(Received for publication, September 11, 1995; and in revised form, September 25, 1995)
From the
We have isolated a cDNA for a novel human amiloride-sensitive
Na channel isoform (called
) which is expressed
mainly in brain, pancreas, testis, and ovary. When expressed in Xenopus oocytes, it generates an amiloride-sensitive
Na
channel with biophysical and pharmacological
properties distinct from those of the epithelial Na
channel, a multimeric assembly of
,
, and
subunits. The Na
current produced by the new
isoform is increased by two orders of magnitude after coexpression of
the
and
subunit of the epithelial Na
channel showing that
can associate with other subunits and
is part of a novel multisubunit ion channel.
Amiloride-sensitive sodium channels (ASCs) ()are
Na
-permeable non-voltage-sensitive ion channels
inhibited by the diuretic amiloride. They are abundant and well
characterized in epithelial tissues such as kidney, colon, and lung
(for review, see (1) ) where they control the rate and extent
of Na
reabsorption under the regulation of steroid
hormones(2, 3, 4, 5) . The same ASCs
also seem to play an important role in taste perception(6) .
Different mammalian forms of ASCs with different biophysical
(conductances, selectivity) and pharmacological properties (sensitivity
to amiloride and derivatives) (for review, see (7) ) have been
characterized recently in thyroid(8) , smooth
muscle(9) , and vascular endothelial cells from
brain(10) . In addition, amiloride-blockable nonselective ion
channels are also important for mechanotransduction(11) .
Molecular cloning of the highly Na
-selective
epithelial Na
channel has demonstrated recently that
it is made of at least three homologous subunits called
,
,
and
(3, 12, 13, 14, 15) .
Each of these subunits has homologies with the degenerins of the
nematode Caenorhabditis
elegans(16, 17, 18, 19) , which
after certain mutations cause neurodegeneration. These degenerins are
thought to be ion channels(17, 18, 22) .
It seems likely that the epithelial amiloride-sensitive
Na channel is the first cloned member of a new family
of ion channels which probably includes mammalian homologues of the C. elegans degenerins which might be involved in certain forms
of neurodegeneration. This paper reports the molecular cloning and
functional expression of a novel human isoform of an
amiloride-sensitive Na
channel expressed in brain,
testis, ovary, and pancreas.
All nucleic acid positions in the text
refer to positions relative to the A in the ATG initiation codon of the
nucleic acid sequence submitted to GenBank (accession
number U38254).
All comparisons of sequences with data bases were done using the Blast network server at the NCBI (National Center for Biotechnology Information).
In order to identify novel homologues of the epithelial
Na channel (NaCh), the sequences of the cloned
subunits (
NaCh,
NaCh,
NaCh) have been compared with the
data base of expressed sequence tags. We found one good matching
partial cDNA sequence (GenBank
accession number T19320) in
this data base. A fragment of this sequence was amplified by PCR from
human kidney cDNA and used to screen a human kidney cDNA library. A
positive clone of 3.4 kb was isolated and sequenced. It contains an
open reading frame of 1914 bases preceded by stop codons in all three
reading frames and codes for a protein of 638 amino acids (Fig. 1a).
Figure 1:
Protein sequence of NaCh and
comparison with
NaCh,
NaCh, and
NaCh. a,
alignment of
NaCh with human
NaCh,
NaCh, and
NaCh.
Residues identical or similar to the corresponding amino acid in the
subunit are printed white on black or black on gray
background, respectively. The putative transmembrane regions for
NaCh are labeled with bars. For MII, the hydrophobic
region is longer than the some 20 amino acids required for an
helix to span the membrane. The sequence which was shown to participate
in the formation of the ionic pore of
NaCh (22) is marked
by a black bar, and flanking hydrophobic regions by gray
bars. The sequence for
NaCh is from GenBank
(accession number X76180), and those for
NaCh and
NaCh
are from EMBL (accession numbers X877159 and X87160). The sequences
were aligned using the GCG Pileup program. b, phylogenetic
tree of the human NaCh subunits. The phylogenetic tree was established
from the alignment shown in a using the Distances program
(GCG) with Kimura substitution followed by the Growtree program (GCG)
with the UPGMA option. c, identity between the cloned human
NaCh subunits and the C. elegans degenerins mec10 and deg1.
The sequences were aligned using the GCG Pileup program, and identities
were calculated with the GCG Distances program without correction for
multiple substitutions. The sequences for the degenerins deg1 and mec10
used are GenBank
accession numbers L34414 and L25312,
respectively.
The homology with the ,
, and
subunit (27-37% identity; Fig. 1c) is
rather low and lies in the same range as observed between
NaCh,
NaCh, and
NaCh (29-36% identity; Fig. 1c). Nevertheless, the homology and phylogenetic
analysis (Fig. 1b) places this new isoform, named
NaCh, closer to the
subunit than to
NaCh and
NaCh.
The
NaCh is, as
NaCh,
NaCh, and
NaCh, about 20%
identical with the degenerins mec10 and deg1 of C. elegans (Fig. 1c).
NaCh has a hydrophobicity
profile similar to
NaCh,
NaCh, and
NaCh and to the
degenerins with two hydrophobic regions (MI and MII, Fig. 1a) long enough to span the plasma membrane.
Together with the sequence homologies, this suggests a transmembrane
topology identical with that proposed for
NaCh (20) with
intracellular amino and carboxyl termini and a large cysteine-rich
extracellular loop between MI and MII.
When expressed alone in Xenopus oocytes, NaCh induced a small (38 ± 5 nA, n = 16) but very reproducible amiloride-sensitive
Na
current (Fig. 2) with macroscopic properties
(pharmacology, selectivity) clearly distinct from those of
NaCh
when expressed in the same conditions(13) .
Figure 2:
Electrophysiology of NaCh. a, effect of amiloride on the whole cell current recorded at
-70 mV on oocytes injected with
NaCh. b,
selectivity of
NaCh. The bars represent the amiloride
(100 µM)-sensitive current. c, dose response
curves for amiloride and benzamil. Each point represents the mean of
the values obtained from 5 oocytes. d, mean i - V relationship of the amiloride (100
µM)-sensitive Na
current with 96 mM Na
or Li
in the external medium.
Points are the mean values from 4 oocytes.
The first
difference concerns the pharmacology. K values (Fig. 2c) for the diuretic amiloride (2.6
µM) and for benzamil (0.27 µM) were about 30
times higher than those for
NaCh (K
= 80 nM; K
= 7 nM). The second difference was the ionic
selectivity. The
NaCh channel was more permeable for Na
than for Li
(I
/I
= 0.6) unlike the human
NaCh or rat
NaCh which
have a higher permeability for Li
than for
Na
(I
/I
2) (Fig. 2b).
NaCh was insensitive to
ethylisopropylamiloride (Fig. 3b), a potent inhibitor
of the Na
/H
exchanger(21) ,
at concentrations below 10 µM.
Figure 3:
Electrophysiology of NaCh. a, amiloride (100 µM)-sensitive Na
currents in oocytes injected with
NaCh alone or together
with human
,
, and/or
NaCh. b, dose-response
curves of
NaCh for amiloride, benzamil, and
ethylisopropylamiloride (EIPA). c, cell-attached
recordings on
NaCh-injected oocytes at different
membrane potentials with 140 mM Na
in the
pipette solution. d, mean i - V relationships with 140 mM Na
or
Li
in the pipette
solution.
Like NaCh,
NaCh
is virtually impermeable for K
. No amiloride-sensitive
current could be detected when Na
was substituted by
K
(Fig. 2b), and the i - V curve shows a positive reversal potential
(+49 ± 7 mV, n = 5) (Fig. 2d).
Since the epithelial Na channel is known to be a multisubunit assembly and since
NaCh alone also induces only small currents when expressed in Xenopus oocytes without
NaCh and
NaCh(3, 12, 13, 14, 15) ,
we examined whether any of the other known human subunits (
,
, or
) increases the
NaCh current (Fig. 3a). Unlike
NaCh for which coexpression of
just the
subunit increases the current by one order of
magnitude(3, 14) , none of the
,
, or
subunits alone altered or increased the
NaCh current when
coexpressed with
NaCh. However, coexpression of both the
and
subunits with the
NaCh increased the Na
current by 50-fold (1.94 ± 0.4 µA, n =
7), an amplification that lies in the same range as that reported after
coexpression of
NaCh with
NaCh and
NaCh (14) .
The macroscopic properties (pharmacology, ionic selectivity) of
NaCh were indistinguishable from those of
NaCh.
Together with the fact that the macroscopic properties of
NaCh are
also not altered by coexpression of
NaCh and
NaCh(3, 14) , this suggests either that
NaCh
and
NaCh are the pore-forming subunits or that low amounts of
endogenous
NaCh- and
NaCh-like subunits are present in the
oocyte and are responsible for the small currents observed after
expression of
NaCh or
NaCh alone.
The single-channel
conductance (Fig. 3) for Na of
NaCh was 11.6 ± 0.4 pS (n = 8).
It was clearly different from that of
NaCh (4.8 ±
0.3 pS, n = 6). The
NaCh conductance for
Li
(6.8 ± 0.5 pS, n = 4) was
nearly identical with that of
NaCh (7.3 ± 0.2 pS, n = 4). The
NaCh channel, like
NaCh, was highly selective for Na
versus K
(pNa
/pK
> 50).
The gating of
NaCh was slow (
= 3.3 ± 1.5 s,
= 1.9
± 0.7 s, n = 5; Fig. 3b), and
the open probability did not show a marked voltage dependence (P
= 0.46 ± 0.05 at -20 mV and
0.49 ± 0.02 at -100 mV, n = 3).
It is
particularly interesting that, despite their low homology (37%
identity), both NaCh and
NaCh can associate with
NaCh
and
NaCh to form a functional channel. Sequence comparisons
between
NaCh and
NaCh (Fig. 1) reveal some motifs that
are well conserved and which are not found in
NaCh and
NaCh.
Those ``common''
and
sequences, and particularly
the sequence just before MI (Fig. 1a), are probably
important elements for the functional association of
NaCh or
NaCh with
NaCh and
NaCh.
The tissue distribution of
NaCh mRNA was analyzed by Northern blot (Fig. 4). The
highest expression levels of the 5.5-kDa mRNA were found in testis,
ovary, pancreas, and brain. Those are tissues in which to our knowledge
no amiloride-sensitive Na
channels have been described
yet. Small amounts of
NaCh-mRNA can be detected in all other
tissues examined except in spleen and small intestine. The dominant
tissue distribution of
NaCh is clearly nonepithelial, and, in
kidney (the tissue we cloned
NaCh from), there are only small
amounts of
NaCh-mRNA present. Therefore, it does not seem likely
that the principal role of this new channel is to be searched in
epithelia.
Figure 4:
Tissue distribution. The tissue
distribution of NaCh-mRNA was analyzed by Northern blot as
described under ``Materials and
Methods.''
The pharmacological and biophysical properties of
NaCh do not really match those of any of the amiloride-sensitive
Na
channels described so far. Like
NaCh, the
major subunit of the epithelial Na
channel,
NaCh,
can associate with
NaCh and
NaCh to form a multisubunit ion
channel. Whether this
subunit combination is the one
actually present in vivo or whether yet unknown subunits form
a channel together with
NaCh requires further investigation.
The presence of NaCh in brain is particularly interesting,
because the C. elegans degenerins (16, 17, 18, 19) which are
homologues to NaCh are expressed in neurons. A more detailed
localization of
NaCh especially in brain might clarify the
physiological role of this new amiloride-sensitive Na
channel.