Академический Документы
Профессиональный Документы
Культура Документы
1122352
, 589 (2006); 312 Science
et al. Mari Sasaki
A Voltage Sensor-Domain Protein Is a Voltage-Gated Proton Channel
This copy is for your personal, non-commercial use only.
clicking here. colleagues, clients, or customers by
, you can order high-quality copies for your If you wish to distribute this article to others
and Na
p
H
D
p
H
i
n
2
4
6
8
10
12
14
-80 -60 -40 -20 0 20 40 60 80 100
Membrane potential (mV)
0
50
100
150
200
250
300
20 40 60 80 100
Membrane potential (mV)
6.5/7.0
6.5/6.5
6.5/6.1
pHin/pHout
N
o
r
m
a
l
i
z
e
d
o
u
t
w
a
r
d
c
u
r
r
e
n
t
T
i
m
e
f
o
r
h
a
l
f
a
c
t
i
v
a
t
i
o
n
(
m
s
)
6.5/7.0
6.5/6.5
6.5/6.1
pHin/pHout
A B
mVSOP Zn
mVSOP Cd
Ci-VSOP Zn
2+
2+
2+
C
D
0.0
0.5
1.0
1.5
2.0
2.5
R
e
l
a
t
i
v
e
e
x
p
r
e
s
s
i
o
n
C
e
r
e
b
r
u
m
C
e
r
e
b
e
l
l
u
m
L
u
n
g
H
e
a
r
t
L
i
v
e
r
S
p
l
e
e
n
K
i
d
n
e
y
T
e
s
t
i
s
B
l
o
o
d
B
o
n
e
m
a
r
r
o
w
M
a
c
r
o
p
h
a
g
e
Fig. 3. pH-dependent gating and inhibition by
divalent metal cations of mVSOP-induced
currents. (A) The current-voltage relationships
evoked by a series of voltage steps in 10-mV
increments (80 to 100 mV) under pH
in
0 6.5
and pH
out
0 7.0, 6.5, or 6.1. The pulse duration
was 500 ms. Currents were measured from the
same sets of cells. Current amplitudes at the
end of the depolarizing pulse obtained under
each condition of pH
out
were normalized by
those at 20 mV recorded under pH
out
0 7.0 for
individual cells. (B) Voltage dependence and
pH dependence of the time required for half-
maximal activation. Maximal current was
measured as the amplitude at the end of
depolarizing pulse. The pulse duration was
500 ms. Representative current traces for (A)
and (B) are shown in fig. S2. Averaged values T
SE are shown (n 0 9) in (A) and (B). (C) Dose-
response curves of inhibition by zinc and
cadmium ions. Small circles are plots from
individual cells, and squares denote average
values. The holding potential was 80 mV. (D)
Tissue distributions of VSOP mRNA examined
by real-time RT-PCR. The expression level of
VSOP mRNA was normalized by expression in
the spleen. L8 ribosomal protein was used as
internal control.
REPORTS
28 APRIL 2006 VOL 312 SCIENCE www.sciencemag.org 590
(I-V) relations measured from the same cell at
three distinct extracellular pHs showed that
when the extracellular pH was decreased from
7.0 to 6.1, the I-V curve shifted in the positive
direction by about 50 mV (Fig. 3A and fig. S2).
When intracellular pH was altered, the I-V
relationship shifted in the opposite direction by
a similar value (fig. S3). Outward currents were
activated at a similar threshold of membrane
potential (V
threshold
), when measured at two
different conditions of extracellular pH 0 6.1
and 6.5, with a small pH gradient across the
cell membrane (fig. S3). These findings are
consistent with the observation that the voltage
activation curve is predicted from the gradient
between the extracellular and the intracellular
pH in native H
v
channels (11). V
threshold
was
always more positive than V
rev
within the range
of pH gradients we examined (1.5 G DpH G
2.5; DpH is the difference of pH across the cell
membrane, pH
out
pH
in
). Therefore, mVSOP
opens at a range of membrane potentials over
the equilibrium potential for protons, thus en-
abling outwardly rectifying property for proton
flow. Activation kinetics were also pH-dependent;
the time for half activation at varied membrane
potentials became slower as the extracellular pH
decreased (Fig. 3B). This pH-dependent gating
of VSOP-induced currents agrees well with a
reported behavior of native H
v
currents (12, 18).
H
v
currents are known to be inhibited by
submillimolar concentrations of Zn
2
, Cd
2
,
and other divalent cations (11, 19, 20). Like-
wise, mVSOP-derived currents were reversibly
decreased by submillimolar concentrations of
Zn
2
and Cd
2
(Fig. 3C and fig. S2). Consistent
with results for native H
v
currents (19, 20),
Zn
2
had a stronger effect (dissociation constant
K
d
0 0.48 mM) than Cd
2
.
Previous electrophysiological studies have
revealed H
v
currents in mammalian blood cells,
alveolar epithelium cells of the lung, and
microglia of the brain (11, 12, 14). Consistent
with this, quantitative reverse transcriptase
polymerase chain reaction (RT-PCR) (Fig.
3D) showed marked gene expression of
mVSOP in the spleen, whole blood, bone
marrow, and macrophages.
Does mVSOP directly encode H
v
channels,
or just activate endogenous H
v
channels? To
address this, we tested whether modification
of molecular structure leads to changes in the
properties of the H
v
current. Changes in volt-
age dependence were examined for mutations
at each of the three positively charged amino
acids of the S4-like segment. The conductance-
voltage (G-V) curve was obtained with the
same pH (7.0) in both the extracellular and
intracellular solutions. In the R204Q mutant,
where Arg
204
is replaced by Gln, detailed
analysis of current kinetics was not possible
due to a low level of protein expression to the
cell surface. The G-V curve for R207Q was
indistinguishable from that of the wild-type
channel (fig. S4), whereas R201Q showed
much faster kinetics of activation (Fig. 4A).
The G-V curve of the R201Q mutant was
shifted in a negative direction by about 50 mV
(Fig. 4B). The steepness of activation (z value)
became slightly sharper (1.9 T 0.25 for R201Q,
n 0 9 cells, versus 1.4 T 0.15 for wild type,
n 0 5). In this mutant, V
theshold
was more
negative than V
rev
, suggesting that inward cur-
rent flows at a membrane potential more neg-
ative than V
rev
. In fact, an inward proton current
was clearly seen at acidic extracellular pH (Fig.
4, C and D). R201 may therefore help the chan-
nel to maintain a V
threshold
that is more positive
than the equilibrium potential for protons,
which enables the outwardly-rectifying prop-
erty of the channel.
Fig. 4. Mutation in the S4-like segment alters the voltage dependence of channel
gating. (A) Current traces recorded from cells expressing wild-type mVSOP (wt) (left)
or R201Q (right). Traces with depolarizing steps (20 to 100 mV) are superimposed.
pH
in
/pH
out
0 7.0/7.0. The pulse protocol is shown in fig. S2. (B) Voltage dependency of
conductance is plotted for wt (n 0 5; circles) and R201Q (n 0 9; squares) and fitted by
the Boltzmann equation G 0
1
1 exp
zeV j V
kT
where k is the Boltzmann constant,
e is the elementary electric charge, T is temperature, and z is the valence. V
values are 63.7 T 7.6 mV and 14.8 T 8.0 mV, and z values are 1.4 T 0.15 and
1.9 T 0.25 for wt and R201Q, respectively. Error bars indicate SD. (C) Current traces under pH
in
/pH
out
0 7.0/6.1 for wt (left) and R201Q (right). Traces with
depolarizing steps (20 to 90 mV) are superimposed. In R201Q, tail currents are scaled out in this magnification. (D) The current-voltage relationships for wt
(n 0 8) and R201Q mutant (n 0 12) in pH
in
/pH
out
0 7.0/6.1. Averaged current densities at the end of depolarization pulses are plotted. Inward current
is evident for R201Q. In [(A) to (D)], the holding potential was 60 mV. Error bars indicate SD.
REPORTS
591 www.sciencemag.org SCIENCE VOL 312 28 APRIL 2006
The ortholog of mVSOP was also isolated
from an ascidian, C. intestinalis (called
Ci-VSOP). Ci-VSOP showed marked ho-
mology to mVSOP in the putative S1 to S4
segments, although the N-terminal and C-
terminal cytoplasmic regions of the two
VSOP proteins were highly divergent (fig.
S1). This ascidian ortholog protein also ex-
hibited outward-rectifying proton currents
activated by membrane depolarization when
expressed in tsA201 cells. Ci-VSOP showed
27 times lower sensitivity to Zn
2
(Fig. 3C)
and significantly faster activation kinetics
than mVSOP (fig. S5). The shifted voltage
dependency of activation in the mutant R201Q
of mVSOP, together with the differences in
kinetics and pharmacology between ascid-
ian and mammalian orthologs, suggest that
VSOP proteins are the principal subunit of
the H
v
channel, rather than a regulator or
accessory subunit of an endogenous proton
channel.
We identified a protein consisting primarily
of a VSD as an H
v
channel, providing the first
example of a protein in which the VSD func-
tions beyond voltage sensing. Proton efflux
through H
v
channels, accompanied by mem-
brane depolarization, facilitates acute produc-
tion of reactive oxygen species in phagocytosis
(11, 14, 21). Further studies of VSOP will not
only provide new clues to general principles of
proton permeation and gating of membrane
proteins (10, 11, 14), but may also open
avenues for advances in understanding biolog-
ical events related to respiratory burst and
phagocytosis.
Note added in proof: D. Clapham_s labora-
tory reports similar properties of a human
ortholog, H
v
1 (22).
References and Notes
1. F. Bezanilla, Physiol. Rev. 80, 555 (2000).
2. S. B. Long, E. B. Campbell, R. Mackinnon, Science 309,
903 (2005).
3. D. M. Starace, F. Bezanilla, Nature 427, 548 (2004).
4. D. M. Starace, F. Bezanilla, J. Gen. Physiol. 117, 469
(2001).
5. S. Sokolov, T. Scheuer, W. A. Catterall, Neuron 47, 183
(2005).
6. F. Tombola, M. M. Pathak, E. Y. Isacoff, Neuron 45, 379
(2005).
7. Y. Murata, H. Iwasaki, M. Sasaki, K. Inaba, Y. Okamura,
Nature 435, 1239 (2005).
8. R. Lopez, V. Silventoinen, S. Robinson, A. Kibria, W. Gish,
Nucleic Acids Res. 31, 3795 (2003).
9. A. Roos, W. F. Boron, Physiol. Rev. 61, 296 (1981).
10. V. V. Cherny, R. Murphy, V. Sokolov, R. A. Levis,
T. E. DeCoursey, J. Gen. Physiol. 121, 615 (2003).
11. T. E. Decoursey, Physiol. Rev. 83, 475 (2003).
12. A. Kapus, R. Romanek, A. Y. Qu, O. D. Rotstein,
S. Grinstein, J. Gen. Physiol. 102, 729 (1993).
13. T. E. DeCoursey, V. V. Cherny, J. Membr. Biol. 152, 131
(1996).
14. C. Eder, T. E. DeCoursey, Prog. Neurobiol. 64, 277
(2001).
15. L. Byerly, R. Meech, W. Moody Jr., J. Physiol. 351, 199
(1984).
16. R. W. Meech, R. C. Thomas, J. Physiol. 390, 433
(1987).
17. R. C. Thomas, R. W. Meech, Nature 299, 826
(1982).
18. V. V. Cherny, V. S. Markin, T. E. DeCoursey, J. Gen. Physiol.
105, 861 (1995).
19. L. Byerly, Y. Suen, J. Physiol. 413, 75 (1989).
20. V. V. Cherny, T. E. DeCoursey, J. Gen. Physiol. 114, 819
(1999).
21. T. E. DeCoursey, D. Morgan, V. V. Cherny, Nature 422,
531 (2003).
22. I. S. Ramsey, M. M. Moran, J. A. Chong, D. E. Clapham,
Nature, published online 22 March 2006; 10.1038/
nature04700.
23. We thank T. Mohri for help with pH imaging; K. Saito,
Y. Murata, and T. Kurokawa for sharing preliminary
electrophysiological data; K. Ikenaka and S. Hitoshi
for help in the analysis of the gene expression profile;
T. McCormack and J. Cui for their critical reading of
the manuscript; and members of the Okamura lab for
helpful discussion. This work was completed in a partial
fulfillment of a Ph.D. degree to M.S. at the Graduate
University for Advanced Studies. This work was
supported by Grants-in-Aid for Scientific Research
and for Creative Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology;
the Naito Foundation; and the Mochida Memorial
Foundation for Medical and Pharmaceutical Research
in Japan. The full-length cDNA sequence of Ci-VSOP is
deposited in the DNA Data Bank of Japan (DDBJ) with
accession number AB218779. The authors declare
no competing financial interests.
Supporting Online Material
www.sciencemag.org/cgi/content/full/1122352/DC1
Materials and Methods
Figs. S1 to S5
References
8 November 2005; accepted 2 March 2006
Published online 23 March 2006;
10.1126/science.1122352
Include this information when citing this paper.
SV2 Is the Protein Receptor for
Botulinum Neurotoxin A
Min Dong,
1
Felix Yeh,
1,2
William H. Tepp,
3
Camin Dean,
1
Eric A. Johnson,
3
Roger Janz,
4
Edwin R. Chapman
1,2
*
How the widely used botulinum neurotoxin A (BoNT/A) recognizes and enters neurons is poorly
understood. We found that BoNT/A enters neurons by binding to the synaptic vesicle protein SV2
(isoforms A, B, and C). Fragments of SV2 that harbor the toxin interaction domain inhibited BoNT/A
from binding to neurons. BoNT/A binding to SV2A and SV2B knockout hippocampal neurons was
abolished and was restored by expressing SV2A, SV2B, or SV2C. Reduction of SV2 expression in
PC12 and Neuro-2a cells also inhibited entry of BoNT/A, which could be restored by expressing SV2
isoforms. Finally, mice that lacked an SV2 isoform (SV2B) displayed reduced sensitivity to BoNT/A.
Thus, SV2 acts as the protein receptor for BoNT/A.
B
otulinum neurotoxin A (BoNT/A) is
one of seven neurotoxins (designated
BoNT/A to BoNT/G) produced by
the bacterium Clostridium botulinum (1).
BoNT/A blocks neurotransmitter release by
cleaving synaptosome-associated protein
of 25 kD (SNAP-25) (2, 3) within presyn-
aptic nerve terminals. Owing to its long-
lasting effects, BoNT/A is used to treat a
wide range of medical conditions and has
also emerged as a potential biological weapon
(4, 5).
It has been proposed that receptors for
BoNTs are composed of both gangliosides
that bind toxins with low affinity and protein
receptors that form high-affinity complexes
with toxins (6, 7). Synaptotagmins I and II,
two homologous synaptic vesicle proteins,
have been shown to function in conjunction
with gangliosides to mediate entry of BoNT/B
into cells (811). Low-affinity interactions
between BoNT/A and gangliosides have also
been reported (1215), and depletion of
gangliosides in neuroblastoma cells prevented
entry of BoNT/A (16). In addition, mice
lacking complex gangliosides display reduced
sensitivity to BoNT/A (10, 17). Protein compo-
nents are certainly required for BoNT/A entry
into nerve terminals but have not been identi-
fied (1, 6, 7).
To identify protein receptors for BoNT/A,
we studied the toxin_s route of entry. Stimu-
lation of neurotransmitter release is known to
reduce the time required for paralysis caused
by BoNT/A at the diaphragm (18). We found
that depolarization with a high-K
solution in
hemidiaphragm preparations increased toxin
binding to neuromuscular junctions (NMJs)
by a factor of 6 (Fig. 1A), suggesting that
1
Howard Hughes Medical Institute and Department of
Physiology,
2
Molecular and Cellular Pharmacology Pro-
gram,
3
Department of Food Microbiology and Toxicology,
University of Wisconsin, Madison, WI 53706, USA.
4
W. M.
Keck Center for Learning and Memory and Department of
Neurobiology and Anatomy, University of TexasHouston
Medical School, Houston, TX 77030, USA.
*To whom correspondence should be addressed: E-mail:
chapman@physiology.wisc.edu
REPORTS
28 APRIL 2006 VOL 312 SCIENCE www.sciencemag.org 592