Whilst many of the residues that form the agonist binding site and channel lining of GABA_A receptors have been identified, the means by which they transduce agonist binding to channel opening remains to be elucidated. To identify domains that mediate activation of murine α1β1 GABA_A receptors, chimeric subunits containing sequences from murine and Drosophila GABA_A receptor subunits were generated, expressed in HEK 293 cells and studied under whole-cell patch-clamp.

The M2-3 loop of the β1 subunit was found to play a critical role in receptor activation. Substituting transmembrane domains M1 and M2 in β1 with the corresponding regions of the Drosophila subunit RDL had no effect on agonist potency (mean ± S.E.M. GABA EC₅₀ of wild-type α1β1 receptors = 1.2 ± 0.1 µM, n = 5). However, GABA (10 mM, n = 30 cells) and pentobarbitone (10 mM n = 18 cells) activation was abolished when RDL domains M1-M3 were introduced into β1 subunits (β1^{RDL M1-M3}). This loss of function was dependent upon multiple residues in the N-terminal half of M3. Indirect immunofluorescence assays demonstrated that subunit assembly was unaffected by these substitutions, consistent with the uncoupling of the channel gate from the agonist-binding domain.

Homology modelling of the N-terminal region of β1 on the structure of the Lymnaea acetylcholine binding protein (Brejc et al. 2001) identified likely points of contact between the extracellular and transmembrane domains. GABA and pentobarbitone activation of receptors containing the non-functional β1^{RDL M1-M3} subunits were restored by introducing RDL-specific sequences into the disulphide loop that is found in all receptors of this class. The GABA EC₅₀ of receptors containing this chimeric subunit (5.1 ± 0.3 µM, n = 6) was close to that of wild-type receptors but the increased degree of desensitisation and reduced whole-cell current amplitude exhibited by these receptors (approximately 20 % of wild-type) indicated that other regions of the N-terminal domain also participate in receptor activation. We are currently engaged in identifying these regions.

Our data show a critical interdependence between the primary sequences of the disulphide and M2-M3 loops of the β subunit in GABA_A receptor activation that may reflect a physical interaction between these domains.

This work was supported by the MRC and The Wellcome Trust.