Structural rearrangements and subunit interactions in P2X receptors [Elektronische Ressource] / von Yogesh Bhargava

Yogesh

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 9 Mo

Extrait

Structural rearrangements and subunit interactions
in P2X receptors



Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften



vorgelegt beim Fachbereich Biochemie, Chemie, Pharmazie
der Johann Wolfgang Goethe – Universität
in Frankfurt am Main



von
Yogesh Bhargava
aus Jabalpur (Indien)




Frankfurt 2009
(D30)






Vom Fachbereich Biochemie, Chemie, Pharmazie der
Johann Wolfgang Goethe – Universität als Dissertation angenommen.


















Deken: Prof. Dr. Dieter Steinhilber
1. Gutachter: Prof. Dr. Bernd Ludwig
2. Gutachter: Prof. Dr. Ernst Bamberg
Datum der Disputation: 20-Nov-2009

Index
Abstract ........................................................................................................iv
1. Introduction .................................................................................. 1
1.1 Study of neurotransmitter gated ion channels ......................................1
1.1.1 Ligand gated ion channels and their functions........................................................ 1
1.1.2 Consequence of the ligand-receptor interactions .................................................... 2
1.2 Historical perspective..............................................................................4
1.2.1 Discovery of purinergic receptors .......................................................................... 4
1.2.2 Classification and nomenclature of purinergic receptors ........................................ 5
1.3 The P2X receptor family.........................................................................7
1.3.1 Gene family........................................................................................................... 7
1.3.2 Protein family........................................................................................................ 7
1.4 P2X receptor channels ............................................................................9
1.4.1 Molecular structure................................................................................................ 9
1.4.1.1 Membrane topology ........................................................................................ 9
1.4.1.2 Amino-terminal tail......................................................................................... 9
1.4.1.3 Extracellular loop......................................................................................... 10
1.4.1.3.1 ATP binding pocket................................................................................ 10
1.4.1.3.2 Conserved amino-acids .......................................................................... 11
1.4.1.4 The pore ....................................................................................................... 12
1.4.1.5 Carboxy-terminal tail.................................................................................... 13
1.4.2 Channel stoichiometry......................................................................................... 13
1.5 Properties of P2X1 receptors................................................................ 15
1.5.1 Ligand binding .................................................................................................... 15
1.5.1.1 Agonists........................................................................................................ 15
1.5.1.2 Antagonists................................................................................................... 18
1.5.2 Desensitization and recovery ............................................................................... 20
1.5.3 Internalization...................................................................................................... 21
1.6 Techniques to study ligand-receptor interactions ............................... 22
1.6.1 Photolabeling....................................................................................................... 22
1.6.1.1 Requirements of the photoprobe.................................................................... 23
1.6.1.2 Photolysable groups...................................................................................... 23
1.6.1.3 Photoaffinity labeling with purine based analogs.......................................... 25
1.6.2 Fluorescence labeling .......................................................................................... 25
1.6.2.1 Strategies of fluorescence labeling................................................................ 25
1.6.2.2 Real time assessment of receptor function ..................................................... 27
1.6.3 Electrophysiology................................................................................................ 29
1.6.3.1 Electrical characteristics of biological membranes....................................... 29
1.6.3.2 The voltage clamp technique......................................................................... 30
2. Materials and Methods............................................................... 34
A) Molecular Biology materials................................................................................ 34
B) Chemicals............................................................................................................ 34
C) Electrophysiology materials ................................................................................ 35
D) Animals and related stuff..................................................................................... 37
2.1 Molecular biology.................................................................................. 37
2.1.1 cDNA construct of P2X2/1 receptor chimera ....................................................... 37
2.1.2 cDNA constructs of cysteine mutants of P2X2 and chimera................................. 38
2.1.3 cDNA constructs of cysteine mutants of P2X1..................................................... 38
2.2 Bacterial culture and cDNA purification............................................. 39
i Index
2.2.1 Culture medium and transformation..................................................................... 39
2.2.2 Plasmid DNA purification ................................................................................... 39
2.2.3 cRNA synthesis ................................................................................................... 39
2.3 Heterologous expression in Xenopus laevis oocytes............................. 40
2.3.1 Frog maintenance ................................................................................................ 40
2.3.2 Surgical preparation............................................................................................. 40
2.3.3 Oocyte preparation and heterologous expression.................................................. 41
2.4 Functional measurement of receptors.................................................. 42
2.4.1 Design of the photolabeling setup ........................................................................ 42
2.4.2 Design of voltage clamp fluorometry setup.......................................................... 43
3. Results.......................................................................................... 47
3.1 Probing allosteric interactions between P2X receptor subunits using
photolabeling............................................................................................... 47
3.1.1 Photolabeling of wild type P2X1 receptors .......................................................... 48
3.1.1.1 Agonist unbinding is required for the recovery from desensitization.............. 48
3.1.1.2 Time course of photolabeling at P2X1 receptors ........................................... 49
3.1.2 Photolabeling of wild type P2X2 receptors .......................................................... 50
3.1.2.1 Efficacy and potency of BzATP and ATP on P2X2 receptors......................... 50
3.1.2.2 Each subunit contributes to the gating process.............................................. 51
3.1.3 Photolabeling of the P2X2/P2X1 receptor chimera .............................................. 52
3.1.3.1 Efficacy and potency of various ligands on the chimera ................................ 53
3.1.3.2 Time course of covalent activation of the chimera......................................... 55
3.1.3.3 Effect of prolonged application of light and BzATP on the chimera............... 57
3.1.3.4 Photolabeling modulates response of the receptors....................................... 58
3.1.3.4.1 Modulation of full agonist response by photolabeling............................. 58
3.1.3.4.2 Modulation of partial agonist response by photolabeling ....................... 60
3.1.3.5 Estimating the number of bound ligands required for the maximal response
generated by TNP-ATP on the receptors................................................................... 61
3.2 Probing allosteric interactions between P2X receptor subunits using
fluorescent ligand........................................................................................ 64
3.2.1 Potency and efficacy of ATP and Alexa-ATP on P2X1 receptors and the ch