Part 4

CAP area (mV*msec)
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Clinical score
180 Days EAE: Protection persists during maintained admin. of phenytoin
Control EAE EAE+ Phen
Cont
EAE
EAE+
Phen
0.8
0.7
0.6
Contr + Phen
EAE + Phen
EAE
4
3.5
3
2.5
180 days C57/BL6
EAE
0.5
0.4
2
1.5
EAE + Phen
0.3
1
0.2
0.1
0.0
* * * * * * *
0.5
0
0 30 60 90 120 150 180
days
Phen
Black et al, Brain, 2006

Na V 1.6 Co-localizes with cytoskeletal F-actin, vimentin in TMP- human
monocytic cell line, and in primary human macrophages
shRNA knockdown of Na V 1.6, and Na channel block with TTX and
phenytoin inhibit macrophage and microglial migration
NaV1.6
F-actin (phalloidin)
Merge+DAPI
Carrithers et al, J. Immunol. 2007
Carrithers et al, JBC, 2008
Microglia/6.3x10 5 m 2
90
80
70
60
50
40
30
20
10
0
*
#
ACM ACM +
ATP
ACM +
ATP +
ACM +
ATP +
0.3 M TTX 10 M TTX
#
Black et al, Glia, 2009

I1848T and L858H Mutations Shift Activation,
Slow Deactivation, and Enhance Response to Small, Slow
Depolarizations to produce a Gain-of-Function in Na V 1.7
0.0
-0.2
-0.4
-0.6
-0.8
-1.0
-80 -60 -40 -20 0 20 40
test potential (mV)
hNa v1.7
l848T
L858H
-100
4ms
-20 -50
hNa v 1.7
l848T
L858H
percentage of peak current
1
0
-1
-2
-3
-100 -80 -60 -40 -20 0
voltage (mV)
hNav1.7
I848T
L858H
0
-100
500 msec
Cummins et al, J.Neurosci. 2004

Mutations in Exon 15 of SCN9A
in Two Chinese families
ATT Isoleucine (I 848)
A CT
Threonine (T)
CTC Leucine (L 858)
C AC
Histidine (H)
Promoter
Exons
Scn9A
5N 5A
Exon 15
I II III IV
7
3 5
15
14 19 20 23 25 26
NH 2
2 4 6
1
Na1.7
v
8
10
9
13
L1
12
11
16
L2
18
17
21
22
L3
24
COOH
J. Med. Genet., March 2004

Molecular Pathophysiology of a Human
Hereditary Pain Syndrome: Inherited Erythromelagia
Sulayman Dib-Hajj, PhD
Lynda Tyrrell, MSc
Ted Cummins, PhD
Tony Rush, PhD
Angelika Lampert, MD
Tanya Fischer, MD, PhD
International Collaborators:
Yong Yang, MD, PhD (China)
Joost Drenth, MD (Netherlands)
Bonnie Wallace, PhD (UK)
Andreas O’Reilly, PhD (UK)
Jin Choi, PhD
Xiaoyang Cheng, PhD
Mark Estacion, PhD
Steve Waxman, MD, PhD
ChongYang Han, PhD

T.M. Highly decorated police officer
Gun shot wound
• College graduate. Mult. Citations for bravery, community service.
• 29 y.o.: GSW to arm. Injury to radial nerve. Distinguished Service
Commendation.
• Motor deficit, partial sensory loss in radial n. distribution.
• Intermittent severe burning pain in area of sensory loss. Exacerbated by
tactile and thermal stimuli.
• No response to NSAIDs, tricyclics.
• Minimal improvement w. phenytoin, gabapentin, lidocaine
patch etc. Minimal response to TENS.
• Partial relief w/ carbamazepine, opiates. Doses limited by SEs.
• Followed by neurologist, pain specialist, psychiatrist
• Unable to work regularly. Disabled

Sodium Channels and Small Fiber Neuropathy

Na V 1.7 mutations in small fiber neuropathy
Clinical:
Janneke Hoeijmakers, MD
Giuseppe Lauria, MD
Joost Drenth, MD, PhD
E.K. Van Houtte, MD
Molecular Biology:
Sulayman Dib-Hajj, PhD
Lynda Tyrrell, MA
Palak Shah, MA
Larry Macala, MA
Peng Zhao, PhD
Cell Biology:
Anna-Karin Persson, PhD
Andreas Gasser, PhD
Joel Black, PhD
Physiology / Biophysics:
Chongyang Han, PhD
Xiaoyang Chen, PhD
Hyesook Ahn, PhD
Jin Choi, PhD
Mark Estacion, PhD
Catherina Faber, MD, PhD
Ingemar Merkies, MD, PhD
Steve Waxman, MD, PhD

Small Fiber Neuropathy
• Dysfunction of small myelinated and unmyelinated nerve fibers:
‣ Burning pain (feet > hands)
‣ Autonomic sx (orthostatic hypotension,
impotence, palpitations etc)
‣ Dx: Normal neurol. exam (DTR’s, Vibr.
sensation, Muscle Str.)
‣ EMG, NCV: wnl
A
Patient
‣ Dx confirmed by:
Reduced IENFD on Cutan. Nerve Bx
QST (warmth,cold sensation, heat pain)
B
Gender- and Age-matched Control

Can be associated with:
Small Fiber Neuropathy
Diabetes mellitus, IGT
Hyperlipidemia, Hemochromatosis
Liver, kidney, thyroid dysfn
Amyloidosis
Fabry’s disease
Neurotoxic drugs (e.g. chemotx)
Anti-phospholipid Ab syndrome
Connective tissue disease
Sarcoidosis
Celiac disease
HIV
EtOH abuse
Idiopathic (No Apparent Cause): 25% - 93% in diff. series.

SCN9A mutations: 8 of 28 pts with biopsyconfirmed
SFN
Patients referred with a
clinical diagnosis of Small
Fiber Neuropathy (SFN)
N=248
No underlying causes
identified
N=63 (25.4%)
Underlying causes
identified
N=185 (74.6%)*
Lost to follow-up or refused
further participation
N=19
Excluded
Patients with
idiopathic SFN
N=44
IENFD + QST normal
N=3
IENFD or QST abnormal
N=13
IENFD + QST abnormal
N=28
Excluded
No mutation
found in SCN9A
N=20
Mutation found
in SCN9A
N=8
Faber et al, Ann. Neurol., 2011

Nav1.7 mutations in Idiopathic SFN
Domain I Domain II Domain III Domain IV
M1532I
+
+
+
+
+
+
+
+
+
+
+
S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6
+
+
+
+
+
+
+
+
+
+
extracellular
cytoplasm
N
R185H*
I228M
L1 L2 L3
I739V
I720K
D623N
M932L – V991L
C
Faber et al, Ann. Neurol., 2011

R M P (m V )
C u rre n t T h re sh o ld (p A )
A P s /5 0 0 m s
A1 A2 A3a
WT
1nA
2ms
M932L/V991L
I /Im a x
1.0
0.8
0.6
0.4
WT
ML/VL
1.0
0.8
0.6
0.4
G /G m a x
I/ Im ax
1.0
0.8
0.6
0.4
M932L / V991L
WT
ML/VL
0.2
0.0
V 1/2,fast-inact
-68.0±0.6mV
-68.5±0.6mV
V 1/2,act
-20.2±0.6mV
-20.4±1.2mV
0.2
0.0
0.2
0.0
V 1/2,slow-inact
-66.1±1.0mV
-63.8±1.7mV
-120 -80 -40 0 40
Voltage (mV)
-140 -120 -100 -80 -60 -40 -20 0 20
Voltage (mV)
A3b
A4
-35
WT
200pA
20ms
1.79% 1/11 (9.1%) 5.111.22% ± 5/10 (50%)
A5
ML/VL
*
A6
4
3
2
1
0
*
WT
ML/VL
*
*
*
*
*
*
*
*
*
*
*
*
250
-40
200
0 50 100 150 200 250 300 350 400 450 500
Stimulus (pA)
-45
-50
150
**
A7
2.0 ± 0.3 Hz
-55
**
100
50
4/2715; %
(0/20; 0%) ; %
-60
WT
ML/VL
0
WT
ML/VL
=+15% (p=0.072)

‘ When Hodgkin and I finished writing the 1952
papers, each of us moved to other lines of work
because we could not see how to make progress on
the mechanism of channel action. Any idea of
analyzing the channels by patch clamp or molecular
biology would have seemed to us to be… science
fiction ‘.
Andrew Huxley, in The Axon, 1995

Na V 1.5 channels, localized on the endosomal membrane,
provide a route for Na efflux that offsets proton influx during
acidification of phagocytosed material
Activation of Na V 1.5 with Veratridine triggers Na efflux and proton influx
(acidification) of purified THP-1 endosomes
Carrithers et al J. Immunol., 2007

Membrane potential (mV)
E Na
+40
+20
-20
V
30
g Na
20
g K
mS/cm 2
E K
-40
-60
-80
10
0
4 msec
I
g
Na
m
3
h(
V
E
Na
)
g
K
n
4
( V
E
K
)
g
leak
( V
E
leak
)
Hodgkin and Huxley, 1952

EFNS guidelines on skin biopsy 2010
Skin biopsy: reduced IENFD
patient
control
A
B
• 3 sections (50 μm) immunostained with PGP 9.5