From the
The highly selective, amiloride-sensitive Na
Sodium transport through the epithelial Na
Expression cloning of the epithelial sodium
channel demonstrated that it was made of at least three homologous
subunits called
The NaCh subunits
and the degenerins have two hydrophobic regions long enough to span the
membrane, and both might participate in the formation of the ionic
pore
(8, 13, 14) . To evaluate their specific
roles, chimeras between the
Goldman, Engelman, and Steitz scores for hydrophobic
transbilayer helices were plotted using the GCG sequence analysis
software, and the putative transmembrane regions were selected.
Mutants were prepared by site-directed mutagenesis using synthetic
oligonucleotides
(15) and verified by dideoxy sequencing.
cRNAs were prepared and oocytes were injected with 100-500 pg
of either NaCh or mutant
Cell-attached recordings were performed on oocytes clamped to zero
mV in high K
The first (MI) or the second (MII) hydrophobic region of
The amino acids that differ between chimera A and
Replacement of Ser-589 by Ile (S589I) caused changes in channel
properties nearly identical to those observed with the MII chimera. The
S589I mutant had a decreased affinity for amiloride
(Fig. 2c, ), a higher conductance for
Na
Substitution of
Ser-589 by Phe, the corresponding residue of the degenerin
Deg-1
(11) , caused the same changes in channel properties as did
S589I, except for a 3-fold lower K
The
mutation S589I had more drastic effects than S593T, but replacement of
a Ser by an Ile causes more structural changes than replacement by a
Thr. Therefore, we also replaced Ser-593 in
Our data suggest that Ser-589 and Ser-593 face
the same side of an
Mutation of
Ala-442 in Mec-4 to Thr (Fig. 1a) causes
neurodegeneration in C. elegans, and it has been speculated
that this could be due to increased ion flow through the putative
channel
(10) . When introduced into the MII chimera, this
mutation (MII chimera A577T; not shown) did not change the properties
of the channel. This finding, together with the fact that degeneration
can occur long after differentiation of neurons
(10) , suggests
that this form of neurodegeneration is more likely caused by impaired
regulation rather than by an alteration of the basic biophysical
properties of the channel.
Other tissues
(1, 2, 17) (i.e. endothelial cells
(18) and smooth
muscle
(19) ) contain a family of amiloride-sensitive channels
with different Na
The table shows mean values ± S.E. n, number
of experiments. Dose-response curves used for K
We are very grateful to Drs. E. Lingueglia, N.
Voilley, and P. Barbry. We thank C. Roulinat and F. Aguila for
technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
channel is formed of three homologous subunits termed
,
, and
. The three subunits exhibit similarities with
Caenorhabditis elegans proteins called degenerins involved in
sensory touch transduction and, when mutated, in neurodegeneration.
Swelling of neurons observed in neurodegeneration suggests an
involvement of ion transport, but the channel function of degenerins
has not yet been demonstrated. We used chimeras to study the functional
relationship between the epithelial sodium channel and the degenerin
Mec-4. Exchange of the hydrophobic domains of the Na
channel
subunit by those of Mec-4 results in a functional
ion channel with changed pharmacology for amiloride and benzamil and
changed selectivity, conductance, gating, and voltage dependence. All
of these differences were also obtained by exchanging Ser-589 and
Ser-593 in the second transmembrane region by the corresponding
residues of Mec-4, suggesting that these two residues are essential for
the ionic pore function of the channel.
channel (NaCh)
(
)
is the limiting step for
Na
reabsorption in various tight
epithelia
(1, 2) . NaCh is also involved in taste
perception
(3) .
,
, and
(4, 5, 6, 7, 8) . While
these three subunits show no significant sequence homology with other
previously cloned channels, significant similarities have been found
with members of the Caenorhabditis elegans degenerin family
(such as Mec-4, Mec-10, and Deg-1) (9-12), a set of proteins
associated with the mechanosensory apparatus that can be mutated to
cause neuronal degeneration. Degenerins may function as channel
proteins, but this has not been demonstrated yet.
subunit of the Na
channel (
NaCh) and Mec-4 were constructed and studied after
co-expression with the
and
subunits in oocytes.
NaCh
and Mec-4 were selected because
NaCh plays a key role in the
formation of NaCh
(4, 5, 6, 7) and Mec-4
in mechanotransduction and neurodegeneration (10).
NaCh cRNA together with the same amount
of cRNA for
and
NaCh as described
(16) .
medium. Pipettes contained (in
mM): NaCl (or LiCl) 140, MgCl
1, CaCl
1, Hepes 10, pH 7.4. Data were sampled at 1 kHz and filtered at
300 Hz for analysis. Single-channel conductances for Na
and Li
were calculated from I-V
relationships from 0 mV to -100 mV with Na
or
Li
as conducting ion. Open probabilities and mean
dwell times were calculated with Biopatch software (Biologic). Values
are means ± S.E.
NaCh were replaced by corresponding sequences of Mec-4
(Fig. 1a). Exchange of either hydrophobic domain caused
significant changes in conductance, gating, and pharmacology. Affinity
for the diuretic amiloride (Fig. 1c) and for benzamil
(), a specific NaCh blocker, decreased by a factor of 3
after exchange of MI and by a factor of 14 after replacement of MII.
Figure 1:
NaCh/Mec-4 chimeras.
a, alignment of the putative transmembrane domains MI and MII
of
NaCh and Mec-4. Boxes indicate sequences of
NaCh
that were replaced by the corresponding Mec-4 residues. Ala-442 in MII
of Mec-4, which causes neurodegeneration after mutation, is
underlined. The symbols used in b-d are:
, NaCh;
, MI chimera;
, MII chimera;
, MIMII
chimera. b, cell-attached recordings at various membrane
potentials with Na
in the pipette. c,
inhibition of the macroscopic current at -70 mV by amiloride.
Each point represents the mean value obtained from 5-7 oocytes.
d, voltage dependence of open probability. Points represent
mean values from 3 (NaCh), 5 (MI chimera), 4 (MII chimera), or 2
recordings (MIMII chimera). e, mean single-channel conductance
for Li
and Na
. ★, significantly
different from NaCh, with p < 0.001 using an unpaired t test. The sequences shown for MI of
NaCh correspond to amino
acids 111-132 (5). MI of Mec-4 is not in the data bases and was
obtained by translating the Mec-4 genomic sequence (10) (bases
1623-1688). For MII, the actual hydrophobic region is longer than
the approximately 20 amino acids required to span the membrane.
Sequences shown are amino acids 567-602 for
NaCh (5) and
433-476 for Mec-4 (10).
Replacement of MI caused only a slight although significant increase
in single-channel conductance for Na and Li
(Fig. 1e). Exchange of MII had more drastic
effects on the ionic pore; the conductivity for Na
nearly tripled, while that for Li
increased only
slightly, resulting in reversed selectivity for Na
over Li
(P
Figure 2:
Mutations in MII of NaCh. a,
partial alignment of MII of
NaCh and Mec-4. Arrows toward
the Mec-4 sequence indicate replacement of a residue in
NaCh by
the corresponding amino acid of Mec-4. Upwardarrows indicate other point mutants of
NaCh, and bars mark
the sequences of
NaCh replaced by corresponding parts of Mec-4.
Mutants labeled with
had properties indistinguishable from
NaCh. The symbols used for b-e are:
,
S589I;
, S593T;
, S593I. b, cell-attached
recordings at various membrane potentials. c, inhibition of
the macroscopic current at -70 mV by amiloride. Each point
represents the mean value obtained from 5-7 oocytes. Dashedlines and icons indicate the log
of
the inhibition constant (pK) values for NaCh and the MII
chimera (Fig. 1b), respectively. d, voltage
dependence of open probability. Points represent mean values from 9
(S589I), 4 (S593T), or 5 (S593I) recordings. e, single-channel
conductance for Li
and Na
. For ease
of comparison, the values for the NaCh and the MII chimera (Fig.
1e) are shown again. f, mean single-channel
current-voltage relationships for S593I with different Ca
concentration in the Na
containing pipette
solution (
,
,
) and for NaCh
(
).
We tested two chimeras
within this part of MII (Fig. 2a, ). Chimera
A (Ser-589 to Val-595) had properties identical to those of the MII
chimera, while chimera B (Met-597 to Phe-602), despite important
sequence changes, had characteristics identical to those of NaCh
().
NaCh were replaced one by one in
NaCh by their Mec-4
counterparts (Fig. 2a, ). Only two of these
mutants (S589I and S593T) had properties different from those of NaCh.
than for Li
(Fig. 2e, ), a low open probability,
and a fast voltage-dependent gating (Fig. 2, b and
d, ). The only minor but significant difference
was a lower single-channel conductance for both Na
(10
pS) and Li
(6.4 pS) than the MII chimera
(Na
: 12.7 pS; Li
: 8.3 pS). Mutation
of Ser-593 to Thr (S593T) significantly increased the conductance for
Na
by 2.5 pS and that for Li
by 1.3
pS compared to NaCh, leaving the other properties unchanged
(Fig. 2, ). Together, these two mutations (S589I and
S593T) account for all of the differences in channel properties
observed between
NaCh and the MII chimera.
for
amiloride (8.9 µM) than S589I ().
NaCh by Ile (S593I).
The differences with NaCh increased and included a fast
voltage-dependent gating (Fig. 2, b and d,
) and a voltage-dependent block by external Ca
(Fig. 2f), suggesting localization of Ser-593
within the ion-conducting pathway, but the affinity for amiloride
remained unchanged (Fig. 2c, ).
Voltage-dependent block by external Ca
was never
observed for NaCh (Fig. 2f) and was a typical behavior
of the S593I mutant.
-helix and line the ionic pore (Fig. 3).
Ser-589 is probably located close to the outer mouth of the pore and is
important for the fixation of amiloride, which blocks from the
outside
(1, 2) . Ser-593 is located on the inner mouth of
the pore and does not participate in the binding of diuretics.
Figure 3:
Topology of MII. The sequence of the
amphiphilic part of NaCh MII is shown in uppercaseletters and the corresponding residues of Mec-4 as
footnotes within the helix. Residues that differ between
NaCh and
Mec-4 and cause significant changes in channel properties after
mutation are numbered. Arrows indicate the
characteristics of the channel that changed after
mutation.
Both
MI and MII play a key role in the formation of the ionic pore of NaCh
and, very likely, that of the degenerins. Our results indicate that
Mec-4 is a subunit of a possibly Na-permeable ion
channel sharing a common pore topology with NaCh but having very
different pharmacological and biophysical properties.
versus K
selectivity, conductance, gating, and pharmacological properties,
but none of these channels has been cloned yet. Since small changes in
the sequence of
NaCh suffice to completely alter channel
properties, it seems possible, if not likely, that some of this
diversity is caused by alternate splicing or editing of the NaCh mRNAs,
as is the case for another multi-subunit ion channel, the ionotropic
glutamate receptor (20, 21).
Table:
Channel characteristics of NaCh and different
mutants
determination were constructed from 5-7 different
experiments. P
was measured at -60 mV.
channel;
NaCh, epithelial Na
channel
subunit; MI, first transmembrane region of the
Na
channel or Mec-4; MII, second transmembrane region
of the Na
channel or Mec-4;
P
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.