THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 279, No. 18, Issue of April 30, pp. 18783–18791, 2004
Printed in U.S.A.
A Family of Acid-sensing Ion Channels from the Zebrafish
WIDESPREAD EXPRESSION IN THE CENTRAL NERVOUS SYSTEM SUGGESTS A CONSERVED
ROLE IN NEURONAL COMMUNICATION*
Received for publication, February 10, 2004
Published, JBC Papers in Press, February 16, 2004, DOI 10.1074/jbc.M401477200
Martin Paukert‡, Samuel Sidi§¶, Claire Russell储, Maria Siba‡, Stephen W. Wilson储**,
Teresa Nicolson§‡‡, and Stefan Gründer‡§§
From the ‡Department of Physiology II and §Max-Planck-Institute of Developmental Biology, 72076 Tübingen, Germany,
and 储Department of Anatomy and Developmental Biology, University College London,
London WC1E 6BT, United Kingdom
Acid-sensing ion channels (ASICs) are excitatory receptors for extracellular Hⴙ. Proposed functions include
synaptic transmission, peripheral perception of pain,
and mechanosensation. Despite the physiological importance of these functions, the precise role of ASICs
has not yet been established. In order to increase our
understanding of the physiological role and basic structure-function relationships of ASICs, we report here the
cloning of six new ASICs from the zebrafish (zASICs).
zASICs possess the basic functional properties of mammalian ASICs: activation by extracellular Hⴙ, Naⴙ selectivity, and block by micromolar concentrations of amiloride. The zasic genes are broadly expressed in the
central nervous system, whereas expression in the peripheral nervous system is scarce. This pattern suggests
a predominant role for zASICs in neuronal communication. Our results suggest a conserved function for receptors of extracellular Hⴙ in the central nervous system
of vertebrates.
Acid-sensing ion channels (ASICs)1 are Na⫹ channels that
are activated by extracellular H⫹ (1). In the central nervous
system, H⫹ is co-released with other transmitters, since synaptic vesicles are acidic (pH 5.7) (2). Thus, ASICs may act as
excitatory receptors. During inflammation, the extracellular
H⫹ concentration can also significantly increase. ASICs may
therefore contribute to the perception of painful stimuli in the
peripheral nervous system. In addition to the activation by H⫹,
the homology of ASICs to mechanosensitive ion channels in
Caenorhabditis elegans and the analysis of knock-out mice for
* This work was supported by grants from the Deutsche Forschungsgemeinschaft (to T. N. and S. G.) and the Wellcome Trust and European
Community (to S. W. W.). The costs of publication of this article were
defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBankTM/EBI Data Bank with accession number(s)
AJ609615–AJ609620.
¶ Present address: Ecole Normale Supérieure, Laboratoire de Biologie Moléculaire du Developpement, 46 rue d’Ulm, 75005 Paris, France.
** A Wellcome Trust Senior Research Fellow.
‡‡ Present address: Oregon Hearing Research Center and Vollum
Institute, Oregon Health and Science University, Portland, OR 97201.
§§ To whom correspondence should be addressed: Dept. of Physiology
II, Gmelinstr. 5, D-72076 Tübingen, Germany. Tel.: 49-7071-29-77357;
Fax: 49-7071-29-5074; E-mail: stefan.gruender@uni-tuebingen.de.
1
The abbreviations used are: ASIC, acid-sensing ion channel; zASIC,
zebrafish ASIC; MES, 2-(N-morpholino)ethanesulfonic acid; RACE,
rapid amplification of cDNA ends; HA, hemagglutinin; VSV, vesicular
stomatitis virus glycoprotein; hpf, hours postfertilization.
This paper is available on line at http://www.jbc.org
individual ASIC subunits have suggested that ASICs are components of mechanically activated ion channels (3).
There are four genes coding for ASICs in the human and
mouse genome (asic1–asic4) (1, 4, 5). Moreover, alternative
splicing of the asic1 and asic2 genes generates the splice variants ASIC1a/ASIC1b and ASIC2a/ASIC2b, respectively, that
differ in the N-terminal third of the protein (6, 7). In rodents,
ASICs show a broad expression pattern in the central and
peripheral nervous system. All ASICs except ASIC4 are expressed in sensory neurons of the dorsal root and trigeminal
ganglia. ASIC1a, -2a, -2b, and -4 are expressed in the central
nervous system (1, 4, 5). ASICs activate rapidly upon H⫹ application (␶act of about 10 ms for ASIC1) (6) and desensitize in
the continuous presence of H⫹. ASIC1 and ASIC3 desensitize
completely (␶inact of about 1 s for ASIC1) (6), whereas ASIC2a
desensitizes incompletely and more slowly (7). ASIC2b and
ASIC4 cannot be activated by H⫹ (4, 5, 7). However, functional
ASICs are probably tetrameric proteins, and ASIC2b forms a
heteromeric channel with ASIC3 (7). Several other combinations of subunits form heteromeric ASICs (8, 9), increasing the
variety of H⫹-gated Na⫹ channels in situ (10, 11).
Despite the progress made in recent years, we are far from a
clear understanding of the physiological role of ASICs. The
zebrafish, Danio rerio, is a model organism for the study of
vertebrate biology that offers some advantages over higher
vertebrates. It has a translucent embryo and develops rapidly,
allowing easy expression analysis of genes and detection of
developmental abnormalities in mutant fish. In order to establish the zebrafish as a model to study the physiological role of
ASICs, we report here the molecular cloning of cDNAs for six
ASICs, their expression pattern during early zebrafish development, and their electrophysiological characterization in a
heterologous expression system. Our results suggest a conserved function for receptors of extracellular H⫹ in the central
nervous system of vertebrates.
EXPERIMENTAL PROCEDURES
Cloning of ASIC cDNAs from Zebrafish—Partial cDNA clones for
zASIC1.2, -1.3, and -4.1 were identified by homology cloning from
embryonic zebrafish brain and eye. Degenerate oligonucleotides and
conditions for PCR were as described elsewhere (4). Partial sequences
for zASIC1.1, -2, and -4.2 were identified by searching the online zebrafish genomic sequence trace data base (ENSEMBL) at the Sanger
institute using the SSAHA program. Different rat ASIC subunits were
used as a probe. These partial sequences were used to design primers
for rapid amplification of 5⬘- and 3⬘-cDNA ends (RACE). Using the
Smart RACE cDNA amplification kit (Clontech), RACE was performed
with poly(A)⫹ RNA from different embryonic and adult zebrafish tissues. PCR products were subcloned with the TOPO-TA cloning kit
(Invitrogen) and sequenced. Full-length zASICs were assembled from
the longest 5⬘ and 3⬘ RACE products. cDNA sequences were deposited
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18784
ASICs from the Zebrafish
FIG. 1. Sequence alignment of zASICs with rat ASIC1a, -2a, -3, and -4. Amino acids showing a high degree of identity are shown as white
letters on a black background. The initiator methionine of each cDNA was assigned to the first ATG in frame. Transmembrane domains are
indicated by bars, conserved cysteines by stars, and a conserved consensus site for N-linked glycosylation in the loop between transmembrane
domains by a branched symbol. Accession numbers are as follows: U94403, ASIC1; U53211, ASIC2; AF013598, ASIC3; AJ271642, ASIC4.
in the EMBL data base under the following accession numbers:
zASIC1.1, AJ609615; zASIC1.2, AJ609616; zASIC1.3, AJ609617;
zASIC2, AJ609618; zASIC4.1, AJ609619; zASIC4.2, AJ609620.
For expression studies in Xenopus oocytes, the entire coding sequences of zASICs were amplified from cDNA of larval brain by PCR
(ExpandHighFidelity PCR system; Roche Applied Science). Using terminal restriction endonuclease recognition sequences (BamHI and
KpnI; zASIC1.1: XhoI and KpnI), the PCR product was ligated in the
oocyte expression vector pRSSP containing the 5⬘-untranslated region
from Xenopus ␤ globin and a poly(A) tail. Several clones from two
independent PCRs were sequenced to exclude PCR errors.
Radiation Hybrid Mapping and Synteny Analyses—PCR primers
designed to amplify single exons from zasic genes were used to screen
pools of zebrafish/hamster radiation hybrid cell lines (Goodfellow radiation hybrid panel) (12). Two independent sets of primers were used per
gene (primer sequences available upon request). The resulting 94-digit
radiation hybrid scores were used to establish physical map positions
using the Instant Mapping program at the Children’s Hospital Zebrafish Genome Project Initiatives Web site (zfrhmaps.tch.harvard.edu/
ZonRHmapper/instantMapping.htm). The same program was used to
calculate Lod scores. Microsatellite markers (Zmarkers) and expressed
sequence tags that flanked each locus were used to establish the putative genetic map positions of the different loci on the integrated genetic/
physical Tübingen map (wwwmap.tuebingen.mpg.de/). zasic-containing
regions were then screened for the presence of expressed sequence tags
that had been used to establish the syntenic correspondence of the
zebrafish and human genomes (13) (zebrafish-human syntenic correspondence map available on the World Wide Web at zfish.wustl.edu/).
Such expressed sequence tags that turned out to be informative were
fb82d01 (zasic1.1), fb36e06 and bact2 (zasic2), and fc17a11 (zasic4.1).
In Situ Hybridization—PCR products comprising 360 –520 bp from
the N-terminal part of zASIC1.1 to zASIC4.2 cDNAs were subcloned in
the TA cloning vector pCRII-TOPO (Invitrogen). Clones were sequenced
to determine orientations relative to T3 and T7 sites and were used as
ASICs from the Zebrafish
18785
TABLE I
Integrated physical and genetic map positions of zasic loci
Gene
LGa
Nearest marker
zasic1.1
zasic1.2
zasic1.3
zasic2
zasic4.1
zasic4.2
22
8
24
3
9
6
Wz12404.1
Zkp113g7ya
unp265
Wz12338.1
bz31c2
fj66a04
Distance
LODb score
Minimal genetic
interval (p-d)c
Genetic Position
(from top of LG)
10.78
5.81
Maximum
15.86
16.54
11.62
Z11379-Z6507
Z7819-Z4323
Z9603-Z6438
Z9664-Z11227
Z7319-Z25375
Z17248-Z12094
⬃15
⬃33
⬃56
⬃68.5
⬃43
39.5–49
cRe
11
43
0g
7
7
24
Human
chromosomed
Synteny
12q12
12q12
12q12
17q11.2–12
2q35–36
2q35–36
Yes
No
No
Yes
Yes
?
cMf
a
Linkage group.
Logarithm of the odds.
Proximal to distal.
d
The physical position of human orthologs; the syntenic region for zasic4.2 was equivocal.
e
Centirays.
f
Centimorgans.
g
Considered as duplicate markers by InstantMapper.
b
c
FIG. 2. Evolutionary relationship of
zASIC subunits and other family
members. Other family members are
ASICs and intestinal Na⫹ channels
(INaCs) from human (h) and rat (r);
ripped pocket (RPK) and pickpocket
(PPK) from Drosophila; DEG-1, MEC-4,
MEC-10, and UNC-8, degenerins from
Caenorhabditis; subunits of the epithelial
Na⫹ channel (ENaC) from rat; and the
FMRFamid receptor (FaNaCh) from the
snail Helix aspersa. Highly divergent sequences at the N and C termini as well as
in the proximal part of the extracellular
loop had been deleted, and the alignment
and the tree for the phylogram have been
established by Neighbor-Joining with
ClustalX. The tree was then imported into
TreeView and rooted with FMRFamid receptor as an outgroup. Maximum likelihood analysis using TreePuzzle gave similar results.
templates for 50-␮l PCRs (Advantage II PCR system; Clontech) using
universal M13 forward and reverse primers. PCR products were precipitated and used as templates (⬃1 ␮g/␮l) for in vitro transcription of
digoxygenin-labeled (Roche Applied Science) sense and antisense riboprobes. Probes were used for whole mount in situ hybridization as
described previously (14). To obtain efficient labeling of internal tissues,
hybridizations of larvae ⱖ3 days postfertilization were carried out
using albino larvae that were treated 18 (3-day postfertilization larvae)
to 22 min (4-day postfertilization larvae) in 10 ␮g/ml proteinase K.
Electrophysiology—Using the mMessage mMachine kit (Ambion,
Austin, TX), capped cRNA was synthesized by SP6 RNA polymerase
from linearized cDNA. We injected 0.2–10 ng of cRNAs; currents ranged
from 0.5 to 50 ␮A. cRNA was injected into stage V or VI oocytes of
Xenopus laevis, and oocytes were kept in OR-2 medium (82.5 mM NaCl,
2.5 mM KCl, 1.0 mM Na2HPO4, 5.0 mM HEPES, 1.0 mM MgCl2, 1.0 mM
CaCl2, and 0.5 g/liter polyvinylpyrrolidone) for 1–7 days. Whole cell
currents were recorded with a TurboTec 03⫻ amplifier (NPI Electronic,
Tamm, Germany) using an automated, pump-driven solution exchange
system together with the oocyte testing carousel controlled by the
interface OTC-20 (NPI Electronic). Data acquisition and solution exchange were managed using the software CellWorks version 5.1.1 (NPI
Electronic). Data were filtered at 20 Hz and acquired at 1 kHz. The bath
solution for two-electrode voltage clamp contained 140 mM NaCl, 1.8
mM CaCl2, 1.0 mM MgCl2, 10 mM HEPES. For the acidic test solutions,
HEPES was replaced by MES buffer. If not otherwise indicated, acidic
application solutions were applied for 3.5 s, and neutral bath solution
(pH 7.4) was applied for 30 s between channel activation. Holding
potential was ⫺60 mV.
For patch clamp experiments, bath solution contained 140 mM NaCl,
1.8 mM CaCl2, 1.0 mM MgCl2, 10 mM HEPES, pH 7.4. Acidic test solutions
were buffered with MES. Patch pipettes contained 140 mM KCl, 2.0 mM
MgCl2, 5.0 mM EGTA, and 10 mM HEPES, pH 7.4. Rapid pH changes in
the outside-out configuration were achieved by placing the patch in front
of a piezo-driven double-barreled application pipette. Time constant for
complete solution exchange is ⬍2 ms (6). Holding potential was ⫺70 mV.
Currents were filtered at 5 kHz and acquired at 50 kHz.
Data were analyzed using IgorPro software (WaveMetrics, Lake Oswego, OR). Dose-response curves were fitted to a Hill equation, and I-V
curves were fitted to a polynomal function. Time constants of desensitization were determined by fitting current traces obtained in outsideout patch clamp experiments to a monoexponential function. The current rise time was evaluated by determining the rise time from 20 to
80% of the peak current (time to peak). All values reported represent
the mean ⫾ S.E. from n individual measurements, if not otherwise
indicated. Statistical analysis was done with Student’s t test.
Immunoprecipitation and Western Blot—zASIC1.2 was epitopetagged by inserting into the cDNA an oligonucleotide encoding the hemagglutinin (HA) epitope (YPYDVPDYA) of influenza virus. zASIC1.3
was epitope-tagged by inserting an oligonucleotide encoding the vesicular stomatitis virus glycoprotein (VSV-G) epitope (YTDIEMNRLGK). Both epitopes were inserted at the C termini of the respective
proteins. To have equal signal intensities in Western blots, half the
amount of cRNA for zASIC1.3 compared with zASIC1.2 was injected
in Xenopus oocytes. Microsomal membranes of oocytes injected with
the tagged proteins were prepared 2 days after injection as described
(15). Proteins from part of the microsomal membranes (equal to two
oocytes) were directly separated on a 9% polyacrylamide-SDS gel and
transferred to a polyvinylidene difluoride membrane (PolyScreen;
PerkinElmer Life Sciences). The polyvinylidene difluoride membrane
was then incubated with either peroxidase-coupled anti-HA antibody
(1:1000; Roche Applied Science) or anti-VSV-G antibody (1 ␮g/ml; Roche
Applied Science) followed by peroxidase-coupled anti-mouse antibody
(1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (Lysate in Fig.
7). Bound antibodies were visualized using the ECL kit (Amersham
Biosciences). The remaining part of the microsomal membranes (equal
to 16 oocytes) was resuspended in immunoprecipitation buffer (20 mM
Tris, pH 7.6, 100 mM NaCl, 2% bovine serum albumin, 0.5% digitonin,
2 mM phenylmethylsulfonyl fluoride, leupeptin, antipain, and pepstatin
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ASICs from the Zebrafish
A) and incubated with an anti-VSV-G antibody coupled to agarose (1:10;
Sigma) for 1 h at 4 °C. The immunoprecipitates were washed several
times and then separated on a gel and subjected to Western blot using
the peroxidase-coupled anti-HA antibody as described above.
RESULTS
Molecular Characterization of ASICs from the Zebrafish—
We isolated six full-length cDNA clones coding for proteins
with strong homology to ASICs from the zebrafish. The open
reading frames of the respective cDNA clones code for proteins
consisting of 501–558 amino acids with similar predicted molecular masses of ⬃60 kDa. The predicted amino acid sequences for all six zebrafish clones are shown together with the
sequence of rat ASICs in Fig. 1. The degree of amino acid
identity between the zebrafish and rat ASICs ranges from 60 to
75%. All six ASICs from zebrafish are encoded by different
genes (Table I). Since only four asic genes are present in the
genome of mouse and humans, the zebrafish has a greater
variety of ASICs, which may be compensated by, at least in
part, alternative splicing of the asic1 and -2 genes in mammals.
Phylogenetic analysis (Fig. 2) predicts that three of the zebrafish ASICs are orthologs of ASIC1, ASIC2, and ASIC4,
respectively. We named these clones zASIC1.1, zASIC2, and
zASIC4.1, respectively. Two other clones, zASIC1.2 and
zASIC1.3, are paralogs of zASIC1.1, and one other clone,
zASIC4.2, is a paralog of zASIC4.1. We did not identify an
ortholog of ASIC3. However, there are genomic sequence data
for additional zebrafish ASICs in the data base. Further studies will show if these genes are transcribed into mRNA coding
for functional ASICs or if these genes are inactive pseudogenes.
All six zASICs show the hallmarks of the degenerin/epithelial Na⫹ channel gene family: two hydrophobic domains, rather
short N and C termini, and a large loop containing conserved
cysteines between the two hydrophobic domains. There is one
consensus site for N-linked glycosylation that is predicted to
have an extracellular location and that is conserved in all
zASICs (Fig. 1). Homology between different ASICs is particularly high in the second transmembrane domain and the region
preceding this domain (Fig. 1). The C-terminal motif ((E/D)(F/
I)(A/T)C) of rat ASIC1 and -2 that mediates the interaction of
these ASICs with the PDZ domain-containing protein PICK1
(16, 17) is conserved in rat ASIC4 and in zASIC1.1, -2, -4.1, and
-4.2 but not in zASIC1.2 and -1.3 (Fig. 1).
Expression Pattern of asic Genes in Zebrafish Embryos and
Larvae—The expression pattern of the six zasic genes in different zebrafish tissues was analyzed by in situ hybridization
at 24, 30, 48, 72, and 96 h postfertilization (hpf). Expression of
the asic genes was restricted mainly to neurons and maybe
some glial cells. In general, each zasic showed spatially localized expression in specific neuronal tissues up until 48 hpf (Fig.
3) and then more widespread expression throughout the central nervous system from 72 hpf. Although overlapping in several cases, the expression patterns of zasic genes were distinguishable, indicating that each pattern was unique.
Hybridization of sense strand cRNAs, which was performed as
controls, did not give hybridization signals above background.
zasic1.1 was first expressed by 30 hpf when its expression was
restricted to the anterior and posterior lateral line ganglia and
the otic sensory neurons (aLL, pLL, and OSN in Fig. 4, A and B).
At 48 hpf, expression also became evident in the trigeminal
ganglia (Tg in Fig. 3, A and B). At 72 and 96 hpf, expression could
be seen throughout most of the central nervous system except for
the eyes (Fig. 4K). It was excluded from the dorsal forebrain
except for the habenula nuclei. Like asic1.3 (see below), asic1.1
expression was stronger in the left habenula in comparison with
the right habenula (Fig. 4K).
Expression of zasic1.2 was first evident at 48 hpf, at which
FIG. 3. zasic genes are widely expressed in the developing
central nervous system. Lateral (A, C, E, G, I, and K) and ventral (B,
D, F, H, J, and L) views of whole brains of embryos at 48 hpf show the
distribution of various ASIC mRNAs (blue labeling). Black and red
dashes indicate the positions of the anterior commissure and optic
chiasm and tracts, respectively. a, anterior; d, dorsal; Hb, hindbrain; h,
hypothalamus; LL, lateral line; Mb, midbrain; OB, olfactory bulb; OSN,
otic sensory neurons; p, posterior; PG, pituitary gland; POA, preoptic
area; RGC, retinal ganglion cells; T, telencephalon; Tg, trigeminal; Th,
thalamus; v, ventral; ⽧, ventral midbrain.
stage weak expression could be observed in the ventral thalamus, ventral midbrain, ventral cerebellum, and ventral hindbrain and in the dorsal thalamus and hypothalamus (Fig. 3, C
and D). There was also expression in the telencephalon, along
the tract of the anterior commissure (black dashes in Fig. 3, C
and D). zasic1.2 was additionally weakly expressed in the dorsal midbrain (dMb) and olfactory bulb (OB) at 48 hpf (Fig. 3, C
and D). The level of staining increased by 96 hpf (Fig. 4, C and
D) when zasic1.2 was also expressed in the tectum (Te in Fig.
4C). Unlike zasic1.1 and -1.3, zasic1.2 was not expressed asymmetrically in the habenulae at 96 h. zasic1.2 was the only zasic
to be faintly expressed in the trunk of the fish (Fig. 4L, 96 hpf).
Presumably, these cell bodies represent dorsal root ganglia.
However, labeling was so faint that we could not unequivocally
attribute it to a certain cell or tissue type.
zasic1.3 was first expressed at 30 hpf in the anterior and
posterior lateral line ganglia (aLL and pLL in Fig. 4, E and F),
where it persisted until at least 48 hpf (Fig. 3, E and F). It was
also expressed between 30 and 72 hpf in the telencephalon,
ASICs from the Zebrafish
18787
FIG. 4. Localized expression of zasic genes in specific central nervous system neurons and cranial ganglia. Shown are lateral (A, C,
E, and I), ventral (B, D, G, H, and J), and dorsal (F, K, and L) views of ASIC mRNA expression (blue) in brains of embryos at the various stages
indicated. The solid line in K represents the midline. Black and red dashes indicate the position of the anterior commisure and of the tract of the
postoptic commisure, respectively. AC, anterior commissure; aLL, anterior lateral line; Cb, cerebellum; dT, dorsal telencephalon; OB, olfactory
bulb; OSN, otic sensory neurons; pLL, posterior lateral line; RGC, retinal ganglion cells; Te, tectum; Tg, trigeminal; TPOC, tract of the postoptic
commissure.
along the tract of the anterior commissure (black dashes in Fig.
3E and data not shown). There was expression in the ventral
thalamus, ventral midbrain, ventral cerebellum, and ventral
hindbrain from 30 hpf. By 48 hpf, expression was also evident
in the dorsal thalamus and hypothalamus (Fig. 3, E and F). At
72 hpf, weak expression was apparent in the habenulae, but by
96 hpf expression was stronger in the left habenula (LH) compared with the right habenula (RH) (not shown). This lateralized expression of zasic1.3 in the habenula has previously been
published under the name habenular expressed sequence tag 1
(hest1) (18).
zasic2 expression was first detected at 30 hpf along the tract
of the anterior commissure, where it persisted until 48 hpf
(data not shown and dashed line in Fig. 3, G and H). At 48 hpf,
zasic2 was also expressed in the preoptic area, ventral thalamus, and ventral midbrain and weakly in the ventral hindbrain (Fig. 3, G and H). At 72 and 96 hpf, expression was seen
throughout most of the brain except for the dorsal forebrain. It
was also expressed in the retinal ganglion cells (RGC in
Fig. 4G).
Expression of zasic4.1 was first evident at 48 hpf, at which
stage the expression pattern was very similar to but stronger
than that of zasic1.2. Of note, zasic4.1 (Fig. 3, I and J) was
expressed in an extra domain in the dorsal midbrain (dMb) and
in the retinal ganglion cells (data not shown).
zasic4.2 was the earliest zasic to be expressed, being evident
along the tract of the anterior commissure between 24 and 30
hpf (black dashes in Fig. 4, I and J, and data not shown). At 30
hpf, cells along the tract of the postoptic commissure also
expressed zasic4.2 (red dashes in Fig. 4, I and J). At 48 hpf,
very localized expression was evident in the preoptic area (POA
in Fig. 3, K and L), which was maintained until at least 96 hpf.
The posterior hypothalamus, ventral midbrain, hindbrain (Fig.
3, K and L), and retinal ganglion cells (Fig. 4H) all expressed
zasic4.2 by 48 hpf. These domains of expression persisted and
strengthened in older embryos. Notably, zasic4.2 was the only
zasic almost devoid of expression in the telencephalon at 96 hpf
(not shown).
Functional Properties of zASICs—The functional properties
of zASICs were investigated in X. laevis oocytes using the
two-electrode voltage clamp technique. Oocytes expressing
zASIC1.1, -1.2, -1.3, or -4.1 showed rapidly desensitizing inward currents when acidic solutions were applied, whereas
zASIC2 and zASIC4.2 could not be activated by acidic test
solutions. All zASIC currents had a similar overall shape (Fig.
5A). They rapidly activated and desensitized completely within
less than 1 s. Occasionally, a small transient current increase
appeared during desensitization of the channels that was
highly variable among oocytes. Prolonged activation of
zASIC4.1 led to the appearance of a sustained current component following the transient component (Fig. 5A, inset). This
behavior is similar to rat ASIC3 (19). In this study, we analyzed
only the transient current component of zASIC4.1 in detail.
Recovery from desensitization of zASICs was complete within
less than 30 s in pH 7.4 (not shown). The pH at which halfmaximal activation of zASICs was reached varied between pH
5.0 and 6.6 (Fig. 5 and Table II). This corresponds to an almost
100-fold difference in agonist concentration. However, these
large differences in agonist affinity are comparable with the
mammalian ASICs, for which pH of half-maximal activation
varies between pH 4.3 (ASIC2a) (20) and 6.7 (ASIC3) (21).
Since zASIC currents showed fast kinetics, we analyzed
the kinetics of channel activation in outside-out patch clamp
recordings using fast solution exchange. To compare the kinetics between different zASICs, all channels were activated
with a saturating H⫹ concentration (pH 4.0). The fast gating
of zASICs is illustrated by a representative recording of a
zASIC1.2 current in Fig. 6A. As is shown in Table II, the mean
time constant of desensitization ranged from 9.5 ms (zASIC1.1)
to 49.5 ms (zASIC1.2). Previously, using outside-out patches, a
time constant of desensitization of 1150 ms was measured for
rat ASIC1 (6), and a time constant of 320 ms was measured for
rat ASIC3 in COS-7 cells (21). These values cannot be directly
compared with the values determined in the present study,
because the previous studies used pH 6.0 to activate channels.
However, the kinetics of zASIC1.2 were not significantly slower
when it was activated with pH 5.7 (not shown), and a fast
desensitization was also consistently observed in the whole cell
measurements. Thus, zASICs desensitize much faster than rat
ASICs. Due to the low current amplitude of zASIC4.1, we did
18788
ASICs from the Zebrafish
FIG. 5. Hⴙ sensitivity of zASICs. A, representative current traces of
zASIC expressing oocytes elicited by varying acidic pH. Acidic application solution was applied for 3.5 s, and neutral bath solution (pH 7.4)
was applied for 20 s (zASIC4.1) or 30 s (zASIC1.1, -1.2, and -1.3)
between activation. Inset, representative current trace of prolonged
activation of zASIC4.1. B, pH-response relationship. Symbols and error
bars represent mean ⫾ S.E. Peak current amplitudes at the most acidic
pH were ⫺24.15 ⫾ 5.57 ␮A for zASIC1.1 (n ⫽ 9), ⫺8.69 ⫾ 1.05 ␮A for
zASIC1.2 (n ⫽ 14), ⫺22.86 ⫾ 3.58 ␮A for zASIC1.3 (n ⫽ 18), and
⫺4.32 ⫾ 0.46 ␮A for zASIC4.1 (n ⫽ 13).
not analyze this channel in outside-out patch clamp recordings.
However, in whole oocytes, zASIC4.1 channels showed fast
kinetics like the other zASICs, desensitizing completely within
less than 200 ms.
A hallmark of mammalian ASICs is their Na⫹ selectivity and
their sensitivity to the antagonist amiloride. We determined
the reversal potential of the zASIC currents in whole oocytes by
repetitive activation with pH 4.0 at different membrane potentials (Fig. 6B). Reversal potentials for zASICs varied between
54 and 61 mV (Table II), revealing a Na⫹-selective ion pore.
Furthermore, all functional zASICs could be blocked by amiloride in a dose-dependent manner (Fig. 6C). Their sensitivity
was in the low micromolar range (Table II), similar to other
ASICs. For zASIC4.1, the sustained component was not affected by 1 mM amiloride (not shown). Thus, as expected from
the high conservation within the second transmembrane domain (Fig. 1), which is supposed to form the ion pore (22),
zASICs share basic pore properties with mammalian ASICs.
zASICs Can Form Heteromeric Channels with Unique Functional Properties—Another property of ASICs is that they
readily form heteromeric ion channels. To analyze whether
zASICs can form heteromeric channels, we co-expressed
zASIC1.2 and -1.3 in Xenopus oocytes. We choose zASIC1.2 and
1.3 as an example, since they showed a broad overlap in expression in the nervous system of zebrafish (Fig. 3). As illustrated in Fig. 7A, oocytes expressing zASIC1.2 and zASIC1.3
together showed a larger ASIC current amplitude than oocytes
expressing either subunit alone. This increase in the expression efficiency is best appreciated when comparing averaged
current traces in Fig. 7A. Since this increase in current amplitude was larger than expected by the addition of the current
amplitude for zASIC1.2 and -1.3 alone, it indicated the formation of a heteromeric channel. Moreover, oocytes expressing
both zASIC1.2 and -1.3 together showed an ASIC current with
a H⫹ sensitivity that did not correspond to either of the homomeric channels (Fig. 7B and Table II) and that could be well
fitted assuming a single H⫹ binding site (Fig. 7B). For two
co-existing, separate populations of homomeric channels, however, one would have expected a biphasic dose-response relationship, which should be well fitted only assuming a twobinding site model. And finally, co-expressing zASIC1.2 and
-1.3 led to a current with unique kinetics (Fig. 7A and Table II).
In whole oocyte measurements (Fig. 7A) as well as in outsideout patches (Table II), zASIC1.2/1.3 desensitized slower than
zASIC1.2 (p ⬍⬍ 0.01 for whole oocytes, p ⫽ 0.06 for patches)
and zASIC1.3 (p ⬍ 0.05 for whole oocytes, p ⫽ 0.09 for patches).
Thus, there was 3-fold evidence for the formation of a heteromeric channel with new electrophysiological properties. In addition, we could directly demonstrate the association between
zASIC1.2 and -1.3 by co-immunoprecipitation. zASIC1.2 and
-1.3 were tagged with short epitopes introduced at their C
termini (zASIC1.2-HA and zASIC1.3-VSV-G), and the tagged
proteins were injected in Xenopus oocytes either alone or in
combination. The detergent-solubilized microsomal membranes were then immunoprecipitated using an antibody directed against the VSV-G epitope, and the immunoprecipitated
fractions were analyzed by Western blot using the HA epitope.
This revealed specific co-immunoprecipitation of zASIC1.2 by
zASIC1.3 (Fig. 7C). This heteromultimeric association occurred
in the intact cell, since zASIC1.2 could not be co-immunoprecipitated by zASIC1.3 when microsomes from cells expressing
either zASIC1.2 or zASIC1.3 alone were mixed in vitro
(Fig. 7C).
DISCUSSION
Similar to mammalian asic genes, the majority of zasic genes
were broadly expressed in the central nervous system, suggest-
ASICs from the Zebrafish
18789
TABLE II
Functional properties of zASICs
Data are mean ⫾ S.E. for the number n of individual oocytes or individual patches indicated in parentheses. pH values at which channels were
half-maximally activated (pH50) were obtained from measurements as shown in Fig. 5A. The speed of channel activation, evaluated as time to peak
(TTP; the current rise from 20 to 80% of the peak), and time constants of desensitization (␶des) were obtained from outside-out patch-clamp
recordings with fast solution exchange. Reversal potential (Vrev) and concentration of half-maximal block by amiloride (IC50, Amil) were obtained
from measurements as shown in Fig. 6, B and C, respectively. ND, not determined.
pH50
TTP/ms
␶des/ms
Vrev/mV
IC50, Amil/␮M
zASIC 1.1
zASIC 1.2
zASIC 1.3
zASIC 4.1
zASIC1.2/1.3
6.19 ⫾ 0.03 (9)
2.72 ⫾ 1.31 (4)
9.5 ⫾ 1.2 (4)
61.2 ⫾ 1.3 (14)
57.8 ⫾ 12.3 (15)
4.99 ⫾ 0.05 (14)
4.85 ⫾ 1.56 (10)
49.5 ⫾ 6.3 (10)
53.6 ⫾ 1.5 (12)
21.7 ⫾ 3.0 (12)
6.62 ⫾ 0.02 (18)
3.76 ⫾ 1.28 (5)
22.5 ⫾ 8.0 (5)
55.5 ⫾ 2.6 (12)
22.9 ⫾ 2.6 (13)
5.71 ⫾ 0.04 (13)
ND
ND
55.7 ⫾ 3.1 (9)
23.2 ⫾ 2.9 (10)
6.24 ⫾ 0.02 (14)
2.6 ⫾ 1.37 (6)
90.1 ⫾ 27.5 (6)
48.5 ⫾ 1.4 (12)
20.5 ⫾ 2.4 (20)
ing that they are involved in neuronal communication. In rodents, ASIC1 is enriched at postsynaptic sites (23), consistent
with the involvement in synaptic transmission. Moreover,
knock-out of the asic1 gene leads to viable mice that show
reduced excitatory postsynaptic potentials and N-methyl-D-aspartate receptor activation during high frequency stimulation
(23). These data suggest a role for ASIC1 in synaptic plasticity.
However, another study found an even distribution of ASIC1
immunreactivity along the soma and along the branches of
axons and dendrites (24), suggesting a more general, and
largely unknown, role in neuronal communication. Further
studies are necessary to reveal the subcellular location of
ASICs in the central nervous system of the zebrafish.
In mice, using Western blot analysis, it was shown that
ASIC1 is expressed early on (at embryonic day 12) in the
central nervous system. Later in development, the abundance
of ASIC1 remains steady (24). Similarly, mRNA for ASIC1a is
detectable in mouse brain at embryonic day 11, and mRNA for
ASIC2a is present at embryonic day 7 (25). Consistent with a
role in early development, all zebrafish asic genes were expressed between 24 and 48 hpf, suggesting a function in the
immature, developing nervous system. Interestingly, several
zasic genes (zasic1.2, -1.3, -2, -4.1, and -4.2) were expressed in
cells adjacent to axonal tracts. Thus, zASICs may play a role in
establishing axonal tracts or in axon guidance or may stabilize
axonal pathways and connections.
Because mammalian ASICs are abundantly expressed in
sensory neurons of the dorsal root and trigeminal ganglia and
have been proposed as receptors for different sensory stimuli,
we expected expression of asic genes in sensory neurons of the
zebrafish. However, in sharp contrast to asic genes from mammals, asic genes were only scarcely expressed in sensory neurons in the zebrafish. asic1.1 was the only asic gene to be
expressed in the trigeminal ganglion during embryonic development (Fig. 3); none of the other asic genes were expressed in
primary sensory neurons at sufficient abundance to be detected
by our in situ hybridization. Moreover, we did not detect expression of any zasic in Rohon-Beard cells, the primary sensory
cells located in the spinal cord of embryos of lower vertebrates
(26, 27). The lack of staining of primary sensory neurons may
be due to the low abundance of zASIC mRNA in these cells that
escaped detection in our whole mount in situ hybridizations.
However, since other ganglia showed specific staining for zASIC mRNA (for example, the lateral line ganglia for zASIC1.1
and zASIC1.3), we think this is an unlikely possibility.
The trigeminal ganglion contains neurons carrying somatosensory information from the head. Receptor types of higher
vertebrates include cutaneous touch receptors, chemoreceptors, and nociceptors. The presence of these principal receptor
types has recently been demonstrated in trigeminal ganglia of
fish (28). In particular, it has been shown that nociceptors
innervate the head (29) and that acid injection elicits a behavior consistent with the perception of pain in fish (29). Thus, as
with mammalian ASIC1 (30, 31), zASIC1.1 may be expressed
in nociceptors and act as a receptor for painful stimuli.
In mice, knock-out of the asic2 gene leads to animals with a
decreased sensitivity for tactile stimuli of rapidly adapting
mechanoreceptors (32). In contrast, mice with a knock-out of
the asic3 gene are characterized by an increased sensitivity of
rapidly adapting mechanoreceptors and a decreased sensitivity
of A fiber mechanonociceptors (33). This suggests a role for
ASIC2 and -3 in the perception of tactile stimuli. We restricted
our expression analysis to the embryonic and larval stages of
the zebrafish up to 96 hpf when whole mount in situ hybridizations are feasible. Therefore, we cannot exclude a broader
expression of zASICs in sensory ganglia of adult fish. However,
Rohon-Beard cells, which are functional in embryos, are mechanosensitive and respond to light touch to the skin (34, 35).
Accordingly, zebrafish embryos respond to touch after 27 hpf
(36, 37). At this stage, we did not observe asic expression in
sensory neurons (Fig. 4). If one assumes a similar molecular
make-up of mechanically activated ion channels in early and
adult stages, a contribution of ASICs to mechanically activated
ion channels in zebrafish is unlikely.
However, the expression pattern suggests that zASICs are
involved in the processing of sensory stimuli. For example,
zasic1.1 and zasic1.3 were expressed in the anterior and posterior lateral line ganglia. Neurons of the anterior lateral line
ganglia innervate mechanoreceptive neuromasts of the head,
and neurons of the posterior lateral line ganglia innervate
trunk neuromasts. zasic1.1 was also expressed in the otic sensory neurons; due to the widespread expression of zasic1.3 in
the hindbrain, we cannot rule out the possibility that zasic1.3
was also expressed in this location. The otic sensory neurons
innervate the mechanosensitive hair cells of the inner ear,
which are evolutionarily related to the hair cells of the lateral
line neuromasts. Thus, zasic1.1 and zasic1.3 were expressed in
neurons innervating sensory cells and may contribute to the
processing of mechanical stimuli perceived by these cells. Interestingly, the mRNAs for all rat ASICs, except ASIC4, are
expressed in the spiral ganglion that innervates the hair cells
in the inner ear of rats,2 suggesting a conserved function in
these two vertebrate species. In addition, three zasic genes
(zasic2, -4.1, and -4.2) were expressed in the retinal ganglion
cells, which receive input from the retinal photoreceptors. Like
zasic4.1 and -4.2, rat ASIC4 is also expressed in a subset of
retinal ganglion cells,3 suggesting again a conserved function.
zasic1.2, -1.3, and -4.1 also had widespread midbrain and hindbrain expression that is likely to encompass nuclei that integrate sensory input. Considering the expression patterns in
these various cell types, it is likely that zASICs contribute to
the processing of diverse sensory stimuli. Besides altered responses of mechanoreceptors, mice with a knock-out of the
2
3
S. Gründer, unpublished results.
T. Gründer and S. Gründer, unpublished results.
18790
ASICs from the Zebrafish
FIG. 6. Kinetics, selectivity, and pharmacology of zASICs. A,
representative current trace from an outside-out patch clamp recording.
zASIC1.2 channels were activated by rapidly exchanging a solution of
pH 7.4 with a solution of pH 4.7. Bottom, enlargement of the activation
phase. B, current-voltage relationship for currents through zASIC1.1,
-1.2, -1.3, and -4.1 elicited by pH 4.0 in whole oocytes. Symbols and error
bars represent mean ⫾ S.E. Peak current amplitudes at ⫺60 mV were
⫺27.26 ⫾ 3.49 ␮A for zASIC1.1 (n ⫽ 14), ⫺2.94 ⫾ 0.45 ␮A for zASIC1.2
(n ⫽ 12), ⫺4.37 ⫾ 0.61 ␮A for zASIC1.3 (n ⫽ 12), and ⫺1.38 ⫾ 0.29 ␮A
for zASIC4.1 (n ⫽ 9). C, dose-response relationship for block of zASIC1.1, -1.2, -1.3, and -1.4 currents by amiloride. The solutions containing varying concentrations of amiloride had a pH of 4.0. Symbols and
error bars represent mean ⫾ S.E. Peak current amplitudes without
amiloride were ⫺36.84 ⫾ 3.11 ␮A for zASIC1.1 (n ⫽ 15), ⫺12.39 ⫾ 1.67
␮A for zASIC1.2 (n ⫽ 12), ⫺7.48 ⫾ 1.68 ␮A for zASIC1.3 (n ⫽ 13), and
⫺1.04 ⫾ 0.15 ␮A for zASIC4.1.
asic3 gene show enhanced behavioral responses to high intensity painful stimuli, regardless of their sensory modality (38).
This finding suggests a role for ASIC3 in the processing of
FIG. 7. Formation of a heteromeric channel by zASIC1.2 and
-1.3. A, current traces representing averaged data from 14 (zASIC1.2),
13 (zASIC1.3), and 12 (zASIC1.2/1.3) individual recordings in whole
oocytes. The amount of cRNA that had been injected into each oocyte
was 10 ng of zASIC1.2 alone, 5 ng of zASIC1.3 alone, or 2 ng of zASIC1.2
together with 0.2 ng of zASIC1.3. Note that despite the lower amount of
cRNA injected for the co-expression experiment, current amplitude was
larger. Note also the slow desensitization of the current from oocytes
expressing both zASIC1.2 and -1.3 (time constants for desensitization of
the averaged traces were 145 ms for zASIC1.2, 101 ms for zASIC1.3,
and 563 ms for zASIC1.2/1.3). B, pH-response relationship for zASIC1.2/1.3 measured in whole oocytes. Data for zASIC1.2 and zASIC1.3
from Fig. 5B are shown for direct comparison. Symbols and error bars
represent mean ⫾ S.E. Peak current amplitude for zASIC1.2/1.3 was
⫺33.35 ⫾ 3.06 ␮A (n ⫽ 13) at pH 5.1. C, co-immunoprecipitation (co-IP)
of zASIC1.2 and -1.3 from Xenopus oocytes. Proteins were immunoprecipitated (IP) using the anti-VSV antibody. The microsomes used for the
co-IP were prepared from either (from left to right) uninjected oocytes,
oocytes injected with zASIC1.2 alone, oocytes injected with zASIC1.3
alone, or oocytes injected with both zASIC1.2 and zASIC1.3 together.
For the last lane on the right, microsomes from oocytes expressing
either zASIC1.2 or zASIC1.3 were mixed in vitro just prior to the
co-immunoprecipitation. The antibody used to detect proteins in the
Western blot is indicated on the right. Western blots of the lysates are
shown at the bottom and demonstrate the presence of the expected
proteins. The molecular masses (kDa) of the size markers are indicated.
ASICs from the Zebrafish
sensory stimuli, such as a tonic inhibition of high intensity pain
signals. Thus, ASICs appear to have a conserved role in the
processing of sensory information in vertebrates.
When analyzed in a heterologous expression system, four of
the six zASICs were activated by extracellular H⫹ with properties similar to the mammalian ASICs. The main difference
was the faster desensitization kinetics of zASICs compared
with mammalian ASICs. A fast kinetics has been previously
described for an ASIC from toadfish (39). In our study, two
zASICs were not activated by low pH, zASIC2 and zASIC4.2.
Like rat ASIC2, zASIC2 may form a heteromeric channel with
new properties as we have, as an example, shown for zASIC1.2
and -1.3. It has been suggested that mammalian ASIC2 may be
a central component of a mechanosensitive ion channel (32). As
described above, the expression pattern in zebrafish embryos
and larvae does not support such a function for zASIC2.
A knock-out mouse for asic4 has not been reported thus far,
and the function of ASIC4 is unknown. Since rat ASIC4 cannot
be activated by H⫹, activation by H⫹ of zASIC4.1 was more
surprising than the H⫹ insensitivity of zASIC4.2. Since
zASIC4.1 is ⬃70% identical to zASIC4.2, a more detailed analysis of these two paralogs may help to identify structures
important for the activation of ASICs by extracellular H⫹. In
addition to H⫹ insensitivity, the restricted expression pattern
of zASIC4.2 is reminiscent of the restricted expression pattern
of mammalian ASIC4. The main site of zASIC4.2 expression
was the preoptic area (Fig. 3, K and L), which is the most
anterior part of the hypothalamus. The preoptic area receives
different kinds of sensory information and participates in the
homeostasis of physiological parameters such as blood pressure
and composition. The main site of ASIC4 expression in humans
as revealed by Northern blot (4) is the pituitary gland (PG in
Fig. 3, K and L), which is in close proximity to the preoptic area.
Given that tissues prepared for a Northern blot may always be
contaminated by surrounding tissues, the possibility cannot be
excluded that there is some overlap in expression and perhaps
function of ASIC4.2 in zebrafish and ASIC4 in humans.
In summary, we have demonstrated that a family of receptors for extracellular H⫹ is broadly expressed in the nervous
system of the zebrafish. These receptors have differential properties, and the formation of heteromeric channels may increase
the repertoire of H⫹ receptors. Our results suggest a role for H⫹
receptors in neuronal communication. Our study represents
the first step toward characterization of these receptors to help
increase our understanding of basic structure-function relationships as well as of their physiological function.
Acknowledgments—We thank H.-S. Geisler for expert technical assistance and M. Mione and M. Kapsimali for help in analysis of ASIC
expression pattern.
REFERENCES
1. Waldmann, R., and Lazdunski, M. (1998) Curr. Opin. Neurobiol. 8, 418 – 424
2. Miesenböck, G., De Angelis, D. A., and Rothman, J. E. (1998) Nature 394,
192–195
3. Welsh, M. J., Price, M. P., and Xie, J. (2002) J. Biol. Chem. 277, 2369 –2372
4. Gründer, S., Geissler, H. S., Bässler, E. L., and Ruppersberg, J. P. (2000)
18791
Neuroreport 11, 1607–1611
5. Akopian, A. N., Chen, C. C., Ding, Y., Cesare, P., and Wood, J. N. (2000)
Neuroreport 11, 2217–2222
6. Bässler, E. L., Ngo-Anh, T. J., Geisler, H. S., Ruppersberg, J. P., and Gründer,
S. (2001) J. Biol. Chem. 276, 33782–33787
7. Lingueglia, E., de Weille, J. R., Bassilana, F., Heurteaux, C., Sakai, H.,
Waldmann, R., and Lazdunski, M. (1997) J. Biol. Chem. 272, 29778 –29783
8. Bassilana, F., Champigny, G., Waldmann, R., de Weille, J. R., Heurteaux, C.,
and Lazdunski, M. (1997) J. Biol. Chem. 272, 28819 –28822
9. Babinski, K., Catarsi, S., Biagini, G., and Séguéla, P. (2000) J. Biol. Chem.
275, 28519 –28525
10. Baron, A., Waldmann, R., and Lazdunski, M. (2002) J. Physiol. (Lond.) 539,
485– 494
11. Benson, C. J., Xie, J., Wemmie, J. A., Price, M. P., Henss, J. M., Welsh, M. J.,
and Snyder, P. M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 2338 –2343
12. Geisler, R., Rauch, G. J., Baier, H., van Bebber, F., Brobeta, L., Dekens, M. P.,
Finger, K., Fricke, C., Gates, M. A., Geiger, H., Geiger-Rudolph, S., Gilmour, D., Glaser, S., Gnugge, L., Habeck, H., Hingst, K., Holley, S., Keenan,
J., Kirn, A., Knaut, H., Lashkari, D., Maderspacher, F., Martyn, U., Neuhauss, S., and Haffter, P. (1999) Nat. Genet. 23, 86 – 89
13. Barbazuk, W. B., Korf, I., Kadavi, C., Heyen, J., Tate, S., Wun, E., Bedell, J. A.,
McPherson, J. D., and Johnson, S. L. (2000) Genome Res. 10, 1351–1358
14. Hauptmann, G., and Gerster, T. (1994) Trends Genet. 10, 266
15. Gründer, S., Firsov, D., Chang, S. S., Jaeger, N. F., Gautschi, I., Schild, L.,
Lifton, R. P., and Rossier, B. C. (1997) EMBO J. 16, 899 –907
16. Hruska-Hageman, A. M., Wemmie, J. A., Price, M. P., and Welsh, M. J. (2002)
Biochem. J. 361, 443– 450
17. Duggan, A., Garcı́a-Añoveros, J., and Corey, D. P. (2002) J. Biol. Chem. 277,
5203–5208
18. Concha, M. L., Russell, C., Regan, J. C., Tawk, M., Sidi, S., Gilmour, D. T.,
Kapsimali, M., Sumoy, L., Goldstone, K., Amaya, E., Kimelman, D., Nicolson, T., Gründer, S., Gomperts, M., Clarke, J. D., and Wilson, S. W. (2003)
Neuron 39, 423– 438
19. Waldmann, R., Bassilana, F., de Weille, J., Champigny, G., Heurteaux, C., and
Lazdunski, M. (1997) J. Biol. Chem. 272, 20975–20978
20. Champigny, G., Voilley, N., Waldmann, R., and Lazdunski, M. (1998) J. Biol.
Chem. 273, 15418 –15422
21. Sutherland, S. P., Benson, C. J., Adelman, J. P., and McCleskey, E. W. (2001)
Proc. Natl. Acad. Sci. U. S. A. 98, 711–716
22. Kellenberger, S., and Schild, L. (2002) Physiol. Rev. 82, 735–767
23. Wemmie, J. A., Chen, J., Askwith, C. C., Hruska-Hageman, A. M., Price, M. P.,
Nolan, B. C., Yoder, P. G., Lamani, E., Hoshi, T., Freeman, J. H., Jr., and
Welsh, M. J. (2002) Neuron 34, 463– 477
24. Alvarez De La Rosa, D., Krueger, S. R., Kolar, A., Shao, D., Fitzsimonds, R. M.,
and Canessa, C. M. (2003) J. Physiol. 546, 77– 87
25. Garcı́a-Añoveros, J., Derfler, B., Neville-Golden, J., Hyman, B. T., and Corey,
D. P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1459 –1464
26. Bernhardt, R. R., Chitnis, A. B., Lindamer, L., and Kuwada, J. Y. (1990)
J. Comp. Neurol. 302, 603– 616
27. Eichler, V. B., and Porter, R. A. (1981) J. Comp. Neurol. 203, 121–130
28. Sneddon, L. U. (2003) Brain Res. 972, 44 –52
29. Sneddon, L. U., Braithwaite, V. A., and Gentle, M. J. (2003) Proc. R. Soc. Lond.
B Biol. Sci. 270, 1115–1121
30. Chen, C. C., England, S., Akopian, A. N., and Wood, J. N. (1998) Proc. Natl.
Acad. Sci. U. S. A. 95, 10240 –10245
31. Olson, T. H., Riedl, M. S., Vulchanova, L., Ortiz-Gonzalez, X. R., and Elde, R.
(1998) Neuroreport 9, 1109 –1113
32. Price, M. P., Lewin, G. R., McIlwrath, S. L., Cheng, C., Xie, J., Heppenstall,
P. A., Stucky, C. L., Mannsfeldt, A. G., Brennan, T. J., Drummond, H. A.,
Qiao, J., Benson, C. J., Tarr, D. E., Hrstka, R. F., Yang, B., Williamson,
R. A., and Welsh, M. J. (2000) Nature 407, 1007–1011
33. Price, M. P., McIlwrath, S. L., Xie, J., Cheng, C., Qiao, J., Tarr, D. E., Sluka,
K. A., Brennan, T. J., Lewin, G. R., and Welsh, M. J. (2001) Neuron 32,
1071–1083
34. Clarke, J. D., Hayes, B. P., Hunt, S. P., Roberts, A., Bixby, J. L., Spitzer, N. C.,
Forehand, C. J., Farel, P. B., Eichler, V. B., and Porter, R. A. (1984)
J. Physiol. 348, 511–525
35. Grunwald, D. J., Kimmel, C. B., Westerfield, M., Walker, C., and Streisinger,
G. (1988) Dev. Biol. 126, 115–128
36. Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., and Schilling, T. F.
(1995) Dev. Dyn. 203, 253–310
37. Ribera, A. B., and Nüsslein-Volhard, C. (1998) J. Neurosci. 18, 9181–9191
38. Chen, C. C., Zimmer, A., Sun, W. H., Hall, J., and Brownstein, M. J. (2002)
Proc. Natl. Acad. Sci. U. S. A. 99, 8992– 8997
39. Coric, T., Zhang, P., Todorovic, N., and Canessa, C. M. (2003) J. Biol. Chem.
278, 45240 – 45247