THE JOURNAL OF BIOLOGICAL CHEMISTRY
18296 –18305, 2004
Acid-sensing Ion Channel 2 (ASIC2) Modulates ASIC1 Hⴙ-activated
Currents in Hippocampal Neurons*
Received for publication, November 5, 2003, and in revised form, February 9, 2004
Published, JBC Papers in Press, February 11, 2004, DOI 10.1074/jbc.M312145200
Candice C. Askwith‡§¶, John A. Wemmie储**, Margaret P. Price‡, Tania Rokhlina‡,
and Michael J. Welsh‡§‡‡§§
From the Departments of ‡Internal Medicine, ‡‡Physiology and Biophysics, and 储Psychiatry,
§Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa,
Iowa City, Iowa 52242 and **Department of Veterans Affairs Medical Center, Iowa City, Iowa 52242
Hippocampal neurons express subunits of the acid-
sensing ion channel (ASIC1 and ASIC2) and exhibit
large cation currents that are transiently activated by
acidic extracellular solutions. Earlier work indicated
that ASIC1 contributed to the current in these neurons
and suggested its importance for normal behavior. How-
ever, the specific contribution of ASIC1 and ASIC2 sub-
units to acid-evoked currents in hippocampal neurons
remained uncertain. To decipher the individual role of
the ASIC subunits, we studied Hⴙ-gated currents in neu-
rons from both ASIC1 and ASIC2 null mice. We found
that much of the current was produced by ASIC1a/2a
heteromultimeric channels, and individual subunits
made distinct contributions. The ASIC1a subunit was
key in establishing current amplitude. The ASIC2a sub-
unit had little effect on amplitude but influenced desen-
sitization, recovery from desensitization, pH sensitivity,
and the response to modulatory agents. We also found
heterogeneity in the contribution of ASIC2 throughout
the neuronal population, with individual neurons ex-
pressing both ASIC1a homomultimeric and ASIC1a/2a
heteromultimeric channels. Studies of neurons het-
erozygous for disrupted ASIC alleles indicated that the
properties of Hⴙ-gated currents are dependent on the
proportion of the individual subunits. These findings
indicate that the absolute and relative amounts of ASIC
subunits determine the amplitude and properties of hip-
pocampal Hⴙ-gated currents and therefore may contrib-
ute to normal physiology and pathophysiology.
Although the extracellular pH of the central nervous system
is tightly controlled, both localized and global reductions in pH
have important physiologic and pathophysiologic consequences
(1). For example, repetitive nerve activity transiently reduces
* This work was supported in part by a Veterans Administration
Research Career Development Award (to J. A. W.) and the Howard
Hughes Medical Institute Biomedical Research Support Program (to
J. A. W.). The In Vitro Models and Cell Culture Core were supported by
the NHLBI, National Institutes of Health (Grant HL61234), the Cystic
Fibrosis Foundation (Grants ENGLH9850 and R458-CR02), and the
NIDDK, National Institutes of Health (Grant DK54759). 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.
¶ Associate of the Howard Hughes Medical Institute. Present ad-
dress: Dept. of Neuroscience, The Ohio State University, Columbus, OH
42310.
§§ Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Howard Hughes Medical Inst.,
Roy J. and Lucille A. Carver College of Medicine, 500 EMRB, Iowa City,
IA 52242. Tel.: 319-335-7619; Fax: 319-335-7623; E-mail:
michael-welsh@uiowa.edu.
18296
Vol. 279, No. 18, Issue of April 30, pp.
Printed in U.S.A.
the local pH, perhaps by releasing the acidic contents of syn-
aptic vesicles (2– 4). Ischemia and seizures also cause extracel-
lular acidosis that may contribute to the pathophysiology of
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those diseases (1, 5). In situations where the extracellular pH
falls, protons may act as ligands, activating H⫹-gated channels
(4, 6). Similarities between the biophysical properties of tran-
sient H⫹-gated cation currents in hippocampal neurons (7, 8)
and currents generated by recombinant ASICs1 suggested they
might be responsible (6, 8).
Three genes, ASIC1, -2, and -3, and their alternatively
spliced transcripts (ASIC1a, -1b, -2a, and -2b) produce subunits
that combine to form multimeric channels activated by extra-
cellular acid (9, 10). When the various ASIC subunits are
expressed individually, they form homomultimeric channels
with distinct electrophysiologic signatures (4, 11–26). When
expressed together, the resulting heteromultimeric channels
manifest specific characteristics that depend on the subunit
composition (12, 14, 19, 24, 26 –30). A fourth gene, ASIC4, has
also been identified, but its heterologous expression has nei-
ther generated nor modified currents from other ASIC subunits
(31, 32).
Because different ASIC subunits confer distinct properties to
H⫹-gated channels, the subunit composition of ASIC channels
in neurons could dramatically affect H⫹-gated currents and
ASIC-induced neuronal signaling. In the peripheral nervous
system, studies of ASIC null animals revealed a contribution of
ASIC1, -2, and -3 to acid-evoked currents and showed their
contribution to mechanosensation and acid-evoked nociception
(24, 33–37). In the brain, neurons express ASIC1a, -2a, and -2b
subunits in variable and overlapping distributions (4, 12, 28,
38 – 40). Studies in mice bearing disruptions of the ASIC1 gene
have demonstrated the importance of ASIC1 for normal central
nervous system function; ASIC1 ⫺/⫺ mice exhibited defective
spatial learning, eye-blink conditioning, fear conditioning, and
hippocampal synaptic plasticity (40, 41).
The contribution of individual ASIC subunits to H⫹-gated
currents in central neurons remains uncertain. In an earlier
study, we found that the loss of ASIC1 abolished pH 5-evoked
currents in hippocampal neurons (41). This result seemed sur-
prising, because ASIC1a is not the only ASIC subunit ex-
pressed in the central nervous system; hippocampal neurons
also express ASIC2a and -2b (12, 28, 38, 39). Although recom-
binant ASIC2b generates no current, heterologous expression
of ASIC2a does elicit acid-evoked cation currents (12, 42, 43). In
addition, Zn2⫹, which modulates ASIC2a-containing channels
but not those produced by ASIC1a, potentiated hippocampal
acid-evoked current (8, 30). Therefore, the goal of this work was
1
The abbreviations used are: ASIC, acid-sensing ion channel; CHO,
Chinese hamster ovary; RT, reverse transcriptase.
This paper is available on line at http://www.jbc.org
 H⫹-activated Currents in Hippocampal Neurons
FIG. 1. Amiloride-sensitive current
in ASIC1 ⴚ/ⴚ neurons. A, representa-
tive recordings of pH 4 and 5 applications
in the presence and absence of amiloride
(250 ␮M). Data are from a wild-type neu-
ron (above) and three ASIC1 ⫺/⫺ neurons
(below) with varying responses to pH 4. B,
peak amiloride-sensitive currents elicited
by pH 5 and 4 for wild-type (n ⫽ 9) and
ASIC1 ⫺/⫺ neurons (n ⫽ 38). The asterisk
indicates p ⬍ 0.05 compared with pH 5. C,
rate of desensitization (␶d) of pH 4-evoked
currents in ASIC1 ⫹/⫹ and ⫺/⫺ neurons
(n ⫽ 7; the asterisk indicates p ⬍ 0.0005).
In all figures, error bars represent S.E.
to learn which ASIC subunits contribute to central H⫹-gated
currents and to determine whether they function as homo-
multimers or heteromultimers. To accomplish this, we exam-
ined the characteristics of whole-cell H⫹-gated currents in
hippocampal neurons from mice with disrupted ASIC1 and
ASIC2 genes. We focused on channel properties that differen-
tiate the individual ASIC subunits including pH sensitivity,
desensitization, recovery from desensitization, and sensitivity
to FMRFamide and zinc. In light of earlier reports, we were
particularly interested in testing the hypothesis that ASIC2
contributes to these currents in hippocampal neurons.
EXPERIMENTAL PROCEDURES
Cells and Culture—Mouse hippocampal neuron cultures were pre-
pared from postnatal day 1–2 mice as described previously (44) with the
addition of insulin-transferrin-sodium selenite (41). Hippocampi from
4 –10 pups were pooled and plated in 24-well dishes on 35-mm collagen-
coated cover slips. Cytosine ␤-D-arabinofuranoside was added at day
3– 4 to inhibit glial growth. Whole-cell patch clamp was performed on
large pyramidal cells maintained in culture for 1 to 3 weeks. Neurons
from ASIC1 ⫺/⫺ (41), ASIC2 ⫺/⫺ (34), and heterozygous mice were
compared with neurons from the corresponding wild-type animals
plated and cultured at the same times. We observed no differences
between the groups of wild-type neurons, and they were combined for
comparison to heterozygous neurons (ASIC1 ⫹/⫺ and ASIC2 ⫹/⫺).
Mouse ASIC1a, -2a, and -2b were cloned into pMT3 for heterologous
expression as described (24). Chinese hamster ovary (CHO) cells were
18297
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transfected with 1–3 ␮g of DNA using the Transfast lipid reagent
(Promega, Madison, WI). CHO cells were used because of the ease of
studying by patch clamp, the efficiency of transfection, and their lack of
native H⫹-gated currents. The properties of ASICs expressed in CHO
cells, COS-7 cells, Xenopus oocytes, and lipid bilayers can vary (4,
11–16, 22, 24, 26, 28, 30, 42, 45). However, mouse ASICs studied in
CHO cells produced properties very similar to those we reported previ-
ously in COS cells (26); compare data in Ref. 24 to that in this manu-
script. The pC1EGFP vector encoding enhanced green fluorescent pro-
tein (Clontech, Palo Alto, CA) was added at 20% the DNA concentration
to identify transfected cells using epifluorescence microscopy. For ex-
periments assessing the function of ASIC2b with ASIC2a, pMT3 was
added to maintain a constant final DNA concentration. Cells were
studied 2–3 days following transfection.
Patch Clamp Electrophysiology—Electrodes were filled with the in-
tracellular solution containing (in mM): 120 KCl, 10 NaCl, 2 MgCl2, 5
EGTA, 10 HEPES, and 2 ATP. The pH was adjusted to 7.3 with
tetramethylammonium hydroxide. Extracellular solutions contained
(in mM): 128 NaCl, 5.4 KCl, 5.55 glucose, 10 HEPES, 10 2-(4-morpho-
lino)-ethanesulfonic acid, 2 CaCl2, and 1 MgCl2. The pH was adjusted
with tetramethylammonium hydroxide, and osmolarity was adjusted
with tetramethylammonium chloride. For neuronal cultures, inhibitors
of synaptic activity were routinely added to the extracellular solution.
These included 1 ␮M tetrodotoxin, 10 ␮M 6-cyano-7-nitroquinoxaline-2,
3-dione, 50 ␮M DL-2-amino-5-phosphonopentanoic acid (dAP5), and 30
␮M (⫺)-bicuculline methiodide. Tetrodotoxin has been shown to alter
the kinetics of H⫹-gated channels in some neurons (46, 47). However,
the concentration of tetrodotoxin used in our experiments had no effect
18298 H⫹-activated Currents in Hippocampal Neurons

FIG. 2. Proton-gated currents from

ASIC2 ⴙ/ⴙ and ⴚ/ⴚ hippocampal
neurons. A, representative trace of pH
5-induced current in ASIC2 ⫹/⫹ and ⫺/⫺
neurons. B, average peak amplitude of pH
5-induced current (ASIC2 ⫹/⫹, n ⫽ 67;
ASIC2 ⫺/⫺, n ⫽ 53). C, relationship be-

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tween pH and current. Holding pH was
7.4. Data were normalized to currents of
flanking pH 5 applications (n ⱖ 7). D,
desensitization rate of pH 5-induced cur-
rents. Data are from ASIC2 ⫹/⫹ (n ⫽ 58)
and ASIC2 ⫺/⫺ (n ⫽ 49) hippocampal
neurons (the asterisk indicates p ⬍
0.00005 compared with ASIC2 ⫹/⫹). The
absolute desensitization rates for wild-
type neurons differ in Fig. 1C and Fig. 2D
as expected based on previous reports of
pH-dependent differences in desensitiza-
tion rates (26).

on H⫹-gated currents in cultured mouse hippocampal neurons. The
peptide FRRFamide was synthesized by Research Genetics (Huntsville,
AL). All other chemicals were obtained from Sigma.
Electrodes had a resistance of 3–5 megohms, and series resistance
was compensated by 70% after establishing the whole-cell configura-
tion. Neurons were held at ⫺70 mV, and solutions were changed by
gravity-fed perfusion pipes. Data were acquired using an AXOPATCH
200B amplifier with pCLAMPex 8.1 software (Axon Instruments, Fos-
ter City, CA). Data were analyzed using Clampfit (Axon Instruments)
and IGOR Pro (WaveMetrics, Lake Oswego, OR). Amplitude was deter-
mined by subtracting the baseline current at pH 7.4 from the peak
current amplitude determined in Clampfit (Axon Instruments). Capac-
itance was measured for each neuron in Clampex (Axon Instruments).
The tau of desensitization (␶d) was calculated by fitting the data to a
single exponential using IGOR Pro (WaveMetrics). Because of the com-
plex nature of current desensitization with FRRFamide application, the
Td1⁄2 of desensitization was used as a quantitative measurement of
desensitization. The Td1⁄2 of desensitization was recorded as the time
from the peak current amplitude to half-maximal peak current (as
described above).
Because of current run-down, data for pH dose-response, zinc poten-
tiation, and recovery from desensitization were normalized to the av-
erage of the preceding and following control pH applications. All data
were obtained from at least two different culture preparations repre-
senting 8 –20 mice. Because current amplitude can vary depending on
the time in culture, neurons of different genotypes were prepared
within 24 h of one another, cultured identically, and analyzed on the
same day after culture. Statistical significance was evaluated using the
paired or unpaired Student’s t test as appropriate.
Northern Analysis and RT-PCR—Hippocampi of 14-day-old mice
were dissected and placed into RNAlater solution (Qiagen, Valencia,
CA). The hippocampi from four animals were pooled, and RNA was
isolated using the RNeasy Mini purification kit (Qiagen) and quanti-
fied. Total RNA (10 ␮g per lane) was run on a formaldehyde-agarose gel
using the NorthernMax system (Ambion, Austin, TX) and transferred to
BrightStar-Plus membrane (Ambion, Austin, TX). The probes used for
these studies were generated from PCR products and included the
following: an 528-bp product specific to ASIC2a (primers m2AF, 5⬘-at-
ggacctcaaggagagcccc-3⬘; and m2AR, 5⬘-ggtgaagtcttgatgcccaca-3⬘), an
495-bp product specific to ASIC2b (primers m2BF, 5⬘-tgtggcccgcacaac-
ttctcc-3⬘; and m2BR, 3⬘-ctggccttctgcacgtccctt-3⬘), and an ⬃900-bp pro-
duct common to both transcripts (m2ABF, 3⬘-gatggcaagcctctgctcacc-3⬘;
and m2ABR, 5⬘-aatctcctccagggtgcccaa-3⬘). The radioactive probes were
generated using the Random Primed DNA Labeling Kit (Roche Applied
Science) and purified using NucAwayTM Spin Columns (Ambion). Blots
were visualized by phosphorscreen (STORM; Amersham Biosciences).
For RT-PCR analysis, first strand cDNA was synthesized with Su-
perscript II (Invitrogen) with random hexamer primers. Primers used
for PCR amplification of sequences specific to ASIC2a or ASIC2b, and
common to ASIC2a/2b, were designed to cross intron/exon boundaries.
The following primers were used: ASIC2a, 5⬘-aacctcaatggcttccggt-
tctcc-3⬘ and 5⬘-cccccccttgaccgtggtgagcag-3⬘ (360-bp band); ASIC2b, 5⬘-
cgagtgcaccgcgagtggagccgc-3⬘ and 5⬘-cccccccttgaccgtggtgagcag-3⬘ (430-
bp band); and ASIC2a/2b, 5⬘-tccgagaacattcttgttctggat-3⬘ and 5⬘-
gttctcatcatggctcccttcctc-3⬘ (210-bp band). The cycling parameters using
the RoboCycler thermocycler (Stratagene, La Jolla, CA) consisted of 40
cycles of 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 2 min.
 H⫹-activated Currents in Hippocampal Neurons 18299
RESULTS
Extracellular pH 4 Solutions Activate H⫹-gated Currents in
Some ASIC1 ⫺/⫺ Hippocampal Neurons—Central neurons ex-
press ASIC1a, -2a, and -2b subunits (4, 12, 25, 28, 38 – 40, 48,
49). To determine the contribution of individual ASICs to hip-
pocampal H⫹-gated currents, we first studied neurons from
ASIC1 ⫺/⫺ animals. Consistent with our earlier work (41), pH
5 solutions failed to activate current (Fig. 1A). However, pre-
vious findings suggested that recombinant ASIC2a homomul-
timeric channels require a very low pH for activation, and
ASIC2a/2b heteromultimeric channels require an even greater
reduction in pH (12, 43). Therefore, we applied a more acidic
solution (pH 4) and uncovered amiloride-sensitive currents in
ASIC1 ⫺/⫺ neurons (Fig. 1A shows some examples). The aver-
age peak current was dramatically reduced compared with
wild-type neurons (Fig. 1, A and B), and not every cell re-
sponded. Approximately 43% of the ASIC1 ⫺/⫺ neurons
showed a transient amiloride-sensitive current during pH 4

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application. In contrast, acidic solutions (pH 4 or 5) elicited
transient currents in ⬎95% of wild-type neurons (41).
The presence of ASIC2a in the hippocampus made it a can-
didate to generate the H⫹-gated currents in ASIC1 ⫺/⫺ cells.
One feature that distinguishes heterologously expressed
ASIC2a and ASIC2a/2b channels from channels containing
ASIC1 subunits is their slower desensitization (4, 11, 12, 42,
43). We found that transient currents from ASIC1 ⫺/⫺ neurons
desensitized more slowly than those in wild-type neurons (Fig.
1, A and C). Thus, the expression of ASIC2a and -2b in the
hippocampus, the failure of ASIC2b expression to generate
current, the requirement for very low pH solutions to activate,
and the slow desensitization (␶d) suggest that ASIC2a (either
alone or as a heteromultimeric channel with ASIC2b) gener-
ated currents in ASIC1 ⫺/⫺ neurons.
Loss of ASIC2 Enhances pH Sensitivity and Slows Desensi-
tization of Acid-evoked Currents in Hippocampal Neurons—To
further test the hypothesis that ASIC2 subunits contribute to
H⫹-gated currents, we compared wild-type and ASIC2 ⫺/⫺
neurons; disruption of the ASIC2 gene eliminates both ASIC2a
and ASIC2b subunits (34). A pH 5 application generated cur-
rents with comparable peak amplitudes in wild-type and
ASIC2 ⫺/⫺ neurons (Fig. 2, A and B). This result suggests that
ASIC2 made little contribution to current amplitude. There-
fore, we studied additional properties. Earlier work showed
that currents from heterologously expressed ASIC2a channels
required more acidic solutions for activation than ASIC1a
channels, and ASIC2a/1a heteromultimers had intermediate
pH sensitivity (24, 28). In wild-type hippocampal neurons, re-
FIG. 3. Proton-gated currents from CHO cells expressing
ducing extracellular pH increased current amplitude (Fig. 2C), ASIC1a and -2a. A, examples of current traces from CHO cells that
consistent with earlier work (8). Eliminating ASIC2 increased were untransfected and that were expressing ASIC1a, ASIC2a, and
the pH sensitivity; for example, pH 6.3 solutions stimulated ASIC1a plus ASIC2a. B, desensitization rate of pH 5-induced currents
from CHO cells expressing ASIC1a ⫹ ASIC2a (n ⫽ 12), ASIC1a (n ⫽
59% of the pH 5-induced current in ASIC2 ⫺/⫺ neurons versus
21), ASIC1a ⫹ ASIC2b (n ⫽ 8), and ASIC2a (n ⫽ 5) (the asterisk
only 26% in wild-type neurons. indicates p ⬍ 0.0005 compared with ASIC1a).
Loss of ASIC2 also prolonged desensitization of H⫹-gated
currents as measured by the desensitization rate (␶d) (Fig. 2, A
and D). Because homomultimeric ASIC2a desensitizes more sum of the components, these data indicated that hetero-
slowly than other ASIC subunits, removing its contribution multimeric channels generated the current. The fact that
might have been expected to shorten, rather than lengthen ␶d. ASIC2 ⫺/⫺ neurons desensitized more slowly than wild-type
To investigate the basis of this change, we measured the ␶d of further suggests that the acid-evoked current in wild-type hip-
recombinant ASIC1a and ASIC2 subunits expressed in heter- pocampal neurons arose, at least in part, from heteromultim-
ologous cells (Fig. 3, A and B). For this and all other studies, we eric channels composed of ASIC1a and ASIC2a. Although
used mouse ASIC subunits to facilitate comparison with the ASIC2b is expressed in hippocampus, earlier reports indicated
properties of ASICs in the mouse neurons. Consistent with that it neither generated current nor altered ASIC1a currents
earlier reports, ASIC1a currents desensitized more rapidly (12). Consistent with that observation, adding ASIC2b failed to
than ASIC2a (24, 28), and ASIC1a/2a currents had a ␶d shorter alter ␶d compared with ASIC1a alone (Fig. 3B). These results
than either subunit alone (24). Because the ASIC1a/2a combi- suggest that the faster desensitization of wild-type neurons is
nation generated currents that desensitized faster than the because of the ASIC2a subunit.
18300 H⫹-activated Currents in Hippocampal Neurons

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FIG. 4. FRRFamide and zinc modulation of Hⴙ-gated currents. A, representative traces showing effect of FRRFamide (200 ␮M) on pH
5-evoked currents in ASIC2 ⫹/⫹ and ⫺/⫺ neurons. B, -fold change in Td1/2 induced by FRRFamide. Left, ASIC2 ⫹/⫹ neurons (n ⫽ 12) and ASIC2
⫺/⫺ neurons (n ⫽ 10). The asterisk indicates p ⬍ 0.01 by unpaired Student’s t test. Right, ASIC1a (n ⫽ 8) and ASIC1a ⫹ ASIC2a (n ⫽ 9) expressed
in CHO cells. The asterisk indicates p ⬍ 0.005. C, representative traces showing effect of zinc (300 ␮M) on pH 6.3-evoked currents in ASIC2 ⫹/⫹
and ⫺/⫺ neurons. D, amplitude of pH 6.3-evoked current in the presence of 300 ␮M zinc as a percentage of current in the absence of zinc in ASIC2
⫹/⫹ (n ⫽ 23) and ASIC2 ⫺/⫺ (n ⫽ 13) neurons. Data were normalized to currents of flanking pH 6.3 applications. The asterisk indicates p ⬍ 0.005
compared with absence of zinc by unpaired Student’s t test.

ASIC2 Contributes to the Modulatory Effects of FRRFamide
and Zinc—To further test the hypothesis that ASIC2 contrib-
utes to acid-evoked currents in hippocampal neurons, we tested
the effect of agents that selectively modulate channels contain-
ing ASIC1a or ASIC2a. In heterologous expression systems,
FRRFamide, FMRFamide, and related neuropeptides potenti-
ate ASIC1a-mediated currents, whereas ASIC2a is unaffected
(20, 29). In wild-type hippocampal neurons, FRRFamide
slowed desensitization and in some cases generated sustained
acid-evoked currents, consistent with the presence of ASIC1a
(Fig. 4, A and B). Surprisingly, eliminating ASIC2 attenuated
this response. These results suggest that the presence of ASIC2
subunits enhanced peptide modulation of ASIC1a-containing
channels. To investigate this possibility, we expressed the sub-
units in CHO cells and found that adding ASIC2a enhanced the
response of ASIC1a (Fig. 4B). These data suggest that hetero-
multimeric channels are responsible for H⫹-gated currents in
hippocampal neurons and that the normal response to FRRF-
amide requires both ASIC1a and ASIC2a subunits. These re-
sults have a parallel in a previous study (29) showing that
heteromultimeric channels composed of ASIC3 and ASIC2a
responded more robustly to FMRFamide-related peptides than
ASIC3 alone.
Earlier work showed that zinc selectively increased H⫹-
gated currents from ASIC2a and ASIC2a-containing hetero-
multimeric channels (8, 30). We found that zinc potentiated
H⫹-gated currents in wild-type neurons, whereas it slightly
inhibited current in ASIC2 ⫺/⫺ neurons (Fig. 4, C and D).
These results support the conclusion that ASIC2 contributes to
hippocampal H⫹-gated currents and is required for zinc
potentiation.
ASIC2a Determines the Fast Recovery from Desensitization in
Wild-type Neurons—Because ASIC2a influences recovery from
desensitization, we also tested this property. ASIC2a and
ASIC1a/2a channels recover rapidly following a pH 5 application,
whereas ASIC1a homomultimers recover more slowly (24). We
found that after 1 s at pH 7.4, wild-type hippocampal neurons
recovered 61% of the pH 5-evoked current (Fig. 5, A and B).
Deleting ASIC2 extended the recovery time, so that in 1 s, only
4% of the current had recovered, and even by 10 s, recovery was
only 58%. Because ASIC2b is reported to not affect ASIC1a
current (12), we assumed that ASIC2a was the subunit respon-
sible for speeding recovery. To test this assumption, we expressed
recombinant ASIC1a with ASIC2a or -2b and measured recovery
rate. Fig. 5C shows that ASIC2a but not -2b accelerated the
recovery of ASIC1a currents. These striking differences indicate
 H⫹-activated Currents in Hippocampal Neurons 18301

FIG. 5. Effect of subunit composi-

tion on recovery from desensitiza-

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tion. Recovery from desensitization was
measured by applying pH 5 for 10 s and
then pH 7.4 for the indicated length of
time (1 to 30 s), followed by a second pH 5
application. A, representative tracings
showing recovery from desensitization
during 1 s at pH 7.4 in ASIC2 ⫹/⫹ and
⫺/⫺ neurons. B, percentage recovery from
desensitization in ASIC2 ⫹/⫹ and ⫺/⫺
hippocampal neurons. Data were normal-
ized to currents of flanking pH 5 applica-
tions (n ⫽ 9 –13). C, percentage recovery
from desensitization in CHO cells ex-
pressing ASIC1a, ASIC1a ⫹ ASIC2b, or
ASIC1a ⫹ ASIC2a (n ⫽ 6 –12).

that ASIC2a determined the fast recovery from desensitization in
hippocampal neurons.
Channels with Heterogeneous Subunit Composition Contrib-
ute to H⫹-gated Hippocampal Currents—The results suggested
that hippocampal H⫹-gated current reflect predominantly
ASIC1a/2a heteromultimers. However, they did not exclude the
presence of ASIC1a homomultimeric channels. Several obser-
vations are consistent with this possibility. First, the peptide
toxin PCTX1, which was reported to selectively inhibit ASIC1a
homomultimeric channels, reduced H⫹-gated current in cen-
tral nervous system neurons (8, 19). Second, recovery from
desensitization by wild-type neurons (Fig. 5B) was slower than
recombinant ASIC1a/2a heteromultimers (Fig. 5C). Third,
wild-type neurons (Fig. 2C) were slightly more pH-sensitive
than ASIC1a/2a heteromultimers (24, 28).
Therefore, we tested for heterogeneity of channel types
within individual neurons. We reasoned that, because ASIC1a
homomultimers recover much more slowly from desensitization
than heteromultimers containing ASIC2a (see Fig. 5C and Ref.
24), we could largely eliminate the contribution of ASIC1a
homomultimers by limiting the time of recovery from desensi-
tization. This would uncover currents from other homo- and
heteromultimeric channels, which we might recognize by a
change in ␶d. To test this, we applied a pH 5 solution, measured
the ␶d (␶d0) and then continued the perfusion for 10 s to desen-
sitize acid-evoked currents. We then stopped the pH 5 solution,
allowed 2 s at pH 7.4 for recovery, and reapplied pH 5 solution
to measure the ␶d a second time (␶d2). Fig. 6A shows the ratio of
␶d after 2 s of recovery relative to that prior to desensitization
(␶d2/␶d0). We expected that if all the channels expressed on a
cell were of identical subunit composition, then the ␶d would be
unchanged after desensitization and the ratio would be 1. To
test this notion, we expressed recombinant ASIC1a, -2a, and
-1a/2a channels and found a ratio of ⬃1 in all cases. Thus, with
a homogeneous population of channels, ␶d was the same irre-
spective of whether the channels showed fast or slow recovery.
18302 H⫹-activated Currents in Hippocampal Neurons
In contrast, wild-type neurons had a ␶d2/␶d0 ratio less than 1,
suggesting that these neurons contain a population of rapidly
recovering channels with a short ␶d (consistent with ASIC1a/2a
FIG. 6. Heterogeneity of Hⴙ-gated currents in individual neu-
rons and in the population of hippocampal neurons. A, data are
ratio of the ␶d of desensitization 2 s after recovery at pH 7.4 (␶d2) versus
before desensitization (␶d0). Measurements were made from pH 5-acti-
vated currents in CHO cells expressing ASIC1a (n ⫽ 12), ASIC2a (n ⫽
5), and ASIC1 ⫹ ASIC2a (n ⫽ 13) and from ASIC2 ⫹/⫹ (n ⫽ 25) and
ASIC2 ⫺/⫺ (n ⫽ 17) hippocampal neurons. The asterisk indicates value
different from 1.0, p ⬍ 0.02. B, recovery from desensitization was
measured as shown in Fig. 5A by applying pH 5 for 10 s, pH 7.4 for 2 s,
and then a second pH 5 application. We measured the percentage
recovery with the second acid stimulus for 32 ASIC2 ⫹/⫹ and 17
ASIC2 ⫺/⫺ neurons. The graph shows percentage current recovery (y
axis) for a series of individual neurons (x axis). Each data point indi-
cates an individual neuron; squares indicate ASIC2 ⫹/⫹, and circles
indicate ASIC2 ⫺/⫺ neurons.
FIG. 7. Effect of ASIC2b on ASIC2a-
induced currents. A, RT-PCR from
ASIC1 ⫹/⫹ and ASIC1 ⫺/⫺ hippocampus
using primers that detected both ASIC2a
and -2b or either alone. B, Northern
analysis of RNA from ASIC1 ⫹/⫹ and
ASIC1 ⫺/⫺ hippocampus using probes
that recognize the indicated transcript. C,
relationship between pH and H⫹-gated
current in CHO cells expressing ASIC2a
alone (n ⫽ 10 –17) or ASIC2a and -2b at
ratios of 1:1 (n ⫽ 9 –14), 1:2 (n ⫽ 6 –13),
and 1:3 (n ⫽ 6 – 8). In all cases the amount
of ASIC2a cDNA was constant, and pMT3
DNA was varied to maintain a constant
amount of total DNA. Data were normal-
ized to response to pH 3 stimulus. D, pH
3-induced current amplitude in cells ex-
pressing ASIC2a with different amounts
of ASIC2b (n ⫽ 17–19).
channels) and a population of slowly recovering channels with
a longer ␶d (consistent with ASIC1a homomultimers). Support-
ing this conclusion, ASIC2 ⫺/⫺ neurons had a ␶d2/␶d0 ratio of 1.
Thus, individual wild-type neurons appeared to express
ASIC1a/2a heteromultimeric channels plus channels with
properties consistent with ASIC1a homomultimers.
We also tested for heterogeneity in the contribution of
ASIC2a throughout the population of neurons. The rate of
recovery from desensitization is one of the characteristics most
strikingly influenced by ASIC2a; ASIC1a/2a heteromultimeric
channels recover quickly, whereas ASIC1a homomultimeric
channels recover more slowly. Therefore, we desensitized cur-
rent with a 10-s pH 5 perfusion and then measured the per-
centage of current recovery 2 s after returning to a pH 7.4
solution. Wild-type neurons showed substantial variability
(Fig. 6B). For example, within 2 s, some neurons had recovered
⬃20% of the initial current, whereas others had recovered all of
the initial current. In contrast, when ASIC2 was eliminated,
Downloaded from http://www.jbc.org/ at JAWAHARLAL NEHRU UNIV on March 22, 2018
none of the neurons showed more than 25% recovery at 2 s.
These results suggest that ASIC2a subunits contributed to the
properties of H⫹-gated currents in the majority of neurons.
ASIC2b Is Expressed in Excess of ASIC2a in Neonatal Hip-
pocampal Neurons—The results described above suggested a
paradox. On one hand, only 43% of ASIC1 ⫺/⫺ neurons pos-
sessed transient currents evoked by pH 4. Yet, on the other
hand, ASIC2a influenced the properties of acid-evoked currents
in the vast majority of wild-type hippocampal neurons. If
ASIC2a were present in most neurons, why were ASIC2a cur-
rents not more common in ASIC1 ⫺/⫺ neurons? A potential
explanation for the discrepancy would be that ASIC1 disrup-
tion reduces ASIC2 expression. Although an earlier study
showed that ASIC2 expression was the same in wild-type and
ASIC1 ⫺/⫺ brains, hippocampal expression was not specifi-
cally measured (41). Using RT-PCR, we found ASIC2a and -2b
transcripts in hippocampi of both wild-type and ASIC1 ⫺/⫺
animals (Fig. 7A). Northern analysis indicated that ASIC2
transcripts were present equally in wild-type and ASIC1 ⫺/⫺
hippocampi (Fig. 7B). Interestingly, the Northern blot also
revealed that ASIC2b transcripts were much more abundant
than ASIC2a transcripts. A greater abundance of ASIC2b
mRNA compared with ASIC2a mRNA has also been reported in
several other brain regions (34, 38, 39, 50). This observation
raised the possibility that ASIC2b might affect ASIC2a current
in ASIC1 ⫺/⫺ neurons.
ASIC2b subunits do not generate H⫹-gated currents when
 H⫹-activated Currents in Hippocampal Neurons
FIG. 8. Hⴙ-gated currents from ASIC1 ⴙ/ⴚ and ASIC2 ⴙ/ⴚ neu-
rons. A, average peak amplitude with pH 5 application in wild-type
(WT; n ⫽ 10), ASIC1 ⫹/⫺ (n ⫽ 24), and ASIC2 ⫹/⫺ (n ⫽ 28) hippocam-
pal neurons. The asterisk indicates p ⬍ 0.002 versus wild-type. B,
desensitization rate (␶d) for wild-type (n ⫽ 24), ASIC1 ⫹/⫺ (n ⫽ 27), and
ASIC2 ⫹/⫺ (n ⫽ 27) hippocampal neurons. The asterisk indicates p ⬍
0.005 versus wild-type. C, relationship between pH and H⫹-gated
current from wild-type (n ⫽ 13–17), ASIC1 ⫹/⫺ (n ⫽ 10 –13), and
ASIC2 ⫹/⫺ (n ⫽ 14 –16) hippocampal neurons. D, recovery from desen-
sitization of wild-type (n ⫽ 16 –18), ASIC1 ⫹/⫺ (n ⫽ 12–15), and
ASIC2 ⫹/⫺ (n ⫽ 14 –17) neurons.
expressed alone, but they were reported to form heteromultim-
eric channels with ASIC2a (12, 42, 43). We reasoned that if
ASIC2b were present in excess of ASIC2a, then in ASIC1 ⫺/⫺
neurons most channels would be ASIC2a/2b heteromultimers.
To test the consequences of such a situation, we expressed
ASIC2a with varying amounts of ASIC2b in CHO cells. Co-
expressing ASIC2b with ASIC2a tended to reduce the pH sen-
sitivity, although the effects were small (Fig. 7C); this result is
consistent with earlier studies (12, 43). However, ASIC2b re-
duced current amplitude produced by pH 3 application (Fig.
7D). Moreover, as the relative amount of ASIC2b increased,
current amplitude fell. These results may explain the low per-
centage of ASIC1 ⫺/⫺ neurons with H⫹-gated currents and
their relatively small current amplitude. In those neurons,
ASIC2b likely dampens current from channels mediated by
ASIC2a, and perhaps currents were only observed in neurons
expressing a higher proportion of ASIC2a.
ASIC1 and ASIC2 Heterozygote Neurons Show Altered H⫹-
gated Currents—The contribution of ASIC1 and -2 subunits to
H⫹-gated currents suggested that hippocampal neurons het-
erozygous for the ASIC1 and ASIC2 alleles might have altered
18303
acid-evoked currents. We found that compared with wild-type
controls, the amplitude of pH 5-evoked currents fell by approx-
imately half in ASIC1 ⫹/⫺ neurons but remained unchanged in
ASIC2 ⫹/⫺ neurons (Fig. 8A). ASIC1 ⫹/⫺ neurons also showed
an accelerated ␶d and ASIC2 ⫹/⫺ neurons a slowed ␶d (Fig. 8B).
H⫹-gated currents from ASIC2 ⫹/⫺ neurons showed little shift
in pH sensitivity, but recovery from desensitization was de-
layed (Fig. 8, C and D). Despite the dramatic reduction in
current amplitude in ASIC1 ⫹/⫺ neurons (Fig. 8A), the pH
dose-response and recovery from desensitization were not al-
tered significantly (Fig. 8, C and D). Together, these results
further support the conclusion that both ASIC1a and ASIC2
subunits influence acid-evoked currents in hippocampal neu-
rons. ASIC1 seems to be important in determining current
amplitude, whereas the predominant role of ASIC2 appears to
be in influencing the properties of the current.
DISCUSSION
Downloaded from http://www.jbc.org/ at JAWAHARLAL NEHRU UNIV on March 22, 2018
It has been known for many years that acidic extracellular
solutions activate transient cation currents in hippocampal
neurons (7). The biophysical properties of those currents (8)
and the expression of ASIC1a and ASIC2 subunits in the hip-
pocampus (4, 12, 26, 28, 38, 39) suggested that ASIC subunits
were responsible. ASIC subunits might generate H⫹-gated cur-
rents in two general ways. ASIC1a and ASIC2a could each form
homomultimeric channels, with net current representing the
sum of the individual currents. Alternatively, the subunits
could combine to form heteromultimeric channels. Our data
indicate that at least two ASIC subunits, ASIC1a and -2a,
contribute to H⫹-activated currents in mouse hippocampal
neurons. They also suggest that both heteromultimeric and
homomultimeric channels generate current, and individual
ASIC subunits make distinct contributions.
Hippocampal Neurons Contain Both Hetero- and Homo-mul-
timeric ASIC Channels—Heteromultimeric ASIC1a/2a chan-
nels produced H⫹-gated current in cultured hippocampal neu-
rons. Support for this conclusion came from the finding that ␶d
in wild-type neurons exceeded that in both ASIC1 ⫺/⫺ and
ASIC2 ⫺/⫺ neurons and in heterologous cells expressing either
ASIC1a or -2a alone. Thus, the ␶d could not be explained by the
sum of currents from homomultimeric channels. FRRFamide
addition also revealed responses that could not be attributed to
an aggregate of homomultimeric channels. Although FRRF-
amide does not alter ASIC2a currents, it produced a greater
response in wild-type neurons and in heterologous cells ex-
pressing ASIC1a/2a heteromultimeric channels than in
ASIC2 ⫺/⫺ neurons or cells expressing ASIC1a homomultim-
ers alone. These results indicate that ASIC1a/2a channels have
unique properties compared with homomultimeric channels,
and those channels generate H⫹-gated currents in hippocam-
pal neurons. An advantage for making this conclusion is that
our studies used neurons with specific gene disruptions. Thus,
they do not depend on comparing absolute values of channel
properties in neurons with those of ASICs expressed in heter-
ologous cells, where values could vary depending on the cell
type and associated proteins. Nevertheless, there was complete
qualitative agreement between data with neurons and recom-
binant channels, and the quantitative responses were very
similar.
Brain also expresses two other DEG/ENaC subunits, ASIC4
(31, 32) and BLINaC (brain-liver-intestine amiloride-sensitive
Na⫹ channel) (51, 52). However, ASIC4 neither generated H⫹-
gated currents on its own nor affected currents produced by
other ASICs (31). BLINaC bearing a gain-of-function mutation
generated a sustained Na⫹ current, but the wild-type protein
did not generate current or alter other DEG/ENaC subunits
(51). Although recombinant ASIC3 can generate H⫹-gated cur-
18304 H⫹-activated Currents in Hippocampal Neurons
rents, it shows little, if any, expression in the mouse brain (15,
16, 36, 53). Thus, ASIC3, ASIC4, and BLINaC would not be
expected to influence the properties of hippocampal acid-
evoked currents. Although our results cannot exclude the
possibility that other DEG/ENaC subunits contribute to H⫹-
gated hippocampal currents in unexpected ways, invoking a
contribution from subunits other than ASIC1a, -2a, and -2b is
not required to explain currents in neurons from wild-type,
ASIC1 ⫺/⫺, or ASIC2 ⫺/⫺ mice. The contribution of the vari-
ous DEG/ENaC subunits to sustained H⫹-gated neuronal cur-
rents is also uncertain. Studying mice with additional targeted
gene disruptions might reveal such contributions.
By forming heteromultimeric ASIC channels, hippocampal
neurons follow a pattern found in other cells expressing DEG/
ENaC subunits. Epithelial cells form heteromultimeric chan-
nels from ␣, ␤, and ␥ ENaC subunits (54 –56). Caenorhabditis
elegans neurons form channels from the combination of Mec4
and Mec10 (57), and dorsal root ganglion neurons combine
ASIC1, -2, and -3 subunits to generate H⫹-gated currents (24,
33). However, differences between subunit desensitization and
recovery from desensitization suggested the presence of
ASIC1a homomultimeric channels in hippocampal neurons.
This conclusion is supported by the observation that the pep-
tide toxin PCTX1 reduced H⫹-gated current in central nervous
system neurons (8). Expression of both heteromultimeric and
homomultimeric channels has not been reported in other cell
types expressing more than one DEG/ENaC subunit. This find-
ing suggests the opportunity for hippocampal neurons to con-
struct channels with a complex variety of subunit compositions
and characteristics.
ASIC Subunits Confer Distinct Properties on Hippocampal
Acid-evoked Currents—ASIC1a and -2a subunits made distinct
contributions to H⫹-activated hippocampal currents. ASIC1a
seemed to be important in establishing current amplitude.
Eliminating ASIC1 from neurons abolished pH 5-induced cur-
rent, and its absence reduced average pH 4-induced current by
⬃95%. Most convincingly, removing one ASIC1 allele approxi-
mately halved H⫹-gated current.
In contrast, ASIC2 ⫺/⫺ and ⫹/⫺ neurons generated the
same current amplitude as wild-type neurons, indicating that
ASIC2 plays a minor role in determining this quantitative
aspect of H⫹-gated current. Instead, ASIC2 influenced the bio-
physical properties, defining, in part, acid sensitivity, desensi-
tization, the rate of recovery from desensitization, and the
response to neuropeptide modulators and zinc. A predomi-
nantly modulatory role for ASIC2a subunits may be analogous
to the contributions of some ␥-aminobutyric acid-A receptor
subunits to that channel complex (58).
The contribution of ASIC2b is less certain. Earlier studies
reported that co-expressing ASIC2b with ASIC1a had no effect
on H⫹-gated currents (12). When expressed with ASIC2a,
ASIC2b reduced pH sensitivity to a small extent (12, 43), con-
sistent with our results. In addition, our data indicate that
ASIC2b reduced ASIC2a current amplitude in response to a
maximal stimulus. This finding, together with a greater rela-
tive abundance of ASIC2b versus -2a transcripts in the hip-
pocampus, provides a potential explanation for the minimal
H⫹-gated current in ASIC1 ⫺/⫺ neurons. An alternative factor
that we cannot exclude, but that could influence current prop-
erties, is the possibility that other channels or proteins might
differentially regulate ASICs in null and wild-type animals
(59).
Contribution of ASIC to Function of Hippocampal Neurons—
Specific ASIC subunits make distinct contributions to hip-
pocampal acid-evoked currents, and there is heterogeneity in
currents within and between individual neurons. This varia-
bility could influence the contribution of ASICs to normal phys-
iology and to pathophysiology. For example, in the peripheral
nervous system relatively small changes in H⫹-gated currents
alter sensory transduction (34 –36). Moreover, in the mouse
brain, disrupting ASIC1 impaired synaptic plasticity and per-
formance in several behavioral tests (40, 41). Pathologic condi-
tions may also skew ASIC levels. For example, ischemia is
reported to increase ASIC2a levels (60); such an increase could
reduce the pH sensitivity of acid-evoked currents, making them
less responsive to an acid insult. Following seizures, the rela-
tive contribution of various subunits to the complex may also
change (50), perhaps altering the responsiveness of hippocam-
pal neurons to seizure-associated acidosis. In addition, if mu-
tations in ASIC genes are discovered in humans, our data
suggest that haplo-insufficiency could have a substantial im-
pact on H⫹-gated currents and perhaps behavior. Finally, these
results suggest that molecules targeting specific ASIC subunits
might have value as therapeutics.
Downloaded from http://www.jbc.org/ at JAWAHARLAL NEHRU UNIV on March 22, 2018
Acknowledgments—We thank Ejvis Lamani, Lisa Momchilov, and
Theresa Mayhew for excellent assistance. We thank Phil Karp, Pary
Weber, and Tami Nesselhauf of the University of Iowa. We thank the
University of Iowa Animal Care Unit.
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J. Cereb. Blood Flow Metab. 21, 734 –740
 Acid-sensing Ion Channel 2 (ASIC2) Modulates ASIC1 H+-activated Currents in
Hippocampal Neurons
Candice C. Askwith, John A. Wemmie, Margaret P. Price, Tania Rokhlina and Michael J.
Welsh
J. Biol. Chem. 2004, 279:18296-18305.
doi: 10.1074/jbc.M312145200 originally published online February 11, 2004

Access the most updated version of this article at doi: 10.1074/jbc.M312145200

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