Differential expression of KvLQT1 and
its regulator IsK in mouse epithelia
Sophie
Demolombe1,*,
Diego
Franco1,*,
Piet
de
Boer1,
Sabina
Kuperschmidt2,
Dan
Roden2,
Yann
Pereon3,
Anne
Jarry3,
Antoon F. M.
Moorman1, and
Denis
Escande3
1 Experimental and Molecular Cardiology Group, Academic
Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The
Netherlands; 2 Departments of Medicine, Pharmacology,
Molecular Physiology & Biophysics, Vanderbilt University School
of Medicine, Nashville, Tennessee 37232; and 3 Laboratoire
de Physiopathologie et de Pharmacologie Cellulaires et
Moléculaires, Institut National de la Santé et de la
Recherche Médicale U533, Hopital Hotel-Dieu, 4093 Nantes,
France
 |
ABSTRACT |
KCNQ1 is the human gene responsible in
most cases for the long QT syndrome, a genetic disorder characterized
by anomalies in cardiac repolarization leading to arrhythmias and
sudden death. KCNQ1 encodes a pore-forming K+
channel subunit termed KvLQT1 which, in association with its regulatory
-subunit IsK (also called minK), produces the slow component of the
delayed-rectifier cardiac K+ current. We used in situ
hybridization to localize KvLQT1 and IsK mRNAs in various tissues from
adult mice. We showed that KvLQT1 mRNA expression is widely distributed
in epithelial tissues, in the absence (small intestine, lung, liver,
thymus) or presence (kidney, stomach, exocrine pancreas) of its
regulator IsK. In the kidney and the stomach, however, the expression
patterns of KvLQT1 and IsK do not coincide. In many tissues, in situ
data obtained with the IsK probe coincide with
-galactosidase
expression in IsK-deficient mice in which the bacterial lacZ
gene has been substituted for the IsK coding region. Because expression
of KvLQT1 in the presence or absence of its regulator generates a
K+ current with different biophysical characteristics, the
role of KvLQT1 in epithelial cells may vary depending on the expression of its regulator IsK. The high level of KvLQT1 expression in epithelial tissues is consistent with its potential role in K+
secretion and recycling, in maintaining the resting potential, and in
regulating Cl
secretion and/or Na+ absorption.
long QT syndrome; Romano-Ward; Jervell-Lange-Nielsen; KCNQ1; KvLQT1; epithelium
 |
INTRODUCTION |
MUTATIONS IN THE
KCNQ1 gene are the most frequent cause of the long QT
syndrome, a genetic disorder characterized by cardiac repolarization
anomalies leading to arrhythmias and sudden death (43). It encodes a pore-forming K+ channel
subunit termed KvLQT1 (43), which, in association with its regulatory
-subunit IsK (also called minK), produces the slow
component of the delayed-rectifier cardiac K+ current, IKs
(3, 34). In mammalian cells investigated at physiological
temperature, the main effect of the regulatory subunit IsK on the
KvLQT1 current is to slow its activation kinetics and also to shift its
activation curve to more depolarized potentials (6, 16).
Thus expression of KvLQT1 in the presence or absence of its regulator
generates a K+ current with different biophysical
characteristics. One physiological implication is to be found in the
human heart muscle where coexpression of IsK and KvLQT1 produces a
delayed-rectifier K+ current that slowly activates within a
few hundred milliseconds (34), i.e., within the same
duration range as the normal action potential.
Expression of KvLQT1 channels is not confined to the heart but has also
been shown in other organs (46). We thus decided to
explore whether KvLQT1 is coexpressed together with its regulator in
these organs. To address this issue, we investigated KvLQT1 and IsK
expression patterns using in situ hybridization in fetal and adult
mice. In situ hybridization data obtained in normal mice were compared
with data obtained in IsK-deficient mice in which the bacterial
lacZ gene was substituted for the IsK coding region
(13). We show that KvLQT1 mRNA is abundant in many
epithelial tissues in the absence (small intestine, lung, liver,
thymus) or presence (kidney, stomach, exocrine pancreas) of IsK. In the kidney, however, KvLQT1 mRNA predominates in the medulla, whereas IsK
mRNA predominates in the cortex. The different expression patterns
between KvLQT1 and its regulator provide important clues with regard to
the physiological role of KvLQT1 in noncardiac tissues.
 |
METHODS |
In situ hybridization.
Control embryos (strain FVB; Charles River, Saint Aubin les Elboeufs,
France) ranging from embryonic day (E) 16.5 to E18.5 were analyzed. The
day of the vaginal plug was scored as 0.5 day of gestation. Organs from
1.5-mo-old adult FVB mice were dissected and processed as follows.
Tissues were fixed in 4% freshly prepared formaldehyde in PBS
overnight at 4°C, rinsed in PBS, dehydrated in a graded series of
ethanol solutions, and paraffin embedded. Serial sections were cut at 7 µm thickness and mounted on RNase-free 3-aminopropyltriethoxysilane-coated slices.
The mouse KvLQT1 probe used for in situ hybridization consisted of a
789-bp segment corresponding to nucleotides 2054-2843 of the
3'-untranslated region, subcloned into the pSK(
) plasmid (3). The plasmid was linearized with the appropriate
restriction enzymes, and sense (T7) or antisense (T3)
double-labeled probes were prepared. The mouse IsK probe corresponded
to nucleotides 1-512 containing the full coding sequence
(14). KvLQT1 and IsK probes were a generous gift from
Jacques Barhanin (IPMC, Sophia Antipolis, France).
Additional probes were used as positive controls to check the
quality of the tissue sections and the quality of the in situ hybridization procedure. These probes corresponded to smooth muscle myosin heavy chain (SM-MHC) cDNA (21); sarcoendoplasmic
reticulum Ca2+-ATPase (SERCA2) cDNA (24);
-subunit of the epithelium Na+ channel
(
-ENaC) cDNA (a generous gift from Paul B. McCray; see Ref.
18); mouse myosin light chain (MLC)-2a (12);
mouse MLC-2v (25); and rat connexin (Cx)-43
(5).
cRNA probes of mouse KvLQT1 and IsK mRNAs were radiolabeled with
[35S]UTP and [35S]CTP by in vitro
transcription according to standard protocols (20).
Hybridization conditions were essentially as described by Moorman et
al. (23-24) with slight modifications. Briefly, the sections were deparaffinized, rinsed in 100% ethanol, and dried in an
air stream. Pretreatment of the sections comprised 10 min with 2× SSC
(70°C), 5 min with bidistilled water, 2-20 min digestion in
0.1% pepsin dissolved in 0.01 M HCl, 30 s in 0.2% glycine/PBS, two 30-s washes in PBS, 5 min in bidistilled water, and 5 min in 10 mM
dithiothreitol (DTT); sections were finally dried in an air stream. The
prehybridization mixture contained 50% formamide, 10% dextran
sulfate, 2× SSC, 2× Denhardt's solution, 0.1% Triton X-100, 10 mM
DTT, and 200 ng/µl heat-denatured herring sperm DNA. The sections
were hybridized overnight at 52°C and washed as follows: one rinse in
1× SSC, 30 min at 52°C in 50% formamide dissolved in 1× SSC, 10 min in 1× SSC, 30 min in RNase A (10 µg/ml), 10 min in 1× SSC, and
10 min in 0.1× SSC; the sections were dehydrated in 50, 70, and 90%
ethanol containing 0.3 M ammonium acetate. The sections were dried and
immersed in nuclear autoradiographic emulsion G5 (Ilford, UK). The
exposure times ranged from 7 to 14 days with development time of 4 min.
Images were taken using a Photometrics camera coupled to a Zeiss
Axiophot microscope, and computerized files were recorded. Panels were
composed using PowerPoint software. Representative sections from each
organ were stained with hematoxiline-azoflozine according to standard procedures.
LacZ staining.
We used the inbred 129SV IsK-deficient mice in which the bacterial
lacZ gene substitutes the IsK coding region
(13). Tissues from IsK(
/
) 1.5-mo-old mice were
dissected and fixed in 4% freshly prepared formaldehyde solution.
Specimens were rinsed in PBS, incubated with increasing sucrose
solutions (10, 20, and 30% in PBS) for 2 h/step, embedded in tissue
freezing medium (optimum cutting temperature compound; Sakura FineTek
Europe, Zouterwoude, The Netherlands), and frozen. Serial sections of
10-15 µm were cut, mounted on polylysine-coated glasses, briefly
dried, rinsed in PBS for 30 min, and incubated in X-Gal solution as
detailed elsewhere (33). Subsequently, sections were
counterstained with azofloxine for 2 min and dehydrated. Coverslips
were mounted with Entellan (Merck, Darmstadt, Germany).
RNase protection assay.
FVB mouse adult tissue samples were dissected out and immediately
frozen in liquid nitrogen. Total RNA was isolated from each sample
using the guanidinium isothiocyanate method (47). RNA samples were quantified by spectrophotometric analysis. Assessment of
the integrity of RNA samples was based on the appropriate
28S-to-18S ribosomal RNA ratios.
For RNase protection assays, the mouse KvLQT1 probe consisted of a
446-bp segment corresponding to nucleotides 2397-2843 of the
3'-untranslated region subcloned into pSK(
) (3). The
mouse IsK probe was similar to that used for in situ hybridization. A
rat cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
Ambion, Austin, TX) was used as an internal marker. Antisense RNA
probes were produced using appropriate polymerase (T3 or T7; Ambion) on
linearized mouse KvLQT1, IsK, and GAPDH templates in the presence of
[
-32P]UTP (NEN, Boston, MA). RNase protection assays
were carried out as previously described (28) using the
RPA II Kit (Ambion). For each tissue source, 50 µg of total RNA were
hybridized to 0.5-1 × 105 counts/min of the RNA
probe. The RNA was run after RNase digestion on a 5% polyacrylamide-8
M urea gel. Sense KvLQT1 RNA was also hybridized and was run on the
same gel as size marker and undigested RNA probes. Negative controls
were run using yeast tRNA hybridized with the KvLQT1 and IsK probes.
Gels were exposed at
80°C to X-ray films (Biomax-MS; Eastman Kodak,
Rochester, NY) with an intensifying screen for 5 days (kidney, small
intestine, stomach, lung, and heart) or for 10 days (skeletal muscle,
liver, and thymus). Signal intensity was in the linear range as
assessed in control experiments.
RT-PCR.
We also used RT-PCR to verify the expression of IsK and KvLQT1 mRNAs.
Total RNA was isolated as described above for RNase protection assay.
Total RNA (1 µg) was reverse transcribed using the SuperScript
One-Step RT-PCR system (Life Technologies, Rockville, MD).
Specific primers for IsK and KvLQT1 cDNAs were designed to the
3'-untranslated region for KvLQT1 and to a portion of the coding sequence for IsK as follows: KvLQT1 forward primer, 5'-CAC CAA CAC CCC
TCT GCC C-3', reverse primer 5'-GGG CTG AGG GTG GAA ACC CC-3' (product
size = 229 pb); IsK forward primer 5'-GCC CAA TTC CAC GAC TGT TCT
GCC C-3', reverse primer 5'-GGT GTG TGG CAG GCT GCT CTA CGG-3' (product
size = 355 pb). PCR conditions were identical for all primer sets
(annealing temperature 60°C).
 |
RESULTS |
In situ hybridization.
Figure 1 shows the expression pattern of
KvLQT1 and IsK mRNA in the kidney. In this organ, KvLQT1 was more
widely distributed than IsK. The antisense probe to IsK hybridized
mainly to the cortex. The IsK expression pattern suggested that IsK
mRNA was most abundant in the distal convoluted tubules and in the
cortical collecting ducts (Fig. 1B). In contrast, the KvLQT1
antisense probe hybridized to the cortical and the medullar areas (Fig. 1A). Serial sections suggested that KvLQT1 was expressed in
the collecting ducts of the outer medulla and in the distal and
collecting tubules of the cortex. Specificity of the KvLQT1 and IsK
antisense probes was demonstrated by the following: 1) sense
probes did not produce any significant signal (Fig.
2); 2) pretreatment of the
sections with RNase prevented hybridization of the antisense probes
(Fig. 2); and 3) a probe for SM-MHC, used as a positive control, only reacted with blood vessels (Fig. 1C). To
further confirm the IsK expression pattern, we used IsK-deficient mice in which the bacterial lacZ gene substituted the IsK coding
region so that
-galactosidase expression was controlled by
endogenous IsK regulatory elements (13). As shown in Fig.
1C,
-galactosidase staining was detected in the cortical
collecting duct region, in concordance with findings obtained in IsK
hybridization experiments. Finally, we also used an antisense probe to
the
-subunit of the amiloride-sensitive epithelial
-ENaC. The
-ENaC probe has been reported to hybridize to the outer medullar and
cortical collecting ducts (8). We observed that
-ENaC
expression (Fig. 1C) localized in the same region as KvLQT1
(Fig. 1A).

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Fig. 1.
In situ hybridization of KvLQT1
(A) and IsK (B) in normal adult mouse kidney.
KvLQT1 probe hybridization to the cortex (C) and to the medulla (M).
IsK probe hybridization confined to the cortex. A and
B were taken with increasing magnification from
left to right. Complementary results are
presented in C [lacZ staining ( -gal) in
kidney sections from IsK-deficient mice in which the bacterial
lacZ gene has been substituted for the IsK coding region; in
situ hybridization of the -subunit epithelial Na+
channel ( -ENaC); in situ hybridization of smooth muscle myosin heavy
chain (SM-MHC)].
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Fig. 2.
Sense probes and RNase treatment in the kidney. Left: in
situ hybridization with KvLQT1 (A) and IsK (B)
sense probes. Right: pretreatment of the kidney sections
with RNase prevented KvLQT1 (A) and IsK (B)
antisense probe hybridization.
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In situ hybridization with KvLQT1 and IsK probes in adult small
intestine are shown in Fig.
3A. KvLQT1 expression was
limited to the basal region of the intestinal villi but was absent in muscularis mucosae stained by the SM-MHC probe. The
hematoxiline-azoflozine staining showed that the base of each gland was
mainly composed of the crypts continuous with the epithelium of the
intestinal villi and of Paneth cells, which have the appearance of
serozymogenic cells. Mitotic activity in the crypts produces a
continuous supply of new cells that progress up to the villi. No
consistent KvLQT1 signal was detected along the entire villi. This does
not exclude that the protein was expressed as previously shown for
lactase protein (31). Specific KvLQT1 signal was found
throughout the length of the small intestine, although the signal
intensity decreased as the sections progressed toward the large
intestine (data not shown). No IsK expression was detected in the small
intestine or in the large intestine. In accordance with this, no
-galactosidase staining was detected in the small or large intestine
in IsK knockout mice. Results obtained in E16.5-E18.5 embryos (Fig.
3B) demonstrated KvLQT1 RNA expression from the base of the
villus up to the midvillus region and thus confirmed its presence in
epithelial cells. The embryonic sections lacked hybridization of the
IsK probe.

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Fig. 3.
In situ hybridization of KvLQT1
and IsK in adult (A) and embryonic (E; B) small
intestine. KvLQT1 staining confined to the basal region of the
intestinal villi (V). Hematoxiline-azoflozine (HA) staining showed the
crypts (C) and the Paneth cells (P). There was a lack of
-galactosidase ( -gal) staining in small intestine of knockout
mice. B: results obtained in E16.5-E18.5 embryos. In the
same section, the pancreas is also visible with the small intestine.
Note pancreatic staining with the KvLQT1 probe. In A and
B, SM-MHC, used as a positive control, stained the
muscularis mucosae (MM).
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In the stomach (Fig. 4), both IsK
and KvLQT1 mRNAs were present at high levels but colocalized only
partially. The KvLQT1 probe hybridized to the first half of the gastric
glands above the muscularis mucosae and extended further to the luminal
side. The resolution of the staining did not allow clear identification of the positive epithelial cells: pepsin-secreting and/or parietal cells. IsK mRNA was detected in the basal portion of the glands. Surprisingly, we did not detect
-galactosidase activity in stomach sections of IsK knockout mice. In the esophagus, different cell layers,
including submucosa, lamina propria, and epithelium, were stained
by both IsK and KvLQT1 antisense probes (data not shown). The
muscularis layers were not stained.

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Fig. 4.
In situ hybridization of KvLQT1 and IsK in adult stomach. KvLQT1
antisense probe hybridization to the first half of the mucosa (M)
above the muscularis mucosae (MM); IsK antisense probe hybridization to
the basal portion of the glands. There was a lack of
-galactosidase staining in stomach sections of IsK knockout mice.
SM-MHC was used as a positive control. L, luminal
side.
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In the lung (Fig. 5), only KvLQT1 mRNA
was detected. The KvLQT1 antisense probe stained the ciliated
epithelial cells of terminal bronchioles but not the alveoli or the
pulmonary veins, which were stained by the SERCA2 probe
(24). The absence of staining under the smooth muscle
(which was negative itself) suggested that there was no expression of
KvLQT1 in serous glands. We looked at the trachea and observed a
tracheal mucosa staining that was preferentially localized in the
respiratory epithelium (data not shown). The submucosa underlying the
lamina propria and which contains numerous seromucous glands seemed to
be unstained. In contrast, we did not detect IsK mRNA in lung and
tracheal sections from normal mice or
-galactosidase activity in
knockout mice.

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Fig. 5.
In situ hybridization of KvLQT1 and IsK in adult lung.
Sections show bronchioles (B) and pulmonary vessels (PV). Ciliated
epithelial cells of the terminal bronchioles were stained by the KvLQT1
antisense probe but not the alveoli (A). Neither IsK mRNA nor
-galactosidase staining in knockout mice was visible in lung
sections. SERCA, sarcoendoplasmic reticulum
Ca2+-ATPase.
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With the use of Northern blot analysis, previous work has revealed high
levels of IsK mRNA in neonatal hearts but a low, albeit detectable,
level of IsK mRNA in the adult heart. In contrast, high levels of
KvLQT1 mRNA that did not change with aging (7, 9) were
observed. In a serial section of adult mouse heart, the atrial and
ventricular walls exhibited a homogenous signal with either IsK or
KvLQT1 probes (Fig. 6, A and
B). Again, signal specificity was demonstrated using sense
probes. The atrioventricular node/His bundle area was identified by the
expression of MLC-2a, MLC-2v, and Cx-43 (Fig. 6C) in serial
sections adjacent to those hybridized with IsK and KvLQT1 probes. This
area is characterized by coexpression of MLC-2a and MLC-2v (D. Franco,
unpublished observations) and the absence of Cx-43
(41). IsK and KvLQT1 staining in the atrioventricular
node/His bundle showed the same intensity as in atria or ventricles. In
contrast, and as previously reported (13), the
-galactosidase staining was confined to the conduction system. In
the skeletal muscle, only KvLQT1 mRNA expression was detected in
myocytes but at very low level compared with the expression level of
SERCA2 (Fig. 7). In agreement with the
absence of IsK mRNA expression, no
-galactosidase activity was
detected in this latter tissue (Fig. 7).

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Fig. 6.
In situ hybridization of KvLQT1 (A), IsK
(B), and control probes [myosin light chain (MLC)-2a,
MLC-2v, and connexin (Cx)-43; C] in the transversal section
of adult mouse hearts at the level of the left atrioventricular
junction. Note that the expression that KvLQT1 and IsK is homogenous
within the atrial (A) and ventricular (V) myocardium and in the His
bundle (arrows) but absent in the valve tissue (arrowheads). Expression
of MLC-2a is confined to the atrial myocardium, whereas MLC-2v is
confined to the ventricular myocardium. Coexpression of MLC-2a and
MLC-2v can be observed in the His bundle branch. The His bundle shows
virtually no expression of Cx-43.
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Fig. 7.
In situ hybridization of KvLQT1
and IsK in adult skeletal muscle. KvLQT1 mRNA was detected in myocyte
sarcolemic membrane (SM). There was a lack of -galactosidase
staining on knockout mice.
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In the pancreas (Fig. 8A), the
secretory acini and Langerhans islets were highly positive for both IsK
and KvLQT1 probes with the exception of the delicate septa between the
lobules and the epithelium that lined the ducts. Pancreatic KvLQT1 but
not the IsK expression was detected from the embryonic life (Fig.
3B).
-Galactosidase staining in knockout mice revealed
only a few blue cells, which were confined to the periphery of
Langerhans islets where
-cells are situated. In the liver (Fig.
8B), there was a weak homogenous staining of the hepatic
lobules but no staining of the connective tissues and vessels of the
portal tracts with the KvLQT1 probe. The IsK mRNA appeared to be absent
from the liver. This observation fits with the lack of
-galactosidase activity in liver sections from IsK knockout mice.

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Fig. 8.
In situ hybridization of KvLQT1
and IsK in adult pancreas (A) and liver (B).
A: in the pancreas, acini and Langerhans islets (L. islets)
were highly positive with both IsK and KvLQT1 antisense probes. The
septa (S) and the ductal epithelium (D) were negative.
-Galactosidase staining in knockout mice revealed few blue cells
confined to the periphery of Langerhans islets. B: in the
liver, there was weak hybridization of KvLQT1 probe to the hepatic
lobules and lack of hybridization of the IsK probe. There was a lack of
-galactosidase activity in liver section from IsK knockout
mice.
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In the adult thymus (Fig. 9A),
KvLQT1 mRNA expression was found in the cortex where thymic lymphocytes
but also epithelial cells were densely aggregated. IsK mRNA and
-galactosidase activity was not detected. In adult spleen sections
(Fig. 9B), neither the red nor the white pulp showed
positive staining with IsK and KvLQT1 probes nor upon X-Gal staining of
the sections from the IsK knockout/lacZ knockin mice.

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Fig. 9.
In situ hybridization of KvLQT1
and IsK in adult lymphatic organs. In the adult thymus (A),
KvLQT1 antisense probe hybridized to the cortex (C) and not to the
medulla (M). IsK mRNA and -galactosidase activity was not detected.
Similar patterns for IsK and -galactosidase were obtained in adult
spleen sections (B). In the spleen, KvLQT1 mRNA was not
detected either in the red (RP) or in the white (WP)
pulp.
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RNase protection and RT-PCR assays.
To independently confirm the data obtained by in situ
hybridization, we investigated the expression of IsK and
KvLQT1 mRNA using more sensitive techniques, including RNase
protection and RT-PCR assays. Using the RNase protection assay, both
IsK and KvLQT1 mRNAs were easily detected in the kidney, the stomach, and the heart from 1.5-mo-old mice (Fig.
10). A strong signal with the specific
KvLQT1 probe but not with the IsK probe was obtained from small
intestine and lung tissues. Quantification of specific KvLQT1 and IsK
signals relative to the GAPDH signal revealed that KvLQT1 RNA was two
times more abundant compared with IsK RNA in the kidney, five times
more abundant in the stomach, and equally abundant in the heart. KvLQT1
expression increased in intensity with the following rank order:
intestine, heart, kidney, lung, and stomach. Exposure of the gels on
X-ray films for >10 days was required to detect KvLQT1 mRNA from
skeletal muscle, liver, and thymus (Fig.
11). Under these conditions, a band
corresponding to KvLQT1 mRNA but not to IsK mRNA was obtained. With
RT-PCR (Fig. 12), KvLQT1 mRNA was
detected in the gut, lung, liver, skeletal muscle, thymus, and spleen.
Thus the RNase protection assay confirmed KvLQT1 in situ data in the
thymus. In the spleen, RT-PCR demonstrated the presence of KvLQT1 mRNA,
which was not detected either by in situ or by the RNase protection.
With IsK-specific primers, no positive signal was detected in gut,
lung, and liver from 1.5-mo-old control mice. Only a faint band was
visible in thymus and spleen tissues, suggesting very low expression
levels and confirming the in situ data. RT-PCR conditions and primers
were positively controlled with total RNA isolated from heart tissue,
and product specificity was warranted by restriction enzyme treatment.

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Fig. 10.
RNase protection assays of IsK and KvLQT1 in mouse adult
tissues after 5 days exposure. Expression of IsK (512-bp protected
fragment) and of KvLQT1 (446-bp protected fragment) mRNA in various
tissues. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe used as
a positive control.
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Fig. 11.
RNase protection assays of IsK and KvLQT1 in mouse adult
tissues after 10 days exposure. Expression of IsK (512-bp protected
fragment) and of KvLQT1 (446-bp protected fragment) mRNA in skeletal
muscle, liver, and thymus. GAPDH probe used as a positive control.
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Fig. 12.
RT-PCR assays in mouse adult tissues using primers
specific for KvLQT1 3'-untranslated region (224-bp fragment) and for
IsK (355-pb fragment). The heart was used as a positive control.
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DISCUSSION |
The present work demonstrates that KvLQT1 is widely distributed in
mouse epithelial tissues. Depending on the organ, the KvLQT1 K+ channel is expressed either in the presence (e.g.,
kidney, stomach, pancreas, heart) or absence (e.g., small intestine,
lung, liver, skeletal muscle) of its regulator IsK. The IsK in situ
data obtained in normal mice were further supported by
-galactosidase staining in IsK knockout mice (13).
-Galactosidase activity agreed with IsK mRNA expression in the
kidney, small intestine, lung, skeletal muscle, liver, thymus, and
spleen. However, in stomach, heart, and pancreas,
-galactosidase
activity was either restricted to a limited area (heart, pancreas) or
not detected (stomach), in discrepancy with in situ data. The
discrepancy between endogenous IsK and lacZ transgene expression has
already been observed in other transgenic models (11, 26, 38,
39). Interference of selection cassette (i.e., neomycin or
thymidine kinase) with neighboring cis-acting elements can
lead to abnormal transcription of the knockout gene or adjacent genes
(see, e.g., Ref. 26). Within the targeting construct used
to invalidate the IsK gene (13), nonspecific recombination
of the phosphoglycerate kinase-thymidine kinase cassette could
lead to abnormal IsK gene regulation if interfering with regulatory
modules located at the 3'-end of the IsK locus. Moreover, the
endogenous promoter regulation could potentially be altered by
substitution of a short coding region by a long one: 427-bp for IsK vs.
3,600-bp for lacZ (39). In line with this
latter hypothesis, the mouse strain that carries a disrupted IsK locus
(7) exhibits a different electrocardiographic phenotype
compared with the mouse strain in which the IsK coding region has been
substituted by the lacZ gene (13).
Coexpression of IsK together with KvLQT1 channels has potential
physiological implications since expression of IsK markedly modifies
the physiological characteristics of the K+ current flowing
through KvLQT1 channels, including its activation kinetics and voltage
dependence (3, 34). Different data have been generated
concerning the effects of IsK expression on KvLQT1 amplitude: IsK has
been shown either to increase the current amplitude related to KvLQT1
expression in Xenopus oocytes (3, 34) or to not
affect KvLQT1 current amplitude in mammalian cells at 37°C (6, 16, 22). This apparent discrepancy may be related to coexpression of the dominant-negative isoform of the KvLQT1 channel (see below). Expression of a time- and voltage-dependent current in
epithelial cells that do not generate action potentials may be
surprising. However, one could hypothesize that activation of the
KvLQT1 K+ channel in an epithelial cell is an efficacious
and economical means to maintain the membrane potential near
50 mV.
It should be stressed, however, that IsK is not necessarily the unique
-subunit to associate with KvLQT1 channels. Other single
transmembrane segment regulators have recently been discovered,
including MIRP1 (1), a 123-amino acid protein that
regulates human ether-a-go-go-related gene channels with no apparent
effect on KvLQT1, more importantly MIRP2 (35), a
103-amino acid protein that provokes a constitutively open
KvLQT1 channel, or channel-inducing factor (2), a
corticosteroid-induced channel regulator with no known partner.
It is thus conceivable that a large family of single transmembrane
segment K+ channel regulators exists. Specificity of these
regulators may be weak, for example, HERG channels are regulated by
MIRP1 (1) but also by IsK (19). It is thus
conceivable that KvLQT1 channels associate either with IsK or with
another channel regulator (MIRP2) that could replace IsK in epithelial
tissue where IsK is not expressed. In accordance with this, MIRP2 has
been shown to colocalize with KvLQT1 in murine crypt cells
(35).
Expression of the dominant negative isoform of KvLQT1,
called isoform 2, must also be taken into account (6).
This isoform, identified in human tissues, strongly downregulates the
amplitude of the KvLQT1 channel. A mouse NH2-terminal
truncated isoform similar to the human isoform 2 has also been
identified (27). We have previously shown that the
dominant-negative property of isoform 2 is strongly reduced by having
IsK present (6). In tissues expressing both isoform 1 (the
channel pore) and isoform 2 (the dominant-negative isoform),
coexpression of IsK should regulate the KvLQT1 current amplitude. The
KvLQT1 probe that we used in the present study recognizes the
3'-untranslated region of mouse KvLQT1 and thus does not discriminate
between isoform 1 and isoform 2. Using RT-PCR, we have explored isoform
2 expression in human epithelial cells (Demolombe, unpublished data).
When grown to confluence, T84 and HT-29-19A colonic cell lines
expressed isoform 1 but not isoform 2. At day 1 postplating,
both T84 and HT-29-19A cells expressed isoform 1 and isoform 2. Primary cultures of respiratory epithelial cells freshly dissociated
from nasal polyps express both KvLQT1 isoform 1 and isoform 2 immediately after dissociation but only isoform 1 at confluence or in
nasal polyp tissue. Patch-clamp experiments in epithelial cells are usually achieved in individual cells soon after plating with the aim to
accurately control the voltage. Under such conditions, the
K+ current related to KvLQT1 isoform 1 would be blunted by
isoform 2 expression. This would explain why the whole cell KvLQT1
current, with its typical tail current, has not previously been
recorded in epithelial cells from human origin (for example, see Ref.
4). However, this may be not applicable to other mammalian
epithelial cells, like murine vestibular dark cells and strial marginal cells.
KvLQT1 expression has been detected previously at the molecular level
in the colonic crypt cells from rabbit (15) and rat (36, 44). In rat and rabbit basolateral membrane of
colonic crypts cells, the chromanol compound 293B, a potent blocker of KvLQT1 channels (16), or azimilide strongly reduces the
forskolin-induced Cl
secretion through the inhibition of
a cAMP-activated K+ conductance (15, 36). This
293B-sensitive current was carried through small-conductance channels
(<3 pS; see Ref. 41) compatible with homomeric single
KvLQT1 channels in the absence of IsK (30, 46). It was
also demonstrated that basolateral cAMP-sensitive K+
channels are an important determinant of the maintained responses to
forskolin-induced secretion in murine nasal and colonic epithelia (17). These results suggest that the K+
conductance supported at the basolateral membrane by KvLQT1 channels is
important for maintaining cAMP-induced Cl
secretion in
colonic epithelia. In the rat tracheal epithelium, a native
K+ current, sharing similarities with IKs, has been
reported previously (10). This time- and voltage-sensitive
K+ current activates at potentials more positive than
50
mV, is sensitive to cAMP stimulation, and is blocked by clofilium. In the kidney, Sugimoto et al. (37) reported a rat membrane
protein that exhibits a voltage-dependent K+ channel
activity and that is confined to the apical membrane portion of the
proximal tubule epithelial cells. These authors suggest that
voltage-dependent K+ channels are involved in
K+ permeation in the apical membrane of epithelial cells
through the depolarizing effect of Na+ entry.
Mutations in the KCNQ1 gene encoding KvLQT1 are responsible
for the most frequent form of the long QT syndrome, a cardiac genetic
disorder leading to cardiac arrhythmias, which is transmitted either as
an autosomal dominant (Romano-Ward) or recessive
(Jervell-Lange-Nielsen) trait. Although KvLQT1 is expressed at a high
level in epithelial tissues, no pathological consequences on epithelial
tissues have been ascribed clinically to the KCNQ1 mutation
in the autosomal dominant Romano-Ward syndrome. This suggests that
either 1) other K+ channels can compensate for
the KvLQT1 defect in epithelia or 2) that the level of
expression of normal proteins encoded by the unaffected allele is
sufficient to ensure its function. In rare cases, biallelic
KCNQ1 mutations lead to the Jervell-Lange-Nielsen syndrome
in which prolonged cardiac repolarization is associated with congenital
deafness. Congenital deafness has been attributed to a defect in the
endolymph secretion (42). The IsK regulator has been
located in the marginal cells of the stria vascularis in the cochlear
duct and in the vestibular dark cells (42). The IsK/KvLQT1
K+ channel in the apical membrane of strial marginal cells
and vestibular dark cells is an essential ion transport pathway for the
secretion of K+ in the endolymph of the inner ear. Apart
from this defect, no other consequences on epithelial tissues have been
related to the biallelic KCNQ1 mutation in the
Jervell-Lange-Nielsen syndrome. The present work provides a basis to
explore functional anomalies associated with KvLQT1/IsK defects in
transgenic animal models [e.g., IsK(
/
) mice] and ultimately in
patients with the long QT syndrome, since a number of these functional
anomalies may remain clinically silent.
 |
ACKNOWLEDGEMENTS |
We thank Patricia Charpentier and Sylvie Leroux for expert
technical assistance with RNase protection assays and RT-PCR.
 |
FOOTNOTES |
*
S. Demolombe and D. Franco contributed equally to this work.
This work was supported by The Netherlands Organization for Scientific
Research (NWO)-Institut National de la Santé et de la Recherche
Médicale exchange program. S. Demolombe was the recipient of a
postdoctorial fellowship from the Ligue Nationale Contre le Cancer. D. Franco was supported by NWO (902-16-219) and the
Dutch Heart Foundation.
Present address for A. Moorman: Dept. of Anatomy & Embryology, Academic
Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
(E-mail: A.F.Moorman{at}amc.uva.nl).
Address for reprint requests and other correspondence: D. Escande, Laboratoire de Physiopathologie et de Pharmacologie
Cellulaires et Moléculaires, INSERM U533, Bat HBN, Hopital
Hotel-Dieu, BP 1005, 44093 Nantes, France (E-mail:
denis.escande{at}nantes.inserm.fr).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 16 March 2000; accepted in final form 7 September 2000.
 |
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