Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

Nicotine acetylcholine receptors (nAChRs) comprise a family of ligand-gated channels widely expressed in the mammalian brain. The β2 subunit is an abundant protein subunit critically involved in the cognitive and behavioral properties of nicotine as well as in the mechanisms of nicotine addiction. In this work, we used matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS/MS) to uncover protein interactions of the intracellular loop of the β2 subunit and components of immunoprecipitated β2-nAChR complexes from mouse brain. Using the β2-knockout mouse to exclude nonspecific binding to the β2 antibody, we identify 21 nAChR-interacting proteins (NIPs) expressed in brain. Western blot analysis confirmed the association between the β2 subunit and candidate NIPs. Based on their functional profiles, the hypothesis is suggested that the identified NIPs can regulate the trafficking and signaling of the β2-nAChR. Interactions of the β2 subunit with NIPs such as G protein α, G protein-regulated inducer of neurite outgrowth 1, and G protein-activated K⁺ channel 1 suggest a link between nAChRs and cellular G protein pathways. These findings reveal intracellular interactions of the β2 subunit and may contribute to the understanding of the mechanisms of nAChR signaling and trafficking in neurons.