THE JOURNAL OF BIOLOGICAL CHEMISTRY
21061–21066, 1998
© 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
HERG Channel Dysfunction in Human Long QT Syndrome
INTRACELLULAR TRANSPORT AND FUNCTIONAL DEFECTS*
(Received for publication, April 29, 1998, and in revised form, June 4, 1998)
Zhengfeng Zhou‡, Qiuming Gong, Miles L. Epstein§¶, and Craig T. Januaryi
From the Departments of Medicine (Cardiology Section) and §Anatomy, University of Wisconsin,
Madison, Wisconsin 53792
Mutations in HERG are associated with human chro-
mosome 7-linked congenital long QT (LQT-2) syndrome.
We used electrophysiological, biochemical, and immu-
nohistochemical methods to study the molecular mech-
anisms of HERG channel dysfunction caused by LQT-2
mutations. Wild type HERG and LQT-2 mutations were
studied by stable and transient expression in HEK 293
cells. We found that some mutations (Y611H and V822M)
caused defects in biosynthetic processing of HERG
channels with the protein retained in the endoplasmic
reticulum. Other mutations (I593R and G628S) were pro-
cessed similarly to wild type HERG protein, but these
mutations did not produce functional channels. In con-
trast, the T474I mutation expressed HERG current but
with altered gating properties. These findings suggest
that the loss of HERG channel function in LQT-2 muta-
tions is caused by multiple mechanisms including ab-
normal channel processing, the generation of nonfunc-
tional channels, and altered channel gating.
The congenital long QT syndrome is a disorder associated
with delayed cardiac repolarization and prolonged electrocar-
diographic QT intervals and the development of ventricular
arrhythmias (torsades de pointes) and sudden death (1). One
cause of congenital long QT syndrome is mutation in the hu-
man ether-a-go-go-related gene (HERG) producing chromosome
7-linked congenital long QT syndrome (LQT-2)1 (2). HERG
encodes a voltage-gated potassium channel (3). HERG channel
current has been shown to have properties similar to the rap-
idly activating delayed rectifier K1 current (IKr), and it plays
an important role in cardiac action potential repolarization in
the mammalian heart (4 – 6). HERG channels are also an im-
portant target for block by many drugs, and suppression of
HERG current causes action potential prolongation and car-
diac arrhythmias (5–12). Therefore, HERG channels have
emerged as an important cardiac ion channel.
More than 30 HERG mutations have been identified in
LQT-2 patients (2, 13–18). The electrophysiological properties
* This study was supported in part by the Oscar Rennebohm Foun-
dation. The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
A brief report of this work has appeared (20).
‡ Recipient of an American Heart Association Scientist Development
Grant.
¶Supported by National Institutes of Health Grant NS31385.
i To whom correspondence should be addressed: Dept. of Medicine
(Cardiology), University of Wisconsin Hospital and Clinics, Room H6/
352, 600 Highland Ave., Madison, WI 53792. Tel.: 608-262-5291; E-
mail: ctj@medicine.wisc.edu.
1
The abbreviations used are: LQT-2, human chromosome 7-linked
congenital long QT; PBS, phosphate-buffered saline; Endo H, endogly-
cosidase H; ER, endoplasmic reticulum; WT, wild type.
This paper is available on line at http://www.jbc.org
Vol. 273, No. 33, Issue of August 14, pp.
Printed in U.S.A.
of a few LQT-2 mutations have been studied in Xenopus oo-
cytes, where they have been shown to result in reduced or
absent HERG current (19). Although the molecular basis for
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some congenital human diseases is known to involve multiple
mechanisms including defective protein processing and abnor-
mal protein function, the molecular basis for long QT syndrome
has not been studied. In the present work, we used electro-
physiological, biochemical, and immunohistochemical methods
to study intracellular protein processing and functional prop-
erties of wild type and five LQT-2 mutant HERG channels. Our
findings show that some mutant HERG proteins are not pro-
cessed to the mature form of the channel. Other mutant HERG
proteins undergo normal processing but do not form functional
channels, or they gate abnormally. These findings provide new
information about the molecular mechanisms for the failure of
mutant LQT-2 channels to generate normal HERG current.
EXPERIMENTAL PROCEDURES
Site-directed Mutagenesis and Transfection—The HERG LQT-2 mu-
tations shown in Table I were generated by site-directed mutagenesis
using the Altered Site II in vitro mutagenesis system (Promega, Mad-
ison, WI). Each mutation was verified by DNA sequencing using auto-
matic DNA sequencer. Wild type and mutant cDNAs were subcloned
into pcDNA3 vector (Invitrogen, Carlsbad, CA), and HEK 293 cells were
transfected transiently or stably with these constructs using a lipo-
fectamine method as described previously (6). After transient transfec-
tion, HEK 293 cells were studied at 48 h. When transiently transfected
cells were used for patch clamp experiments, green fluorescent protein
cDNA (1 mg) was co-transfected with HERG cDNA (5 mg) to serve as an
indicator. In our experiments, .90% of green fluorescent protein-posi-
tive cells express HERG channels. For LQT-2 mutants, we established
four stable cell lines (T474I, I593R, Y611H, and G628S). LQT-2 mutant
stably transfected cell lines were selected by their G418 resistance
and identified by the presence of HERG protein on Western blot. A
mock-transfected (pcDNA3 vector) cell line was selected by its G418
resistance.
HERG Antibody—The HERG protein antibody was generated using
a fusion protein as antigen. The cDNA fragment of HERG encoding 181
amino acid residues from the carboxyl terminus was subcloned into the
pET-32a vector (Novagen, Madison, WI) to make a histidine-tagged
thioredoxin-HERG fusion construct. This construct was expressed in
Escherichia coli AD494(DE3)pLysS strain (Novagen). The histidine-
tagged thioredoxin-HERG fusion protein was purified using the His-
Bind Buffer Kit (Novagen). The purified fusion protein was injected into
rabbits to generate polyclonal antibody using a standard method (21).
The specificity of the polyclonal anti-HERG antibody was tested by
Western blot, immunohistochemical, and immunoprecipitation assays.
Western blot showed that the HERG antibody recognized HERG pro-
tein bands only in HERG-transfected HEK 293 cells and not in mock-
transfected or untransfected HEK 293 cells, and this reaction was
inhibited when the HERG antibody was preincubated with the fusion
protein. In addition, preimmune rabbit serum did not recognize HERG
protein on Western blot. Immunofluorescence experiments demon-
strated positive fluorescence staining only in HERG-transfected HEK
293 cells and not in mock-transfected and untransfected cells. Immu-
noprecipitation experiments also showed the absence of detectable
HERG protein bands in mock-transfected cells.
Patch Clamp Recordings—Membrane currents were recorded in
whole cell configuration using suction pipettes as described previously
21061
21062 Mechanisms of HERG Channel Dysfunction in LQT-2
TABLE I
LQT-2 mutations
Mutation HERG protein domain
T474I S2–S3 intracellular loop or S2
I593R S5 pore region
Y611H S5 pore region
G628S Pore region
V822M Cyclic nucleotide binding domain
(6, 22). Cells were superfused with HEPES-buffered Tyrode solution
containing (in mM) 137 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 glucose, and
10 HEPES (pH 7.4 with NaOH). The internal pipette solution contained
130 mM KCl, 1 mM MgCl2, 5 mM EGTA, 5 mM MgATP, 10 mM HEPES
(pH 7.2 with KOH). All experiments were performed at 22–23 °C. Data
are presented as mean 6 S.E. Student’s t test was used for statistical
analysis.
Western Blot Analysis—Membrane protein preparation and Western
blot procedures were previously described (6). The membrane proteins
were subjected to SDS-polyacrylamide gel electrophoresis and then
electrophoretically transferred onto nitrocellulose membranes. The ni-
trocellulose membranes were incubated with the HERG antiserum
(1:20,000 dilution) at room temperature overnight, and the antibody
was detected with an ECL detection kit (6).
For proteinase K treatment of cells, wild type HERG- and LQT-2
mutant-transfected cells were washed with PBS and incubated with 2
ml of buffer containing 10 mM HEPES, 150 mM NaCl, and 2 mM CaCl2
(pH 7.4) with or without 200 mg/ml proteinase K (Sigma) at 37 °C for 30
min. The proteinase K activity was stopped by adding 1.3 ml of ice-cold
PBS containing 6 mM phenylmethylsulfonyl fluoride and 25 mM EDTA.
The cells were then harvested and washed three times with ice-cold
PBS. The membrane proteins were isolated for Western blot analysis.
For experiments using endoglycosidase H (Endo H) treatment, 30 mg
of cell membrane protein was dissolved in 30 ml of 50 mM sodium citrate
buffer (pH 5.5) containing 15 mM b-mercaptoethanol and 0.12% SDS by
boiling for 2 min. Phenylmethylsulfonyl fluoride was added to a final
concentration of 0.5 mM followed by the addition of 15 milliunits of Endo
H. The mixture was incubated at 37 °C for 24 h. The reaction was
stopped by adding sample buffer (23) and boiling for 2 min.
Pulse-Chase Metabolic Labeling and Immunoprecipitation—Wild
type or mutant HERG-transfected cells were starved for 1 h in serum-
free Dulbecco’s modified Eagle’s medium lacking methionine and cys-
teine and containing 0.25% bovine serum albumin. Cells were then
incubated in the same medium containing [35S]methionine and [35S]cys-
teine (400 mci/ml). After 1 h of labeling, the medium was removed, and
cells were washed and chased in Dulbecco’s modified Eagle’s medium
with 2 mM unlabeled methionine and cysteine. At different time inter-
vals, the cells were washed and lysed in 500 ml of immunoprecipitation
buffer (50 mM Tris-HCl, pH 8.0 containing 150 mM NaCl, 1% Triton
X-100, 1% sodium deoxycholate, and 0.1% SDS) with a protease inhib-
itor mixture (6). After centrifugation at 14,000 rpm for 5 min at 4 °C,
the cell lysate was precleared by incubation with protein A-agarose
beads (Pierce). HERG antiserum (1:100 dilution) was then added, and
the mixture was incubated at 4 °C overnight. The antigen-antibody
complexes were isolated with protein A-agarose beads. The immuno-
precipitates were washed with the immunoprecipitation buffer. The
bound antigen was eluted from the protein A-agarose beads by sample
buffer (23), subjected to 7.5% SDS-polyacrylamide gel electrophoresis,
and visualized with autoradiography.
Immunofluorescence Microscopy—Wild type or mutant HERG-trans-
fected cells were fixed with 4% paraformaldehyde for 20 min at room
temperature. The cells were blocked with a buffer containing 5% goat
serum, 0.2% Triton X-100, and 0.05% azide in PBS and then incubated
with the HERG antiserum (1:3000 dilution) at 4 °C overnight. Follow-
ing washing with PBS, the cells were incubated with fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG secondary antibody
(Jackson, West Grove, PA) and washed with PBS. For double immuno-
fluorescence staining experiments, cells were also incubated with a
monoclonal antibody to BiP (Stressgen, Victoria, Canada) and Texas
Red-conjugated goat anti-mouse IgG secondary antibody (Jackson,
West Grove, PA). Immunofluorescence staining was viewed with a
Nikon fluorescence microscope (Nikon, Tokyo, Japan).
RESULTS
Electrophysiological Properties of Wild Type and Mutant
Channels— As shown in Table I, we performed site-directed
mutagenesis of HERG to generate five reported LQT-2 muta-
tions, T474I, I593R, Y611H, G628S, and V822M (2, 13–15) and
expressed these mutant HERG channels in HEK 293 cells. To
study the functional expression of the HERG wild type and
mutant channels, we performed patch clamp studies on tran-
siently and stably transfected cells. Patch clamp recordings
using transient transfection are shown in Fig. 1. Wild type
HERG current showed voltage-dependent activation with in-
ward rectification at more positive voltages as previously
shown (6, 10). For mutants I593R, Y611H, G628S, and V822M,
no HERG current was recorded; rather, only a small amplitude
current endogenous to HEK 293 cells was present (6, 10).
Similar results were obtained in stably transfected cells with
the mutants I593R, Y611H, and G628S (data not shown). In
contrast, the T474I mutation expressed functional channels
but with altered gating properties. The T474I mutation acti-
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vated at more negative voltages compared with wild type
HERG channels. Similar results were obtained with stably
transfected cells (data not shown). Fig. 1B shows the activation
curves for wild type and T474I mutant currents. When fit as a
Boltzmann function, the half-maximal activation voltages for
wild type and the T474I mutation were 215.9 6 1.1 mV (n 5 17
cells) and 243.2 6 1.2 mV (n 5 16 cells), respectively (p , 0.05).
The slope factors were 7.6 6 0.3 and 6.8 6 0.6, respectively (p .
0.05). Fig. 1C shows the I-V plots of wild type and T474I
mutant current density measured at the end of the depolariz-
ing step. It shows that the maximal outward current in the
T474I mutation was reached at 220 mV and that for wild type
it was reached at 0 mV. The maximal outward current densi-
ties for wild type and T474I were 34.7 6 4.0 and 26.6 6 5.6
pA/pF, respectively (p . 0.05). Fig. 1C also shows marked
inward rectification for both wild type and T474I currents at
more positive voltages, and in the voltage range 0 – 60 mV the
current amplitudes for the T474I mutation were decreased
compared with wild type current amplitude (p , 0.05 at each
voltage). Fig. 1, B and C, also shows that the threshold voltage
for eliciting current was shifted negatively for the T474I
mutation.
Protein Processing Studied by Western Blot Analysis—We
have shown previously that wild type HERG channel protein
expressed in HEK 293 cells consists of two forms on Western
blot, a 135-kDa (lower) band and a 155-kDa (upper) band, and
that both species involve N-linked glycosylation (6). We pro-
posed that the upper band was the complexly glycosylated,
mature form of the HERG channel and that the lower band was
a core-glycosylated, precursor form of the HERG channel. In
order to study the mechanisms accounting for dysfunctional
mutant channels, we analyzed HERG channel proteins by
Western blot as shown in Fig. 2. The HERG antibody did not
recognize protein in mock-transfected cells, whereas it recog-
nized HERG proteins in wild type-transfected and all five mu-
tant-transfected cells. Wild type HERG, as well as the T474I,
I593R, and G628S mutations, expressed two protein bands, a
lower band at 135 kDa and an upper broad band at 155 kDa.
These findings suggest that the T474I, I593R, and G628S mu-
tations undergo protein processing similar to that of wild type
HERG channels. In contrast, the Y611H and V822M mutations
expressed only the lower protein band at 135 kDa without the
upper protein band. The absence of the complexly glycosylated
form of the channel in the Y611H and V822M mutants strongly
suggests that a defect in protein processing causes failure of
channel maturation.
To study cell surface expression of wild type and LQT-2
mutant channels, intact cells stably transfected with wild type,
Y611H, and G628S were exposed to proteinase K to digest the
extracellular domains of HERG protein on the cell surface.
Proteinase K is a serine protease that cleaves peptide bonds
 Mechanisms of HERG Channel Dysfunction in LQT-2 21063

FIG. 1. Voltage clamp records of

wild type and LQT-2-associated mu-
tants expressed in HEK 293 cells. A,
representative currents are shown for
HEK 293 cells transfected with wild type
HERG and T474I, I593R, Y611H, G628S,
and V822M mutants (the numbers of cells
studied were 17, 16, 12, 12, 13, and 14,
respectively). In these experiments,
HERG current was activated by 4-s-long
depolarizing steps between 270 and 60
mV from a holding potential of 280 mV.
Cells were then clamped to 250 mV for

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6 s to record a tail current. B, activation
curves for wild type HERG (●) and T474I
(Œ) tail current (normalized to peak tail
current). C, I-V plot HERG current den-
sity (normalized to cell capacitance) of
wild type (●) and T474I (Œ) measured at
the end of the end of depolarizing steps.

FIG. 2. Western blot analysis of wild type and five LQT-2 mu-
tant HERG channels. HEK 293 cells were stably (mock, wild type,
T474I, I593R, Y611H, and G628S) or transiently (V822M) transfected.
30 mg of crude membrane proteins from stably transfected cells and 60
mg from transiently transfected cells were separated by a 7.5% SDS-
polyacrylamide gel. Similar results were obtained when wild type,
T474I, I593R, Y611H, and G628S were transiently transfected.
adjacent to the carboxylic group of aliphatic and aromatic
amino acids (24). Under our experimental conditions, the cells
remained intact after proteinase K treatment. Membrane pro-
teins were then isolated and analyzed by Western blot. As
shown in Fig. 3, the upper band of wild type HERG was sen-
sitive to the proteinase K treatment with the complete disap-
pearance of the band. This was associated with the appearance
of lower molecular mass bands of 60 –75 kDa that should rep-
resent degraded C terminus-containing fragments from the
upper band. Thus, HERG channels are sensitive to digestion by
extracellular proteases. In contrast, the 135-kDa band was
resistant to proteinase K treatment. These results support the
hypothesis that the upper band represents the mature form of
the HERG channel located in the surface membrane and that
the lower band is the precursor form located inside the cell.
Similar to wild type HERG, the upper band of the G628S
mutant was also sensitive to proteinase K treatment, indicat-
ing that the G628S mutant protein traffics normally into the
surface membrane. The lack of HERG current is probably due
to the functional defects in the channel protein. For Y611H, the
single lower band was resistant to proteinase K treatment.
Similar findings with proteinase K were obtained with the
V822M mutation. This suggests that these mutant channels
are not transported to the cell surface membrane.
In Fig. 4, we used Endo H to study further the intracellular
FIG. 3. Western blot analysis of HERG channel after treatment
of cells with proteinase K. The intact cells stably transfected with
wild type, Y611H, and G628S were treated (1) or not treated (2) with
proteinase K. The isolated membrane proteins were then analyzed on
7.5% SDS-polyacrylamide gel.
biochemical processing of HERG channel protein. Endo H di-
gests high mannose oligosaccharides that are added during
core glycosylation of newly synthesized proteins in the endo-
plasmic reticulum (ER), leaving a single GlcNAc residue at-
tached to the protein. Once proteins reach the medial Golgi,
they undergo complex oligosaccharide modification to become
Endo H-resistant (25). As shown in the Western blot analyses
in Fig. 4, the upper band of wild type HERG was resistant to
Endo H digestion. In contrast, the lower band of wild type
HERG was sensitive to Endo H treatment and was reduced in
molecular mass from 135 to about 132 kDa. This band is sim-
ilar in molecular mass to the smaller protein band observed
following N-glycosidase F treatment of wild type HERG and
may represent the deglycosylated HERG protein (6). These
findings provide further evidence that the 155-kDa band is the
complexly glycosylated, mature form of HERG channel and
that the 135-kDa band is a core-glycosylated precursor form of
the HERG channel. Fig. 4 also shows data for the Y611H
mutation. The 135-kDa band is sensitive to Endo H treatment
and showed a decreased molecular mass of about 132 kDa. This
suggests that Y611H mutant HERG protein undergoes core
glycosylation in the ER but then fails to be transported to the
medial Golgi to undergo complex glycosylation.
21064 Mechanisms of HERG Channel Dysfunction in LQT-2
FIG. 4. Western blot analysis of wild type and Y611H mutant
HERG proteins treated with Endo H. 30 mg of crude membrane
proteins from stably transfected cells of wild type and Y611H mutant
were treated (1) or not treated (2) with Endo H and separated on a
7.0% SDS-polyacrylamide gel.
Protein Processing Studied Using Metabolic Labeling—To
study the biosynthesis of wild type HERG and transport-defi-
cient LQT-2 mutant proteins, we performed pulse-chase exper-
iments using metabolic labeling. As shown in Fig. 5, the wild
type HERG protein was initially synthesized as a precursor
form of 135 kDa, which was gradually converted to a larger
form of 155 kDa. These findings provide direct evidence for the
precursor and product relationship of the 135- and 155-kDa
bands of HERG channel protein found in Western blot experi-
ments. The pulse-chase data also show that the rate of conver-
sion of the precursor form to the mature form is relatively slow
when compared with other membrane channel proteins ex-
pressed in mammalian cells (26, 27). For the Y611H, the mu-
tant protein was initially synthesized as a 135-kDa form; how-
ever, it was not converted to a larger molecular mass form for
chase times up to 24 h. Rather, the mutant protein underwent
progressive degradation with the appearance of smaller molec-
ular mass bands and with the nearly complete disappearance of
the 135-kDa band by 24 h. These findings show that the Y611H
mutation fails to generate the mature form of the channel
protein and that the immature form of the channel protein is
rapidly degraded.
Immunolocalization of Wild Type and Mutant HERG Chan-
nels—We studied the subcellular localization of HERG protein
in wild type and the five LQT-2 mutants by immunostaining
permeabilized transfected HEK 293 cells. As shown in Fig. 6,
the mock-transfected HEK 293 cells showed no detectable im-
munofluorescence staining. In cells transfected with wild type
HERG, the immunofluorescence staining pattern is visible
throughout the cells and their processes, showing the wide-
spread distribution of HERG protein. Similar immunofluores-
cence patterns were obtained with the T474I, I593R, and
G628S mutations. In contrast, cells transfected with the
Y611H and V822M mutations display an immunofluorescence
staining pattern that is more restricted to a perinuclear region.
This restricted intracellular distribution pattern is consistent
with the results of the Western blot experiments and shows
that the mutant proteins are retained intracellularly.
To study whether transport-deficient HERG mutant protein
is trapped in the ER, we performed double immunofluorescence
staining of HERG proteins and the ER-resident chaperone
protein BiP (28 –30). Fig. 7 shows results of cells transfected by
the Y611H mutation (A–D) and by wild type HERG (E–H). For
the Y611H mutant, the green HERG and red BiP immunore-
activities are restricted to the same perinuclear distribution.
This is confirmed in the superimposed image, which shows a
uniform yellow color, indicating that the mutant HERG and
BiP are colocalized. In contrast, for the wild type-transfected
cell, HERG protein is widely distributed, whereas BiP shows a
more restricted intracellular localization. These findings sug-
FIG. 5. Pulse-chase analysis of wild type and Y611H mutant
HERG proteins. Stably transfected cells with wild type and Y611H
mutant were metabolically labeled with [35S]methionine and [35S]cys-
teine for 1 h and chased with 2 mM unlabeled methionine and cysteine
for times between 0 and 24 h. The cell lysates were then immunopre-
cipitated by the HERG antibody and subjected to 7.5% SDS-polyacryl-
amide gel for analysis. Mock-transfected cells were analyzed after 1 h of
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labeling with [35S]methionine and [35S]cysteine at 0 h of chase time.
gest that some LQT-2 mutations result in intracellular protein
processing defects with retention of mutant HERG channel
protein in the ER.
DISCUSSION
Our results provide new data about the biosynthesis pat-
terns of a human, voltage-gated ion channel protein studied in
a heterologous mammalian expression system. Wild type
HERG channel protein is initially synthesized in the ER as the
core-glycosylated precursor form with a molecular mass of 135
kDa. It is then modified in the Golgi apparatus, where it
becomes the mature form of the channel with a molecular mass
of about 155 kDa. As we noted previously, much of the in-
creased protein size results from the addition of complex oligo-
saccharides by N-linked glycosylation, although other post-
translational modification may occur (6). The 155-kDa protein
is then transported into the plasma membrane. The fact that
only 155-kDa protein is sensitive to externally applied protein-
ase K suggests that 155-kDa proteins form functional HERG
channels in the cell surface membrane. Therefore, the 155-kDa
band observed on Western blot serves as a useful marker to
assess HERG protein maturation.
An important finding of the present study is that some
LQT-2 disease-causing mutations result in protein processing
defects that lead to failure of the channel protein to undergo
normal transport to the cell surface. Rather, mutant channels
are retained in the ER, where they are rapidly degraded. The
Y611H and V822M mutations are examples of this defect.
These mutations are most easily identified on Western blot
analysis, where they generate only the single lower molecular
mass precursor band. Several lines of evidence show that these
mutant channels are located intracellularly. They are not sen-
sitive to externally applied proteinase K. The Y611H mutation
is not converted to the mature form, as shown in the pulse-
chase experiments. The immunolocalization experiments con-
firm a restricted intracellular distribution for both mutations
and suggest that the mutant channels are retained in the ER.
The mechanism of the retention of the mutant HERG channel
protein in the ER is not identified in these experiments. It is
well recognized that export of newly synthesized proteins from
the ER to the Golgi is regulated by a “quality control” mecha-
nism (31, 32). This mechanism ensures that only properly
folded and assembled proteins leave the ER. Misfolded, unas-
sembled, and incompletely assembled subunit proteins are re-
tained in the ER and undergo degradation without reaching
the Golgi complex (33, 34). Thus, the LQT-2 mutations Y611H
and V822M may cause structural abnormalities with protein
misfolding or improper assembly, and they are retained and
degraded in the ER. Whether these channel mutations are
capable of forming functional channels is unknown.
 Mechanisms of HERG Channel Dysfunction in LQT-2
FIG. 6. Immunofluorescence stain-
ing for localization of wild type and
mutant HERG channels. HEK 239 cells
were stably (mock, wild type, T474I,
I593R, Y611H and G628S) or transiently
(V822M) transfected. For each transfec-
tion, the upper and lower photomicro-
graphs show the phase contrast light
transmission and immunofluorescence
images, respectively, for the same cell.
The magnification is identical to that in
Fig. 7.
Defective protein processing has been recognized as an im-
portant mechanism in some congenital human diseases. For
cystic fibrosis transmembrane conductance regulator chloride
channels, the most common mutation is the deletion of pheny-
lalanine at position 508 (DF508), which causes 70% of cystic
fibrosis cases. This mutation leads to retention of the channel
protein within the cell and failure of channel trafficking to the
plasma membrane (26). Protein processing defects have also
been shown to be important in the low density lipoprotein
receptor in familial hypercholesterolemia (35), in the Na1/
glucose symporter in glucose-galactose malabsorption (36), and
in several other congenital human diseases (37). Our results
with LQT-2 mutations are the first to show this mechanism for
a human, voltage-gated K1 channel.
In addition to transport-deficient mutants, other mecha-
nisms for HERG channel dysfunction in LQT-2 are shown by
21065
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FIG. 7. Double immunofluorescence
staining of HERG channels and the
ER-resident chaperone protein BiP.
Staining patterns are shown for the
Y611H (A–D) and wild type HERG (E–H)
channels. For each cell, a phase contrast
photomicrograph (A and E) and HERG
antibody immunofluorescence staining (B
and F, green images) and BiP antibody
immunofluorescence staining (C and G,
red images) patterns are shown. The two
immunofluorescence color images were
then superimposed (D and H, summed
colors giving yellow images). Calibration
bar, 20 mm.
our findings. The I593R and G628S mutations generate chan-
nel proteins that are processed similarly to wild type HERG
protein, yet these proteins fail to form functional ion channels.
The experiments showing sensitivity of the G628S mutant
protein to digestion by proteinase K confirm its cell surface
location. The immunolocalization experiments also show im-
munofluorescence staining patterns throughout the cell and its
processes. These mutations are close to the pore region of
HERG, which may result in defective channel gating or ion
permeation; hence, we conclude that these channels cannot
open or conduct ions normally. In a brief report, Nie et al. (38)
have suggested that the I593R mutation also is able to insert
into the plasma membrane, since epitope-tagged mutant pro-
tein can be detected on the cell surface. It is also interesting in
our experiments that the I593R mutation causes a weakly
stained upper band on Western blot. This could suggest that
21066 Mechanisms of HERG Channel Dysfunction in LQT-2
intracellular protein transport may not be completely normal
and that some LQT-2 mutations could have both protein proc-
essing and functional defects.
The T474I mutation represents another mechanism of chan-
nel dysfunction. This mutation generates HERG current with
altered gating properties. The channels activate at more neg-
ative voltages (activation V1⁄2 shifted negatively by 27.3 mV).
Although maximum current amplitude was similar to that of
wild type current, the peak of the I-V plot was shifted nega-
tively by about 20 mV as shown in Fig. 1C. At more positive
voltages, inward rectification was present for both the wild
type and T474I mutation, and there was a reduction in outward
current for the mutation compared with wild type current. This
decrease in current at more positive voltages may account for
its QT-prolonging phenotype. This mutation has been proposed
to be in the S2-S3 intracellular loop (3) or in the S2 intramem-
brane domain (39) of the putative channel protein structure.
Interestingly, another LQT-2 mutation in the S2 region
(N470D) also results in functional channels that gate at more
negative voltages (19). These results suggest that the S2 region
or adjacent S2-S3 loop contribute to the voltage dependence of
HERG channel activation. A role of the S2 region in channel
gating also has been reported in other voltage-gated K1
channels (40, 41).
LQT-2 is an autosomal dominant inherited disease with both
normal and mutant genes present in patients. The present
results were obtained by expressing LQT-2 mutations and
wild-type HERG protein as homomultimeric channels. Since
HERG proteins are thought to form tetrameric channels, the
co-expression of some LQT-2 mutations with wild type HERG
is thought to result in dominant negative suppression of HERG
current (19). These investigators have shown in Xenopus oo-
cytes that the G628S mutation caused marked dominant neg-
ative suppression of HERG current, suggesting co-assembly of
the G628S mutant protein with wild type protein. In a prelim-
inary report, Nie et al. (38) also have shown that the I593R
mutant is able to co-assemble with the wild type protein. As
shown in our experiments, these mutations are processed sim-
ilarly to wild type HERG channels. Whether the transport-
deficient mutants such as Y611H and V822M can co-assemble
with wild type HERG protein to cause similar dominant nega-
tive suppression is not known and is presently under
investigation.
In summary, our data identify three types of defects for
HERG mutants in LQT-2: 1) some mutations result in intra-
cellular transport defects with channel proteins retained in the
ER; 2) some mutations result in channel proteins that are
processed similarly to wild type channels but do not have
electrophysiological function; and 3) some mutations result in
channel proteins that gate abnormally. These findings provide
new information about the molecular mechanisms for HERG
channel dysfunction in LQT-2 patients. The mutations with
these different mechanisms may result in different severities of
LQT clinical phenotypes. More detailed clinical studies are
needed to permit correlation of the LQT-2 phenotypes with our
molecular findings.
Acknowledgments—We thank Drs. Gail A. Robertson, James D.
Bangs, Shetuan Zhang, Jonathan C. Makielski, and Timothy J. Kamp
for helpful advice and discussion.
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HERG Channel Dysfunction in Human Long QT Syndrome: INTRACELLULAR
TRANSPORT AND FUNCTIONAL DEFECTS
Zhengfeng Zhou, Qiuming Gong, Miles L. Epstein and Craig T. January
J. Biol. Chem. 1998, 273:21061-21066.
doi: 10.1074/jbc.273.33.21061

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