(Received for publication, December 26, 1995)
From the
Small neurons of the dorsal root ganglia (DRG) are known to play
an important role in nociceptive mechanisms. These neurons express two
types of sodium current, which differ in their inactivation kinetics
and sensitivity to tetrodotoxin. Here, we report the cloning of the
-subunit of a novel, voltage-gated sodium channel (PN3) from rat
DRG. Functional expression in Xenopus oocytes showed that PN3
is a voltage-gated sodium channel with a depolarized activation
potential, slow inactivation kinetics, and resistance to high
concentrations of tetrodotoxin. In situ hybridization to rat
DRG indicated that PN3 is expressed primarily in small sensory neurons
of the peripheral nervous system.
Voltage-gated sodium channels play a fundamental role in the
regulation of neuronal excitability. In addition to differences in
primary structure and kinetic properties(1) , these channels
can be distinguished pharmacologically on the basis of their relative
sensitivity to the neurotoxin, tetrodotoxin
(TTX)()(2) . Two types of sodium currents are
expressed by sensory neurons within the dorsal root ganglion (DRG), a
fast inactivating TTX-sensitive current and a slow inactivating,
TTX-resistant current that appears to be expressed by a high proportion
of the small afferent
neurons(3, 4, 5, 6, 7, 8) .
Of the large and small neurons of the DRG, the latter is of primary
importance in the processing of nociceptive information within the
somatosensory system(9, 10) . In order to define the
molecular basis of sodium channel conductance in sensory neurons, we
have attempted to identify and clone novel sodium channel
-subunits. As described here, this work has led to the isolation
and functional expression of PN3, a novel voltage-gated, TTX-resistant
sodium channel expressed predominantly by small sensory neurons within
the peripheral nervous system.
Figure 5: Expression of PN3 in Xenopus oocytes. A, currents produced by step depolarizations of an oocyte injected with 18 ng of PN3 cRNA from a holding potential of -100 to -30 mV through +50 mV in 10-mV increments. The Geneclamp hardware leak subtraction was used. No inward current was observed in oocytes injected with water. B, current-voltage relationship of the data in A. C, concentration dependence for TTX block of PN3 sodium current. Each oocyte was exposed to the full range of TTX concentrations shown, beginning with the lowest concentration and proceeding to the highest. For each concentration, the effect was allowed to attain steady state. Each point represents the mean ± range; n = 2.
Nucleotide sequence analysis of the PN3 cDNA identified a 5868-base
open reading frame, coding for a 1956-amino acid protein. In common
with other sodium channels, there is an ATG 5 base pairs upstream of
the genuine ATG. The deduced amino acid sequence of PN3 (Fig. 1)
exhibited the primary structural features of an -subunit of a
voltage-gated sodium channel. PN3 contains four homologous domains
(I-IV), each consisting of six putative
-helical
transmembrane segments (S1-S6). The positively charged residues
in the voltage sensor (S4 segments) and the inactivation gate between
IIIS6 and IVS1 are highly conserved in PN3; sites for cAMP-dependent
phosphorylation and N-linked glycosylation shown to exist in
other sodium channels (1) are also present in PN3. In PN3,
however, there are two unique consensus sites for cAMP-dependent
phosphorylation sites, one in domain II between S3 and S4 and another
in the interdomain II-III (Fig. 1). Modulation of rBIIa
channel function by cAMP-dependent protein kinase A has been
demonstrated(1, 17, 18) . The significance of
the unique cAMP-dependent protein kinase A consensus sites in PN3 in vitro and in vivo remains to be demonstrated. In
addition, there is an insertion of an additional Gln between
Pro
and Ala
in several partial clones. The
significance of the glutamine insertion has not been determined.
Figure 1:
Deduced amino acid sequence of
peripheral nerve sodium channel type 3 (PN3) showing putative
transmembrane domains. , potential cAMP-dependent phosphorylation
site;
, potential N-linked glycosylation site;
,
TTX resistance site; *, termination codon; ⇑, site of an
additional Gln insertion.
Fig. 2A shows an amino acid homology comparison of PN3 with other cloned rat sodium channels. The higher sequence homology between PN3 and the TTX-insensitive cardiac channel and their slow inactivation kinetics (discussed below) suggest that they belong to a unique subfamily of sodium channels. Indeed, the Distance paradigm of the GCG program classifies PN3 and the cardiac sodium channel as a subfamily of sodium channels (Fig. 2A).
Figure 2: A, amino acid homology comparison of PN3 with selected sodium channels. Numbers indicate percent amino acid similarity between each channel and PN3. B, tissue distribution profile of PN3 by RT-PCR analysis after 35 cycles of amplification. SCG, superior cervical ganglia.
In situ hybridization using PN3-specific oligonucleotide probes showed
that PN3 mRNA was expressed predominantly by small neurons in rat DRG (Fig. 3). Approximately 76% of the small cell population
(400-1000 µm) and 33% of the large cell
population (1400-2000 µm
) were hybridized with
probes for PN3 (Fig. 4). Recently, Akopian and Wood (28) isolated a partial cDNA clone, G7, from a rat DRG cDNA
library with homology to known sodium channels and specific expression
in subsets of sensory neurons. Whether PN3 and G7 are
the same gene is unknown at present.
Figure 3: Emulsion autoradiography illustrating the distribution of PN3 mRNA in rat DRG. Dark field photomicrographs of sections hybridized with antisense (A) and sense (B) strand PN3-specific radiolabeled oligomers. C, bright field enlargement of a section hybridized with a radiolabeled antisense probe mixture and counterstained with hematoxylin. Labeled neurons (arrow) and unlabeled neurons (double arrowhead) were distributed throughout the DRG. Scale: A and B, 250 µm; C, 50 µm.
Figure 4: Frequency distribution histogram of somal areas from sections through lumbar DRG. The number of neurons hybridized with PN3 is shown by filled bars (n = 246). The number of non-hybridized neurons is shown by hatched bars (n = 215).
The currents produced by
injection of PN3 cRNA had slow inactivation kinetics (Fig. 5A). rBIIa, rBIII, and rSkM1 sodium channels also
produce currents with slow inactivation kinetics when injected into Xenopus oocytes; coexpression of the 1-subunit greatly
accelerates the inactivation kinetics of these
channels(22, 32, 33, 34, 35) .
However, coinjection of 1.3 ng of human sodium channel
1-subunit
(hSCN
1) (36) cRNA, which is homologous to the rat brain
and DRG SCN
1, with PN3 cRNA did not accelerate the inactivation
kinetics (data not shown). In contrast, coexpression of this quantity
of hSCN
1 cRNA with rSkM1 cRNA was sufficient to accelerate the
inactivation kinetics of rSkM1 maximally. Therefore, PN3 may possess
inherently slow kinetics.
When expressed in Xenopus oocytes, the PN3 sodium current is highly resistant to TTX
(IC
100 µM) (Fig. 5C).
The TTX-sensitive brain and skeletal muscle sodium channels are blocked
by nanomolar TTX concentrations, whereas the TTX-insensitive cardiac
sodium channels are blocked by micromolar TTX
concentrations(2) . In rat heart sodium channel 1 (rH1),
Cys
is a critical determinant of TTX insensitivity (37, 38, 39) ; in the TTX-sensitive rBI,
rBII, rBIII, and rSkM1, the corresponding residue is an aromatic amino
acid, either Phe or Tyr, the aromatic ring of which facilitates the
binding of TTX to the protein. In PN3, this position is occupied by a
Ser residue (Ser
), which may explain the unique response
to TTX. Site-directed mutagenesis of this residue to Phe/Tyr or Cys
will determine whether this amino acid residue is solely responsible
for TTX resistance.
TTX-resistant sodium currents have been implicated in peripheral and central neuronal sensitization mediated by the C-fibers of small neurons following peripheral tissue damage and nerve injury. The biophysical and pharmacological properties of PN3 suggest that it contributes to the TTX-resistant sodium currents in small neurons of DRG. PN3 may, therefore, play a role in the the sensory function and dysfunction that is characteristic of pathophysiological pain processing. In addition, we suggest that PN3 may conduct TTX-resistant sodium currents in other sensory ganglia of the peripheral nervous system such as nodose ganglia(40) .