From the Laboratoire de Physiopathologie et de Pharmacologie
Cellulaires et Moléculaires, INSERM CJF96-01, Hôpital
Hôtel-Dieu, Nantes, France, the Institut de Pharmacologie
Moléculaire et Cellulaire,
UPR CNRS 411, Sophia-Antipolis, France, and the Academic Medical
Center, University of Amsterdam, Amsterdam, The Netherlands
 |
INTRODUCTION |
Mutations in the KvLQT1 gene are the most frequent
cause of the congenital long QT syndrome, a familial disorder
characterized by prolonged cardiac repolarization, syncope, and a high
incidence of sudden death (1). KvLQT1 is also responsible
for the Jervell and Lange-Nielsen cardioauditory syndrome in which
prolonged cardiac repolarization is associated with bilateral
congenital deafness (2). In a recent study, the KvLQT1 gene
was found to encompass chromosomal rearrangements associated with the
Beckwith-Wiedemann syndrome which causes prenatal overgrowth and cancer
(3). Thus, the same gene at locus 11p15.5 could be implicated in at
least three distinct genetic disease entities.
The KvLQT1 gene product is a six-transmembrane domain
protein which, in association with a membranous regulator termed IsK (previously known as minK), produces a K+ current with
physiological characteristics similar to those of the major component
of the delayed rectifier sustained K+ current in cardiac
myocytes (4, 5). KvLQT1 expression is not limited to the
heart muscle but can also be observed in human pancreas, lung, kidney,
inner ear, and placenta (4-6). In autosomal dominant long QT syndrome
1, mutations in KvLQT1 supposedly decrease the amount of
K+ current available for repolarization, thereby prolonging
the cardiac action potential and inducing arrhythmias. In the
recessively inherited Jervell and Lange-Nielsen syndrome, bi-allelic
mutations in KvLQT1 (2) not only alter cardiac
electrophysiology but also inner ear endolymph homeostasis, leading to
congenital deafness.
In the present work, we have cloned an N-terminal truncated isoform of
the KvLQT1 gene product corresponding to KvLQT1 isoform 2 simultaneously identified by Lee et al. (3) and Yang
et al. (7). It was determined that KvLQT1 isoform 2, which
is constitutively expressed in adult heart muscle, is a strong dominant
negative of KvLQT1 isoform 1. Our results demonstrate that the cardiac K+ channel responsible for the long QT syndrome 1 is a
complex protein assembly composed of isoform 1 (the channel pore),
isoform 2 (a dominant negative), and IsK (the channel regulator). These
findings could have important physiopathological implications.
 |
EXPERIMENTAL PROCEDURES |
Cloning of KvLQT1 Isoform 2--
To obtain the 5' sequence of
KvLQT1, a rapid amplification of cDNA ends
(RACE)1 experiment was
performed using a Marathon cDNA Amplification kit and Marathon
ready cDNA from human adult heart (CLONTECH). Two nested primers (LQTN1, 5' GAT GTA CAG GGT GGT TAT CAG C 3'; and
LQTN2, 5' TGA GGT CGA TGA TGG AAA TGG G 3') were derived from the
previously published partial KvLQT1 sequence (1), and RACE was performed according to the manufacturer's protocol. The fragment of 320 bp obtained was cloned in pUC 18 using the Sure Clone Ligation kit (Pharmacia Biotech Inc.). Sequencing reactions were subsequently performed on two independent clones using the dideoxy terminator cycle
sequencing kit (Applied Biosystems). Sequencing reaction products were
analyzed on 4.0% polyacrylamide gels using an ABI-377 automated DNA
sequencer (Applied Biosystems). The sequence of both clones was
identical, revealing a start codon 6 bp upstream from the previously
published sequence (1).
Molecular Expression of KvLQT1 Isoforms 1 and 2--
Human heart
RNA was prepared using left or right ventricular tissues from the
explanted heart of a patient with cystic fibrosis. Right atrial
appendage biopsies were also obtained during open heart surgery from
six patients with coronary heart disease. Tissue samples were
quick-frozen in liquid nitrogen, and total RNA was then isolated
separately from each sample using the classical guanidinium
isothiocyanate method (8). RNA samples were quantified by
spectrophotometric analysis. The lack of degradation of RNA samples was
monitored by the observation of appropriate 28 S to 18 S ribosomal
RNA ratios as determined by ethidium bromide staining of the agarose
gel.
Reverse transcriptase-polymerase chain reactions (RT-PCR) were
performed on total RNA. cDNA was synthesized from approximately 1 µg of RNA. RT-PCR reactions contained 5 mM
MgCl2, 1× PCR Buffer II, 1 mM dNTP, 1 unit/µl RNase inhibitor, 2.5 units/µl murine leukemia virus reverse
transcriptase (Perkin-Elmer), and 0.75 µM primers in a
final volume of 20 µl. Controls without reverse transcriptase were
performed to check that amplification did not proceed from residual
genomic DNA. cDNA was used for PCR reactions with 2 mM
MgCl2, 1× PCR Buffer II, 2.5 units/100 µl AmpliTaq DNA polymerase (Perkin-Elmer), and 0.5 µM primers (Genosys)
in a final volume of 100 µl. PCR reactions were performed as follows
with a Peltier Thermal Cycler (PTC-200; MJ Research). cDNA was
heated to 95 °C for 105 s and then amplified by 40 cycles
(95 °C for 15 s and 60 °C for 30 s) before primer
extension at 72 °C for 7 min. Ten microliters of each PCR product
were analyzed by electrophoresis on a 2% agarose NA (Pharmacia) gel,
followed by ethidium bromide staining. Sequences of the sense (F1) and
antisense (R) oligonucleotide primers for human KvLQT1 isoform 1 are
shown in Fig. 1. The sense primer (F2) used for specific detection of
human KvLQT1 isoform 2 is also shown in Fig. 1. Sense
5'-TCCTGTCTAACACCACAGCG-3' and antisense 5'-TTCAACGACATAGCACGACC-3'
were used for IsK. Sense 5'-GGCATCGTGATGGACTCCG-3' and antisense
5'-GCTGGAAGGTGGACAGCGA-3' were used for human
-actin. The expected
fragment lengths calculated from cDNA sequences were 274, 245, 326, and 615 bp for KvLQT1 isoform 1, isoform 2, IsK, and
-actin,
respectively.
Three different cDNAs were used as hybridization probes for RNase
protection assays. The first was a human 274-bp KvLQT1 isoform 1 cDNA corresponding to the 5' 37 nucleotides upstream and 237 nucleotides downstream from the start codon (Fig. 1). This cDNA contains 62 nucleotides specific for isoform 1, which in an RNase protection assay provides a 274-nucleotide isoform 1-specific protected
fragment and a 212-nucleotide protected fragment that hybridizes
non-isoform 1 transcripts. The probe was PCR-cloned from the expression
vector pCI-KvLQT1 (see below) using tailed primers between the
BamHI and HindIII sites of a pBluescript SK vector (Stratagene). The second probe was a human 347-bp KvLQT1 isoform
2 cDNA corresponding to the 5' 27 nucleotides upstream and the 320 nucleotides downstream from the start codon. This cDNA contains 33 nucleotides specific for isoform 2, which in an RNase protection assay
provides a 347-nucleotide isoform 2-specific protected fragment and a
314-nucleotide non-isoform 2 protected fragment. This probe was
subcloned from the expression vector pECE-KvLQT1 between the
HindIII and PstI sites of a pBluescript SK vector
(Stratagene). The third probe was a human 393-bp cDNA corresponding
to the full-length IsK sequence (clone pCMV/HIsK).
Antisense RNA probes were produced using T7, T3, and SP6 polymerases
(Ambion) on linearized KvLQT1 isoform 1, KvLQT1 isoform 2, and IsK
templates, respectively, in the presence of [
-32P]rUTP
(NEN Life Science Products). RNase protection assays were carried out
as described previously (9) using the RPA II Kit (Ambion). For each
tissue source, 15 µg of total RNA were hybridized to 0.5-1 × 105 cpm of the RNA probe. The RNA was run after RNase
digestion on a 5% polyacrylamide, 8 M urea gel. Sense
KvLQT1 isoform 1 RNA was also hybridized and was run on the same gel
for use as a size marker (not illustrated), as well as undigested RNA
probes. Negative controls were run using t-yeast RNA hybridized with
the isoform 1 and isoform 2 probes. Gels were exposed for 48 h at
80 °C on x-ray films (Biomax-MS, Eastman Kodak Co.) with an
intensifying screen. Autoradiograms of RNase protection assays were
subjected to densitometric analysis using the ImageQuant program
(Molecular Dynamics). The ratios of KvLQT1 isoform 1 and KvLQT1 isoform
2 mRNA levels were determined directly from specific signals since respective protected bands were hybridized with the same probe.
Intranuclear Injection of Plasmids--
The kidney-derived COS-7
cell line was obtained from the American Type Culture Collection. Cells
grown on plastic Petri dishes were microinjected with plasmids at day 1 after plating. Our protocol to microinject cultured cells using the
Eppendorf ECET microinjector 5246 system has been reported in detail
elsewhere (10). Plasmids were diluted at a final concentration of 1-5
µg/ml in a buffer composed (in mM): HEPES 50, NaOH 50, NaCl 40, pH 7.4, supplemented with fluorescein isothiocyanate-dextran
0.5%. Human cardiac KvLQT1 isoform 1 (Ref. 26; GenBankTM
accession number AF000571), KvLQT1 isoform 2, as well as IsK cDNAs
were subcloned into the mammalian expression vector pCI (Promega,
Madison, WI) under the control of a cytomegalovirus enhancer/promoter.
The KvLQT1 isoform 2 construct was derived from the isoform 1 pCI
plasmid using polymerase chain reaction and tailed primers. A green
fluorescence protein pCI plasmid (a kind gift from Dr. Rainer Waldmann,
Sophia-Antipolis, France) was used as an inert plasmid to ensure that
cells were always injected with a constant 15 µg/ml plasmid
concentration.
Patch-clamp Recordings--
Whole cell currents were recorded as
described previously (10). A Petri dish containing cells was placed on
the stage of an inverted microscope and continuously superfused with
the standard extracellular solution containing (in mM) NaCl
145, KCl 4, MgCl2 1, CaCl2 1, HEPES 5, glucose
5, pH adjusted to 7.4 with NaOH. Patch pipettes with a tip resistance
2.5-5 M
were electrically connected to a patch-clamp
amplifier (Axon Instruments). The intracellular medium contained (in
mM): potassium gluconate 145, HEPES 5, EGTA 2, 1/2 magnesium gluconate 2 (free Mg2+, 0.1), K2ATP
2, pH 7.2, with KOH, whereas the extracellular medium used to record
pure K+ currents contained (in mM): sodium
gluconate 145, potassium gluconate 4, 1/2 calcium gluconate 7 (free
Ca2+, 1), 1/2 magnesium gluconate 4 (free Mg2+,
1), HEPES 5, glucose 5, pH 7.2 with NaOH. Stimulation, data recording,
and analysis were performed through an A/D converter (Labmaster). A
microperfusion system allowed local application and rapid change of the
different experimental solutions warmed at 37 °C. Patch-clamp
measurements are presented as the mean ± S.E. The statistical
significance of the observed effects was assessed by means of the
standard t test.
Immunocytochemistry and Immunoblotting of Epitope-tagged KvLQT1
Constructs--
The sequence encoding the influenza hemagglutinin (HA)
epitope YPYDVPDYA (11) was inserted in frame with the KvLQT1 isoform 1 coding sequence after nucleotide position 654 (see
GenBankTM accession number AF000571). This position
corresponds to the second external loop of the protein, between serine
218 and lysine 219. pCI-KvLQT1 isoform 1 plasmid was digested with
EcoRI and AflIII, and the 716-bp fragment so
produced was subcloned into pUC 19 and then digested with
BanII and PstI. A sense oligonucleotide containing the tag sequence 5'-TACCCATACGATGTTCCAGATTACGCT-3' was
annealed with its reverse oligonucleotide and inserted between BanII and PstI sites. The resulting product was
digested with EcoRI and AflIII and subcloned into
the pCI-KvLQT1 isoform 1 plasmid. To insert the HA tag into KvLQT1
isoform 2 coding sequence, a PCR reaction was performed using the
epitope-tagged KvLQT1 isoform 1 plasmid as a template. Epitope-tagged
KvLQT1 plasmids were checked by sequencing. Functional expression of
the epitope-tagged plasmids was verified by patch-clamp analysis of
transfected cells (data not shown).
For immunoblotting experiments, COS-7 cells cultured in
25-cm2 flasks were transfected with tagged pCI-KvLQT1
plasmids using 22-kDa polyethyleneimine as a polycationic transfection
vector. Twenty four hours post-transfection, cells were washed twice
with cold phosphate-buffered saline (PBS), incubated with 200 µl of SDS gel-loading buffer (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10%
glycerol), and scraped. The samples were placed in a boiling water bath
for 10 min, and the chromosomal DNA was sheared by sonication for 2 min. Samples were centrifuged at 10,000 × g for 10 min
at room temperature, and the supernatant was used for immunoblotting.
Protein concentration was determined using the Bradford assay (bovine
serum albumin as a standard). Proteins (15 µg/well) were separated by
electrophoresis on 7.5% polyacrylamide gels using the Laemmli system.
The separated proteins were electrotransferred to an Immobilon-P
polyvinylidene difluoride membrane (Sigma). Electroblotting was carried
out in a Tris buffer containing 40.7 mM glycine and 20%
(v/v) methanol, pH 8.3, at 100 V for 1.5 h. For immunodetection of
the proteins, blots were blocked with PBS supplemented with 5% (w/v)
non-fat dry milk and 0.1% Tween for 1 h at 37 °C. The
monoclonal anti-HA antibody (clone 12CA5, Boehringer Mannheim) was
diluted 500-fold in PBS containing 2.5% (w/v) non-fat dry milk and
0.1% (v/v) Tween 20 and incubated with the blots for 1 h at room
temperature. After primary antibody reaction, the filters were rinsed 5 times with quench buffer and incubated for 1 h at room temperature
in a 1:1000 dilution of a biotin-conjugated goat anti-mouse antibody
(Sigma). The blots were then incubated for 30 min with conjugated
extravidin peroxidase and finally developed by the enhanced
chemiluminescence Western blotting system. Biomax-MR Kodak films were
used to detect protein in the blot.
For immunocytochemistry, transfected COS-7 cells seeded on 12-mm
collagen-coated coverslips were washed three times with PBS supplemented with 1 mM Ca2+ and 0.5 mM Mg2+ and incubated with the monoclonal
anti-HA antibody diluted 1:10 in PBS, 0.5% ovalbumin for 1 h at
37 °C. Cells were washed three times and incubated for 30 min at
room temperature with a fluorescein isothiocyanate-conjugated goat
anti-mouse IgG (Sigma) diluted at 1:25 in PBS, 0.5% ovalbumin. The
cells were subsequently washed and then fixed using a 15-min incubation
with 4% formaldehyde. The glass coverslips were further washed and
then mounted in a mounting medium (Citifluor) to prevent
photobleaching. Staining was observed using a standard epifluorescence
oil-immersion microscope (Axioskop; Carl Zeiss, Oberkochen,
Germany).
 |
RESULTS |
Cloning--
5'-RACE experiments performed on human heart cDNA
identified a clone predicted to encode a truncated KvLQT1 protein
lacking an intracytoplasmic N-terminal tail. The predicted N-terminal sequence (Fig. 1) was only 2 amino acids
(Met and Asp) longer than the previously published partial sequence (1)
and 131 amino acids shorter than the cardiac KvLQT1 isoform 1 (6). A
27-bp sequence identified 5'-upstream from the start codon matched the
KvLQT1 exon 1b sequence recently published by Lee et
al. (3).

View larger version (73K):
[in this window]
[in a new window]
|
Fig. 1.
5' sequence alignment of human cardiac KvLQT1
isoform 2 (Iso 2) and isoform 1 (Iso 1;
GenBankTM accession number AF000571). Identical
nucleotides are denoted by an asterisk. The primer sequences
used for RT-PCR are indicated. The start codon for isoform 2 is
boxed. The gray line indicates the RNase
protection assay hybridization probes used to detect isoform 1 and 2 RNA.
|
|
RNA Expression in the Heart--
When RT-PCR primers specific for
each isoform (Fig. 1) were used, both full-length and N-terminal
truncated KvLQT1 isoform RNAs were detected in human ventricular
tissues from an explanted heart (Fig. 2).
Isoform 2 mRNA was also detected in six right atrial appendages
sampled during open heart cardiac surgery (Fig. 2). KvLQT1
expression was further quantified in adult human cardiac muscle using
an RNase protection assay (Fig. 3). The
probe used for isoform 2 mRNA detection spanned 347 bp and covered
a portion of isoform 2-specific 5'-coding and non-coding sequences as
well as a 314-bp sequence common to isoforms 1 and 2. Thus, with our isoform 2 probe, two RNA fragments were RNase-protected as follows: a
347-bp sequence specifically corresponding to isoform 2 and a 314-bp
sequence corresponding to non-isoform 2 (Fig. 3). For isoform 1, the
RNase protection hybridization probe covered a 274-bp segment which
comprised a 62-bp fragment of isoform 1-specific 5' sequence and a
212-bp segment common to isoform 1 and 2 sequences (Fig. 1). With this
probe, the following two RNA fragments were RNase-protected: a 274-bp
sequence corresponding to isoform 1 and a 212-bp sequence corresponding
to non-isoform 1 (Fig. 3). This approach allowed direct comparison of
the expression level of the different isoforms without the need for an
internal marker. Densitometric analysis was used to compare isoform
1/non-isoform 1 bands and isoform 2/non-isoform 2 bands from cardiac
tissues. This analysis revealed that isoform 1 and isoform 2 represented 71.9 ± 0.6 and 28.1 ± 0.6%, respectively, of
the total KvLQT1 expression in the ventricle and 79.3 ± 2.1 and 20.7 ± 2.1% in the atrium (mean ± S.E.,
n = 6). Relative expression of isoform 2 in the auricle
was significantly lower than in the ventricle (p < 0.05).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 2.
RT-PCR performed in total RNA extracted from
human ventricle (left panel) and atrium (right
panel). Oligonucleotide-specific primers used to detect
KvLQT1 isoform 1 (Iso 1), isoform 2 (Iso 2),
IsK and -actin ( -act) are indicated under
"Experimental Procedures." In the left panel, + and indicate experiments performed with or without reverse
transcriptase, respectively.
|
|

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 3.
Expression of mRNA for KvLQT1 isoform 1 and isoform 2 in human ventricle and atrium. Representative RNase
protection assays demonstrate the presence of the following: (i) a
393-bp protected fragment when ventricular or auricular RNA was
hybridized with the IsK probe (lane A); (ii) 274- and 212-bp
protected fragments when cardiac RNA was hybridized with the isoform 1 probe (lane B); (iii) 347- and 314-bp protected fragments
when RNA was hybridized with the isoform 2 probe (lane C)
(see "Experimental Procedures").
|
|
Functional Expression--
We first investigated whether KvLQT1
isoform 2 is fully processed to the cell membrane. To this end, COS-7
cells were transfected with epitope-tagged plasmids encoding KvLQT1
isoform 1 or KvLQT1 isoform 2. In our tagged constructs, the influenza
hemagglutinin (HA) flag was placed within the second extracellular loop
of the channel protein so that immunodetection of the tag could be
performed in non-permeabilized, non-fixed cells. Fig.
4A shows that specific immunostaining appeared as dots situated at the membrane surface of
cells transfected with tagged isoform 1 and isoform 2 but not at the
surface of cells transfected with an untagged plasmid. Western blot
experiments performed in COS-7 cells transfected with HA-tagged
plasmids confirmed that KvLQT1 isoform 1 and KvLQT1 isoform 2 encode
proteins at the molecular mass (e.g. 75 and 61 kDa,
respectively) predicted by their coding sequences. However, as shown in
Fig. 4B, two different bands for each isoform were visible
on the immunoblot. We hypothesized that the lower band may correspond
to the unglycosylated form of the protein, whereas the upper band
may correspond to the glycosylated form (1).

View larger version (64K):
[in this window]
[in a new window]
|
Fig. 4.
Immunodetection and immunoblots of HA-tagged
KvLQT1 in transfected COS-7 cells. A, immunolabeling of
non-permeabilized COS-7 cells transfected with HA-tagged KvLQT1 isoform
1 (Iso 1) or isoform 2 (Iso 2) plasmids or with
an untagged KvLQT1 isoform 1 plasmid (Cont). B,
immunoblots of cell lysates from COS-7 cells transfected with HA-tagged
KvLQT1 isoform 1 (Iso 1) or isoform 2 (Iso 2)
plasmids or with an untagged KvLQT1 isoform 1 plasmid (Cont). Molecular mass standards indicated are
-galactosidase (114 kDa) and bovine serum albumin
(81 kDa).
|
|
Functional expression experiments were conducted using untagged
plasmids. In these experiments, we used the intranuclear plasmid injection technique to ensure precise control of the relative expression of the different transgenes. COS-7 cells intranuclearly injected with 50 µg/ml KvLQT1 isoform 2 exhibited no detectable voltage-activated K+ current either in the absence (Fig.
5A) or presence (Fig.
6A) of the IsK regulator. To
investigate a possible effect related to isoform 2 expression, isoform
2 was co-injected with isoform 1 in the absence or presence of IsK. In
these experiments, a green fluorescence protein plasmid (pGFP) was used
as an inert plasmid substitute to ensure that the total plasmid
concentration injected was always 15 µg/ml. Cells injected with 5 µg/ml KvLQT1 isoform 1 (plus 10 µg/ml pCI-GFP) exhibited a
voltage-activated K+ current characterized by fast
activation kinetics and slow deactivation kinetics (Fig. 5A
and Table I). Cells co-injected with 5 µg/ml isoform 1 plus 5 µg/ml isoform 2 (plus pCI-GFP, 5 µg/ml)
displayed no detectable K+ current (not illustrated). Cells
co-injected with 5 µg/ml isoform 1 plus 1 µg/ml isoform 2 (plus
pCI-GFP, 9 µg/ml) exhibited a K+ current with similar
kinetics (Table I) but with markedly reduced amplitude (Fig. 5,
A and B). Fig. 5C shows that the
activation curve for the isoform 1-related K+ current was
not affected by the presence of isoform 2. In six different experiments
during which current tail protocols were applied, the ionic selectivity
of the KvLQT1 isoform 1 current was not modified by the presence of
isoform 2 (not illustrated). A series of experiments was also performed
in the presence of IsK (Fig. 6). Cells co-injected with 5 µg/ml
isoform 1 and 5 µg/ml IsK (plus pCI-GFP, 5 µg/ml) exhibited a
voltage-activated K+ current with a similar amplitude but
slower activation kinetics than cells injected with isoform 1 alone
(Table I). Again, co-injection of isoform 2 (1 µg/ml) together with
isoform 1 (5 µg/ml) consistently reduced the K+ current
amplitude in the presence of IsK (Fig. 6, A and
B). As shown in Fig. 6C, the presence of isoform
2 did not change the half-activation voltage but shifted the activation
threshold by
20 mV to a more depolarized voltage. Again, in the
presence of IsK, isoform 2 did not alter the activation and
deactivation kinetics of the isoform 1-related K+ current
(Table I). As summarized in Fig. 7,
isoform 2 reduced average current tail amplitude
dose-dependently. In the presence of 1, 2, and 5 µg/ml
isoform 2 but without IsK, the current tail amplitude induced by 5 µg/ml isoform 1 was reduced to 12.6, 4.7, and 0%, respectively, of
its control value. In the presence of 5 µg/ml IsK, the same
concentrations of isoform 2 reduced current tail amplitude to 47, 33, and 1.8% of its control value, respectively. Thus, the current related
to isoform 1 expression was more sensitive to the negative dominance of
isoform 2 in the absence than in the presence of IsK. Fig. 7 also shows
that the dominant negative effects produced by 1 µg/ml isoform 2 were
partially reversed by increasing the IsK plasmid concentration in the
injected mixture from 5 to 9 µg/ml.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5.
Whole cell K+ currents in COS-7
cells injected with KvLQT1 cDNA in the absence of IsK.
A, typical current traces obtained in a control COS-7 cell
(C), in a cell injected with 50 µg/ml pCI-KvLQT1 isoform 2 plasmid (Iso 2), in a cell injected with 5 µg/ml
pCI-KvLQT1 isoform 1 plasmid (Iso 1), and in a cell
co-injected with 5 µg/ml pCI-KvLQT1 isoform 1 plus 1 µg/ml
pCI-KvLQT1 isoform 2 plasmids (Iso 1 + Iso 2). In these
experiments, cells were also co-injected with a pCI-GFP plasmid, so the
total plasmid concentration was always 15 µg/ml. Superimposed current
traces were obtained by voltage steps, 20-mV increment, applied from
100 to +60 mV. Holding potential: 80 mV. Current scale: 5 pA/pF.
Time scale: 200 ms. B, tail current voltage relations for
KvLQT1 current. Circles are from a 5 µg/ml pCI-KvLQT1
isoform 1 plasmid-injected cell (same cell as in A).
Squares are from a 5 µg/ml pCI-KvLQT1 isoform 1 and 1 µg/ml pCI-KvLQT1 isoform 2 co-injected cell (different cell from
A). Triangles are from the 50 µg/ml pCI-KvLQT1
isoform 2-injected cell shown in A. C, averaged
KvLQT1 activation curves in the presence (squares,
n = 5) and absence (circles,
n = 5) of isoform 2. V0.5
denotes the potential for half-maximal activation.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 6.
Whole cell KvLQT1 K+ currents in
COS-7 cells co-injected with 5 µg/ml pCI-IsK plasmid. The same
abbreviations and protocols as in Fig. 5 are used. A,
current traces obtained in a cell injected with 50 µg/ml pCI-KvLQT1
isoform 2 plasmid (Iso 2), in a cell injected with 5 µg/ml
pCI-KvLQT1 isoform 1 plasmid (Iso 1), and in a cell
co-injected with 5 µg/ml pCI-KvLQT1 isoform 1 plus 1 µg/ml
pCI-KvLQT1 isoform 2 plasmids (Iso 1 + Iso 2). Time scale: 200 ms for Iso 2 and 500 ms for the Iso 1 and
Iso 1 + Iso 2. Current scale: 5 pA/pF. B,
corresponding current voltage relations in cells injected with isoform
1 (circles), isoform 1 plus isoform 2 (squares),
and with isoform 2 (triangles) cDNA. Same cells as in
A. C, averaged KvLQT1 activation curves in the
presence (squares, n = 4) and absence
(circles, n = 5) of isoform 2. V0.5 denotes the potential for half-maximal
activation.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Activation and deactivation time constants for the
time-dependent K+ current related to KvLQT1 isoform
1 expression and recorded in the presence or absence of KvLQT1 isoform
2 without and with IsK
The respective concentrations in the injection mixture for isoform 1 (Iso 1), isoform 2 (Iso 2), and IsK are given in µg/ml. In the
absence of IsK, current activation at +40 mV was fitted by a
biexponential with time constant, act fast, for the fast
component which accounted for 83% of the total current and act slow for the slow component. In the presence of IsK, current activation at +40 mV was fitted by a monoexponential relation. Both in the presence and absence of IsK, deactivation at 40 mV was
fitted by a single exponential. None of the differences reached statistical significance.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of KvLQT1 isoform 2 on the averaged
current tail density related to isoform 1 expression. The
left panel summarizes data obtained in the absence of IsK,
and the right panel summarizes data obtained in the presence
of IsK. The tail amplitude of the current related to isoform 1 expression was determined for variable isoform 1/isoform 2 cDNA
ratios in the injected mixture. Respective concentrations (µg/ml) in
the injection mixture for isoform 1 (Iso 1), isoform 2 (Iso 2), and IsK plasmids are indicated on the
x axis. Cells were also co-injected with a pCI-GFP plasmid so the total plasmid concentration was always 15 µg/ml. * denotes p < 0.05, and *** denotes p < 0.001 when comparison is made with cells injected with KvLQT1 isoform 1 alone. NS is non-significant. Numbers in
parentheses indicate n values.
|
|
 |
DISCUSSION |
Our results demonstrate that an N-terminal truncated isoform of
the KvLQT1 gene product is constitutively expressed in adult human heart and that this truncated isoform is a strong dominant negative of KvLQT1 isoform 1 K+ channels. The isoform 2 coding sequence reported here corresponds to that identified by Yang
et al. (7) and is referred to as variant B. In Northern blot
analysis, variant B expression was demonstrated in the heart but not in
other organs (7). At the genomic level (3), isoform 2 comprises exon
1b, exon 1, and exon 2 but lacks exon 1c of the KvLQT1 gene.
Exon-trapping and exon-connection experiments (3) clearly demonstrate
that isoforms 1 and 2 are splice variants. The KvLQT1 gene
spans a large region of at least 300 kilobase pairs and comprises a
minimum of 14 exons (3). Although the KvLQT1 promoter region
has not yet been identified, it can be inferred that isoform 1 and
isoform 2 use either the same promoter or, alternatively, a different
promoter as is the case for insulin-like growth factor 2, for example,
which has multiple promoters indicative of developmental and tissue
dependence (12).
The N-terminal end of KvLQT1 appears to be essential for the function
but not for the mediation of the subunit association to form the
tetrameric K+ channel complex. This situation differs
notably from the classic status for the voltage-dependent
K+ channel in which a domain designed as "NAB" domain,
conserved in the cytoplasmic N terminus, is crucial for
subunit-specific multimerization (13). Such a NAB domain is not found
in the long N-terminal sequence of KvLQT1 isoform 1. If it is assumed that KvLQT1 subunits form tetrameric channels, the coexpression of
full-length isoform 1 and truncated isoform 2 will lead to the
formation of homotetramers and of three classes of heterotetramers (with one, two, and three isoform 2 subunits). A binomial equation (14)
can be used to predict the percentage of channels expected for each
class of tetrameric channel by assuming the following: (i) the ratio of
the cDNA injected linearly predicts their resultant protein ratio;
(ii) the amplitude of induced current is dose-dependent on
the amount of injected cDNA; (iii) isoform 1 and isoform 2 subunits
assemble equally well as homo- or heteromeric channels; and (iv)
inclusion of at least one dominant negative subunit suppresses channel
function. According to this prediction, the remaining current amplitude
should be 47, 25, and 6.25% at isoform 1/isoform 2 ratios of 5/1, 5/2,
and 5/5 respectively. These values agree reasonably well with the
experimental data obtained in the presence of IsK but not in its
absence. Indeed, in the absence of IsK, the dominant negative effects
of isoform 2 were much more pronounced than in its presence and also
more marked than predicted with the binomial equation. Thus, our
results suggest the following: (i) the affinity of an isoform 1 subunit
for an isoform 2 subunit is higher than the affinity of an isoform 1 subunit for another isoform 1 subunit; (ii) the affinity of isoform 1 for isoform 2 subunits is decreased in the presence of the IsK
regulator; and (iii) inclusion of at least one isoform 2 subunit
suppresses channel function. These possibilities have important
physiological implications since the level of isoform 2 expression in a
tissue affect channel function differently depending on the
coexpression of the IsK regulator.
In our experimental conditions, the amplitude of the current induced by
KvLQT1 isoform 1 expression approximated that induced by coexpression
of isoform 1 plus IsK. However, previous findings for transfected COS
and Chinese hamster ovary cells as well as Xenopus oocytes
(4, 5, 15) showed that the channel regulator not only affected
activation kinetics (as with our cells) but also markedly increased its
density. Differences in experimental procedures may account for this
discrepancy, particularly with Xenopus oocytes in which
IsK-induced KvLQT1 current is highly temperature-dependent
(16). Previous experiments were performed at 20-22 °C, whereas in
our study a physiological temperature (37 °C) was chosen. Moreover,
we used direct cDNA injection, whereas previous studies chose
classical synthetic vector-mediated gene transfer.
In the heart ventricle, we observed that
30% KvLQT1 mRNA
expression is linked to isoform 2, and
70% is linked to isoform 1. This corresponds to the 5/2 isoform 1/isoform 2 cDNA ratio used in
COS-7 expression experiments. In the absence of IsK, this ratio
produced only 4.7% of the current detected in the absence of isoform
2. However, in the presence of 5 µg/ml IsK cDNA, injection of the
5/2 ratio produced 33% of the current detected in the absence of
isoform 2. Our findings suggest that the characteristics of endogenous
K+ current available for cardiac repolarization depend on
the relative expression of the following three different proteins:
isoform 1, isoform 2, and IsK. In the presence of IsK, isoform 2 expression shifted the KvLQT1 activation voltage toward more
depolarized potentials (see Fig. 6C), providing a better
representation of the activation curve of the slow component of the
endogenous cardiac delayed rectifier K+ current (17).
Moreover, the dominant negative effects of isoform 2 were increased in
the absence of IsK. This property may contribute to the inner ear
defect observed in IsK knock-out mice (18), although isoform 2 expression has not yet been demonstrated in the inner ear during
embryonic development or after birth.
Mutations in the following three genes are considered responsible for
the dominantly inherited Romano-Ward long QT syndrome: (i)
KvLQT1 localized to chromosome band 11p15.5, responsible for the most frequent LQT1 phenotype (19); (ii) HERG, a
K+ channel encoding gene at 7q35-36 and responsible for
the LQT2 phenotype (20); and (iii) SCN5A, a
voltage-dependent Na+ channel encoding gene at
3p21-24 responsible for the LQT3 phenotype (21). A fourth locus (LQT4)
has been identified by our laboratory at 4q25-27 (22). The vast
majority of the mutations responsible for LQT1 lie within the KvLQT1
transmembrane domains and the pore region (Refs. 1 and
2)2 and thus supposedly
affect both isoform 1 and isoform 2. In the heart of a Romano-Ward LQT1
patient, the delayed rectifier K+ current available for
repolarization would thus result from the association within
heterotetrameric channel protein complexes composed of the following:
(i) mutated isoform 1, (ii) wild-type isoform 1, (iii) mutated isoform
2, (iv) wild-type isoform 2, and (v) IsK proteins. This implies that
the functional effects of KvLQT1 mutations on isoform 2 negative dominance also need to be evaluated. Indeed, a mutation within
isoform 2 could very well reduce or even suppress its dominant negative
effect on wild-type isoform 1. Paradoxically, this would increase the
K+ current related to KvLQT1 isoform 1 expression from the
unaffected allele since less dominant negative isoform 2 transcripts
would be available. Therefore, depending on the effects of a familial mutation on isoform 2, the amplitude of the delayed rectifier K+ current available for cardiac repolarization (and hence
the phenotype) may vary. Current research programs in our laboratory
are exploring the consequences of familial mutations on isoform 2 negative function.
We thank Béatrice Leray and
Marie-Joseph Louerat for expert technical assistance with cell cultures
and plasmid amplifications.