Synaptic membrane freezing affects modulatory sites in avian central nervous system GABA(A) receptor

Studies were carried out to determine whether barbiturates and neurosteroids share common recognition sites at the GABA(A) receptor complex in avian CNS. To achieve this, differentially prepared fresh and frozen synaptic membranes were used. Both the barbiturate, pentobarbital, and the neurosteroid, 3alpha-hydroxy-5alpha-pregnan-20-one, were able to stimulate GABA binding in both types of membranes. Stimulation differed markedly when both drugs were added jointly to different treated tissue. In frozen membranes drugs acted synergistically and were differentially displaced by picrotoxinin, while in fresh ones, where both compounds were inhibited by the convulsant, this additivity was absent. Post-freezing wash supernatants were collected and used as a source of putative endogenous factors involved in the above mentioned membrane differences. Addition of a high molecular weight fraction from supernatants to frozen synaptic membranes led to an inhibition of barbiturate and neurosteroid potentiation, as well as a loss of their additive effect. Our results indicate that GABA(A) receptor modulation by barbiturates and neurosteroids is affected by synaptic membrane treatment, with a common modulatory site in fresh membranes and separate recognition sites after a freeze-thawing procedure. There may also be endogenous factors involved in overlapping of modulatory sites, which would thus regulate GABA(A) receptor functionality by direct interaction with the complex.