Responses of γ-aminobutyrate receptor from rat brain: Similarity of different preparation methods; muscimol induced desensitization and chloride exchange

Abstract

Chloride-36 exchange into three different membrane vesicle preparations from rat brain homogenate was followed. The different preparations all contained the same sealed vesicular components characterized by their rates of chloride exchange. The GABA-mediated³⁶Cl⁻ exchange in all the preparations occurred in two phases shown to be mediated by two distinguishable receptors present in the activity ratio of 5∶1 as previously described (Cash, D.J., Subbarao, K. 1987.Biochemistry 26:7556, 7562). Reported differences do not result from differences in the membrane preparations used or from the use of a GABA-mimetic instead of GABA, but from experimental differences. The preparations compared were made with mild or vigorous homogenization and with different extents of purification from solutes or membrane components: (i) a synaptoneurosome preparation, (ii) a Ficoll gradient preparation, and (iii) a washed P₂ preparation. In each preparation the same four populations of membrane vesicles were characterized by their³⁶Cl⁻ influx rates: (i) a major population (40–50%) (t _1/2=1.4 min), (ii) a slower exchanging major population (40–55%) (t _1/2=24 min), (iii) a minor population (5–12%) containing active GABA receptor and having the GABA-independent permeability of the slower exchanging population, and (iv) a very small exchange (∼2%) (t _1/2∼0.2 sec). The GABA-independent³⁶Cl⁻ exchange processes were kinetically first order. The relative quantities of the different vesicle populations varied slightly with the preparation and purification technique. The identity of these components, observed in the different preparations, was attributed to the vesicle formation being dependent on the morphology and properties of the membrane rather than the preparation method. The soluble brain extract was GABA-mimetic with the two observed receptors, causing channel opening and desensitization. But little washing of the membrane was required to observe the function of both receptors. Muscimol was GABA-mimetic with both receptors. With muscimol, channel opening occurred at 2.6-fold lower concentrations while desensitization was unaltered relative to GABA. This is additional evidence that these responses are mediated by different pairs of binding sites. The dependence of desensitization rate on muscimol concentration indicated that there are two binding sites mediating desensitization, as described with GABA.