Structural and functional studies of {_k63M-conotoxin [kappa-M-conotoxin] RIIIK interaction with Shaker-related potassium channels from trout fish (TSha1) [Elektronische Ressource] / vorgelegt von Ahmed Nafi'(Sulaiman) al-Sabi ́

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• Publié par: universitat_bremen
• Publié le: 01 janvier 2005
• Nombre de lectures: 33
• Langue: English
• Poids de l'ouvrage: 9 Mo

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Structural and functional studies of kM-conotoxin
RIIIK interaction with Shaker-related potassium
channels from trout fish (TSha1)
Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften
-Dr. rer. nat.-
dem Fachbereich Biologie/Chemie der
Universität Bremen
vorgelegt von
Ahmed Nafi' (Sulaiman) Al-Sabi'
Bremen
November 2004Die vorliegende Arbeit wurde in der Zeit von Oktober 2001 bis November 2004 am Max-
Planck-Institut für Experimentelle Medizin in Göttingen angefertigt.
1. Gutachter: Prof. Dr. Venugopalan Ittekkot
2. Gutachter: Prof. Dr. Heinrich Terlau
Tag des Promotionskolloquiums: 04. November 2004To my father & mother
sisters & brothersLIST OF CONTENTS i
LIST OF CONTENTS
LIST OF CONTENTS i
LIST OF TABLES AND FIGURES iv
ABBREVIATIONS vi
CHAPTER ONE: INTRODUCTION 1
1.1. Voltage-gated ion channels 1
1.1.1. Voltage-gated potassium channels (VGKC) 3
1.1.2. Structural and functional studies of VGKC 6
1.2. Conopeptides 8
1.2.1. Cone snails: the source of conopeptides 8
1.2.2. Conopeptides and conotoxins 10
1.2.3. Conotoxins targeting VGKC and dyad hypothesis 12
1.3. Objectives 15
CHAPTER TWO: MATERIALS AND METHODS 16
2.1. Electrophysiological methods 16
2.1.1. Xenopus oocyte preparation 16
2.1.1.1. Xenopus oocyte solutions 16
2.1.1.2. oocytes handeling 16
2.1.2. Two-electrode voltage clamp (TEVC) 20
2.1.2.1. Instrumentation 21
2.1.2.2. Micropipettes and electrodes 22
2.1.2.3. TEVC stimulation protocols 23
2.1.2.3.1. Single pulse (I-V) protocol 23
2.1.2.3.2. Double pulse (DP) protocol 24
2.1.2.3.3. Leakage and capacitive current correction 24
2.2. Molecular Biology 25
2.2.1. Vector ligation 26
2.2.2. TSha1 DNA Transformation 27
2.2.3. Bacterial culture 27
2.2.4. Plasmid isolation 28LIST OF CONTENTS ii
2.2.5. Restriction enzyme digestion 28
2.2.6. Agarose gel electrophoresis 28
2.2.7. Mutagenic primer design and PCR amplification 29
2.2.8. Plasmid transformation 30
2.2.9. Plasmid isolation and linearization 30
2.2.10. In vitro transcription 32
2.3. Data analysis 33
CHAPTER THREE: RESULTS 36
+3.1. kM-RIIIK interacting with Shaker-type K channels 36
3.1.1. kM-RIIIK specificity interacts with TSha1 channels 36
3.1.1.1. Determining IC of open state block 3650
+3.1.1.2. Interaction at different extracelluar K concentrations 36
3.1.2. kM-RIIIK interacts with mammalian Kv1.2 38
3.1.2.1. Determining IC of open state block of Kv1.2 3950
3.1.2.2. Inhibition of Kv1.2 mediated currents is state dependent 40
3.2. Alanine scanning mutagenesis 44
3.2.1. Identification of the residues important for kM-RIIIK 44
3.2.2. Evaluation of additional substitution mutations at Leu1 47
3.2.3. kM-RIIIK analogs affect the binding kinetics 49
3.2.4. kM-RIIIK mutants do not interact with Nav1.4 sodium channel 53
3.3. Mutant cycle analysis 55
3.3.1. TSha1 mutant channels 56
3.3.2. Determining IC values of the mutant cycle analysis 5950
3.3.3. Docking model of kM-RIIIK interaction with TSha1 63
CHAPTER FOUR: DISCUSSION 64
+4.1. kM-RIIIK: a peptide blocking K channels 64
4.1.1. kM-RIIIK specificity interacts with the pore TSha1channels 64
+4.1.2. kM-RIIIK binding is altered by [K ] 64o
4.1.3. kM-RIIIK: first conotoxin targeting mammalian Kv1.2 65
4.1.4. kM-RIIIK mutants affect kinetics of binding 67LIST OF CONTENTS iii
4.1.5. kM-RIIIK mutants do not interact with Nav1.4 sodium channel 68
4.2. kM-RIIIK: novel structure among conotoxins 69
4.2.1. Structure of kM-conotoxin RIIIK 69
4.2.2. Comparison between kM-RIIIK and members of M-superfamily 69
4.3. Identification of kM-RIIIK pharmacophore 71
4.3.1. kM-RIIIK pharmacophore is not organized around a dyad motif 71
4.3.2. kM-RIIIK pharmacophore is a plannar ring of four residues 73
4.4. Mutant cycle analysis 75
4.4.1. TSha1 mutant channels 76
4.4.2. Determinin IC and DDG of the mutant cycle analysis 7750
4.4.3. The lid docking model of kM-RIIIK-TSha1 77
CHAPTER FIVE: SUMMARY 82
CHAPTER SIX: REFERENCES 84
ACKNOWLEDGEMENTS 93
Curriculum Vitae et studiorum 94LIST OF TABLES AND FIGURES iv
LIST OF TABLES AND FIGURES
LIST OF TABLES
Table 2.1. Primer sequences of TSha1 point mutation. 31
Table 2.2. The transcription mixture 32
+Table 3.1. The effect of kM-RIIIK interaction with TSha1 K channels
+under different extracelluar K concentrations 38
Table 3.2. Summary of K , k and k values for open and close statesD on off
+ of human Kv1.2 and TSha1 K channels 42
+Table 3.3. IC Values for TSha1 K block of kM-RIIIK and Analogs 4450
+Table 3.4. Mutations of Leu1 affect the affinity of kM-RIIIK for the TSha1 K Channel 48
Table 3.5. Summary of Association and dissociation rate constants of kM-RIIIK analogs 50
Table 3.6. kM-RIIIK Hyp15 mutants 52
+Table 3.7. IC Values for TSha1 mutant K block of kM-RIIIK WT 5750
+Table 3.8. IC Values for TSha1 mutant K block of kM-RIIIK WT and analogs 5950
Table 3.9. Summary of IC , W and DDG values to identify the toxin binding orientation 6350
LIST OF FIGURES
Figure 1.1. Schematic representation of the proposed transmembrane topology
of voltage gated ion channels. 2
Figure 1.2. Inactivation mechanisms 3
Figure 1.3. The three-dimensional structures of potassium channels. 5
Figure 1.4. Cone snails. 9
Figure 1.5. Organizational diagram for Conus peptides. 10
Figure 1.6. Sequence alignment of kM-RIIIK with other M-superfamily peptides. 14
Figure 2.1. The artificial expression of ion channels on the membrane of
Xenopus laevis oocytes 19
Figure 2.2. Schematic diagram of the main components of the two-electrode
voltage clamp (TEVC). 21LIST OF TABLES AND FIGURES v
Figure 2.3. Single pulse (IV) protocol. 24
Figure 2.4. Double pulse protocol. 24
Figure 2.5. Schematic representation of the site-directed mutagenesis to produce
cRNA of TSha1 channel with selected mutation. 25
Figure 2.6. Schematic representation of the pSGEM vector and TSha1 ORF DNA 26
Figure 3.1. kM-RIIIK reversibly blocks TSha1-mediated currents 37
Figure 3.2. The effect of external potassium concentration ([K] ) on the binding0
affinity of kM-RIIIK to TSha1-channels 38
Figure 3.3. kM-RIIIK reversibly blocks TSha1-mediated currents 39
Figure 3.4. Dose–response curve of the block of Kv1.2 mediated
steadystate currents by kM-RIIIK 40
Figure 3.5. The block of kM-RIIIK of Kv1.2 is state dependent 41
Figure 3.6. Relaxation of kM-RIIIK binding to closed Kv1.2 channels
measured by a double pulse stimulation 43
Figure 3.7. The summary of the alanine mutagenesis assay 45
Figure 3.8. Mutation of residues L1, R10, K18, and R19 results in
isoforms of kM-RIIIK with low affinity for TSha1 channels 46
Figure 3.9. Changes in the affinity of the mutants of kM-RIIIK at Leu1 position 49
Figure 3.10. kM-RIIIK mutants do not block Nav1.4 mediated currents 53
+Figure 3.11. The amino acid alignment of the pore region of the Shaker K channel (S5-S6 linker)
+and the corresponding region of TSha1 K channel 56
Figure 3.12. Influence of single point mutations in the pore region of TSha1 on kM-RIIIK binding 58
Figure 3.13. Effect of point mutations of pore region of TSha1 channels
on kM-RIIIK binding. Whole-cell 58
Figure 3.14. Examples for mutant cycles of TSha1 E354 with kM-RIIIK-Arg10 60
Figure 3.15. Summary of mutant cycle analysis 62
Figure 4.1. Superimposition of the 13 lowest-energy structures of kM-RIIIK 69
Figure 4.2. The three-dimensional NMR structures of M superfamily representatives 70
Figure 4.3. Electrostatic surface potential for kM-RIIIK 74
Figure 4.4. Close view of kM-RIIIK-TSha1 docking model obtained from mutant cycle analysis 78
Figure 4.5. Orientation of kM-RIIIK-TSha1 (lid) docking model obtained from mutant cycle analysis 80ABBREVIATIONS vi
EDTA Ethylenediamine tetraceticA adenine
acidA, Ala Alanine
EGTA Ethylenglycol-bis(b-A Ampere
-10 aminoethylether)Å Angstrom (10 )
N,N,N’,N’-Tetraacetic acidaa amino acid
et al. et aliibp base pair
F, phe phenyalanine°C degree Celsius
Fig, fig. FigureC cytosine
g gramC-, COOH- carboxy terminus of
G guanineterminus protein
G, Gly glycineC, Cys cysteine
2+ H, His HistidineCa calcium ion
h, hrs hour(s)cDNA complementary
H O waterdesoxyribonucleic acid 2
- Hepes N-2- Hydroxyethylpiperazine-Cl chloride ion
N’-2-ethanesulfonic acidCNS central nervous system
Hyp hydroxyprolinecRNA complementary RNA
Hz hertz(ribonucleic acid)
I currentCTX Charybdotoxin (a-KTx)
I, Ile IsoleucineD, asp asp