GABAAρ receptors are a subfamily of the GABAA receptor family of pentameric ligand-gated ion channels (pLGICs). Each subunit has a common structure, including a transmembrane domain of four α-helices (M1–M4). The aim of this study was to identify important M1 residues in the GABAAρ receptor (GABAAρR), using mutagenesis and functional assays combined with bioinformatic approaches. Alanine substitution of 12 of the 23 M1 residues yielded receptors with altered functional parameters, indicating these residues contribute to GABAAρR function. Further mutations reveal the properties that are important for function in critical residues, and, using a GABAAρR homology model, we suggest amino acid interactions that could be important. Phylogenetic analysis comparing GABAAR and other pLGICs subunits reveals most M1 residue properties linked to GABAAρR function are ancestrally ancient, but some are more recent acquisitions. Multiple sequence alignment of M1 residues across GABAAR subunits reveal three residues are well conserved except in GABAAR α subunits. Substitution of ρ1 subunit residues to their α1 subunit equivalents showed one alters functional parameters. Overall, the data provide a comprehensive picture of M1 residues that contribute to GABAAρR function, and illustrate how they might do so.