The dramatic correlation of hydrophobicity with anesthetic potency predated an understanding of protein interiors, so initial focus fell on the obvious oily candidate: the lipid bilayer (Fig. 37-6). The idea was that solubilization of the hydrophobic anesthetic into the thin lipid membrane altered a property sensed by the cell or by proteins wholly or partially embedded in the lipid. The crucial property was (and still is) unclear, so surrogate properties that could be easily measured, such as the gel–liquid phase transition, were studied extensively. Changes induced by the anesthetics in these properties could be demonstrated, but several features gradually emerged that dampened enthusiasm in the lipid membrane as an important transducer of anesthetic effects. First, measurable changes in phase transition at clinical concentrations of anesthetic were small, mimicked by only 1° to 2°C. Second, other compounds that dissolved well and produced similar effects in these bilayers (eg, nonimmobilizers) did not produce anesthesia. Finally, the emergence of protein-centered theories shifted attention away from the lipid bilayer.
Snapshot of a lipid membrane in a molecular dynamics simulation. This gives an idea of the complexity and organization of this essential and widely distributed structure. Our poor understanding of its communication with proteins embedded in it and of effects of solutes distributed in it have conspired to make it difficult to eliminate the lipid bilayer as a viable potential target for transducing some anesthetic effects.
A shift in attention, however, does not suffice to eliminate the potential for an important contribution. New theories, coupled with improved understanding of the lipid bilayer and its interaction with anesthetics and membrane protein, call for a return to the lipid bilayer at some point to test whether anesthetic modulation contributes to in vivo anesthetic effects. For example, it is clear that phase transitions are not a relevant feature of biologic membranes, so conclusions based on their measurement must be viewed cautiously. It now is clear that anesthetics distribute in the lipid bilayer heterogeneously, less in the center (acyl trough) and more toward the outer edges. This asymmetric distribution can influence lipid properties not easily measured, such as lateral pressure (Fig. 37-7) but posited to have a potent effect on membrane protein conformational transitions.10 Interestingly, even though nonimmobilizers partition well into the lipid bilayer, the distribution within the bilayer is very different than that of an anesthetic,11 yet again opening the door for lipid theories. Furthermore, lipid microdomains exist (eg, lipid rafts) that appear to organize specific proteins into functional groups may have a higher affinity for inhaled anesthetics (because of unique lipids or unique proteins), increasing the opportunity for anesthetic effects on signaling pathways.12 Finally, in most assays of membrane protein activity (eg, ion channels), protein is functionally inseparable from the lipid. Cholesterol, for example, may contribute structurally essential roles in some membrane proteins by binding deep within the protein matrix.13 Therefore, mutagenesis of membrane protein by itself cannot be directly used to implicate a protein target because the mutation also might influence the interaction with lipid. Thus, it is premature to rule out the biologic lipid membrane as an important direct target for the inhaled anesthetics.
Lateral pressure hypothesis of membrane–protein communication. A. Membrane protein in 2 conformations: R for resting, and A for activated. The A conformer requires some expansion in the membrane center. B. Membrane lateral profile of vectors that favors the R state. C. Pressure profile that favors the A conformer. A solute that distributes unevenly across the membrane will, of necessity, alter this lateral pressure profile, resulting in a modulation of conformational state and therefore of activity. Molecular dynamics simulations predict that anesthetic molecules do distribute unevenly across the lipid bilayer. [Reprinted with permission from Gompf H, Chen J, Sun Y, et al. Halothane-induced hypnosis is not accompanied by inactivation of orexinergic output in rodents. Anesthesiology. 2009;111:1001-1009.]
Proteinaceous components of the cell as targets for anesthetics were first proposed by Claude Bernard in 1875 but did not gain favor until the demonstration that the activity of lipid-free preparations of protein were reversibly influenced by anesthetics and in a way that reproduced the correlation with hydrophobicity.14 Because this paralleled the understanding that the interior of proteins is as hydrophobic as that of lipid membranes, this association should not have been surprising. When combined with enantioselectivity and an ability to measure and alter protein function, especially in ion channels, protein-centered theories rapidly became favored. Many protein targets have been proposed and studied, and we review some of them briefly. However, at this time it is safe to conclude that not a single protein target or even class or family of protein targets has been proven to play the central role in inhaled anesthetic action. This is not to say, however, that favored candidates have not emerged, especially for the injectable induction agents. We first review some essential basics of protein–ligand interactions as they relate to anesthetics and then present the evidence for specific targets.
The submolecular mechanism by which the anesthetic causes a change in protein function or activity must first involve a binding event—formation of drug–target complex. This often is the initial criterion for establishing the relevance of any proposed drug target but has not been used for inhaled anesthetics because the affinity for protein targets is low, and they appear to be fairly promiscuous, binding to many targets. Both nuclear magnetic resonance spectroscopy and photolabeling have suggested a large number of protein binding targets (ie, 27) and no particular regional preference in the brain (Fig. 37-8).15,16 This renders untenable the conventional radioligand binding approach for discovering targets. Whether anesthetics also significantly alter the function of a large number of proteins is not clear, but even if only a minority of the binding target pool, the number of contributing targets is still large.
A. Autoradiogram of rat brain sagittal section photoaffinity labeled with 14C-halothane. The degree of halothane binding is indicated by the level of darkness; no other stain has been applied to this section. There appears to be no regional preference, and excess unlabeled halothane reduces incorporation by approximately 70% (image not shown), indicating that this distribution represents specific binding. B.19F-Nuclear magnetic resonance coronal section of in vivo rat brain after equilibration with sevoflurane. C. Orientation of B. Both experiments demonstrate widespread anesthetic distribution in mammalian brain. [Part B from Eckenhoff MF, Eckenhoff RG. Quantitative autoradiography of halothane binding in rat brain. J Pharmacol Exp Ther.1998;285; Fig. 2c.]
Nature of the Binding Site
Until recently, the nature of anesthetic binding sites has been inferred from correlations with anesthetic structural or physicochemical features. For example, the Overton-Meyer relationship suggested that sites are in the hydrophobic interior of proteins, and the relationship of halogen size and asymmetric hydrogens with potency suggested that polar amino acids must contribute to these sites. High-resolution structures of a clinically used inhaled anesthetic complexed to a protein target have been revealed for at least 3 protein models: serum albumin,17 apoferritin (Fig. 37-9),18 and integrin lymphocyte function-associated antigen 1 (LFA-1).19 Although unlikely to transduce desirable effects of anesthesia, the affinity of these proteins (see later discussion) suggests substantial occupancy at clinical concentrations of anesthetic (˜0.2 mmol/L), and thus the binding site architecture is likely to bear resemblance to those in targets underlying effects relevant to anesthesia. These structures show occupancy of preexisting internal packing defects, also known as cavities. The apoferritin example goes further to suggest that a packing density of just over half full represents an optimal balance between enthalpic and entropic forces. Finally, all of these examples suggest that polar (but uncharged) amino acids contribute to the strength of binding. In the apoferritin cavity, 2 serine–tyrosine–leucine triads interact with either halothane or isoflurane atoms to produce the highest affinity binding yet described for these compounds (Fig. 37-9). The stringency of these features is not yet clear, but preliminary estimates based on current entries in the Protein Data Bank (http://www.rcsb.org/pdb) suggest that less than 1% of the proteome (˜3000 proteins) have comparable binding sites for volatile anesthetics.
High-resolution structure of the high-affinity anesthetic binding site in an interface between 2 subunits of apoferritin. Halothane is shown in the middle, coordinated by 2 serine–tyrosine–leucine (SYL) triads (see text for details). Note that the parallel helical bundle motif of this binding domain is found in the transmembrane region of the ligand-gated ion channel subunits and in many receptors (see Figs. 37-14 and 37-15).
Affinity and Stoichiometry
Affinity of a drug for a site, indicated by the association constant (Ka) or, more commonly, the dissociation constant (Kd = 1/Ka), is experimentally defined as the drug concentration at which half the sites are occupied. It is more properly called "apparent" affinity because other aspects of the mixture can modify the true affinity of the ligand–site complex. Relevant to this is whether the binding event "causes" a conformational change (see later discussion) in the protein to which it binds. If so, the apparent affinity will be lowered by the amount of free energy used to change the protein conformation (Fig. 37-10). Thus, a set of binding site features that produces a 10 μmol/L absoluteKd with an anesthetic perhaps will have an apparentKd of 1 mmol/L if approximately 2 kcal/mol of free energy is required to change the protein conformation.
Sigmoid-shaped dose–response curve for a typical anesthetic-like drug. The effect curve often most left shifted is the steepest. The binding effects that underlie that the effect usually is shifted to the right and may or may not have the same slope, depending on the number targets, the number of sites per target, and whether the sites interact (cooperativity). This indicates that molecular sites underlying a specific effect do not necessarily need to be fully occupied to reach full (saturation) effect. Nonspecific binding (that generally not associated with the primary effect) is most right shifted and may appear to be linear in many experiments.
The slope of the relationship between drug concentration and occupancy, the Hill slope, is an indication of both the number of sites on a target and whether they interact. For example, a single site should always have a Hill slope of 1, reflecting a relationship in which the site goes from 0% to 100% occupancy over an approximately 100-fold change in drug concentration (Fig. 37-10). Multiple sites on a single target also can demonstrate a Hill slope of 1, or they can "cooperate," increasing the slope. In other words, if occupancy of the first site enhances occupancy of the second (as in oxygen binding by hemoglobin), the slope will rise, although rarely over 2 or 3. Although difficult to measure as indicated, anesthetic Kd values in model proteins typically are approximately 1 mmol/L, and Hill slopes are generally 1 or slightly greater even though more than 1 binding site has been found in most proteins studied. Serum albumin has at least 6 sites,17 apoferritin 12,18 and the intact nicotinic acetylcholine receptor (nAChR) 15 to 20.20-23 As might be deduced from the preceding paragraph, occupancy of multiple sites is a potentially powerful method of donating free energy to the protein to alter its conformation. Occupancy of only 1 additional binding site in a protein contributes as much free energy as an approximately 10-fold increase in affinity at a single site.
An issue frequently causing confusion has been what affinity an important anesthetic target should display for anesthetics. Prevailing logic has suggested that for a target to be relevant to a particular endpoint (eg, immobility), the dissociation constant for the anesthetic–target complex should approximate that achieved at MAC. However, this logic holds only if one assumes that this target is alone capable of producing the endpoint, an assumption that rarely is valid. Furthermore, even if the target is wholly responsible for the endpoint, it is difficult to know the expected Kd because drug efficacy at this target is not known. It is rare for the relationship between receptor occupancy and median effective concentration (EC50) to be linear; thus, predicting more than that some degree of occupancy should be anticipated at clinical concentrations is difficult. As our discussion on Hill slopes indicates, this limits the expected Kd to an approximately 100-fold range.
As discussed earlier, the anesthetic can only alter the protein's activity through a contribution of binding energy—through either the affinity or the stoichiometry of the complex. This binding energy alters protein activity through at least 3 potential and partially overlapping mechanisms. The first mechanism, competition, occurs when the drug–protein complex is strong enough to compete with the binding of an endogenous ligand, therefore inhibiting the associated effect (Fig. 37-11). This was the mechanism initially proposed for anesthetic inhibition of firefly luciferase.14 There are 2 forms of competition: isosteric and allosteric. In isosteric competition, the drug occupies the same binding site as the endogenous ligand, physically preventing binding. An anesthetic example is halothane occupancy of the retinal cavity in the G-protein coupled receptor (GPCR) rhodopsin.24 In allosteric competition, the drug binds elsewhere in the protein, altering the structure sufficiently to disfavor ligand binding in its otherwise unoccupied site. A clear example is integrin LFA-1, in which isoflurane binds in a cavity that stabilizes a conformation with low affinity for the intercellular adhesion molecule (ICAM) receptor protein.19 In a closely related form of allosterism, cooperativity, allosteric binding of anesthetic favors a conformation that binds the endogenous ligand more tightly. Thus, in firefly luciferase, the binding of adenosine triphosphate (ATP) and anesthetic is cooperative.25 Cooperative effects of agonist and anesthetic in some receptors may also be attributable to binding cooperativity.22,26
A. Example of isosteric competition in which the anesthetic (red oval) achieves a high enough concentration to actually replace the endogenous ligand (green trapezoid) in its site of action. B. Example of allosteric competition in which the anesthetic binds preferentially to a conformer that disfavors binding of the endogenous ligand, but at a distant site.
The second general category of drug–effect coupling is ensemble modulation (Fig. 37-12). Functional proteins exist in an ensemble of conformations, each of which is associated with some aspect of activity. Resting, active, desensitized, and so on all describe states of activity with a specific underlying protein conformation. Some conformers may possess binding sites for anesthetics that are more attractive than others and therefore are populated to a greater extent when the anesthetic is present. The anesthetic changes protein activity by selecting the most favorable conformer for binding and increasing its stability and therefore prevalence. One can immediately see the overlap with allosterism. Similar to allosterism, ensemble modulation can explain either an enhancement or an inhibition in protein activity, depending on which conformer has the most attractive anesthetic binding sites.
Simple ensemble of protein conformers, with the relative free energy shown beneath. A. Anesthetic favors the conformer with the most attractive cavities, lowering its free energy (red line) and increasing its concentration. B. Same schematic demonstrating cooperativity, but in this case, the anesthetic-preferred conformer also preferentially binds an endogenous ligand. Thus, free energy is lowered even further in the presence of both ligands, more dramatically enhancing the population and therefore activity associate with this conformer. [Part A reprinted with permission from Gompf H, Chen J, Sun Y, et al. Halothane-induced hypnosis is not accompanied by inactivation of orexinergic output in rodents. Anesthesiology. 2009;111:1001-1009.]
The final general mechanism for coupling binding to a change in protein function is oligomerization modulation (Fig. 37-13). Much protein activity and signaling is controlled by interactions with other proteins. In general, these interactions are highly specific but relatively weak so that they can be readily reversed. The protein–protein interface may include features attractive to an anesthetic, such as cavities. If these features are optimal when the proteins are linked, then the anesthetic will enhance the interaction or oligomerization; if they are optimal when separated, anesthetics will disfavor oligomerization. Examples of the former appear to be the sarcoplasmic reticular calcium adenosine triphosphatase (ATPase)27 and of the latter the PSD-95 PDZ domain proteins (see later discussion).28 The synaptic vesicle machinery is highly dependent on these oligomerization events, and components that might be modulated by anesthetics in this way have been identified.29 It should be apparent that this mechanism has important implications as to the number of potential effects these drugs might have. Whereas the set of all proteins expressed—the proteome—is very large (˜300 000 proteins), the number of protein–protein interactions, the so-called interactome, is much larger. Thus, anesthetic effects at the interactome level produced by effects at the interface could dwarf those at the individual protein level.
Example of oligomerization modulation. A. Anesthetic binding site is created by dimerization of a monomer that otherwise has no binding site. Thus, the anesthetic will be observed to increase the population of dimer. B. On the other hand, occupancy of an anesthetic site in the monomer disfavors binding of the second protein because of a steric clash. In this case, the anesthetic will be observed to decrease the population of dimer. Although both mechanisms may occur in vivo, the situation shown in A probably is more likely.
An important caveat in this discussion is that all binding interactions do not necessarily result in an important change in protein activity.30 It is possible that an anesthetic binding site is precisely preserved across the entire conformation or oligomerization ensemble, in which case anesthetic binding will not alter the distribution of conformers and therefore will have no effect on activity. How commonly this form of "unproductive" binding occurs is not clear.
Any or all of these general coupling mechanisms may be involved in anesthetic-induced protein dysfunction. The distinction often is difficult but is important if we are to intelligently modify the drugs to favor or disfavor specific interactions.
Potential Protein Targets
As suggested earlier, the anesthetic sensitivity of a number of protein systems has been studied over the past few decades, and, remarkably, many are altered within only a 10-fold range of clinical concentrations. We briefly review some of these systems and the evidence for inclusion. This discussion is not intended to be a comprehensive list of those proteins studied; many have come and gone. Rather, we focus on some of the more recent and compelling candidates, especially those with associated in vivo evidence for a contribution. Furthermore, we focus on targets associated with the desirable effects, such as hypnosis, rather than the many that might underlie less desirable side effects.
As pointed out earlier, proteins have domains as hydrophobic as those of the lipid bilayer, and it now is clear that anesthetics bind to and influence the activity of many soluble proteins. Much of the published work involved model systems, such as firefly luciferase, serum albumin, and apoferritin, which are unlikely to contribute directly to anesthesia. However, plausible candidates, centrally located in signal transduction cascades or cellular machinery, have emerged. For example, protein kinase C (PKC) transduces receptor activation to phosphorylation of key membrane proteins, such as certain ion channels and other receptors, and has been shown to bind general anesthetics.31 Alteration of its activity by anesthetics would be expected to have generalized and widespread cellular effects that might contribute to anesthesia, although controversy exists as to both the expected and observed directions of effect (reviewed by Rebecchi and Pentyala32 and Gomez and Guatimosim33). Initially thought to be inhibited by general anesthetics, later studies performed under physiologic conditions showed PKC to be activated. This one system demonstrates the complexity of sorting out anesthetic targets and effects. The observed effects in this relatively simple physiologic assay can result from anesthetic effects on membrane lipid, stimulus–kinase coupling, kinase–target coupling, or the phosphorylated target itself. Anesthetics might interact with any component. Studies in intact, genetically altered animals are in progress but to date have not provided unambiguous answers. Further studies on PKC isoform distribution and activity are required to sort out the influence of anesthetics and potential contribution to the anesthetic state.
Another attractive anesthetic target is any of the components of the cellular cytoskeleton, motility apparatus, and vesicle transport systems. These systems subserve a variety of basic cellular functions, the alteration of which would manifest as a decrease in cellular activity or communication. For example, tubulin forms microtubules, which are crucial elements of cellular scaffolding and motility. Anesthetics bind specifically to tubulin,34 and the assembly of the monomer into the large tubular oligomer is altered by anesthetics, albeit at high concentration.35 Entire mechanisms for general anesthesia based on microtubular dynamics and organization have been proposed,36 and some experimental support has been reported.37 Oligomerization of actin, a protein acting with myosin and other proteins to subserve cellular motion, is inhibited by anesthetics and is another relatively unexplored potential general mechanism of anesthesia.38
Anesthetics also affect the synaptic vesicle machinery. A decrease in synaptic transmission is generally agreed to accompany general anesthesia; therefore, inhibition of synaptic vesicle transport, fusion, and release is a plausible general mechanism of central nervous system (CNS) dysfunction. Evidence comes from several disparate sources. In a genetic screen of the nematode Caenorhabditis elegans, mutations in the vesicle fusion soluble N-ethylmaleimide sensitive attachment factor (SNARE) proteins were found to produce resistance to some anesthetic endpoints.29 Furthermore, 1 component, syntaxin, and various SNARE assemblies have been shown to bind isoflurane using nuclear magnetic resonance approaches.39 Transcript profiling40 in mammals has shown an upregulation of synaptotagmin, another vesicle-release protein. The latter does not necessarily implicate synaptotagmin as a direct anesthetic target, but it lends credence to the idea that synaptic vesicle release is inhibited during general anesthesia. Other synaptic targets implicated are the PDZ domain proteins. These domains are responsible for fusion events (oligomerization) between proteins and are intimately involved in synaptic vesicle trafficking. Work has shown that anesthetics disrupt the oligomerization, and the probable anesthetic binding site responsible has been identified in a truncated version of PSD-95.28
A variety of proteins embedded in, and dependent on, the lipid bilayer for their activity are altered by general anesthetics in in vitro assays. Most studied are the ion channels, largely because of the availability of electrophysiologic approaches for measurement of function. Of the ion channels, most emphasis has been placed on the ligand-gated cys-loop receptor–channel complex, the prototype of which is the nicotinic acetylcholine receptor (nAChR). These proteins are transmembrane heterooligomers of 5 subunits arranged around a central ion channel. A variety of anesthetics inhibit this excitatory channel, perhaps by promoting the desensitized state via cooperative binding with acetylcholine; the probable basis for the muscle relaxation associated with many general anesthetics. Volatile anesthetics bind this receptor specifically, and a site underlying cooperative binding behavior has been tentatively identified.20,22 The nAChRs are also found in the brain, adding credibility to the possibility that alterations in their activity contribute to anesthesia. In vivo support of an important role for these receptors is still lacking.
γ-Aminobutyric acid (GABA)ergic neurotransmission was identified 20 years ago as a plausible substrate for anesthetic effects,41 so recent emphasis has been placed on the inhibitory GABA type A (GABAA) and glycine receptor–ion channel complex as potential contributors to at least the sedative or amnestic component of general anesthesia. Similar to the nAChR, many general anesthetics (most volatile ones and alcohols) probably bind cooperatively with agonist (either GABA or glycine). However, because these receptors undergo desensitization more slowly that the nAChR, the final effect is enhanced agonist-stimulated currents instead of inhibition (Fig. 37-14). These ion channels are inhibitory (produce hyperpolarization of neuronal membrane by opening of chloride channels), so enhancement of their activity is expected to inhibit synaptic transmission. The difficulty of isolating or expressing sufficient GABAA receptor of any subunit composition for biochemical studies so far has precluded the demonstration of specific volatile anesthetic binding, although an etomidate site has been identified at an intersubunit interface.42
Enhancement of activity of γ-aminobutyric acid type A (GABAA) chloride conductance by isoflurane and halothane. In both cases, the effect of an endogenous ligand, GABA, is significantly enhanced by the presence of the anesthetic. The probable basis for this is shown in Fig. 37-12B, and the region of the protein thought to transduce this effect is shown on the right by the hypothetical placement of an anesthetic in the interhelical space. Note the similarity of this hypothetical structural motif and binding site to the crystal structure shown in Fig. 37-9. [Reprinted with permission from Jenkins A, Greenblatt EP, Faulkner HJ, et al. Evidence for a common binding cavity for three general anesthetics within the GABAA receptor. J Neurosci 2001;21:RC136]
Most studies to date have focused on the action of volatile anesthetics on GABAA receptors mediating rapid synaptic transmission. However, volatile anesthetics also act on GABAA channels mediating tonic inhibition. Tonic GABAA channels are found in many areas of the nervous system, including the hippocampus and the spinal cord interneurons. Although the precise receptor subunits composing the tonic GABAA channels remain unknown, the α5- and δ-subunits likely are involved because tonic currents inhibited by bicuculline are not seen in null-mutant mice devoid of these subunits. These GABAA channels are more sensitive to agonists than the rapid type, requiring only low micromolar concentrations of GABA for opening, concentrations found in even extrasynaptic regions of a nerve cell. Similarly, tonic GABAA channels are more sensitive to volatile anesthetics than those mediating rapid synaptic transmission.43 Because of this sensitivity and expression in the hippocampus, it is reasonable to hypothesize that these channels mediate the amnestic component of volatile anesthetic action. A thorough characterization of the volatile anesthetic effects on the various tonic GABAA null mutant mice has not been reported.
In an attempt to both find sites of action as well as to implicate anesthetic targets, site-directed mutagenesis has been coupled with electrophysiology. For example, an asparagine to methionine mutation at position 265 in the GABAA β3 subunit largely eliminated enflurane enhancement of GABA action in vitro, but the knock-in mouse, in which the wild-type β3-subunit was replaced by the point mutant, had minimally altered righting reflex sensitivity or MAC.44,45 Greater success was obtained with the injectable general anesthetics (induction agents) in this same model. For example, similar to enflurane, etomidate and propofol failed to enhance GABA-evoked currents in vitro, but in this case, the whole-animal sensitivity to both injectable drugs was significantly diminished. The technology of mutant receptor knock-in is far superior to an overall elimination of the receptor produced by the knock-out technology because the normal cellular process that regulates expression of the wild-type receptor is preserved. Based on structure–activity studies demonstrating the role of both α- and β-GABAA subunits46 as well as other ligand-gated channels in determining volatile anesthetic responsiveness in in vitro expressed receptors, a multiple knock-in mutant mouse in which many sites are simultaneously altered is more likely to exhibit relative resistance to the volatile anesthetics.
Interestingly, in contrast to prototypical volatile anesthetics such as isoflurane and injectable anesthetics such as propofol, etomidate and barbiturates, a group of agents called dissociative anesthetics, similar to ketamine and nitrous oxide (N2O),47 as well as xenon,48 at clinically relevant concentrations, do not significantly affect GABAA currents, but they inhibit a major excitatory drive in the CNS via blockade of N-methyl-D-aspartate receptors (NMDAr). Thus, when ligand-gated ion channels are concerned, general anesthetics may inhibit CNS neurons either by potentiating inhibitory GABAA currents or inhibiting excitatory NMDAr-mediated currents. Because NMDA receptors play a key role in pain pathways in CNS, their blockade may contribute to the prominent analgesic properties of dissociative anesthetics.
Another hypothesis is that the 2-pore domain family of background K (KCNK) channels is involved in volatile anesthetic action. This gene family encodes for K+-selective ion channels with a common structural feature of 2 protein domains putatively lining the ion channel and 4 transmembrane domains. Two 2-pore domain proteins are thought to assemble as a dimer, creating a complete protein complex with a functional K+-permeable ion channel normally open at all physiologic membrane potentials, thus contributing to the background leak K channel critical in determining the resting membrane potential and regulating neuronal excitability.49 Opening of channels encoded by 2 members of the KCNK family, TREK-1 and TREK-2, and more recently a third member, TRESK, are enhanced by volatile anesthetics, thereby hyperpolarizing the cell membrane. However, the enhancement is not seen in all the family TRESK because the channel activity of a closely related member, TRAAK, is not affected. As in the case for GABAA receptors, the hypothesis that the anesthetic-responsive KCNK channels play a role in the general anesthetic action at the whole animal level was examined and confirmed in TREK-1 knock-out mice. For example, mice without the TREK-1 gene were approximately 20% less sensitive to halothane, sevoflurane, desflurane, and chloroform (but not pentobarbital) as defined by the loss of righting reflex (LORR) and withdraw to tail clamp. Interestingly, an invertebrate analog of this channel41 was discovered a decade earlier in the pond snail,49 pointing out the importance of information derived regardless of the model organism used. Voltage-gated potassium channels are generally insensitive to anesthetics, although a couple variants found in Drosophila and in mammalian orthologs are sensitive to clinical concentrations of both alcohols and anesthetics.50
Voltage-gated calcium channels (VGCCs) have been studied for years because of the central role of calcium in intracellular signaling. VGCCs, which are heteromeric complexes in the plasma membrane of virtually all cell types, show a high level of electrophysiologic and pharmacologic diversity. These channels consist of a pore-forming α1-subunit and ancillary subunits β, γ, and α2–δ.51 On the basis of the membrane potential at which they activate, these channels are subdivided into high voltage-activated (HVA) and low voltage-activated (LVA) or transient T-type Ca2+ channels (T channels). These channels in nerve tissue have a central function in sensory, cognitive, and motor pathways and controlling cell excitability and neurotransmitter release. These channels, which are products of different genes, give rise to α1 subunits that form the pore of the neuronal VGCCs. The HVA VGCCs are members of different families: CaV1 (α1C) encoding L-type, CaV2.1 (α1A) encoding P/Q-type, CaV2.2 (α1B) encoding N-type, and CaV2.3 (α1E) encoding R-type HVA current. Similarly, cloning of T-type channels has established that at least 3 isoforms exist based on the structure of α1 subunits: CaV3.1 (α1G), CaV3.2 (α1H), and CaV3.3 (α1I). Because of its importance, intracellular calcium is tightly controlled via many systems, including voltage- and ligand-gated channels, exchangers, ATPases, and soluble binding proteins. Many of these systems have been shown to be influenced by anesthetics, although early studies have shown that, in general, the magnitude of effect is modest at clinical concentrations.33 However, in most instances, previous studies used only 1 general anesthetic; did not pharmacologically separate subtypes of VGCCs in native cells; and in many cases, did not obtain careful concentration-response curves. An important issue is that even small blockade of a particular VGCC by any general anesthetic may produce profound physiologic effects. When this has been examined, small changes in Ca2+ influx into presynaptic terminals can result in profound changes in transmitter release and synaptic efficacy.51 Indeed, presynaptic transmitter release is proportional up to the fourth power of Ca2+ entry.51 Thus, even if VGCCs are only partially inhibited by anesthetics in the clinically relevant range, this can profoundly alter neuronal signaling. The effects of volatile anesthetics on even 1 subset of VGCCs are variable, probably reflecting the molecular heterogeneity of these channels. Of the functionally characterized VGCCs, the N- and P/Q-type channels, which are major putative presynaptic channels regulating neurotransmitter release, were only partially inhibited by clinically relevant concentrations of inhaled anesthetics.52,53 The recent evidence supporting a possible role of inhibition of N-type VGCCs in the pharmacology of inhaled anesthetics comes from the observation that N-type VGCCs knock-out mice exhibited increased sensitivity to halothane,54 although as noted elsewhere in this chapter, this is an extremely common observation regardless of the target manipulated. Another recent study using in vitro recordings from intact thalamic slices and in vivo recordings using CaV2.3 R-type knock-out mice has established that presynaptic R-type VGCCs in the thalamus are potently inhibited by clinically relevant concentrations of isoflurane.55 The T-type VGCCs channels, although not involved in synaptic neurotransmitter release, play an important role in controlling neuronal excitability and in generating spontaneous oscillatory bursting of groups of neurons in the thalamus thought to be involved in regulating the state of arousal and sleep. The thalamic T-type channels are significantly blocked by clinically relevant concentrations of the inhaled anesthetics.56 The molecular mechanism of how these drugs inhibit the VGCCs channels is not known but appears to involve acceleration of channel inactivation and slowing of the recovery from this nonconducting state.56 There is no evidence of anesthetic binding to these targets, so whether this electrophysiologic effect is mediated via anesthetic interactions with lipid or protein is uncertain.
Voltage-gated sodium channels (NaVs) play a key role in regulating neuronal excitability and therefore are a plausible target for the inhaled volatile anesthetics. However, NaVs were largely dismissed as a relevant target because initial studies indicated that high concentrations were required for inhibition in squid axons (reviewed by Elliott et al57). However, later studies of mammalian brain NaVs indicated that these channels are significantly inhibited by lower, more clinically relevant concentrations of volatile anesthetics.58 In fact, these anesthetics influence specific ligand binding to GABAA receptors and the NaVs with approximately equal potency,59 and rank-order effects appear to qualify the presynaptic NaV as a feasible target for contributing to general anesthesia (Box 37-1).
Box 37-1 ||Download (.pdf)
Isoform and State Dependence of Anesthetic Action
It now is clear that voltage-gated sodium channels (NaVs) constitute a family of closely related proteins with distinct physiologic and pharmacologic properties, although which isoforms are present in the presynaptic terminal remains unknown. Systematic comparison of the sensitivity of different NaV isoforms with isoflurane confirmed the differential sensitivity of the different isoforms to this volatile anesthetic.60 Of curiosity is the observation that volatile anesthetics had no effect on the NaV1.8 tetrodotoxin-resistant isoform predominantly expressed in the primary afferent neurons thought to play a critical role in pain signaling, which could explain why volatile anesthetics exhibit no analgesic property. An additional confounder that may explain some of the inconsistencies reported in the literature is the fact that halothane inhibition of NaV was dependent on coexpression of protein kinase C.48 Therefore, both the state of the NaV itself (resting vs inactivated) and the presence or absence of other modulatory proteins most likely influence the effect of volatile anesthetics on NaVs. Similar arguments can be used when studies concerning voltage-gated calcium channels are interpreted.
The dominant receptor type in the brain is the GPCR. This enormous family includes most of the neurotransmitter and sensory receptors, whether small molecule, peptide, or lipid. These receptors are monomeric and have 7 transmembrane domains arranged like an envelope around a central cleft, or cavity. On the cytoplasmic face, GPCRs couple to the heterotrimeric (3 different subunits) G-protein messenger systems. The native ligand binding site tends to be at various depths in the interhelical cavity, and although some features are conserved, the site can accommodate a wide variety of ligand structure and chemistry (Fig. 37-15). Ligands range from small molecules (volatile odorants, catecholamines) to small peptides (endogenous opioids). That anesthetics act on these receptors is suggested by the anesthetic-sparing effect of agonists for the dopamine and α2-adrenergic receptors in addition to alterations of activity of the eicosanoid receptors.61 Finally, volatile anesthetics have been shown to bind the conserved agonist site in many of these receptors (Fig. 37-15),24 producing either inhibition or excitation.62 Effects downstream of these receptors (eg, on the G-protein transduction pathway) are also possible. Evidence is ample for anesthetic effects on G-protein–mediated signaling32,61 and the importance to function in vivo. For example, C. elegans with diminished Go activity (a negative modulator of synaptic activity) was 2-fold resistant to isoflurane.63 These effects probably are related to effects on the receptor, or the receptor–G interface, because evidence for direct binding to any G-protein subunit or the heterotrimeric complex has not yet emerged.32 This work is highly intricate because of the multiple complexes and states and at this time is unresolved.
Structure of the prototypical G-protein coupled receptor (mammalian rhodopsin, PDB No. 1F88) showing the anesthetic binding site (blue object) located through photolabeling experiments. Occupancy of this interhelical hydrophobic site competes with native ligand (retinal), a mechanism shown in Fig. 37-11. The general motif of an interhelical site in a parallel bundle (see Figs. 37-9 and 37-14) is retained. [Reprinted with permission from Eckenhoff RG. An inhalational anesthetic binding domain in the nicotinic acetylcholine receptor. Proc Natl Acad Sci U S A. 1996;93:2807-2810.]
Mitochondria have long been suspected as contributing to anesthetic action because of their obvious role in cellular energy production. However, the anesthetic concentrations required for inhibition of ATP production and the apparently slow kinetics for ATP depletion seemed to obviate their role. Recent evidence has renewed interest in the mitochondrion. First, signaling and feedback between mitochondria and cellular consumers of ATP is considerably more precise and rapid than originally thought, effectively eliminating the kinetic argument against their contribution to the anesthetic state. Second, mitochondrial genes have been implicated in unbiased genetic screens in simple organisms (see later discussion). For example, mutations in a mitochondrial complex I subunit gene dramatically increased sensitivity to halothane, enflurane, and isoflurane. As expected, mitochondria isolated from this mutant had a reduced rate of oxidative phosphorylation in the presence of halothane.64 Furthermore, several subunits of the oxidative phosphorylation complexes, including complex I, were found to bind halothane specifically.65 Perhaps most importantly, children with biopsy-proven complex I disease were found to be extraordinarily sensitive to the anesthetic sevoflurane,66 rendering this anesthetic target the only to date that has supportive evidence from the gene, the binding interaction, to the human. The underlying mechanism for how the altered mitochondrial function affects anesthetic sensitivity is unclear, but given the recent evidence that mitochondria modulates synaptic transmission through the regulation of presynaptic Ca2+ dynamics,67 it is possible that previously described mutations affect synaptic transmission. Another potential mitochondrial target is the voltage-dependent anion channel 1 (VDAC-1), an anion channel also related to mitochondrial steroid synthesis and synaptic activity and implicated through halothane and neurosteroid binding assays, although knock-out animals had unaltered anesthetic sensitivity.65,68,69
A wide variety of molecular candidates have been examined, and although several are compelling, proof for dominant involvement in the in vivo anesthesia endpoint is still lacking. But how does one determine the relevance of a molecular interaction to the in vivo effect, especially when it is as complex as consciousness?