Vol. 61, Issue 4, 861-869, April 2002

Tracazolate Reveals a Novel Type of Allosteric Interaction with Recombinant -Aminobutyric Acid_A Receptors

Sally-Anne Thompson, Peter B. Wingrove, Linda Connelly, Paul J. Whiting, and Keith A. Wafford

Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Essex, United Kingdom (S.-A.T., P.B.W., P.J.W., K.A.W.); and Wolfson Institute for Biomedical Research, University College London, London, United Kingdom (L.C.)

Tracazolate, a pyrazolopyridine, is an anxiolytic known to interact with -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 2s subunit within the 132s receptor with the  subunit caused a dramatic change in the functional response to tracazolate from potentiation to inhibition. The 2s subunit was not critical for potentiation because 13 receptors were also potentiated by tracazolate. 2/ chimeras revealed a critical N-terminal domain between amino acids 206 and 230 of 2, governing the nature of this response. Replacement of the 3 subunit with the 1 subunit within 132s and 13 receptors also revealed selectivity of tracazolate for 3-containing receptors, determined by asparagine at position 265 within transmembrane 2. Replacement of 2s with 1 or 3 revealed a profile intermediate to that of 11 and 112s. 11 receptors were also potentiated by tracazolate; however, the maximum potentiation of the EC₂₀ was much greater than on 112. Concentration-response curves to GABA in the presence of tracazolate for 11 and 112s revealed a concentration-related decrease in maximum current amplitude, but a leftward shift in the EC₅₀ only on 112. Like 112s, GABA concentration-response curves on 11 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  subunit; but more importantly, its intrinsic efficacy, potentiation, or inhibition is determined by the nature of the third subunit (1-3, , or ) within the receptor complex.