Interaction Between Allopregnanolone and Amiloride Binding Sites on the GABA_A Receptor

Allopregnanolone (Allo) is a positive allosteric modulator of the GABA_A receptor, and amiloride (Ami) is a competitive antagonist of the GABA_A receptor. The purpose of this work was to study the combined effect of Allo and Ami on functional activity of GABA_A receptor. The GABA-induced chloride current (I_GABA) was measured in isolated Purkinje cells of rat cerebellum using the patch-clamp technique and a system of fast application. Our results indicate that Allo suppresses the inhibitory effect of Ami on I_GABA, the IC₅₀ value of Ami concentration-response curve was increased from 164 to 547 µM (P < 0.001) in the presence of Allo. Next, GABA concentration-response curves (EC₅₀ = 5.8 µM) were constructed in the presence of Allo (EC₅₀ = 1.2 µM), Ami (EC₅₀ = 25.5 µM), and the combination of Allo+Ami (EC₅₀ = 3.2 µM). Changes in EC₅₀ values as a percentage relative to the control were calculated. The blocking effect of Ami is reduced in the presence of Allo (340% vs 150%, P < 0.01) and the potentiating effect of Allo does not change in the presence of Ami (78% vs 87%, P > 0.05). The results suggest that there is an allosteric relationship between the Allo and Ami binding sites on GABA_A receptor that operates in one direction, from Allo sites to Ami site, but not vice versa.