Molecular targets underlying general anaesthesia

Abstract

The discovery of general anaesthesia, over 150 years ago, revolutionised medicine. The ability to render a patient unconscious and insensible to pain made modern surgery possible and general anaesthetics have become both indispensible as well as one of the most widely used class of drugs. Their extraordinary chemical diversity, ranging from simple chemically inert gases to complex barbiturates, has baffled pharmacologists, and ideas about how they might work have been equally diverse. Until relatively recently, thinking was dominated by the notion that anaesthetics acted 'nonspecifically' by dissolving in the lipid bilayer portions of nerve membranes. While this simple idea could account for the chemical diversity of general anaesthetics, it has proven to be false and it is now generally accepted that anaesthetics act by binding directly to sensitive target proteins. For certain intravenous anaesthetics, such as propofol and etomidate, the target has been identified as the GABA(A) receptor, with particular subunits playing a crucial role. For the less potent inhalational agents, the picture is less clear, although a relatively small number of targets have been identified as being the most likely candidates. In this review, I will describe the work that led up to the identification of the GABA(A) receptor as the key target for etomidate and propofol and contrast this with current progress that has been made in identifying the relevant targets for other anaesthetics, particularly the inhalational agents.

Figure 1

(a) A playbill from 1824 advertising an entertainment at the Savoy Theatre in London in which a demonstration of the ‘exhilarating effects' of laughing gas (nitrous oxide) was promised to ‘any of the audience who may chuse [sic] to inhale it'. It was at a similar occasion in Hartford Connecticut in December 1844 that Horace Wells had seen nitrous oxide demonstrated and where he observed that a volunteer, Samuel Cooley, a clerk at the local drugstore, had injured his leg without any apparent pain. Reproduced with permission from the Victoria and Albert Museum. (b) A re-enactment of the first public demonstration of the use of ether in surgery. This photograph, dated 1847 is a staging of the original event at the Massachusetts General Hospital, in October 1846 by some of the participants. William Morton is at the head of the patient holding his flask of ether. The surgeon, John Collins Warren, is facing the camera with his hands on the patient's leg. The photograph is a daguerreotype by Albert Sands Southworth and Josiah Johnson Hawes and is in the J. Paul Getty Museum, Los Angeles, © The J. Paul Getty Museum.

Figure 2

A selection of general anaesthetics that are used clinically shown as space-filling models. The top row are intravenous anaesthetics, the middle row are volatile inhalational agents and the bottom row are inorganic gases. The older agents are on the left and the most recently introduced are on the right. (A distinction is sometimes made between ‘vapours' such as isoflurane and ‘gases' such as nitrous oxide. However, the physical states of a vapour and a gas are indistinguishable; strictly speaking, a vapour only exists lower than a ‘critical temperature', below which a high enough pressure can condense it into a liquid. The critical temperature of nitrous oxide, for example, is 36.4°C, which strictly makes it a vapour at room temperature, but a gas at body temperature.) The molecular models were drawn using PyMol (DeLano Scientific, San Carlos, CA, U.S.A.).

Figure 3

General anaesthetics act by binding directly to proteins. (a) The famous Meyer/Overton correlation has traditionally been interpreted as meaning that the primary target sites are the lipid portions of nerve membranes. In its modern form, shown here, a good correlation is seen to exist between the potency of an anaesthetic (≡reciprocal of its molar EC50 concentration for anaesthesia) and its lipid/water partition coefficient. (b) General anaesthetic potencies in animals can be correlated equally well with their ability to inhibit the activity of certain soluble enzymes, such as firefly luciferase, whose crystal structure is shown in the inset (Franks & Lieb, 1994). Reprinted with permission from Nature.

Figure 4

Propofol-binding sites on human serum albumin (Bhattacharya et al., 2000). Approximately 98% of propofol binds to blood constituents following administration. The major binding is to the carrier protein serum albumin. A crystallographic analysis shows two possible binding sites (PR1 in subdomain IIIA and PR2 in subdomain IIIB), but it is likely that only site PR1 is occupied under physiological conditions because of the strong binding of fatty acids to the PR2 site. The dashed line in the close-up view on the right represents a hydrogen bond. Propofol binds to a pre-formed cavity with very little perturbation of the local structure.

Figure 5

(a) In vitro data showing the potentiating actions of the intravenous anaesthetics, propofol (PROP) and etomidate (ETOM) on α₂β₃γ₂ GABA_A receptors (Siegwart et al., 2002), and the effects of a β-subunit mutation, β₃(N265M). This mutation substantially, although not completely, reduces the potentiations by etomidate and propofol (but has little, if any, effect on the actions of alphaxalone – not shown). Reproduced with permission from Blackwell Publishing. (b) Behavioural responses to the same two anaesthetics in wild-type and β₃(N265M) knock-in mice (Jurd et al., 2003). Durations for LORR and LHWR were determined. ETOM and PROP had essentially no effect on LHWR in the β₃(N265M) mice and a reduced effect on LORR (corresponding to a rightward shift in the dose–response curve of about three-fold or less). Alphaxalone had the same effect for both end points in the wild-type and β₃(N265M) mice – not shown. Wild-type mice (black shading), β₃(N265M) mice (gray shading). ^**P<0.01, ^***P<0.001 compared to wild type. Reproduced with permission from FASEB.

Figure 6

Two likely targets for inhalational general anaesthetics. (a) The GABA_A receptor (Nishikawa et al., 2002). Reprinted with permission from Elsevier. These data show typical potentiations by halothane and isoflurane of α₁β₂γ₂ GABA_A receptors, and how these potentiations are virtually abolished by a single mutation in the α subunit (S270W). (b) The glycine receptor. These data show percentage potentiations by volatile agents acting on glycine receptors expressed in Xenopus oocytes (Mascia et al., 1996) and (c) in medullary neurons (Downie et al., 1996).

Figure 7

The most recently identified target for inhalational anaesthetics – two-pore domain potassium channels. (a) These data are from rat motoneurons showing the extent of halothane-induced hyperpolarisation and (b) halothane-activated currents due to the anaesthetic activation of a TASK-like potassium channel (Sirois et al., 2000). Copyright 2000 by the Society of Neuroscience. (c) In vivo data showing how TREK-1 knock-out mice are significantly less sensitive to halothane than wild-type controls (Heurteaux et al., 2004). Reprinted with permission.