The binding of the neurotransmitter GABA induces conformational changes in the GABAA receptor (GABAAR), leading to the opening of a gate that controls ion permeation through an integral transmembrane pore. A number of structural elements within each subunit, located near the membrane interface, are believed to undergo relative movements during this activation process. In this study, we explored the functional role of the β-10 strand (pre-M1 segment), which connects the extracellular domain to the transmembrane domain. In α1β2γ2s GABAARs, analysis of the 12 residues of the β-10 strand in the α1 subunit proximal to the first transmembrane domain identified two residues, α1V212 and α1K220, in which mutations produced rightward shifts in the GABA concentration–response relationship and also reduced the relative efficacy of the partial agonist, piperidine-4-sulphonic acid. Ultra-fast agonist techniques were applied to mutant α1(K220A)β2γ2s GABAARs and revealed that the macroscopic functional deficit in this mutant could be attributed to a slowing of the opening rate constant, from ∼1500 s−1 in wild-type (WT) channels to ∼730 s−1 in the mutant channels, and a reduction in the time spent in the active state for the mutant. These changes were accompanied by a decrease in agonist affinity, with half-maximal activation rates achieved at 0.77 mM GABA in WT and 1.4 mM GABA in the α1(K220A)β2γ2s channels. The β-10 strand (pre-M1 segment) emerges, from this and other studies, as a key functional component in the activation of the GABAAR.