Nano-LC-FTMS Analysis of the Primary Structure and Post-translational Modifications of the α1 Subunit of the GABAA Receptor

GABAAR is one of the most significant drug targets in the treatment of neuropsychiatric disorders such as epilepsy, insomnia, anxiety, as well as anesthesia in surgical operations. However, complete information on the regulation of GABAA receptor activity is lacking. Therefore, studies are needed on post-translational modifications of receptor subunits that exhibit different pharmacological and physiological properties. GABAAR has been immunopurified from rat brain membranes on protein A agarose beads immobilized with complex anti-GABAAR antibody. Primary structure and post-translational modifications of the α1 subunit of GABAAR have been characterized by nano-LC-FTMS peptide mapping by direct in situ–gel digestion on the one-dimensional gel. The primary structure of the α1 subunit of GABAAR has been identified with high sequence coverage (81%, 371 from 455 amino acids). The extracellular domains, N-terminal and the C-terminus, have been identified with extensive sequence coverage, 85 and 100 % (214 from 249 amino acids), respectively. Other soluble domains, including the M1-M2 linker, and the M3-M4 linker, have also been determined with 100 and 85 % (75 from 88 amino acids), respectively. Transmembrane domains, including M1, M2, and M4, were identified almost completely. Post-translational modifications of the α1 subunit peptides have been found, such as phosphorylation, methionine oxidation, and carbamidomethylation.