Download Mainstream- Fringe- and Patho- Physiology of Voltage Dependent

Mainstream- Fringe- and Patho- Physiology of
Voltage Dependent Na+ (Nav) Channels
Alon Meir, Ph.D.
Voltage gated Na+ (Nav) channels mediate action potential genesis and propagation in
most excitable cells. Other roles played by Nav channels in both excitable but also in
non-excitable cells highlight their role as Na+ influx mediators in response to stimuli
other than strong and brief depolarization of the cell’s membrane.
The lipid membrane that surrounds the cell is
impermeable to charged ions that are unequally
distributed across it and this unequal distribution
of charged particles generates polarization of the
membrane potential (more negative inside). Ion
channels are proteins that span the membrane
and form channels through which these ions
can pass and generate ionic electrical currents.
Most channels exist in either an open or shut
conformation and the transition between these
two main conformations is highly controlled.
Voltage sensitive channels respond to changes in
membrane potential by transition to a conducting
conformation.1
Voltage dependent Na+ (Nav) channels are
sensitive molecular devices that respond to
membrane depolarization with a transient Na+
influx.2 This activity switches on the action
potential (AP), the principal neuronal and
muscular electrical signal.1 This review focuses
on Nav channels and their role in other cellular
process ranging from neuronal modulation to
cancer.
Structure, Nomenclature and
TTX - Classical Blocker of Some Nav Channels
Distribution of Nav Channels
Nav channels belong to the larger protein
superfamily of voltage dependent channels
that also include the Kv and Cav channels.3
The archetypical voltage sensing Kv channel
is formed by tetramerization of similar pore
forming subunits and is also true for bacterial
Na+ channels.4 However, in higher organisms,
Nav genes encode a protein with four similar
domains, each corresponding to a single subunit
in the Kv channel tetramer.3 Nine related genes
encoding Nav channels have been identified in
mammals, all forming one protein family (See
Table).5 In addition, a structurally similar gene
corresponding to a Na+ activated channel (NaX)
which lacks the voltage sensor element, might
form a second subfamily within Nav .5 However this
will not be discussed in this review.
Reversible inhibition of Nav channels current in ND7-23 cells
by 60 nM TTX (#T-500). Top: time course of inward current
amplitude, recorded in a typical cell. Holding potential –100
mV, test pulse to –20 mV was delivered every 10 seconds.
Period of TTX bath perfusion is indicated by the horizontal
bar. Bottom: Traces before (blue) and during application of
Nav channels are expressed primarily in excitable
TTX.
Summary of Nomenclature and Tissue Distribution of Nav Channels
Channel
Gene
Chromosome
TTX sensitivity
Main tissue distribution
Nav1.1
SCN1A
2q23-24
Yes
CNS
Nav1.2
SCN2A
2q23-24
Yes
CNS
Nav1.3
SCN3A
2q23-24
Yes
CNS (embryonic)
Nav1.4
SCN4A
Yes
Skeletal muscle
Nav1.5
SCN5A
Nav1.6
SCN8A
Nav1.7
SCN9A
Nav1.8
SCN10A
3p21-24
No
PNS
Nav1.9
SCN11A
3p21-24
No
PNS
17q23-25
3p21-24
No
12q13
2q23-24
Heart muscle
Yes
CNS (neuron, glia), PNS
Yes
PNS (neuron, Schwan), endocrine cells
CNS = central nervous system, PNS = peripheral nervous system.
Modulator No.20 Fall 2005 www.alomone.com
15
tissues (nerve and muscle), with certain isoformtissue/area specificity (See Table).5, 6 This point
is nicely illustrated by the detection of the
cardiac isoform Nav1.5 in human embryonic stem
cell derived cardiomyocytes.7 However, recent
findings demonstrate that neuronal Nav1.1 and
Nav1.3 channels control the pacing of the mouse
heart8, 9 and that the cardiac isoform Nav1.5 is
expressed in limbic regions of the rat brain.10 In
addition, several reports describe the expression
of Nav channels in non-excitable cells.11-13 This
demonstrates that the tissue specificity of Nav
channels is a broad observation, but important
deviations from this pattern exist.
by all but the Nav1.5, 1.8 and 1.9 isoforms (See
Table).5, 6
Nav Channels as AP Initiators
The nine human Nav channel genes are clustered
on different chromosomes. Apparently structural,
functional and pharmacological characteristics
separates the channels encoded on chromosome
3 from all the others. This is manifested mainly
by Tetrodotoxin (TTX) sensitivity demonstrated
Depolarization of the resting membrane potential
is caused by the accumulation of positive
charges on the inner interface of the plasma
membrane. Thus, Na+ inflow results in membrane
depolarization. This positively charged ion influx
causes further depolarization of the membrane,
which activates neighboring channels. This
mechanism forms a positive feedback loop that
lies at the heart of membrane excitability, as
it forms the rising phase of most APs.1 AP is a
regenerative voltage spike, traveling along the
cell membrane, by means of ionic currents and
activation of voltage dependent ion channels (Kv
and KCa channels, co-localized with Nav , repolarize
the cells’ membrane, terminating the signal). In
neurons, AP travel rapidly for long distances along
Expression of Nav1.1 in Mouse Cerebellum
Expression of Nav1.2 in Mouse Hippocampus
axons, transmitting cellular coded information.
Thus, most of the nine human genes encoding
Nav channels participate in AP generation in
neurons and muscle cells.2, 5 The combination of
Nav channel isoforms expressed by an excitable
cell determines parameters such as AP firing
frequency, which are in the basis of neuronal
coding. Indeed, different cell types express
different combinations of Nav channels (See
Table) and alterations in such expression patterns
occur in different pathophysiological conditions.
For example, changes in the levels of functional
Nav channel expression forms the basis of
changes in conduction properties of DRG neurons
associated with chronic pain phenomena.6
Recently, it was shown that the ubiquitin system
participates in the regulation of Nav1.5 channel
functional membrane expression in the heart,14
adding another dimension to the regulation of
cardiac excitability.
Expression of Nav1.3 in Rat Brain
A
B
Immunohistochemical staining of Nav1.1 channel with Anti-
Immunohistochemical staining of Nav1.2 channel with Anti-
Immunohistochemical staining of Nav1.3 channel in rat
Na v1.1 antibody (#ASC-001) in mouse cerebellum. (A) The
Na v1.2 antibody (#ASC-002) in mouse hippocampus. (A)
brain using Anti-Na v1.3 antibody (#ASC-004). Na v1.3 was
distribution of Nav1.1 (red) forms a band (arrows) in the
Nav1.2 channel (red) in dendrites of pyramidal neurons in the
visualized with immuno-peroxidase methods and final
molecular layer (Mol), close to the Purkinje cell bodies. (B)
CA3 region. (B) staining of Purkinje nerve cells with mouse
brown-black diaminobenzidine color product. There was
Purkinje nerve cells are stained with mouse anti Parvalbumin
anti Parvalbumin (green) demonstrates the restriction of
strong staining of some axonal groups such as the mossy
(green). (C) Confocal merge of Na v1.1 and Parvalbumin.
Nav1.2 to dendrites (arrows) outside the pyramidal layer (P).
fibers in hippocampus (A) and cortico-striatal fibers (B).
(C) Confocal merge of Nav1.2 and Parvalbumin.
Western blotting of rat newborn
Western blotting of rat brain
Western blotting of rat newborn
membranes:
brain membranes:
(1:200).
1. Anti-Na v1.2 antibody (#ASC-002)
1. Anti-Na v1.3 antibody (#ASC-004)
2. Anti-Nav1.1 antibody,
(1:200).
(1:200).
preincubated with the control
2. Anti-Nav1.2 antibody, preincubated
2. Anti-Nav1.3 antibody, preincubated
peptide antigen.
with the control peptide antigen.
with the control peptide antigen.
brain membranes:
1. Anti-Na v1.1 antibody (#ASC-001)
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No.20 Fall 2005 www.alomone.com
Nav Channel Influxes Near the
Resting Membrane Potential
and in Non-Excitable Cells
Usually Nav channel influxes are brief and
transient, generally activating within 1ms after
depolarization and terminating within 10 ms
(while the stimulus is still on, a process called
inactivation).5 Thus, AP arise from the brief
activity of Nav channels, which respond to
the extreme depolarization generated by AP.
Nevertheless, several mechanisms and Nav
channels may give rise to different activities,
when the membrane voltage is at rest or during
mild depolarization. This corresponds both to
neuronal excitability and to the emerging role of
Nav channels in non-excitable tissue physiology
and pathophysiology. Both of these roles are
briefly discussed below.
Neuronal modulation by Nav channels at rest
and mild depolarization may arise from voltage
dependent kinetic properties of channels such
as inactivation,15 persistence16 and resurgence17.
These phenomena control the availability of Nav
channels before and after an AP has passed,
thus, influencing the probability of an AP to
emerge.18 Nav channel isoforms differ in the finetuning of their kinetic properties. Differences
may arise from intrinsic properties, or from
the ability to interact with other factors.19 The
expression patterns of the different isoforms
gives rise to a large diversity of neurons with
different AP firing behavior. Indeed, long lasting
changes in expression patterns of Nav channels
under pathological conditions for example, is
strongly correlated with changes in such coding
capabilities of neurons.20
Neuronal modulation may also arise from
activation of channels by means other than
voltage sensitivity. In mouse DRG neurons, Nav1.9
Expression of Nav1.5 in Rat Heart Cardiomyocytes
A
currents were found to be up-regulated by the
inflammatory agent prostaglandin E2, in a Gprotein dependent manner.21 Another important
example is the activation of the TTX-resistant
Nav1.9 channel by neurotrophic factors (NT).22, 23
This was shown in hippocampal neurons, where
BDNF via TrkB receptors and Nav1.9 channels
activated a long lasting Na+ current (about 200
ms) and a similar current was fourfold longer
in neuroblastoma cells.23 In frog sympathetic
neurons, NGF upregulated both TTX-sensitive
and TTX–resistant Nav currents.24 On the
other hand, NGF and GDNF reduced the Nav1.3
expression that accompanied DRG axotomy.25
These interactions may contribute to pain
sensation and pathophysiology, with differential
specific interactions between sets of NTs and
Nav channels.6 Such interactions lie at the base
of differential analgesic effects of different NTs,
mediated by regulation of Nav channels in the
peripheral nervous system.25
Nav1.9 Expression in DRG
B
Immunohistochemical staining of Nav1.5 channel with Anti-Na v1.5 antibody (#ASC-005) in rat heart.
Transversal section of the ventricular wall in the epicardium area also shows a big artery and myocardial
area. DAB product is brown and counterstaining is Cresyl Violet. (A) Shows strong staining of myocytes
(green arrow); no staining is evident in smooth muscle (red arrow), endothelium (black arrow), connective
tissue (fibroblasts) (yellow arrow) or epicardial epithelium (blue arrows). (B) Enlargement of (A).
Immunohistochemical staining of Nav1.9 channel
in rat dorsal root ganglion (DRG) with Anti-Na v1.9
antibody (#ASC-017). Cells within the DRG were
Expression of Nav1.8 in Subpopulations of DRG Neurons
Nav1.8
Merge
stained (see solid line frame enlarged in (B)) as well
as fibers and the area of entry of dorsal root into
Neurofilament 200
spinal cord (see dashed line frame enlarged in (C)).
The counterstain in (B) and (C) is DAPI, a fluorescent
dye visualized in the UV range.
Western blotting of rat DRG lysates:
1. Anti-Na v1.9 antibody, (#ASC-017)
(1:200).
2. Anti-Nav1.9 antibody, preincubated
with the control peptide antigen.
Immunohistochemical staining of Nav1.8 in adult rat dorsal root ganglion (DRG) with Anti-Na v1.8 antibody (#ASC-016).
Nav1.8 Staining (red) was cytoplasmic and the intensity varied among DRG cells. There was a partial overlap in the
distribution of Nav1.8 and neurofilament 200 (green).
Modulator No.20 Fall 2005 www.alomone.com
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All these subthreshold activities may be the link
and explanation for the expression of these highly
voltage sensitive channels in non-excitable cells
that do not fire AP.
Ion channel activity is an important factor in
volume regulation and the associated motility of
cells. A related mechanism is suggested for the
ability to migrate, acquired by transformed glial
cells, on their way to spread brain cancer.26
Several Nav channels were reported to be
upregulated in prostate cancer cell lines.12, 27, 28
Interestingly, the magnitude of such Nav current
was shown to be dependent on environmental
factors that are present in the cell culture serum.29
The role played by Nav channels, is suggested
by the positive correlation between the rate
of invasiveness and the level of Nav current or
expression.12, 30 Most importantly, TTX, a highly
specific (for Nav, but not very selective between
the six sensitive isoforms), Nav channel blocker,
reduced these cells’ invasiveness.12 A possible
role for Nav channels in volume regulation was
demonstrated in Jurkat T cells, where Saxitoxin
(STX) sensitive Nav channels play a role in
apoptosis related cell shrinkage and death.13, 31
In addition, the Nav auxiliary subunit β3 (SCNB3)
was found to be up-regulated in cancer cell lines
in a p53 dependent manner, suggesting a role
for Nav channels in induced apoptosis of cancer
cells.32
These observations highlight the notion that
voltage activated channels might be expressed
in non-excitable cells, possibly under extreme
cellular conditions.
Nav Channelopathies
Involvement of Nav channels in pathophysiological conditions may arise either from
genetic alterations in channel activity or from
changes in functional expression levels.
ATX-II Potently Enhances Nav Currents by
Reducing Inactivation
Mutations in coding for Nav channel proteins
cause of several hereditary neuronal,33 cardiac34
and muscular35 diseases. In addition, the
mutation may occur de-novo (neither parent
QX-314 - A Local Anesthetic Blocker of Nav
Channels
Enhancement of NaV1.5 channel currents by increasing
concentrations of ATX-II (#A-700). Currents were recorded
from Xenopus oocytes injected with NaV1.5 mRNA and were
elicited every 10 seconds from holding potential of –100 mV,
by 50 ms pulses to –20 mV.
Dose response of QX-314 (#Q-150) on inward currents in
Xenopus oocytes injected with Nav1.5 mRNA. Using two
electrode voltage clamps, oocyte membrane potential
was held at -100 mV and currents were elicited by 25 ms
depolarization to -20 mV, delivered every 5 seconds. Traces
before (blue) and during bath application of different
(indicated with arrow) QX-314 concentrations.
Top: Superimposed NaV current responses in control
conditions (red) and during applications of increasing
concentrations of ATX-II up to 1µM (lowest trace).
Bottom: Dose response of NaV currents along their typical
waveform. Note that the current enhancement is greater once
the channel is inactivated.
Specific Expression of Nav1.6 and Nav1.7 in Smooth Muscle Cells
A
B
C
Immunocytochemical staining of murine portal vein smooth muscle cells for Na v1.6 and Na v1.7 voltage-dependent Na + channels.
Fluorescence images of a single confocal plane of the cell labelled with (A) Anti-Na v1.6 antibody (#ASC-009); (B) Anti-Na v1.7 antibody (#ASC-008). (incubation with antigen peptide
eliminated staining, data not shown). Calibration bar: 10 µm. (C) Summary data on intensity of fluorescence, expressed as average pixel fluorescence. The values of all pixels in the
cell’s confocal plane were added up and then divided by the number of pixels (gray). The specificity of labeling was confirmed for both antibodies by greatly reduced fluorescence after
pre-incubation with respective antigenic peptide (dark gray) or by virtual lack of fluorescence in the absence of primary antibodies (white).
*: statistically significant.
Acknowledgements: Data were generated by Vladimír Pucovský in association with Sohag Saleh, S.Y.M. Yeung, Sally Prestwich and Iain Greenwood, St. Geoge’s Hospital Medical
School, London, UK.
18
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No.20 Fall 2005 www.alomone.com
carries it) and such a mutant Nav1.5 channel was
linked to Sudden Infant Death Syndrome.36 In
many of these mutations, the channel activity is
up-regulated due to reduced inactivation, thus
resulting in elevated baseline activity.33
Altered expression patterns and level of Nav
channels in pathological states is demonstrated
in Multiple Sclerosis (MS) and may contribute to
specific phenotypes of this disease.20 In addition,
it was shown using specific antibodies that colocalization of Nav with the Na+ -Ca2+ exchanger,
plays a role in the increased axonal degradation
seen in a mouse model of MS.37 Nav channel
expression is also changed at sites of axonal
injury, conferring altered firing behavior to these
injured neuronal branches.6 Such a mechanism is
the basis of abnormal pain phenomena as well.
Pharmacology of Nav Channels
Nav channels are involved in several different
diseases and mediation of pain and indeed,
these proteins are targets for many drugs ranging
from anti-epileptic to local anesthetic agents.5 In
addition, many venomous organisms target their
prey’s or attacker’s Nav channels.38 In general,
Nav pharmacophores are classified according to
their receptor site on the channel. Among these,
several molecules block the channel, while others
support increased activity, either by enhancement
of activation or by slowing inactivation.5
TTX, STX and µ-Conotoxins, modulate Nav
channels by blocking the pore from outside.
TTX (See Table) and STX are largely nonselective between the different Nav isoforms ,5
while µ-Conotoxins are more specific and may
differentiate between Nav current components.
39, 40
These agents are widely used to block
conduction in excitable tissue in a range of
experimental procedures.
Local anesthetics and related compounds, such
as QX-222 and QX-314, block Nav channels by
binding to an intracellular site near the pore.
These agents are capable of blocking all Nav
isoforms when applied intracellularly. However,
once applied from outside, it blocks channels
differentially. This is probably correlated to the
ability of these agents to penetrate the cell via the
open channel.41 Since these compounds can exert
their blocking effect only once the channel has
opened, they are called open channel blockers or
activity dependent blockers.
exert similar effects on inactivation.4 Two small
peptide toxins found in wasp venom, α and
β-Pompilidotoxin, and promote Nav currents by
slowing the inactivation process.19 Such toxins
are used to mimic, or specifically up-regulate, Nav
subthreshold activities such as persistent and
resurgent currents. Therefore, these Nav channels
toxin activators might be used as powerful tools
in Nav current induction at rest or in non-excitable
cells.
Anthopleurin-C (APE2-1) - An Enhancer of
NaV Currents
Activation of Nav channels current in mNGF 2.5S (#N-100)
treated PC12 cells by 5 nM APE2-1. example current traces
recorded in a typical cell before (blue) and during (black)
application of Anthopleurin-C (APE2-1) (#A-400). Holding
potential –120 mV, test pulse to –20 mV was delivered every
10 seconds.
References:
1. Hille, B. (2001) Ion Channels in Excitable Membranes. 3rd edition.
2. Catterall, W. A. (2000) Neuron 26, 13.
3. Yu, F. H. and Catterall, W. A. (2004) Sci. STKE. 2004, re 15.
4. Koishi, R. et al. (2004) J. Biol. Chem. 279, 9532.
5. http://www.iuphar-db.org/iuphar-ic/sodium.html
6. Ogata, N. and Oshini, Y. (2002) Jpn. J. Pharmacol. 88, 365.
7. Satin, J. et al. (2004) J. Physiol. 559, 479.
8. Maier, S. K. et al. (2003) P. N. A. S. USA. 100, 3507.
9. Lei, M. et al. (2004) J. Physiol. 559, 835.
11. Fraser, S. P. et al. (2004) FEBS Lett. 569, 191.
Related Products
12. Bennett, E. S. et al. (2004) Pflugers. Arch. 447, 908.
Compound
10. Hartmann, H. A. et al. (1999) Nature Neurosci. 2, 593.
13. Bortner, C. D. and Cidlowski, J. A. (2003) J. Biol. Chem. 278,
39176.
14. Van Bemmelen, M. X. et al. (2004) Circ. Res. 95, 284.
15. Goldin, A. L. (2003) Curr. Opin. Neorobiol. 13, 284.
16. Fleidervish, I. A. and Gutnick, M. J. (1996) J. Neurophysiol. 76,
2125.
17. Afshari, F. S. et al. (2004) J. Neurohysiol. 92, 2831.
18. Do, M. T. H. and Bean, B. P. (2003) Neuron 39, 109.
19. Grieco, T. M. and Raman, I. M. (2004) J. Neurosci. 24, 35.
20. Black, J. A. et al. (2000) P. N. A. S. USA. 97, 11598.
21. Rush, A. M. and Waxman, S. G. (2004) Brain Res. 1023, 264.
22. Barde, Y. A. (2002) Nature 419, 683.
23. Blum, R. et al. (2002) Nature 419, 687.
24. Lei, S. et al. (2001) J. Neurophysiol. 86, 641.
25. Leffler, A. et al. (2002) J. Neurophysiol. 88, 650.
26. Sontheimer, H. (2003) Trends Neurosci. 26, 543.
27. Bronstein-Sitton, N. (2004) Modulator 17, 4. http://
www.alomone.com
28. Bogin, O. (2004) Modulator 18, 24. http://www.alomone.com
29. Ding, Y. and Djamjoz, M. B. (2004) Int. J. Biochem. Cell. Biol. 36,
1249.
30. Grimes, J. A. et al. (1995) FEBS. Lett. 369, 290.
31. Meir, A. (2004) Modulator 18, 2. http://www.alomone.com
32. Adaci, K. et al. (2004) Oncogene 23, 7791.
33. Lossin, C. et al. (2002) Neuron 34, 877.
Product #
Antibodies
Anti-Na v1.1 (Brain Type I)
Anti-Na v1.2 (Brain Type II)
Anti-Na v1.3 (Brain Type III)
Anti-Na v1.5
Anti-human Na v1.5
Anti-Na v1.6 (Scn8a (PN4))
Anti-Na v1.7 (PN1)
Anti-Na v1.8
Anti-Na v1.9
Anti-Pan Na v (SP-19)
Anti-Na vβ2
ASC-001
ASC-002
ASC-004
ASC-005
ASC-013
ASC-009
ASC-008
ASC-016
ASC-017
ASC-003
ASC-007
Blockers
µ-Conotoxin GIIIB
QX-222
QX-314 (Bromide Salt)
QX-314 (Chloride Salt)
Tetrodotoxin (citrate free)
Tetrodotoxin (with citrate)
C-270
Q-200
Q-100
Q-150
T-500
T-550
Activators
Anthopleurin-C
APE 1-2
ATX II
α-Pompilidotoxin
β-Pompilidotoxin
A-400
A-470
A-700
P-170
P-180
34. Maraban, E. (2002) Nature 415, 213.
Many venoms from different animals contain
peptides that are Nav channel activators. Scorpion
venoms are the largest source described so
far.38 Both α and β scorpion toxins bind to
extracellular moieties on domain IV and II of the
channel to slow inactivation or enhance activation
kinetics, respectively.5 Sea anemone Nav toxins
such as Anthopleurin-C, APE 1-242 or ATX II43,
44
bind to the α scorpion toxin binding site and
Modulator No.20 Fall 2005 www.alomone.com
35. Lehmann-Horn, F . and Jurkat-Rott, K. (1999) Phys. Rev. 79, 1317.
Neurotrophic Factors
36. Wedekind, H. et al. (2001) Circulation 104, 1158.
mNGF 2.5S (Grade I)
mNGF 2.5S (Grade II)
mNGF 7S
hβ-NGF
hBDNF
hCardiotrophin-1
hGDNF
hNeurotrophin-3 (NT-3)
hNeurotrophin-4 (NT-4)
hPersephin
37. Craner, M. J. et al. (2004) Brain 127, 294.
38. Possani, L. D. et al. (1999) Eur. J. Biochem. 264, 287.
39. West, P. J. et al. (2002) Biochemistry 41, 15388.
40. Terlau, H. and Olivera, B. M. (2004) Phys. Rev. 84, 41.
41. Sunami, A. et al. (2000) P. N. A. S. USA. 97, 2326.
42. Bruhn, T. et al. (2001) Toxicon 39, 693.
43. Mantegazza, M. et al. (1998) J. Physiol. 507, 105.
44. Mee, C. J. et al. (2004) J. Neurosci. 24, 8695.
N-240
N-100
N-130
N-235
B-250
C-200
G-240
N-260
N-270
P-320
19