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H Lüddens

Publications and source records attributed to H Lüddens.

54 records · Page 3Linked to original sources
PubMed1994

Impact of beta and gamma variants on ligand-binding properties of gamma-aminobutyric acid type A receptors.

We expressed in cultured cells recombinant gamma-aminobutyric acid type A (GABAA) receptors of the subunit compositions alpha 1 beta j gamma k and alpha 5 beta j gamma k (j = 1, 2, or 3 and k = 2 or 3). A comparison of ligand-binding properties revealed a functional role for individual beta variants, which depended on the alpha subunit in the GABAA receptor. Recombinant alpha 5 beta x gamma 2/3 receptors recognized the cage convulsant t-butylbicyclophosphoro[35S]thionate, as well as the benzodiazepine (BZ) receptor inverse agonist [3H]Ro 15-4513, only with the beta 3 variant. In contrast, the exchange of beta variants in alpha 1 beta x gamma 2 receptors imparted differential modulation of t-butyl-bicyclophosphoro[35S]thionate binding by BZ receptor ligands. The BZ site of gamma 3-containing receptors was partially independent of the accompanying alpha and beta variants. alpha 1/5 beta 3 gamma 3 receptors were zolpidem insensitive but distinguished from alpha 5 beta 3 gamma 2 receptors by high affinity for the partial BZ receptor agonist CI 218,872. The distinct affinities of recombinant receptors for CI 218,872 suggested that the alpha 5 beta 3 gamma 2 receptor is the dominant zolpidem-insensitive GABAA receptor in the brain. Hence, alpha 5 beta 3 gamma 3 recpetors are not a major fraction of the native zolpidem-insensitive receptors, even though their genes are colocalized on mouse chromosome 7 and on human chromosome 15.

Azides↗
PubMed1993

Regional gamma-aminobutyric acid sensitivity of t-butylbicyclophosphoro[35S]thionate binding depends on gamma-aminobutyric acidA receptor alpha subunit.

gamma-Aminobutyric acid (GABA) modulates the convulsant binding site on GABAA receptors labeled by t-butylbicyclophosphoro[35S] thionate ([35S]TBPS). The modulation varies between different brain regions, reflecting the molecular heterogeneity of the GABAA receptors. In rat brain cryostat sections, the main sensitivity difference to GABA between brain regions was observed within the cerebellum. [35S]TBPS binding in the granule cell layer was more sensitive to GABA than was that in the molecular layer and was detected only after blockade of the GABA agonist sites by the specific GABAA antagonists SR 95531, RU 5135, and bicuculline. This indicates that the [35S]TBPS binding sites in cerebellar granule cells were blocked by endogenous GABA. In contrast, the internal rim of the granule cell layer had a small amount of binding that was largely insensitive to 50 microM GABA. The molecular basis for the sensitivity difference could be traced to the alpha subunits of the GABAA receptor. Expression in human embryonic kidney 293 cells of alpha 6 beta 2 gamma 2 receptors produced [35S] TBPS binding sites that were about 10-fold more sensitive to inhibition by GABA than were those inherent to alpha 1 beta 2 gamma 2 receptors. Coexpression of alpha 6 and beta 2 subunits produced [35S]TBPS binding sites that were largely insensitive to GABA inhibition, resembling in their pharmacological profile the sites in the internal granule cell layer. Furthermore, the differences between alpha 6 beta 2 and alpha 6 beta 2 gamma 2 receptors stress the importance of the gamma 2 subunit for the proper pharmacological fingerprint of the rest of the granule cell layer. The neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one affected the binding in both alpha 1 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptors, but inhibition was greater in alpha 6-containing than in alpha 1-containing receptors, suggesting differential coupling of both GABA and neurosteroid sites with the convulsant site. These data might serve as a platform for additional studies to assess the amino acid residues in the two alpha subunits that are critically involved in the allosteric interactions between the GABAA agonist/antagonist or neurosteroid domains and the convulsant site.

Animals↗
PubMed1992

GABAA antagonists reveal binding sites for [35S]TBPS in cerebellar granular cell layer.

Autoradiography revealed picrotoxin-sensitive [35S]TBPS ([35S]t-butylbicyclophosphorothionate) binding sites in the cerebellar granule cell layer only in the presence of GABAA receptor antagonists. However, under the same conditions, the binding was decreased in other brain regions, including the cerebellar molecular cell layer. Thus, the convulsant binding sites at GABAA receptors in the granule cell layer are highly sensitive to down-regulation by GABA, which may be due to a unique combination of receptor subunits.

Androstanes↗
PubMed1992

The third gamma subunit of the gamma-aminobutyric acid type A receptor family.

Cloned cDNAs encoding a member of the gamma-aminobutyric acid type A receptor gamma-subunit class were isolated from rat-brain-mRNA-derived libraries. The gamma 3 mRNA is present in cortex, claustrum, caudate putamen, and some thalamic nuclei, particularly the medial geniculate nucleus, where it is the predominant gamma-subunit transcript. The gamma 3 gene is expressed at very low levels in cerebellum and hippocampus. In coexpression experiments with the alpha 1 and beta 2 subunits, gamma 3 imparts benzodiazepine binding to gamma-aminobutyric acid type A receptors and forms gamma-aminobutyric acid-gated benzodiazepine-modulated chloride channels that exhibit a larger conductance than alpha 1 beta 2 receptor channels. Furthermore, the presence of gamma 3 in place of gamma 2 in alpha 1 beta 2 gamma x receptors generates a marked decrease in the affinity of agonists while leaving the affinity of antagonists or negative modulators largely unaffected.

Amino Acid Sequence↗
PubMed1992

A single histidine in GABAA receptors is essential for benzodiazepine agonist binding.

Benzodiazepines (BZ) modulate neurotransmitter-evoked chloride currents at the gamma-aminobutyric acid type A (GABAA) receptor, the major inhibitory ion channel in the mammalian brain. This receptor is composed of structurally distinct subunits whose numerous molecular variants underlie the observed diversity in the properties of the BZ site. Pharmacologically distinct BZ sites can be recreated by the recombinant coexpression of any one of six alpha subunits, a beta subunit variant, and the gamma 2 subunit. In these receptors the alpha variant determines the affinity for ligand binding of the BZ site. Notably, the alpha 1 and alpha 6 variants impart on alpha chi beta 2 gamma 2 receptors high and negligible affinity, respectively, to BZ ligands with sedative as well as anxiolytic activities. By exchanging domains between the alpha 1 and alpha 6 variants, we show that a portion of the large extracellular domain determines sensitivity toward these ligands. Furthermore, we identify a single histidine residue in the alpha 1 variant, replaced by an arginine in alpha 6, as a major determinant for high affinity binding of BZ agonists. This residue also plays a role in determining high affinity binding for BZ antagonists. Hence, this histidine present in the alpha 1, alpha 2, alpha 3, and alpha 5 subunits appears to be a key residue for the action of clinically used BZ ligands.

Affinity Labels↗
PubMed1991

Cloning, pharmacological characteristics and expression pattern of the rat GABAA receptor alpha 4 subunit.

A cDNA of rat brain encoding the GABAA receptor alpha 4 subunit has been cloned. Recombinant receptors composed of alpha 4, beta 2 and gamma 2 subunit bind with high affinity the GABA agonist [3H]muscimol and the benzodiazepine 'alcohol antagonist' [3H]Ro 15-4513, but fail to bind benzodiazepine agonists. The alpha 4 subunit is expressed mainly in the thalamus, as assessed by in situ hybridization histochemistry, and may participate in a major population of thalamic GABAA receptors. The alpha 4 mRNA is found at lower levels in cortex and caudate putamen, and is rare in cerebellum.

Affinity Labels↗
PubMed1991

More than one alpha variant may exist in a GABAA/benzodiazepine receptor complex.

GABAA/benzodiazepine receptors are formed by the assembly of presumably five polypeptides with unknown stoichiometry. Six alpha, three beta, two gamma, and one delta subunit have been characterized on the molecular level. In analogy to the nicotinic acetylcholine receptor, and supported by functional analysis of recombinantly expressed GABAA receptor subunits, a structure containing at least three different polypeptides has been proposed for the functional GABAA and benzodiazepine regulated Cl(-)-channel. Using an alpha 1 subunit specific antiserum we could show that additional alpha variants are present in alpha 1 subunit containing GABAA/Benzodiazepine receptor complexes. This suggests that the diversity of GABAA/Benzodiazepine receptors may be larger than previously thought.

Animals↗
PubMed1990

Cerebellar GABAA receptor selective for a behavioural alcohol antagonist.

Benzodiazepines are widely prescribed anxiolytics and anticonvulsants which bind with high affinity to sites on the GABAA receptor/Cl- channel complex and potentiate the effect of the neurotransmitter GABA (gamma-aminobutyric acid). The heterogeneity of benzodiazepine recognition sites in the central nervous system was revealed by studies showing different classes of GABAA receptor subunits (classes alpha, beta and gamma) and variant subunits in these classes, particularly in the alpha-class. Expression of recombinant subunits produces functional receptors; when certain alpha-variants are coexpressed with beta- and gamma-subunits the resulting receptors have pharmacological properties characteristic of GABAA-benzodiazepine type I or type II receptors. The alpha-variants are differentially expressed in the central nervous system and can be photoaffinity-labelled with benzodiazepines. Here we report a novel alpha-subunit (alpha 6) of cerebellar granule cells. We show that recombinant receptors composed of alpha 6, beta 2 and gamma 2 subunits bind with high affinity to the GABA agonist [3H]muscimol and the benzodiazepine [3H]Ro15-4513 but not the other benzodiazepines or beta-carboniles. The same distinctive pharmacology is observed with GABAA receptors from rat cerebellum immunoprecipitated by an antiserum specific for the alpha 6 subunit. We conclude that this alpha-subunit is part of a cerebellar receptor subtype, selective for Ro15-4513, an antagonist of alcohol-induced motor incoordination and ataxia.

Amino Acid Sequence↗
PubMed1990

Subunit selectivity and epitope characterization of mAbs directed against the GABAA/benzodiazepine receptor.

mAbs bd 17, bd 24, and bd 28 raised against bovine cerebral gamma-aminobutyric acid (GABAA)/benzodiazepine receptors were analyzed for their ability to detect each of 12 GABAA receptor subunits expressed in cultured mammalian cells. Results showed that mAb bd 17 recognizes epitopes on both beta 2 and beta 3 subunits while mAb bd 24 is selective for the alpha 1 subunit of human and bovine, but not of rat origin. The latter antibody reacts with the rat alpha 1 subunit carrying an engineered Leu at position four, documenting the first epitope mapping of a GABAA receptor subunit-specific mAb. In contrast to mAbs bd 17 and bd 24, mAb bd 28 reacts with all GABAA receptor subunits tested but not with a glycine receptor subunit, suggesting the presence of shared epitopes on subunits of GABA-gated chloride channels.

Amino Acid Sequence↗
PubMed1989

Type I and type II GABAA-benzodiazepine receptors produced in transfected cells.

GABAA (gamma-aminobutyric acid A)-benzodiazepine receptors expressed in mammalian cells and assembled from one of three different alpha subunit variants (alpha 1, alpha 2, or alpha 3) in combination with a beta 1 and a gamma 2 subunit display the pharmacological properties of either type I or type II receptor subtypes. These receptors contain high-affinity binding sites for benzodiazepines. However, CL 218 872, 2-oxoquazepam, and methyl beta-carboline-3-carboxylate (beta-CCM) show a temperature-modulated selectivity for alpha 1 subunit-containing receptors. There were no significant differences in the binding of clonazepam, diazepam, Ro 15-1788, or dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) to all three recombinant receptors. Receptors containing the alpha 3 subunit show greater GABA potentiation of benzodiazepine binding than receptors containing the alpha 1 or alpha 2 subunit, indicating that there are subtypes within the type II class. Thus, diversity in benzodiazepine pharmacology is generated by heterogeneity of the alpha subunit of the GABAA receptor.

Cell Line↗
PubMed1986

Characterization of the porcine ACTH receptor with the aid of a monoclonal antibody.

Monoclonal antibodies were raised against the ACTH receptor by immunization of BALB1 c mice with porcine adrenal cortex cell membranes. Competition and binding experiments confirmed that one of these antibodies, IV/14/9, reacted with the ACTH receptor in competition with ACTH and caused a definite ACTH-like effect. This antibody was used to characterize the hormone receptor of the porcine adrenal cortex. The number of antibody binding sites per cell was calculated from a Scatchard plot to be 124,100 +/- 10,000. The curve was linear indicating the existence of a single class of receptors. The finding that a high concentration of IV/14/9 totally suppressed maximally ACTH-induced steroidogenesis confirms the view that only a single class of ACTH receptors exists. The antibody IV/14/9 neither reacted with intact porcine thymus cells nor with normal porcine lymphocytes but was bound to the mouse adrenal tumor cell line Y1 and to normal rat adrenal cortex cells with a low affinity. Two dimensional electrophoresis of lysates of porcine adrenal cortex cells and subsequent blotting of the proteins on nitrocellulose, using IV/14/9 as a primary and anti-mouse Ig as a secondary reagent, permitted the detection of three forms of the receptor, two of which had an identical apparent molecular mass of about 85,000 Da (isoelectric points: 6.14 and 6.27). The third was somewhat larger (94,000 Da and pI = 6.21).

Adrenal Cortex↗
PubMedDate not supplied

Biological function of GABAA/benzodiazepine receptor heterogeneity.

gamma-Aminobutyric acid (GABA) is the most prominent of the inhibiting neurotransmitters in the brain. It exerts its main action through GABAA receptors. The receptors respond to the presence of GABA by the opening of an intrinsic anion channel. Hence, they belong to the molecular superfamily of ligand-gated ion channels. There exist in the brain multiple GABAA receptors that show differential distribution and developmental patterns. The receptors presumably form by the assembly of five proteins from at least three different subunits (alpha 1-6, beta 1-3 and gamma 1-3). The regulation of functional properties by benzodiazepine (BZ) receptor ligands, neurosteroids, GABA and its analogs differs dramatically with the alpha variant present in the complex. Additional variation of the GABAA receptors comes with the exchange of the gamma subunits. No clear picture exists for the role of the beta subunits, though they may play an important part in the sensitivity of the channel-receptor complex. The effects of BZ receptor ligands on animal behavior range from agonist effects, e.g. anxiolysis, sedation, and hypnosis, to inverse agonist effects, e.g. anxiety, alertness, and convulsions. The diversity of effects reflects the ubiquity of the GABAA/BZ receptors in the brain. Recent data provide some insight into the mechanism of action of BZ ligands, but no clear delineation can be drawn from a single ligand to a single behavioral effect. This may be due to the fact that intrinsic efficacies of the ligands differ between receptor subtypes, so that the diversity of native receptors is further complicated by the diversity of the mode the ligands act on GABAA receptor subtypes. The behavioral actions of alcohol (ethanol) are similar to those produced by GABAA receptor agonists. In agreement, alcohol-induced potentiation of GABAergic responses has often been observed at behavioral, electrophysiological and biochemical levels. Thus, there is clearly a GABAA-dependent component in the actions of alcohol. However, the site and mode of action of ethanol on GABAA/BZ receptors remain controversial.

Alcohol Drinking↗