REPORTS

generated amino acids are imported to the LCV

through SLC1A5 and other host amino acid transporters (20, 22). However, generation of amino
acids through promoting proteasomal degradation
may not be the only virulence strategy dedicated to
obtain carbon and energy sources from the host,
because the type IIsecreted degradation enzymes
(21) may provide an additional strategy to generate sources of carbon and energy. Our work shows
a microbial strategy that exploits conserved eukaryotic proteasomal degradation of K48-linked
polyubiquitinated proteins to generate amino acids
for use as sources of carbon and energy in vivo.
This microbial strategy is required for intracellular
bacterial growth in evolutionarily distant host cells
and for manifestation of disease in mammals.
References and Notes
1. R. Appelberg, J. Leukoc. Biol. 79, 1117 (2006).
2. L. Rohmer, D. Hocquet, S. I. Miller, Trends Microbiol.
19, 341 (2011).
3. M. Molmeret, M. Horn, M. Wagner, M. Santic,
Y. Abu Kwaik, Appl. Environ. Microbiol. 71, 20 (2005).
4. R. R. Isberg, T. J. OConnor, M. Heidtman, Nat. Rev.
Microbiol. 7, 13 (2009).
5. Z.-Q. Luo, Front Microbiol. 2, 31 (2011).
6. C. T. Price, T. Al-Quadan, M. Santic, S. C. Jones,
Y. Abu Kwaik, J. Exp. Med. 207, 1713 (2010).
7. M. Lomma et al., Cell. Microbiol. 12, 1272 (2010).
8. C. T. Price et al., PLoS Pathog. 5, e1000704 (2009).
9. T. Al-Quadan, Y. A. Kwaik, Front. Microbiol. 2, 23 (2011).
10. M. S. Dorer, D. Kirton, J. S. Bader, R. R. Isberg, PLoS
Pathog. 2, e34 (2006).
11. T. P. Al-Quadan, C. T. Price, N. London, O. SchuelerFurman, Y. Abu Kwaik, Trends Microbiol., published
online 7 October 2011 (10.1016/j.tim.2011.08.003).

The Molecular Basis

of Acid Insensitivity in the
African Naked Mole-Rat
Ewan St. John Smith,* Damir Omerbaic, Stefan G. Lechner, Gireesh Anirudhan,
Liudmila Lapatsina, Gary R. Lewin*
Acid evokes pain by exciting nociceptors; the acid sensors are proton-gated ion channels that
depolarize neurons. The naked mole-rat (Heterocephalus glaber) is exceptional in its acid
insensitivity, but acid sensors (acid-sensing ion channels and the transient receptor potential
vanilloid-1 ion channel) in naked mole-rat nociceptors are similar to those in other vertebrates.
Acid inhibition of voltage-gated sodium currents is more profound in naked mole-rat nociceptors
than in mouse nociceptors, however, which effectively prevents acid-induced action potential
initiation. We describe a species-specific variant of the nociceptor sodium channel NaV1.7,
which is potently blocked by protons and can account for acid insensitivity in this species.
Thus, evolutionary pressure has selected for an NaV1.7 gene variant that tips the balance from
proton-induced excitation to inhibition of action potential initiation to abolish acid nociception.
cid is a noxious and painful stimulus in
all vertebrates examined, with one known
exception among mammals: the African
naked mole-rat (1, 2). The absence of acid nociception can be explained by the acid-insensitivity

Department of Neuroscience, Max-Delbrck Center for Molecular

Medicine, Robert-Rssle-Strasse 10, 13125 Berlin-Buch, Germany.
*To whom correspondence should be addressed. E-mail:
glewin@mdc-berlin.de (G.R.L.); ewan.smith@mdc-berlin.
de (E.St.J.S.)

of C-fiber nociceptors and may have resulted from

exposure to high-CO2 conditions that accompanies the communal living of many naked mole-rats
underground (2, 3). We sought to identify factors
that render naked mole-rat nociceptors insensitive
to acid. Two main acid sensors are proposed to
participate in acid-mediated nociception: the acidsensing ion channel (ASIC) family and the transient
receptor potential vanilloid-1 ion channel (TRPV1)
(1, 4). We first asked whether naked mole-rat nociceptors simply lack proton-gated ion channels by

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VOL 334

12. S. Al-Khodor, C. T. Price, F. Habyarimana, A. Kalia,

Y. Abu Kwaik, Mol. Microbiol. 70, 908 (2008).
13. N. Gallastegui, M. Groll, Trends Biochem. Sci. 35, 634
(2010).
14. T. Saric, C. I. Graef, A. L. Goldberg, J. Biol. Chem. 279,
46723 (2004).
15. K. Falk, O. Rtzschke, H. G. Rammensee, Nature 348,
248 (1990).
16. E. Reits et al., Immunity 18, 97 (2003).
17. H. Ashida et al., Nat. Cell Biol. 12, 66, 1 (2010).
18. Z. D. Dalebroux, R. L. Edwards, M. S. Swanson,
Mol. Microbiol. 71, 640 (2009).
19. E. Eylert et al., J. Biol. Chem. 285, 22232 (2010).
20. H. Wieland, S. Ullrich, F. Lang, B. Neumeister, Mol.
Microbiol. 55, 1528 (2005).
21. S. DebRoy, J. Dao, M. Sderberg, O. Rossier, N. P. Cianciotto,
Proc. Natl. Acad. Sci. U.S.A. 103, 19146 (2006).
22. J. D. Sauer, M. A. Bachman, M. S. Swanson, Proc. Natl.
Acad. Sci. U.S.A. 102, 9924 (2005).
Acknowledgments: We thank C. Sasakawa for the
generous gift of providing all the ubiquitin mutant plasmid
DNA and J. Cox at the University of Utah Metabolomics
Core Facility for the free amino acids analyses. Y.A.K. is
supported by Public Health Service awards R01AI43965
and R01AI069321 from National Institute of Allergy and
Infectious Diseases and by the commonwealth of Kentucky
Research Challenge Trust Fund.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1212868/DC1
Materials and Methods
Figs. S1 to S6
Tables S1 and S2
References (2338)
18 August 2011; accepted 27 October 2011
Published online 17 November 2011;
10.1126/science.1212868

using whole-cell patch-clamp to compare protongated currents in nociceptors isolated from naked
mole-rat and mouse dorsal root ganglia (DRG)
[supporting online material (SOM) methods].
Surprisingly, both ASIC-like currents, which are
benzamil-sensitive and transient, as well as TRPV1like currents that are sustained-only and blocked
by N-(4-tertiarybutylphenyl)-4-(3-chloropyridin2-yl)tetrahy-dropyrazine-1(2H)-carboxamide
(BCTC) (5) were present both in naked mole-rat
and mouse nociceptors (Fig. 1, A and B, and fig.
S1). Only minor differences were noted in the incidence and kinetic properties of proton-gated currents between mouse and naked mole-rat (fig. S1
and table S1). Real-time polymerase chain reaction
(PCR) analysis revealed that mRNA encoding both
TRPV1 and ASIC subunits were similarly abundant in mouse and naked mole-rat DRGs (fig. S2).
Therefore, the acid insensitivity of naked mole-rat
nociceptors cannot be explained by minor differences observed in proton-gated ion currents in this
species as compared with mice (6, 7).
We next cloned and expressed recombinant ASIC1a, ASIC1b, and TRPV1 channels to
determine whether naked mole-rat channels exhibit unusual pH sensitivity. The proton sensitivity and pharmacology of naked mole-rat ASIC1a
(nmrASIC1a), ASIC1b (nmrASIC1b), and TRPV1
(nmrTRPV1) were essentially the same as their
mouse counterparts (Fig. 1, C and D, fig. S3, and
table S2). Both mouse and naked mole-rat ASICs
displayed steady-state inactivation (SSI) (Fig. 1C,

16 DECEMBER 2011

Downloaded from www.sciencemag.org on August 11, 2015

and cysteine are converted by Legionella to pyruvate, followed by acetyl coenzyme A (CoA)
that feeds the TCA cycle (fig. S6). Similar to
supplementation of cysteine and serine, supplement of pyruvate or citrate specifically rescued
the ankB but not the dotA mutant for intracellular
proliferation in human cells (Fig. 4D) and amoeba (fig. S5C). Thus, rescue of the ankB mutant
upon amino acid supplementation was direct and
was not due to a secondary effect on host cells by
the excess amino acids. Rescue of the ankB mutant for intracellular proliferation by cysteine,
serine, pyruvate, or citrate indicates that the ankB
mutant has sufficient levels of nutrients in the
host cell with the exception of higher levels of a
needed major source of carbon and energy
production through the TCA cycle.
The high demand for amino acids by Legionella is demonstrated by the presence of ~12
classes of adenosine triphosphatebinding cassette transporters, amino acid permeases, and
many proteases and by the requirement to supplement the rich medium for in vitro growth of
Legionella with 3.3 mM cysteine (21, 22). Our
data suggest that cysteine is a major source of
carbon and energy for Legionella within amoeba
and human cells. To satisfy its high demands for
amino acids within amoeba and human cells,
Legionella uses the AnkB bona fide F-box effector
to exploit the conserved eukaryotic processes of
K48-linked polyubiquitination and the proteasome machineries to generate amino acids. The

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REPORTS
fig. S3, and table S2), a phenomenon in which
mild acidification (pH 7.0) inactivates the channels, impairing their ability to open after challenge with much lower pH (8). The rate at which
recombinant nmrASIC1a- and nmrASIC1bmediated currents inactivated was similar to that
found for native naked mole-rat ASIC-like currents (fig. S1) and was significantly faster than
their mouse orthologs (table S2). nmrTRPV1
had properties characteristic of other mammalian
TRPV1s; it was capsaicin-sensitive, outwardly
rectifying, and heat-gated (threshold 42.6 T
0.5C, n = 15 cells) (Fig. 1D and fig. S3). Based
on BCTC-sensitivity, ~52% of sustained-only currents in naked mole-rat DRG neurons were mediated via nmrTRPV1 channels (Fig. 1B).
Consistent with these results, increasingly
acidic stimuli evoked graded increases in inward current and increasing depolarization in
both mouse and naked mole-rat DRG neurons
(fig. S4). However, despite pronounced membrane depolarization few neurons in the mouse
(4.3%) or naked mole-rat (13.3%) fire an action
potential (AP) after acid stimulation (Fig. 2, A
and B), although the depolarization was sufficient to activate voltage-gated sodium channels
(NaVs) to trigger APs. Approximately 20% of
C-fibers are excited by acid in skin-nerve studies
(9, 10), and so we made additional recordings
from identified skin DRG neurons, retrogradely
labeled from mouse skin. Stimulation with pH
5.0 evoked APs more commonly in identified
skin neurons (18.5%), and these neurons exhibited
larger peak current densities and larger, transient, ASIC-like currents at pH 5.0 than did nonidentified mouse neurons (fig. S5 and table S1).
Thus, larger current/bigger depolarization increases
AP probability, but there does not appear to be
a voltage threshold above which neurons always

spike (fig. S5). AP spiking DRG neurons were,

however, on average more depolarized by acid
than were non-spikers (10.4 T 1.3 mV versus
26.0 T 1.3 mV, P < 0.001, unpaired t test) (fig.
S6). In both mouse and naked mole-rat DRG
neurons, we observed that acid-evoked transient
currents were significantly more likely to generate an AP than were sustained-only currents
(P < 0.05 and 0.01 respectively, Fishers exact
test). Why can acid evoke APs in naked mole-rat
DRG neurons but still not excite C-fiber nociceptors? (2). This discrepancy may arise because
in our in vitro experiments, pH stimuli are applied
rapidly (<0.5 ms), but tissue acidification is probably buffered so that pH falls only gradually.
Gradual acidification will favor SSI of ASICmediated currents (Fig. 1C), and indeed, transient currents in naked mole-rats DRG neurons
inactivated with a half-maximal inhibitory concentration (IC50) of pH 7.31 (Fig. 2C). In current-clamp
mode, gradual acidification to pH 5.0 produced a
similar absolute depolarization as step acidification from pH 7.4 to pH 5.0 (Fig. 2D), but gradual
acidification never evoked APs (n = 0 of 30
cells), whereas step acidification did (n = 12 of 69
cells, P < 0.05 Fishers exact test). This suggests
that ASIC-mediated APs only occur when there
are rapid shifts in pH. Moreover, in the same
mouse DRG neurons large acid-induced depolarization produced no AP, but 50 mM adenosine
5-triphosphate (ATP) triggered a small depolarization and many APs (Fig. 2E). Thus, during
acid-evoked depolarization a concomitant inhibitory process prevents AP firing.
Protons can inhibit NaV channels (11, 12), and
so we next examined acid inhibition of macroscopic, voltage-gated inward currents in mouse
DRG neurons. At pH 6.0, there was a decrease in
inward current amplitude and a depolarizing shift in

the half-maximal voltage for activation (V1/2); peak

inward conductance was inhibited by 42% (Fig. 3,
A and B, and fig. S7). The IC50 for this inhibition
was pH 5.95, which was accompanied by a pHdependent depolarizing shift in the V1/2 for activation (fig. S7 and table S3). Unlike voltage-gated
inward currents, macroscopic outward currents
only showed a large inhibition at pH 5.0 (fig. S7).
Pharmacologically isolated NaV currents exhibited
a 46% reduction in conductance at pH 6.0 (fig.
S7)a pH sensitivity similar to the macroscopic
current. These data predict that acid would raise
AP firing thresholds, and AP threshold was increased significantly in neurons exposed to pH
6.0, and AP amplitudes decreased (fig. S8). The
characteristic hump on the falling phase of the AP
in nociceptors (1, 13, 14) was often absent at low
pH, so that 41% lacked a hump at pH 6.0, and all
APs at pH 5.0 were humpless (fig. S8).
We next tested whether voltage-gated inward
currents in naked mole-rat are more susceptible
to acid inhibition than are those in mice. Macroscopic voltage-gated inward currents in naked molerat neurons were inhibited by pH 6.0, coupled with
a depolarizing shift in V1/2 for activation, resulting
in a 63% decrease in conductance (Fig. 3C). The
63% inhibition of conductance at pH 6.0 was significantly greater than the 42% inhibition observed
in mouse neurons (P < 0.01, unpaired t test) (Fig.
3D). If acid is more potent in inhibiting voltagegated inward currents in naked mole-rat neurons,
then low pH should also inhibit nociceptor firing
to natural stimuli more profoundly than in the
mouse. Indeed, pH 4.0 and pH 6.0 solutions inhibited mechanically evoked C-fiber nociceptor firing
more potently in naked mole-rat than in mouse [P <
0.05, 2-way analysis of variance (ANOVA)] (Fig.
3, E and F). Furthermore, the overall effect of
exposure to pH 6.0 in mouse C-fibers was one of

Fig. 1. Cloned and native

naked mole-rat acid sensors are similar to those of
mice. (A) Acid evokes transient(T),benzamil-sensitive,
inward currents followed
by a small sustained phase
(S) and sustained-only currents, (B) 52% of which
are capsaicin-sensitive and
inhibited by the TRPV1selective antagonist BCTC.
(C)nmrASIC1aandmASIC1a
are acid-sensitive, pH50
6.03 (n = 12 to 18 cells)
and 6.28 (n = 12 to 13
cells), respectively, and
undergo SSI. nmrASIC1a,
IC50 = 7.38 (n = 3 to 22
cells); mASIC1a, IC50 =
7.46 (n = 10 cells). (D)
nmrTRPV1 and mTRPV1
are acid-sensitive, pH50
5.31 (n = 11 to 13 cells)
and 5.22 (n = 10 cells).
**P < 0.01, ***P < 0.001.

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REPORTS
Fig. 2. Proton-gated currents poorly drive AP spiking in DRG neurons. In
(A) mouse and (B) naked
mole-rat, larger protongated currents produce
larger depolarizations, but
no threshold exists above
which all cells spike. (C)
Transient currents undergo SSI in naked mole-rat
DRG neurons (IC50 =
7.31, n = 17 cells). (D)
Gradual acidification does
not produce spiking, although the same depolarization is reached with
a step from pH 7.4 to 5.0
(compare black curve, n =
30 cells, with red point,
n = 69 cells). (E) 50 mM
ATP evokes APs in cells,
whereas pH 5.0 does not.

Fig. 3. Increased acid block of voltage-gated inward currents and AP initiation in naked mole-rat neurons. (A) pH 6.0 inhibits voltage-gated inward current
amplitude and produces a depolarizing shift in V1/2 for activation [outward
currents digitally removed (fig. S7)]. (B) pH 6.0 inhibits macroscopic voltagewww.sciencemag.org

SCIENCE

gated inward conductance by 42% in mouse and (C) 63% in naked mole-rat,
which is (D) a significant difference. (E) Mechanically evoked APs in C-fibers
are inhibited by acid (red traces) in (left) mouse and (right) naked mole-rat,
but (F) significantly more in naked mole-rat. **P < 0.01.
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REPORTS
sensitization, as has been observed in the rat (10),
whereas most naked mole-rat C-fibers were inhibited at pH 6.0 (fig. S9). Electrically evoked APs
were also blocked by acid (fig. S9). Thus, proton
inhibition of NaV currents in nociceptors directly
inhibits natural activation of nociceptors in vivo.
The threshold for AP generation in nociceptors
is largely determined by NaV1.7 (15); nonsense
mutations in the human SCN9A gene encoding
NaV1.7 produce complete insensitivity to pain
(16, 17); and extreme pain disorders are associated with activating NaV1.7 mutations (1820). The
outer carboxylate ring in the pore loop of NaV
channels is responsible for determining acid sensitivity (11). Therefore, we sequenced the naked
mole-rat NaV1.7 transcript (nmrNaV1.7, 94.7%
sequenced based on human NaV1.7) and looked
for variants in regions that might alter the pH sensitivity of NaV1.7. In nmrNaV1.7, domain IVa
highly conserved, positively charged amino acid
motif (KKV, amino acids 1718 to 1720 in human
NaV1.7)was negatively charged (EKE) (Fig.
4A). We introduced the EKE motif into hNaV1.7
and compared acid sensitivity of hNaV1.7 and
hNaV1.7EKE. In the presence of acid (pH 6.0),

hNaV1.7EKE was significantly more inhibited, so

that the conductance was inhibited by 73% compared with 58% for hNaV1.7 (P < 0.001) (Fig. 4,
B to D). Thus, naked mole-rats harbor a distinct
amino acid motif within the NaV1.7 gene, and the
exchange of two amino acids renders the channel
more sensitive to proton block than other vertebrate NaV channels. The degree of proton inhibition of naked mole-ratlike hNaV1.7EKE channels
was similar to that of macroscopic voltage-gated inward currents in naked mole-rats. Sequencing of the
nmrNaV1.8 transcript did not identify any chargechanging motifs around the outer carboxylate ring.
Lack of proton sensors in naked mole-rat nociceptors cannot explain acid insensitivity in this
species, but rather, protons act to put a brake on
AP initiation by potently blocking nmrNaV1.7.
Our data supports a new model for understanding how acid excites mammalian nociceptors:
Depolarizing input (from TRPV1 and ASICs)
must overcome the concurrent inhibition of NaV
channels (Fig. 4E). This balance is shifted in naked
mole-rat neurons because nmrNaV1.7 is more potently blocked by acid, resulting in no acid-evoked
APs. In their subterranean niche, naked mole-rats

Fig. 4. A sequence variation in naked mole-rat NaV1.7 enhances acid block. (A) Alignment of NaV1.7
domain IV. nmrNaV1.7 harbors a negatively charged EKE motif, in contrast to more positively charged
residues in other mammalian NaV1.7s (green boxes, charges next to alignment; orange square, outer
carboxylate ring; and yellow star, EKE motif). (B) Wild-type hNaV1.7 and (C) hNaV1.7EKE currents are
inhibited by pH 6.0; (D) hNaV1.7EKE is significantly more inhibited. (E) Proton-mediated C-fiber nociceptor
activation is a balance between (red arrow) depolarizing input and (gray bar) proton-mediated inhibition of
NaV1.7. In mouse, acid can evoke nociceptor AP firing; however, the increased nmrNaV1.7 proton
sensitivity in naked mole-rats largely prevents acid-driven AP initiation and behavior. ***P < 0.001.

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are exposed to an unusually high-CO2/low-O2

environment (3), and high CO2 can drive tissue
acidosis (21). Another vertebrate species exposed
to a high-CO2 environment is the cave-roosting
microbat Myotis lucifugus, and it carries a motif
charged similarly to the naked mole-rat EKE
motif in its NaV1.7 gene. In contrast, the treeroosting nonCO2-challenged megabat Pteropus
vampyrus carries a more typical, less negatively
charged motif NKV in its NaV1.7 gene (Fig. 4A).
Thus, convergent evolution on species with similar high-CO2 living conditions may have selected for NaV1.7 gene variants that reduce
acid nociception. Our study illustrates that the
extreme physiology of the naked mole-rat offers
particular insights into normal physiology relevant to inflammatory pain processes in which
tissue acidosis plays a role (22).
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Acknowledgments: E.St.J.S. carried out DRG electrophysiology.
E.St.J.S. and D.O. carried out CHO cell electrophysiology. S.L.
carried out skin-nerve electrophysiology. D.O., L.L., G.A., and
E.St.J.S. carried out molecular biology. E.St.J.S. and G.R.L.
designed experimental studies and wrote the paper. E.St.J.S.
was supported by the Alexander von Humboldt foundation,
and D.O. was supprted by the MolNeuro Helmholtz Research
School. Additional funding was obtained from Deutsche
Forschungsgemeinschaft collaborative research center 665 and
Exc 257 (NeuroCure). We thank H. Thrnhardt, A. Wegner,
and K. Barda for technical support. Sequences have been
submitted to GenBank (SOM).

Supporting Online Material

www.sciencemag.org/cgi/content/full/334/6062/1557/DC1
Materials and Methods
Figs. S1 to S9
Tables S1 to S3
References (23, 24)
8 September 2011; accepted 1 November 2011
10.1126/science.1213760

www.sciencemag.org

The Molecular Basis of Acid Insensitivity in the African Naked

Mole-Rat
Ewan St. John Smith et al.
Science 334, 1557 (2011);
DOI: 10.1126/science.1213760

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