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E Sigel

Publications and source records attributed to E Sigel.

At least 37 records · Page 2Linked to original sources
PubMed1998

The antiepileptic drug AWD 131-138 stimulates different recombinant isoforms of the rat GABA(A) receptor through the benzodiazepine binding site.

Recombinant gamma-aminobutyric acid A (GABA(A)) receptors of the subunit compositions alpha1beta2gamma2, alpha1beta3gamma2, alpha2beta2gamma2, alpha3beta2gamma2 and alpha5beta2gamma2 were expressed in Xenopus oocytes in a functionally active form. At all subunit combinations, AWD 131-138 dose-dependently stimulated GABA currents. At 10 microM AWD 131-138, this allosteric stimulation amounted in average to about 12-21% of the maximal stimulation achieved using diazepam. The threshold of stimulation was about 0.3-1.0 microM. One micrometer of the benzodiazepine antagonist flumazenil (Ro 15-1788) counteracted the current stimulation by 10 microM AWD 131-138, indicating that this drug acts at the binding site for benzodiazepines.

Animals↗
PubMed1998

The benzodiazepine binding pocket of recombinant alpha1beta2gamma2 gamma-aminobutyric acidA receptors: relative orientation of ligands and amino acid side chains.

Wild-type alpha1beta2gamma2 gamma-aminobutyric acid (GABA)A receptors and receptors containing a point-mutated subunit gamma2F77Y were expressed by transient transfection in human embryonic kidney 293 cells. Mutant receptors bound the benzodiazepine binding site ligand [3H]flumazenil with similar, subnanomolar affinity as wild-type receptor. Displacement studies with diazepam showed that the affinity for this compound was reduced 250-fold on mutation, indicating that the tyrosine hydroxyl group interferes with diazepam binding. This differential behavior then was used to find the chemical entity presumably interacting with the phenyalanine residue in position 77 of the gamma2 subunit of wild-type receptors. Thirty-four substances were analyzed in this respect. Our results suggest that the phenyl substituent of diazepam is located close to gammaF77. Similarly, we investigated the possible location of alpha1T206 and gamma2M130. Electrophysiological data obtained with the wild-type receptor furthermore suggest a simple overlap between positive allosteric modulators acting at the benzodiazepine binding site with its antagonists.

Allosteric Regulation↗
PubMed1997

A point mutation in the gamma2 subunit of gamma-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity.

Benzodiazepines allosterically modulate gamma-aminobutyric acid (GABA) evoked chloride currents of gamma-aminobutyric acid type A (GABAA) receptors. Coexpression of either rat gamma2 or gamma3, in combination with alpha1 and beta2 subunits, results both in receptors displaying high [3H]Ro 15-1788 affinity. However, receptors containing a gamma3 subunit display a 178-fold reduced affinity to zolpidem as compared with gamma2-containing receptors. Eight chimeras between gamma2 and gamma3 were constructed followed by nine different point mutations in gamma2, each to the homologous amino acid residue found in gamma3. Chimeric or mutant gamma subunits were coexpressed with alpha1 and beta2 in human embryonic kidney 293 cells to localize amino acid residues responsible for the reduced zolpidem affinity. Substitution of a methionine-to-leucine at position 130 of gamma2 (gamma2M130L) resulted in a 51-fold reduction in zolpidem affinity whereas the affinity to [3H]Ro 15-1788 remained unchanged. The affinity for diazepam was only decreased by about 2-fold. The same mutation resulted in a 9-fold increase in Cl 218872 affinity. A second mutation (gamma2M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and gamma2M130L-containing receptors were functionally expressed in Xenopus oocytes. Upon mutation allosteric coupling between agonist and modulatory sites is preserved. Dose-response curves for zolpidem and for diazepam showed that the zolpidem but not the diazepam apparent affinity is drastically reduced. The apparent GABA affinity is not significantly affected by the gamma2M130L mutation. The identified amino acid residues may define part of the benzodiazepine binding pocket of GABAA receptors. As the modulatory site in the GABAA receptor is homologous to the GABA site, and to all agonist sites of related receptors, gamma2M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.

Animals↗
PubMed1997

Subtle changes in residue 77 of the gamma subunit of alpha1beta2gamma2 GABAA receptors drastically alter the affinity for ligands of the benzodiazepine binding site.

Recombinant alpha1beta2gamma2 gamma-aminobutyric acid type A (GABAA) receptors were functionally expressed in Xenopus oocytes. Upon the mutation F77L, diazepam and Ro 15-1788 retained the ability to interact with the benzodiazepine binding site, but zolpidem lost this ability. To quantify these data, radioligand binding experiments were performed using membrane preparations of transiently transfected human embryonic kidney 293 cells. The amino acid gamma77, phenylalanine, was also mutated to tyrosine, tryptophan, and isoleucine. Although there was little effect on Ro 15-1788 binding upon mutation to tyrosine, the loss in affinity for diazepam was from 12 to 2,720 nM. The change to leucine, in contrast, resulted in little change in the diazepam affinity, whereas there was a strongly reduced affinity for zolpidem from 17 to 4,870 nM and for methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) from 1.9 to 1,780 nM, respectively. The change to tryptophan resulted in two-phasic displacement curves, and only about 50% of the [3H]flunitrazepam binding could be displaced by zolpidem, DMCM, and Ro 15-1788, respectively, whereas midazolam and diazepam still resulted in 100% displacement, indicating the presence of two sites upon expression of this mutant receptor. Functional expression in Xenopus oocytes showed that all mutant channels displayed a comparatively small change (<4.3-fold) in their apparent agonist affinity and that these channels could still be functionally modulated by ligands of the benzodiazepine binding site. We conclude that subtle changes in gammaF77 drastically affect benzodiazepine pharmacology and that this residue probably interacts directly with most ligands of the benzodiazepine binding site and therefore defines part of the benzodiazepine binding pocket.

Animals↗
PubMed1997

The benzodiazepine binding site of GABAA receptors.

The GABAA receptor belongs, along with the nicotinic acetylcholine receptor, the glycine receptor and the 5-HT3 receptor, to a family of homologous transmitter-gated ion channels mediating fast synaptic transmission. Many classes of drug interact with the GABAA receptor, which is the major inhibitory ion channel in the mammalian brain. Among these drugs are the allosteric modulators acting at the benzodiazepine binding site. In this article, Erwin Sigel and Andreas Buhr discuss recent studies that have identified amino acid residues that are thought to form the binding pocket for these compounds. These residues are probably located at subunit interfaces of the protein pentamer and at least some of them are homologous to residues implicated in channel agonist binding. This implies pseudosymmetry of channel agonist and channel modulatory sites, which may be, as recent data indicate, a general principle realized in other pseudosymmetric protein complexes.

Amino Acid Sequence↗
PubMed1997

Proteins associated with alpha 1-subunit-containing GABAA receptors from bovine brain.

In contrast to some other ion channels, there are at present no proteins known that bind specifically to mature GABAA receptor channels. Such proteins may be important for the structural organization and cytoskeletal anchoring of GABAA receptors and could also be expected to have channel modulatory effects. To identify proteins that are associated with naturally occurring GABAA receptors we immunoprecipitated these receptors from detergent-solubilized bovine brain membranes by an antibody directed against the alpha 1-subunit. Tubulin and actin were observed to coprecipitate specifically with the receptors. Nine additional proteins were detected, hinting at a complex protein network associated with alpha 1-subunit-containing GABAA receptors. Results of a biochemical characterization of these GABAA receptor-tubulin complex-associated proteins (GTAPs) are presented here. Peptide mass fingerprinting analysis and microsequencing of tryptic peptides indicated that at least three GTAPs have not been described until the present.

Actins↗
PubMed1997

Residues at positions 206 and 209 of the alpha1 subunit of gamma-aminobutyric AcidA receptors influence affinities for benzodiazepine binding site ligands.

Ligands of the benzodiazepine binding site allosterically modulate gamma-aminobutyric acidA receptors. Their binding pocket is made up of amino acid residues located on both alpha and gamma subunits. We transiently expressed wild-type alpha1beta2gamma2 and mutant GABAA receptors in human embryonic kidney 293 cells and determined their binding properties. Receptors containing the mutant alphaY209A showed approximately 40-fold decrease in affinity for [3H]Ro 15-1788 and diazepam, whereas zolpidem displayed no measurable affinity. Receptors containing the mutant alphaY209F showed a small-to-moderate decrease in affinity for [3H]Ro 15-1788, diazepam, zolpidem, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, and Cl 218872, amounting to 2-8-fold. Receptors containing the mutant alphaY209Q appeared in the surface membrane of transfected cells, bound [3H]muscimol with wild-type affinity, but failed to bind [3H]Ro 15-1788 or [3H]flunitrazepam with detectable affinity. If these mutant receptors were expressed in Xenopus laevis oocytes, the apparent affinity for GABA was only slightly decreased, whereas the ability of the currents to be stimulated by low concentrations of flunitrazepam was abolished. Receptors containing a point mutant of another amino acid residue, alphaT206A, surprisingly showed an increase in affinity of 5- and 16-fold, for the negative allosteric modulator methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate and the partial positive allosteric modulator Cl 218872, respectively, whereas there was only a small decrease in affinity for Ro 15-1788, diazepam, and zolpidem, amounting to 2-, 4-, and 5-fold. Both alpha206 and alpha209 are thus both important in determining the binding affinities for ligands of the benzodiazepine binding site. The residues are spaced at an interval of three amino acids and may be part of an alpha helix.

Animals↗
PubMed1996

Subunit stoichiometry of oligomeric membrane proteins: GABAA receptors isolated by selective immunoprecipitation from the cell surface.

GABAA receptors are hetero-oligomeric proteins of unknown subunit stoichiometry. In this study alpha 1 beta 3 GABAA receptor channels were functionally expressed in Xenopus oocytes. Direct immunoprecipitation from the oocyte surface was used to exclusively isolate mature GABAA receptors. The subunit ratio was determined by quantitation of the amount of [35S]methionine incorporated into individual receptor subunits. Antibody released from the antigen or antibody not reacted was prevented from reassociation with labeled antigen by addition of excess unlabeled antigen. Variation of the alpha 1 beta 3 ratio of injected cRNAs only slightly affected the subunit ratio in mature receptors. This indicates that the subunit stoichiometry generated is independent of the pools of newly synthesized subunit monomers and supports the view that the receptor assembly is a regulated process. The ratio of alpha 1/beta 3 subunits was found to be 1.1 +/- 0.1 (SEM, n = 6). Our data are in best agreement with a tetrameric receptor with the composition 2 alpha 2 beta. For a pentameric receptor the ratio found slightly favors a receptor with the composition 3 alpha 2 beta. The method developed here is applicable to the determination of the subunit stoichiometry of other recombinant oligomeric membrane proteins.

Animals↗
PubMed1996

Point mutations of the alpha 1 beta 2 gamma 2 gamma-aminobutyric acid(A) receptor affecting modulation of the channel by ligands of the benzodiazepine binding site.

Clinically relevant benzodiazepines allosterically stimulate neurotransmitter-evoked chloride currents at the gamma-aminobutyric acid type A(GABAA) receptor. Rat wild-type or mutated alpha 1, beta 2, and gamma 2S subunits were coexpressed in Xenopus oocytes and investigated with electrophysiological techniques. Point mutations in two subunits were identified that affect the response of gamma-aminobutyric acid (GABA)-induced currents by benzodiazepines. Mutation of one of three amino acid residues to alanine (alpha Tyr161 and alpha Thr206) or leucine (gamma Phe77) resulted in a approximately 3-fold increase in potentiation by diazepam. The response to zolpidem was increased in two mutant channels containing the mutated alpha subunit but was nearly absent in channels containing the mutated gamma subunit. In the former cases, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) acted as a negative allosteric modulator of the channel, much stronger than in the wild-type channel, whereas there was no significant difference to the wild-type channel in the latter case. Thus, the mutant gamma subunit has different functional consequences for the various types of ligand of the benzodiazepine binding site. All three amino acid residues, alpha Tyr161, alpha Thr206, and gamma Phe77, are close or identical to homologous residues that are implicated in GABA binding. If the residues binding the channel agonist GABA are located at subunit interfaces, the residues influencing the benzodiazepine effects must also be located at subunit interfaces.

Amino Acid Sequence↗
PubMed1995

Cellular prion protein and GABAA receptors: no physical association?

The so-called prion diseases are probably caused by the conformational conversion of the cellular prion protein (PrPc) into an abnormal, pathological form (PrPsc). PrPc is widely expressed in neuronal tissues, but its function is not known. From electrophysiological measurements in prion-less mice it was proposed that PrPc may contribute to the structural integrity of central synapses containing gamma-aminobutyric acid type A (GABAA) receptors. We tried to substantiate this hypothesis by obtaining evidence for a structural link between the GABAA receptor and PrPc. Preparations of PrPc and GABAA receptors, respectively, from cow brain were analysed for PrPc-GABAA receptor complexes. No evidence for such complexes could be obtained in our experiments, although the protein purification schemes used should favour the preservation of intermolecular linkages. We conclude that further data concerning interactions of PrPc with other proteins are needed to obtain insight into its normal functional role.

Animals↗
PubMed1994

Protein kinase C transiently activated heteromeric N-methyl-D-aspartate receptor channels independent of the phosphorylatable C-terminal splice domain and of consensus phosphorylation sites.

We have expressed dual subunit combinations of isoforms of the N-methyl-D-aspartate receptor, NR1A-NR2A and NR1C-NR2A, in Xenopus oocytes. We show that both forms of the receptor are stereospecifically activated by low concentrations (10 nM) of the phorbol ester 4-beta-phorbol 12-myristate 13-acetate, known to activate protein kinase C (PKC). The activation is transient, and, after reaching a maximum in about 10 min, it decreases rapidly in spite of the continuous presence of phorbol ester. The addition of 2 microM oleoylacetylglycerol had similar consequences. NR1C differs from NR1A by a deletion of 37 amino acids that include four consensus phosphorylation sites for PKC in the C-terminal region. The corresponding peptide has been shown to become phosphorylated upon activation of PKC in neurons (Tingley, W. G., Roche, K. W., Thompson, A. K., and Huganir, R. L. (1993) Nature 364, 70-73). However, the activity of NR1C-NR2A receptors was stimulated 7-fold, twice the potentiation observed for NR1A-NR2A. By site-specific mutagenesis of NR1C and NR2A, we removed additional consensus PKC phosphorylation sites located between TM3 and TM4. Coexpression of these mutant subunits showed a similar response to phorbol esters as wild type receptors. Our results indicate that neither the predicted consensus phosphorylation sites between transmembrane sequences TM3 and TM4 nor the phosphorylatable C-terminal splice domain is essential for the modulation of N-methyl-D-aspartate receptors by PKC.

Amino Acid Sequence↗
PubMed1993

Recombinant GABAA receptor function and ethanol.

Different combinations of cloned subunits of the rat brain GABAA receptor were expressed in Xenopus oocytes. Possible effects of ethanol on the expressed GABA-induced chloride current were determined. The consequence of replacing the gamma 2S subunit by the alternatively spliced variant gamma 2L was specifically tested on the responsiveness to ethanol. A significant stimulation of the GABA response was only observed at very high concentrations (> 60 mM) of ethanol. No differential response was observed between subunit combinations containing different gamma 2 subunit splice variants.

Alternative Splicing↗
PubMed1992

Function of the alpha 1 beta 2 gamma 2S gamma-aminobutyric acid type A receptor is modulated by protein kinase C via multiple phosphorylation sites.

Activation of protein kinase C (PKC) results in down-modulation of the gamma-aminobutyric acid type A (GABAA) receptor. In this study, the recombinant subunit combination alpha 1 beta 2 gamma 2S was expressed in Xenopus oocytes. The resulting channel was shown to be modulated by 2 microM oleoylacetylglycerol or, stereo-specifically, by low concentrations (10 nM) of the phorbol ester 4 beta-phorbol 12-myristate 13-acetate. By site-specific mutagenesis, we altered the serine or threonine residues of consensus phosphorylation sites for PKC in the large, intracellular domain of alpha 1, beta 2, and gamma 2S. Mutant subunits were co-expressed with wild type subunits to yield alpha 1 beta 2 gamma 2S combinations. All of the tested 14 mutations did not affect the level of expression of GABA current. Two of these mutations, Ser-410 in beta 2 and Ser-327 in gamma 2S, resulted in a significant reduction of the effect of the activator of PKC, 4 beta-phorbol 12-myristate 13-acetate, on the GABA current amplitude. Thus, we have identified two single serine residues, Ser-410 in the subunit beta 2 and Ser-327 in gamma 2S, as phosphorylation sites of a PKC endogenous to Xenopus oocytes. Co-expression of the mutant subunits suggests that phosphorylation of both sites is required for a full, PKC-mediated down-regulation of GABA currents.

Animals↗
PubMed1992

Point mutations affecting antagonist affinity and agonist dependent gating of GABAA receptor channels.

Two variant amino acid sequences, which differ in a single amino acid residue, have been reported for the alpha 1-subunit of the rat brain GABAA receptor. We separately co-expressed these two variants in Xenopus oocytes, in combination with beta 2 and gamma 2. This experiment showed that substitution of alpha 1-Phe64 by Leu strongly decreases the apparent affinity for GABA dependent channel gating from 6 microM to 1260 microM. Starting from this observation, we used in vitro mutagenesis to obtain information relevant for the localization of the agonist/antagonist binding site in the GABAA receptor. Homologous mutation in alpha 5 had similar consequences for alpha 5 beta 2 gamma 2. Homologous mutation in beta 2 and gamma 2 resulted in intermediate and small shifts in EC50, respectively. The apparent affinities of the competitive antagonists bicuculline methiodide and SR95531, the latter sharing close structural similarity with the agonist GABA, were decreased 60- to 200-fold by these mutations in alpha-subunits. Interestingly, these affinities remained nearly unaffected upon introduction of the homologous mutations in beta 2 and gamma 2, or upon mutation of the neighbouring amino acid in alpha 1, Phe65 to Leu. These results suggest close functional and structural association of alpha-subunits with the agonist/antagonist binding site, and involvement of N-terminal portions of the extracellular domains of all subunits in the gating of the channel.

Amino Acid Sequence↗
PubMed1991

The gamma 3-subunit of the GABAA-receptor confers sensitivity to benzodiazepine receptor ligands.

The gamma 3-subunit of the GABAA-receptor in rat brain has been identified by molecular cloning. When co-expressed with the alpha 5- and beta 2-subunits in transfected cells a high potency for GABA (Ka = 4.9 +/- 1.2 microM) and a strong cooperativity in gating the channel (H = 1.9 +/- 0.2) was observed. The GABA response was potentiated in the presence of flunitrazepam and reduced by beta CCM. An analogous bi-directional modulation of the GABA response was observed with diazepam and DMCM as tested with the subunit combinations alpha 1 beta 2 gamma 3 and alpha 3 beta 2 gamma 3 expressed in Xenopus oocytes. Since the benzodiazepine receptor ligands were virtually inactive in the absence of the gamma 3-subunit, as tested with the alpha 3 beta 2- and alpha 5 beta 2-subunit combinations, the gamma 3-subunit is a prerequisite for the benzodiazepine receptor sensitivity of the expressed GABAA-receptors. The gamma 3-subunit could functionally replace the gamma 2-subunit with regard to the bi-directional allosteric drug modulation.

Amino Acid Sequence↗
PubMed1991

Activation of protein kinase C results in down-modulation of different recombinant GABAA-channels.

Different combinations of cloned rat brain subunits of the GABAA receptor were expressed in Xenopus oocytes. The effect of the phorbol ester PMA, an activator of protein kinase C, on the expressed GABA-gated ion current was determined. Ion currents were diminished by beta-PMA, but not by the control substance alpha-PMA, irrespective of the subunit combination studied. The mechanism of current decrease was investigated in more detail for the subunit combination alpha 5 beta 2 gamma 2. The reversal potential of the current remained unaffected, while the maximal current amplitude was decreased and the apparent Ka for GABA-dependent channel gating was shifted to higher concentrations.

Animals↗
PubMed1991

Ionic channels: modulation by G proteins and by phosphorylation.

The gating of ion channels may be modulated by G proteins or by phosphorylation. Direct coupling between G proteins and ion channels has been shown in excised patches of membrane. Steps must now be taken to study the protein domains of G proteins and ion channels involved in the mutual interaction. The concept of channel modulation by protein kinases has recently been extended to include additional types of ion channel.

Animals↗