A general anaesthetic (or anesthetic) is a drug that brings about a reversible loss of consciousness. These drugs are generally administered by an anaesthetist/anesthesiologist to induce or maintain general anaesthesia to facilitate surgery.
General anaesthetics have been widely used in surgery since 1842 when Crawford Long for the first time administered diethyl ether to a patient and performed a painless operation. It has long been believed that general anaesthetics exert their effects (analgesia, unconsciousness, immobility) through a membrane mediated mechanism or by directly modulating the activity of membrane proteins in the neuronal membrane. In general, different anaesthetics exhibit different mechanisms of action such that there are numerous non-exclusionary molecular targets at all levels of integration within the central nervous system. However, for certain intravenous anaesthetics, such as propofol and etomidate, the main molecular target is believed to be GABAA receptor, with particular β subunits playing a crucial role.
The concept of specific interactions between receptors and drugs first introduced by Paul Ehrlich in 1897 states that drugs act only when they are bound to their targets (receptors). The identification of concrete molecular targets for general anaesthetics was made possible only with the modern development of molecular biology techniques for single amino acid mutations in proteins of genetically engineered mice.
Lipid solubility-anaesthetic potency correlation (the Meyer-Overton correlation)
A nonspecific mechanism of general anaesthetic action was first proposed by Emil Harless and Ernst von Bibra in 1847. They suggested that general anaesthetics may act by dissolving in the fatty fraction of brain cells and removing fatty constituents from them, thus changing activity of brain cells and inducing anaesthesia. In 1899 Hans Horst Meyer published the first experimental evidence of the fact that anaesthetic potency is related to lipid solubility. Two years later a similar theory was published independently by Charles Ernest Overton.
Meyer compared the potency of many agents, defined as the reciprocal of the molar concentration required to induce anaesthesia in tadpoles, with their olive oil/water partition coefficient. He found a nearly linear relationship between potency and the partition coefficient for many types of anaesthetic molecules such as alcohols, aldehydes, ketones, ethers, and esters. The anaesthetic concentration required to induce anaesthesia in 50% of a population of animals (the EC50) was independent of the means by which the anaesthetic was delivered, i.e., the gas or aqueous phase.
Meyer and Overton had discovered the striking correlation between the physical properties of general anaesthetic molecules and their potency: the greater the lipid solubility of a compound in olive oil, the greater its anaesthetic potency. This correlation is true for a wide range of anaesthetics with lipid solubilities ranging over 4-5 orders of magnitude if olive oil is used as the oil phase. This correlation can be improved considerably in terms of both the quality of the correlation and the increased range of anaesthetics if bulk octanol or a fully hydrated fluid lipid bilayer is used as the "oil" phase. It was also noted that volatile anaesthetics are additive in their effects. (A mixture of a half dose of two different volatile anaesthetics gave the same anaesthetic effect as a full dose of either drug alone.)
The best characterized anesthetics site accounting for the Meyer-Overton correlation resides in ordered lipid domains. Anesthetics adhere non-specifically to the surface of a palmitate specific binding site within the lipid membrane, displacing the palmitate from ordered GM1 lipids. The process gives rise to a component of membrane-mediated anesthesia. A similar mechanism was shown for luciferase. The anesthetics bound non-specifically to a hydrophobic surface and out-competed the specific binding of luciferin. However luciferase is not physiologically relevant to vertebrates as it is not endogenously expressed in vertebrates.
Early lipid hypotheses of general anaesthetic action
From the correlation between lipid solubility and anaesthetic potency, both Meyer and Overton had surmised a unitary mechanism of general anaesthesia. They assumed that solubilization of lipophilic general anaesthetic in lipid bilayer of the neuron causes its malfunction and anaesthetic effect when critical concentration of anaesthetic is reached. Later in 1937 Miller and Smith suggested the critical volume hypothesis also called lipid bilayer expansion hypothesis. They assumed that bulky and hydrophobic anaesthetic molecules accumulate inside the hydrophobic (or lipophilic) regions of neuronal lipid membrane causing its distortion and expansion (thickening) due to volume displacement. Accumulation of critical amounts of anaesthetic causes membrane thickening sufficient to reversibly alter function of membrane ion channels thus providing anaesthetic effect. Actual chemical structure of the anaesthetic agent per se is not important, but its molecular volume plays the major role: the more space within membrane is occupied by anaesthetic - the greater is the anaesthetic effect. Based on this theory, in 1954 Mullins suggested that the Meyer-Overton correlation with potency can be improved if molecular volumes of anaesthetic molecules are taken into account. This theory existed for over 60 years and was supported by experimental fact that increases in atmospheric pressure reverse anaesthetic effect (pressure reversal effect).
Then other, physicochemical theories of anaesthetic action emerged that took into account the diverse chemical nature of general anaesthetics and suggested that anaesthetic effect is exerted through some perturbation of the lipid bilayer. Several types of bilayer perturbations were proposed to cause anaesthetic effect, including (1) changes in phase separation, (2) changes in bilayer thickness, (3) changes in order parameters, or (4) changes in curvature elasticity.
According to the lateral phase separation theory anaesthetics exert their action by fluidizing nerve membranes to a point when phase separations in the critical lipid regions disappear. This anaesthetic-induced fluidization makes membranes less able to facilitate the conformational changes in proteins that may be the basis for such membrane events as ion gating, synaptic transmitter release, and transmitter binding to receptors. More recent techniques with super resolution imaging show that the anesthetics do not overcome phase separation—the phase separation persists. Rather saturated lipids within the phase separation can undergo a transition from ordered to disordered which is dramatically affected by anesthetics. Nonetheless the concept of proteins moving between phase separated lipids in response to anesthetic has now been shown to be correct.
All these early lipid theories were generally thought to suffer from four weaknesses (full description with rebuttals see in sections below): Stereoisomers of an anaesthetic drug have very different anaesthetic potency whereas their oil/gas partition coefficients are similar. Certain drugs that are highly soluble in lipids, therefore expected to act as anaesthetics, exert convulsive effect instead (and therefore were called nonimmobilizers). A small increase in body temperature affects membrane density and fluidity as much as general anaesthetics, yet it does not cause anaesthesia. Increasing the chain length in a homologous series of straight-chain alcohols or alkanes increases their lipid solubility, but their anaesthetic potency stops increasing beyond a certain cutoff length.
The correlation between lipid solubility and potency of general anaesthetics was thought to be a necessary but insufficient condition for inferring a lipid target site. General anaesthetics could equally well be binding to hydrophobic target sites on proteins in the brain, but given the chemical diversity of anesthetics this would likely need to include more than one site and those sites would not inherently preclude a site in the membrane. For proteins, one reason that more polar general anaesthetics could be less potent is that they have to cross the blood–brain barrier to exert their effect on neurons in the brain.
Modern lipid hypothesis
There are two modern lipid hypotheses which are non-exclusionary with direct protein binding. The most recent hypothesis postulates that ordered lipids in the plasma membrane contain a structured binding site for the lipid palmitate. It is a lipid binding site within a lipid structure, not a protein structure. Proteins that contain a covalently attached palmitate (palmitoylation) are targeted to the ordered lipids through a specific lipid-lipid interaction. The binding of palmitate to the lipid domain is cholesterol dependent and the cell regulates the protein by nanoscopic localization.
Anesthetics work by binding non-specifically to the palmitate binding site, which disrupts the ability of the cholesterol to bind to and sequester the protein into an inactive state. This membrane mediated mechanism was demonstrated experimentally by Pavel and colleagues in 2020. They showed that the enzyme phospholipase D2 (PLD2) is anesthetic sensitive and activates the potassium channel TREK-1 through a membrane mediated mechanism. The anesthetics displaced PLD2 from ordered lipid domains, allowing the enzyme to be activated by substrate presentation and activate the channel.
The second lipid hypothesis states that anaesthetic effect happens if solubilization of general anaesthetic in the bilayer causes a redistribution of membrane lateral pressures.
Each bilayer membrane has a distinct profile of how lateral pressures are distributed within it. Most membrane proteins (especially ion channels) are sensitive to changes in this lateral pressure distribution profile. These lateral stresses are rather large and vary with depth within the membrane. According to the modern lipid hypothesis a change in the membrane lateral pressure profile shifts the conformational equilibrium of certain membrane proteins known to be affected by clinical concentrations of anaesthetics such as ligand-gated ion channels. This mechanism is also nonspecific because the potency of the anaesthetic is determined not by its actual chemical structure, but by the positional and orientational distribution of its segments and bonds within the bilayer.
In 1997, Cantor suggested a detailed mechanism of general anesthesia based on lattice statistical thermodynamics. It was proposed that incorporation of amphiphilic and other interfacially active solutes (e.g. general anaesthetics) into the bilayer increases the lateral pressure selectively near the aqueous interfaces, which is compensated by a decrease in lateral pressure toward the centre of the bilayer. Calculations showed that general anaesthesia likely involves inhibition of the opening of the ion channel in a postsynaptic ligand-gated membrane protein by the following mechanism: A channel tries to open in response to a nerve impulse, thus increasing the cross-sectional area of the protein more near the aqueous interface than in the middle of the bilayer; Then the anaesthetic-induced increase in lateral pressure near the interface shifts the protein conformational equilibrium back to the closed state, since channel opening will require greater work against the higher pressure at the interface.
This is the first hypothesis that provided not just correlations of potency with structural or thermodynamic properties, but a detailed mechanistic and thermodynamic understanding of anaesthesia.