From the Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique-UPR 411, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France
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ABSTRACT |
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The acid-sensing ion channel (ASIC) subunits
ASIC1, ASIC2, and ASIC3 are members of the amiloride-sensitive
Na+ channel/degenerin family of ion channels. They form
proton-gated channels that are expressed in the central nervous system
and in sensory neurons, where they are thought to play an important role in pain accompanying tissue acidosis. A splice variant of ASIC2,
ASIC2b, is not active on its own but modifies the properties of ASIC3.
In particular, whereas most members of the amiloride-sensitive Na+ channel/degenerin family are highly selective for
Na+ over K+, ASIC3/ASIC2b heteromultimers
show a nonselective component. Chimeras of the two splice variants
allowed identification of a 9-amino acid region preceding the first
transmembrane (TM) domain (pre-TM1) of ASIC2 that is involved in ion
permeation and is critical for Na+ selectivity. Three amino
acids in this region (Ile-19, Phe-20, and Thr-25) appear to be
particularly important, because channels mutated at these residues
discriminate poorly between Na+ and K+. In
addition, the pH dependences of the activity of the F20S and T25K
mutants are changed as compared with that of wild-type ASIC2. A
corresponding ASIC3 mutant (T26K) also has modified Na+
selectivity. Our results suggest that the pre-TM1 region of ASICs participates in the ion pore.
ASICs1 belong to the
NaC/DEG family of sodium channels whose members are involved in
physiological functions as diverse as Na+ reabsorption in
epithelia (epithelial Na+ channel), mechanotransduction
(degenerins of Caenorhabditis elegans), and
neurotransmission (Phe-Met-Arg-Phe-amide peptide-gated Na+
channel (FaNaC)). ASIC1, ASIC2, and ASIC3 (previously named ASIC (1), MDEG (2), and DRASIC (3), respectively) are located in the central
nervous system (ASIC1 and ASIC2) or in sensory neurons (ASIC1 and
ASIC3). They are activated by a drop in extracellular pH. It has
been proposed that ASICs in sensory neurons mediate the pain
accompanying tissue acidosis (3).
ASIC2b (previously named MDEG2) is a splice variant of ASIC2 found in
the brain and in sensory neurons (4). It does not form a pH-activated
channel by itself but acts as a modulatory subunit when associated with
other ASIC subunits such as ASIC3. The ASIC3 response to a drop in pH
is biphasic, with both current components being carried mainly by
Na+ (3). Co-expressing ASIC3 with ASIC2b does not change
the characteristics of the transient current, but the sustained current
becomes nonselective (4). The native proton-gated current in dorsal
root ganglia also shows a transient Na+-selective component
and a sustained nonselective component (5). Therefore, the ASIC2b/ASIC3
association appears to be a good candidate for the H+-gated
channel involved in pain sensation (4).
The ASIC2 and ASIC2b splice variants differ only in the first 185 N-terminal amino acids of ASIC2. Chimeric constructs of these splice
variants led us to identify a region before the first transmembrane
(TM1) domain of ASIC2 that controls the ion selectivity and the pH
dependence of the channel. Point mutations in this particular region of
the ASIC2 protein have led to identification of amino acids that play a
key role in defining the biophysical properties of the channel.
Corresponding mutations in ASIC3 induce similar changes in ion
selectivity. These results indicate the important role of the region
preceding the first transmembrane domain (pre-TM1) in the pore
formation of ASICs.
Molecular Biology--
We used the previously described
constructions of FaNaC in pRC/CMV vector (Invitrogen) (6) and of ASIC2,
ASIC2b, and ASIC3 in pCI vector (Promega) (1-4). For the generation of
chimeras, two complementary primers containing the sequence of the
desired junction between ASIC2 and ASIC2b were chosen. Two different
PCRs were performed, one on the ASIC2 plasmid with a junction primer and a specific ASIC2 primer, and the other on the ASIC2b plasmid with
the other junction primer and a specific ASIC2b primer. Fragments were
purified with the Wizard PCR Preps DNA purification system (Promega).
Both fragments were mixed with the specific primers, and another PCR
was performed. The PCR product was digested and subcloned in ASIC2 or
ASIC2b expression plasmids. The PCR was carried out using the Expand
High Fidelity PCR system (Boehringer Mannheim), and the subcloned
fragments were sequenced. Five percent Me2SO or GC Melt
(CLONTECH) was added for PCR on the GC-rich ASIC2b 5' translated region.
Site-directed mutagenesis was performed by PCR according to the
QuickChange Site-Directed Mutagenesis Kit protocol (Stratagene) or with
a variation of the method used for chimeras.
Expression in COS Cells--
COS cells, at a density of 20,000 cells/35-mm diameter Petri dish, were co-transfected with expression
vectors containing CD8 receptor cDNA and the PCI-ASIC vectors (a
1:5 ratio) using the DEAE-dextran method. Cells were used for
electrophysiological measurements 1-3 days after transfection.
Successfully transfected cells were recognized by their ability to bind
to CD8 antibody-coated beads (7).
Electrophysiology--
Ion currents were recorded using either
the whole cell or outside-out patch-clamp technique and stored on a
hard disk for later off-line analysis. The pipette solution contained
120 mM KCl, 30 mM NaCl, 2 mM
MgCl2, 5 mM EGTA, and 10 mM
HEPES/KOH (pH 7.2). The bath solution contained 140 mM
NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, and 10 mM HEPES/NaOH, pH
7.3 (Na+-rich solution) or 145 mM KCl, 2 mM MgCl2, 2 mM CaCl2,
and 10 mM HEPES/KOH, pH 7.3 (K+-rich solution).
Changes in extracellular pH were induced by shifting one of six outlets
of a microperfusion system in front of the cell or patch. Test
solutions with a pH < 6 were buffered with 10 mM MES
rather than HEPES but were identical to the control solution in all
other respects. Experiments were carried out at room temperature
(20 °C to 24 °C).
The ASIC2 and ASIC2b splice variants are different in their first
half, which includes the N-terminal region, the first transmembrane domain, and one-third of the extracellular loop. They are identical from amino acid 186 of ASIC2 onward (Fig.
1A). Replacement of the
N-terminal intracellular region of ASIC2 (amino acids 1-42) with that
of ASIC2b led to functional chimeras that displayed different
properties compared with wild-type ASIC2 (Fig. 1, B and
C, chimera C1). ASIC2 was selective for sodium over
potassium (4, 8), as confirmed by the current reversal potential of 32 ± 2 mV (n = 19;
pNa+:pK+ = 9.8), which is close to the sodium
equilibrium potential of 41 mV. Chimera C1 did not discriminate well
between Na+ and K+ because the reversal
potential (Erev) was 7 ± 1 mV
(n = 6), corresponding to a
pNa+:pK+ ratio of 1.5. Chimera C2, with a
junction at amino acid 26 of the ASIC2 sequence, also had a lower
selectivity for Na+ over K+ as compared with
ASIC2 (Erev = 11 ± 3 mV; n = 4; pNa+:pK+ = 1.9). Chimera C3, with a
junction at amino acid 17, was at least as selective for
Na+ as the wild-type ASIC2 channel
(Erev = 39 ± 2 mV; n = 5).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Reversal potentials of chimeric ASIC2/ASIC2b
channels. A, topological organization of ASIC2 and its
splice variant, ASIC2b. TM1 and TM2 indicate the
transmembrane domains. The region common to ASIC2 and ASIC2b is
indicated in light gray. B, horizontal
bars in the schematic representation of chimeras indicate the
junction positions between ASIC2b (gray) and ASIC2
(white). Numbers beneath the bars indicate amino
acid position in ASIC2. This corresponds to the following positions in
ASIC2b: 86 (C1), 66 (C2), and 57 (C3).
I/V (current/voltage) curves shown on the right were
obtained from whole cell recordings. Currents were normalized with
respect to the peak current recorded at 60 mV. C, the
change in current reversal potential depends on the junction position.
Peak currents reversed at 32 ± 2 mV for wild-type ASIC2, at
7 ± 1 mV for C1, at 11 ± 3 mV for C2, and at 39 ± 2 mV for C3.
The modification in ion selectivity observed with chimeras can thus be associated with a 9-amino acid region of ASIC2, from Ile-17 to Thr-25, located before the TM1. These nine amino acids were then systematically replaced in ASIC2 by those that are present in the ASIC2b sequence, and the reversal potential was determined for each mutant (Table I). Shifting the pH from pH 7.4 to pH 5 activated all mutant channels, with current amplitudes similar to that of wild-type ASIC2 (data not shown). Six mutants had reversal potentials that were significantly lower than that of ASIC2 (Table I). The largest changes in selectivity were observed for the I19P, F20S, and T25K mutants, with pNa+:pK+ values of 3.3 (n = 7), 1.7 (n = 5), and 1.4 (n = 8), respectively, whereas the pNa+:pK+ value calculated for wild-type ASIC2 was 9.8, as described previously (8). A similar change in selectivity occurred when Thr-25 was mutated for an apolar Val or a negatively charged Asp residue instead of a Lys residue. The pNa+:pK+ ratio was 2.7 for T25D (n = 4) and 1.5 for T25V (n = 4) (Table I).
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The ASIC2a current inactivates exponentially and completely after a change in pH. The time constant for inactivation, which is 3.2 s for the wild-type, was also modified for some mutants. Mutants Q18R, N22S, and T25K had a time constant of inactivation that was twice as large as that for the wild-type (Table I).
The two mutants that are particularly altered in their ion selectivity,
F20S and T25K, were analyzed in more detail. Unlike ASIC2, unitary
currents recorded from the T25K and F20S mutants were very noisy, so
that single-channel properties could not be determined. Reversal
potentials for outside-out patches (ASIC2-F20S, 8 ± 2 mV,
n = 4; ASIC-T25K, 12 ± 2 mV, n = 4) were similar to these for the whole cell (Table I; Fig.
2, A and B). When
extracellular Na+ was replaced by K+,
proton-gated currents elicited by a pH step from pH 7.3 to pH 5 were
almost as large as in the presence of Na+ (Fig. 2,
C and D). The currents recorded in the
K+-rich extracellular solution reversed at 2 and 4 mV for
the ASIC2-F20S and ASIC2-T25K mutants, respectively.
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The pH of half-maximal activation (pH0.5) of the F20S and
T25K mutants was shifted to a more alkaline pH as compared with that of
wild-type ASIC2. Whereas the pH0.5 value was 4.4 for
wild-type ASIC2 and 4.7 for ASIC2-I19P, it was 5.7 and 5.4 for
ASIC2-F20S and ASIC2-T25K, respectively (Fig.
3).
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Mutations corresponding to ASIC2-I19P, F20S, and T25K were then
introduced in other channels of the NaC/DEG family (Fig.
4A). This led to a loss of
function for some of the mutants such as FaNaC-N51K, ASIC1-F21S,
ASIC1-T26K, and ASIC3-F21S. However, two ASIC3 mutants, ASIC3-V20P and
ASIC3-T26K, could still be activated by a drop in extracellular pH.
ASIC3 normally displays biphasic kinetics with a rapidly inactivating
current and a sustained current (3), and both currents are carried
essentially by Na+ (Ref. 3; Fig. 4C; reversal
potential, 32 ± 3 mV (n = 5),
pNa+:pK+ = 9.8). Both components of ASIC3-V20P
were also selective for Na+. Their reversal potentials of
26 ± 2 mV (n = 6) correspond to a
pNa+:pK+ ratio of 5.2 (Fig. 4C). On
the contrary, ASIC3-T26K, like ASIC2-T25K, displayed a reduced
Na+/K+ selectivity. The reversal potential for
both the transient current and the sustained current was 9 ± 3 mV
(n = 7), corresponding to a
pNa+:pK+ ratio of 1.7 (Fig. 4C).
Neither component of the ASIC3-T26K mutant showed a change in
pH0.5 with respect to the wild-type (Fig.
4B).
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DISCUSSION |
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ASICs are a new class of cation channels activated by extracellular acidification. They belong to the NaC/DEG superfamily of ion channels (9). Subunits of this family have a topological organization comprising two transmembrane segments and a large extracellular loop (10-13) and assemble into tetramers, as demonstrated for both FaNaC (6) and ENaC (14, 15). Selective functional domains in control of the pore selectivity, the sensitivity to amiloride, and the gating properties have been identified for some members of the family (16-19), but there are few structural data on the ASICs (8).
The participation of the transmembrane domain TM2 and perhaps TM1 in the ion pore has not been directly demonstrated for ASICs but is well documented for ENaC (20) and another ligand-gated channel, FaNaC (21). The region located just before TM2 has been shown to be important for gating and for the pharmacological properties of ASIC2. The ASIC2 mutations G430T, G430V, or G430F, which correspond to mutations leading to gain of function or neurodegeneration in C. elegans degenerins (22-25), cause large changes in pH dependence and the inactivation process as well as changes in amiloride sensitivity (8). Participation of the pre-TM2 region in ion selectivity and conductance has also been established for ENaC (17, 18).
The present study focuses on the pre-TM1 domain of ASICs. This region
is highly conserved among NaC/DEG family members and is involved in the
gating properties of the epithelial sodium channel. Mutations of a
conserved glycine in the pre-TM1 domain of -ENaC is associated with
type I pseudohypoaldosteronism (26) and leads to a reduction of ENaC
activity. ENaC channels containing
-ENaC or
-ENaC subunits
mutated at this glycine residue display a reduction in open
probability, with no change in ion conductance and selectivity (27).
However, a participation of this region in the ion permeation or the
selectivity filter was never reported.
Our results demonstrate an involvement of the pre-TM1 domain of ASIC2 in both the pH dependence and the ion pore properties. F20S and T25K mutations in ASIC2 change the pH dependence from a pH0.5 of 4.4 to 5.7 (F20S) and 5.4 (T25K). The pH dependence of ASICs is associated with several regions of the protein because mutations in the pre-TM2 position can also modify the pH0.5 value (8).
Of particular interest is the modification of ASIC2 ion selectivity after mutations in the pre-TM1 domain. The I19P, F20S, and T25K mutations cause a 3- to 7-fold reduction in the Na+:K+ permeability ratio. The large changes in selectivity are not restricted to ASIC2 mutants because both the peak and sustained components of the ASIC3-T26K mutant show a 6-fold reduction in the Na+:K+ permeability ratio.
This loss of ion selectivity is associated with the replacement of ASIC2 and ASIC3 pre-TM1 residues by amino acids present in ASIC2b. In sensory cells, ASIC2b associates with ASIC3 and then confers a nonselective character to its sustained component (4). The pre-TM1 domain of ASIC2b may thus be one of the structural elements responsible for the loss of Na+ selectivity of this sustained component. The T26K mutation in ASIC3 changes the reversal potential of the peak and sustained currents in parallel, whereas co-expression of ASIC3 and ASIC2b influences the sustained current but not the peak current. This discrepancy between the properties of the heteromeric ASIC3/ASIC2b and the mutated ASIC3 T26K channel could be explained by the existence of two channel modes for ASIC3, one associated with the transient current, and one associated with the sustained current. The ASIC2b subunit would associate only with the conformation corresponding to the channel mode responsible for the sustained component and would modify its properties by changing its ionic selectivity. The T26K mutation introduced in ASIC3 would change the ion selectivity of both modes (transient and sustained currents) of the ASIC3 channel.
This study suggests that the pre-TM1 domain contributes to the ion pore
of ASICs and plays a crucial role in the selectivity filter, possibly
as a re-entrant loop from the cytoplasmic side. The present data
suggest a model for the ion pore of ASICs that involves the
transmembrane domains (TM2 and perhaps TM1) and the pre-TM2 and pre-TM1
regions. This model could possibly be suitable for other members of the
NaC/DEG family.
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ACKNOWLEDGEMENTS |
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We are very grateful to Drs. P. Barbry and R. Waldmann for helpful discussions; to N. Leroudier, V. Friend, and M. Jodar for excellent technical assistance; and to V. Briet for secretarial assistance.
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FOOTNOTES |
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* This work was supported by the Centre National de la Recherche Scientifique, the Association Française contre les Myopathies, and the Association pour la Recherche sur le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A recipient of Grant 91815-48/A000 from the Délégation
Générale pour l'Armement.
§ To whom correspondence should be addressed. Tel.: 33-04-93-95-77-02 or -03; Fax: 33-04-93-95-77-04; E-mail, ipmc{at}ipmc.cnrs.fr.
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ABBREVIATIONS |
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The abbreviations used are: ASIC, acid-sensing ion channel; ENaC, epithelial Na+ channel; FaNaC, Phe-Met-Arg-Phe-amide-gated channel; PCR, polymerase chain reaction; TM, transmembrane; NaC/DEG, amiloride-sensitive Na+ channel/degenerin.
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REFERENCES |
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