हम करने के लिए मस्तिष्क प्रोटीन है कि एटीपी gated P2X2 रिसेप्टर्स की पूरी लंबाई सी टर्मिनस करने के लिए बाध्य की पहचान के लिए एक सरल प्रोटोकॉल का वर्णन. एक्सटेंशन और सभी P2X रिसेप्टर्स करने के लिए इस दृष्टिकोण के व्यवस्थित आवेदन P2X रिसेप्टर संकेतन का एक बेहतर समझ के लिए नेतृत्व की उम्मीद है.
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 ions3, 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.
आयन चैनलों अभिन्न झिल्ली प्रोटीन का एक प्रमुख वर्ग हैं. वे पानी भरा pores कि चुनिंदा उनके विद्युत gradients के नीचे आयनों के प्लाज्मा झिल्ली भर में आंदोलन की अनुमति होते हैं. आयन चैनल गेट खुला और बंद राज्यों के ब?…
दप और TMV NCRR और NHLBI द्वारा स्वास्थ्य के राष्ट्रीय संस्थान में समर्थित हैं. BSK और एचएस NINDS और राष्ट्रीय स्वास्थ्य संस्थान के NIGMS द्वारा समर्थित हैं.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Acetonitrile | Reagent | JT Baker | 9829-02 | |
Acrylamide | Reagent | BIO-RAD | 161-0156 | |
Ampicillin | Reagent | VWR | VW1507-01 | |
Ammonium Bicarbonate | Reagent | Fluka | 09830 | |
Ammonium Persulphate (APS) | Reagent | Sigma | A3678 | |
Adenosine Triphosphate (ATP) | Reagent | Sigma | A7699 | |
Bradford reagent | Reagent | BIO-RAD | 500-0006 | |
Bromophenol blue | Reagent | Fisher Scientific | B-392 | |
Commassie blue R-250 | Reagent | Santa Cruz Biotechnology | Sc-24972 | |
Dithiotritol (DTT) | Reagent | EMD | 3860 | |
Ethylenediaminetetraacetic acid (EDTA) | Reagent | VWR | VW1474-01 | |
Ethylene Glycol tetraacetic acid (EGTA) | Reagent | Sigma | E8145 | |
Formic acid | Reagent | EMD | 11670-1 | |
Glutathione Sepharose 4B beads | Reagent | GE Healthcare | 17-5132-01 | |
Hydrochloric acid (HCl) | Reagent | Sigma | H1758 | |
Isopropyl-beta-D-thiogalactopyranoside (IPTG) | Reagent | Sigma | 15502 | |
Iodoacetamide | Reagent | Sigma | I1149 | |
Luria-Bertani (LB) Media | Reagent | EMD | 1.00547.5007 | |
Leupeptin | Reagent | Sigma | L8511 | |
Lysozyme | Reagent | Sigma | 62971 | |
Magnesium Sulphate (MgSO4) | Reagent | Sigma | S7653 | |
Sodium Chloride (NaCl) | Reagent | Sigma | S3014 | |
Sodium Flouride (NaF) | Reagent | Sigma | S7920 | |
Sodium Orthovanadate (Na3VO4) | Reagent | Sigma | S6508 | |
Nonidet P40 | Reagent | Fluka | 74385 | |
Phenylmethanesulphonylfluoride (PMSF) | Reagent | Sigma | P7626 | |
Protease inhibitor tablet | Reagent | Sigma | S8820 | |
Protein standard | Reagent | BIO-RAD | 161-0305 | |
Sarkosyl | Reagent | Acros | 61207 | |
Screw top vial | Tool | Agilent Technologies | 5182-0866 | |
Sodium dodecyl sulfate | Reagent | Sigma | L4509 | |
SYPRO® Ruby protein gel stain | Reagent | BIO-RAD | 170-3125 | |
N,N,N’,N’-Tetramethylethylenediamine (TEMED) | Reagent | Sigma | T9281 | |
Tris base | Reagent | Sigma | T1503 | |
Triton X-100 | Reagent | Sigma | T9284 | |
Trypsin | Reagent | Promega | V5111 | |
Tween 20 | Reagent | Sigma | P5927 | |
Water | Reagent | Burdick&Jackson | 365-4 | |
LTQ-Orbitrap tandem mass spectrometer | Tool | ThermoFisher Scientific | ||
Nano Liquid Chromatography System | Tool | Eksigent | ||
B-Mercaptoethanol | Reagent | Sigma | M6250 | |
Glycerol | EMD | GX0185-6 |