Residues in the 1st Transmembrane-Spanning Helix Are Important for GABA_Aρ Receptor Function

GABA_Aρ receptors are a subfamily of the GABA_A 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 GABA_Aρ receptor (GABA_AρR), 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 GABA_AρR function. Further mutations reveal the properties that are important for function in critical residues, and, using a GABA_AρR homology model, we suggest amino acid interactions that could be important. Phylogenetic analysis comparing GABA_AR and other pLGICs subunits reveals most M1 residue properties linked to GABA_AρR function are ancestrally ancient, but some are more recent acquisitions. Multiple sequence alignment of M1 residues across GABA_AR subunits reveal three residues are well conserved except in GABA_AR 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 GABA_AρR function, and illustrate how they might do so.