Aus der Klinik für Anästhesiologie
der
Friedrich-Alexander-Universität Erlangen-Nürnberg
Direktor: Prof. Dr. med. Dr. h.c. Jürgen Schüttler
Effect of tolperisone on rat skeletal muscle, human heart
and sensory neuron specific sodium channels
Inaugural-Dissertation
zur Erlangung der Doktorwürde
der Medizinischen Fakultät
der
Friedrich-Alexander-Universität
Erlangen-Nürnberg
Vorgelegt von
Doris Rohde
aus
Erlangen
Gedruckt mit Erlaubnis der
Medizinischen Fakultät der Friedrich-Alexander-Universität
Erlangen-Nürnberg
Dekan:
Prof. Dr. J. Schüttler
Referent:
Prof. Dr. C. Nau
Korreferent:
Prof. Dr. P. Reeh
Tag der mündlichen Prüfung:
15. Dezember 2010
Für meine Mutter und Luigi
Inhaltsverzeichnis
Seite
1.
Zusammenfassung
1
2.
Introduction
4
2.1.
Mechanisms of pain and nociception
4
2.2.
Voltage-gated sodium channels
8
2.3.
Aims of the study
9
3.
4.
Methods
14
3.1.
Site-directed mutagenesis and transient transfection
14
3.2.
Chemicals and solutions
15
3.3.
Patch-clamp technique
16
3.4.
Electrophysiology and data acquisition
22
Results
23
4.1.
Comparison of Tolperisone and Lidocaine Action on
µ1 Wild-Type Na+ Channels
23
4.2.
Block of Mutant Nav 1.4 Channels by Tolperisone
26
4.3.
Voltage-dependent Block of Nav 1.5 and Nav 1.8
Wild-Type Channels
29
4.4.
Inactivation Kinetics of Nav 1.8 Channels and Recovery
from Block by Tolperisone
30
4.5.
State-Dependent Block of Nav 1.4, Nav 1.5 and Nav 1.8
Channels by Tolperisone
31
4.6.
Tolperisone and Lidocaine Block of Inactivation-deficient
Nav 1.4-WCW sodium channels
33
5.
Discussion
35
6.
References
38
7.
Abkürzungsverzeichnis
43
8.
Danksagung
45
9.
Lebenslauf
46
-1-
1.
D i e W i r k u n g v o n To l p e r i s o n , e i n e m o r a l a p p l i z i e r t e n
Natriumkanalblocker zur Behandlung schmerzhafter Muskelverspannungen,
auf muskuläre, kardiale und neuronale Natriumkanäle
Hintergrund und Ziele
Tolperison (Mydocalm®) wird als „Lokalanästhesie zum Schlucken“ vermarktet und
ist zugelassen zur Behandlung von schmerzhaften Muskelverspannungen bei
degenerativen Veränderungen des Bewegungsapparates sowie zur Behandlung von
Spastizität bei neurologischen Erkrankungen. Tolperison weist strukturelle
Ähnlichkeiten mit Lidocain auf, trägt allerdings eine Ketongruppe anstelle einer
Amidgruppe. Ziel dieser Studie war zu untersuchen, ob Tolperison mit der
Bindungsstelle für Lokalanästhetika spannungsabhängiger Natriumkanäle interagiert,
ob Tolperison unterschiedliche Wirkungen auf muskuläre Nav1.4, kardiale Nav1.5
und neuronale Nav1.8 Kanäle hat und ob sich die Wirkung von Tolperison und
Lidocain auf Nav1.4 Kanäle unterscheidet.
Methoden
Heterologe transiente Expression.
HEK 293t bzw. ND7/23 Zellen wurden mit Hilfe
der Kalzium-Phosphat Methode transient transfiziert mit den Wild-Typen der Na+Kanäle Nav1.4-WT, Nav1.5-WT und Nav1.8-WT bzw. mit den Mutationen Nav1.4F1579K, Nav1.4-L1280K, Nav1.4-N434K und Nav1.4-WCW (10µg). Als Reporter
Antigen wurde CD8-pih3m (1-2µg) co-transfiziert, so dass transfektions-positive
Zellen mittels anti-CD8 Immunperlen (CD8-Dynabeads, Dynal A.S., Oslo, Norway)
zu identifizieren waren.
Lösungen.
Tolperison und Lidocain wurden in DMSO gelöst, um Stocklösungen
in einer Konzentration von 100 mM zu erhalten. Die Extrazellulärlösung enthielt (in
mM) 65 NaCl, 85 Choline Cl, 2 CaCl2 und 10 Hepes (pH 7.4, eingestellt mit TMA-
-2-
OH). Die Pipettenlösung enthielt (in mM) 100 NaF, 30 NaCl, 10 EGTA und 10 Hepes
(pH 7.2, eingestellt mit CsOH).
Elektrophysiologie und Datenerhebung.
Die Natriumströme wurden bei
Zimmertemperatur mit Hilfe der Whole-Cell Konfiguration der Patch-Clamp
Methode gemessen. Das Haltepotential betrug –140 mV. Natriumströme wurden mit
einem Axopatch 200B Patch Clamp Verstärker (Axon Instruments, Union City, CA)
aufgezeichnet. Die Filter-Frequenz betrug 5 kHz, die Sample-Frequenz betrug 20
kHz. Das Programm pCLAMP 8.0 (Axon Instruments) diente zur Erhebung und
Analyse der Daten. Für die statistische Auswertung und die Erstellung von Graphiken
wurde das Programm Origin 6.1 (Origin Lab Corporation, Northampton, MA)
angewendet.
Ergebnisse und Beobachtungen
Die Wirkung von Tolperison auf den muskulären Nav1.4-WT-Kanal und auf dessen
Mutationen F1579K, L1280K und N434K wurden untersucht. Diese Mutationen
befinden sich im Bereich der Lokalanästhetikabindungsstelle spannungsabhängiger
Natriumkanäle. Es wurde der spannungsabhängige Block auf ruhende und
inaktivierte Kanäle bestimmt. Dabei wurden ruhende Kanäle durch negativere
Vorpulspotentiale und inaktivierte Kanäle durch positivere Vorpulspotentiale
charakterisiert. Es zeigte sich, dass der Block für ruhende Kanäle keinen signifikanten
Unterschied zwischen dem WT-Kanal und den Mutationen aufweist, während der
Block für inaktivierte Kanäle bei den Mutationen deutlich geringer ausfällt als am
WT-Kanal. Weiterhin wurde mittels Dosis-Wirkungs-Experimenten die Wirkung von
Tolperison auf ruhende und inaktivierte Nav1.4, Nav1.5 und Nav1.8 Kanäle
untersucht. Es stellte sich heraus, dass die ermittelten IC50 Werte sowohl für ruhende
als auch für inaktivierte Kanäle keinen signifikanten Unterschied zwischen den
Natriumkanal Isoformen zeigen. Zuletzt wurde wiederum mittels DosisWirkungsexperimenten die Wirkung von Tolperison und Lidocain am muskulären
Nav-1.4 Kanal verglichen. Die Experimente ergaben, dass Tolperison mit 143 µM
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einen deutlich niedrigeren IC50 Wert hat als Lidocain, dessen IC50 Wert 528 µM
beträgt.
Praktische Schlussfolgerungen
Tolperison interagiert mit der Lokalanästhetikabindungsstelle spannungsabhängiger
Natriumkanäle und zeigt dabei keine Isoform Spezifität zwischen muskulären,
kardialen und neuronalen Natriumkanälen. Zudem ist Tolperison ca. 4-fach potenter
als Lidocain am muskulären Natriumkanal.
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2.
INTRODUCTION
2.1. Mechanisms of pain and nociception.
Pain experience is a highly complex process involving physiological, cognitive as
well as emotional aspects. In clinical practice, pain is generally classified as acute and
chronic. Acute pain is elicited by a noxious stimulus applied on healthy tissue. Pain
can be seen as a warning system which is meant to preserve the organism from
damage and injury by triggering protective reflexes. In the case of chronic pain, the
sensation of pain looses its protective function and becomes a debilitating, pathologic
process with no useful purpose. States of persistent pain can be initiated and
maintained by peripheral as well as central mechanisms. In chronic pain syndromes a
normally painful stimulus can lead to a painful sensation of greater intensity. This
phenomenon is called hyperalgesia. A normally nonpainful stimulus causing a painful
sensation, however, is referred to as allodynia. Generally chronic pain can be
subdivided into two classes: nociceptive and neuropathic pain. Nociceptive pain is a
result of direct activation of nociceptors caused by tissue damage leading to an
inflammatory process in the surrounding tissue. The inflammation will cause
persistent pain which can be associated with hyperalgesia and allodynia. Neuropathic
pain, on the other side, results from direct injury or disease of neuronal structures
either in the peripheral or central nervous system. It may have burning or electrical
character and can be combined with hyperalgesia and allodynia. In the periphery
direct nerve injury but also indirect damage through metabolic diseases may cause
neuropathic pain. Central pain may occur as a result of damage to central structures
such as the thalamus. Examples for neuropathic pain syndromes are reflex
sympathetic dystrophy, postherpetic neuralgia and phantom limb pain following
traumatic or surgical amputation of a limb.
The physiological processes involved in the experience of pain are transduction,
transmission, modulation and perception. Transduction describes the process of
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converting a noxious stimulus into an electrical impulse in form of an action
potential. This occurs in peripheral tissues where specialized sensory receptors called
nociceptors are activated by a noxious stimulus. These nociceptors are free nerve
endings of primary sensory neurons whose cell bodies are located in the dorsal root
ganglia of the spinal cord and the trigeminal ganglia for the body and the face,
respectively. These nociceptors are excited by a variety of stimuli which can be of
either mechanical, thermal or chemical nature. Most nociceptors are polymodal,
meaning that they detect more than one form of stimulus. Furthermore, nociceptors
can be distinguished from other sensory neurons as they exhibit distinct thresholds
allowing only high intensity stimuli to be transducted into an action potential.
Transmission defines the conduction of the electrical impulse in form of an action
potential from the peripheral nociceptor to the dorsal root ganglion in the spinal cord
and further to different regions of the brain. Fibers innervating the body and the head
originate from cell bodies in the trigeminal and dorsal root ganglia. There are three
main groups of fibers. Aβ fibers are myelinated, large diameter fibers with rapid
conduction velocity of mostly innocuous stimuli which do not result in painful
sensation. Medium diameter, myelinated Aδ fibers have conduction velocities of
approximately 5-30 m/s and are thought to mediate the acute sharp pain, whereas
unmyelinated, small diameter C fibers (conduction velocitiy ~ 1 m/s) mediate the
more delayed and dull pain after a noxious stimulus. Aδ fibers can be divided further
into two subclasses according to their threshold properties. Type I Aδ fibers respond
to chemical and mechanical stimuli and exhibit a high threshold for thermal stimuli at
approximately 53 °C. In contrast, type II Aδ fibers have a much lower threshold for
heat at approximately 43 °C, but they also detect noxious mechanical and chemical
stimuli. The unmyelinated C fibers are very heterogenous, detecting a wide range of
noxious mechanical, chemical and thermal stimuli. C fibers have a thermal threshold
at ~ 43 °C like the type II Aδ fibers. The cell bodies of these fibers lie within the
dorsal horn of the spinal cord where the signal is transmitted synaptically to the
-6-
ascending pathways. Six laminae can be distinguished in the dorsal horn based on
different cytological properties of the cells predominantly found in each lamina.
Primary afferent sensory fibers end in distinct layers of the dorsal horn based on
different modalities of these neurons. Nociceptive fibers of Aδ and C fibers end in the
superficial laminae I and II. Neurons in laminae III and IV mainly receive input from
Aβ fibers in form of nonnoxious stimuli. Lamina V plays a particular role as so-called
“wide dynamic range” (WDR) neurons receive a broad range of input directly from
Aβ and Aδ fibers and indirectly from C fibers. Moreover, these WDR neurons
respond to nociceptive input from visceral tissues. This convergence of peripheral
somatic as well as visceral nociceptive input in WDR neurons is thought to be the
origin of the so-called referred pain. In this case, pain resulting from an injury or
disease of a visceral tissue is projected to a somatic structure (e.g. in case of
myocardial infarction pain is referred to the left shoulder and arm). The major
neurotransmitter involved in synaptic transmission of nociceptive signals is the
excitatory amino acid glutamate which leads to activation of the AMPA-type
glutamate receptor. The major ascending pathways for pain are the spinothalamic,
spinoreticular, spinomesencephalic, cervicothalamic and spinohypothalamic tract,
respectively. The spinothalamic tract ascends on the contralateral side of the spinal
cord and terminates in the thamalus which acts as a relay for afferent information to
the central cortex. The perception of pain mainly occurs in the thamalus and cerebral
cortex. Regions involved in the affective component of pain are the amygdalae and
the gyrus cinguli as part of the limbic system. The spinohypothalamic tract ends in
the hypothalamus leading to endocrine reaction of the body to painful stimulus.
The alteration of pain transmission through inhibitory and excitatory mechanisms is
called modulation. Nociceptors do not only have an afferent function, but also exhibit
efferent properties leading to peripheral modulation and sensitization. In response to
tissue damage and inflammation a variety of mediators is released at the peripheral
end of the nociceptor including extracellular protons, arachidonic acid,
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prostaglandins, serotonin, bradykinin, potassium, and neuropeptides such as
substance P and calcitonin-gene-related peptide. These substances lead to the socalled neurogenic inflammation. This process leads to drop of activation thresholds in
nociceptors so that even normally innocuous stimuli may result in a painful sensation.
Central mechanisms of sensitization lead to a state of hyperexcitability within the
central nervous system following painful stimuli. This can occur through alterations
in glutamatergic transmission, loss of inhibitory controls as well as through glial
interactions with the neurons in the dorsal horn. Noxious stimuli may increase the
release of neurotransmitters in the dorsal horn which results in activation of silent
NMDA receptors, consequently increasing the calcium influx in secondary
postsynaptic neurons. Also the activation of second messenger pathways (PKA, PKC,
MAP-kinase) will lead to a further increase of neuronal excitability by modulating
receptor function. The loss of GABA-ergic and glycinergic inhibition in the dorsal
horn will also contribute to states of increased pain by enhancing depolarization of
postsynaptic neurons. Finally, after nerve injury microglia and astrocytes can be
inceasingly found in the dorsal horn where they produce cytokines such as TNF-α
and different interleukins which contribute to central sensitization.
-8-
Figure 1. Membrane topology of the alpha-subunit of the voltage-gated sodium channel. The diagram
shows the four homologous domains each consisting of six transmembrane segments. Intra- and
extracellular linkers connect the segments as well as the domains with each other. Positively charged
amino acid residues in the S4 segments serve for voltage sensing. S5 and S6 segments form the inner
pore of the channel. The intracellular linker between D3-S6 and D4-S1 is involved in the inactivation
of the channel. Diagram from Catterall W.A. (5).
2.2. Voltage-gated sodium channels.
The conduction of a pain signal along nerve fibers occurs by activation of a variety of
voltage-gated ion channels. Especially voltage-gated sodium and potassium channels
are involved in the generation and propagation of action potentials along nerve fibers.
At present, nine different isoforms of voltage-gated sodium channels have been
identified, which are expressed in different tissues such as cardiac muscle, skeletal
muscle or nerve cells. All isoforms of the voltage-gated sodium channel family
consist of one larger α-subunit (~ 260 kDa) containing approximately 2000 amino
acids and one or more β-subunits (β1-β3; ~33-36 kDa; see Figure 1). The nine
-9-
isoforms of voltage-gated sodium channels are identical in more than 50 % of the
amino acid sequence. Heterologous expression of the α-subunit of various isoforms
indicates that the α-subunit alone is essential for channel function forming the ionconducting channel pore. Coexpression of β-subunits modulates voltage-dependence
as well as kinetics of gating and increases the level of expression of the alpha-subunit.
Four transmembrane domains (D1-D4), each consisting of six alpha-helical
transmembrane segments (S1-S6), form the functional alpha-subunit. The various
segments are connected by intra- and extracellular linkers which are in part
glycosylated (linker D1-S5 to D1-S6). The four domains are linked by larger
intracellular loops. These domains assemble about the axis to form the functional unit
of the channel. Several amino acid residues have been identified to play important
roles in channel function. The S5 and S6 segments of each domain form the inner
wall of the channel pore (4) with the extracellular linker between S5 and S6, also
called P-loop, functioning as a selectivity filter. Positively charged amino acid
residues in S4 segments in each domain create the voltage sensor (23, 35).
Hydrophobic amino acid residues (isoleucine, phenylalanine, methionine) in the
intracellular D3-D4 linker can block the inner mouth of the pore, putatively forming
the inactivation gate of the channel (9, 36, 40). Sodium channel function can be
characterized by three key features first described and recorded by Hodgkin and
Huxley (17): voltage-dependent activation, rapid inactivation and sodium ion
selectivity. Sodium channels change their conformation depending on membrane
potential. The channel exists in three different conformational states: on the one hand
the open state which is ion-conducting, on the other hand the resting and the
inactivated state which both are not ion-conducting.
2.3. Aims of the study.
In this study, one of the predominant aims was to analyze tolperisone interaction with
the local anesthetic binding site of voltage-gated sodium channels. Furthermore,
- 10 -
tolperisone action on three different isoforms of the voltage-gated sodium channel
family, namely the rat skeletal muscle Nav 1.4 , the human heart Nav 1.5 and the
sensory neuron specific Nav 1.8 sodium channel was investigated. Finally, the
tolperisone and lidocaine block of the rat skeletal muscle sodium channel was
described in detail.
Tolperisone is a centrally acting muscle relaxant (23) which is used for the
treatment of muscle spasm and spasticity occuring in degenerative and inflammatory
diseases of the musculoskeletal and nervous system. In previous clinical studies,
tolperisone has been reported to be a reliable adjuvant to physiotherapy in alleviating
muscular pain (21). Other studies have also demonstrated the positive effects of
tolperisone on painful reflex muscle spasm (32) and spasticity following cerebral
stroke (34). Typical side effects of centrally acting muscle relaxants such as sedation,
dizziness and impairment of coordination did not appear during treatment with
tolperisone (8). Therefore, this drug is considered to be an alternative to other muscle
relaxants.
The pharmacological targets of tolperisone are voltage-gated sodium channels.
Previously, it has been shown that tolperisone inhibits mono- and polysynaptic reflex
pathways in animal spinal motoneurons (10). Moreover, it decreased sodium
permeability in single intact ranvier nodes (15). Voltage-gated sodium channels play
an important role in the origin of acute and chronic pain as they underlie modulation
and sensitization through inflammatory mediators.
The rat skeletal muscle Nav 1.4 sodium channel was included in our study because
tolperisone acts as a muscle relaxant, the Nav 1.5 channel was included because it
may be the cause of cardiac side effects such as arrhythmias. As the sensory neuron
specific SNS (Nav 1.8) sodium channels are of great significance concerning chronic
pain disorders, they were also included in this study (1, 24).
- 11 -
Figure 2. Molecular structure of tolperisone (Fig. 2A) and the local anesthetic lidocaine (Fig. 2B). The
two molecules have common chemical features such as a tertiary nitrogen, a carbonyl group as well as
an aromatic ring. Tolperisone contains a ketone group instead of an amide group.
The chemical structure of tolperisone is very similar to that of the local
anesthetic lidocaine (see Figure 2). In fact, a study of Fels (11) has demonstrated that
both drugs exhibit a closely related three-dimensional geometry. It is therefore
conceivable that tolperisone acts on the same receptor site as lidocaine. The common
structure of voltage-gated sodium channels consists of an α subunit, forming the
functional unit of the channel, combined with one or more smaller β subunits. The
complex α subunit is composed of four domains (D1-D4), each one having six
transmembrane segments (42). The putative local anesthetic binding site of voltagegated sodium channels consists of the amino acid sequence of the S6 segments of
- 12 -
domains 1, 3 and 4 (38). We investigated whether tolperisone interacts with the local
anesthetic binding site by creating three lysine point mutations of the rat skeletal
muscle Nav 1.4 sodium channel. These point mutations at position 434, 1280 and
1579 in amino acid sequence lie within the sixth segments of domains 1, 3 and 4,
respectively. They are thus part of the putative local anesthetic binding site. By
substituting amino acid residues with lysine, we introduced a positive charge at
positions 434, 1280 and 1579 in amino acid sequence, thus altering the interaction
between the amino acid residue and the local anesthetic. It has been shown that the
local anesthetic block by bupivacaine was reduced in the inactivated state of the
mutant N434K (27). Moreover, the mutant L1280K displays a strong reduction in
bupivacaine affinity for the inactivated state (39). Finally, lysine mutation at F1579
also reduces the local anesthetic block of inactivated channels (41).
- 13 -
Figure 3. This diagram schematically shows the steps of site-directed mutagenesis using the
TransformerTM Site-Directed-Mutagenesis Kit (Clontech Laboratories, Inc.). Diagram from
TranformerTM Site-Directed Mutagenesis Kit User Manual.
- 14 -
3.
METHODS
3.1. Site-directed mutagenesis and transient transfection.
Site-directed mutagenesis of rat skeletal muscle Nav1.4-cDNA was performed with
the TransformerTM Site-Directed-Mutagenesis Kit (Clontech Laboratories, Inc.) to
create point mutations at specific sites of amino acid sequence. This method was first
described in 1978 by Hutchinson CA et al (18). Figure 3 shows the steps of sitedirected mutagenesis. This technique allows introducing specific mutations (single or
multiple base changes, deletions or insertions) into a defined region in a DNA
molecule. Double strand plasmids containing the gene of interest and a unique
restriction enzyme site are denatured into single-strand plasmids. The unique
restriction enzyme site is required to allow selection between mutated and unmutated
parental plasmids. The single-strand plasmids are re-annealed with a selection primer
as well as the mutagenic primer containing the mutation to be introduced. With DNA
polymerase the second strand is synthesized and gaps are sealed with DNA ligase
creating a hybrid mutated-parental plasmid. After undergoing DNA elongation and
ligation the mutated and unmutated plasmids are transformed into E.coli strain which
lacks mismatch repair thus preventing repair of the mutant DNA strand. Then, the
plasmid DNA is isolated from the bacterial pool and subjected to digestion with a
selective restriction enzyme. As the mutated DNA plasmids lack the selective
restriction enzyme they are resistant to digestion and will be preserved whereas
parental DNA will be eliminated. Final transformation is performed to amplify and
clone the mutated plasmid. This technique will yield a mutation efficiency of >
70-90%. The culture of human embryonic kidney (HEK) 293t cells and their
transfection by the calcium phosphate precipitation method have been described
previously (3). Calcium phosphate precipitation is widely used for transfection. This
method was first systematically examined and described by Graham and van der Eb
in 1973 (12). In their study the effect of different cations, cationic and phosphate
- 15 -
solutions on transfection parameters was analyzed. This method can be used for
transfection of many different types of cultured cells. The DNA which is to be
transfected is mixed with calcium chloride and added to a buffered saline/phosphate
solution. Then this mixture incubates at room temperature generating a precipitate
which is added to the cultured cells. Via endocytosis and phagocytosis the cells will
take up the DNA precipitate.
In this study, the alpha subunits of Nav 1.4, Nav 1.5 as well as the mutants Nav 1.4F1579K, L1280K, N434K, WCW and reporter plasmid CD8-pih3m were transiently
transfected in HEK 293t cells. The alpha subunit of the Nav 1.8 channel was
transiently transfected together with reporter plasmid CD8-pih3m and the β1-subunit
in the dorsal root ganglion neuroblastoma cell line ND7-23. This cell line was chosen
as it has been proven to reliably express Nav 1.8-cDNA in comparison to other
heterologous expression systems (19). Approximately 10 µg of Nav 1.4, Nav 1.5, Nav
1.8 and mutant plasmids, 5 µg of β1-subunit plasmid and 1-2 µg of reporter plasmid
CD8 were used for transfection. Cells were grown to 50 % confluence in Ti25 flask
before transfection in Dulbecco’s modified Eagle’s medium (Life Technologies,
GIBCO BRL), containing 10 % fetal bovine serum (Life Technologies, GIBCO
BRL), 1 % penicillin and streptomycin solution (Life Technologies, GIBCO BRL), 3
mM taurine, and 25 mM HEPES (Sigma Chemicals, St. Louis, MO). For transfection,
cells rested for 12 hours and were then replated in 35 mm culture dishes. Transfected
cells were used for experiments within 1-3 days. Transfection positive cells were
identified with CD8-dynabeads (Dynal A.S., Oslo, Norway).
3.2. Chemicals and Solutions.
Tolperisone (CHEMOS GmbH, Regenstauf, Germany) and lidocaine (Sigma
Chemicals, St. Louis, MO) were dissolved in dimethylsulfoxide (DMSO) to obtain
100 mM stocks. For electrophysiological experiments, the substances were diluted to
the concentration required. The internal solution for pipettes contained 100 mM NaF,
- 16 -
30 mM NaCl, 10 mM EGTA and 10 mM HEPES adjusted to pH 7.2 with CsOH. The
external solution consisted of 65 mM NaCl, 85 mM choline chloride, 2 mM CaCl2
and 10 mM HEPES titrated with TMA-OH (tetramethylammonium-hydroxide) to pH
7.4.
3.3 Patch-Clamp Technique.
Today the patch-clamp technique represents one of the fundamental experimental
methods in electrophysiological research allowing detailed analysis of ion channel
function in cell membranes. In history, the beginnings of electrophysiology go back
to the mid 16th century when the dutch scientist Jan Swammerham (6) first developed
a neuromuscular preparation from a frog’s thigh. He found that the stimulation of a
nerve would trigger the contraction of the adherent muscle. Less than a century later,
Luigi Galvani (Italian physician and scientist) published his study “De viribus
electricitatis in motu muscularis commentarius”, in which he presents his theory on
“animal electricity” (37). This was based on observations of nerve-muscle
preparations from frog legs and their isolated sciatic nerves. He postulated that
“animal electricity” results from a discrepancy of positive and negative charges on
the in- and outside of the nerve’s surface. Only in 1838 could the Italian Carlo
Matteucci measure directly with a galvanometer the electrical current generated by
muscle tissue (37). Subsequently, Emil du Bois-Reymond (Physiologist at Charité,
Berlin, Germany) detected an electrical response to stimulation of nerve or muscle
tissue with zinc/zinc-sulfate electrodes. He called this response “negative
Schwankungen” or “negative fluctuations” (37). In the years 1850-1852, Hermann
von Hemholtz (German physicist and physiologist) first determined the speed by
which electrical impulses are propagated along nerve fibres (14). In nerve-muscle
preparations, he measured the latency between an electrical stimulus of nerve fibre
and muscle contraction. The values he determined were about 25-40 m/sec. Julius
Bernstein later confirmed Hemholtz’s data with the first recordings of resting and
action potentials with a new experimental setup. The so-called “differential
- 17 -
rheotome” allowed the recording of very fast electrical impulses with a sampling rate
of approximately a few tens of milliseconds. For the first time ever, it was thus
possible to display the time course of action potentials. On the basis of his
experiments, Bernstein calculated the resting potential at approximately -60 mV and
postulated that potassium selectivity across a permeable membrane was responsible
for maintaining resting potential. Bernstein also determined the rising time of action
potential at about 0,3 ms and its duration at about 0,8-0,9 ms (2). Bernstein’s theory
of potassium ions being involved in stabilizing resting potential was further
developed by Charles Ernst Overton, who suggested that sodium ions are required for
action potential and that this process results from the exchange of sodium and
potassium ions (30). Overton also first proposed that the cell membrane had a lipidlike structure as he found that lipid soluble dyes would enter cells much more easily
than water soluble dyes. The lipid bilayer structure of cell membranes was finally
confirmed in the 1920’s and 1930’s by Gorter and Grendel (37) as well as Danielli
and Dawson (37). They also postulated that the lipid bilayer would contain proteins
as well as pores allowing the passage of hydrophilic substances such as ions.
Therefore by that time, the basic structure of cell membranes had been found, yet no
one had proven directly the existence of ion channels nor shown that channels were
responsible for the excitability of membranes.
In the late nineteen thirties Kenneth S. Cole and H.J. Curtis introduced the voltage
clamp technique. They used this new method to perform impedance measurements on
isolated axons from squid axons by using extracellular electrodes (7). With these
experiments they demonstrated that action potential leads to a rapid decrease in
membrane resistance. This indicated that the conductance of a nerve membrane was
significantly increased during the development of action potential. Between 1949 and
1952, Alan Hodgkin and Andrew Huxley employed the voltage clamp technique to
study the generation of action potentials in the giant squid axon. They performed
these experiments to demonstrate their hypothesis of a “carrier-model” being
- 18 -
responsible for the generation of action potentials. However, their experiments
yielded data that proved their first hypothesis to be false. They clearly found that
neuronal excitability was due to specific potassium and sodium currents. In several
experiments, the “carrier-model” could not explain the results, such as the almost
linear correlation between current and voltage. Finally, Hodgkin and Huxley
suggested a voltage-dependent gate to be involved in ion conductance across the
membrane (17).
Experiments performed by Hladky and Haydon (1970) with artificial membranes
resembling the phosholipid bilayer of living cell membranes (the so-called “black
film” or “Müller-Rudin-Membrane”) brought evidence of single pore-like structures
being involved in conductance changes in membranes (16). However, this could not
be applied to biological membranes of living cells. The methods available in the
1960’s and 1970’s were not suited for recordings of currents in living cells as
background noise levels were very high. Katz and Miledi (1972) used the method of
noise analysis to provide further information about ion conductance by analyzing the
properties of nicotinic acetylcholine receptors at the neuromuscular junction (20).
However, their experiments were based on the measurement of a large number of
channels and could not deliver detailed information about the function of single ion
channels.
The major limitation preventing detailed measurements of single-channel currents in
excitable cells was the background electrical noise which in conventional methods
was about 100 times higher than the current through a single ion channel. Glass
micropipettes had already been used since 1919 (31) for current measurements and
cell stimulations, but were associated with a background noise of at least 100 pA. In
the 1970’s, Erwin Neher and Bert Sakmann made efforts to monitor single
acetylcholine-activated channels in patches of frog skeletal muscle membranes by
improving the use of glass micropipettes. They first treated the surface of denervated
muscle fibres enzymatically in order to be able to place the tip of the micropipette
- 19 -
directly onto the cell membrane’s surface. The process of denervation garanteed that
acetylcholine channels would be dispersed all over the muscle fibre. Yet, the seal
resistances at the beginning were only about 10-20 megaohm and thus still too low to
decrease leakage and background noise enough to get good recordings. By reducing
the size of the pipette tip even further and by optimizing the tip’s shape, Neher and
Sakmann finally managed to record square pulses which could be interpreted as
signals of single acetylcholine channels. However, the pipette-to-membrane seal still
had quite a low resistance (megaohms), not permitting the recording of smaller and
shorter signals of other channel types. The amplitude of the different signals varied
significantly allowing only partial recording of channel signals. Almost by chance,
Neher and Sakmann noticed that by applying slight suction with a cleaned and
smooth micropipette onto a clean membrane surface the seal suddenly ranged in the
gigaohms (33). This seal – the so-called “gigaseal” – highly reduced the background
noise. From this point onwards, the gigaseal allowed reliable high resolution
recordings of all kinds of channels by providing electrical stability and a tight
mechanical connection between the pipette and the membrane surface. In the
following years, the patch clamp method became fundamental for the discovery of
many ion channel families.
The physical explanation for the so-called gigaseal has not been given until today.
The cell membrane is attached very tightly to the tip of the glass micropipette
allowing several variations of the initial technique settings without ever loosing the
tight contact between the micropipette and the membrane (see Figure 4). Generally,
cell-attached configurations can be distinguished from excised membrane patches. In
the cell-attached configuration, the tip of the micropipette is placed with slight
suction tightly onto the cell membrane in order to achieve the gigaseal. The cell
membrane remains intact under the micropipette so that all intracellular functions are
not altered. Also, the cell’s resting potential is not altered except directly in the area
under the micropipette where the potential can be controlled via the amplifier.
- 20 -
Figure 4. Schematic diagram of different patch clamp configurations. By creating a high resistance
seal (“gigaseal”) cell-attached configuration is established. By suction the membrane area under the
micropipette is ruptured and an electrical connection to the interior of the cell can be achieved. This
configuration is named “whole-cell recording”. Diagram from Hamill et al. (13).
In the inside-out configuration, the cytoplasmatic surface of the membrane is exposed
to the external bath solution. Starting with the cell-attached configuration, the pipette
is slowly pulled away from the cell surface until a piece of the membrane will
disconnect from the cell without influencing the seal resistance. In a similar manner,
- 21 -
the outside-out configuration is achieved starting out with the whole cell
configuration (see below). The inside of the micropipette is now connected with the
inner surface of the membrane and the bath solution corresponds to the extracellular
area. With this configuration ligand-gated channels can be analyzed easily by
applying substances from the extracellular side of the membrane to the patch. Today,
the whole-cell configuration is the most widely used technique of the patch clamp
method. To achieve the whole-cell configuration, the membrane in the cell-attached
configuration is ruptured under the micropipette by slight suction or short voltage
transients. The gigaseal must remain intact after rupturing the membrane in order to
allow the measurement of currents from the whole interior of the cell. The
intracellular side of the cell is connected directly with the micropipette, quickly
bringing the internal solution of the micropipette into equilibrium with the interior of
the cell.
The main components for the experimental setup for the patch clamp technique will
be described in the following section. An inverse microscope is needed to study
cultured cells consisting of one single layer. In this case, the object lens point
upwards so that the cell preparation is observed from underneath. In this way, more
space is left above the cell plate to place the micropipette, the perfusion system as
well as other apparatus. The table holding the cell plate must be fixed in order to
allow for better control of the pipette micromanipulator. To focus onto the cell
preparation, the object lens must be moved in relation to the table. The apparatus
must be placed onto a special table which attenuates vibrations from the environment.
To reduce background noise caused by the electromagnetic field (∼ 50 Hertz), a
faraday cage should surround the apparatus. A micromanipulator guarantees exact and
stable placing of the micropipette onto the cell membrane surface. The function
principle of the micromanipulator may be either mechanical, hydraulic or electrical.
To apply substances to the bath solution, a stable pipette holder combined with an
amplifier and a perfusion system are required. The electronical components of the
- 22 -
patch-clamp setup mainly consist of the headstage, the main amplifier, a low pass
filter as well as an AD/DA converter. The principle of voltage clamp allows to hold
the membrane potential stable at a certain level. This can be achieved by creating a
compensatory current which corresponds exactly to the current across the cell
membrane, but flowing in the counterwise direction. By a negative feedback
mechanism, the membrane potential is measured and compared to the reference
voltage. If there is a difference between the actual membrane and the reference
potential, a current will flow into the cell in order to compensate this difference. This
compensatory current can be measured with the patch-clamp technique. Thus, one
can determine the membrane conductance which is associated directly with the
activity of ionic channels. The headstage is fundamental for the control of the
membrane potential and to measure the compensatory currents. Essentially, the
headstage is a current-to-voltage converter with a high gain (operational amplifier or
OPA) and a feedback resistor. The OPA measures at its entrance into the cell the
voltage of the pipette as well as the reference voltage given by the experimenter. If
there is a difference between these two voltages, this difference will be amplified with
a high gain at the exit of the OPA, creating a current that will flow through the
feedback resistor to compensate voltage difference. This current will only flow into
the pipette because the resistance at the entry of the OPA is very high. The current
will persist until the pipette potential and the reference potential are equalized.
3.4. Electrophysiology and Data Acquisition.
Sodium currents were recorded with the whole-cell configuration of the patch-clamp
technique at room temperature ranging from 21 to 23°C (13). For experiments,
holding potential was stabilized at –140 mV. For experiments with ND7-23 cells, 200
nM tetrodotoxin was added to solutions in the glass perfusion system to block TTXsensitive sodium currents constitutively expressed in this cell line. Patch pipettes
were pulled from borosilicate glass tubes (TW150F-3, World Precision Instruments,
- 23 -
Sarasota, FL) and heat polished at the tip to give a resistance of 1.0-1.6 MΩ when
filled with internal solution. Cells were dialyzed for 10 to 20 minutes to equilibrate
with the pipette solution before data were acquired. Whole cell sodium currents were
recorded with Axopatch 200 B amplifier (Axon Instruments, Union City, CA),
filtered at 5 kHz and sampled at 20 kHz. All experiments were conducted during
capacitance cancellation and series resistance compensation of 60-70%. To acquire
and analyze currents, Clampex 8.1 software (Axon Instruments) was used. Curve
fitting was performed by Origin 6.1 software (OriginLab Corp., Northampton, MA).
4.
RESULTS
4.1. Comparison of Tolperisone and Lidocaine Action on µ1 Wild-Type Na+
Channels.
In this study we aimed at a detailed comparison between the tolperisone and lidocaine
block of Nav 1.4 wild-type channels. We therefore initially assessed concentration
dependence of block of resting Nav 1.4 channels by tolperisone (Fig. 5A) and
lidocaine (Fig. 5B). From a holding potential of -140 mV, test pulses to + 50 mV were
delivered to determine the block of resting Nav 1.4 channels. The IC50 values for the
block by tolperisone (A) and lidocaine (B) were 143.7 ± 3.6 µM and 528 ± 26 µM,
respectively. The IC50 value for tolperisone is therefore ~ 3.7-fold higher than that of
lidocaine. The Hill coefficients were close to unity which suggests a single binding
site for tolperisone as well as for lidocaine in Nav 1.4 wild-type channels.
Consecutively, we analyzed fast inactivation of Nav 1.4 wild-type channels in the
presence of equipotent doses of tolperisone and lidocaine. By referring to the
previously assessed concentration-inhibition experiments (Fig. 5A and 5B), we found
100 µM tolperisone and 300 µM lidocaine to be approximately equipotent
concentrations. We used a standard two-pulse protocol to determine voltage-
- 24 -
dependence of fast inactivation. Conditioning prepulses of 100 ms duration were
applied between -160 and -15 mV with steps of 5 mV before sodium currents were
evoked by a 5-ms test pulse to + 50 mV. Holding potential was -140 mV and pulses
were delivered at 15-s intervals. The experiments yielded V0.5 values of -79.6 ± 0.1
mV for tolperisone and 85.7 ± 0.1 mV for lidocaine. These V0.5 values showed no
statistically significant difference.
Finally, we determined the recovery time course from the block evoked by
tolperisone and lidocaine, respectively. We measured currents in the absence of drugs
and in the presence of 100 µM tolperisone (Fig. 5E) or lidocaine, respectively (Fig.
5F). These currents recovered from a 10-s prepulse to -70 mV with fast (T1) and slow
time constants (T2). In the presence of 100 µM lidocaine, a large fraction of the
current recovered with a fast time constant of 6.5 ± 1.4 ms and a smaller fraction with
a slow time constant of 0.20 ± 0.01 s. However, in the presence of 100 µM
tolperisone the recovery time course changed significantly: a small fraction of the
current (25%) recovered with a fast time constant of 2.9 ± 0.7 ms while a greater
fraction (73%) recovered with a slow time constant of 1.3 ± 0.1 s.
- 25 -
Figure 5. Comparison of the block of rat skeletal muscle Nav 1.4 channels by tolperisone and
lidocaine. (A, B) Concentration dependence of the block of resting Nav 1.4-WT channels by
tolperisone and lidocaine. To characterize the block of resting channels, test pulses up to +50 mV were
applied at 30-s intervals to evoke sodium currents. Holding potential was –140 mV. Sodium currents
- 26 -
were measured in different drug concentrations (A, tolperisone; B, lidocaine), normalized with respect
to the peak amplitude in control, and plotted against the drug concentration. Solid lines represent fits to
the data with the Hill equation. Hill coefficients are given in the diagram. (C, D) Fast inactivation of
µ1 wild-type sodium channels in the presence of tolperisone and lidocaine. Sodium currents were
evoked by a 5-ms test pulse to +50 mV after 100-ms conditioning prepulses between –160 and –15
mV in 5 mV increments. Pulses were delivered at 15-s intervals. Holding potential was –140 mV.
Sodium currents were measured in control and in the presence of 100 µM tolperisone (C) or 300 µM
lidocaine (D). Control currents were normalized to the current with a prepulse to –160 mV. Currents
measured in the presence of 100 µM tolperisone or 300 µM lidocaine were normalized to the current
obtained in control with the corresponding prepulse potential. Solid lines represent fits of the data to a
Boltzmann equation. V0.5 is the voltage at which 50 % of channels are inactivated and k is the slope
factor. (E, F) Recovery from the block of inactivated µ1 wild-type Na+ channels by tolperisone and
lidocaine. 10-s conditioning prepulses to –70 mV were applied from a holding potential of –140 mV.
Recovery was determined by a 5-ms test pulse to +50 mV applied at various times after conditioning
prepulse. Control currents (E, F; open circles) and currents in the presence of 100 µM tolperisone (E;
filled circles) or 100 µM lidocaine (F; filled squares) were normalized to the peak amplitude of the test
pulse obtained after 50-s recovery time. The data were best fitted by the sum of two exponentials (solid
lines). Fast (T1) and slow time constants (T2) are given in the diagram.
4.2. Block of Mutant Nav 1.4 Channels by Tolperisone.
To determine whether tolperisone shares a common receptor site on voltagedependent sodium channels with local anesthetics, we used site-directed mutagenesis
(see Materials and Methods) to create lysine point mutations at positions N434,
L1280 and F1579 in amino acid sequence of rat skeletal muscle Nav 1.4 channel.
These point mutations are known to be critical for LA binding affinity, especially in
the depolarized state of the channel (25). We compared the tolperisone voltagedependent block of the mutants F1579K (Fig. 6E), L1280K (Fig. 6F), and N434K
(Fig. 6G) with that of wild-type Nav 1.4 channels (Fig. 6D). To characterize both
resting and inactivated channels, we used a two-pulse protocol with 10-s conditioning
prepulses varying from -180 mV to -50 mV (Fig. 6C). Experiments were assessed for
mutant and wild-type channels in the absence of drug and in the presence of 100 µM
tolperisone. 100 µM tolperisone blocked approximately 33% of Nav -1.4 wild-type
- 27 -
channels at hyperpolarized prepulse voltages below -150 mV and approximately 82%
of Na+ currents by reaching a steady-state level at depolarized prepulse voltages
above -90 mV (Fig. 6D). However, the block of tolperisone of mutant Nav 1.4F1579K channels (Fig. 6E) showed a highly reduced binding affinity at depolarized
prepulse voltages with approximately only 50% of channels being blocked at
prepulse voltage of -50 mV. In a similar manner, the block of tolperisone of mutant
Nav L1280K (Fig. 6F) and Nav N434K (Fig. 6G) was reduced at depolarized prepulse
voltages. In mutant Nav L1280K channels tolperisone block of the inactivated state
showed almost no difference to the block of resting state. Also in mutant Nav-N434K
channels tolperisone block of inactivated channels at depolarized prepulse voltages
was reduced compared to wild-type channels with approximately only 60% of
channels being blocked. At depolarized prepulse voltages steady-state level was not
reached, indicating that tolperisone does not have two distinguishable binding
affinities in mutant Nav 1.4-F1579K, -L1280K and -N434K channels. This suggests
that tolperisone interacts with the local anesthetic binding site of voltage-gated
sodium channels. Moreover, tolperisone binding affinity in wild-type Nav 1.4
channels is low at hyperpolarized and high at depolarized prepulse voltages, showing
that tolperisone binding with Nav 1.4 wild-type channels is highly voltage-dependent.
- 28 -
- 29 -
Figure 6. Voltage-dependent block of Nav 1.4 wild-type and mutant Nav 1.4-F1579K, L1280K, and
N434K channels by tolperisone. (A, B) Exemplary sodium currents of µ1 wild-type (A) and mutant
µ1-F1579K (B) channels measured in control and in the presence of 100 µM tolperisone at a
conditioning prepulse of –70 mV. The pulse protocol as shown in (C) was used. (D, E, F, G) A 10-s
conditioning prepulse varying from –180 mV to –50 mV was applied, followed by a 100-ms interval at
holding potential of –140 mV. A 5-ms test pulse to +50 mV was then delivered to evoke Na+ currents.
The interval between pulses was 30 s. Control currents (open circles) were normalized to the current
with a prepulse to –180 mV. Currents measured in the presence of 100 µM tolperisone (filled circles)
were normalized to the current obtained in control with the corresponding prepulse potential. Solid
lines represent fits of the data to a Boltzmann function.
4.3. Voltage-dependent Block of Nav 1.5 and Nav 1.8 Wild-Type Channels.
To determine the voltage-dependent block of Nav 1.5 channels by 100 µM
tolperisone, we used the same two-step protocol as shown in Fig. 6C. As previous
studies have shown that sensory neuron specific Nav 1.8 channels exhibit different
electrophysiological properties concerning activation and inactivation kinetics (28,
29), we varied the two-step protocol by inserting conditioning prepulse potentials
ranging from -160 to -30 mV for assessing voltage-dependent block of Nav 1.8
channels by 100 µM tolperisone. At hyperpolarized prepulse voltages below -140
mV, tolperisone block of human cardiac Nav 1.5 channels (Fig. 7A) reached a steady
state-level with approximately 40% of channels being blocked. 100 µM tolperisone
blocked approximately 82% of Nav 1.5 channels by reaching a plateau at depolarized
prepulse voltages above -90 mV. In the experiments with Nav 1.8 channels (Fig. 7B),
a plateau was reached at hyperpolarized prepulse voltages below -140 mV with
approximately 43% of channels being blocked. In comparison to Nav 1.4 and Nav 1.5
channels (Fig. 6D and Fig. 7A, respectively) where a plateau was reached at
depolarized prepulse voltages above -90 mV, tolperisone block of Nav 1.8 channels
only reached a steady-state level at depolarized prepulse voltages above – 50 mV
with approximately 82% of channels being blocked.
- 30 -
4.4 Inactivation Kinetics of Nav 1.8 Channels and Recovery from Block by
Tolperisone.
To characterize the inactivation properties of Nav 1.8 channels in the presence of 100
µM tolperisone, we applied a two-step protocol with conditioning prepulses varying
from -120 to +40 mV. The midpoint potential of inactivation (V0.5) in the presence of
tolperisone was -60.1 ± 0.8 mV and was thus shifted by 6.7 mV in the hyperpolarized
direction in comparison to V0.5 in control. The slope factor k is given in Fig. 7C. We
then assessed the recovery time course of Nav 1.8 channels from a block by 100 µM
tolperisone (Fig. 7D). The channels recovered with fast (T1) and slow (T2) time
constants of 20.4 ± 5.6 ms and 2.25 ± 0.17 s, respectively.
Figure 7. (A, B) Voltage-dependent block of wild-type Nav 1.5 and Nav 1.8 sodium channels by
tolperisone. Sodium currents were measured in control (open circles) and in the presence of 100 µM
- 31 -
tolperisone (filled squares). (A) Cells were conditioned by a 10-s prepulse varying from –180 to –50
mV. After a 50-ms interval at holding potential of –140 mV, a 5-ms test pulse to +50 mV was delivered
to evoke sodium currents. (B) A 10-s conditioning prepulse, varying from –160 to –30 mV, was applied
followed by a 100-ms interval at holding potential of –140 mV. Then, a test pulse as described before
in (A), was delivered to evoke Na+ currents. In both diagrams (A) and (B) control currents were
normalized to the current with a prepulse to –180 mV and –160 mV, respectively. Currents measured in
the presence of 100 µM tolperisone were normalized to the current obtained in control with the
corresponding prepulse potential. Solid lines represent fits of the data to a Boltzmann equation. (C)
Fast inactivation of Nav 1.8 sodium channels in the presence of tolperisone. After a 100-ms
conditioning prepulse ranging from –120 to +40 mV in 5 mV increments, sodium currents were
evoked by a 5-ms test pulse to +50 mV. Pulses were delivered at 15-s intervals. Holding potential was
–140 mV. Sodium currents were measured in control (open circles) and in the presence of 100 µM
tolperisone (filled circles). Control currents were normalized with respect to the current obtained after
a prepulse to –120 mV. Currents measured in the presence of 100 µM tolperisone were normalized to
the current in control with the corresponding prepulse potential. Solid lines represent fits of the data to
a Boltzmann equation. V0.5 represents the voltage at which 50 % of channels are inactivated. K is the
slope factor. (D) Recovery of inactivated Nav 1.8 channels from block by tolperisone. From a holding
potential of –140 mV, a 10-s depolarizing prepulse to –40 mV was applied to condition cells. Recovery
was determined by applying a test pulse to +50 mV at various times after the conditioning prepulse.
Currents were measured in control (open circles) and in the presence of 100 µM tolperisone (filled
circles). The data were normalized to the amplitude of the test pulse obtained after 50-s recovery time.
The data were best fitted by the sum of two exponentials.
4.5. State-Dependent Block of Nav 1.4, Nav 1.5 and Nav 1.8 Channels by
Tolperisone.
In concentration-inhibtion experiments, we characterized more precisely the potency
of tolperisone action on the three sodium channel isoforms, namely the rat skeletal
muscle Nav 1.4 (Fig. 8A), the human heart Nav 1.5 (Fig. 8B), and the sensory neuron
specific Nav 1.8 (Fig. 8C) sodium channel. Based on the results from Fig. 6D and Fig.
7A, prepulse voltages of -140 mV and -70 mV were applied to characterize resting
and inactivated Nav 1.4 and Nav 1.5 channels, respectively. For analysis of Nav 1.8
channels, prepulse voltages of -140 mV for resting and -40 mV for inactivated
channels were used, based on the results of voltage-dependence experiments from
- 32 -
Fig. 7B. The IC50 values for resting Nav 1.4, Nav 1.5 and Nav 1.8 channels were 143.7
± 3.6 µM, 164.1 ± 9.2 µM, and 173.5 ± 6.9 µM, respectively. For inactivated Nav 1.4,
Nav 1.5 and Nav 1.8 channels IC50 values were 26.9 ± 4.1 µM, 27.0 ± 1.8 µM and
20.2 ± 1.9 µM.
Figure 8. State-dependent block of Nav 1.4, Nav 1.5 and Nav 1.8 wild-type channels by tolperisone. To
characterize block of resting channels (open squares), a test pulse to +50 mV was applied to evoke Na+
currents. Holding potential was stabilized at –140 mV. In order to characterize the inactivated state
(filled squares), 10-s conditioning prepulses to –70 mV (Nav 1.4, Nav 1.5) or to –40 mV (Nav 1.8) were
applied followed by a 100-ms interval at holding potential before a test pulse to +50 mV was delivered
to evoke Na+ currents. Pulses were delivered at 30-s intervals. Na+ currents were measured in different
drug concentrations and normalized with respect to the peak amplitude in control and plotted against
the drug concentration. Solid lines represent fits to the data with the Hill equation. Hill coefficients are
given in the diagrams.
- 33 -
For both resting and inactivated channels, 50% inhibitory concentrations showed no
statistically significant difference. Tolperisone was 5.3 times, 6 times and 8.5 times
more potent at depolarizing potentials than at hyperpolarizing potentials in Nav 1.4
channels, Nav 1.5 channels, and in Nav 1.8 channels, respectively. Hill coefficients
were calculated for resting and inactivated channels and were close to unity for all
three isoforms, suggesting that one tolperisone molecule is sufficient to block one
voltage-gated sodium channel. Tolperisone thus blocks resting and inactivated Nav
1.4, Nav 1.5, and Nav 1.8 channels with similar 50% inhibitory concentrations and
exhibits low affinity binding at hyperpolarized and high affinity binding at
depolarized potentials in the three sodium channel isoforms analyzed.
4.6. Tolperisone and Lidocaine Block of Inactivation-deficient Nav 1.4-WCW
sodium channels.
Wild-type sodium channels open only very shortly (~ 0,5 ms) during depolarization
before turning to the inactivated state. In order to directly assess open channel block
by tolperisone and lidocaine, we used inactivation-deficient mutant Nav1.4-WCW
channels. This channel exhibits three point mutations, namely L435W, L437C, and
A438W, located at the COOH terminus of D1S6. In this mutation, inactivation occurs
only minimally while peak current is maintained largely during the depolarization
phase. The open channel block was therefore determined by applying a 50 ms test
pulse to +50 mV with holding potential stabilized at –140 mV and interpulse intervals
being 30 seconds. Peak currents at the beginning of the test pulse represent the resting
channel block. We used various concentrations of tolperisone and lidocaine to create
the dose-response curves shown in Figs. 9A and 9B (see next page). For tolperisone,
IC50 values were 12.4 ± 0.9 µM and 93.8 ± 7.3 µM, for the open and resting channel
block, respectively. For lidocaine, IC50 were 20.4 ± 1.1 µM for open channel block
and 313.1 ± 16.1 µM for the resting channel block.
- 34 -
Figure 9. Block of inactivation-deficient Nav1.4-WCW channels by tolperisone and lidocaine. (A)
Exemplary currents of Nav1.4-WCW channels at various tolperisone concentrations. (B, C) To create
dose-response curves with tolperisone (B) and lidocaine (C), a test pulse to +50 mV was delivered for
a duration of 50 ms with interpulse intervals being 30 seconds. Holding potential was stabilized at –
140 mV. An open channel block was obtained by measuring the current amplitudes at the end of the 50
ms test pulse to +50 mV. For resting channel block peak currents were measured at the beginning of
the test pulse to +50 mV. Both were normalized to the highest current under control and then plotted
against the drug concentration. Solid lines represent fits to the data with the Hill equation. (C) The
decay phase of Na+ currents was fitted with a single exponential function. The corresponding time
constant (τ) was inverted and plotted against the corresponding drug concentration. Data were fitted
with a linear function y = kon ⋅ x + koff (solid line). Kon is the on-rate corresponding to the slope of the
fitted line, Koff is the off-rate corresponding to the y-intercept. The equilibrium dissociation constant
was determined by the equation KD = koff / kon. KD equalled 67 µM for tolperisone and 203 µM for
lidocaine.
- 35 -
5.
DISCUSSION
This study showed evidence for the following assumptions:
(1) The effect of tolperisone on Nav 1.4 sodium channels is approximately four times
stronger than lidocaine.
(2) The three mutations of Nav 1.4 channel, F1579K, L1280K, and N434K, show a
clear reduction in tolperisone affinity of inactivated channels, thus indicating that
tolperisone interacts with the local anesthetic binding site of voltage gated sodium
channels.
(3) The tolperisone block of voltage gated sodium channels is highly state dependent
and does not alter significantly between the Nav 1.4, Nav 1.5, and Nav 1.8 isoforms.
Tolperisone is a potent blocker of voltage-gated sodium channels.
Tolperisone has been described before to block voltage-gated sodium channels (15).
Yet, there are few studies which explore thoroughly the molecular mechanisms of the
interaction of tolperisone with sodium channels. In our study, we analyzed in detail
how tolperisone affects voltage gated channel action. We could show that tolperisone
is indeed a very potent voltage gated sodium channel blocker. In fact, compared to the
local anesthetic lidocaine, the tolperisone tonic block of resting Nav 1.4 channels is
approximately four fold stronger, with IC50 values for lidocaine of 528 ± 26 µM and
for tolperisone 143.7 ± 3.6 µM. Recovery time course from block by tolperisone of
µ1-channels also differs exceedingly from the lidocaine block. In the presence of
lidocaine, a large portion of the current showed a fast recovery from inactivation of
unblocked channels, whereas in the presence of tolperisone, the major portion (73%)
showed a slow recovery. This reflects the slower dissociation of tolperisone from
channels that were blocked during the conditioning prepulse.
Within the amino acid sequence of voltage-gated sodium channels, the sites for local
anesthetic binding have been studied in detail (26). We chose three mutations within
the amino acid sequence of the Nav-channel, namely F1579K, L1280K, and N434K,
- 36 -
to analyze tolperisone interaction with the local anesthetic binding site. As already
mentioned above (see introduction), the similar structure of lidocaine and tolperisone
may lead to the conclusion that both drugs interact with the same molecular targets
within the voltage-gated sodium channel (11). Our experiments showed that the
tolperisone affinity of inactivated channels is significantly reduced in all three
mutations. This new finding indicates that tolperisone indeed interacts with the
putative local anesthetic binding site of voltage gated sodium channels.
In concentration-dependence experiments we could show that the tolperisone block of
sodium channels is clearly state dependent. This effect is well known for local
anesthetics in general. We found that tolperisone blocks the inactivated state of
Nav1.4-wild-type sodium channels more effectively than the resting state. By
analyzing the block of mutant, inactivation-deficient Nav1.4-WCW channels we
could demonstrate that the open channel block evoked by tolperisone is also stronger
than the block of resting channels.
Tolperisone shows no selectivity between skeletal muscle, cardiac and neuronal
sodium channel isoforms.
We could demonstrate that the state-dependent block of voltage gated sodium channel
isoforms does not alter significantly. For resting state, 50% inhibitory concentrations
of tolperisone were for the skeletal Nav 1.4 channel 143.7 ± 3.6 µM, for the cardiac
Nav 1.5 channel 164.1 ± 9.2µM, and for the neuronal Nav 1.8 channel 173.5 ± 6.9
µM. The tolperisone block of inactivated Nav 1.4, Nav 1.5, Nav 1.8 channels also
showed no significant difference with IC50 values being 26.9 ± 4.1 µM, 27.0 ± 1.8
µM and 20.2 ± 1.9 µM, respectively. The similar potency of tolperisone in different
isoforms may on the one hand lead to undesired side effects such as on cardiac
muscle, on the other hand it may also implicate that the clinical range of tolperisone
is wider than originally expected with possibly new medical indications in the field of
cardiac arrhythmias or epileptic disease.
- 37 -
Clinical aspects of tolperisone action.
On a molecular basis, tolperisone has a potent effect on different isoforms of voltagegated sodium channels. This may indeed be a desired effect with respect to skeletal
Nav 1.4 and neuronal Nav 1.8 channels, yet severe side effects can occur due to block
of cardiac Nav 1.5 channels. The possible antiepileptic and antiarrhythmic effects of
tolperisone through block of Nav 1.8 and Nav 1.5 channels, respectively, have to be
discussed, too. Moreover, there is a clear discrepancy between the potent molecular
action of tolperisone and the mild clinical effects being achieved, as tolperisone is
only used as an adjuvant to pain therapy in most cases. It also does not exhibit cardiac
side effects as one might expect on the basis of the experimental data. This can be
explained by the fact that tolperisone does not reach concentrations high enough to
achieve significant effects at the site of molecular targets in the body. In fact, oral
bioavailability is very low and varies between 22.3 ± 6.3% for Mydeton tablets and
16.7 ± 8.9% for Mydocalm tablets (25). This, however, can only be studied by
assessing further experimental and clinical data.
- 38 -
6.
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7.
Abkürzungsverzeichnis (alphabetisch)
AMPA
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
CA
California
CD
cluster of differentiation
cDNA
complementary desoxyribonucleic acid
DMSO
dimethylsulfoxide
e.g.
exempli gratia
EGTA
ethylene glycol tetraacetic acid
Fig
Figure
GABA
gamma aminobutyric acid
HEK
human embryonic kidney
HEPES
N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonic acid
IC50
50 % inhibitory concentration
kHZ
kilohertz
MA
Massachusetts
MAP
mitogen activated protein
mM
millimolar
ms
millisecond
mV
millivolt
MΩ
Mega-Ohm
- 44 -
Nav
voltage-gated sodium channel
pH
potentia hydrogenii
PKA
protein kinase A
PKC
protein kinase C
TMA
tetramethylammonium-hydroxide
TNF
tumor necrosis factor
TTX
tetrodotoxin
V0.5
voltage at which 50 % of the channels are inactivated
WDR
wide dynamic range
WT
wild-type
µM
micromolar
- 45 -
8.
Danksagung
Ein großer Dank gilt dem gesamten Team des Labors, das mich mit Rat und Tat über
die Jahre immer unterstützt hat.
Danke an meine Doktormutter für die fundierte, wissenschaftliche Unterstützung, die
grundlegend war für das Gelingen dieser Arbeit. Danke auch dafür, dass ich die
Materialen und Versuchsstände im Labor für die Experimente der Arbeit so
problemlos nutzen konnte.
Zuletzt möchte ich meinen Eltern und meinem Mann danken, die über die Zeit der
Promotion eine große Stütze waren.
- 46 -
9.
Lebenslauf
Persönliche Daten:
Name
Vorname
Geburtsdatum/-ort
Familienstand
Eltern
Ehemann
Rohde, geb. Weigand
Doris
02.12.1981 in Erlangen
verheiratet
Dr. Werner Weigand und Prof. Dr. Edda Weigand
Dirk Rohde
Schulbildung:
1987 - 1990
1990 - 1991
1991 - 1995
1995 - 1997
1997 - 1998
1998 - 2000
2000
Scharrer Grundschule, Nürnberg, 1.-3. Klasse
Grundschule Giacomo Venezian, Trieste, 4. Klasse
International School of Trieste, Italien, 5.-8. Klasse
Pascal Gymnasium, Münster, 9.-10. Klasse
Hebron Academy, Maine, USA, 11. Klasse
Pascal Gymnasium, Münster, 12.-13. Klasse
Abitur
Studium:
WS 2000/2001 – WS 2006/2007 Studium der Humanmedizin an der FriedrichAlexander-Universtität Erlangen-Nürnberg
SS 2002
SS 2003
WS 2005/2006
SS 2006 – WS 2006/2007
SS 2007
Ärztliche Vorprüfung
Erster Abschnitt der Ärztlichen Prüfung
Zweiter Abschnitt der Ärztlichen Prüfung
Praktisches Jahr
Dritter Abschnitt der Ärztlichen Prüfung; Erlangen
der Approbation
Praktisches Jahr:
Innere Medizin: Medizinische Klinik 3, FAU
Chirurgie: Chirurgische Klinik, FAU
Anästhesie: Anästhesiologische Klinik, FAU
- 47 -
Beruflicher Werdegang:
Seit 01.07.2007
Ärztin in Weiterbildung an der Anästhesiologischen
Klinik des Universitätsklinikums Erlangen
Famulaturen:
14.07. - 29.07.2003
03.09. - 02.10.2003
10.03. - 26.03.2004
01.08. - 31.08.2004
01.03. - 31.03.2005
Promotion:
Institut für Diagnostische Radiologie, FAU
Klinik für Anästhesiologie und operative Intensivmedizin, Klinikum Nürnberg
Medizinische Klinik 1, Clemenshospital, Münster
Klinik für Anästhesie und Intensivmedizin,
Allgemeines
Regionalkrankenhaus Bozen, Italien
Praxis Dr. med. B. Rohde, Allgemeinmedizin,
Nürnberg
Promotionsthema: „Effect of tolperisone on Nav1.4,
Nav1.5, and Nav1.8 sodium channels” unter Leitung
von Prof. Dr. Carla Nau, Anästhesiologische Klinik,
Universität Erlangen-Nürnberg
Weitere Kenntnisse/
Tätigkeiten:
1999
2000
12/2004
Teilnahme am Bundeswettbewerb Fremdsprachen,
3. Platz in den Sprachen Englisch, Italienisch und
Französisch
Teilnahme am Bundeswettbewerb Fremdsprachen,
2. Platz in den Sprachen Englisch und Italienisch
Teilnahme am Palliativseminar, Klinik für
Anästhesiologie, FAU
- 48 -
Seit 04/2004
Tätigkeit im Rettungsdienst als aktives Mitglied der
Bergwacht Bayern
04/2005
DAC 2005, München: Posterpräsentation „Die
Wirkung von Tolperison, einem oral applizierten
Natriumkanalblocker zur Behandlung schmerzhafter
Muskelverspannungen, auf muskuläre, kardiale und
neuronale Natriumkanäle“
SS 2004 – WS 2006/2007
Studentische Hilfskraft am Institut für
Experimentelle und Klinische Pharmakologie und
Toxikologie der FAU, Tätigkeit als Tutorin im
Seminar für Allgemeine Pharmakologie für
Humanmediziner im 6. Semester