Tagetone modulates the coupling of flunitrazepam and GABA binding sites atGABA(A) receptor from chick brain membranes

The effects of tagetone on flunitrazepam (FNTZ) binding to synaptosomal mem branes from chick brains in the presence and absence of allosteric modulati ons induced by gamma-aminobutyric acid (GABA) were investigated. Tagetone, at 50 mu g/ml (final concentration), decreased the binding affinity of [H-3 ]FNTZ to synaptosomal membranes form chick brain (K-d = 3.34 +/- 0.36 nM wi thout tagetone and K-d,K-t = 5.86 +/- 0.86 nM with tagetone; p < 0.05, two tailed Student's t-test) without affecting maximal binding (B-max = 488 +/- 24 fmoles/mg protein, and B-max,B-t = 500 +/- 25 fmoles/ mg protein in the absence and in the presence of tagetone respectively). The potency of GABA to stimulate [H-3]FNTZ binding increased in the presence of tagetone (EC50 values were 2.78 and 1.12 mu M with and without tagetone respectively). GA BA was able to decrease merocyanine Delta A(570-610) values in a concentrat ion dependent manner; half maximal effect was attained at a GABA concentrat ion of 34 +/- 13 mu M. Tagetone, at a concentration of 50 mu g/ml and in th e presence of GABA 30 mu M or 60 mu M, enhanced the ability of GABA alone o n decreasing Delta A(570-610).Tagetone alone did not change Delta A570-610 values. FNTZ, a well known GABA modulator, could also potentiate the effect of GABA. Theoretical calculations indicate that the effects on merocyanine Delta A(570-610) Value are mainly exerted at the membrane potential level (Delta Psi(m)). The present results strongly suggest that tagetone affected the function of GABA, receptor in a complex way: on the one hand it impair ed FNTZ binding; on the other hand tagetone improved both the coupling betw een FNTZ and GABA binding sites and it enhanced GABA-induced chloride perme ability. Changes in the geometrical and electrostatic properties of the sel f-organized membrane structure may account for these effects of tagetone.