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• W1501712086 abstract "Early one Monday morning in the 1980s, I was chased around the car park of a Dundee slaughterhouse by an angry bullock. In those days I would regularly set up shop in the gentlemen’s toilet of the abattoir waiting to be handed a steaming mass of bovine fatty tissue, within which was buried an adrenal gland. The gland first had to be isolated, cannulated and flushed with saline before transportation on ice to the lab at Ninewells Hospital where I worked as a PhD student. Chromaffin cells within the adrenal medulla are derived from the neural crest and express neurotransmitter receptors and ion channels similar to those found in the brain [1]. The incident in the car park caused me momentarily to question the utility of bovine chromaffin cells in my anaesthetic research. This is one in a long line of what, at first glance, appear to be esoteric models. Some may argue that research using such a model is unsuitable to grace the pages of an ostensibly clinical journal, such as this one. However, a recent editorial articulated this Journal’s strategy of including relevant animal studies [2]. This is encouraging because most of what we know about anaesthetic mechanisms comes from studies using a panoply of animal models. We used bovine chromaffin cells in electrophysiological experiments to investigate how general anaesthetics affect the activity of the γ-aminobutyric acid type A (GABAA) receptor, the major inhibitory neurotransmitter-activated ion channel in the brain. The patch-clamp technique enables recordings of ionic currents through ion channels either in the membrane of an individual chromaffin cell or in a tiny patch of excised membrane [3]. Using this approach, we discovered that the then fledgling intravenous agent, propofol, potentiates chloride currents activated by GABA and at higher concentrations, directly activates GABAA receptors [4]. Similar experiments in the same study with cultured mouse neurones revealed that propofol also modulates glycine receptors, evolutionary ‘cousins’ of GABAA receptors, which mediate inhibitory neurotransmission in the spinal cord. The textbook view of the molecular mechanism of anaesthesia at the time was the ‘unitary lipid theory’, first proposed at the turn of the 20th century by Meyer and Overton. Based on a correlation between the potency of anaesthetics in tadpoles and their coefficients of partition between olive oil and water, this theory implied that anaesthetics exert their actions primarily by dissolving in the lipid membrane (reviewed in [5]). When the existence of ion channels became incontrovertible, they became candidate mediators of the putative narcotic effects of membrane lipid perturbation (i.e. membrane deformation as a result of anaesthetic dissolution indirectly affects ion channels contained in the membrane) [6]. However, research in the 1980s and 1990s caused the lipid theory to become an anecdote in the history of anaesthesia research. A key development was the demonstration by Franks and Lieb of a striking correlation between the potencies of a series of alcohols as anaesthetics and their potencies of direct inhibition of the soluble luciferase protein isolated from fireflies, in the absence of any lipid [7]. Luciferase is not found in vertebrates and is obviously not a candidate for the molecular target of anaesthesia, but this work established an important point. There is no lipid in the luciferase assay and therefore anaesthetics must be able to modulate proteins through direct interactions. Subsequently, the GABAA receptor became a leading contender for the site of action of intravenous general anaesthetics [8]. In addition to propofol, anaesthetic barbiturates, etomidate and anaesthetic steroids all enhance neuronal GABAA receptor function [4, 9]. Inhalational anaesthetics also positively modulate GABAA receptor activity at relevant concentrations [10]. Anaesthetic steroids are chiral molecules. Their mirror image structures exhibit markedly different anaesthetic potencies and this behavioural stereospecificity is matched by their selectivities as positive allosteric modulators of GABAA receptor function, and this hints at a highly coordinated interaction within the receptor protein [11, 12]. The years between the late 1980s and mid-1990s was a time of frenzied gene cloning. Many ion channel genes, including 19 encoding GABAA receptors, were cloned ahead of the official completion of the human genome project [13]. The ability to express GABAA receptors as recombinant proteins in mammalian cell lines and in oocytes from the African clawed frog, Xenopus leavis, was a major dividend of cloning. Receptors of known composition could now be studied electrophysiologically in isolation and this approach revealed the functional effects of experimental mutations introduced within cloned DNA sequences of GABAA receptors. This research revealed that not all GABAA receptor subtypes respond in the same way to anaesthetics [14]. Furthermore, mutations that cause the substitution of specific amino acids render these receptors unresponsive to certain inhalational anaesthetics, to alcohol, or to the intravenous agent etomidate [15, 16]. These amino acids are prime candidates for the sites through which these drugs allosterically modulate GABAA receptor function. Advances in mouse genetic manipulation enabled the introduction of mutant genes encoding etomidate resistant GABAA receptors [17, 18]. The mutant mice were resistant to the immobilising and sedative effects of etomidate but responded normally to the anaesthetic steroid alphaxalone, which acts elsewhere within the GABAA receptor complex [19]. Anaesthetics have evolved through trial and error since the early discoveries of the immobilising effects of nitrous oxide and ether, without knowledge of their sites of action [5, 8]. Now we know that GABAA receptors are important anaesthetic targets, there is a realistic possibility of designing new drugs with more desirable characteristics. To do this we need to visualise the receptor’s structure in the absence and presence of anaesthetics. Unfortunately, it is not easy to solve the structure of a membrane protein and the GABAA receptor is no exception. A well-ordered protein crystal suitable for interrogation by X-rays is required for the production of a high resolution structural model. The first glimpse of the nicotinic acetylcholine (nACh) receptor, another evolutionary ‘cousin’ of the GABAA receptor, was provided by cryo-electron microscopy applied to protein isolated from the electric organ of the marine ray Torpedo marmorata [20]. This 4-Å resolution structural model has been extremely influential in shaping our view of the GABAA receptor. By aligning GABAA receptor amino acid residues with those of the T. marmorata’s nACh receptor, ‘homology model structures’ of the former can be produced. The method of homology modelling is based on the fact that evolutionarily related proteins often share a common tertiary (three dimensional) structure even when their amino acid sequences are only weakly conserved. Because it is difficult to determine the tertiary structures of membrane proteins directly using methods such as X-ray crystallography and cryo-electron microscopy, homology modelling using a template structure provides a useful surrogate approach for identifying key regions of interest within a protein. These models are the best maps that we currently have for locating amino acids that form the putative anaesthetic-binding sites [19, 21]. The water soluble acetylcholine-binding protein (AChBP) is another useful high quality template structure. First isolated from the pond snail Lymneae stagnalis, AChBP is homologous to the extracellular portion of the GABAA receptor where GABA binds to the agonist site [22]. The AChBP structure was solved at 2.2-Å resolution in the presence and absence of bound nicotine. While the protein provides valuable insight into the neurotransmitter binding site of the GABAA receptor, it lacks the membrane-spanning domains where anaesthetics exert their effects. The structure of a pentameric ligand-gated ion channel (termed GLIC) of the bacterium Gloeobacter violaceus was recently solved in the presence and absence of propofol and desflurane, revealing specific residues involved in anaesthetic binding [23]. These amino acids are located in the first (M1), second (M2) and third (M3) of the four transmembrane domains within each of the five subunits that combine to form GLIC. The binding site is at the interface between subunits, involving M2 and M3 of one subunit and M1 of the adjacent subunit. Photo-affinity labelling of the GABAA receptor with an etomidate analogue reveals a similar inter-subunit binding site that appears to be shared by several anaesthetics, including propofol [24, 25]. Visualising the anaesthetic target, albeit on a receptor that is an ancestral relative of our own GABAA receptors, brings us a step closer to realising the ultimate goal of this type of research. The next frontier is to solve the structure of human GABAA receptors and other neurotransmitter receptors and ion channels that are modulated by general anaesthetics. This will enable the design of new drugs that interact with ion channels to change their function in predictably beneficial ways. There are a couple of morals to this story. First, science moves in mysterious ways. Some of the most esoteric of animal models have made crucial contributions to our understanding of the mechanisms of anaesthetics and the likelihood is that they will continue to do so. In my experience, anaesthetists are often fascinated to learn about the extraordinary arsenal of approaches used to understand the scientific basis of their discipline. In these days of diminishing resources and extraordinary barriers placed in the way of clinical research projects, we will have to rely more heavily on different model systems to strengthen the academic basis of our field. The second moral is less evangelical: when visiting an abattoir, be sure that there are no angry bullocks in the car park before exiting your vehicle. No external funding and no competing interests declared." @default.
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• W1501712086 date "2011-03-31" @default.
• W1501712086 modified "2023-09-29" @default.
• W1501712086 title "Mechanisms of anaesthetics: lessons learned from creatures great and small" @default.
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