Nicholas P. Franks

Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites.

Human serum albumin (HSA) is the most abundant protein in the circulatory system. Its principal function is to transport fatty acids, but it is also capable of binding a great variety of metabolites and drugs. Despite intensive efforts, the detailed structural basis of fatty acid binding to HSA has remained elusive. We have now determined the crystal structure of HSA complexed with five molecules of myristate at 2.5 Å resolution. The fatty acid molecules bind in long, hydrophobic pockets capped by polar side chains, many of which are basic. These pockets are distributed asymmetrically throughout the HSA molecule, despite its symmetrical repeating domain structure.

General anaesthesia: from molecular targets to neuronal pathways of sleep and arousal

The mechanisms through which general anaesthetics, an extremely diverse group of drugs, cause reversible loss of consciousness have been a long-standing mystery. Gradually, a relatively small number of important molecular targets have emerged, and how these drugs act at the molecular level is becoming clearer. Finding the link between these molecular studies and anaesthetic-induced loss of consciousness presents an enormous challenge, but comparisons with the features of natural sleep are helping us to understand how these drugs work and the neuronal pathways that they affect. Recent work suggests that the thalamus and the neuronal networks that regulate its activity are the key to understanding how anaesthetics cause loss of consciousness.

The α2-adrenoceptor Agonist Dexmedetomidine Converges on an Endogenous Sleep-promoting Pathway to Exert Its Sedative Effects

Background The authors investigated whether the sedative, or hypnotic, action of the general anesthetic dexmedetomidine (a selective &agr;2-adrenoceptor agonist) activates endogenous nonrapid eye movement (NREM) sleep-promoting pathways. Methods c-Fos expression in sleep-promoting brain nuclei was assessed in rats using immunohistochemistry and in situ hybridization. Next, the authors perturbed these pathways using (1) discrete lesions induced by ibotenic acid, (2) local and systemic administration of &ggr;-aminobutyric acid receptor type A (GABAA) receptor antagonist gabazine, or (3) &agr;2-adrenoceptor antagonist atipamezole in rats, and (4) genetic mutation of the &agr;2A-adrenoceptor in mice. Results Dexmedetomidine induced a qualitatively similar pattern of c-Fos expression in rats as seen during normal NREM sleep, i.e., a decrease in the locus ceruleus (LC) and tuberomammillary nucleus (TMN) and an increase in the ventrolateral preoptic nucleus (VLPO). These changes were attenuated by atipamezole and were not seen in mice lacking functional &agr;2A-adrenoceptors, which do not show a sedative response to dexmedetomidine. Bilateral VLPO lesions attenuated the sedative response to dexmedetomidine, and the dose–response curve to dexmedetomidine was shifted right by gabazine administered systemically or directly into the TMN. VLPO lesions and gabazine pretreatment altered c-Fos expression in the TMN but in not the LC after dexmedetomidine administration, indicating a hierarchical sequence of changes. Conclusions The authors propose that endogenous sleep pathways are causally involved in dexmedetomidine-induced sedation; dexmedetomidines sedative mechanism involves inhibition of the LC, which disinhibits VLPO firing. The increased release of GABA at the terminals of the VLPO inhibits TMN firing, which is required for the sedative response.

Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes

BACKGROUND Firefly luciferase is a 62 kDa protein that catalyzes the production of light. In the presence of MgATP and molecular oxygen, the enzyme oxidizes its substrate, firefly luciferin, emitting yellow-green light. The reaction proceeds through activation of the substrate to form an adenylate intermediate. Firefly luciferase shows extensive sequence homology with a number of enzymes that utilize ATP in adenylation reactions. RESULTS We have determined the crystal structure of firefly luciferase at 2.0 A resolution. The protein is folded into two compact domains. The large N-terminal domain consists of a beta-barrel and two beta-sheets. The sheets are flanked by alpha-helices to form an alphabetaalphabetaalpha five-layered structure. The C-terminal portion of the molecule forms a distinct domain, which is separated from the N-terminal domain by a wide cleft. CONCLUSIONS Firefly luciferase is the first member of a superfamily of homologous enzymes, which includes acyl-coenzyme A ligases and peptide synthetases, to have its structure characterized. The residues conserved within the superfamily are located on the surfaces of the two domains on either side of the cleft, but are too far apart to interact simultaneously with the substrates. This suggests that the two domains will close in the course of the reaction. Firefly luciferase has a novel structural framework for catalyzing adenylate-forming reactions.

How does xenon produce anaesthesia

Since the discovery that the gas xenon can produce general anaesthesia without causing undesirable side effects, we have remained surprisingly ignorant of the molecular mechanisms underlying this clinical activity of an ‘inert’ gas. Although most general anaesthetics enhance the activity of inhibitory GABAA (γ-aminobutyric acid type-A) receptors,, we find that the effects of xenon on these receptors are negligible. Instead, xenon potently inhibits the excitatory NMDA (N-methyl-D-aspartate) receptor channels, which may account for many of xenons attractive pharmacological properties.

Fatty acid binding to human serum albumin: new insights from crystallographic studies

Human serum albumin possesses multiple fatty acid binding sites of varying affinities, but the precise locations of these sites have remained elusive. The determination of the crystal structure of human serum albumin complexed with myristic acid recently revealed the positions and architecture of six binding sites on the protein. While the structure of the complex is consistent with a great deal of the biochemical and biophysical data on fatty acid binding, it is not yet possible to provide a completely rigorous correlation between the structural and binding data. The challenge now is to use the new structural information to design experiments that will identify the physiologically important binding sites on HSA and provide a much richer description of fatty acid interactions with the protein.

Molecular targets underlying general anaesthesia

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

Xenon and Hypothermia Combine to Provide Neuroprotection from Neonatal Asphyxia

Perinatal asphyxia can result in neuronal injury with long‐term neurological and behavioral consequences. Although hypothermia may provide some modest benefit, the intervention itself can produce adverse consequences. We have investigated whether xenon, an antagonist of the N‐methyl‐D‐aspartate subtype of the glutamate receptor, can enhance the neuroprotection provided by mild hypothermia. Cultured neurons injured by oxygen‐glucose deprivation were protected by combinations of interventions of xenon and hypothermia that, when administered alone, were not efficacious. A combination of xenon and hypothermia administered 4 hours after hypoxic‐ischemic injury in neonatal rats provided synergistic neuroprotection assessed by morphological criteria, by hemispheric weight, and by functional neurological studies up to 30 days after the injury. The protective mechanism of the combination, in both in vitro and in vivo models, involved an antiapoptotic action. If applied to humans, these data suggest that low (subanesthetic) concentrations of xenon in combination with mild hypothermia may provide a safe and effective therapy for perinatal asphyxia. Ann Neurol 2005;58:182–193

Differential sensitivities of mammalian neuronal and muscle nicotinic acetylcholine receptors to general anesthetics.

Background: Nicotinic acetylcholine receptors (nAChRs) are members of a superfamily of fast neurotransmitter‐gated receptor channels that includes the gamma‐aminobutyric acidA (GA‐BAA), glycine and serotonin type 3 (5‐HT3) receptors. Most previous work on the interactions of general anesthetics with nAChRs has involved the muscle‐type receptor. The authors investigate the effects of general anesthetics on defined mammalian neuronal and muscle nAChRs expressed in Xenopus oocytes. Methods: Complementary deoxyribonucleic acid (cDNA) or messenger ribonucleic acid (mRNA) encoding for various neuronal or muscle nAChR subunits was injected into Xenopus oocytes, and the resulting ACh‐activated currents were studied using the two‐electrode voltage‐clamp technique. The effects of halothane, isoflurane, sevoflurane, and propofol on the peak acetylcholine‐induced currents were investigated, and concentration‐response curves were constructed. Results: The neuronal nAChRs were found to be much more sensitive to general anesthetics than were the muscle nAChRs, with IC50 concentrations being 10‐ to 35‐fold less for the neuronal receptors. For the inhalational general anesthetics, the IC50 concentrations were considerably less than the free aqueous concentrations that cause general anesthesia in mammals. In addition, qualitative (dependence on acetylcholine concentration) and quantitative (steepness of concentration‐response curves) differences in the anesthetic interactions between the neuronal and muscle nAChRs suggest that different mechanisms of inhibition may be involved. Conclusions: Although there is considerable uncertainty about the physiologic roles that neuronal nAChRs play in the central nervous system, their extraordinary sensitivity to general anesthetics, particularly the inhalational agents, suggests they may mediate some of the effects of general anesthetics at surgical, or even subanesthetic, concentrations.

Xenon mitigates isoflurane-induced neuronal apoptosis in the developing rodent brain.

Background:Anesthetics, including isoflurane and nitrous oxide, an antagonist of the N-methyl-d-aspartate subtype of the glutamate receptor, have been demonstrated to induce apoptotic neurodegeneration when administered during neurodevelopment. Xenon, also an N-methyl-d-aspartate antagonist, not only lacks the characteristic toxicity produced by other N-methyl-d-aspartate antagonists, but also attenuates the neurotoxicity produced by this class of agent. Therefore, the current study sought to investigate xenons putative protective properties against anesthetic-induced neuronal apoptosis. Method:Separate cohorts (n = 5 or 6 per group) of 7-day-old rats were randomly assigned and exposed to eight gas mixtures: air, 75% nitrous oxide, 75% xenon, 0.75% isoflurane, 0.75% isoflurane plus 35% or 75% nitrous oxide, 0.75% isoflurane plus 30% or 60% xenon for 6 h. Rats were killed, and cortical and hippocampal apoptosis was assessed using caspase-3 immunostaining. In separate cohorts, cortices were isolated for immunoblotting of caspase 3, caspase 8, caspase 9, and cytochrome c. Organotypic hippocampal slices of postnatal mice pups were derived and cultured for 24 h before similar gas exposures, as above, and subsequently processed for caspase-3 immunostaining. Results:In vivo administration of isoflurane enhances neuronal apoptosis. When combined with isoflurane, nitrous oxide significantly increases whereas xenon significantly reduces apoptosis to a value no different from that of controls. In vitro studies corroborate the ability of xenon to attenuate isoflurane-induced apoptosis. Isoflurane enhanced expression of indicators of the intrinsic and common apoptotic pathways; this enhancement was increased by nitrous oxide but attenuated by xenon. Conclusions:The current study demonstrates that xenon prevents isoflurane-induced neonatal neuronal apoptosis.