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Ligand Gated Ion channels: crucial targets for anaesthetics?
Current Anaesthesia & Critical Care (2002) 13, 334 ^342
c 2003 Elsevier Science Ltd. All rights reserved. doi:10.1054/cacc.2003.0425
FOCUS ON: MOLECULAR PHARMACOLOGY
Ligand Gated Ion channels: crucial targets for anaesthetics? L. Coyne and G. Lees Institute of Pharmacy, Chemistry and Biomedical Sciences, School of Science and Social Sciences, University of Sunderland, Sunderland SRI 3SD, UK
KEYWORDS ion channels, anesthetic, general; neurotransmitters, receptors, GABA, neurons
Summary Anaesthetic agents range from simple inert gases to complex synthetic compounds. It is di⁄cult to envisage a unifying mechanism by which all of these agents induce anaesthesia.Thisreviewis focused onligand-gatedion channels.Weintroduce the molecular classi¢cation (plus the concept of superfamilies and receptor isomerism) and nomenclature ofthe pore-forming proteins, then overview thelarge bodyof recentdata suggesting theymay be selective anaesthetic targets. Awide varietyof anaesthetics (volatiles, propofol, etomidate, neurosteroids and barbiturates) have been shown to interact with GABA A receptors.Point mutationsin membrane spanning subunits comprising circa 450 amino acids can ablate the sensitivity of recombinant channels suggesting that the site of actionis notin bulk membrane orinterfaciallipids.Such drugs act stereoselectively to enhance the amplitude and or duration of inhibitory synaptic currents. In contrast, ketamine, nitrous oxide and xenon produce their anaesthetic e¡ects (and untoward side e¡ects) by depressing activity in the glutamatergic NMDA receptor. A minority of energetic researchers suggest that NMDA receptor activity is crucial for arousal (plays a more pivotal role in anaesthesia) and others suggest that voltage-gated channels are equally important in depressant drug action. Molecular biology and electrophysiology have been crucial for our understanding of channel function, but no concensus mechanism for anaesthetic action has yet emerged.
c 2003 Elsevier Science Ltd. All rights reserved.
NEURONAL SIGNALLING Ion channels are crucial for generating action potentials and for modulating neuronal activity at synaptic contacts. The purpose of this article is to outline the emerging evidence that such channels are important targets for depressant drugs. Action potentials are crucial in carrying fast electrical signals to nerve terminals wherevoltage gated calcium channels and potassium channels regulate transmitter release. Postsynaptically located ligand-gated ion channels (LGIC) respond di¡erentially: the net e¡ect is dependent on the identity of the neurotransmitter released. Excitatory transmitters (e.g. glutamate and acetylcholine) bind to their receptor and cause cation in£ux, resulting in further action potential propagation. Inhibitory neurotransmitters (e.g. g-aminobutyric acid or ‘GABA’ and glycine) induce the in£ux of negatively charged chloride ions, which prevents further Correspondence to: GL.Tel.: +44 191515 3428; Fax: +44 191515 3405; E-mail: [email protected] 0953-7112/03/$ - see front matter
action potentials from ¢ring. The ‘current concensus’, often dogmatic, view is that anaesthetics preferentially interact with such fast ligand-gated channels on the postsynaptic side of the membrane.
PUTATIVE CELLULAR TARGETS FOR ANAESTHETICS Over 100 years ago, Meyer and Overton proposed a model for anaesthetic action derived from their lipid solubility/potency relationship.1,2 This correlation suggested that perturbation of neuronal membrane £uidity was responsible for the depressant actions of anaesthetic agents (reviewed in Ref. 3). The advancement of gene cloning and elegant expression systems, in the 1980s, suggested to a new generation of researchers that it was in fact membrane proteins rather than lipids which were the cellular sensors transducing the characteristic e¡ects of general anaesthetics.4 In summary, this evidence pivots on stereoselectivity of anaesthetic
LIGAND GATED ION CHANNELS
action and the relative lack of e¡ect of temperature (heating or cooling by 1^21C, has large e¡ects on membrane structure and £uidity, but is not anaesthetic). A key issue is the identi¢cation of signalling targets (proteins) embedded in the lipid matrix which respond to clinical levels of anaesthetic drugs. Minimum alveolar concentration (MAC) equivalent concentrations of anaesthetics are similar to those required to modulate LGIC in vitro.4 In£uential pioneers in this ¢eld like Harrison, Lambert and Franks 4 ^ 6 stress that the preferential targets are LGIC.Voltage-gated ion channels respond to slightly higher concentrations of anaesthetics, but many studies report interactions in the low MAC range which may be equally physiologically relevant (reviewed recently in Ref. (7)) and potentially clinically important, as highlighted in the next paragraph.
Voltage gated ion channels Voltage-gated sodium channels (VGSC) are activated by depolarization, but can be blocked tonically by clinically relevant concentrations of anaesthetics.8 Depolarising shifts inVGSC activation and hyperpolarizing shifts in inactivation can be induced by anaesthetics.4 Although most voltage-gated potassium channels (VGPC) are only sensitive to high doses of anaesthetic agents, the recently discovered ‘twin-pore’ potassium channels are sensitive to much lower concentrations.9,10 The controversial issue of di¡erential susceptibility (ligand vs voltage-gated channels) to anaesthetic action can only be resolved by paired recordings from pre- and postsynaptic elements in a relevant CNS synapse. Forsythe and Sakmann have revealed such a preparation in the rat brain (Calyx of Held) which might de¢nitively solve this controversial issue in the medium term future.11,12
Ligand-gated ion channels The consensus ‘fashionable’ targets for low concentrations of anaesthetics are the fast postsynaptic ligand-gated ion channels. Such channels are classi¢ed into families based primarily on their structure.
Nicotinic acetylcholine receptor (nACHr) superfamily The nACHr superfamily (classi¢cation is based on conservation of primary structure) can be subdivided into cation channels and anion channels. Cation channels (excitatory) are nACHr and 5-hydroxytryptamine (5HT)3 receptors. Anion channels (inhibitory) are GABAA receptors and glycine receptors. All members of this family are thought to share a pentameric structure, made up from ¢ve separate subunits. Each subunit is potentially a unique gene product which can be combined to make
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many receptor isoforms, leading to structural diversity and functional complexity. Each subunit contains four hydrophobic transmembrane regions (Ml ^M4), a long N-terminal and a large intracellular loop between M3 and M4. The second of these transmembrane domains, M2, is thought to line the pore of the ion channel. It is therefore, important in selectivity. Rings of amino acids at the inner and outer end of the M2 domain contain amino acids which carry the opposite charge to the permeant ion, i.e. a cation channel will contain a selectivity ¢lter, £anking M2, with a ring of negative charge. The traditional model for ion channel structure (produced from the pioneering work of Unwin) is that the ¢ve subunits surround a centrally located channel pore (which is blocked by a hydrophobic amino acid when the channel is closed). This pore is lined with relatively polar amino acids allowing for ion permeation and hydration. When the channel is activated, this induces a conformational change resulting in central helices ‘twisting’ to open the pore (Fig. 1). This allows a rapid signal to be transmitted across the cell membrane. Importantly for anaesthetics, which are likely to partition into lipids, this gating event is con¢ned to the core of the protein (the subunits per se do not twist in the lipid matrix). This model has evolved recently to show that the channel is blocked by a protein (rapsin) which is not part of the channel and that ions move into the cell through small openings surrounding this ‘blocking protein’15. Although this work has only been carried out in the nicotinic acetylcholine receptor, it could have implications for other receptors in this superfamily. By using electron microscopy of torpedo stingray postsynaptic membranes, Miyazawa et al. discovered that nicotinic acetylcholine channel pore is blocked by a protein and that narrow ‘tunnels’ allow ions to £ow through when the receptor is open.The binding pocket for acetylcholine is envisaged to be inside the channel pore rather than on the outside, with acetylcholine entering the pore mouth before it can reach its binding site.15 A pentameric structure consisting of various subunits allows receptor diversity, creating the possibility of many di¡erent receptor subtypes.This isomerism allows di¡erent subtypes of receptors to react di¡erently to chemical transmitters and hormones, and hence extend the repertoire of signalling roles. Much anaesthetic research has been carried out on LGIC, but which receptor represents the most likely target to produce general anaesthesia? Table 1 shows the pharmacology and toxicology of LGIC. Over and above this textbook pharmacological pro¢le, most LGIC in the nAchr superfamily show sensitivity to anaesthetics at clinically relevant concentrations. In general, cation channels are blocked (e.g. CNS nAchr) and inhibitory channels potentiated (by an allosteric mechanism) leading to larger more prolonged currents.4
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CURRENT ANAESTHESIA & CRITICAL CARE
Figure 1 Nicotinic acetylcholine receptor superfamily structure. (A) 65 KD submits assemble to produce a pentameric structure within the cell membrane. Note that the extracellular segments constitute the bulk of the quatemary structure which has been con¢rmed by electron microscopy. (B and C) Each subunit has four transmembrane regions (Ml^M4) of which M2 lines the pore. Each subunit bears a large aminoterminal and a shortcarboxylterminal, which are both extracellular.The circlesrepresentregions that have been found to be important in volatile anaesthetic recognition (based on data from Refs.13,14)
Nicotinic acetylcholine receptors Nicotinic acetylcholine receptors are the most studied of all LGICs.They are mainly located on the postsynaptic membranes of muscle cells on the neuromuscular junction, but di¡erent subtypes of this receptor (mainly a4 b2 and the homo-oligomeric a7 receptor) are also found in the brain. General anaesthetics do synergize muscle relaxants. Some of this may re£ect a weak blockade of neuromuscular junction receptors. Volatile anaesthetics and ketamine inhibit central nicotinic acetylcholine receptors at clinically relevant doses, but higher concentrations of barbiturates are required to produce the same e¡ect.16 Central receptors are much more sensitive to anaesthetics, and although
they are not likely to be the sole contributor to the anaesthetic state, they may be involved in the production of several features of anaesthesia such as amnesia and delirium.16
5HT3 receptors The 5HT3 receptor is the only ionotropic member of the 5HT receptor family (the others, 5HT1^7, are G-protein coupled). It is a homo-oligmeric cation channel which is activated by serotonin. The 5HT3 receptor has a limited distribution in the CNS, but is expressed in the gastrointestinal tract and in brain stem/pontine nuclei: it has an acknowledged role in anaesthetic-induced nausea and
LIGAND GATED ION CHANNELS
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Table 1 LGIC (ionotrophic receptor) classi¢cation and pharmacology Receptor
Subunit Types
Conductance
Agonists
Antagonists
Na+/excitatory
Nicotine, lobeline
Cl /inhibitory Cl /inhibitory Na+/excitatory
Muscimol Taurine Biguanide,Cl-phenyl
Hexamethonium, tubocurarine, trimetaphaan Bicuculline,Picrotoxin Strychnine Ondansetron, tropisetron, granisetron
Glutamate receptor family AMPA GluR1-4 Kainate GluR 5-7 NMDA NRi,NR2A-D,NR3
Na+/excitatory Na+/excitatory Ca2+/excitatory
AMPA Kainate NMDA
CNQX CNQX D-AP5
Other LGICs Vanilloid
Excitatory
Capsaicin, anandamide Heat, osmolarity TRP3,5-7 by oleylacetylglycerol ATP, ab-MeATP
Capsazepine
Nicotinic receptor superfamily Nicotinic acetylcholine a1-10,b1-4,g,d,E
GABA A Glycine 5HT3
TRP P2X
a1-6,b1-3,g1-3,d,Er1-3p, a1-3, b1-2 5HT3A and B
VR1, VRL1-3 TRP1-7 P2X1-7
Excitatory Excitatory
vomiting.17 Experiments carried out so far reveal that volatile anaesthetics enhance 5HT3 -mediated currents.17 More recent studies have shown that barbiturates decrease 5HT3A-receptor-mediated currents in HEK cells.18 Few studies have been carried out on 5HT3 receptors within real neuronal circuits so their role in anaesthetic sensing remains obscure.
Glycine receptors Glycine receptors are predominantly found in the brain stem and spinal cord, but are also distributed throughout the brain.These inhibitory ion channels are also sensitive to anaesthetics, but not as strongly as GABAA receptors.19 Glycine receptors have a pentameric structure formed from a(1^ 4) and b1 subunits. These receptors are strychnine sensitive. Strong modulation of glycine receptors is noteworthy for volatile anaesthetics, compared to only modest e¡ects produced by barbiturates, alphaxalone and propofol.20
GABAA receptors The GABAA receptor is a particularly good model anaesthetic target and is made up from 6a, 3b, 3g, d, E p, y, and 3r subunits, leading to well over 100 000 possible combinations.21 In reality, there are probably only about 20 functional combinations expressed in mammalian CNS according to recent evidence based on RNA expression or immunoprobing.22 These receptors are found at one third of all CNS synapses and are widely distributed in almost all nuclei. Hence GABA (which also activates the metabotropic
Suramin,TNP-ATP,IP5Ib
GABAB receptor) is the most important inhibitory neurotransmitter in the brain. Concentrations of anaesthetics required to modulate GABAA receptors correlate with concentrations required to produce anaesthesia in vivo.23 Many depressant drugs such as hypnotics and anticonvulsants have acknowledged e¡ects on the GABAA receptor, and it is modulated by a broad spectrum of anaesthetic agents. Ketamine, xenon and nitrous oxide are thought to act elsewhere (see below).24,25 Not only do anaesthetics modulate the duration of GABAA receptor opening times, but they can also increase GABA release pre-synaptically (e.g. Ref 26). Since the 1980s most of the ion channels have been cloned. This facilitates work on human proteins. Many studies have looked at the selectivity of anaesthetics for certain subunits as a means of ¢nding a binding site. The discovery that the r subunit was absolutely insensitive to anaesthetics, was a pivotal disclosure. Chimeras were made (large chunks of protein from a sensitive subunit were spliced at the appropriate segment into cDNAs for the r homologue). This showed that segments which span the membrane were crucial. Further mutations in the M2 and M3 domains revealed that single amino acids conferred sensitivity to etomidate and loreclezole.27,28 Other work has shown that the M2 domain on both a and b subunits is vital for the binding of anaesthetics and alcohol.13 Several other amino acid mutations can also reduce or eliminate anaesthetic sensitivity. The size of the anaesthetic molecule determines the sensitivity of various mutations.23 This suggests that there is a ‘pocket’ where anaesthetics bind, and slight variations in the amino acids creating this pocket
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could lead to anaesthetic sensitivity. Further evidence to support the binding pocket theory was produced 1999 when Belelli et al. showed that by mutating a single amino acid in the rho subunit (normally refractory to barbiturates), they could produce sensitivity to pentobarbitone.29 Variations in subunit composition are obviously important in anaesthetic action, so it is conceivable that selectivity for certain receptor isoforms in discrete nuclei account for the diverse pharmacological pro¢les of structurally heterogeneous anaesthetics?
CURRENT ANAESTHESIA & CRITICAL CARE
Glutamate receptor superfamily Glutamate receptors are a distinctive group of LGICs which are structurally di¡erent from other LGICs. The ¢rst structural di¡erence is simply molecular mass (see Fig. 2), glutamate receptor subunits are much larger than the nicotinic family members. Receptor channels are made up from four or ¢ve subunits.30,31 Further diversity and complexity is introduced in the glutamate families by splice variation (alternative exons can be transcribed from the DNA) or by RNA editing (see Fig. 2). Examples
Figure 2 Schematic diagram of amino acid sequences of glutamate receptor subunits.Glutamate receptor subunits range from 95 to 163 kDa in size. Note they are much larger than the other ligand-gated channels depicted approximately to scale. Further diversity is conferred uponthe glutamate family by RNA editing (unique to the glutamate superfamily) and by splice variation (alternative exons are transcribed from the gene). Splice variation is only seen in one of the eight variants of GABAa subunit (hatched triangle) and has not been detected in other members of the nicotinic superfamily.
LIGAND GATED ION CHANNELS
of this are the £ip and £op variations of the AMPA receptors. These two splice variations of subunits are expressed during di¡erent stages of development, the £ip form is expressed in pre- and postnatal brains, whereas the £op is expressed later. The two variations of the receptor have slightly di¡erent kinetics.30 Each subunit is made up of only three transmembrane domains, Ml, M3 and M4, with an intramembrane loop (M2).The M2 loop lines the channel pore (see Fig. 3). They can be divided into three major groups, AMPA (DL-a-amino-3-hydroxyS-methylisoxazole- 4 -propionate), kainate and NMDA (n-methyl, D aspartate) receptors, based on their pharmacology31 (see Table 1). AMPA and kainate receptors are primarily permeable to sodium and potassium ions. AMPA receptor subunits are GluR 1- 4: they have a similar structure to the kainate receptor subunits which are named GluR 5^7.30 The NMDA receptor is a Ca++ permeant channel involved in a process called long term potentiation (LTP), which is a cellular model for memory formation in hippocampus. Activation of NMDA receptors (by L glutamate) requires the concurrent binding of glycine at an allosteric co-agonist site. At rest, NMDA channels are also blocked by Mg++ ions, but they can be unblocked by depolarization. If there is enough repetitive stimulation of AMPA receptors and NMDA receptors are activated, this is thought to trigger a chain of intracellular events (secondary to Ca++ entry), resulting in a long-lived change in the intensity of synaptic signalling.32 Functional NMDA receptors comprise NRl, NR2A-D and NR3. It is widely accepted that ketamine, nitrous oxide and xenon induce anaesthesia by blocking the NMDA receptor21,25,33 Anaesthetic agents which block NMDA receptors clearly have very di¡erent pro¢les in vivo (although xenon is still relatively poorly characterized in man) which may be di⁄cult to align with a single molecular target. Conscious functions only are impaired, and re£ex actions are una¡ected by anaesthetics targeting only the NMDA receptor.34 However, nitrous oxide is strongly analgesic but is weakly anaesthetic. Animal models suggest that some of the potential side e¡ects are common to NMDA channel block. Psychotomimetic e¡ects can be seen with amantadine (another NMDA channel blocker) in neurological therapies. Recent papers suggest that the NMDA receptor may also be inhibited indirectly by anaesthetic agents whose primary ‘consensus’ target is the GABAA receptor. Fig. 4 (reproduced with Professor Flohr’s consent 34) shows that ketamine and drugs classically believed to be GABA-selective anaesthetics all block the binding of a radiolabelled NMDA antagonist which is dependent upon channel activation for access to its binding site. Most anaesthetics are unable to produce an e¡ect on NMDA receptors 4, but the recent work by Flohr suggests NMDA receptors may have a much more important role in the action of anaesthetics and in arousal per se.
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Figure 3 NMDA receptor structure. (A) NMDA receptor activation requires repetitive depolarization (usually supplied by activation of adjacent AMPA receptors) to dislodge the blocking Mg2+ ion and also requires both glutamate and glycine to bind to distinct recognition sites. (B) Subunits are composed of four transmembrane regions, the second of which is an intramembrane loop. The boxed region (black) indicates a putative glutamate-binding motif projecting into the synaptic cleft. The carboxyl terminal di¡ers from subunitto subunit, with the smaller NRl receptor subunits terminating atthe point marked X
Miscellaneous novel ion channels With the revolution in gene cloning, new classes of ion channels are emerging, e.g. the ATP-activated P2X re-
340
CURRENT ANAESTHESIA & CRITICAL CARE
Figure 4 (Replicated from Ref. (34)) This shows the uptake of a radiolabelled NMDA receptor antagonist (MK-801) in an awake animal brain (A), in an animal under ketamine anaesthesia (B), and in an animal under pentobarbital anaesthesia (C).Equivalent coronal sections ofcortex are depictedinthe top and bottomrows.The authorsinfer fromthis work andrelated modelling that NMDAchannel activation is a necessary condition for consciousness.
ceptor superfamily, the transient receptor potential (TRP) family and the vanilloid receptors (reviewed in Refs. 35^37 ).
Vanilloid receptors The active ingredient in chilli peppers, capscaisin, creates its burning sensation by activating the vanilloid receptors. Vanilloid receptors (VRl) are LGICs whose activation causes primarily the in£ux of Ca2+ ions, but are also highly permeable to Na+ and K+ ions. The ion channels are tetrameric. Each subunit contains six transmembrane domains (Sl ^S6), with a pore lining transmembrane loop between S5 and S6. Both the C terminal and the N terminal are intracellular, suggesting that ligands activate the receptor from the cytoplasm.37 Channel opening can be activated also by a decrease in pH and by an increase in temperature.VRl receptors located in the periphery are responsible for burning sensations induced by acids, temperature changes and nootropic chemicals which activate these receptors.VRl receptors which are centrally located but much more dif¢cult to study may also be involved in pain processing.37 Little work has been carried out to determine whether or not vanilloid receptors are involved in the analgesic e¡ects of anaesthetics, however, recent work has suggested that propofol is a VRl receptor agonist (which may underpin the painful response at the injection site)38
Vanilloid-receptor-like (VRL) ion channels are also subtypes of the vanilloid receptor family. There are three known types (VRLl ^3). All are capsaicin insensitive.VRLl is activated by high temperatures (of 501c and above). VRL2 is osmotically sensitive, and is activated by hypotonic solutions.36 VRL3 receptors are heat sensitive (activated by temperatures of X391C).These receptors can associate with VRl receptors to produce heterooligmers.39 VRLl and VRL3 may also be important in nociception.
TRP receptors TRP receptors resemble vanilloid receptors in their structural make up.They are tetrameric.There are seven di¡erent TRP subunits,TRPl ^7, and each has seven transmembrane regions. M6 is an intramembrane loop.36 Activation of these receptors results in calcium in£ux. Although many of these receptors are found outside of the nervous system, there is also a high expression in neuronal cells. They may be involved in the modulation of the cell’s resting potential and are thought to be activated by intracellular second messengers.36
P2X receptors P2X receptors are ionotrophic purinergic receptors. There are seven types of P2X subunit (P2X1^7 ), each with
LIGAND GATED ION CHANNELS
two membrane spanning regions.Either three or four assemble to produce a functional receptor. Their roles range from reproduction in males to fast synaptic signalling.They are found predominantly in the peripheral nervous system, but are also present in the brain. Many autonomic neurones express both nAChr and P2X receptors, which may interact with one another.35 During in£ammation, ATP is released and acts at the P2X receptors, indicating that they have a role in pain. Also, P2X7 receptor antagonists are antinociceptive.40
Concluding remarks Ligand-gated ion channels, particularly GABAA receptors and NMDA receptor subtypes, are excellent model targets for anaesthetic action in vitro. Most scientists now believe that the ‘anaesthetic receptor’ resides on proteins rather than in bulk lipid.The drugs are undoubtedly low a⁄nity and non-selective in action F only when selective knock-outs of implicated proteins in vivo impair drug action, in transgenic animals, will these authors be convinced.
ACKNOWLEDGEMENTS Thanks to the Northern Anaesthetics Research Consortium (NARC) for ¢nancial support and constant encouragement.
GLOSSARYOF TERMS Action potential the ‘nerve spike’, a transient depolarization of nerve or skeletal muscle propagated by voltage-gated ion channels. Chimera a recombinant protein containing complementary segments of two distinct receptors spliced together. Combining di¡erent segments allows the neuroscientist to recognize key functional domains or drugbinding sites. Depolarization reduction in the resting membrane potential (normally 60 to 80 mV) towards or beyond 0 mV. Hyperpolarization A negative shift in the transmembrane resting potential. Immunoprobing Fluorescent antibodies are used to label and anatomically localize antigenic segments of speci¢c receptor proteins on the cell surface. Long-term potentiation (LTP) Activity-dependent potentiation of synaptic signalsF an excellent model for memory formation in mammalian CNS. Neurotransmitter a chemical which is released from the nerve terminal which di¡uses across a narrow synaptic cleft to activate receptors on adjacent cells. Postsynaptic ‘dendritic’ regions of cells beyond the synaptic cleft which bear receptors to sense neurotransmitter binding.
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Pre-synaptic The terminal region of the neurone before the synapse with capacity to release neurotransmitter. RNA-editing Lengths of RNA can be di¡erentially translated resulting in alteration of amino acid composition at de¢ned sites on the protein. Splice-variations Alternative exons are transcribed from the gene which again results in insertion of a new cassette of amino acids in the protein. Synapse The cleft between neurones across which signals can be transmitted to turn neighbouring cells on (excitation) or o¡ (inhibition)
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c 2003 Elsevier Science Ltd. All rights reserved. doi:10.1054/cacc.2003.0425
FOCUS ON: MOLECULAR PHARMACOLOGY
Ligand Gated Ion channels: crucial targets for anaesthetics? L. Coyne and G. Lees Institute of Pharmacy, Chemistry and Biomedical Sciences, School of Science and Social Sciences, University of Sunderland, Sunderland SRI 3SD, UK
KEYWORDS ion channels, anesthetic, general; neurotransmitters, receptors, GABA, neurons
Summary Anaesthetic agents range from simple inert gases to complex synthetic compounds. It is di⁄cult to envisage a unifying mechanism by which all of these agents induce anaesthesia.Thisreviewis focused onligand-gatedion channels.Weintroduce the molecular classi¢cation (plus the concept of superfamilies and receptor isomerism) and nomenclature ofthe pore-forming proteins, then overview thelarge bodyof recentdata suggesting theymay be selective anaesthetic targets. Awide varietyof anaesthetics (volatiles, propofol, etomidate, neurosteroids and barbiturates) have been shown to interact with GABA A receptors.Point mutationsin membrane spanning subunits comprising circa 450 amino acids can ablate the sensitivity of recombinant channels suggesting that the site of actionis notin bulk membrane orinterfaciallipids.Such drugs act stereoselectively to enhance the amplitude and or duration of inhibitory synaptic currents. In contrast, ketamine, nitrous oxide and xenon produce their anaesthetic e¡ects (and untoward side e¡ects) by depressing activity in the glutamatergic NMDA receptor. A minority of energetic researchers suggest that NMDA receptor activity is crucial for arousal (plays a more pivotal role in anaesthesia) and others suggest that voltage-gated channels are equally important in depressant drug action. Molecular biology and electrophysiology have been crucial for our understanding of channel function, but no concensus mechanism for anaesthetic action has yet emerged.
c 2003 Elsevier Science Ltd. All rights reserved.
NEURONAL SIGNALLING Ion channels are crucial for generating action potentials and for modulating neuronal activity at synaptic contacts. The purpose of this article is to outline the emerging evidence that such channels are important targets for depressant drugs. Action potentials are crucial in carrying fast electrical signals to nerve terminals wherevoltage gated calcium channels and potassium channels regulate transmitter release. Postsynaptically located ligand-gated ion channels (LGIC) respond di¡erentially: the net e¡ect is dependent on the identity of the neurotransmitter released. Excitatory transmitters (e.g. glutamate and acetylcholine) bind to their receptor and cause cation in£ux, resulting in further action potential propagation. Inhibitory neurotransmitters (e.g. g-aminobutyric acid or ‘GABA’ and glycine) induce the in£ux of negatively charged chloride ions, which prevents further Correspondence to: GL.Tel.: +44 191515 3428; Fax: +44 191515 3405; E-mail: [email protected] 0953-7112/03/$ - see front matter
action potentials from ¢ring. The ‘current concensus’, often dogmatic, view is that anaesthetics preferentially interact with such fast ligand-gated channels on the postsynaptic side of the membrane.
PUTATIVE CELLULAR TARGETS FOR ANAESTHETICS Over 100 years ago, Meyer and Overton proposed a model for anaesthetic action derived from their lipid solubility/potency relationship.1,2 This correlation suggested that perturbation of neuronal membrane £uidity was responsible for the depressant actions of anaesthetic agents (reviewed in Ref. 3). The advancement of gene cloning and elegant expression systems, in the 1980s, suggested to a new generation of researchers that it was in fact membrane proteins rather than lipids which were the cellular sensors transducing the characteristic e¡ects of general anaesthetics.4 In summary, this evidence pivots on stereoselectivity of anaesthetic
LIGAND GATED ION CHANNELS
action and the relative lack of e¡ect of temperature (heating or cooling by 1^21C, has large e¡ects on membrane structure and £uidity, but is not anaesthetic). A key issue is the identi¢cation of signalling targets (proteins) embedded in the lipid matrix which respond to clinical levels of anaesthetic drugs. Minimum alveolar concentration (MAC) equivalent concentrations of anaesthetics are similar to those required to modulate LGIC in vitro.4 In£uential pioneers in this ¢eld like Harrison, Lambert and Franks 4 ^ 6 stress that the preferential targets are LGIC.Voltage-gated ion channels respond to slightly higher concentrations of anaesthetics, but many studies report interactions in the low MAC range which may be equally physiologically relevant (reviewed recently in Ref. (7)) and potentially clinically important, as highlighted in the next paragraph.
Voltage gated ion channels Voltage-gated sodium channels (VGSC) are activated by depolarization, but can be blocked tonically by clinically relevant concentrations of anaesthetics.8 Depolarising shifts inVGSC activation and hyperpolarizing shifts in inactivation can be induced by anaesthetics.4 Although most voltage-gated potassium channels (VGPC) are only sensitive to high doses of anaesthetic agents, the recently discovered ‘twin-pore’ potassium channels are sensitive to much lower concentrations.9,10 The controversial issue of di¡erential susceptibility (ligand vs voltage-gated channels) to anaesthetic action can only be resolved by paired recordings from pre- and postsynaptic elements in a relevant CNS synapse. Forsythe and Sakmann have revealed such a preparation in the rat brain (Calyx of Held) which might de¢nitively solve this controversial issue in the medium term future.11,12
Ligand-gated ion channels The consensus ‘fashionable’ targets for low concentrations of anaesthetics are the fast postsynaptic ligand-gated ion channels. Such channels are classi¢ed into families based primarily on their structure.
Nicotinic acetylcholine receptor (nACHr) superfamily The nACHr superfamily (classi¢cation is based on conservation of primary structure) can be subdivided into cation channels and anion channels. Cation channels (excitatory) are nACHr and 5-hydroxytryptamine (5HT)3 receptors. Anion channels (inhibitory) are GABAA receptors and glycine receptors. All members of this family are thought to share a pentameric structure, made up from ¢ve separate subunits. Each subunit is potentially a unique gene product which can be combined to make
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many receptor isoforms, leading to structural diversity and functional complexity. Each subunit contains four hydrophobic transmembrane regions (Ml ^M4), a long N-terminal and a large intracellular loop between M3 and M4. The second of these transmembrane domains, M2, is thought to line the pore of the ion channel. It is therefore, important in selectivity. Rings of amino acids at the inner and outer end of the M2 domain contain amino acids which carry the opposite charge to the permeant ion, i.e. a cation channel will contain a selectivity ¢lter, £anking M2, with a ring of negative charge. The traditional model for ion channel structure (produced from the pioneering work of Unwin) is that the ¢ve subunits surround a centrally located channel pore (which is blocked by a hydrophobic amino acid when the channel is closed). This pore is lined with relatively polar amino acids allowing for ion permeation and hydration. When the channel is activated, this induces a conformational change resulting in central helices ‘twisting’ to open the pore (Fig. 1). This allows a rapid signal to be transmitted across the cell membrane. Importantly for anaesthetics, which are likely to partition into lipids, this gating event is con¢ned to the core of the protein (the subunits per se do not twist in the lipid matrix). This model has evolved recently to show that the channel is blocked by a protein (rapsin) which is not part of the channel and that ions move into the cell through small openings surrounding this ‘blocking protein’15. Although this work has only been carried out in the nicotinic acetylcholine receptor, it could have implications for other receptors in this superfamily. By using electron microscopy of torpedo stingray postsynaptic membranes, Miyazawa et al. discovered that nicotinic acetylcholine channel pore is blocked by a protein and that narrow ‘tunnels’ allow ions to £ow through when the receptor is open.The binding pocket for acetylcholine is envisaged to be inside the channel pore rather than on the outside, with acetylcholine entering the pore mouth before it can reach its binding site.15 A pentameric structure consisting of various subunits allows receptor diversity, creating the possibility of many di¡erent receptor subtypes.This isomerism allows di¡erent subtypes of receptors to react di¡erently to chemical transmitters and hormones, and hence extend the repertoire of signalling roles. Much anaesthetic research has been carried out on LGIC, but which receptor represents the most likely target to produce general anaesthesia? Table 1 shows the pharmacology and toxicology of LGIC. Over and above this textbook pharmacological pro¢le, most LGIC in the nAchr superfamily show sensitivity to anaesthetics at clinically relevant concentrations. In general, cation channels are blocked (e.g. CNS nAchr) and inhibitory channels potentiated (by an allosteric mechanism) leading to larger more prolonged currents.4
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CURRENT ANAESTHESIA & CRITICAL CARE
Figure 1 Nicotinic acetylcholine receptor superfamily structure. (A) 65 KD submits assemble to produce a pentameric structure within the cell membrane. Note that the extracellular segments constitute the bulk of the quatemary structure which has been con¢rmed by electron microscopy. (B and C) Each subunit has four transmembrane regions (Ml^M4) of which M2 lines the pore. Each subunit bears a large aminoterminal and a shortcarboxylterminal, which are both extracellular.The circlesrepresentregions that have been found to be important in volatile anaesthetic recognition (based on data from Refs.13,14)
Nicotinic acetylcholine receptors Nicotinic acetylcholine receptors are the most studied of all LGICs.They are mainly located on the postsynaptic membranes of muscle cells on the neuromuscular junction, but di¡erent subtypes of this receptor (mainly a4 b2 and the homo-oligomeric a7 receptor) are also found in the brain. General anaesthetics do synergize muscle relaxants. Some of this may re£ect a weak blockade of neuromuscular junction receptors. Volatile anaesthetics and ketamine inhibit central nicotinic acetylcholine receptors at clinically relevant doses, but higher concentrations of barbiturates are required to produce the same e¡ect.16 Central receptors are much more sensitive to anaesthetics, and although
they are not likely to be the sole contributor to the anaesthetic state, they may be involved in the production of several features of anaesthesia such as amnesia and delirium.16
5HT3 receptors The 5HT3 receptor is the only ionotropic member of the 5HT receptor family (the others, 5HT1^7, are G-protein coupled). It is a homo-oligmeric cation channel which is activated by serotonin. The 5HT3 receptor has a limited distribution in the CNS, but is expressed in the gastrointestinal tract and in brain stem/pontine nuclei: it has an acknowledged role in anaesthetic-induced nausea and
LIGAND GATED ION CHANNELS
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Table 1 LGIC (ionotrophic receptor) classi¢cation and pharmacology Receptor
Subunit Types
Conductance
Agonists
Antagonists
Na+/excitatory
Nicotine, lobeline
Cl /inhibitory Cl /inhibitory Na+/excitatory
Muscimol Taurine Biguanide,Cl-phenyl
Hexamethonium, tubocurarine, trimetaphaan Bicuculline,Picrotoxin Strychnine Ondansetron, tropisetron, granisetron
Glutamate receptor family AMPA GluR1-4 Kainate GluR 5-7 NMDA NRi,NR2A-D,NR3
Na+/excitatory Na+/excitatory Ca2+/excitatory
AMPA Kainate NMDA
CNQX CNQX D-AP5
Other LGICs Vanilloid
Excitatory
Capsaicin, anandamide Heat, osmolarity TRP3,5-7 by oleylacetylglycerol ATP, ab-MeATP
Capsazepine
Nicotinic receptor superfamily Nicotinic acetylcholine a1-10,b1-4,g,d,E
GABA A Glycine 5HT3
TRP P2X
a1-6,b1-3,g1-3,d,Er1-3p, a1-3, b1-2 5HT3A and B
VR1, VRL1-3 TRP1-7 P2X1-7
Excitatory Excitatory
vomiting.17 Experiments carried out so far reveal that volatile anaesthetics enhance 5HT3 -mediated currents.17 More recent studies have shown that barbiturates decrease 5HT3A-receptor-mediated currents in HEK cells.18 Few studies have been carried out on 5HT3 receptors within real neuronal circuits so their role in anaesthetic sensing remains obscure.
Glycine receptors Glycine receptors are predominantly found in the brain stem and spinal cord, but are also distributed throughout the brain.These inhibitory ion channels are also sensitive to anaesthetics, but not as strongly as GABAA receptors.19 Glycine receptors have a pentameric structure formed from a(1^ 4) and b1 subunits. These receptors are strychnine sensitive. Strong modulation of glycine receptors is noteworthy for volatile anaesthetics, compared to only modest e¡ects produced by barbiturates, alphaxalone and propofol.20
GABAA receptors The GABAA receptor is a particularly good model anaesthetic target and is made up from 6a, 3b, 3g, d, E p, y, and 3r subunits, leading to well over 100 000 possible combinations.21 In reality, there are probably only about 20 functional combinations expressed in mammalian CNS according to recent evidence based on RNA expression or immunoprobing.22 These receptors are found at one third of all CNS synapses and are widely distributed in almost all nuclei. Hence GABA (which also activates the metabotropic
Suramin,TNP-ATP,IP5Ib
GABAB receptor) is the most important inhibitory neurotransmitter in the brain. Concentrations of anaesthetics required to modulate GABAA receptors correlate with concentrations required to produce anaesthesia in vivo.23 Many depressant drugs such as hypnotics and anticonvulsants have acknowledged e¡ects on the GABAA receptor, and it is modulated by a broad spectrum of anaesthetic agents. Ketamine, xenon and nitrous oxide are thought to act elsewhere (see below).24,25 Not only do anaesthetics modulate the duration of GABAA receptor opening times, but they can also increase GABA release pre-synaptically (e.g. Ref 26). Since the 1980s most of the ion channels have been cloned. This facilitates work on human proteins. Many studies have looked at the selectivity of anaesthetics for certain subunits as a means of ¢nding a binding site. The discovery that the r subunit was absolutely insensitive to anaesthetics, was a pivotal disclosure. Chimeras were made (large chunks of protein from a sensitive subunit were spliced at the appropriate segment into cDNAs for the r homologue). This showed that segments which span the membrane were crucial. Further mutations in the M2 and M3 domains revealed that single amino acids conferred sensitivity to etomidate and loreclezole.27,28 Other work has shown that the M2 domain on both a and b subunits is vital for the binding of anaesthetics and alcohol.13 Several other amino acid mutations can also reduce or eliminate anaesthetic sensitivity. The size of the anaesthetic molecule determines the sensitivity of various mutations.23 This suggests that there is a ‘pocket’ where anaesthetics bind, and slight variations in the amino acids creating this pocket
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could lead to anaesthetic sensitivity. Further evidence to support the binding pocket theory was produced 1999 when Belelli et al. showed that by mutating a single amino acid in the rho subunit (normally refractory to barbiturates), they could produce sensitivity to pentobarbitone.29 Variations in subunit composition are obviously important in anaesthetic action, so it is conceivable that selectivity for certain receptor isoforms in discrete nuclei account for the diverse pharmacological pro¢les of structurally heterogeneous anaesthetics?
CURRENT ANAESTHESIA & CRITICAL CARE
Glutamate receptor superfamily Glutamate receptors are a distinctive group of LGICs which are structurally di¡erent from other LGICs. The ¢rst structural di¡erence is simply molecular mass (see Fig. 2), glutamate receptor subunits are much larger than the nicotinic family members. Receptor channels are made up from four or ¢ve subunits.30,31 Further diversity and complexity is introduced in the glutamate families by splice variation (alternative exons can be transcribed from the DNA) or by RNA editing (see Fig. 2). Examples
Figure 2 Schematic diagram of amino acid sequences of glutamate receptor subunits.Glutamate receptor subunits range from 95 to 163 kDa in size. Note they are much larger than the other ligand-gated channels depicted approximately to scale. Further diversity is conferred uponthe glutamate family by RNA editing (unique to the glutamate superfamily) and by splice variation (alternative exons are transcribed from the gene). Splice variation is only seen in one of the eight variants of GABAa subunit (hatched triangle) and has not been detected in other members of the nicotinic superfamily.
LIGAND GATED ION CHANNELS
of this are the £ip and £op variations of the AMPA receptors. These two splice variations of subunits are expressed during di¡erent stages of development, the £ip form is expressed in pre- and postnatal brains, whereas the £op is expressed later. The two variations of the receptor have slightly di¡erent kinetics.30 Each subunit is made up of only three transmembrane domains, Ml, M3 and M4, with an intramembrane loop (M2).The M2 loop lines the channel pore (see Fig. 3). They can be divided into three major groups, AMPA (DL-a-amino-3-hydroxyS-methylisoxazole- 4 -propionate), kainate and NMDA (n-methyl, D aspartate) receptors, based on their pharmacology31 (see Table 1). AMPA and kainate receptors are primarily permeable to sodium and potassium ions. AMPA receptor subunits are GluR 1- 4: they have a similar structure to the kainate receptor subunits which are named GluR 5^7.30 The NMDA receptor is a Ca++ permeant channel involved in a process called long term potentiation (LTP), which is a cellular model for memory formation in hippocampus. Activation of NMDA receptors (by L glutamate) requires the concurrent binding of glycine at an allosteric co-agonist site. At rest, NMDA channels are also blocked by Mg++ ions, but they can be unblocked by depolarization. If there is enough repetitive stimulation of AMPA receptors and NMDA receptors are activated, this is thought to trigger a chain of intracellular events (secondary to Ca++ entry), resulting in a long-lived change in the intensity of synaptic signalling.32 Functional NMDA receptors comprise NRl, NR2A-D and NR3. It is widely accepted that ketamine, nitrous oxide and xenon induce anaesthesia by blocking the NMDA receptor21,25,33 Anaesthetic agents which block NMDA receptors clearly have very di¡erent pro¢les in vivo (although xenon is still relatively poorly characterized in man) which may be di⁄cult to align with a single molecular target. Conscious functions only are impaired, and re£ex actions are una¡ected by anaesthetics targeting only the NMDA receptor.34 However, nitrous oxide is strongly analgesic but is weakly anaesthetic. Animal models suggest that some of the potential side e¡ects are common to NMDA channel block. Psychotomimetic e¡ects can be seen with amantadine (another NMDA channel blocker) in neurological therapies. Recent papers suggest that the NMDA receptor may also be inhibited indirectly by anaesthetic agents whose primary ‘consensus’ target is the GABAA receptor. Fig. 4 (reproduced with Professor Flohr’s consent 34) shows that ketamine and drugs classically believed to be GABA-selective anaesthetics all block the binding of a radiolabelled NMDA antagonist which is dependent upon channel activation for access to its binding site. Most anaesthetics are unable to produce an e¡ect on NMDA receptors 4, but the recent work by Flohr suggests NMDA receptors may have a much more important role in the action of anaesthetics and in arousal per se.
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Figure 3 NMDA receptor structure. (A) NMDA receptor activation requires repetitive depolarization (usually supplied by activation of adjacent AMPA receptors) to dislodge the blocking Mg2+ ion and also requires both glutamate and glycine to bind to distinct recognition sites. (B) Subunits are composed of four transmembrane regions, the second of which is an intramembrane loop. The boxed region (black) indicates a putative glutamate-binding motif projecting into the synaptic cleft. The carboxyl terminal di¡ers from subunitto subunit, with the smaller NRl receptor subunits terminating atthe point marked X
Miscellaneous novel ion channels With the revolution in gene cloning, new classes of ion channels are emerging, e.g. the ATP-activated P2X re-
340
CURRENT ANAESTHESIA & CRITICAL CARE
Figure 4 (Replicated from Ref. (34)) This shows the uptake of a radiolabelled NMDA receptor antagonist (MK-801) in an awake animal brain (A), in an animal under ketamine anaesthesia (B), and in an animal under pentobarbital anaesthesia (C).Equivalent coronal sections ofcortex are depictedinthe top and bottomrows.The authorsinfer fromthis work andrelated modelling that NMDAchannel activation is a necessary condition for consciousness.
ceptor superfamily, the transient receptor potential (TRP) family and the vanilloid receptors (reviewed in Refs. 35^37 ).
Vanilloid receptors The active ingredient in chilli peppers, capscaisin, creates its burning sensation by activating the vanilloid receptors. Vanilloid receptors (VRl) are LGICs whose activation causes primarily the in£ux of Ca2+ ions, but are also highly permeable to Na+ and K+ ions. The ion channels are tetrameric. Each subunit contains six transmembrane domains (Sl ^S6), with a pore lining transmembrane loop between S5 and S6. Both the C terminal and the N terminal are intracellular, suggesting that ligands activate the receptor from the cytoplasm.37 Channel opening can be activated also by a decrease in pH and by an increase in temperature.VRl receptors located in the periphery are responsible for burning sensations induced by acids, temperature changes and nootropic chemicals which activate these receptors.VRl receptors which are centrally located but much more dif¢cult to study may also be involved in pain processing.37 Little work has been carried out to determine whether or not vanilloid receptors are involved in the analgesic e¡ects of anaesthetics, however, recent work has suggested that propofol is a VRl receptor agonist (which may underpin the painful response at the injection site)38
Vanilloid-receptor-like (VRL) ion channels are also subtypes of the vanilloid receptor family. There are three known types (VRLl ^3). All are capsaicin insensitive.VRLl is activated by high temperatures (of 501c and above). VRL2 is osmotically sensitive, and is activated by hypotonic solutions.36 VRL3 receptors are heat sensitive (activated by temperatures of X391C).These receptors can associate with VRl receptors to produce heterooligmers.39 VRLl and VRL3 may also be important in nociception.
TRP receptors TRP receptors resemble vanilloid receptors in their structural make up.They are tetrameric.There are seven di¡erent TRP subunits,TRPl ^7, and each has seven transmembrane regions. M6 is an intramembrane loop.36 Activation of these receptors results in calcium in£ux. Although many of these receptors are found outside of the nervous system, there is also a high expression in neuronal cells. They may be involved in the modulation of the cell’s resting potential and are thought to be activated by intracellular second messengers.36
P2X receptors P2X receptors are ionotrophic purinergic receptors. There are seven types of P2X subunit (P2X1^7 ), each with
LIGAND GATED ION CHANNELS
two membrane spanning regions.Either three or four assemble to produce a functional receptor. Their roles range from reproduction in males to fast synaptic signalling.They are found predominantly in the peripheral nervous system, but are also present in the brain. Many autonomic neurones express both nAChr and P2X receptors, which may interact with one another.35 During in£ammation, ATP is released and acts at the P2X receptors, indicating that they have a role in pain. Also, P2X7 receptor antagonists are antinociceptive.40
Concluding remarks Ligand-gated ion channels, particularly GABAA receptors and NMDA receptor subtypes, are excellent model targets for anaesthetic action in vitro. Most scientists now believe that the ‘anaesthetic receptor’ resides on proteins rather than in bulk lipid.The drugs are undoubtedly low a⁄nity and non-selective in action F only when selective knock-outs of implicated proteins in vivo impair drug action, in transgenic animals, will these authors be convinced.
ACKNOWLEDGEMENTS Thanks to the Northern Anaesthetics Research Consortium (NARC) for ¢nancial support and constant encouragement.
GLOSSARYOF TERMS Action potential the ‘nerve spike’, a transient depolarization of nerve or skeletal muscle propagated by voltage-gated ion channels. Chimera a recombinant protein containing complementary segments of two distinct receptors spliced together. Combining di¡erent segments allows the neuroscientist to recognize key functional domains or drugbinding sites. Depolarization reduction in the resting membrane potential (normally 60 to 80 mV) towards or beyond 0 mV. Hyperpolarization A negative shift in the transmembrane resting potential. Immunoprobing Fluorescent antibodies are used to label and anatomically localize antigenic segments of speci¢c receptor proteins on the cell surface. Long-term potentiation (LTP) Activity-dependent potentiation of synaptic signalsF an excellent model for memory formation in mammalian CNS. Neurotransmitter a chemical which is released from the nerve terminal which di¡uses across a narrow synaptic cleft to activate receptors on adjacent cells. Postsynaptic ‘dendritic’ regions of cells beyond the synaptic cleft which bear receptors to sense neurotransmitter binding.
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Pre-synaptic The terminal region of the neurone before the synapse with capacity to release neurotransmitter. RNA-editing Lengths of RNA can be di¡erentially translated resulting in alteration of amino acid composition at de¢ned sites on the protein. Splice-variations Alternative exons are transcribed from the gene which again results in insertion of a new cassette of amino acids in the protein. Synapse The cleft between neurones across which signals can be transmitted to turn neighbouring cells on (excitation) or o¡ (inhibition)
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