Mass spectrometric analysis of GABAA receptor subtypes and phosphorylations from mouse hippocampus

Abstract

The brain GABA(A) receptor (GABA(A) R) is a key element of signaling and neural transmission in health and disease. Recently, complete sequence analysis of the recombinant GABA(A) R has been reported, separation and mass spectrometrical (MS) characterisation from tissue, however, has not been published so far. Hippocampi were homogenised, put on a sucrose gradient 10-69% and the layer from 10 to 20% was used for extraction of membrane proteins by a solution of Triton X-100, 1.5 M aminocaproic acid in the presence of 0.3 M Bis-Tris. This mixture was subsequently loaded onto blue native PAGE (BN-PAGE) with subsequent analysis on denaturing gel systems. Spots from the 3-DE electrophoretic run were stained with Colloidal Coomassie Brilliant Blue, and spots with an apparent molecular weight between 40 and 60 kDa were picked and in-gel digested with trypsin, chymotrypsin and subtilisin. The resulting peptides were analysed by nano-LC-ESI-MS/MS (ion trap) and protein identification was carried out using MASCOT searches. In addition, known GABA(A) R-specific MS information taken from own previous studies was used for searches of GABA(A) R subunits. β-1, β-2 and β-3, θ and ρ-1 subunits were detected and six novel phosphorylation sites were observed and verified by phosphatase treatment. The method used herein enables identification of several GABA(A) R subunits from mouse hippocampus along with phosphorylations of β-1 (T227, Y230), β-2 (Y215, T439) and β-3 (T282, S406) subunits. The procedure forms the basis for GABA(A) R studies at the protein chemical rather than at the immunochemical level in health and disease.