Fig 1: The endogenous a1 subunit interacts with inhibitory scaffolding proteins and cytoskeletal proteins. (A) The 720 kDa band of a1-containing GABAARs purified from PM was subjected to LC-MS/MS. Label-free normalized quantification and Welch’s t-test against non-immune control IgG revealed 93 significantly enriched binding proteins of the a1 subunit. The heatmap shows the top 10 binding proteins of a1-containing GABAARs by abundance (SIGI value) across all replicates. The most abundant proteins detected with the a1 subunit are Sptan1, Sptbn1, and Sptbn2 (n = 5 replicates). (B) Pie chart shows the relative abundance of the proteins that are significantly enriched with a1-containing GABAARs. Sptan1, Sptbn1, and Sptbn2 together constitute more than 70% of the total amount of proteins. Proteins that make up less than 2% of the total amount of proteins (86 proteins) were grouped together as ‘Other.’ (C) Immunopurified a1-containing GABAARs were resolved on BN-PAGE and probed for the a1 subunit, high-abundance proteins (Sptan1, Sptbn1, and Sptbn2), and low-abundance proteins (Erc2, Kcc2, and gephyrin). Sptan1, Sptbn1, and Sptbn2 are observed at around 720 kDa with a1-containing GABAARs. Erc2 and Kcc2 are present slightly below 720 kDa and gephyrin is observed higher than 720 kDa. The immunoreactivity of these proteins is specific to purified a1-containing GABAARs. (D) The network analysis shows known inhibitory scaffolding proteins such as Actn1, Cyfip2, and Gphn (gephyrin), and a subnetwork of spectrins (Sptan1, Sptbn1, Sptbn2, and Sptb) and ankyrins (Ank2 and Ank3). GO analysis reveals that most proteins are involved in cytoskeletal organization, ion transport, or signal transduction.