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

Expression patterns of GABAA receptor subtypes in developing hippocampal neurons

Developing cultured hippocampal neurons were studied for the expression of GABAA receptor subunits using anti-peptide antibodies. The antibodies decorated the plasma membrane of cell bodies and all neurites in immature neurons, whereas the immunoreactivity was restricted to somata and dendrites in fully developed cells. Most receptor subunits were expressed throughout the entire culture period. However, the α1 and γ2 subunits were detected only late in culture, while α6 disappeared around this time. These findings were partly corroborated by in situ hybridization data in the developing rat hippocampus. Our results reveal a temporal control of gene expression for subsets of receptor subunits in hippocampal neurons and suggest a change in the subunit composition of GABAA receptors in the developing brain

Two novel GABAA receptor subunits exist in distinct neuronal subpopulations

Two cDNAs encoding novel GABAA receptor subunits were isolated from a rat brain library. These subunits, gamma 2 and delta, share approximately 35% sequence identity with alpha and beta subunits and form functional GABA-gated chloride channels when expressed alone in vitro. The gamma 2 subunit is the rat homolog of the human gamma 2 subunit recently shown to be important for benzodiazepine pharmacology. Cellular localization of the mRNAs encoding the gamma 2 and delta subunits in rat brain revealed that largely distinct neuronal subpopulations express the two subunits. The delta subunit distribution resembles that of the high affinity GABAA receptor labeled with [3H]muscimol; the gamma 2 subunit distribution resembles that of GABAA/benzodiazepine receptors labeled with [3H]flunitrazepam. These findings have implications for the composition of two different GABAA receptor subtypes and for information processing in networks using GABA for signaling

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

Polyethylenimine (PEI) is a simple, inexpensive and effective reagent for condensing and linking plasmid DNA to adenovirus for gene delivery

A simple and inexpensive method of condensing and linking plasmid DNA to carrier adenovirus particles is described. The synthetic polycation polyethylenimine is used to condense plasmid DNA into positively charged 100 nm complexes. These PEI–DNA complexes are then bound to adenovirus particles through charge interactions with negative domains on the viral hexon. The resulting transfection complexes deliver plasmid DNA to cells by the adenovirus infectious route without interference from virus gene expression because psoralen-inactivated virus is employed. The PEI–DNA–adenovirus complexes display DNA delivery comparable to more sophisticated DNA virus complexes employing streptavidin/biotin linkage, but require no special reagents and are much easier to prepare

Antigen processing and class II MHC peptide-loading compartments in human B-lymphoblastoid cells

The peptide/class II major histocompatibility complex (MHC) complexes recognized by CD4+ T cells have been characterized at the structural and biochemical levels and studies on the transport and maturation of class II MHC indicate that specialized sites may be involved in peptide acquisition. Here we report the characterization of the compartments involved in antigen processing and class II MHC loading relative to distinct functional domains of the endocytic pathway in antigen-specific human B lymphocytes. Peroxidase-mediated crosslinking analysis in intact cells demonstrates that peptide loading of class II MHC takes place in a compartment accessible to membrane immunoglobulin but not to transferrin receptors, although processing may be initiated within the latter domain. The density of membrane vesicles carrying newly assembled class II MHC complexes was distinct from early and late endosomes and dense lysosomes. Endocytosed antigen-gold complexes enter a class II MHC-rich compartment morphologically very similar to that described previously and within the time frame of biochemically detectable peptide loading