Matthew D. Krasowski

Sites of alcohol and volatile anaesthetic action on GABA A and glycine receptors

Volatile anaesthetics have historically been considered to act in a nonspecific manner on the central nervous system. More recent studies, however, have revealed that the receptors for inhibitory neurotransmitters such as γ-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABAA and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA ρ1 receptor is reduced. We have used this difference in pharmacology to investigate the molecular basis for modulation of these receptors by anaesthetics and alcohols. By using chimaeric receptor constructs, we have identified a region of 45 amino-acid residues that is both necessary and sufficient for the enhancement of receptor function. Within this region, two specific amino-acid residues in transmembrane domains 2 and 3 are critical for allosteric modulation of both GABAA and glycine receptors by alcohols and two volatile anaesthetics. These observations support the idea that anaesthetics exert a specific effect on these ion-channel proteins, and allow for the future testing of specific hypotheses of the action of anaesthetics.

General anaesthetic actions on ligand-gated ion channels.

Abstract. The molecular mechanisms of general anaesthetics have remained largely obscure since their introduction into clinical practice just over 150 years ago. This review describes the actions of general anaesthetics on mammalian neurotransmitter-gated ion channels. As a result of research during the last several decades, ligand-gated ion channels have emerged as promising molecular targets for the central nervous system effects of general anaesthetics. The last 10 years have witnessed an explosion of studies of anaesthetic modulation of recombinant ligand-gated ion channels, including recent studies which utilize chimeric and mutated receptors to identify regions of ligand-gated ion channels important for the actions of general anaesthetics. Exciting future directions include structural biology and gene-targeting approaches to further the understanding of general anaesthetic molecular mechanisms.

Bile salts of vertebrates: structural variation and possible evolutionary significance

Biliary bile salt composition of 677 vertebrate species (103 fish, 130 reptiles, 271 birds, 173 mammals) was determined. Bile salts were of three types: C27 bile alcohols, C27 bile acids, or C24 bile acids, with default hydroxylation at C-3 and C-7. C27 bile alcohols dominated in early evolving fish and amphibians; C27 bile acids, in reptiles and early evolving birds. C24 bile acids were present in all vertebrate classes, often with C27 alcohols or with C27 acids, indicating two evolutionary pathways from C27 bile alcohols to C24 bile acids: a) a ‘direct’ pathway and b) an ‘indirect’ pathway with C27 bile acids as intermediates. Hydroxylation at C-12 occurred in all orders and at C-16 in snakes and birds. Minor hydroxylation sites were C-1, C-2, C-5, C-6, and C-15. Side chain hydroxylation in C27 bile salts occurred at C-22, C-24, C-25, and C-26, and in C24 bile acids, at C-23 (snakes, birds, and pinnipeds). Unexpected was the presence of C27 bile alcohols in four early evolving mammals. Bile salt composition showed significant variation between orders but not between families, genera, or species. Bile salt composition is a biochemical trait providing clues to evolutionary relationships, complementing anatomical and genetic analyses.

Human dopamine transporter gene: coding region conservation among normal, Tourette's disorder, alcohol dependence and attention-deficit hyperactivity disorder populations.

The dopamine transporter (DAT) provides major regulation of the synaptic levels of dopamine and is a principal target of psychostimulant drugs. Associations between DAT gene polymorphisms and human disorders with possible links to dopaminergic neurotransmission, including attention-deficit/hyperactivity disorder (ADHD) and consequences of cocaine and alcohol administration, have been reported. We now report approximately 60000 bp of genomic sequence containing the entire DAT gene. This sequence was used to amplify each of the 15 DAT gene exons and several introns and analyze these amplification products by single-stranded sequence conformation (SSCP) and/or direct sequencing. These results define silent allelic single nucleotide sequence variants in DAT gene exons 2, 6, 9 and 15. Rare conservative mutations are identified in amino acids encoded by DAT exons 2 and 8. Analyses of the common nucleotide variants and the previously reported VNTR in the non-coding region of exon 15 define the pattern of linkage disequilibrium across the DAT locus. These comprehensive analyses, however, fail to identify any common protein coding DAT sequence variant in more than 150 unrelated individuals free of neuropsychiatric disease, 109 individuals meeting City of Hope criteria for Tourettes syndrome, 64 individuals with DSM-IV diagnoses of ethanol dependence, or 15 individuals with ADHD. These data are consistent with substantial evolutionary conservation of the DAT protein sequence. They suggest that gene variants that alter levels of DAT expression provide the best current candidate mechanism for reported associations between DAT gene markers, ADHD and other more tentatively associated neuropsychiatric disorders.

The actions of ether, alcohol and alkane general anaesthetics on GABAA and glycine receptors and the effects of TM2 and TM3 mutations

The actions of 13 general anaesthetics (diethyl ether, enflurane, isoflurane, methoxyflurane, sevoflurane, chloral hydrate, trifluoroethanol, tribromoethanol, tert‐butanol, chloretone, brometone, trichloroethylene, and α‐chloralose) were studied on agonist‐activated Cl− currents at human GABAA α2β1, glycine α1, and GABAC ρ1 receptors expressed in human embryonic kidney 293 cells. All 13 anaesthetics enhanced responses to submaximal (EC20) concentrations of agonist at GABAA and glycine receptors, except α‐chloralose, which did not enhance responses at the glycine α1 receptor. None of the anaesthetics studied potentiated GABA responses at the GABAC ρ1 receptor. Potentiation of submaximal agonist currents by the anaesthetics was studied at GABAA and glycine receptors harbouring mutations in putative transmembrane domains 2 and 3 within GABAA α2, β1, or glycine α1 receptor subunits: GABAA α2(S270I)β1, α2(A291W)β1, α2β1(S265I), and α2β1(M286W); glycine α1(S267I) and α1(A288W). For all anaesthetics studied except α‐chloralose, at least one of the mutations above abolished drug potentiation of agonist responses at GABAA and glycine receptors. α‐Chloralose produced efficacious direct activation of the GABAA α2β1 receptor (a ‘GABA‐mimetic’ effect). The other 12 anaesthetics produced minimal or no direct activation of GABAA and glycine receptors. A non‐anaesthetic isomer of α‐chloralose, β‐chloralose, was inactive at GABAA and glycine receptors and did not antagonize the actions of α‐chloralose at GABAA receptors. The implications of these findings for the molecular mechanisms of action of general anaesthetics at GABAA and glycine receptors are discussed.

Human Pregnane X Receptor Antagonists and Agonists Define Molecular Requirements for Different Binding Sites

The pregnane X receptor (PXR) is an important transcriptional regulator of the expression of xenobiotic metabolism and transporter genes. The receptor is promiscuous, binding many structural classes of molecules that act as agonists at the ligand-binding domain, triggering up-regulation of genes, increasing the metabolism and excretion of therapeutic agents, and causing drug-drug interactions. It has been suggested that human PXR antagonists represent a means to counteract such interactions. Several azoles have been hypothesized to bind the activation function-2 (AF-2) surface on the exterior of PXR when agonists are concurrently bound in the ligand-binding domain. In the present study, we have derived novel computational models for PXR agonists using different series of imidazoles, steroids, and a set of diverse molecules with experimental PXR agonist binding data. We have additionally defined a novel pharmacophore for the steroidal agonist site. All agonist pharmacophores showed that hydrophobic features are predominant. In contrast, a qualitative comparison with the corresponding PXR antagonist pharmacophore models using azoles and biphenyls showed that they are smaller and hydrophobic with increased emphasis on hydrogen bonding features. Azole antagonists were docked into a proposed hydrophobic binding pocket on the outer surface at the AF-2 site and fitted comfortably, making interactions with key amino acids involved in charge clamping. Combining computational and experimental data for different classes of molecules provided strong evidence for agonists and antagonists binding distinct regions on PXR. These observations bear significant implications for future discovery of molecules that are more selective and potent antagonists.

PXR: a xenobiotic receptor of diverse function implicated in pharmacogenetics.

The pregnane X receptor (PXR; NR1I2), a member of the nuclear receptor superfamily, regulates the expression of drug-metabolic enzymes and transporters involved in the responses of mammals to their chemical environment. The same enzyme and transporter systems are also involved in the homeostasis of numerous endogenous chemicals. The regulatory function of PXR is implicated in normal physiology and diseases, such as drug-drug interactions, hepatic steatosis, vitamin D homeostasis, bile acids homeostasis, steroid hormones homeostasis and inflammatory bowel diseases. As such, any genetic variations of this receptor could potentially have widespread effects on the disposition of xenobiotics and endobiotics. Knowledge concerning the genetic polymorphisms of PXR may help to understand the variations in human drug response and ensure safe drug use. The correlation of PXR genetic polymorphisms with several disease conditions also suggests that this receptor may represent a valid therapeutic for hepato-intestinal disorders such as inflammatory bowel disease and primary sclerosing cholangitis.

Evolution and Function of the NR1I Nuclear Hormone Receptor Subfamily (VDR, PXR, and CAR) with Respect to Metabolism of Xenobiotics and Endogenous Compounds

The NR1I subfamily of nuclear hormone receptors includes the 1,25-(OH)(2)-vitamin D(3) receptor (VDR; NR1I1), pregnane X receptor (PXR; NR1I2), and constitutive androstane receptor (CAR; NR1I3). PXR and VDR are found in diverse vertebrates from fish to mammals while CAR is restricted to mammals. Current evidence suggests that the CAR gene arose from a duplication of an ancestral PXR gene, and that PXR and VDR arose from duplication of an ancestral gene, represented now by a single gene in the invertebrate Ciona intestinalis. Aside from the high-affinity effects of 1,25-(OH)(2)-vitamin D(3) on VDRs, the NR1I subfamily members are functionally united by the ability to bind potentially toxic endogenous compounds with low affinity and initiate changes in gene expression that lead to enhanced metabolism and elimination (e.g., induction of cytochrome P450 3A4 expression in humans). The detoxification role of VDR seems limited to sensing high concentrations of certain toxic bile salts, such as lithocholic acid, whereas PXR and CAR have the ability to recognize structurally diverse compounds. PXR and CAR show the highest degree of cross-species variation in the ligand-binding domain of the entire vertebrate nuclear hormone receptor superfamily, suggesting adaptation to species-specific ligands. This review examines the insights that phylogenetic and experimental studies provide into the function of VDR, PXR, and CAR, and how the functions of these receptors have expanded to evolutionary advantage in humans and other animals.

Intravenous anesthetics differentially modulate ligand-gated ion channels

Background Heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) are potently inhibited by volatile anesthetics, but it is not known whether they are affected by intravenous anesthetics. Ketamine potentiates &ggr;-aminobutyric acid type A (GABAA) receptors at high concentrations, but it is unknown whether there is potentiation at clinically relevant concentrations. Information about the effects of intravenous anesthetics with different behavioral profiles on specific ligand-gated ion channels may lead to hypotheses as to which ion channel effect produces a specific anesthetic behavior. Methods A heteromeric nAChR composed of &agr;4 and &bgr;4 subunits was expressed heterologously in Xenopus laevis oocytes. Using the two-electrode voltage clamp technique, peak ACh-gated current was measured before and during application of ketamine, etomidate, or thiopental. The response to GABA of &agr;1&bgr;2&ggr;2s GABAA receptors expressed in human embryonic kidney cells and Xenopus oocytes was compared with and without coapplication of ketamine from 1 &mgr;m to 10 mm. Results Ketamine caused potent, concentration-dependent inhibition of the &agr;4&bgr;4 nAChR current with an IC50 of 0.24 &mgr;m. The inhibition by ketamine was use-dependent; the antagonist was more effective when the channel had been opened by agonist. Ketamine did not modulate the &agr;1&bgr;2&ggr;2s GABAA receptor response in the clinically relevant concentration range. Thiopental caused 27% inhibition of ACh response at its clinical EC50. Etomidate did not modulate the &agr;4&bgr;4 nAChR response in the clinically relevant concentration range, although there was inhibition at very high concentrations. Conclusions The &agr;4&bgr;4 nAChR, which is predominantly found in the central nervous system (CNS), is differentially affected by clinically relevant concentrations of intravenous anesthetics. Ketamine, commonly known to be an inhibitor at the N-methyl-d-aspartate receptor, is also a potent inhibitor at a central nAChR. It has little effect on a common CNS GABAA receptor in a clinically relevant concentration range. Interaction between ketamine and specific subtypes of nAChRs in the CNS may result in anesthetic behaviors such as inattention to surgical stimulus and in analgesia. Thiopental causes minor inhibition at the &agr;4&bgr;4 nAChR. Modulation of the &agr;4&bgr;4 nAChR by etomidate is unlikely to be important in anesthesia practice based on the insensitivity of this receptor to clinically used concentrations.

Normal electrophysiological and behavioral responses to ethanol in mice lacking the long splice variant of the γ2 subunit of the γ-aminobutyrate type A receptor

The γ subunit of the γ-aminobutyric acid type A receptor (GABAA-R) is essential for bestowing both normal single channel conductance and sensitivity to benzodiazepines on native GABAA-Rs. The long splice variant of the γ2 subunit (γ2L) has been postulated to be essential in mediating the modulatory actions of ethanol at the GABAA-R. In order to evaluate this hypothesis, gene targeting was used to delete the 24bp exon which distinguishes γ2L from the short splice variant (γ2S). Mice homozygous for this exon deletion (γ2L−/−) are viable and indistinguishable from wild-type (γ2L+/+) mice. No γ2L mRNA was detected in these mice, nor could γ2L-containing GABAA-R protein be detected by specific antibodies. Radioligand binding studies showed the total amount of γ2 subunit protein to be not significantly changed, suggesting that γ2S replaces γ2L in the brains of the knockout animals. Electrophysiological recordings from dorsal root ganglion neurons revealed a normal complement of functional receptors. There was no difference in the potentiation of GABA currents by ethanol (20–200 mM) observed in neurons from γ2L+/+or γ2L−/− mice. Several behavioral effects of ethanol, such as sleep time, anxiolysis, acute functional tolerance, chronic withdrawal hyperexcitability and hyperlocomotor activity were also unaffected by genotype. It is concluded that γ2L is not required for ethanol’s modulatory action at the GABAA-R or whole animal behavioral effects.