The GABAA receptor α subunit β1 strand runs anti-parallel to the β2 strand, which contains loop D, known to participate in receptor activation and agonist binding. However, a role for the β1 strand has yet to be established. We used molecular dynamics simulation to quantify the solvent accessible surface area (SASA) of β1 strand residues in the GABAA β3 homopentamer structure. Residues in the complementary interface equivalent to those between Asp43 and Thr47 in the a1 subunit have an alternating pattern of high and low SASA consistent with a β strand structure. We investigated the functional role of these β1 strand residues in the α1 subunit by individually replacing them with Cys residues. D43C and T47C substitutions reduced the apparent potency of GABA at α1β2γ2 receptors by around 50-fold and 8-fold, respectively, whereas the F45C substitution caused a biphasic GABA concentration-response relationship and increased spontaneous gating. Receptors with D43C or T47C substitutions were sensitive to MTSEA modification. However, GABA-evoked currents mediated by α1(F45C)β2γ2 receptors were unaffected by MTSEA, suggesting that this residue is inaccessible. Both GABA and the allosteric agonist propofol reduced MTSEA modification of α1(D43C)β2γ2 and α1(T47C)β2γ2 receptors indicating movement of the β1 strand even during allosteric activation. This is in contrast to α1(F64C)β2γ2 receptors where only GABA, but not propofol reduced MTSEA modification. These findings provide the first functional evidence for movement of the β1 strand during gating of the receptor and identify residues that are critical for maintaining GABAA receptor function. This article is protected by copyright. All rights reserved.