Interaction of transient receptor potential Vanilloid 1 (TRPV1) with G-protein coupled receptors and TRP ion channels [Elektronische Ressource] / vorgelegt von Viola Spahn
109 Pages
English

Interaction of transient receptor potential Vanilloid 1 (TRPV1) with G-protein coupled receptors and TRP ion channels [Elektronische Ressource] / vorgelegt von Viola Spahn

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Interaction of Transient Receptor Potential Vanilloid 1 (TRPV1) with G-protein coupled receptors and TRP ion channels Inauguraldissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) am Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin vorgelegt von Viola Spahn Berlin 2011 Erstgutachter: Herr Prof. Dr. Christian Zöllner Zweitgutachterin: Frau Prof. Dr. Monika Schäfer-Korting Tag der Disputation: 28.01.2011 Table of contents Table of contents Abbreviations…………………...……………………...1 1. Introduction……….………………...………………....4 1.1. Pain………...……………………….………………...….……..5 1.2. Transient receptor potential ion channel family………...…..6 1.2.1. TRPV1…………………………………………...……………………….7 1.2.2. Sensitization of TRPV1………………………………..………………...9 1.2.3. TRPA1………………...……………………………...…………………11 1.3. Opioids……………………….…………………...…………...14 1.3.1. μ-opioid receptor……………………………..………………………...15 1.3.2. Opioid withdrawal-induced hyperalgesia………….…...…………….17 2. Objectives……………………………...……………...19 3. Animals, material and methods………….…………..20 3.1. Materials……………..……………………………….…….…20 3.1.1. Cell lines and bacteria…………………………………..…...…………20 3.1.2. Animals and animal housing………………………………….……….20 3.1.3. Chemicals…………………………………...…………………………..20 3.1.4. Media, buffer………………………..………………………………….22 3.1.5. Reaction systems………………………..………………………….…...23 3.1.6.

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Published 01 January 2011
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Language English
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Interaction of Transient Receptor Potential Vanilloid 1
(TRPV1) with G-protein coupled receptors and
TRP ion channels


Inauguraldissertation
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
(Dr. rer. nat.)



am Fachbereich Biologie, Chemie, Pharmazie
der Freien Universität Berlin



vorgelegt von
Viola Spahn



Berlin 2011






















Erstgutachter: Herr Prof. Dr. Christian Zöllner
Zweitgutachterin: Frau Prof. Dr. Monika Schäfer-Korting
Tag der Disputation: 28.01.2011
Table of contents
Table of contents

Abbreviations…………………...……………………...1
1. Introduction……….………………...………………....4
1.1. Pain………...……………………….………………...….……..5
1.2. Transient receptor potential ion channel family………...…..6
1.2.1. TRPV1…………………………………………...……………………….7
1.2.2. Sensitization of TRPV1………………………………..………………...9
1.2.3. TRPA1………………...……………………………...…………………11
1.3. Opioids……………………….…………………...…………...14
1.3.1. μ-opioid receptor……………………………..………………………...15
1.3.2. Opioid withdrawal-induced hyperalgesia………….…...…………….17
2. Objectives……………………………...……………...19
3. Animals, material and methods………….…………..20
3.1. Materials……………..……………………………….…….…20
3.1.1. Cell lines and bacteria…………………………………..…...…………20
3.1.2. Animals and animal housing………………………………….……….20
3.1.3. Chemicals…………………………………...…………………………..20
3.1.4. Media, buffer………………………..………………………………….22
3.1.5. Reaction systems………………………..………………………….…...23
3.1.6. Expendable materials……………………………………….…….……24
3.1.7. Technical equipment………………………………………………..….24
3.1.8. Antibodies……………………………………………………………….25
3.2. Methods…………………….………………………………....25
3.2.1. Experimental procedure of animals………………...………………...25
Culture of dorsal root ganglion (DRG) neurons………………..……25
Behavioural experiments……………………….……………………...26 Table of contents
3.2.2. Cell biological techniques…………………………….………….…….27
Culture of HEK 293 and HEK 293 Tet-On cells…………..….……...27
Transient transfection…………………………………………....…….27
Transformation and amplification of plasmid-DNA…….…….….….30
Small interference RNA………………………….………………….....31
3.2.3. Calcium Imaging experiments………………………....……………...31
3.2.4. Electrophysiology………………………………………..……………..33
Patch Clamp experiments………………………….…………………..33
3.2.5. Radioligand receptor binding studies…………………………………35
3.2.6. Immunoprecipitation / co-immunoprecipitation………………..……37
3.2.7. Western Blot analysis……………………………..……………………38
3.2.8. cAMP Enzyme-linked Immunosorbant Assay (ELISA)……….…….39
3.2.9. Statistical analysis……………………………...……...………………..40
4. Results…………………………………………………42
4.1. Interaction of TRPV1 and μ-opioid receptor during
opioid withdrawal……………………………………….……42
4.1.1. TRPV1 activity and expression during opioid withdrawal …………42
Phosphorylation of TRPV1 during opioid withdrawal………...…….44
4.1.2. Mutant TRPV1 activity during opioid withdrawal…………………..44
4.1.3. Role of adenylylcyclases during opioid withdrawal………………….47
4.1.4. Effects of opioid withdrawal in vivo……………………………..…….49
Thermal hypersensitivity during opioid withdrawal………..……….49
Nocifensive behaviour during opioid withdrawal…………...……….50
4.2. Interaction of TRPV1 and TRPA1……………………….…51
4.2.1. Physical interaction of TRPV1 and TRPA1………………….……….51
4.2.2. Interaction of TRPV1 and TRPA1 via signalling pathways……...….53
Modulation of TRPV1 activity after MuO induced
TRPA1 stimulation……………………………………………………..54
Table of contents
Change of the intracellular cAMP concentration after TRPA1
activation…………………………………………………….…...……..56
Phosphorylation of TRPV1………………..….……………………….56
Modulation of mutant TRPV1 activity after MuO induced TRPA1
activation………………………………………………………………..57
Modulation of TRPV1 activity after MuO pretreatment in DRG
neurons………………………………………………...……………….58
5. Discussion……………………………………………..60
5.1. Hypothesis 1: Increased TRPV1 activity during opioid
withdrawal is dependent on the presence of adenylylcyclases
and on phosphorylation of TRPV1 at specific
phosphorylation sites……………………………………...….61
5.1.1. Increased TRPV1 activity during opioid withdrawal………..………61
5.1.2. TRPV1 expression and opioid withdrawal ………………..…………63
5.1.3. Increased phosphorylation of TRPV1 during opioid withdrawal.….64
5.1.4. Mutation of threonine 144 and serine 774, but not serine 116 and
serine 502, resulted in a loss of increased TRPV1 activity during
opioid withdrawal....................................................................................65
5.1.5. Downregulation of AC 3, but not 5, led to a reversal of the enhanced
TRPV1 activity during opioid withdrawal……………………………66
5.1.6. Paw withdrawal latency and nocifensive behaviour during opioid
withdrawal in male Wistar rats…………………………………...…..68
5.2. Hypothesis 2: TRPA1 stimulation modulates TRPV1
activity……………………………………………………..….70
5.2.1. TRPA1 stimulation does not alter the expression of TRPV1…...…..70
5.2.2. TRPV1 and TRPA1 do not form complexes in transfected
HEK Tet-On cells………………………………..……………………..71
Table of contents
5.2.3. TRPA1 stimulation increases TRPV1 activity in a calcium and
cAMP dependent manner …………………………………….……….72
5.2.4. TRPV1 is phosphorylated after TRPA1 stimulation …………..…....73
5.2.5. Mutation of TRPV1 phophorylation sites reversed the increased
TRPV1 activity after TRPA1 activation ………………………..…....74
5.2.6. TRPA1 stimulation enhanced TRPV1 currents in native DRG
neurons in a calcium and PKA-dependent manner…….……………75
5.3. Limitations, future prospects and clinical relevance …...…75
6. Summary……….……………...………………………79
7. References……………………….…………………….82
8. Curriculum vitae……………………………...………99
9. Publications and presentations………… ………….100
Acknowledgment……………………………...…………..102
Selbstständikeitserklärung...…………………………….103





Abbreviations
Abbreviation

A alanine
AC adenylylcyclase(s)
ASIC acid sensing ion channel
AgCl silver chloride
AKAP A kinase anchoring protein
AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
ANKTM1 / p120 old names for TRPA1
ANOVA analysis if variance
ATP adenosine triphosphate
BSA bovine serum albumin
CA cinnamaldehyde
CaCl calcium chloride 2
cAMP cyclic adenosine monophosphate
capsa capsaicin
CB cannabinoid receptor
Cdk5 cyclin-dependent kinase 5
cDNA copy desoxyribonucleic acid
CFA Complete Freund´s Adjuvant
CGRP calcitonin gene-related peptide
CIB Calcium Imaging Buffer
CREB cAMP response element binding protein
CTRL control
Da dalton
DAG diacylglycerol
2 4 5DAMGO D-Ala , N-MePhe , Gly -ol-enkephalin
DMSO dimethyl sulphoxide
DNA desoxyribonucleic acid
DOPA dihydroxyphenylalanine
DOR δ-opioid receptor
DRG dorsal root ganglion
DTT dithiothreitol
E. coli Escherichia coli
1 Abbreviations
ECS extracellular buffer
EDTA ethylene diamine tetraacetic acid
EGTA ethylene glycol tetraacetic acid
ERK extracellular signal regulated kinase
F340/F380 ratio of fluorescence at 340 nm to that at 380 nM
-15
f femto (10 )
FBS fetal bovine serum
g gram (s)
GFP green fluorescent protein
GPCR G-protein coupled receptor
HEK 293 human embryonic kidney cells 293
HEPES 4-2hydroxyethyl-1-piperazineethanesulfonic acid
I current
IB4 isolectin B4
IBMX isobutylmethylxanthin
ICS intracellular buffer
IP inositol triphosphate 3
JNK c-Jun N-terminal kinase
k kilo
K dissociation constant D
KCl potassium chloride
LB Luria-Bertani
LC Locus coeruleus
LTP long-term potentiation
M molar
MAPK mitogen activated protein kinase
MgCl magnesium chloride 2
min minute
ml milliliter
mM millimolare
MOR μ-opioid receptor
mRNA messenger RNA
MuO mustard oil
mV millivolt
2 Abbreviations
n number
nA nanoampere
NA nucleus accumbens
NGF nerve growth factor
nM nanomolar
nm nanometer
n.s. not significant
NLX nalaxone
NMDA N-methyl-D-asparate
OIH opioid induced hyperalgesia
OWIH opioid withdrawal induced hyperalgesia
pA picoampere
PBS phosphate buffered saline
PIP phosphatidylinositol bisphosphate 2
PKA protein kinase A
PKC protein kinase C
PLC phospholipase C
PTX pertussis toxin
PUFA polyunsaturated fatty acid
PVD polyvinylidene fluoride
RTX resiniferatoxin
s second
S serine
SP substance P
TG trigeminal ganglion
TM transmembrane domain
TNFα tumor necrosis factor alpha
TRIS tris (hydroxymethyl) amino-methane
TRP transient receptor potential
TRPA1 transient receptor potential ankyrin 1
TRPV1 transient receptor potential vanilloid 1
V volt
YFP yellow fluorescent protein
3 1. Introduction
1. Introduction

Injury and inflammation of peripheral tissue stimulates electrical activity of sensory dorsal
root ganglion (DRG) neurons (“nociceptors”). These impulses can be modulated by excitatory
and inhibitory ion channels and receptors, and are eventually transmitted to the central
nervous system where they are translated into the perception of “pain”. Among the most
prominent nociceptor membrane proteins are excitatory transient receptor potential (TRP)
channels and inhibitory opioid receptors. The interplay between these membrane proteins and
their signalling pathways shall be elucidated here.
The aims of this doctoral thesis are, first, to investigate the involvement of the excitatory ion
channel TRPV1 (Transient Receptor Potential Vanilloid 1) during withdrawal from inhibitory
(analgesic) drugs (opioids) and second, the influence of a related ion channel TRPA1
(Transient Receptor Potential Ankyrin 1) on TRPV1 activity. Besides inflammatory
mediators, both channels are activated by pungent components such as capsaicin (TRPV1)
and mustard oil (TRPA1), and play a critical role in pain sensation and in the development of
enhanced sensitivity to painful stimuli (“hyperalgesia”) typically associated with tissue injury.
Both channels are co-expressed in nociceptors.
Opioids produce analgesia (pain inhibition) by activation of G -protein-coupled opioid i
receptors and subsequent dampening of neuronal excitability. However, after prolonged
opioid treatment and abrupt withdrawal, paradoxical hyperalgesia can arise. Although the
precise molecular mechanism is not yet fully understood, this is generally thought to result
from neuroplastic changes in the peripheral and central nervous systems that lead to
sensitization of pronociceptive pathways. In the following, the role of TRPV1 in opioid
withdrawal-induced hyperalgesia will be investigated in the peripheral nervous system.
Behavioural studies indicated that, in addition to TRPV1, the TRPA1 channel also plays a key
role in pain transduction, especially during pathological conditions triggered by tissue damage
and inflammation. TRPV1-mediated responses in neurons have a characteristic voltage
dependency that is influenced by extracellular Ca2+ and by the type and concentration of
TRPV1-specific agonists. Because of the prominent role of both TRP channels in
inflammatory pain, we decided to investigate the functional relevance of interactions between
TRPA1 and TRPV1, and whether TRPV1-mediated responses can be modulated by TRPA1.


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