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Biomedical subjects

Richard W Olsen

Publications and source records attributed to Richard W Olsen.

At least 19 recordsLinked to original sources
PubMed2006

Identification of a GABAA receptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog.

General anesthetics, including etomidate, act by binding to and enhancing the function of GABA type A receptors (GABA(A)Rs), which mediate inhibitory neurotransmission in the brain. Here, we used a radiolabeled, photoreactive etomidate analog ([(3)H]azietomidate), which retains anesthetic potency in vivo and enhances GABA(A)R function in vitro, to identify directly, for the first time, amino acids that contribute to a GABA(A)R anesthetic binding site. For GABA(A)Rs purified by affinity chromatography from detergent extracts of bovine cortex, [(3)H]azietomidate photoincorporation was increased by GABA and inhibited by etomidate in a concentration-dependent manner (IC(50) = 30 microm). Protein microsequencing of fragments isolated from proteolytic digests established photolabeling of two residues: one within the alphaM1 transmembrane helix at alpha1Met-236 (and/or the homologous methionines in alpha2,3,5), not previously implicated in etomidate function, and one within the betaM3 transmembrane helix at beta3Met-286 (and/or the homologous methionines in beta1,2), an etomidate sensitivity determinant. The pharmacological specificity of labeling indicates that these methionines contribute to a single binding pocket for etomidate located in the transmembrane domain at the interface between beta and alpha subunits, in what is predicted by structural models based on homology with the nicotinic acetylcholine receptor to be a water-filled pocket approximately 50 A below the GABA binding site. The localization of the etomidate binding site to an intersubunit, not an intrasubunit, binding pocket is a novel conclusion that suggests more generally that the localization of drug binding sites to subunit interfaces may be a feature not only for GABA and benzodiazepines but also for etomidate and other intravenous and volatile anesthetics.

Animals↗
PubMed2006

GABAA receptor associated proteins: a key factor regulating GABAA receptor function.

gamma-Aminobutyric acid (GABA), an important inhibitory neurotransmitter in both vertebrates and invertebrates, acts on GABA receptors that are ubiquitously expressed in the CNS. GABA(A) receptors also represent a major site of action of clinically relevant drugs, such as benzodiazepines, barbiturates, ethanol, and general anesthetics. It has been shown that the intracellular M3-M4 loop of GABA(A) receptors plays an important role in regulating GABA(A) receptor function. Therefore, studies of the function of receptor intracellular loop associated proteins become important for understanding mechanisms of regulating receptor activity. Recently, several labs have used the yeast two-hybrid assay to identify proteins interacting with GABA(A) receptors, for example, the interaction of GABA(A) receptor associated protein (GABARAP) and Golgi-specific DHHC zinc finger protein (GODZ) with gamma subunits, PRIP, phospholipase C-related, catalytically inactive proteins (PRIP-1) and (PRIP-2) with GABARAP and receptor gamma2 and beta subunits, Plic-1 with some alpha and beta subunits, radixin with the alpha5 subunit, HAP1 with the beta1 subunit, GABA(A) receptor interacting factor-1 (GRIF-1) with the beta2 subunit, and brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) with the beta3 subunit. These proteins have been shown to play important roles in modulating the activities of GABA(A) receptors ranging from enhancing trafficking, to stabilizing surface and internalized receptors, to regulating modification of GABA(A) receptors. This article reviews the current studies of GABA(A) receptor intracellular loop-associated proteins.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Synthesis of trifluoromethylaryl diazirine and benzophenone derivatives of etomidate that are potent general anesthetics and effective photolabels for probing sites on ligand-gated ion channels.

To locate the binding sites of general anesthetics on ligand-gated ion channels, two derivatives of the intravenous general anesthetic etomidate (2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate), in which the 2-ethyl group has been replaced by photoactivable groups based on either aryl diazirine or benzophenone chemistry, have been synthesized and characterized pharmacologically. TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate) and BzBzl-etomidate (4-benzoylbenzyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate are both potent general anesthetics with half-effective anesthetic concentrations of 700 and 220 nM, respectively. Both agents resembled etomidate in enhancing currents elicited by low concentrations of GABA on heterologously expressed GABAA receptors and in shifting the GABA concentration-response curve to lower concentrations. They also allosterically enhanced the binding of flunitrazepam to mammalian brain GABAA receptors. Both agents were also effective and selective photolabels, photoincorporating into some, but not all, subunits of the Torpedo nicotinic acetylcholine receptor to a degree that was allosterically regulated by an agonist or a noncompetitive inhibitor. Thus, they have the necessary pharmacological and photochemical properties to be useful in identifying the site of etomidate-induced anesthesia.

Allosteric Regulation↗
PubMed2006

Low dose acute alcohol effects on GABA A receptor subtypes.

GABA(A) receptors (GABA(A)Rs) are the main inhibitory neurotransmitter receptors and have long been implicated in mediating at least part of the acute actions of ethanol. For example, ethanol and GABAergic drugs including barbiturates and benzodiazepines share many pharmacological properties. Besides the prototypical synaptic GABA(A)R subtypes, nonsynaptic GABA(A)Rs have recently emerged as important regulators of neuronal excitability. While high doses (> or =100 mM) of ethanol have been reported to enhance activity of most GABA(A)R subtypes, most abundant synaptic GABA(A)Rs are essentially insensitive to ethanol concentrations that occur during social ethanol consumption (< 30 mM). However, extrasynaptic delta and beta3 subunit-containing GABA(A)Rs, associated in the brain with alpha4 or alpha6 subunits, are sensitive to low millimolar ethanol concentrations, as produced by drinking half a glass of wine. Additionally, we found that a mutation in the cerebellar alpha6 subunit (alpha6R100Q), initially reported in rats selectively bred for increased alcohol sensitivity, is sufficient to produce increased alcohol-induced motor impairment and further increases of alcohol sensitivity in recombinant alpha6beta3delta receptors. Furthermore, the behavioral alcohol antagonist Ro15-4513 blocks the low dose alcohol enhancement on alpha4/6/beta3delta receptors, without reducing GABA-induced currents. In binding assays alpha4beta3delta GABA(A)Rs bind [(3)H]Ro15-4513 with high affinity, and this binding is inhibited, in an apparently competitive fashion, by low ethanol concentrations, as well as analogs of Ro15-4513 that are active to antagonize ethanol or Ro15-4513's block of ethanol. We conclude that most low to moderate dose alcohol effects are mediated by alcohol actions on alcohol/Ro15-4513 binding sites on GABA(A)R subtypes.

Acute Disease↗
PubMed2006

Ethanol potently and competitively inhibits binding of the alcohol antagonist Ro15-4513 to alpha4/6beta3delta GABAA receptors.

Although GABA(A) receptors have long been implicated in mediating ethanol (EtOH) actions, receptors containing the "nonsynaptic" delta subunit only recently have been shown to be uniquely sensitive to EtOH. Here, we show that delta subunit-containing receptors bind the imidazo-benzodiazepines (BZs) flumazenil and Ro15-4513 with high affinity (K(d) < 10 nM), contrary to the widely held belief that these receptors are insensitive to BZs. In immunopurified native cerebellar and recombinant delta subunit-containing receptors, binding of the alcohol antagonist [(3)H]Ro15-4513 is inhibited by low concentrations of EtOH (K(i) approximately 8 mM). Also, Ro15-4513 binding is inhibited by BZ-site ligands that have been shown to reverse the behavioral alcohol antagonism of Ro15-4513 (i.e., flumazenil, beta-carbolinecarboxylate ethyl ester (beta-CCE), and N-methyl-beta-carboline-3-carboxamide (FG7142), but not including any classical BZ agonists like diazepam). Experiments that were designed to distinguish between a competitive and allosteric mechanism suggest that EtOH and Ro15-4513 occupy a mutually exclusive binding site. The fact that only Ro15-4513, but not flumazenil, can inhibit the EtOH effect, and that Ro15-4513 differs from flumazenil by only a single group in the molecule (an azido group at the C7 position of the BZ ring) suggest that this azido group in Ro15-4513 might be the area that overlaps with the alcohol-binding site. Our findings, combined with previous observations that Ro15-4513 is a behavioral alcohol antagonist, suggest that many of the behavioral effects of EtOH at relevant physiological concentrations are mediated by EtOH/Ro15-4513-sensitive GABA(A) receptors.

Animals↗
PubMed2006

Contributions of the GABAA receptor alpha6 subunit to phasic and tonic inhibition revealed by a naturally occurring polymorphism in the alpha6 gene.

GABAA receptors (GABARs) are heteromultimeric proteins composed of five subunits. The specific subunit composition determines critical properties of a GABAR such as pharmacological sensitivities and whether the receptor contributes to synaptic or extrasynaptic forms of inhibition. Classically, synaptic but not extrasynaptic GABARs are thought to respond to benzodiazepines, whereas the reverse has been suggested for ethanol. To examine the effects of subunit composition on GABAR function in situ, we took advantage of two naturally occurring alleles of the rat gene for GABAR subunit alpha6 (Gabra6(100R) and Gabra6(100Q)). Depending on their subunit partners, these two variants of alpha6 can lead to differential sensitivities to benzodiazepines and ethanol. An examination of synaptic and extrasynaptic GABA-mediated currents in cerebellar granule cells from Gabra6(100R/100R) and Gabra6(100Q/100Q) rats uncovered marked allele-dependent differences in benzodiazepine sensitivity. Unexpectedly, we found that the benzodiazepines flunitrazepam and diazepam enhanced extrasynaptic inhibition mediated by delta subunit-containing GABARs in Gabra6(100Q/100Q) rats. Complementary experiments on recombinant GABARs confirmed that, at subsaturating [GABA], flunitrazepam potentiates alpha6/delta subunit-containing GABARs. Based on data and a simple theoretical analysis, we estimate that the average extrasynaptic [GABA] is approximately 160 nm in perfused slices. These results (1) demonstrate contributions of alpha6 subunits to both synaptic and extrasynaptic GABA responses, (2) establish that delta subunit-containing GABARs are benzodiazepine sensitive at subsaturating [GABA] and, (3) provide an empirical estimate of extrasynaptic [GABA] in slices.

Animals↗
PubMed2006

GABA(A)-R alpha1 subunit knockin mutation leads to abnormal EEG and anesthetic-induced seizure-like activity in mice.

Gamma-aminobutyric acid-type A receptors (GABA(A)-Rs) have been proposed as a target for many general anesthetics. We recently created knockin (KI) mice harboring a point mutation (serine 270 to histidine) in the GABA(A)-R alpha1 subunit. This mutation abolishes sensitivity of recombinant GABA(A)-Rs to isoflurane while maintaining normal sensitivity to halothane and increasing the potency of GABA. KI mice showed abnormalities in the EEG baseline, including occasional spike-wave activity and spindle-like bursts. When anesthetized with isoflurane, the KI mice but not the control mice revealed repetitive 4-5 Hz slow wave discharges in the cortical EEG. KI mice did not differ from controls in response to isoflurane or halothane in the standard tail clamp/withdrawal and loss of righting reflex assays. We recorded miniature inhibitory postsynaptic currents (mIPSCs) from hippocampal interneurons and pyramidal cells in brain slices. mIPSCs in neurons from KI mice were of normal amplitude, but decayed more slowly than controls. Hippocampal mIPSCs in control mice were significantly prolonged by 0.4 and 0.9 MAC isoflurane, and by 0.5 MAC halothane. In KI mice, the effect of isoflurane on mIPSC decay was dramatically reduced, while halothane prolonged mIPSCs as for controls. We conclude that the kinetic and pharmacological properties of hippocampal GABA(A)-Rs in the KI mouse recapitulate many features of mutant alpha1beta2gamma2 GABA(A)-Rs observed in vitro. GABA(A)-Rs containing alpha1 subunits do not appear to contribute to the actions of isoflurane in the spinal cord, but both EEG and synaptic recordings provide evidence for effects of isoflurane on these GABA(A)-R isoforms in cortical structures.

Anesthetics↗
PubMed2006

Chronic intermittent ethanol-induced switch of ethanol actions from extrasynaptic to synaptic hippocampal GABAA receptors.

Alcohol withdrawal syndrome (AWS) symptoms include hyperexcitability, anxiety, and sleep disorders. Chronic intermittent ethanol (CIE) treatment of rats with subsequent withdrawal of ethanol (EtOH) reproduced AWS symptoms in behavioral assays, which included tolerance to the sleep-inducing effect of acute EtOH and its maintained anxiolytic effect. Electrophysiological assays demonstrated a CIE-induced long-term loss of extrasynaptic GABAA receptor (GABAAR) responsiveness and a gain of synaptic GABAAR responsiveness of CA1 pyramidal and dentate granule neurons to EtOH that we were able to relate to behavioral effects. After CIE treatment, the alpha4 subunit-preferring GABAAR ligands 4,5,6,7 tetrahydroisoxazolo[5,4-c]pyridin-3-ol, La3+, and Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5alpha][1,4]benzodiazepine-3-carboxylate) exerted decreased effects on extrasynaptic currents but had increased effects on synaptic currents. Electron microscopy revealed an increase in central synaptic localization of alpha4 but not delta subunits within GABAergic synapses on the dentate granule cells of CIE rats. Recordings in dentate granule cells from delta subunit-deficient mice revealed that this subunit is not required for synaptic GABAAR sensitivity to low [EtOH]. The profound alterations in EtOH sensitivity and alpha4 subunit localization at hippocampal GABAARs of CIE rats suggest that such changes in these and other relevant brain circuits may contribute to the development of tolerance to the sleep-inducing effects and long-term dependence on alcohol.

Animals↗
PubMed2005

GABAA receptor beta3 subunit gene-deficient heterozygous mice show parent-of-origin and gender-related differences in beta3 subunit levels, EEG, and behavior.

The homozygous knockout mouse for the beta3 subunit of the GABAA receptor has been proposed as a model for the neurodevelopmental disorder, Angelman syndrome, based on phenotypic similarities of craniofacial abnormalities, cognitive defects, hyperactivity, motor incoordination, disturbed rest-activity cycles, and epilepsy. Since most children with Angelman syndrome are autosomal heterozygotes of maternal origin, apparently through genomic imprinting, we used gabrb3-deficient heterozygote mice of defined parental origin to investigate whether this phenotype is also maternally imprinted in mouse. Whole brain extracts showed greatly reduced beta3 subunit levels in male mice of maternal origin but not in male mice of paternal origin. Females of both parental origin showed greatly reduced beta3 subunit levels. Heterozygotes did not exhibit hyperactive circling behavior, convulsions, or electrographically recorded seizures. EEGs showed qualitative differences among heterozygotes, with male mice of maternal origin demonstrating more abnormalities including increased theta activity. Ethosuximide inhibited theta bursts, suggesting an alteration in the thalamocortical relay. Carbamazepine induced EEG slowing in males and EEG acceleration in females, with a larger effect in paternal-origin heterozygotes. Evidence thus suggests both parent-of-origin and gender-related components in developmental regulation of beta3 expression, in particular, that the maternally-derived male heterozygote may carry a developmental modification resulting in less beta3 protein, which may reflect partial genomic imprinting of the gabrb3 gene in mice.

Angelman Syndrome↗
PubMed2005

GABAA receptor-associated protein regulates GABAA receptor cell-surface number in Xenopus laevis oocytes.

GABA(A) receptor-associated protein (GABARAP) was isolated previously in a yeast two-hybrid screen using the intracellular loop of the gamma2 subunit of the GABA(A) receptor as bait. GABARAP has been shown to participate in the membrane-clustering and intracellular-trafficking of GABA(A) receptors, including a stimulation of the surface expression of GABA(A) receptors. To assess this quantitatively, we used Xenopus laevis oocytes expressing alpha1beta2gamma2S-containing GABA(A) receptors to demonstrate that coexpression of GABARAP increased net surface levels of GABA(A) receptors as shown by both increased GABA currents and surface-expressed protein. This GABARAP stimulation of GABA currents required the receptor gamma2 subunit and full-length GABARAP: deletion of the microtubule-binding domain (amino acids 1-22) or disrupting the polymerization of microtubules abolished the enhancement, indicating that the effect of GABARAP was derived from the interaction with microtubules. GABARAP coexpression did not alter the general properties of GABA(A) receptors such as sensitivity to GABA or benzodiazepines, but it increased surface levels of receptor protein in oocytes. Rather, it seems to supplement inadequate amounts of endogenous GABARAP to support optimum trafficking and/or stabilization of surface GABA(A) receptors.

Animals↗
PubMed2005

Alcohol-induced motor impairment caused by increased extrasynaptic GABA(A) receptor activity.

Neuronal mechanisms underlying alcohol intoxication are unclear. We find that alcohol impairs motor coordination by enhancing tonic inhibition mediated by a specific subtype of extrasynaptic GABA(A) receptor (GABAR), alpha6beta3delta, expressed exclusively in cerebellar granule cells. In recombinant studies, we characterize a naturally occurring single-nucleotide polymorphism that causes a single amino acid change (R100Q) in alpha6 (encoded in rats by the Gabra6 gene). We show that this change selectively increases alcohol sensitivity of alpha6beta3delta GABARs. Behavioral and electrophysiological comparisons of Gabra6(100R/100R) and Gabra6(100Q/100Q) rats strongly suggest that alcohol impairs motor coordination by enhancing granule cell tonic inhibition. These findings identify extrasynaptic GABARs as critical targets underlying low-dose alcohol intoxication and demonstrate that subtle changes in tonic inhibition in one class of neurons can alter behavior.

Action Potentials↗
PubMed2005

Plasticity of GABAA receptors in brains of rats treated with chronic intermittent ethanol.

The study of alcohol dependence mechanisms has been aided by work in rodents, where regimens of intermittent chronic administration with repeated episodes of intoxication and withdrawal can be coupled with controlled timing of in vitro studies and the possibility of relating them to behavior. The chronic intermittent ethanol (CIE) model in the rat has been found to be a good model of human alcohol dependence, showing persistent signs of withdrawal and self-administration. Studies in CIE rats suggest that plastic changes in GABA-mediated inhibition involving the GABAA receptor system may be responsible for the behavioral alterations. Here we summarize a combination of evidence that the alcoholic rat CIE model demonstrates changes in GABAA receptor subunit levels, in receptor localization, and in physiology and pharmacology, leading to alterations in behavior that contribute to the hyperexcitable alcohol withdrawal state (anxiety, insomnia, seizure susceptibility) and alcohol dependence.

Animals↗
PubMed2005

Pharmacologic evidence for abnormal thalamocortical functioning in GABA receptor beta3 subunit-deficient mice, a model of Angelman syndrome.

PURPOSE: gamma-Aminobutyric acid receptor (GABA(A)r) subunit beta3-deficient mice model Angelman syndrome by displaying impaired learning, abnormal EEG with interictal spikes and slowing, myoclonus, and convulsions. The beta3-subunit deficiency causes a failure of intrathalamic reticular nucleus inhibition, leading to abnormally synchronized thalamocortical oscillations. We postulated that this pathophysiology underlies the abnormal cortical EEG and triggers interictal spikes and seizures, but extrathalamic regions also contribute to interictal spikes and seizures, so that the EEG slowing should reveal an absence-like response profile, whereas spikes and seizures have dual responsiveness to absence and partial-seizure drugs. METHODS: Recording electrodes were implanted over the parietal cortices of wild-type, heterozygotes, and homozygous null mice. In each experiment, EEG was recorded for 45 min, either drug or vehicle administered, and EEG recorded for another 3 h. Each EEG was scored for slow-wave activity, interictal spikes, and seizures by a reader blinded to treatments. RESULTS: Interictal spiking and percentage of time in EEG slowing in heterozygotes were increased by the proabsence drug baclofen (GABA(B)-receptor agonist), whereas CGP 35348 (GABA(B)-receptor antagonist) had the opposite effect. The antiabsence drug ethosuximide markedly suppressed EEG slowing and interictal spiking in heterozygote and null mice. Broad-spectrum clonazepam and valproate were more effective on interictal spiking than on EEG slowing, and fosphenytoin suppressed only interictal spiking. CONCLUSIONS: The results suggest that this model of Angelman syndrome, although not expressing typical absence seizures, is characterized by hypersynchronous thalamocortical oscillations that possess absence-like pharmacologic responsiveness and promote EEG slowing, interictal spikes, and convulsive seizures.

Angelman Syndrome↗
PubMed2004

GABAA receptor-associated protein traffics GABAA receptors to the plasma membrane in neurons.

The trafficking of GABA(A) receptors is an important component of the pathway that regulates plasticity of inhibitory synapses. The 17 kDa GABA(A) receptor-associated protein (GABARAP) has been implicated in the trafficking of GABA(A) receptors because of its ability to interact not only with the gamma2 subunit of the receptor but also with microtubules and the N-ethylmaleimide-sensitive factor (NSF). To elucidate the role of GABARAP in the trafficking of GABA(A) receptors, we have constructed a yellow fluorescent protein (YFP) fusion protein of GABARAP and expressed it in neurons using adenovirus, so that its function may be examined. YFP-GABARAP colocalized with gamma2 subunit-containing GABA(A) receptors and NSF to the perinuclear cytoplasm in cultured hippocampal neurons and to the proximal regions of dendrites that are making synaptic contact. Expression of YFP-GABARAP in Cos7 cells and cultured hippocampal neurons was able to increase the level of GABA(A) receptors detected at the plasma membrane, even at low levels of YFP-GABARAP expression. This effect is specific to the function of GABARAP on GABA(A) receptor trafficking, because point mutations in the gamma2-binding domain of YFP-GABARAP interfered with the ability of YFP-GABARAP to increase GABA(A) receptor surface levels. These mutations also disrupted the colocalization of YFP-GABARAP with the gamma2 subunit and with NSF in hippocampal neurons. The results of this study show for the first time that GABARAP has a functional effect on the trafficking of GABA(A) receptors and provide decisive evidence for the role of GABARAP in transporting GABA(A) receptors to the plasma membrane in neurons.

Amino Acid Sequence↗
PubMed2004

Alcohol effects on gamma-aminobutyric acid type A receptors: are extrasynaptic receptors the answer?

GABA(A) receptors have long been implicated in mediating at least part of the actions of ethanol in mammalian brain. However, until very recently, reports of the actions of EtOH on recombinant receptors have required very high doses of ethanol and animals lacking receptor subunits shown to be important for ethanol actions in vitro did not support the view that these subunits are crucial in ethanol actions. Recombinant alpha4beta3delta and alpha6beta3delta GABA(A) receptors are uniquely sensitive to ethanol, with a dose-response relationship mirroring the well known effects of alcohol consumption on the human brain. Receptors containing the delta subunit are thought to be located extrasynaptically and it will be important to determine if these extrasynaptic GABA(A) receptor subunit combinations mediate low dose alcohol effects in vivo.

Animals↗
PubMed2004

Fishing for allosteric sites on GABA(A) receptors.

GABA(A) receptors have structural and functional homology with a super-family of cys-loop ligand-gated ion channel receptors including the nicotinic acetylcholine receptors. Amino acid residues involved in ligand-binding pockets are homologous among super-family members, leading to the multiple-loop model of binding sites situated at subunit interfaces, validated by structural studies on the nicotinic acetylcholine receptor and water-soluble snail acetylcholine binding protein. This article will briefly review the literature on the agonist binding sites on the receptor super-family, and then describe the current situation for attempts to identify sites for allosteric modulators on the GABA(A) receptors. A combination of mutagenesis and photoaffinity labeling with anesthetic ligands has given some leads in this endeavor. Current work by others and ourselves focuses on three putative sites for modulators: (1) within the ion channel domain TM2, near the extracellular end; (2) the agonist binding sites and homologous pockets at other subunit interfaces of the pentameric receptor; and (3) on the linker region stretching from the agonist site loop C to the top of the TM1 region. It is likely that concrete structural information will be forthcoming soon.

Allosteric Site↗