Abstract

Tracazolate, a pyrazolopyridine, is an anxiolytic known to interact with γ-aminobutyric acid (GABA)_A receptors, adenosine receptors, and phosphodiesterases. Its anxiolytic effect is thought to be via its interaction with GABA_A receptors. We now report the first detailed pharmacological study examining the effects of tracazolate on a range of recombinant GABA_A receptors expressed in Xenopus laevis oocytes. Replacement of the γ2s subunit within the α1β3γ2s receptor with the ε subunit caused a dramatic change in the functional response to tracazolate from potentiation to inhibition. The γ2s subunit was not critical for potentiation because α1β3 receptors were also potentiated by tracazolate. γ2/ε chimeras revealed a critical N-terminal domain between amino acids 206 and 230 of γ2, governing the nature of this response. Replacement of the β3 subunit with the β1 subunit within α1β3γ2s and α1β3ε receptors also revealed selectivity of tracazolate for β3-containing receptors, determined by asparagine at position 265 within transmembrane 2. Replacement of γ2s with γ1 or γ3 revealed a profile intermediate to that of α1β1ε and α1β1γ2s. α1β1δ receptors were also potentiated by tracazolate; however, the maximum potentiation of the EC₂₀ was much greater than on α1β1γ2. Concentration-response curves to GABA in the presence of tracazolate for α1β1ε and α1β1γ2s revealed a concentration-related decrease in maximum current amplitude, but a leftward shift in the EC₅₀ only on α1β1γ2. Like α1β1γ2s, GABA concentration-response curves on α1β1δ receptors were shifted to the left with increased maximum responses. Tracazolate has a unique pharmacological profile on recombinant GABA_A receptors: its potency (EC₅₀) is influenced by the nature of the β subunit; but more importantly, its intrinsic efficacy, potentiation, or inhibition is determined by the nature of the third subunit (γ1–3, δ, or ε) within the receptor complex.