Proteomics to identify proteins interacting with P2X2 ligand-gated cation channels

Ligand-gated ion channels underlie synaptic communication in the nervous system(1). In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channel family(2). In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions(3, 4), which have downstream signaling roles(5) (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes(6). In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain. P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release(7). P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function(8). P2X2 receptors are abundant in the nervous system(9) and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 & TM2) separated by an extracellular region(7) and intracellular N and C termini (Fig 1a)(7). P2X subunits(10) (P2X1-P2X7) show 30 50% sequence homology at the amino acid level(11). P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors(9) no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C terminus of P2X2 receptors.