Molecular mechanisms of inwardly rectifying Kir2 potassium channels modulation:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2008
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | X, 120 S. Ill., graph. Darst. 21 cm |
Internformat
MARC
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| 245 | 1 | 0 | |a Molecular mechanisms of inwardly rectifying Kir2 potassium channels modulation |c von Nicoleta Oana Ureche (geb. Juravle) |
| 264 | 1 | |c 2008 | |
| 300 | |a X, 120 S. |b Ill., graph. Darst. |c 21 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
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| 502 | |a Tübingen, Univ., Diss., 2008 (Nur beschränkt für den Austausch) | ||
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Datensatz im Suchindex
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|---|---|
| adam_text | Contents II_
CONTENTS
Acknowledgements I
Contents II
Abbreviations V
Summary VIII
1. Introduction 1
1.1 Ion channels 1
1.2 Potassium channels 3
1.2.1 The selectivity Filter and ion permeation 4
1.2.2 Potassium channels gating 5
1.2.3 Overall K* channel architecture 6
1.3 Inwardly rectifying potassium channels and their physiological role 8
1.3.1 The Mechanism of Strong Inward Rectification 9
1.3.2 Structural determinants of inward rectification 12
1.3.3 Physiological role of rectification 14
1.3.4 Channelopaties resulting form Kir channel mutations 14
1.3.5 Structural and functional diversity of Kir Channels 15
1.3.5.1 Kir2 subfamily 17
1.4 Modulation/regulation of Kir channels 18
1.4.1 Regulation by internal and external pH 18
1.4.1.1 Internal pH 19
1.4.1.2 External pH 21
1.4.2 Regulation by phosphoinositides and phosphorylation 22
1.4.2.1 Phosphoinositides 22
1.4.2.2 Phosphorylation 25
1.4.3 Regulation by Grb proteins 26
1.4.3.1 The Src homology 2 domain 26
1.4.3.2 Pleckstrin homology domain 27
1.4.3.3 The Grb7 family 28
1.5 Aim of the study 31
Contents SL
2. Material and methods 32
2.1 Vectors and bacterial strains 32
2.2 Quantitation of nucleic acid concentration 33
2.3 Restriction digests of DNA 34
2.4 Agarose gel electrophoresis 34
2.5 Fragment isolation 35
2.6 In vitro site-directed mutagenesis 35
2.7 Dephosporylation of linearized plasmid DNA 39
2.8 Ligation of DNA fragments 40
2.9 Heat shock transformation of competent bacteria with plasmid DNA 40
2.10 Isolation of Plamid DNA from 2 ml bacterial cultures 41
2.11 Isolation of Plamid DNA from 50 ml bacterial cultures 42
2.12 The SDS polyacrylamid gel electrophorese (PAGE) 43
2.13 Western Blotting 44
2.14 Cell transfection 44
2.15 Protein detection 45
2.16 Detection of Cell Surface Expression by Chemiluminescence 45
2.17 In vi rro-Synthesis of cRNA 46
2.18 The Xenopus laevis oocytes expression system 48
2.18.1 Oocytes preparation 49
2.19 cRNA injection 49
2.20 Electrophysiological measurements 50
2.21 Substituted cysteine accessibility method 52
2.22 Molecular modeling of Kir2.3 outer pore 53
2.23 Data analysis 53
3. Resuls 54
3.1 Regulation of Inward rectifier channels Kir2.3 by external pH 54
3.1.1 Different pHeMenwi sensitivity among related Kir potassium channels 54
3.1.2 Histidine 117 is involved in Kir2.3 pHeMernai sensitivity 55
3.1.3 Histidine introduction into Kir2.1 brings only mild pH sensitivity 57
3.1.4 Construction of Co2+-coordination sites in Kir2.3 59
3.1.5 Construction of putative disulfide bonds in Kir2.3 60
3.1.6 Introduction of TEA binding sites into Kir2.3 and Kir2.1 channels 62
Contents IV^
3.1.7 Analysis of the distances between residue Kir2.3-Hl 17C and 63
the TEA binding site
3.1.8 Determination of distances between native and engineered 65
cysteines in Kir2.3
3.1.9 Molecular modeling of the Kir2.3 outer pore domain 66
3.2 Regulation of Kir2 channels by SGK3 via phosphorylation of 68
PIKfyve and PI(3^)P2
3.2.1 Upregulation of Kir2.2 by active SGK1-3 68
3.2.2 PIKfyve phosphorylation by SGK3 is crucial for Kir2.2 stimulation 70
3.2.3 Kir2.2 is stimulated by PI(3,5)P2 but not by PI(4,5)P2 72
3.2.4 Defining Kir2 regions which confer SGK3 sensitivity 73
3.2.5 Heteromeric Kir2.1/Kir2.2/Kir2.3 channels are activated 74
by SGK3, an effect absent in PIP2-insensitive Kir2.1 mutant channels
3.2.6 Stimulation of native SGK increases plasma membrane 76
localisation of EGFP-Kir2.2 in HEK cells
3.2.7 Kir2.2 transfers SGK3-sensitivity to several mutant 77
heteromeric Kir2 channel complexes
3 J Regulation of the heart Kir2 channels by adaptor proteins 79
3.3.1 Regulation of Kir2 channels by the Grb7 protein family 79
3.3.2 GrblO downregulate various heart potassium channels 81
3.3.3 Heteromeric Kir2.1/Kir2.2/Kir2.3 channels are activated 81
by GrblO when coexpressed with SGK3
3.3.4 GrblO downregulate various heart potassium channels 82
4. Discussions 84
4.1 Modulation of Kir2.3 inward rectifier channels by extracellular pH 84
4.2 Modulation of Kir2.2 channels by phosphorylation and phosphoinositides 88
4.3 Grb7 family proteins as regulators for cardiac potassium ion channels 92
References 96
Curriculum vitae 119
|
| adam_txt |
Contents II_
CONTENTS
Acknowledgements I
Contents II
Abbreviations V
Summary VIII
1. Introduction 1
1.1 Ion channels 1
1.2 Potassium channels 3
1.2.1 The selectivity Filter and ion permeation 4
1.2.2 Potassium channels gating 5
1.2.3 Overall K* channel architecture 6
1.3 Inwardly rectifying potassium channels and their physiological role 8
1.3.1 The Mechanism of Strong Inward Rectification 9
1.3.2 Structural determinants of inward rectification 12
1.3.3 Physiological role of rectification 14
1.3.4 Channelopaties resulting form Kir channel mutations 14
1.3.5 Structural and functional diversity of Kir Channels 15
1.3.5.1 Kir2 subfamily 17
1.4 Modulation/regulation of Kir channels 18
1.4.1 Regulation by internal and external pH 18
1.4.1.1 Internal pH 19
1.4.1.2 External pH 21
1.4.2 Regulation by phosphoinositides and phosphorylation 22
1.4.2.1 Phosphoinositides 22
1.4.2.2 Phosphorylation 25
1.4.3 Regulation by Grb proteins 26
1.4.3.1 The Src homology 2 domain 26
1.4.3.2 Pleckstrin homology domain 27
1.4.3.3 The Grb7 family 28
1.5 Aim of the study 31
Contents SL
2. Material and methods 32
2.1 Vectors and bacterial strains 32
2.2 Quantitation of nucleic acid concentration 33
2.3 Restriction digests of DNA 34
2.4 Agarose gel electrophoresis 34
2.5 Fragment isolation 35
2.6 In vitro site-directed mutagenesis 35
2.7 Dephosporylation of linearized plasmid DNA 39
2.8 Ligation of DNA fragments 40
2.9 Heat shock transformation of competent bacteria with plasmid DNA 40
2.10 Isolation of Plamid DNA from 2 ml bacterial cultures 41
2.11 Isolation of Plamid DNA from 50 ml bacterial cultures 42
2.12 The SDS polyacrylamid gel electrophorese (PAGE) 43
2.13 Western Blotting 44
2.14 Cell transfection 44
2.15 Protein detection 45
2.16 Detection of Cell Surface Expression by Chemiluminescence 45
2.17 In vi'rro-Synthesis of cRNA 46
2.18 The Xenopus laevis oocytes expression system 48
2.18.1 Oocytes preparation 49
2.19 cRNA injection 49
2.20 Electrophysiological measurements 50
2.21 Substituted cysteine accessibility method 52
2.22 Molecular modeling of Kir2.3 outer pore 53
2.23 Data analysis 53
3. Resuls 54
3.1 Regulation of Inward rectifier channels Kir2.3 by external pH 54
3.1.1 Different pHeMenwi sensitivity among related Kir potassium channels 54
3.1.2 Histidine 117 is involved in Kir2.3 pHeMernai sensitivity 55
3.1.3 Histidine introduction into Kir2.1 brings only mild pH sensitivity 57
3.1.4 Construction of Co2+-coordination sites in Kir2.3 59
3.1.5 Construction of putative disulfide bonds in Kir2.3 60
3.1.6 Introduction of TEA binding sites into Kir2.3 and Kir2.1 channels 62
Contents IV^
3.1.7 Analysis of the distances between residue Kir2.3-Hl 17C and 63
the TEA binding site
3.1.8 Determination of distances between native and engineered 65
cysteines in Kir2.3
3.1.9 Molecular modeling of the Kir2.3 outer pore domain 66
3.2 Regulation of Kir2 channels by SGK3 via phosphorylation of 68
PIKfyve and PI(3^)P2
3.2.1 Upregulation of Kir2.2 by active SGK1-3 68
3.2.2 PIKfyve phosphorylation by SGK3 is crucial for Kir2.2 stimulation 70
3.2.3 Kir2.2 is stimulated by PI(3,5)P2 but not by PI(4,5)P2 72
3.2.4 Defining Kir2 regions which confer SGK3 sensitivity 73
3.2.5 Heteromeric Kir2.1/Kir2.2/Kir2.3 channels are activated 74
by SGK3, an effect absent in PIP2-insensitive Kir2.1 mutant channels
3.2.6 Stimulation of native SGK increases plasma membrane 76
localisation of EGFP-Kir2.2 in HEK cells
3.2.7 Kir2.2 transfers SGK3-sensitivity to several mutant 77
heteromeric Kir2 channel complexes
3 J Regulation of the heart Kir2 channels by adaptor proteins 79
3.3.1 Regulation of Kir2 channels by the Grb7 protein family 79
3.3.2 GrblO downregulate various heart potassium channels 81
3.3.3 Heteromeric Kir2.1/Kir2.2/Kir2.3 channels are activated 81
by GrblO when coexpressed with SGK3
3.3.4 GrblO downregulate various heart potassium channels 82
4. Discussions 84
4.1 Modulation of Kir2.3 inward rectifier channels by extracellular pH 84
4.2 Modulation of Kir2.2 channels by phosphorylation and phosphoinositides 88
4.3 Grb7 family proteins as regulators for cardiac potassium ion channels 92
References 96
Curriculum vitae 119 |
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| spelling | Ureche, Nicoleta Oana 1977- Verfasser (DE-588)136264735 aut Molecular mechanisms of inwardly rectifying Kir2 potassium channels modulation von Nicoleta Oana Ureche (geb. Juravle) 2008 X, 120 S. Ill., graph. Darst. 21 cm txt rdacontent n rdamedia nc rdacarrier Tübingen, Univ., Diss., 2008 (Nur beschränkt für den Austausch) Regulation (DE-588)4049075-0 gnd rswk-swf Kaliumkanal (DE-588)4258505-3 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Kaliumkanal (DE-588)4258505-3 s Regulation (DE-588)4049075-0 s DE-604 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016756321&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Ureche, Nicoleta Oana 1977- Molecular mechanisms of inwardly rectifying Kir2 potassium channels modulation Regulation (DE-588)4049075-0 gnd Kaliumkanal (DE-588)4258505-3 gnd |
| subject_GND | (DE-588)4049075-0 (DE-588)4258505-3 (DE-588)4113937-9 |
| title | Molecular mechanisms of inwardly rectifying Kir2 potassium channels modulation |
| title_auth | Molecular mechanisms of inwardly rectifying Kir2 potassium channels modulation |
| title_exact_search | Molecular mechanisms of inwardly rectifying Kir2 potassium channels modulation |
| title_exact_search_txtP | Molecular mechanisms of inwardly rectifying Kir2 potassium channels modulation |
| title_full | Molecular mechanisms of inwardly rectifying Kir2 potassium channels modulation von Nicoleta Oana Ureche (geb. Juravle) |
| title_fullStr | Molecular mechanisms of inwardly rectifying Kir2 potassium channels modulation von Nicoleta Oana Ureche (geb. Juravle) |
| title_full_unstemmed | Molecular mechanisms of inwardly rectifying Kir2 potassium channels modulation von Nicoleta Oana Ureche (geb. Juravle) |
| title_short | Molecular mechanisms of inwardly rectifying Kir2 potassium channels modulation |
| title_sort | molecular mechanisms of inwardly rectifying kir2 potassium channels modulation |
| topic | Regulation (DE-588)4049075-0 gnd Kaliumkanal (DE-588)4258505-3 gnd |
| topic_facet | Regulation Kaliumkanal Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016756321&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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