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Voltage-gated calcium fluxes studied in skeletal muscle fibers of genetically engineered mice [Elektronische Ressource] / Zoita Andronache

103 pages
Thesis for doctoral degree (Dr.biol.hum) Institute of Applied Physiology Ulm 2008 Zoita Andronache Voltage-Gated Calcium Fluxes Studied in Skeletal Muscle Fibers of Genetically Engineered Mice Thesis for doctoral degree (Dr.biol.hum) Institute of Applied Physiology Faculty of Medicine, Ulm University, Germany Prof. Dr.med. Dr.hc. Frank Lehmann-Horn Voltage-Gated Calcium Fluxes Studied in Skeletal Muscle Fibers of Genetically Engineered Mice Zoita Andronache Ulm 2008 Amtierender Dekan: Prof. Dr. Klaus-Michael Debatin 1. Berichterstatter: PD.Dr. Werner Melzer 2. Beichterstatter: Prof. Dr. Paul Dietl Tag der Promotion: 21.07.2008 Pentru parintii mei, fratele si Eduard  TABLE OF CONTENTS LIST OF ABBREVIATION 1. INTRODUCTION...................................
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Thesis for doctoral degree (Dr.biol.hum)
Institute of Applied Physiology
Ulm 2008



Zoita Andronache




Voltage-Gated Calcium Fluxes Studied in
Skeletal Muscle Fibers of Genetically
Engineered Mice






















Thesis for doctoral degree (Dr.biol.hum)





Institute of Applied Physiology
Faculty of Medicine, Ulm University, Germany
Prof. Dr.med. Dr.hc. Frank Lehmann-Horn




Voltage-Gated Calcium Fluxes Studied in Skeletal
Muscle Fibers of Genetically Engineered Mice



Zoita Andronache

Ulm 2008















Amtierender Dekan: Prof. Dr. Klaus-Michael Debatin
1. Berichterstatter: PD.Dr. Werner Melzer
2. Beichterstatter: Prof. Dr. Paul Dietl
Tag der Promotion: 21.07.2008














































Pentru parintii mei, fratele si Eduard







TABLE OF CONTENTS


LIST OF ABBREVIATION


1. INTRODUCTION......................................................................................... 1
1.1 Excitation-contraction coupling machinery................................................................. 1
1.2 Signals in EC coupling under voltage-clamp condition. ............................................. 2
1.3 Dihydropyridine receptor ............................................................................................ 2
1.3.1 Modulatory subunits of the skeletal muscle dihydropyridine receptor ................ 4
1.3.2 γ subunit............................................................................................................... 5 1
2+1.3.3 Ca antagonists in skeletal EC-coupling ............................................................ 6
1.4 Ryanodine receptor...................................................................................................... 7
2+ 2+1.4.1 Modulation of RyR by Ca and Mg .................................................................. 8
1.5 Malignant Hyperthermia.............................................................................................. 9
1.5.1 Ryanodine Receptor 1 Y522S knock-in mouse ................................................... 11
1.6 Aims of the study....................................................................................................... 12

2. MATERIALS AND METHODS................................................................ 13
2.1 Animal preparation.................................................................................................... 13
2.2 Solutions .................................................................................................................... 13
2.3 Voltage-clamp on isolated single fibers .................................................................... 14
2.3.1 Voltage-clamp and data acquisition................................................................... 14
2.3.2 Fluorescence recordings .................................................................................... 16
2+2.3.3 Ca current analysis ......................................................................................... 16
2+2.3.4 Ca input flux analysis...................................................................................... 17
2+2.3.5 Ca removal analysis during prolonged fiber perfusion .................................. 20

3. RESULTS 26
2+3.1 Interaction between Ca antagonist D888 and DHPR γ subunit ............................ 26 1
3.1.1 Voltage-dependent activation............................................................................. 26
3.1.2 Voltage-dependent inactivation.......................................................................... 28
3.1.3 Slow recovery from inactivation......................................................................... 31
3.2 EC coupling in the mouse heterozygous for the Y522S mutation in RyR1.............. 40
2+ 2+3.2.1 Voltage-dependent activation of Ca release and Ca current....................... 40
2+3.2.2 Voltage-activated Ca release permeability ..................................................... 43
2+ 2+3.2.3 Time course of Ca release and Ca current activation ................................. 44
3.2.4 Voltage-dependent inactivation 47
2+ 2+3.2.5 Window Ca release and window Ca current................................................ 48
2+ 2+3.2.6 Time course of Ca release and Ca current inactivation .............................. 51
2+3.2.7 Recovery from fast Ca -induced inactivation of release .................................. 52



4. DISCUSSION ............................................................................................. 55
2+ 4.1 Ca antagonist D888 and γ subunit interaction....................................................... 55 1
4.2 Functional effects of RyR mutation Y522S on EC - coupling................................. 60 1

5. SUMMARY ................................................................................................ 67
6. REFERENCES............................................................................................ 69
7. ACKNOWLEDGEMENTS ........................................................................ 86
8. APPENDIX ................................................................................................. 88






































List of Abbreviations

4-AP 4-Aminopyridine
ATPAdenosinetriphosphate
BTS N-benzyl-p-toluene sulphonamide
CCDCentral coredesease
CICR Calcium-induced calcium release
DHPR Dihydropyridine receptor
DMSODimethyl sulfoxide
ECC Excitation-contraction coupling
EGTAEhylenglycol-bis-(2-aminoethyl)-N,N,N’,N’,tetraacetic acid
HEPES N-[1-Hydroxyethyl]-piperazin-N’[2-Ethansulfonic acid]
K Dissociation constant D
ktion rate constant off
k Binding rate constant on
L-type Long lasting calcium channels
MH Malignant hyperthermia
PAAPhenylankyamine
RyRRyanodinereceptor
SEM Standard error of the mean
SR Sarcoplasmatic reticulum
TEATetraethylammonium
TEVC Two electrode voltage clamp
TTX Tetrodotoxin
γ-/- knockout mice for the DHPR γ subunit 1
Y522S+/- transgenic mice heterozygous for the Y522S mutation in the
RyR1
WT Wild type


1. Introduction



1. INTRODUCTION


1.1 Excitation-contraction coupling machinery

Excitation-contraction (EC) coupling is the physiological process that describes the
converting of the electrical stimulus to mechanical response. In muscle physiology, the
electrical signal is usually an action potential and the mechanical response is contracture.
The muscle cell membrane invaginates to form a network of transverse (or T-) tubules that
span the cross section of the muscle fiber, transmitting the depolarization signal uniformly
2+throughout the cell. Contraction is regulated by the cytoplasmic calcium (Ca ) ion
2+concentration. In the resting state, a fiber keeps most of its intracellular Ca sequestered in
an extensive system of vesicles known as the sarcoplasmic reticulum (SR). Once released,
2+ 2+ Ca binds to troponin, and force is produced. The basis for the control of Ca release in
skeletal muscle is a direct functional and structural interaction between the dihydropyridine
receptor (DHPR) located in the T-tubules and the ryanodine receptor (RyR) in the terminal
cisternae of the SR.
The DHPR is a voltage sensor that undergoes conformational changes in response to
2+depolarization of the plasmalemma. DHPR is also a voltage-dependent Ca channel which
2+ 2+mediates the entry of extracellular Ca . In cardiac muscle, entry of Ca through the
DHPR activates ryanodine receptor 2, RyR2 (cardiac isoform of RyR), causing the release
2+ 2+ 2+of Ca from the SR (Ca -induced Ca release mechanism) (Nabauer et al., 1989). In
2+skeletal muscle, however, entry of Ca through the DHPR is not required for E-C coupling
(Tanabe et al., 1988). Instead, voltage-dependent conformational changes in the DHPR
produce a signal (orthograde signal) which has been hypothesized to allosterically activate
ryanodine receptor 1, RyR1 (skeletal muscle isoform of RyR) (Schneider & Chandler,
1973, Tanabe et al., 1990, Adams et al., 1990, Rios & Brum, 1987). This signal is thought
to be transmitted through a physical, possibly direct link between DHPR and RyR1
2+ 2+(“mechanical-link” mechanism) (Protasi, 2002). Ca -induced Ca release was proposed
to be supplementary to the direct molecular coupling in skeletal muscle (Pizarro et al.,
2+1991). The presence of the RyR1 protein promotes the Ca conducting activity and
1 Zoita Andronache

2+accelerates the activation of skeletal L-type Ca channels ("retrograde signal") (Fleig et
al., 1996, Nakai et al., 1996, avila & Dirksen, 2000). The precise mechanism for the
retrograde signaling in skeletal muscle is still not clear. Thus, the signaling between the
skeletal muscle DHPR and RyR1 is bidirectional, such that the channel activity associated
with each protein is strongly dependent upon this unique interaction.


1.2 Signals in EC coupling under voltage-clamp condition.

2+The physiological stimulus for Ca release is the action potential, a brief membrane
depolarization that is sensed by the voltage-sensitive calcium channel, the DHPR located
in the TT membrane. Due to the intimate DHPR-RyR1 interaction, state changes of the
2+ 2+ 2+DHPR control both Ca fluxes, i.e. Ca entry from the extracellular space and Ca
release from the SR. To assess EC coupling function under voltage control condition
2+several signals can be measured including: voltage sensor charge movements, Ca -current
2+and Ca release. Upon membrane depolarization, the DHPR exhibits a rapid intra-
membrane charge movement measurable as a non-linear component of the capacitive
2+current (Melzer et al., 1986). This results in massive Ca release from the SR. The flux of
2+Ca from the SR peaks within less than 10 ms during depolarization. The peak is followed
2+by a rapid, presumably Ca -induced partial inactivation (Melzer et al., 1987, Simon et al.,
2+ 2+1991, Jong et al., 1993). The Ca -inward current through L-type Ca -channel of skeletal
muscle exhibits slow activation kinetics reaching its maximum conductance in about 100
ms of depolarization (Ursu et al., 2005) (Results, Fig.3.1). During long lasting
2+depolarization, the DHPR enters slowly into an inactivated state affecting both Ca current
2+and Ca release (Brum et al., 1988, Pizarro et al., 1988, and Ríos & Pizarro, 1991)
(Results, Fig.3.3). The “availability” of the channels is studied by applying a secondary test
pulse and measuring the residual signals (Results, Fig.3.5). As seen later in the results, the
speed of this inactivation and the extent of recovery from the inactive state are functions of
voltage.


1.3 Dihydropyridine receptor

2+L-Type Ca channels, also known as dihydropyridine receptors (DHPRs) due to their high
affinity to dihydropyridine drugs, are hetero-oligomeric membrane protein complexes with
2