High level production, characterization and structural analysis of neuronal calcium-activated potassium channels [Elektronische Ressource] / von Luana Licata

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• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2005
• Nombre de lectures: 49
• Poids de l'ouvrage: 30 Mo

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High level production, characterization and structural analysis of neuronal
calcium-activated potassium channels
Dissertation
Zur Erlangung des Doctorgrades der Naturwissenschaften
Vorgelect beim Fachbereich
Biochemie, Pharmazie, und Lebensmittelchemie
Der Johann Wolfgang Goethe-Universität
In Frankfurt am Main
Von
Luana Licata
Aus Sciacca
Italien
Frankfurt am Main, im Jahre 2004Vom Fachbereich Biochemie, Pharmazie, und Lebensmittelchemie der Johann Wolfgang
Goethe-Universität
Als Dissertation angenommen.
Dekan: Prof. Dr. Walter Müller
Gutachter Prof. Dr. Ernst Bamberg
Gutachter Prof . Dr. Werner KühlbrandtTo Accursio, the nicest, sincere, vital, wonderful and lovely father that I could have wished
for and to his infinite love for me.
To Alessandro, my great master of science and life and to his elegant mind.
To their love for life that I keep with me.Muere lentamente
quien se transforma en esclavo del hábito,
repitiendo todos los días los mismos trayectos,
…y no le habla a quien no conoce.
Muere lentamente
quien evita una pasión,
quien prefiere el negro sobre blanco
y los puntos sobre las "íes" a un remolino de emociones,
justamente las que rescatan el brillo de los ojos,
sonrisas de los bostezos,
corazones a los tropiezos y sentimientos.
Muere lentamente
quien no voltea la mesa cuando está infeliz en el trabajo,
quien no arriesga lo cierto por lo incierto para ir detrás de un sueño,
quien no se permite por lo menos una vez en la vida,
huir de los consejos sensatos.
Muere lentamente
quien no viaja,
quien no lee,
quien no oye música,
quien no encuentra gracia en si mismo.
Muere lentamente
quien destruye su amor propio,
quien no se deja ayudar.
Muere lentamente,
quien pasa los días quejándose de su mala suerte
o de la lluvia incesante.
quien abandona un proyecto antes de iniciarlo,
no preguntando de un asunto que desconoce o
no respondiendo cuando le indagan sobre algo que sabe…
Pablo NerudaTable of contents
Abstract 1
Chapter 1. Introduction 3
1.1 The anatomy of the brain 3
1.2 The synapse 4
1.3 Ionic basis of the action potential 6
1.4 Ion Channels: Biophysical characteristics 7
and ionic distribution and action potential
1.5 Characteristic of potassium channels 9
1.5.1 Potassium channels families 10
1.5.2 Regulation of potassium channel assembly and trafficking 11
1.5.3 X-ray structure of potassium channels 12
1.5.4 Voltage-gated potassium channels 13
1.5.5 Calcium-activated potassium channels 15
1.5.5.1 Small calcium-activated potassium channels 16
1.5.5.1.2 Biophysical characteristics of small calcium 17
activated potassium channels
1.5.5.1.3 SK2 channels and its interaction with calmodulin 18
1.5.5.2 Large calcium-activated potassium channels 20
1.5.5.2.1 X-ray structure of the bacterial calcium activated 21
potassium channel
1.5.5.2.2 Physiological and functional characteristics 23
of BK channels
1.6 Heterologous expression Systems 23
1.6.1 Pichia pastoris expression System 24
1.6.2 Semliki Forest virus expression system 25
1.7 Aim of present study 26Chapter 2. Materials and methods 29
2.1 Materials 29
2.1.1 Chemicals, antibodies, detergents and lipids 29
2.1.2 Solutions 30
2.1.3 Oligonucleotide primers 35
2.2 Methods 36
2.2.1 Molecular biological methods 36
2.2.1.1 DNA cloning 37
2.2.1.2 Transformation of E.coli Strain Top 10’ by plasmid DNA 39
2.2.1.3 Transformation of Pichia pastoris cells 39
2.2.1.4 Preparation of mRNA in vitro 40
2.2.1.5 Electroporation of RNA into BHK-21 cells 41
2.2.1.6 Transfection of mammalian cells using Lipofectamine 2000 42
2.2.2 Protein production 42
2.2.2.1 Small scale production in Pichia pastoris 42
2.2.2.2 Large scale production in 42
2.2.2.3 Small scale production in BHK cells 43
2.2.2.4 Large scale production in BHK cells 43
2.2.3 Membrane preparation 44
2.2.3.1 Small scale membrane preparation with glass beads 44
2.2.3.2 Larger-scale preparation of membranes using a microfluidiser 44
2.2.3.3 Preparation of synaptosomal plasma membranes 45
2.2.4 Polyacrylamide gel analysis 45
2.2.4.1 Staining of SDS-PAGE gels 46
2.2.5 Western blot 46
2.2.6 Solubilisation 47
2.2.7 Purification of solubilized membrane proteins 47
2.2.7.1 Affinity Chromatography 472.2.7.1.1. Immobilised metal affinity chromatography 47
2.2.7.1.2 Affinity chromatography on streptactin column 48
2.2.7.2 Ion Exchange Chromatography 48
2.2.7.3 Size exclusion chromatography 49
2.2.8 Immunofluorescence analysis 49
2.2.9 Immunogold electron microscopy 50
1252.2.10 Measurement of I-apamin binding to SK2 channels 50
2.2.11 Electron microscopy of single particle 51
Chapter three Results and Discussion: 53
3.1 Expression of a small calcium activated potassium 53
channel in Pichia pastoris.
3.1.1 Cloning and expression of rSK2 53
3.1.2 Localisation of expressed SK2 channels 56
3.1.3 Binding of specific ligands to Pichia pastoris expressed SK2 channels 57
3.1.4 Solubilisation of SK2 channels from Pichia pastoris membranes 59
3.1.5 Large scale protein production 62
3.1.6 Purification of SK2 64
3.1.6.1 Immobilised metal affinity chromatography 64
3.1.6.2 Affinity chromatography on Streptactin column 65
3.1.6.3 Ion exchange chromatography 66
3.1.6.4 Size exclusion chromatography 67
3.2 Expression of small calcium activated potassium 68
channel-FCYENE in Pichia pastoris SMD1163 cells
3.2.1 Cloning and expression of SK2-FCYENE in Pichia pastoris cells 69
1253.2.2 Binding of I-apamin to membranes of SK2-FCYENE expressing cells 70
3.2.3 Solubilisation of SK2-FCYENE 70
3.2.4 Localisation of SK2-FCYENE mutant channel 71
3.2.5 Purification 733.3 Co-expression of SK2 and calmodulin in Pichia pastoris SMD1163 cells 74
3.3.1 Cloning and expression 76
1253.3.2 Specific I -apamin binding to Pichia pastoris membrane 77
expressing SK2-q-Calmodulin tandem construct and localization studies
3.3.3 Solubilisation of the tandem linked SK2-q-calmodulin 80
3.3.4 Large scale production and purification of 81
SK2-q-calmodulin tandem construct
3.3.4.1 Detergent pre-extraction experiments 82
3.3.4.2 Ammonium sulphate precipitation 82
3.4 Over-expression and characterisation of 83
small calcium-activated potassium channel (SK2) using Semliki Forest virus
3.4.1 Cloning of SK2 channel DNA into the 83
Semliki Forest virus vector pSFV2gen
3.4.2 SK2 channel production and localization 83
3.4.3 Specific apamin binding in BHK cells expressing SK2 85
3.4.4 Solubilisation of SK2 channels from BHK membranes 90
3.4.5 Purification of SK2 channels over-expressed in BHK cells 91
3.4.5.1 Size exclusion chromatography 92
3.4.6 Blue native gel electrophoresis 93
3.4.7 Single particles analysis 94
3.5 Over-expression and characterization of a 96
large calcium-activated potassium channel (BK) in Pichia pastoris
3.5.1 Cloning and expression of BK alpha subunit in cells 96
3.5.2 Localisation of BK alpha subunit in Pichia pastoris cells 98
3.5.3 Solubilisation of BK channel 100
3.5.4 Cloning of the BK beta subunit and co-expression 101
with the BK channel into Pichia pastoris cells
3.5.5 Solubilisation of membranes containing the BK/BKb channels 102