Pharmacological and Biochemical Properties of the γ‐Aminobutyric Acid–Benzodiazepine Receptor Protein from Codfish Brain

Abstract

The gamma-aminobutyric acidA (GABAA) receptor of codfish brain has been purified to homogeneity and contains a single polypeptide band of 56 kDa molecular mass. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) of codfish GABA receptor photoaffinity-labeled by both [3H]flunitrazepam ([3H]Flu) and [3H]muscimol showed a single radioactive peak with molecular mass of 56 kDa, in contrast to the multiple subunits found in other vertebrate species. The codfish receptor, purified using benzodiazepine (BZ, Ro 7-1986/1) affinity chromatography, contains an apparent single band both by isoelectric focussing and on a silver-stained SDS gel. The receptor density and affinity constants for [3H]muscimol and [3H]Flu binding are comparable to those in mammalian brain, and the specific activity (greater than 1,000 pmol/mg of protein) is comparable to that of preparations purified from those sources. The pharmacological specificity of the codfish GABA-BZ receptor is generally similar to that of mammalian brain, including GABA-BZ coupling. The BZ binding exhibits homogeneous kinetic properties resembling those of the mammalian BZ2 receptor type, and shows strong GABA enhancement of [3H]Flu binding and weaker pentobarbital potentiation. This is consistent with other observations of an earlier phylogenetic, as well as ontogenetic, emergence in mammals of the BZ2 receptor subtype than the BZ1. Codfish GABA receptor is postulated to be a homo-oligomer in which the conformation of GABA and BZ recognition sites is very similar to that in the mammalian hetero-oligomeric GABAA receptor. The codfish receptor appears to be encoded by an ancestral gene and indicates an early development of BZ-GABA coupling.

Distribution and Pharmacological Properties of the GABAA/ Benzodiazepine/Chloride Ionophore Receptor Complex in the Brain of the Fish Anguilla anguilla

“…At variance with this study is the finding of Deng et al (1987) on the presence in both fish and mammalian brain of a major radioactive band of 53 kD after photoaffinity labeling with [3H]FNT. However, the same authors, by using irradiation inactivation analysis, have also found important variations between fishes and mammals in the molecular weight of GABA and benzodiazepine receptors (Deng et al, 1987). These findings are consistent with the view that relevant functional and biochemical differences in the GABAA receptor complex may exist between fishes and mammals.…”

“…In fact, photoaffinity labeling of brain membranes with [3H]flunitrazepam ([3H]FNT) followed by sodium dodecyl sulfate-gel electrophoresis and fluorography has shown mainly a single protein band of 55 kilodaltons (kD) in fishes and two major bands in mammals (Hebebrand et al, 1987). At variance with this study is the finding of Deng et al (1987) on the presence in both fish and mammalian brain of a major radioactive band of 53 kD after photoaffinity labeling with [3H]FNT. However, the same authors, by using irradiation inactivation analysis, have also found important variations between fishes and mammals in the molecular weight of GABA and benzodiazepine receptors (Deng et al, 1987).…”

“…Therefore, it may be proposed that the development of the full functional relationship between the different components of the GABAA receptor complex may require further evolutionary steps from lower to higher vertebrates. Accordingly, the works of Hebebrand et al (1987) and Deng et al (1987) have shown differences in the subunit composition of the GABAA receptor complex in the eel and the rat brain. This finding may explain the differences in the biochemical properties of the GABAA receptor complex observed in the present study.…”

Differential distribution of GABAA receptor subunits in soma and processes of cerebellar granule cells: Effects of maturation and a GABA agonist

“…1l In cerebellum, the receptor complexes most likely consist of at least one a subunit together with [3 and ~/subunits. 1,17,20,43,47 Notably, it has been shown that a, 13 and ~/subunits are necessary to form GABAA receptors with the classical functional and regulatory properties. 48 However, the exact subunit composition forming functionally pentameric receptor complexes is still a matter of debate.…”

Effect of avermectin (AVM) on the expression of γ -aminobutyric acid A receptor (GABAA R) in Carassius gibelio (Bloch, 1782)

“…These processes attenuate sensitivity to excitatory or inhibitory signal transfer reactions in insects, affect normal neural activities, and eventually cause paralysis and death (Wang, ). While the effects of AVM on small animals such as mice (Soderlund et al., ; Krflgek and Zemkovfi, ), rats (Abalis et al., ), guinea pigs (Coccini et al., ), finfish (Deng et al., ; Lumaret et al., ), and mammals (Robertson, ) and the distribution of GABA A R in C. gibelio have been studied (Ruan et al., ), the effects of AVM on C. gibelio remain unknown. Therefore, this paper investigated the effects of different medium lethal concentrations of AVM on the protein expression of GABA A R in C. gibelio by Western blot and immunohistochemistry methods.…”