Expression of TWIK-1, a novel weakly inward rectifying
potassium channel in rat kidney
F.
Cluzeaud1,
R.
Reyes3,
B.
Escoubet2,
M.
Fay1,
M.
Lazdunski3,
J. P.
Bonvalet1,
F.
Lesage3, and
N.
Farman1
1 Unité 478 and
2 Unité 426, Institut
National de la Santé et de la Recherche Médicale,
Faculté de Médecine X. Bichat, F-75870 Paris cedex 18;
and 3 Institut de Pharmacologie
Moléculaire et Cellulaire, Centre National de la Recherche
Scientifique, 06560 Valbonne, France
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ABSTRACT |
Several
K+ conductances have been
identified in the kidney, with specific properties and localization in
distinct cell types and membrane domains. On the other hand, several
K+ channels have been
characterized at the molecular level. By immunolocalization, we show
that a new inward rectifying K+
channel, TWIK-1, is specifically expressed in distinct tubular segments
and cell types of the rat kidney. In the proximal tubule, TWIK-1
prevails in the initial portions (convoluted part), where it is
restricted to the apical (brush-border) membrane. In the collecting
duct, immunofluorescence was intracellular or confined to the apical
membrane and restricted to intercalated cells, i.e., in cells lacking
aquaporin-2, as shown by double immunofluorescence. TWIK was also
expressed in medullary and cortical parts of the thick limb of the loop
of Henle, identified with an anti-Tamm-Horsfall protein antibody
(double immunofluorescence). The intensity of TWIK-1 immunolabeling was
unchanged in rats fed a low-Na+ or
a low-K+ diet. Because TWIK-1
shares common properties with the low-conductance apical
K+ channel of the collecting duct,
we propose that it could play a role in
K+ secretion, complementary to
ROMK, another recently characterized K+ channel located in principal
cells of the cortical collecting duct and in the loop of Henle.
potassium secretion; collecting duct; loop of Henle; proximal
tubule; aquaporin-2; Tamm-Horsfall protein; immunolocalization
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INTRODUCTION |
CONTROL OF K+
permeation through the cell membrane is a ubiquitous phenomenon (22).
Multiple K+ channels are expressed
in cells, with distinct biophysical, physiological, and pharmacological
properties (2, 11, 22). These channels play a critical role in
excitable cells by determining action potential firing or muscle
contraction. In epithelial cells, selective expression in distinct
membrane domains and regulation of
K+ channels lead to control of
resting membrane potential, cell volume, and transepithelial
K+ transport, as well as
K+ recycling (8). Recently,
several renal K+ channel subunits
have been characterized at the molecular level (3, 4, 12,
26).
Two main families of pore-forming
K+ channel subunits sharing common
structural motifs have been identified (5, 13, 21). Voltage-gated
K+ channels have six transmembrane
segments (TMS) and a so-called P domain, which forms part of
the conduction pore. Among them, Shaker-like channels have been widely
studied. Inward rectifying K+
channels have only two TMS separated by a P domain. The ROMK channels
belong to this category (1, 10, 12). Importantly, these channels are
likely to form multimers to yield a functional K+ pore (2). Recently, a new
structural family of K+ channels
has been discovered in mammals. These channel subunits have four TMS
and two P domains (6, 16, 17). TWIK-1 (16) is the first identified
member in this family. It exhibits weak inward rectifying properties
when expressed in Xenopus oocytes. Its
unitary conductance is 30-40 pS, and it can be blocked by quinidine. Ba2+ can also block its
activity, while it is relatively insensitive to tetraethylammonium ion.
Interestingly, TWIK-1 (16) is downregulated by internal acidification
and upregulated by protein kinase C (PKC) (16, 17). TWIK-1 mRNA has
been shown to be expressed mainly in brain, although other tissues were
positive, such as lung, skeletal muscle, and kidney (16, 17). TWIK-1
proteins self-associate to form functional covalent dimers (17, 18).
Several K+ conductances have been
reported in the different tubular epithelia lining the renal nephron,
with distinct biophysical and pharmacological properties (8, 30).
Attempts to correlate these conductances to the growing number of
cloned K+ channels require
comparison of their functional properties and their precise cellular
specificity of expression (cell type and apical vs. basolateral membrane).
The aim of this study was to determine precisely the cellular
expression of TWIK-1 within kidney tubular cells and its membrane domain of expression compared with other
K+ channels that are known to be
expressed in kidney cells such as ROMKs.
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MATERIALS AND METHODS |
Animals.
Adult male Sprague-Dawley rats were fed a normal diet (control rats), a
low-K+ diet (UAR, Epinay sur Orge,
France) containing 120 mg K+/kg
for 10 days, or a low-Na+ diet
(UAR) containing 90 mg Na+/kg for
8 days.
Antibodies.
Affinity-purified rabbit polyclonal antibodies directed against the
COOH-terminal region of TWIK-1 (amino acids 264-336) fused to
glutathione S-transferase (GST) (17,
18) were used. Anti-TWIK-1 antibodies were raised against a GST fusion
protein containing the COOH terminus of TWIK-1 (amino acids
264-336). Female New Zealand White rabbits were immunized with 300 µg of purified fusion protein in the presence of complete Freund's
adjuvant and boosted 1 mo later with 150 µg of the immunogen in the
presence of incomplete Freund's adjuvant. Rabbits were bled 15 days
after the boost. The antibodies were affinity purified by using His-Tag
fusion proteins containing the same domains of TWIK-1 as the GST fusion proteins used for the immunization. Briefly, the crude antisera were
incubated for 4 h at 4°C with 100-200 µg of purified His-Tag fusion proteins previously transferred to Hybond C-extra nitrocellulose membranes (Amersham). After three washes in PBS [10 mM phosphate buffer (pH 7.2) and 0.15 M NaCl] and 0.1% Tween 20, the
anti-TWIK-1 antibodies were recovered by a 1-min elution of each strip
with 0.1 M glycine and 0.5% BSA (pH 2.8). After the elution the
purified antibodies were rapidly brought to pH 7.6 with 1 M Tris (pH
8.0) and 0.5% BSA.
Western blotting was performed on crude rat kidney homogenates
extracted in 50 mM Tris · HCl (pH 8), 150 mM NaCl,
0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, and 100 µg/ml
phenylmethylsulfonyl fluoride. The affinity-purified
antibody (1:600) revealed a protein with a molecular ratio
(Mr) of 87,000 (Fig. 1), in agreement with previous
reports on brain expression of TWIK-1 (17; present study); there was no
signal in the presence of the immunizing protein (1 µg/ml).

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Fig. 1.
Western blots of TWIK-1 and aquaporin-2 (AQP-2) in kidney homogenates.
A: crude rat kidney homogenates were
blotted in presence of affinity-purified anti-TWIK-1 antibody, in
absence ( ) or presence (+) of immunizing fusion protein, and
with an anti-actin antibody as an internal control.
B: signal observed with unpurified
anti-AQP-2 antibody, alone ( ) or in presence of immunizing
peptide (+); actin was used as an internal control.
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Rabbit polyclonal anti-aquaporin-2 (AQP-2) antibodies (against a
peptide corresponding to amino acids 250-271 of AQP-2, conjugated to keyhole limpet hemocyanin) were generated (Neosystem, Strasbourg, France), as described elsewhere (19). Western blot with this nonpurified antiserum (1:1,000) revealed a protein with an
Mr of ~29,000
in rat kidney (Fig. 1), corresponding to the nonglycosylated form of
AQP-2; the signal was largely reduced by preadsorption of the antibody
with the immunizing peptide (250 µg/ml).
Sheep polyclonal anti-Tamm-Horsfall protein (THP) antibody (sheep
antiuromucoid) was purchased from Biodesign (Kennebunk, ME) and used at
1:100 dilution.
Mouse monoclonal anti-
-actin (AC74 clone, Sigma Chemical) was used
at 1:5,000 dilution.
Detection of TWIK-1 in COS cells.
The TWIK-1 sequence was excised from the pEXO-TWIK-1 plasmid (16) and
subcloned into the pIRES-cd8 vector to obtain pIREScd8-TWIK-1. The
pIRES-cd8 vector was obtained by replacing the neo gene in pIRESneo
(Clontech) by the CD8 gene. COS cells were seeded at a density of 3 × 104 cells/35-mm dish 24 h
before transfection. Cells were transiently transfected by the
classical DEAE-dextran method with 1 µg of pIREScd8-TWIK-1 plasmid
per dish. After 1 day, cells were dissociated and plated on
polylysine-coated coverslips in a 24-well cluster. Transfected cells
were visualized 48 h after transfection by application of anti-CD8
antibody-coated beads (Dynabeads, Dynal). TWIK-1 immunodetection (Fig.
2) was then performed as previously described (18), except cells were
permeabilized by addition of 0.1% Triton X-100 in the blocking
solution (PBS supplemented with 2% BSA and 5% normal goat serum). For
control, immunodetection was performed on cells transfected in the same
manner but by using affinity-purified anti-TWIK-1 antibodies
preincubated over 30 min with the GST-TWIK-1 fusion protein that has
been used for rabbit immunization (25 µg GST-TWIK-1/ml detection
solution). For Western blot, proteins from COS cells and synaptic
membranes from adult rat brain were prepared and analyzed in the
absence of reducing agents, as previously described (17, 18).
Immunolocalization of TWIK or AQP-2 on kidney sections.
Kidneys from male Sprague-Dawley rats were obtained after in vivo
perfusion (at 80 mmHg) of the aorta with 2% paraformaldehyde, then
4-6 h of immersion in the same fixative at 4°C. The tissue was
then cryoprotected by immersion in 30% sucrose in phosphate buffer
(120 mM
Na2HPO4-NaH2PO4,
pH 7.2) overnight at 4°C, frozen in liquid nitrogen, and stored at
80°C. Cryostat sections (10 µm) were deposited on
Superfrost slides, dried in air, and immersed in phosphate buffer.
Immunolocalization was performed by incubating sections with the first
antibody for 4 h at room temperature (or overnight at 4°C). The
following antisera were used: 1)
affinity-purified anti-TWIK-1 (diluted 1:100), alone or in the presence
of the fusion protein (15 µg/ml) used to generate the antibody, for
competition studies, 2) nonpurified
anti-AQP-2 antiserum (diluted 1:100), alone or in the presence of the
immunizing peptide (250 µg/ml), and
3) the respective preimmune sera of
each rabbit, used as control. After several washes with phosphate
buffer, the secondary antibody [goat anti-rabbit coupled to the
fluorochrome CY3 (Jackson), diluted 1:100] was incubated for 1 h
at room temperature. After washes with phosphate buffer, slides were
mounted using Vectashield (Vector) and examined under a confocal
microscope (model TCS 4D, Leica). This protocol was used for
Figs. 3, 4, and 6.
In some experiments, immunodetection of TWIK and AQP-2 was performed on
serial sections (see Fig. 6, E and
F) or by double immunofluorescence
(see Fig. 6D) as follows: sections
were incubated with TWIK antibody, then with the Fab fraction of goat
anti-rabbit antibody coupled to CY3; subsequently, the sections were
treated with AQP-2 antibody, biotinylated goat anti-rabbit antibody
(Fab fraction), and streptavidin-FITC detection system.
To achieve immunolocalization of TWIK and THP on serial sections by
double immunofluorescence (see Fig. 5), it was necessary to use unfixed
frozen kidneys (liquid nitrogen); the cryostat sections were then fixed
in methanol. Anti-TWIK and anti-THP antibodies were used at 1:100
dilution, then the secondary antibodies, i.e., goat anti-rabbit coupled
to CY3 for TWIK-1 and donkey anti-goat FITC for THP, were applied.
 |
RESULTS |
Specificity of TWIK-1 antibody.
Antibody specificity was tested on transiently transfected COS cells.
Cells were transfected with a polycistronic vector to coexpress TWIK-1
and the CD8 protein from the same transcript. Expressing cells were
visualized by application of anti-CD8 antibody-coated beads (Fig.
2, A and
B). As expected, a positive signal
was obtained by incubating TWIK-1-expressing cells with anti-TWIK-1
antibodies. TWIK-1 channels were not detected from mock-transfected
cells (not shown) or from TWIK-1-expressing cells when anti-TWIK-1
antibodies were preincubated with the immunizing fusion protein before
the detection step (Fig. 2B). Figure
2C shows that TWIK-1 channels were
specifically detected from TWIK-1-expressing COS cells
(lane 2) and from rat brain synaptic
membranes (lane 3). The
Mr of the rat
brain TWIK-1 (~80,000) is identical to the previously reported Mr of the mouse
brain TWIK-1 (17). It probably corresponds to a disulfide-bridged
dimeric form of the protein, as shown for the human channel (18). The
Mr of TWIK-1 is
lower (70,000-75,000) in transiently transfected COS cells than in
brain (Fig. 2C, lane 2). This could
be due to differences in posttranslational modifications of the
protein. A similar lower
Mr has been
observed in another transient expression system (18).

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Fig. 2.
Immunodetection of TWIK-1 in transfected COS cells and in rat brain.
A and
B: TWIK-1 was coexpressed in
transfected COS cells together with CD8 protein. Transfected COS cells
were visualized with anti-CD8-coated beads (arrowheads). Then cells
were fixed, permeabilized, and incubated successively with
affinity-purified anti-TWIK-1 antibodies in absence
(A) or presence
(B) of immunogenic glutathione
S-transferase-TWIK-1 fusion protein
and with FITC-conjugated goat anti-rabbit IgG. Immunocomplexes (stained
green) were visualized by fluorescence microscopy at ×400
magnification. C: Western blot
analysis of TWIK-1 protein in mock-transfected COS cells
(lane 1), TWIK-1-expressing COS
cells (lane 2), and synaptic
membranes of adult rat brain (lane
3).
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Western blot on whole kidney homogenate revealed a band with an
Mr of 87,000, which was abolished when the antibody was preincubated with immunizing
fusion protein (Fig. 1).
Immunolocalization of TWIK-1.
Immunofluorescence was apparent in several cell types of the nephron,
as illustrated in Fig. 3, which shows the
results obtained in the superficial cortex, the deep cortex, the
medulla, and the papilla of paraformaldehyde-fixed kidneys. In the
cortex (Fig. 3A) the apical membrane
of the proximal convoluted tubule showed a strong signal that was
clearly limited to the brush-border membrane (Fig.
3E). The expression of TWIK-1
decreased along the length of the proximal tubule and appeared much
higher in its initial portion (proximal convoluted tubule) than in its
terminal portion, i.e., the pars recta, located in the deep cortex, as
shown in Fig. 3B (cf. Fig.
3A). However, the brush-border
membranes were clearly positive in these paraformaldehyde-fixed
kidneys, whereas the signal was more diffuse after methanol fixation
(see Fig. 5). The distal tubules and early collecting ducts (Fig.
3A) were also positive, with
cytoplasmic as well as apical staining, whereas glomeruli were
negative. In the collecting duct, some cells were positive (Fig.
3F), with a cellular pattern of
expression that varies from cell to cell: in some cases, TWIK-1 appears
intracellular; in other cases, it is clearly restricted to the apical
membrane (Fig. 3G). The reason for
this variable expression in the collecting duct is unclear, and the
cytoplasmic signal may represent a storage compartment of TWIK-1. To
see whether this pattern of expression could vary with the
K+ status of the animal,
immunofluorescence studies were performed in rats fed a
low-K+ diet (to enhance renal
K+ reabsorption) and in rats fed a
low-Na+ diet, a condition that is
known to enhance plasma aldosterone concentration and thus
K+ secretion in the collecting
duct. Neither of these manipulations altered the apparent level of
expression or the cellular pattern of expression of TWIK-1 in the
collecting duct (not shown). In the outer medulla (Fig.
3C), TWIK-1 was found in the
medullary collecting duct and in the thick ascending limbs of the loop
of Henle. Identification of cortical thick ascending limbs was
difficult on these sections. TWIK-1 immunofluorescence was also present in the papillary collecting duct (Fig.
3D), where the signal was diffuse
throughout the cell; the loops of Henle (thin limbs) were negative.

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Fig. 3.
Immunofluorescence of TWIK-1 in kidney sections. Confocal microscopy
was used to show expression of TWIK-1 at protein level in renal
superficial cortex (A), deep cortex
(B), medulla
(C), and papilla
(D). Immunofluorescence was
restricted to brush-border (apical) membrane of initial parts of
proximal tubules (pt in A, and
E) and to cells of distal tubule and
early collecting duct (dct), whereas glomerulus (g) was negative.
Collecting duct cells (cd) remain positive along its entire length,
including its papillary portion (D).
Signal observed in proximal tubule was strongly reduced in its terminal
part, i.e., pars recta (pr in B). In
cortical collecting duct, immunofluorescence was intracellular or
apical (F and
G) in a minority of cells. No clear
signal was visible over cells of thick ascending limb of loop of Henle
(hl in C) or thin limbs of loop of
Henle (hl in D). Magnification
×170 in A-D, ×240 in
E and
F, and ×360 in
G.
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The specificity of the observed immunofluorescence with TWIK-1 antibody
has been assessed by using the preimmune serum (Fig. 4). In each kidney zone (outer cortex,
inner cortex, outer medulla, and papilla), some nonspecific staining
was apparent in some cells (essentially in the glomerulus and papillary
collecting duct). When TWIK antibody was incubated in the presence of
the immunizing fusion protein, the resulting immunostaining was clearly
reduced, as illustrated in Fig. 4,
E-G, in outer cortex, inner
cortex, and papilla.

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Fig. 4.
Control immunofluorescence. Kidney sections from outer cortex
(A), inner cortex
(B), medulla
(C), and papilla
(D) incubated with preimmune serum
show nonspecific signal in some cells, mainly in cortex (glomerulus)
and papillary collecting duct cells. Signal is clearly reduced compared
with that observed with TWIK-1 antibody (Fig. 3). TWIK-1
immunofluorescence is displaced in presence of immunizing fusion
protein in outer cortex (E), inner
cortex (F), and papilla
(G). Magnification ×100.
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TWIK-1 and THP immunostainings are illustrated in Fig.
5 in methanol-fixed cryostat sections of
kidney cortex and medulla. The loop of Henle was identified by its
immunostaining with an antibody against THP (Fig. 5,
A and
D). Clear labeling was observed in
the loop of Henle (in its cortical as well as its medullary portion)
with the TWIK-1 antibody: double immunofluorescence shows that TWIK-1
and THP clearly colocalize in this nephron segment (Fig. 5,
C and
F). The medullary collecting ducts
(which are TWIK-1 positive and THP negative) and the medullary thick
ascending limbs of the loop of Henle are closely apposed and very
difficult to discriminate morphologically by using methanol-fixed
sections, underlining the interest of double immunofluorescence.

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Fig. 5.
Immunolocalization of TWIK-1 and Tamm-Horsfall protein (THP) in kidney.
Methanol-fixed cryostat cortex
(A-C) and medulla
(D-F) sections were incubated
with anti-TWIK-1 (B and
E, stained red) or anti-THP
(A and
D, stained green) antibody (to
identify thick ascending limbs of loop of Henle).
C and
F: double immunofluorescence showing
colocalization of TWIK-1 and THP (stained yellow) in cortical and
medullary parts, respectively, of thick ascending limb of loop of
Henle. Magnification ×160.
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To gain some insight into the cell type(s) that expresses TWIK in the
cortical collecting duct, we have compared its expression with that of
the water channel AQP-2, a marker of principal cells (Fig.
6). The antipeptide antibody against AQP-2
labels the collecting duct (Fig.
6A); no labeling was observed in the
presence of immunizing peptide (Fig.
6B) or with the preimmune serum
(Fig. 6C). Double immunofluorescence
with both antibodies (Fig. 6D) shows
the apical staining of collecting duct principal cells for AQP-2,
whereas TWIK immunofluorescence is clearly on a distinct cell
population, i.e., intercalated cells. Such a distinct pattern of
expression is also visible on serial sections incubated with AQP-2
antibody (Fig. 6E) and TWIK antibody
(Fig. 6F). These results indicate that TWIK is expressed in intercalated (not principal) cells of the
cortical collecting duct.

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Fig. 6.
Immunolocalization of TWIK-1 and AQP-2 in kidney cortex.
Paraformaldehyde-fixed kidney sections were incubated with anti-AQP-2
antibody alone (A) or in presence of
immunizing peptide (B); preimmune
serum gave low background signal
(C).
D: coimmunolocalization of TWIK-1
(stained red) and AQP-2 (stained green) showing distinct cellular
expression. Such distinct cellular expression is also illustrated by
incubating serial sections with anti-AQP-2 antibody
(E), a marker of principal cells of
cortical collecting duct, or anti-TWIK antibody
(F). Magnification ×100 for
A-C and ×250 for
D-F. Arrows, intercalated cells.
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DISCUSSION |
Several K+ conductances have been
reported in kidney cells over the last few years (for review see Refs.
8, 9, and 30). Detailed information is now available on their
biophysical and pharmacological properties in distinct cell types. They
are involved in the establishment of a
K+ concentration gradient (which
can be used to generate membrane potential of tubular cells and
Na+-coupled transport), in
regulation of cell volume, in recycling of
K+ across apical and basolateral
membranes, and in K+ secretion in
the collecting duct (8, 9, 30). Low-conductance (30-pS)
K+ channels have been identified
in the apical membrane of collecting duct principal cells (7, 8, 28,
31); these channels share some properties with TWIK-1. The
low-conductance apical K+ channel
of the cortical collecting duct and TWIK-1 are weakly inward
rectifying, with a low conductance (30-35 pS) and a low sensitivity to tetraethylammonium ion, and are downregulated by internal acidification (7, 28, 30, 31). Their sensitivity to PKC
activation appears to differ, since PKC upregulates TWIK-1 activity in
oocytes (16, 17) but reduces apical
K+ channel activity in the
collecting duct (8, 29); protein kinase A activates apical
K+ conductance (29), whereas
TWIK-1 seems to be insensitive to cAMP (16). Whether these differences
are real or depend on the cellular context of expression (oocytes
injected with TWIK-1 mRNA vs. native cells of the collecting duct)
remains unknown. Finally, apparent divergences may be due to more
complex phenomena; the notion of possible heteromultimerization of
K+ channels (compared with
homomultimerization) has been proposed and may suggest a new diversity
of function among cloned K+
channels (2).
To gain some insight into the involvement of TWIK-1 in
K+ handling along the nephron, its
expression at the protein level in the rat kidney was characterized.
Results show that TWIK-1 renal expression is in the proximal tubule,
the thick ascending limb of the loop of Henle, and collecting duct
intercalated cells. This pattern of expression along the nephron is
close to that observed with another class of low-conductance
K+ channels, the ATP-sensitive
ROMKs, which belong to the inward rectifying
K+ channel family, characterized
by two membrane-spanning segments (10, 12). ROMK mRNAs have been shown
(10, 15) to be expressed essentially in the distal half of the nephron,
with distinct expression according to the isoform. In particular,
ROMK-2 and ROMK-3 mRNAs were found in the ascending limb of the loop of
Henle and in the distal tubule and collecting duct, whereas ROMK-1 mRNA
was absent in the loop of Henle and present in the distal nephron (1). In the cortical collecting duct, ROMKs are restricted to the apical membrane domain of principal cells (32), at variance with TWIK-1, which
is in intercalated cells. Thus it appears that these two K+ channels have complementary
patterns of expression in the cortical collecting duct.
Expression of apical K+
conductances along the nephron is interesting to discuss in view of
genetic disorders such as those observed in Bartter's syndrome. This
syndrome was initially attributed to a defect in the Na-K-2Cl
cotransporter (NKCC2) in the loop of Henle (23), leading to impaired
NaCl reabsorption in this epithelium, responsible for
Na+ wasting, secondary
hyperaldosteronism and hypokalemic alkalosis. More recently (14, 24),
mutations of ROMK have been identified in some cases of Bartter's
syndrome. The loss of ROMK function results in the inability to recycle
K+ from the cells of the ascending
limb of the loop of Henle, leading to severe impairment of the activity
of the Na-K-2Cl cotransporter (all mutations identified are in the core
peptide shared by all known ROMK isoforms; consequently, activity of
all isoforms is expected to be affected by these mutations). Because
ROMK isoforms are also expressed more distally along the nephron, i.e.,
in the distal tubule and collecting duct (which are major sites for net renal K+ secretion), it was
expected that ROMK mutations would also impair distal
K+ secretion and prevent the
hypokalemia secondary to hyperaldosteronism. Of interest, hypokalemia,
although less severe than in NKCC2 mutations, was also present, despite
expected ROMK-dependent impairment in distal
K+ secretion (24). This suggests
that the ROMK K+ conductance plays
a major role in the loop of Henle, not in the collecting tubule. We
propose that TWIK-1 activity may compensate for the loss of function of
ROMK in the collecting duct. Such functional compensation may not exist
(or is not sufficient) in the loop of Henle, despite the presence of TWIK.
Two other nephron segments express TWIK-1, but not ROMKs: the proximal
tubule and the inner medullary collecting duct. In the brush-border
membrane of the proximal tubule, TWIK-1 may participate to maintain the
negative potential of tubule cells or to regulate cellular volume (8),
together with another K+ channel
subunit, minK (also expressed in the brush-border membrane of the
proximal tubule) (25). In the inner medullary (papillary) collecting
duct, a basolateral Shaker-like
K+ channel has been reported (27);
together with TWIK-1, it may play a role in the final adjustments of
K+ secretion in the urine.
In a recent report (20) a cDNA named KCNK1 has been cloned from human
kidney, with complete identity to TWIK-1. A partial rabbit clone was
also amplified, and rabbit-specific primers were used to probe the
expression of KCNK1 along the nephron. Positive signals were obtained
in the cortical part of the thick ascending limb of the loop of Henle
and in the cortical and outer medullary collecting duct. The proximal
tubule, the medullary thick ascending limb of the loop of Henle, and
papillary collecting ducts were referred to as negative. If this cDNA
is identical to TWIK-1, its expression at the mRNA level along the
rabbit nephron is clearly distinct from our findings at the protein
level in the rat. However, because no sequence information has been
provided for the amplified rabbit sequences and because data originate
from a single experiment using nonquantitative RT-PCR, it is difficult
to interpret these findings in terms of specific
K+ channel expression.
In conclusion, we have shown that TWIK-1, a new
K+ channel with four transmembrane
domains, is selectively expressed in the brush-border membrane of the
proximal convoluted tubule, in the thick ascending limb of the loop of
Henle, and in collecting duct intercalated cells, with intracellular
and apical localization. TWIK-1 may play a significant role in
K+ secretion, thus participating
in the final adjustments of K+
handling in the kidney.
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FOOTNOTES |
Address for reprint requests: N. Farman, INSERM U478, Faculté
de Médecine X. Bichat, BP. 416, 75870 Paris cedex 18, France.
Received 21 October 1997; accepted in final form 21 August 1998.
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