Residues in the 1st Transmembrane-Spanning Helix Are Important for GABA(A)ρ Receptor Function

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