(Received for publication, January 2, 1995; and in revised form, February 6, 1996)
From the
We have cloned a novel cDNA from human brain which encodes a
non-voltage-dependent Na channel (BNC1). BNC1 has some
sequence similarity (24-28%) with a new channel family that
includes subunits of the mammalian epithelial Na
channel, the Caenorhabditis elegans degenerins, and the Helix aspersa FMRF-amide-gated Na
channel.
Like other family members it is inhibited by amiloride. However, its
predicted structure differs from other family members, its
discrimination between Na
and Li
is
different, and in contrast to other mammalian family members,
coexpression with other cloned subunits of the family does not increase
current. BNC1 has a unique pattern of expression with transcripts
detected only in adult human brain and in spinal cord. Thus, BNC1 is
the first cloned member of a new subfamily of mammalian
Na
channels. The function of BNC1 as a
non-voltage-gated Na
channel in human brain suggests
it may play a novel role in neurotransmission.
Recent studies have identified a new family of Na channels whose characteristic features include Na
selectivity, inhibition by amiloride, and a conserved primary
structure (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) .
Family members contain 500 to 800 residues. Sequence analysis and
studies of topology suggest that the amino and carboxyl termini are
intracellular, that there are two hydrophobic regions that traverse the
membrane (M1 and M2), (
)and that between M1 and M2 there
lies a large cysteine-rich extracellular domain (12, 13, 14) .
The best characterized
members of this family are the amiloride-sensitive epithelial
Na channels (ENaC) that control Na
and fluid absorption in the kidney, colon, and lung. ENaC
channels are constructed from at least three homologous subunits
(
-,
-, and
ENaC)(4, 5, 6, 7, 8, 9) .
These channels may also be involved in detection of salty
taste(15) . A closely related subunit,
NaCh, is expressed
in pancreas, testis, ovary, and brain.
NaCh generates
Na
channels when coexpressed with
- and
ENaC(10) , suggesting that it may be part of the ENaC
subfamily of channels. Several family members have also been discovered
in C. elegans, including MEC-4, MEC-10, and DEG-1, which when
mutated produce a touch-insensitive
phenotype(1, 2, 3) . Although the function of
these gene products has not been established, several observations
suggest that they form ion channels: their sequences are similar to the
ENaC subunits; genetic evidence suggests that three gene products are
required for function(3) ; M2 of
ENaC can substitute for
M2 of MEC-4 (16) ; and specific mutations cause ballooning
cellular degeneration (1, 2) similar to that found
with overexpression of active ENaC subunits (17) . Based on
this ability to produce cell degeneration, family members in C.
elegans are called ``degenerins.'' The most recent
addition to this family is a Phe-Met-Arg-Phe-NH
(FMRF-amide)-stimulated Na
channel (FaNaCh)
cloned from Helix(11) .
Here we report the cloning
and expression of a novel member of the family which is expressed in
human brain. We name it BNC1 for Brain Na Channel, and
1 with the expectation that additional subunits will be discovered in
the future.
Figure 1:
Analysis of BNC1 sequence. A,
predicted amino acid sequence of the open reading frame of BNC1. Underlined sequence refers to predicted hydrophobic,
membrane-spanning segments. Conserved cysteines are indicated with asterisks. Potential glycosylation sites in the extracellular
domain are indicated with squares. Potential protein kinase C
phosphorylation site in the intracellular domain is indicated with a circle. The nucleic acid sequence was submitted to GenBank
(accession number U50352). B, structural comparison of cloned
family members. MEC-4 (2) and hENaC (6) were
chosen as representative members of the degenerin and ENaC/
NaCh
proteins, respectively. Black areas identify transmembrane
segments (M1 and M2), shaded areas indicate cysteine-rich
domains (CRD), cross-hatched area indicates
additional region of conserved sequence, and thin black line indicates regions which are missing in some family members. C, phylogenetic tree of family
members.
Fig. 1B shows
that BNC1 has a predicted structure with some features similar to that
of other cloned amiloride-sensitive Na channels and
the degenerins. Of particular interest are the two hydrophobic
transmembrane segments and the extracellular cysteine-rich domains.
There is also an area with limited sequence conservation between two
cysteine-rich domains (cross-hatched area in Fig. 1B). However, there are also significant
differences between BNC1 and other cloned members of the family (Fig. 1B). In the amino-terminal half of the
extracellular domain, BNC1 seems more similar to FaNaCh because it
lacks sequences found in degenerins and ENaC. Yet, in the
carboxyl-terminal half of the extracellular domain, BNC1 is more
similar in length to ENaC and the degenerins than to FaNaCh. BNC1 has a
relatively short carboxyl-terminal intracellular tail. It lacks the
conserved proline-rich sequences of ENaC that may be involved in
protein-protein interactions(20) . It also lacks the
PPPXYXXL motif which determines the amount of cell
surface protein and which is deleted from
hENaC or
hENaC in
patients with Liddle's syndrome (17) . BNC1 has consensus N-linked glycosylation sequences in the extracellular domain (Fig. 1A). The amino-terminal intracellular sequence
contains one consensus sequence for protein kinase C phosphorylation.
Although absolute homology is relatively low, BNC1 shares slightly
greater overall amino acid sequence identity with FaNaCh than with
other members of the family. BNC1 is 28.4% identical with FaNaCh,
24.2-26.6% identical with -,
-, and
ENaC and
NaCh, and 24.4-25.4% identical with the degenerins. Despite
the species difference, phylogenetic analysis placed BNC1 closest to
FaNaCh, rather than to other mammalian members of the family (Fig. 1C).
Figure 2:
Northern blot analysis of BNC1 expression
in adult human tissues (A) and in specific regions of adult
human brain (B). Each lane contains approximately 2
µg of poly(A) RNA; the amount of RNA in each lane
was adjusted to observe identical levels of
-actin expression.
Filters were hybridized with a probe corresponding to the coding
sequence of BNC1 as described under ``Experimental
Procedures.'' Blots were exposed to film for 4 days; a 7-day
exposure of B showed that both transcripts were evident to
some extent in every lane.
The expression
pattern of BNC1 is unique; expression primarily in the central nervous
system contrasts with previously identified mammalian members of the
family. Although transcripts of - and
ENaC and
NaCh have
been detected in brain, they are much more prevalent in other tissues.
- and
ENaC are most abundant in epithelia of kidney, colon,
and
lung(4, 5, 6, 7, 8, 9) ,
and
NaCh is most abundant in testis, ovary, and
pancreas(10) . Expression of nonmammalian members of the family
has been reported in excitable tissue. Transcription of FaNaCh occurs
in muscle and nervous tissue of Helix(11) , and the
degenerins are expressed in the peripheral and central nervous system
of C. elegans(1, 2, 3) .
When the
entire coding region of BNC1 was used as a probe, we detected two
transcripts, 2.7 and 3.7 kb in length (Fig. 2, A and B). In general, relative hybridization to the two transcripts
was similar in most brain regions, although there was a greater
relative abundance of the large transcript in the cerebellum, medulla,
spinal cord, corpus collosum, hypothalamus, substantia nigra, and
thalamus. To investigate the relationship between the two transcripts,
we prepared probes from the 5` and 3` regions of BNC1 cDNA
(corresponding to the amino and carboxyl termini of the predicted
protein) and hybridized them to a Northern blot containing human brain
poly(A) RNA (Fig. 3). Whereas the 3` probe
hybridized with both transcripts, the 5` probe hybridized with the
2.7-kb transcript only. These data indicate that the cDNA reported here
is produced by the smaller transcript. There are at least two possible
explanations for the presence of two transcripts. First, alternative
splicing at the amino terminus might generate two transcripts from a
single gene. Second, there may be two genes with very similar sequences
corresponding to the 3` end of BNC1. Further investigation is necessary
to distinguish between these alternatives. In either case, the data
suggest the possibility of structural and thus functional complexity
with multimeric channel proteins.
Figure 3:
Northern blot analysis of human brain RNA
using 5` and 3` specific BNC1 probes. 5 µg of adult human brain
poly(A) RNA were run on a 1.2% agarose-formaldehyde
gel, transferred to a nitrocellulose filter, and hybridized with
labeled probes prepared from either the 5` or 3` ends of the BNC1 cDNA
as shown at bottom.
Figure 4:
Expression of BNC1 in Xenopus oocytes. Oocytes were injected with cDNA encoding BNC1, and
current was measured by two-electrode voltage clamp 1 day after
injection at a holding potential of -60 mV. A,
representative current trace. Amiloride (100 µM) was
present during time indicated by bar. B, current-voltage
relationships for amiloride-sensitive current from representative
oocytes expressing BNC1 or injected with HO (Control). Oocytes were bathed in Na
- or
K
-containing solution, as indicated. C,
effect of increasing concentrations of amiloride, plotted as fraction
of response to 100 µM amiloride (n = 4). D, amiloride-sensitive current measured in presence of
Na
, Li
, or K
as
indicated. Data are plotted relative to current in NaCl. Oocytes
expressed BNC1 (n = 9) or
hENaC
(``hENaC,'' n = 4), as indicated. E,
amiloride-sensitive current in oocyte expressing ENaC subunits with or
without BNC1, as indicated. n = 5-16 for each
except
hENaC where n =
3.
When we replaced
Na with Li
, we measured equal
currents through BNC1 channels (Fig. 4D). This differs
from
ENaC and
ENaC which are 2-fold more conductive
to Li
than to Na
(Fig. 4D), and from FaNaCh and
NaCh which
are more conductive to Na
than to
Li
(9, 10, 11) . It was
previously shown that Ser
in
rENaC was important for
Na
/Li
selectivity; mutation to Ile
increased Na
conductance relative to
Li
(21) . The analogous residue in BNC1 (and
NaCh) is alanine (Ala
), suggesting that this residue
might help determine relative Na
/Li
conductivity in BNC1 as well as in other family members.
Na current generated by BNC1 was not significantly
increased by coexpression with combinations of
-,
-, and/or
hENaC subunits (Fig. 4E). In contrast,
coexpression of the three ENaC subunits significantly increased current
compared with expression of only two subunits. This suggests that BNC1
functions as a novel member of the Na
channel family.
The data with BNC1 also contrast with
NaCh in which coexpression
with
- and
hENaC markedly increased current.
We considered the possibility that BNC1 current might be stimulated by an agonist, much as FaNaCh requires activation by the Helix aspersa neuropeptide FMRF-amide. However, BNC1 was not activated by FMRF-amide or the related mammalian peptides F-8-F-amide or A-18-F-amide. Although this suggests that BNC1 is not the mammalian homologue of the Helix FaNaCh, it does not exclude the possibility that BNC1 could be a receptor for another neurotransmitter.
BNC1 is a novel member of the ENaC/degenerin family. However,
it has several significant differences from other cloned members of the
family: it has a different predicted structure; it does not
discriminate between Na and Li
as
current carriers; expression was detected only in the central nervous
system; and BNC1 current is not augmented when it is coexpressed with
subunits of ENaC. These considerations suggest that BNC1 may be the
first cloned member of a new subfamily of mammalian Na
channels.
Although expression of BNC1 generated a
Na current, the magnitude was small. There are at
least two explanations. It is possible that other, as yet unidentified,
subunits might be required to produce a fully functional channel
complex. The situation may be analogous to
ENaC which generates
small currents when expressed alone, but produces large currents when
coexpressed with
and
ENaC(5, 7) . It is
also possible that BNC1 might require activation by an agonist or
neurotransmitter, just as FaNaCh is stimulated by
FMRF-amide(11) . This latter possibility seems particularly
attractive because of its expression in brain. Moreover,
ligand-regulated activity rather than constitutive activity would be
more consistent with neuronal expression, because constitutive
non-voltage-dependent Na
channel activity could
depolarize the cell, thereby disrupting signal transduction, or it
could cause cell toxicity. It is interesting to speculate that the
large cysteine-rich extracellular domain of BNC1 might have a receptor
function. Certainly the large size and presence of multiple cysteine
residues is reminiscent of other receptor proteins. Identification of
other brain Na
channel subunits and/or receptor
ligands should help us better understand the function of BNC1 and the
role of non-voltage-gated Na
channels in the central
nervous system.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U50352[GenBank].