossref PubMed Scopus (33) Google Scholar From a screen of mutants known to be altered in neural function of Caenorhabditis, Crowder and colleagues88vanSwinderen B Saifee O Shebester L Roberson R Nonet ML Crowder CM A neomorphic syntaxin mutation blocks volatile anesthetic action in Caenorhabditis elegans.Proc Natl Acad Sci USA. 1999; 96: 2479-2484Crossref PubMed Scopus (96) Google Scholar identified an allele (md130) of unc-64, of the nematode ortholog of syntaxin as conferring substantial resistance to the effects of isoflurane and halothane on the ability of C. elegans to disperse toward a source of food (Fig. 1). Other alleles of unc-64 were associated with an increased potency of these agents, i.e. the opposite anaesthetic phenotype to md130. This was surprising because many of these alleles shared with md130 a resistance to a cholinesterase inhibitor, suggesting that they all had reduced presynaptic function. The possibility that volatiles directly interact with syntaxin (or other components of complexes containing syntaxin) provides a plausible explanation for the different phenotypes of the alleles; subtle differences in syntaxin protein could raise or lower the affinity for volatiles. However, it should be pointed out that syntaxin is now known to have no less than twenty interacting partners,46Lewis JL Dong M Earles CA Chapman ER The transmembrane domain of syntaxin 1A is critical for cytoplasmic domain protein–protein interactions.J Biol Chem. 2001; 276: 15458-15465Crossref PubMed Scopus (33) Google Scholar specific alleles might affect interactions with some partners more than others. If so, rather than resulting from an altered contact between the anaesthetic and a syntaxin-containing complex, anaesthetic resistance or sensitivity could reflect altered function of an anaesthetic-sensitive circuit as a consequence of a particular imbalance between partners of syntaxin. Nevertheless, it seems likely that syntaxin is ‘close to the action’ of anaesthetics in the nematode and therefore provides a valuable lead. Accordingly, Crowder and colleagues have been exploiting a powerful genetic strategy: examining for genes that interact with the gene for syntaxin. They have reported that mutants of a subunit of a neuronally expressed G-protein alter the sensitivity of unc-64 mutants87vanSwinderen B Metz LB Shebester LD Mendel JE Sternberg PW Crowder CM Goalpha regulates volatile anesthetic action in Caenorhabditis elegans.Genetics. 2001; 158: 643-655PubMed Google Scholar and at the Bonn conference they reported the preliminary identification of several more suppressors. Lipid rafts are microdomains of the cell membrane rich in sphingolipids, cholesterol, s