(Received for publication, May 2, 1997, and in revised form, June 6, 1997)
From the Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France
We have cloned and expressed a novel proton-gated Na+ channel subunit that is specific for sensory neurons. In COS cells, it forms a Na+ channel that responds to a drop of the extracellular pH with both a rapidly inactivating and a sustained Na+ current. This biphasic kinetic closely resembles that of the H+-gated current described in sensory neurons of dorsal root ganglia (1). Both the abundance of this novel H+-gated Na+ channel subunit in sensory neurons and the kinetics of the channel suggest that it is part of the channel complex responsible for the sustained H+-activated cation current in sensory neurons that is thought to be important for the prolonged perception of pain that accompanies tissue acidosis (1, 2).
Many painful inflammatory and ischemic conditions are accompanied by a decrease of the extracellular pH (2, 3). H+-gated cation channels are present in sensory neurons (1, 4-6), and it is likely that those acid-sensing ion channels are the link between tissue acidosis and pain. We recently cloned a rapidly inactivating H+-gated cation channel ASIC1 (7) (acid-sensing ion channel). Fast inactivating H+-gated cation currents were described in neurons of the central nervous system (6, 8, 9) and in sensory neurons (4-6), tissues where ASIC is well expressed (7). However, rapidly inactivating H+-gated cation channels cannot account solely for the prolonged sensation of pain that accompanies tissue acidosis. Sensory neurons respond to a drop in pH with a rapidly inactivating followed by a sustained current, which is thought to mediate the non-adaptive pain caused by acids (1). Here we describe the cloning of a H+-gated cation channel specific for sensory neurons that has both a rapidly inactivating and a sustained component.
We used an anchored PCR approach to
identify the sequences upstream and downstream of the expressed
sequence tag (W62694). An double stranded adapter (anchor) was
prepared by annealing the oligonucleotides
GATTTAGGTGACACTATAGAATCGAGGTCGACGGTATCCAGTCGACGAATTC and
PO4-GAATTCGTCGACTG-NH2. The shorter
oligonucleotide was protected with a 3 NH2 group to avoid
extension during the PCR reaction. This adapter was ligated to double
stranded rat brain cDNA resulting in a cDNA with known
sequences (the anchor) on both extremities. The so prepared anchored
cDNA was used to amplify the 5
and the 3
end of the coding
sequence by PCR. This was done using either the primer
GATTTAGGTGACACTATAGAA or TAGAATCGAGGTCGACGGTATC, which are identical to
parts of the longer of the two adapter oligonucleotides together with
either the sense primer (CACTACACGCTATGCCAAGG, for amplification of the
3
end) or the antisense primer (CCCAGCAACTCCGACACTTC, for
amplification of the 5
end) complementary to the expressed sequence
tag (W62694). The PCR products were subcloned into Bluescript, and five
clones each for the 5
PCR and for the 3
PCR were sequenced. The
anchored PCR allowed us to identify the sequences upstream of the first
ATG codon and downstream of the stop codon. However all clones isolated
from brain contained introns with in frame stop codons and code for
truncated proteins lacking the second transmembrane domain that was
found to be essential for channel function (10). Analysis of the tissue
distribution showed that high levels of the mRNA are only found in
DRG. Primers flanking the coding sequence (sense:
ACGAATTCTCCCTGGTCCAGCCATGAAAC, antisense:
CCTCGAGCTAGAGCCTTGTGACGAGGTAA) that contained an EcoRI site
(sense) or an XhoI site (antisense) were used to amplify the
full-length coding sequence from DRG cDNA. The PCR product was
digested with EcoRI and XhoI and subcloned into
the EcoRI/SalI-digested PCI expression vector.
One clone was sequenced on both strands, and two independent clones
were sequenced on one strand using an Applied Biosystems sequencer.
Unlike in brain, the three clones isolated from DRG code for
full-length proteins.
COS7 cells were co-transfected with DRASIC cDNA in the PCI expression vector and an expression vector containing the CD8 receptor cDNA using DEAE-dextran. 3 days later, cells binding CD8 antibody-coated beads (11) were used for experiments. Ion currents were recorded using either the whole cell or the patch-clamp technique. The pipette solution contained (in mM): KCl 120, NaCl 30, MgCl2 2, EGTA 5, HEPES 10 (pH 7.2). For the "0 sodium" solution NaCl was replaced by KCl. The bath solution contained in mM: NaCl 140, KCl 5, MgCl2 2, CaCl2 2, HEPES 10 (pH 7.3). Rapid changes in extracellular pH were induced by opening an outlet of a microperfusion system at a distance of ~50 µm from the cell. Test solutions having a pH of less then 6 were buffered with 10 mM MES rather than HEPES. Experiments were carried out at room temperature (20-24 °C).
Northern Blot and in Situ Hybridization4 µg of total RNA from dorsal root ganglia of 7-day-old rats and 4 µg of poly(A+) RNA from adult rat brain were separated on a 1% formaldehyde-agarose gel and subsequently transferred to nylon membranes. The blots were hybridized with a random prime 32P-labeled fragment of the DRASIC cDNA corresponding to nucleotide 141-1145 in 6 × SSC, 10 × Denhardt's solution, 0.1% SDS, 100 µg/ml herring sperm DNA, washed with 0.1 × SSC, 0.1% SDS at 70 °C, and subsequently exposed to a Fuji phosphoimager screen. For the in situ hybridizations on frozen fixed 10-µm brain sections from adult Wistar rats, we used a 33P-random prime-labeled fragment of DRASIC corresponding to nucleotide 141-1145. Brain sections from adult rats were hybridized with the 33P-end-labeled probes overnight at 37 °C in 50% formamide, 2 × SSC, and subsequently washed at room temperature in 1 × SSC. Sections (6 µm) and primary cultures of rat dorsal root ganglia were hybridized with double-stranded DNA fragments labeled by PCR with DIG-dUTP (sections), or fluorescein-12-dUTP (primary cultures). The probes used correspond to nucleotide 141-1145. Probe labeling, sample preparation, hybridization, and visualization of DIG nucleic acids with alkaline phosphatase-conjugated anti-DIG antibodies was carried out following the protocols from Boehringer Mannheim. Primary cultures of DRG neurons from 4-day-old rats were prepared essentially as described (1) and used for in situ hybridization after 7 days in culture. The in situ hybridization results shown were confirmed with one additional probe. Sequence positions given refer to the sequence submitted to GenBankTM.
Computer AnalysisThe sequence alignment was computed with the GCG (Genetics Computer Group, Madison, WI) software package. All comparisons of sequences with data bases were done using the Blast network server at the National Center for Biotechnology Information (NCBI).
Comparison of the ASIC protein sequence with the data base of
expressed sequence tags identified one novel member of this family of
ion channels. We used anchored PCR to clone the complete coding
sequence from rat DRG. The DRASIC cDNA has an open reading frame of
1599 base pairs preceded by stop codons and codes for a protein of 533 amino acids. DRASIC belongs to the amiloride-sensitive Na+
channel (12-18)/degenerin (19-21) family of ion channels and shares 53% sequence identity with its closest relative ASIC (Fig.
1). A DRASIC transcript of 2.6
kilobases was detected in total RNA of DRG (Fig.
2a). In brain
poly(A+) RNA where ASIC mRNA is abundant (7), no DRASIC
transcript was detectable. Furthermore a mouse multitissue Northern
blot (CLONTECH) with poly(A+) RNA from
brain, heart, spleen, lung, liver, skeletal muscle kidney, and testis
did not give any signal (not shown) with the probe that labeled the
DRASIC mRNA in total RNA from DRG, indicating that DRASIC is
specific for sensory neurons. In situ hybridization confirmed those results (Fig. 2, b-d). DRASIC is expressed
in DRG neurons and absent in brain. The small sensory neurons are thought to carry the nociceptive signals from polymodal sensory nerve
endings and interestingly small neurons are intensely labeled. The
specific expression in sensory neurons suggests that the DRASIC channel
has properties required for a specific function of this type of neuron.
Expression of DRASIC in COS cells induced a H+-gated cation
channel with properties clearly distinct from those of ASIC (7). A
rapid decrease of the extracellular pH from pH 7.4 to pH 4 induces a
fast rising, rapidly inactivating current followed by a much slower
activating sustained inward current (Fig.
3a). Surprisingly, expression
of DRASIC can induce both a rapidly and a slowly activating current.
The kinetics of the DRASIC current very closely resemble the biphasic
H+-gated cation current described in sensory neurons (1).
Both the transient and the sustained DRASIC current reverse at +32 ± 3 mV (n = 5), which is close to the Na+
equilibrium potential of +40 mV in the experimental conditions concerned (Fig. 3b). This indicates that the two components
are highly selective for Na+
(gNa+/gK+ = 13.5). Unitary currents were
recorded from outside-out patches in the absence of Na+ in
the pipette (Fig. 3, c and d). The slope
conductance of DRASIC is with 12.6 ± 0.2 picosiemens
(n = 3) (Fig. 3d), close to that reported
for ASIC (14.3 picosiemens) (7). The unitary current has a reversal
potential of +62 mV (Fig. 3d), indicating an 11.5-fold higher selectivity of the channel for Na+ over
K+. Amiloride inhibits the transient current with a
K0.5 of 63 ± 2 µM (Fig. 3,
e and f). The effect of amiloride on the
sustained DRASIC current is complex. In the presence of 200 µM amiloride where the transient current is inhibited by
68 ± 5% (Fig. 3, e and f), the sustained
current is higher than in the absence of amiloride (Fig.
3e). A closer examination of the pH dependence of the DRASIC
current shows that the transient and the sustained phase can be clearly
separated (Fig. 3, g-i). The transient current is activated
when the pH drops only slightly (half-maximal activation at pH 6.5 when
stepping from pH 7.3; Fig. 3h) but requires an initial pH
above 7 for full activation (Fig. 3i). On the contrary, the
sustained current needs more important acidification (below pH 4) for
activity (Fig. 3h) but may still be activated if the resting
pH is far below pH 7 (Fig. 3i). The situation is similar in
sensory neurons (1) where a slight acidification activates only the
transient current, while both the transient and the sustained current
are activated after more important drops of the extracellular pH. A
H+-gated cation channel capable of mediating a prolonged
sensation of pain during tissue acidosis should not only be activated
when the pH drops rapidly but also when the pH decreases slowly, since this is likely to happen during the onset of a tissue acidosis. Unlike
ASIC, that requires a rapid (
1 s) drop of the pH (not shown),
DRASIC responds to slow decreases of the pH (Fig. 3j). If
the extracellular pH is decreased gradually by approaching the cell
slowly with the perfusion outlet, the first transient current
disappears, while the sustained component still develops to its full
size (Fig. 3j). The kinetics of the DRASIC channel and the
fact that DRASIC mRNA is only present in sensory neurons, where it
is abundant, suggest that DRASIC is part of the channel complex
responsible for the sustained H+-gated current in sensory
neurons. However, there are important differences between the
non-inactivating DRASIC current and the sustained current described in
sensory neurons (1). To activate the sustained DRASIC current, the pH
has to become very acidic (pH 4; Fig. 3h), while a tonic
response in sensory neurons is already obtained at pH 6 (1).
Furthermore, both the rapidly inactivating and the sustained phase of
the DRASIC current are highly selective for Na+, while in
sensory neurons a transient Na+-selective current is
followed by a sustained current that discriminates only poorly between
Na+ and K+ (1). Those differences between the
DRASIC current and the native current indicate that more than just
DRASIC is required to form the non-inactivating H+-gated
cation channel in sensory neurons. H+-gated sustained
Na+-selective currents were never reported in sensory
neurons, where DRASIC is well expressed, suggesting that DRASIC in
sensory neurons has indeed properties distinct from the DRASIC channel
expressed in COS cells. This might be due either to a specific
posttranslational modification, such as phosphorylation, or to an
association with other subunits. Heteromultimeric association of
homologous subunits is commonly found with ion channels (22, 23) and
might be the link between the DRASIC subunit and the sustained
non-selective current recorded in sensory neurons. Furthermore,
relatives of DRASIC, the amiloride-sensitive Na+ channel
(13, 15, 16, 18) and the degenerins of Caenorhabditis elegans (19), even require several homologous subunits for correct function, and it would be surprising if this would not be the case for
the H+-gated cation channels. ASIC, that is also expressed
in sensory neurons (7), is not the missing partner of DRASIC since
co-expression of both subunits yields currents that can be explained by
two independent channels (not shown).
A diversity of H+-gated cation channels is described in both sensory neurons (1, 4-6) and in neurons of the central nervous system (6-9). It is therefore likely that new members of this ion channel family will be discovered in the near future. The localization of their mRNAs and proteins should allow studies about the interaction of different H+-gated cation channel subunits expressed in the same type of neuron and might lead to the identification of subunit combinations with properties identical to the native H+-gated channels. The identification of subunits that associate with DRASIC is of particular interest because of the potential importance of sustained H+-gated cation currents for the prolonged sensation of pain caused by acids. The development of blockers that are selective for a H+-gated cation channel specific for sensory neurons, such as DRASIC, might lead to the discovery of new non-addictive analgesics.
We are very grateful to Dr. Eric Lingueglia for helpful discussions and to Catherine Widmann, Gisèle Jarretou, Catherine Le Calvez, Martine Jodar, and Nathalie Leroudier for their skilful technical assistance, Dahvya Doume for secretarial assistance, and Frank Aguila for help with the artwork.