Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Summary

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Abstract

Ligand-gated ion channels underlie synaptic communication in the nervous system¹. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels². 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 roles⁵ (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 complexes⁶. 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 release⁷. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function⁸. P2X2 receptors are abundant in the nervous system⁹ and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 & TM2) separated by an extracellular region⁷ and intracellular N and C termini (Fig 1a)⁷. P2X subunits¹⁰ (P2X1-P2X7) show 30-50% sequence homology at the amino acid level¹¹. 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 receptors⁹ 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.

Protocol

EXPERIMENTAL PROCEDURES

The experimental procedure (Fig 2) consists of four parts that are described in a step-wise manner below.

Part 1: Subcloning and expression of the C-terminus of P2X2 receptors.

We have expressed the full length C-terminus of P2X2 receptors in bacteria to identify the brain proteins to which it binds.

1. The C-terminus (residues 353-472) of the P2X2 receptor (Fig 1) was amplified by PCR, cloned into pGEX 4NT1 (GE Life Sciences) and verified with sequencing.
2. The recombinant plasmid was transformed into E. Coli (BL21) for expression of rec....

Discussion

Ion channels are a major class of integral membrane proteins. They contain water filled pores that selectively permit the movement of ions down their electrochemical gradients across the plasma membrane. Ion channels gate between open and the closed states. The gating step is triggered by transmitters (e.g. ATP) in case of P2X ligand gated ion channels, or it may be regulated by interactions with other proteins. The last decade has witnessed an increase in our understanding of how P2X receptors bind ATP¹³, but.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetonitrile	Reagent	JT Baker	9829-02
Acrylamide	Reagent	Bio-Rad	161-0156
Ampicillin	Reagent	VWR international	VW1507-01
Ammonium Bicarbonate	Reagent	Fluka	09830
Ammonium Persulphate (APS)	Reagent	Sigma-Aldrich	A3678
Adenosine Triphosphate (ATP)	Reagent	Sigma-Aldrich	A7699
Bradford reagent	Reagent	Bio-Rad	500-0006
Bromophenol blue	Reagent	Fisher Scientific	B-392
Commassie blue R-250	Reagent	Santa Cruz Biotechnology, Inc.	Sc-24972
Dithiotritol (DTT)	Reagent	EMD Millipore	3860
Ethylenediaminetetraacetic acid (EDTA)	Reagent	VWR international	VW1474-01
Ethylene Glycol tetraacetic acid (EGTA)	Reagent	Sigma-Aldrich	E8145
Formic acid	Reagent	EMD Millipore	11670-1
Glutathione Sepharose 4B beads	Reagent	GE Healthcare	17-5132-01
Hydrochloric acid (HCl)	Reagent	Sigma-Aldrich	H1758
Isopropyl-beta-D-thiogalactopyranoside (IPTG)	Reagent	Sigma-Aldrich	15502
Iodoacetamide	Reagent	Sigma-Aldrich	I1149
Luria-Bertani (LB) Media	Reagent	EMD Millipore	1.00547.5007
Leupeptin	Reagent	Sigma-Aldrich	L8511
Lysozyme	Reagent	Sigma-Aldrich	62971
Magnesium Sulphate (MgSO4)	Reagent	Sigma-Aldrich	S7653
Sodium Chloride (NaCl)	Reagent	Sigma-Aldrich	S3014
Sodium Flouride (NaF)	Reagent	Sigma-Aldrich	S7920
Sodium Orthovanadate (Na3VO4)	Reagent	Sigma-Aldrich	S6508
Nonidet P40	Reagent	Fluka	74385
Phenylmethanesulphonylfluoride (PMSF)	Reagent	Sigma-Aldrich	P7626
Protease inhibitor tablet	Reagent	Sigma-Aldrich	S8820
Protein standard	Reagent	Bio-Rad	161-0305
Sarkosyl	Reagent	Acros Organics	61207
Screw top vial	Tool	Agilent Technologies	5182-0866
Sodium dodecyl sulfate	Reagent	Sigma-Aldrich	L4509
SYPRO® Ruby protein gel stain	Reagent	Bio-Rad	170-3125
N,N,N’,N’-Tetramethylethylenediamine (TEMED)	Reagent	Sigma-Aldrich	T9281
Tris base	Reagent	Sigma-Aldrich	T1503
Triton X-100	Reagent	Sigma-Aldrich	T9284
Trypsin	Reagent	Promega Corp.	V5111
Tween 20	Reagent	Sigma-Aldrich	P5927
Water	Reagent	Burdick & Jackson	365-4
LTQ-Orbitrap tandem mass spectrometer	Tool	Thermo Fisher Scientific, Inc.
Nano Liquid Chromatography System	Tool	Eksigent
B-Mercapt–thanol	Reagent	Sigma-Aldrich	M6250
Glycerol		EMD Millipore	GX0185-6