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R W Olsen

Publications and source records attributed to R W Olsen.

At least 19 recordsLinked to original sources
PubMed2006

GABAA receptor alpha 4 subunits mediate extrasynaptic inhibition in thalamus and dentate gyrus and the action of gaboxadol.

The neurotransmitter GABA mediates the majority of rapid inhibition in the CNS. Inhibition can occur via the conventional mechanism, the transient activation of subsynaptic GABAA receptors (GABAA-Rs), or via continuous activation of high-affinity receptors by low concentrations of ambient GABA, leading to "tonic" inhibition that can control levels of excitability and network activity. The GABAA-R alpha4 subunit is expressed at high levels in the dentate gyrus and thalamus and is suspected to contribute to extrasynaptic GABAA-R-mediated tonic inhibition. Mice were engineered to lack the alpha4 subunit by targeted disruption of the Gabra4 gene. alpha4 Subunit knockout mice are viable, breed normally, and are superficially indistinguishable from WT mice. In electrophysiological recordings, these mice show a lack of tonic inhibition in dentate granule cells and thalamic relay neurons. Behaviorally, knockout mice are insensitive to the ataxic, sedative, and analgesic effects of the novel hypnotic drug, gaboxadol. These data demonstrate that tonic inhibition in dentate granule cells and thalamic relay neurons is mediated by extrasynaptic GABAA-Rs containing the alpha4 subunit and that gaboxadol achieves its effects via the activation of this GABAA-R subtype.

Animals↗
PubMed2006

Low-dose alcohol actions on alpha4beta3delta GABAA receptors are reversed by the behavioral alcohol antagonist Ro15-4513.

Although it is now more than two decades since it was first reported that the imidazobenzodiazepine Ro15-4513 reverses behavioral alcohol effects, the molecular target(s) of Ro15-4513 and the mechanism of alcohol antagonism remain elusive. Here, we show that Ro15-4513 blocks the alcohol enhancement on recombinant "extrasynaptic" alpha4/6beta3delta GABA(A) receptors at doses that do not reduce the GABA-induced Cl(-) current. At low ethanol concentrations (< or =30 mM), the Ro15-4513 antagonism is complete. However, at higher ethanol concentrations (> or =100 mM), there is a Ro15-4513-insensitive ethanol enhancement that is abolished in receptors containing a point mutation in the second transmembrane region of the beta3 subunit (beta3N265M). Therefore, alpha4/6beta3delta GABA receptors have two distinct alcohol modulation sites: (i) a low-dose ethanol site present in alpha4/6beta3delta receptors that is antagonized by the behavioral alcohol antagonist Ro15-4513 and (ii) a site activated at high (anesthetic) alcohol doses, defined by mutations in membrane-spanning regions. Receptors composed of alpha4beta3N265Mdelta subunits that lack the high-dose alcohol site show a saturable ethanol dose-response curve with a half-maximal enhancement at 16 mM, close to the legal blood alcohol driving limit in most U.S. states (17.4 mM). Like in behavioral experiments, the alcohol antagonist effect of Ro15-4513 on recombinant alpha4beta3delta receptors is blocked by flumazenil and beta-carboline-ethyl ester (beta-CCE). Our findings suggest that ethanol/Ro15-4513-sensitive GABA(A) receptors are important mediators of behavioral alcohol effects.

Animals↗
PubMed2006

Basis of the gabamimetic profile of ethanol.

This article summarizes the proceedings of a symposium held at the 2005 Research Society on Alcoholism meeting. The initial presentation by Dr. Wallner provided evidence that selected GABA(A) receptors containing the delta subunit display sensitivity to low intoxicating ethanol concentrations and this sensitivity is further increased by a mutation in the cerebellar alpha6 subunit, found in alcohol-hypersensitive rats. Dr. Mameli reported that ethanol affects gamma-aminobutyric acid (GABA) function by affecting neural circuits that influence GABA release. Dr. Parsons presented data from electrophysiological and microdialysis investigations that ethanol is capable of releasing GABA from presynaptic terminals. Dr. Morrow demonstrated that systemic ethanol increases neuroactive steroids in brain, the absence of which alters various functional responses to ethanol. Dr. Criswell presented evidence that the ability of ethanol to increase GABA was apparent in some, but not all, brain regions indicative of regional specificity. Further, Dr. Criswell demonstrated that neurosteroids alone and when synthesized locally by ethanol act postsynaptically to enhance the effect of GABA released by ethanol in a region specific manner. Collectively, this series of reports support the GABAmimetic profile of acutely administered ethanol being dependent on several specific mechanisms distinct from a direct effect on the major synaptic isoforms of GABA(A) receptors.

Alcoholism↗
PubMed2003

Ethanol enhances alpha 4 beta 3 delta and alpha 6 beta 3 delta gamma-aminobutyric acid type A receptors at low concentrations known to affect humans.

gamma-Aminobutyric acid type A receptors (GABARs) have long been implicated in mediating ethanol (EtOH) actions, but so far most of the reported recombinant GABAR combinations have shown EtOH responses only at fairly high concentrations (> or = 60 mM). We show that GABARs containing the delta-subunit, which are highly sensitive to gamma-aminobutyric acid, slowly inactivating, and thought to be located outside of synapses, are enhanced by EtOH at concentrations that are reached with moderate, social EtOH consumption. Reproducible ethanol enhancements occur at 3 mM, a concentration six times lower than the legal blood-alcohol intoxication (driving) limit in most states (0.08% wt/vol or 17.4 mM). GABARs responsive to these low EtOH concentrations require the GABAR delta-subunit, which is thought to be associated exclusively with alpha 4- and alpha 6-subunits in vivo, and the beta 3-subunit, which has recently been shown to be essential for the in vivo anesthetic actions of etomidate and propofol. GABARs containing beta 2-instead of beta 3-subunits in alpha 4 beta delta- and alpha 6 beta delta-receptor combinations are almost 10 times less sensitive to EtOH, with threshold enhancement at 30 mM. GABARs containing gamma 2-instead of delta-subunits with alpha 4 beta and alpha 6 beta are three times less sensitive to EtOH, with threshold responses at 100 mM, a concentration not usually reached with social EtOH consumption. These combined findings suggest that "extrasynaptic" delta-subunit-containing GABARs, but not their "synaptic" gamma-subunit-containing counterparts, are primary targets for EtOH.

Alcohol Drinking↗
PubMed2001

Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2.

Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma2 and the remainder of the gamma2 or alpha1 subunits, respectively, were expressed with beta2 and beta2gamma2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (alpha1/gamma2)beta2 and (alpha1/gamma2)beta2gamma2 but not the (gamma2/alpha1)beta2 and (gamma2/alpha1)beta2gamma2 subunit combinations formed functional receptor complexes as shown by whole-cell patch-clamp recordings and [3H]muscimol and [3H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (alpha1/gamma2)-containing receptors was pronounced, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch-clamp recordings, which was independent of whether the chimera was expressed in combination with beta2 or beta2gamma2. Surprisingly, the (alpha1/gamma2)(gamma2/alpha1)beta2 subunit combination did desensitize, indicating that the C-terminal segment of the alpha1 subunit may be important for desensitization. Moreover, desensitization was observed for the (alpha1/gamma2)beta2gamma2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.

Animals↗
PubMed2001

Chronic benzodiazepine treatment of cells expressing recombinant GABA(A) receptors uncouples allosteric binding: studies on possible mechanisms.

Functional and behavioral tolerance to chronic benzodiazepine (BZ) exposure has been associated with an uncoupling of the BZ and GABA binding sites. As in rats exposed to BZ for periods of a week or longer, recombinant GABA(A) receptors (GABARs) expressed in Sf9 cells lose the normally observed allosteric enhancement of [3H]flunitrazepam binding by GABA agonists, which is measured in homogenized membranes after a few hours exposure to pharmacological doses of agonist BZ. Treatment of Sf9 cells expressing recombinant GABAR with various drugs that inhibit protein kinase A (PKA), but not protein kinase C (PKC), resulted in an uncoupling of the BZ and GABA binding sites; whereas promotion of phosphorylation by PKA, but not PKC, favored coupling and recoupling. However, mutation of the only PKA phosphorylation site expressed from among the subunits proved that direct phosphorylation of the GABAR was not involved in either coupling after chronic BZ exposure or reversal of uncoupling after exposure to the competitive BZ antagonist, flumazenil. Osmotic-shock of cell membrane homogenates to lyse intracellular compartments reversed uncoupling, and uncoupling can be replicated in untreated cells by performing membrane binding assays in an acidic environment, suggesting that GABARs become internalized into an acidic intracellular environment where normal BZ binding occurs, but that potentiation by GABA is hindered. The internalization of receptors was shown by immunofluorescence after chronic exposure to either BZ or the PKA inhibitor H-89.

Animals↗
PubMed2001

Altered gabaa receptor subunit and splice variant expression in rats treated with chronic intermittent ethanol.

BACKGROUND: Intermittent chronic administration of ethanol to rats has been shown previously to produce a hyperexcitable, kindling-like state, accompanied by reduced inhibitory synaptic transmission in the hippocampus and changes in gamma-aminobutyric acid type A (GABAA) receptors. Further information is needed on the detailed changes in GABAA receptors and their time course and persistence, as is comparison to changes after chronic, continuous ethanol. METHODS: GABAA receptors were analyzed in the rat brain after chronic intermittent ethanol (CIE) by using radioligand binding, photoaffinity labeling of polypeptides, and estimates of messenger RNA (mRNA) levels of receptor subunits by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. RESULTS: CIE rats were confirmed to have increased GABAA receptor binding of the benzodiazepine partial inverse agonist and ethanol antidote ligand Ro15-4513, due to increased expression of the alpha6 subunit polypeptide in the cerebellum, shown by photoaffinity labeling. Estimates of mRNA levels by use of RT-PCR did not reveal any significant increase in alpha6 or in several other receptor subunits in several brain regions, but a decrease in the ratio of the long and short splice variants (L/S) of the gamma2 subunit was detected in the hippocampus, especially the CA1 region. CONCLUSIONS: Changes in GABAA receptors were found in rats given CIE. Increased alpha6 subunit in the cerebellum was demonstrated by using both the binding to diazepam-insensitive sites for [3H]Ro15-4513 and increased levels of the 57-kDa alpha6 polypeptide after photoaffinity labeling with this ligand. This increase appeared after 30 doses of ethanol and decayed to normal 1 week after ethanol was discontinued. The transient change in cerebellar alpha6 subunit-containing receptors, also reportedly seen after chronic continuous ethanol, is thus unlikely to account for the persistently hyperexcitable, kindled, seizure-susceptible state seen in CIE. However, the significant decrease in gamma2 subunit L/S splice variant ratio in the hippocampus implies changes in GABAA receptor function, possibly involving protein phosphorylation by protein kinase C. Altered receptor trafficking and turnover associated with synaptic plasticity may contribute to the observed reduced inhibition in the hippocampus and other signs of alcohol dependence produced by CIE.

Alcoholism↗
PubMed2000

The gamma-aminobutyric acid type A (GABAA) receptor-associated protein (GABARAP) promotes GABAA receptor clustering and modulates the channel kinetics.

A microtubule-associated protein, gamma-aminobutyric acid type A (GABA(A)) receptor-associated protein (GABARAP), was previously identified as binding to the intracellular domain of GABA(A) receptors by using the yeast two-hybrid screen. In the present work, immunofluorescent staining and green fluorescent protein-tagged receptor subunits showed that GABARAP is associated with and promotes the clustering of GABA(A) receptors in QT-6 quail fibroblasts. The tubulin-binding motif of GABARAP and the gamma2 subunit of the receptor are required. Disruption of microtubules prevents the clustering in a time-dependent manner. When green fluorescent protein-tagged alpha1 or gamma2 subunit coexpressed with beta2, gamma2L, and GABARAP was used, recordings from visually identified cells revealed that clustered GABA(A) receptor had an EC(50) of about 20 microM, vs. 5.7 microM for the diffuse receptor. Clustered receptors deactivated faster and desensitized slower than the diffuse receptors, because of decrease in the apparent affinity of GABA binding. Different properties for clustered receptors relative to unclustered receptors in heterologous cells suggest that homologous differences between extrasynaptic and synaptic clustered receptors in neurons may be due to the organization of the postsynaptic machinery.

Adaptor Proteins, Signal Transducing↗
PubMed2000

The gamma-aminobutyric acid type A receptor (GABAAR)-associated protein GABARAP interacts with gephyrin but is not involved in receptor anchoring at the synapse.

gamma-Aminobutyric acid type A receptors (GABA(A)Rs) are ligand-gated chloride channels that exist in numerous distinct subunit combinations. At postsynaptic membrane specializations, different GABA(A)R isoforms colocalize with the tubulin-binding protein gephyrin. However, direct interactions of GABA(A)R subunits with gephyrin have not been reported. Recently, the GABA(A)R-associated protein GABARAP was found to bind to the gamma2 subunit of GABA(A)Rs. Here we show that GABARAP interacts with gephyrin in both biochemical assays and transfected cells. Confocal analysis of neurons derived from wild-type and gephyrin-knockout mice revealed that GABARAP is highly enriched in intracellular compartments, but not at gephyrin-positive postsynaptic membrane specializations. Our data indicate that GABARAP-gephyrin interactions are not important for postsynaptic GABA(A)R anchoring but may be implicated in receptor sorting and/or targeting mechanisms. Consistent with this idea, a close homolog of GABARAP, p16, has been found to function as a late-acting intra-Golgi transport factor.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Stable GABA(A) receptor intermediates in SF-9 cells expressing alpha1, beta2 and gamma2 subunits.

Spodoptera frugiperda insect cells (Sf-9 cells) were used to study GABA(A) receptor assembly. Time courses of the expression level of alpha1beta2 and alpha1beta2gamma2 receptor protein showed [(3)H]muscimol binding to appear 2 hr before [(3)H]flunitrazepam and [(35)S]TBPS binding. This indicates that muscimol may bind to pentamers with an immature conformation or to molecules smaller than the pentamer. Binding studies performed on fractions from sucrose gradients loaded with solubilized alpha1beta2 or alpha1beta2gamma2 containing membranes revealed no binding other than to the pentameric fractions. Western blotting on fractionated sucrose gradients, however, clearly revealed the existence of GABA(A) receptor intermediates. The alpha1 subunit was seen in fractions corresponding to molecules smaller than the pentamer only when co-expressed with gamma2, indicating that the gamma2 subunit is needed for the alpha1 to form relatively long lasting intermediates. Moreover, Western blots revealed multiple isoforms for each subunit. In general, it was primarily the lower molecular weight forms that were detected in the pentameric fractions. The exception being for the alpha1 and gamma2 forms in subunit combinations that did not contain both of these subunits (i.e., alpha1, gamma2, alpha1beta2, beta2gamma2), where higher molecular weight forms were strongly represented. These findings show that alpha1 and gamma2 prefer specific protein forms when expressed together.

Animals↗
PubMed2000

Deduction of amino acid residues in the GABA(A) receptor alpha subunits photoaffinity labeled with the benzodiazepine flunitrazepam.

Peptide mapping and microsequencing were used to infer the site of photoaffinity labeling by the gamma-aminobutyric acidA receptor modulator [3H]flunitrazepam. Peptide mapping with and without N-deglycosylation was used to restrict the domain for photoaffinity labeling to residues 74-123 of the bovine alpha1 subunit, in agreement with a previously predicted labeling domain between residues 59-148 based on cyanogen bromide fragmentation. Edman degradation of partially purified photolabeled peptides gave release of 3H counts in the ninth cycle of a tryptic peptide sequence. A second V8/chymotryptic peptide produced an impure sequence with release of 3H counts in the seventh through ninth cycle of sequence. The combined data support those previously reported, i.e., that the primary site for photoaffinity labeling by [3H]flunitrazepam is His102 of the bovine alpha1 subunit. In addition we also detected possible secondary labeling of Pro97.

Affinity Labels↗
PubMed2000

Binding of the GABA(A) receptor-associated protein (GABARAP) to microtubules and microfilaments suggests involvement of the cytoskeleton in GABARAPGABA(A) receptor interaction.

GABA(A) receptor-associated protein (GABARAP) was isolated on the basis of its interaction with the gamma2 subunit of GABA(A) receptors. It has sequence similarity to light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. This suggests that GABARAP may link GABA(A) receptors to the cytoskeleton. GABARAP associates with tubulin in vitro. However, little is known about the mechanism for the interaction, and it is not clear whether the interaction occurs in vivo. Here, we report that GABARAP interacts directly with both tubulin and microtubules in a salt-sensitive manner, indicating the association is mediated by ionic interactions. GABARAP coimmunoprecipitates with tubulin and associates with both microtubules and microfilaments in intact cells. The cellular distribution is altered by treatment with taxol, nocodazole, and cytochalasin D. The tubulin binding domain was located at the N terminus of GABARAP by using synthetic peptides and deletion constructs and is marked by a specific arrangement of basic amino acids. The interaction between GABARAP and actin might be mediated by other proteins. These results demonstrate the GABARAP interacts with the cytoskeleton both in vitro and in cells and suggest a role of GABARAP in the interaction between GABA(A) receptors and the cytoskeleton. Such interactions are presumably needed for receptor trafficking, anchoring, and/or synaptic clustering. The structural arrangement of the basic amino acids present in the tubulin binding domain of GABARAP may aid in recognition of the potential of tubulin binding activity in other known proteins.

Actin Cytoskeleton↗
PubMed2000

A single glycine residue at the entrance to the first membrane-spanning domain of the gamma-aminobutyric acid type A receptor beta(2) subunit affects allosteric sensitivity to GABA and anesthetics.

Site-directed mutagenesis of the gamma-aminobutyric acid type A (GABA(A)) receptor beta(2) subunit has demonstrated that conversion of a conserved glycine residue located at the entrance to the first transmembrane domain into the homologous rho(1) residue phenylalanine alters the modulating effects of four different i.v. anesthetics: pentobarbital, alphaxalone, etomidate, and propofol. Using the baculovirus expression system in Spodoptera frugiperda 9 cells, anesthetic-induced enhancement of [(3)H]muscimol and [(3)H]flunitrazepam binding in receptors containing the beta(2)(G219F) point mutation displayed a significantly reduced efficacy in modulation by all four i.v. anesthetics tested. Furthermore, GABA(A) receptors containing the alpha(1)(G223F) point mutation also significantly decreased the maximal effect of etomidate- and propofol-induced enhancement of ligand binding. Conversely, the homologous point mutation in rho(1) receptors (F261G) changed the i.v. anesthetic-insensitive receptor to confer anesthetic modulation of [(3)H]muscimol binding. Consistent with the binding, functional analysis of pentobarbital-enhanced GABA currents recorded with whole-cell patch clamp demonstrated the beta(2)(G219F) subunit mutation eliminated the potentiating effect of the anesthetic. Similarly, propofol-enhanced GABA currents were potentiated less in alpha(1)beta(2)(G219F)gamma(2) receptors than in alpha(1)beta(2)gamma(2) receptors. Although ligand binding displayed comparable K(D) values for muscimol among wild-type, alpha(1)beta(2)gamma(2), and mutant receptors, patch-clamp recordings showed that alpha(1)beta(2)(G219F)gamma(2) receptors had a significantly more potent response to GABA than did alpha(1)beta(2)gamma(2) or alpha(1)(G223F)beta(2)gamma(2). The alpha(1)beta(2)(G219F)gamma(2) receptors also were more sensitive to direct channel activation by pentobarbital and propofol in the absence of GABA. These results suggest that the first transmembrane glycine residue on the beta(2) subunit may be important for conformational or allosteric interactions of channel gating by both GABA and anesthetics.

Allosteric Regulation↗
PubMed2000

Differential sensitivity of recombinant GABA(A) receptors expressed in Xenopus oocytes to modulation by topiramate.

PURPOSE: This study evaluated the modulatory effects of topiramate (TPM) on various subtypes of recombinant rat gamma-aminobutyric acid A (GABA(A)) receptors expressed in Xenopus oocytes. METHODS: Specific subunits of GABA(A) receptors were expressed in Xenopus oocytes. Voltage-clamp recordings of currents were performed after application of TPM (1-100 microM) to these oocytes in the presence or absence of GABA. RESULTS: In a concentration-dependent fashion, TPM (1-100 microM) reversibly inhibited GABA-evoked Cl- currents in oocytes expressing either alpha1beta2gamma2S and alpha2beta2gamma2S recombinant GABA(A) receptors and reduced the current-fading rate in alpha1beta2gamma2S-expressing oocytes. Topiramate was effective at GABA concentrations of 1-10 microM but not at 100 microM. Topiramate (1-100 microM) potentiated GABA-evoked Cl- currents and increased the fading rate in oocytes expressing the alpha6beta2gamma2S GABA(A) receptor. It had no effect on Cl- currents mediated through the alpha4beta2gamma2S receptor or through the mixed population of GABA(A) receptors expressed from rat brain mRNA. In general, the observed effects of TPM were more pronounced on fading rates than on peak Cl- currents. CONCLUSIONS: These results indicate that TPM may affect desensitization of GABA(A) receptors as assessed by changes in the fading rates of GABA-evoked Cl- currents, possibly by effects on second-messenger systems.

Animals↗
PubMed1999

Attenuated sensitivity to neuroactive steroids in gamma-aminobutyrate type A receptor delta subunit knockout mice.

gamma-Aminobutyric acid (GABA) type A receptors mediate fast inhibitory synaptic transmission and have been implicated in responses to sedative/hypnotic agents (including neuroactive steroids), anxiety, and learning and memory. Using gene targeting technology, we generated a strain of mice deficient in the delta subunit of the GABA type A receptors. In vivo testing of various behavioral responses revealed a strikingly selective attenuation of responses to neuroactive steroids, but not to other modulatory drugs. Electrophysiological recordings from hippocampal slices revealed a significantly faster miniature inhibitory postsynaptic current decay time in null mice, with no change in miniature inhibitory postsynaptic current amplitude or frequency. Learning and memory assessed with fear conditioning were normal. These results begin to illuminate the novel contributions of the delta subunit to GABA pharmacology and sedative/hypnotic responses and behavior and provide insights into the physiology of neurosteroids.

Animals↗
PubMed1999

Two invariant tryptophans on the alpha1 subunit define domains necessary for GABA(A) receptor assembly.

Two invariant tryptophan residues on the N-terminal extracellular region of the rat alpha1 subunit, Trp-69 and Trp-94, are critical for the assembly of the GABA(A) (gamma-aminobutyric acid, type A) receptor into a pentamer. These tryptophans are common not only to all GABA(A) receptor subunits, but also to all ligand-gated ion channel subunits. Converting each Trp residue to Phe and Gly by site-directed mutagenesis allowed us to study the role of these invariant tryptophan residues. Mutant alpha1 subunits, coexpressed with beta2 subunits in baculovirus-infected Sf9 cells, displayed high affinity binding to [(3)H]muscimol, a GABA site ligand, but no binding to [(35)S]t-butyl bicyclophosphorothionate, a ligand for the receptor-associated ion channel. Neither [(3)H]muscimol binding to intact cells nor immunostaining of nonpermeabilized cells gave evidence of surface expression of the receptor. When expressed with beta2 and gamma2 polypeptides, the mutant alpha1 polypeptides did not form [(3)H]flunitrazepam binding sites though wild-type alpha1 polypeptides did. The distribution of the mutant receptors on sucrose gradients suggests that the effects on ligand binding result from the inability of the mutant alpha1 subunits to form pentamers. We conclude that Trp-69 and Trp-94 participate in the formation of the interface between alpha and beta subunits, but not of the GABA binding site.

Amino Acid Sequence↗
PubMed1999

Synthesis and properties of 3-(2-hydroxyethyl)-3-n-pentyldiazirine, a photoactivable general anesthetic.

To overcome the difficulties of locating the molecular sites of general anesthetic action, we synthesized a novel photoactivable general anesthetic, 3-(2-hydroxyethyl)-3-n-pentyldiazirine (3-diazirinyloctanol), which anesthetized tadpoles with an ED(50) of 160 microM. Subanesthetic concentrations of 3-diazirinyloctanol enhanced GABA-induced currents in GABA(A) receptors, an effect that has been implicated in general anesthetic action. It also enhanced [(3)H]muscimol binding to this receptor. In muscle nicotinic acetylcholine receptors (nAcChoR), it inhibited the response to acetylcholine with an IC(50) of 33 microM. 3-Diazirinyloctanol's pharmacological actions were comparable to those of octanol. 3-(2-Hydroxyethyl)-3-[4,5-(3)H(2)]-n-pentyldiazirine photoincorporated into Torpedo nAcChoR-rich membranes mainly in the alpha subunit with 70% being in a proteolytic fragment containing the M4 transmembrane segment. Agonist enhanced the photolabeling 10-fold in a fragment containing the M1, M2, and M3 transmembrane segments. Thus, 3-diazirinyloctanol is a novel general anesthetic that acts on, and can be photoincorporated into, postsynaptic receptors.

Allosteric Regulation↗