Anaesthetic molecules act on synaptic transmission via the allosteric modulation of ligand-gated chloride channels, such as hetero-oligomeric α1β2γ2 GABAA receptors. To elucidate the overall activation paradigm via allosteric versus orthosteric sites, we used highly homologous, but homo-oligomeric, ρ1 receptors that are contrastingly insensitive to anaesthetics and respond partially to several full GABA α1β2γ2 receptor agonists. Here, we coexpressed varying ratios of RNAs encoding the wild-type and the mutated ρ1 subunits, which are anaesthetic-sensitive and respond with full efficacy to partial GABA agonists, to generate distinct ensembles of receptors containing five, four, three, two, one, or zero mutated subunits. Using these experiments, we then demonstrate that, in the pentamer, three anaesthetic-sensitive ρ1 subunits are needed to impart full efficacy to the partial GABA agonists. By contrast, five anaesthetic-sensitive subunits are required for direct activation by anaesthetics alone, and only one anaesthetic-sensitive subunit is sufficient to confer the anaesthetic-dependent potentiation to the GABA current. In conclusion, our data indicate that GABA and anaesthetics holistically activate the GABAA ρ1 receptor through distinct subunit level rearrangements and suggest that in contrast to the global impact of GABA via orthosteric sites, the force of anaesthetics through allosteric sites may not propagate to the neighbouring subunits and, thus, may have only a local and limited effect on the ρ1 GABAA receptor model system.