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INTRODUCTION |
Long QT syndrome is an inherited cardiac disorder that causes
sudden death from tachyarrhythmias. Five loci have been mapped to
11p15.5 (LQT1), 7q35-36 (LQT2), 3p21-24 (LQT3), 4q25-27 (LQT4), and
21q22-23 (LQT5 and LQT6) (1). Several other kindreds have not been
fully mapped. The gene responsible for LQT2 is the human ether-a-go-go-related gene
(HERG),1 which encodes the
pore-forming subunit of the rapidly activating delayed rectifier
potassium channel (Ikr) in cardiac myocytes (2, 3). MirP1,
the gene product of KCNE2 (LQT6) and minK, the gene product of KCNE1
(LQT5), are small membrane proteins that are capable of assembling with
HERG and regulating its function (4, 5).
The HERG protein contains a putative cyclic nucleotide binding domain
(CNBD) in its cytoplasmic carboxyl terminus (2). A splice-acceptor
mutation resulting in a C-terminal truncation lacking the entire CNBD
was one of the HERG mutants that was originally identified as a cause
of LQT2 (3). More recently, new LQT2 mutations have been discovered in
the putative CNBD (1, 6, 7).
Although the cardiac Ikr was originally thought to be
insensitive to cAMP-dependent regulation (3, 8), recent
studies provided evidence that HERG channels can be modulated via
cAMP-dependent protein kinase (PKA) phosphorylation pathway
(9-11). We reported that cAMP could regulate HERG by direct
interaction or through PKA-mediated phosphorylation of the channel
(12). In this study, we further investigate the direct effect of cAMP
on HERG channel properties through mutagenesis designed to disrupt the
ability of HERG to bind cAMP.
Previous studies of the CNBD in cyclic nucleotide-dependent
protein kinase and bacterial cAMP-regulated catabolic gene activator protein (CAP) identified six invariant key amino acid residues for
cyclic nucleotide binding (13). Three of these are glycine residues
that are essential for maintenance of the
-barrel structure that is
required to form a pocket for cyclic nucleotide binding. A glutamic
acid residue forms a hydrogen bond with the ribose 2'-OH of cAMP and an
arginine residue interacts with the phosphate of cAMP to form a salt
bridge (14). Mutagenesis of any of these six invariant amino acid
residues in CAP or in type I cAMP-dependent kinase impairs or
eliminates cAMP binding (15-20). Homologous amino acid residues are
also present in the CNBD of HERG. Here we describe the functional
consequences of mutations at these homologous residues (G806D,
E807(K/Q), and R823(W/Q)) designed to alter the ability of the channel
to bind cAMP. Because HERG assembles in a tetramer to form functional
channels, we also investigated the effects of co-expression of CNBD
mutants with wild-type HERG. We found that CNBD mutants lose specific
binding affinity for cAMP. They do not express currents as
homotetrameric channels. Moreover, the loss of function is not because
of defective protein biosynthesis or trafficking. Mutant HERG proteins
do not have a dominant-negative effect on wild-type current but do
alter voltage-dependent gating. Co-expression with the
accessory subunit KCNE2, converts the CNBD mutants into partially
dominant-negative suppressors.
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MATERIALS AND METHODS |
Mutagenesis of HERG cDNA--
Epitope tagging of HERG with
c-Myc has been described (4). Site-directed missense mutagenesis was
performed by an overlap extension PCR strategy (21). The mutated
XhoI-FseI fragments were subcloned into a
HERG-myc-pCI-neo plasmid. An inframe deletion mutation
NBD was constructed using a one-step PCR. The forward primer begins
at 2662 of HERG cDNA (5'-TACTGTGACCTACACAAGATCCATC) and the reverse
primer spanned an EcoRI site within the psp64 vector
(5'-GCTCGTATGTTGTGTGGAATTGTGAGCGG). The resulting PCR product was
digested with FseI and the ~300-base pair fragment was
purified and ligated into the parent construct HERG-psp64 digested with StuI and FseI sites. The resulting
NBD
mutation lacks the coding sequence for amino acids 815-825. N-terminal
GFP-tagged HERG plasmids were constructed by inserting WT-HERG or CNBD
mutants into pEGFPC3 at BamHI-HindIII sites.
Mutant constructs were verified by DNA sequence analyses.
Transfection of cDNA into Cultured Cells--
CHO cells were
maintained in Ham's F-12 media supplemented with 10% fetal calf serum
and penicillin/streptomycin at 37 °C under 5% CO2. Gene
transfer was performed using 1-5 µg of Qiagen Midiprep purified
plasmid DNA. Cells were electroporated at 225 V, 72 ohms, and 1800 microfarads in cytomix media (22). Cells were studied after
36-72 h after transfection. A plasmid containing the cDNA for GFP
was combined with HERG plasmids (in a ratio of 1:5 GFP/HERG) to
allow identification of transfected cells as previously described
(4). In co-transfection experiments current amplitudes were
normalized each day to a group of cells transfected with HERG alone to
control for day-to-day variations in transfection efficiency.
Electrophysiology--
Whole-cell patch clamp (23) membrane
currents were recorded as previously described (12). Extracellular
solution was NaCl 150 mM, 1.8 mM
CaCl2, 4 mM KCl, 1 mM
MgCl2, 5 mM glucose, and 10 mM
HEPES, pH 7.4, osmolarity ~300-330 mM. Pipette solution was 120 mM KCl, 4 mM Mg-ATP, 2 mM
MgSO4, 5 mM EGTA, 0.5 mM
CaCl2, 10 mM HEPES, pH 7.2. Osmolarity was
adjusted to maintain the pipette solution at 20-40 mM less
than the extracellular solution. Currents were evoked by 2-4 s
depolarizing steps to various levels from a holding potential of
70
mV followed by a repolarizing step to
40 mV and then to
120 mV
briefly to measure outward and inward tail currents. Current densities
were calculated by dividing maximal tail current peaks by cell
capacitance measurements. Voltage activation data were plotted as peak
tail current amplitudes against the test potential values and were
fitted to a Boltzmann function, I = 1/(1 + exp[(V1/2
V)/k]), where I is the measured tail current, V is the applied
membrane voltage, V1/2 is the voltage at
half-maximal activation, and k is the slope factor.
Immunoblot Analysis--
CHO or HEK293 cells were transiently
transfected using LipofectAMINE 2000 according to the manufacturer's
instructions (Life Technologies, Inc.). Cells were harvested 24 h
after transfection for biochemical analyses. Membrane preparations were
enriched for plasma membrane proteins and Western blots were carried
out as previously described (4, 24). To control for transfection efficiency between groups of cells, cDNA for Myc-tagged connexin43 was included. Proteins were separated by SDS-PAGE on a 7% gel. Anti-Myc 9E10 monoclonal antibody ascites fluid was used for Western blots to detect Myc-tagged HERG proteins.
Antibodies--
Anti-HERG polyclonal antisera was generated
against a GST fusion protein constructed from a PCR fragment encoding
amino acids 979-1159 ligated into pGEX-KG. The GST·HERG
fusion protein was produced in Escherichia coli strain BL21
and was partially purified on GSH-agarose. The protein was then
identified by Coomassie Blue staining after SDS-PAGE, and the
appropriate gel band was excised, electroeluted, and injected into New
Zealand White rabbits. The resulting antisera was assayed for ability
to recognize heterologously expressed HERG·Myc protein on immunoblot
and immunoprecipitation and was verified by comparison to anti-Myc
staining. Anti-Myc monoclonal antibody was used as ascites fluid
diluted into immunoblot blocking solution (Tris-buffered saline, 10%
nonfat dry milk, and 0.1% Tween 20).
Microscopy--
CHO cells were transiently transfected with
LipofectAMINE (as above) with GFP·HERG plasmids. After 24-48 h, the
transfected cells were resuspended and replated on glass coverslip
chambers (Nunc) and allowed to attach for at least 24 h prior to
imaging. GFP fluorescent images were collected using an Olympus I×70
microscope with 12 bit cooled Photometrics Sensys CCD camera. Image
analysis was performed using NIH Image, ImagePro, and Photoshop 7.0 software.
cAMP Binding Assay--
For [3H]cAMP binding,
HERG·Myc was immunoprecipitated from HEK293 cell lysates with
anti-Myc polyclonal antibody A-14 (Santa Cruz Biotechnology., Inc.) and
Ultralink immobilized protein G (Pierce). Precipitated proteins were
washed with ice-cold NDET (150 mM NaCl, 0.4% deoxycholic
acid, 5 mM EDTA, 25 mM Tris, 1% Nonidet P-40)
and suspended in cAMP binding buffer (50 mM Tris, 120 mM potassium phosphate, 10 mM
MgCl2, 1 mg/ml bovine serum albumin, 0.5 mg/ml Histone IIA,
2 mM EGTA, 1 mM
isobutylmethylxanthine (IBMX), pH 7.0). Determination of cyclic
nucleotide binding was performed as previously described (12).
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RESULTS |
Targeted Mutations of HERG CNBD Result in Functionally Defective
Channels--
To investigate the importance of the HERG nucleotide
binding domain, we constructed eight CNBD mutations targeted to disrupt cAMP binding (Fig. 1a). The
selected mutant amino acid residues are homologous to invariant
residues in other cyclic nucleotide-binding proteins that have been
identified as essential for normal conformation and cAMP binding
ability. Among these mutations, V822M and R823W are naturally occurring
LQT2 mutations (1, 6).
NBD is an inframe deletion mutant HERG
lacking 11 amino acids in the cyclic nucleotide binding site
(
815-825).

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Fig. 1.
Mutations in the cyclic nucleotide binding
domain cause loss of HERG function. a, mutations in
cAMP binding sites. Single-letter amino acid sequences are shown for
the region of mutations in the cAMP binding domain of HERG. The sites
of point mutations are indicated in bold. Both Glu-807
and Arg-823 have two different point mutations, respectively. Mutant
E807K/R823W was a double mutation. A deletion mutant NBD lacked 11 amino acid residues from 815 to 825. b, whole cell current
recordings in response to depolarizing steps in CHO cells transfected
with either wild-type HERG or CNBD mutants. CNBD mutations produce
little (R823Q) or no HERG current.
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We measured the whole cell K+ current from CHO cells
transiently transfected with each HERG mutant. Typical whole cell
current tracings are seen in Fig. 1b. Except for mutant
R823Q, none of the mutations could produce voltage-gated K+
current when expressed alone. We also did not detect any K+
current when mutant expressing cells were exposed to 1 mM
CPT-cAMP. Our results confirmed previous reports that LQT2
homotetramers involving the cyclic nucleotide binding domain (V822M and
S818L) do not form functional channels (25, 26).
One of the CNBD mutants, however, R823Q, did produce HERG current.
Comparison of current-voltage and voltage-dependent
activation showed that the current amplitude was greater for wild-type
HERG than R823Q homotetramers at all voltages (Fig.
2a). The maximal tail current
density measuring at
40 mV after a 2 s depolarizing test for
wild-type HERG is 54.5 ± 5.8 pA/pF (n = 8);
whereas R823Q is 11.3 ± 1.37 pA/pF (n = 33;
p < 0.001). When the data were independently normalized to unity (Fig. 2a, inset), the voltage
to achieve half-activation (V1/2) for R823Q was
shifted to more negative potentials when compared with wild-type HERG,
(V1/2 WT =
12.33 ± 0.23 mV; V1/2 R823Q =
20.09 ± 0.31 mV). There was no apparent change in the slope factor
(kWT = 10.13 ± 0.21 mV;
kR823Q = 9.21 ± 0.29 mV).

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Fig. 2.
Electrophysiological properties of mutant
R823Q. a, comparison of current density of wild-type
(WT) HERG ( ), and mutant R823Q ( ). Both
current-voltage (left graph) and voltage-activation curves
(right graph) show that current density is greater for
wild-type HERG than mutant R823Q at all voltages. Inset,
data are normalized to unity showing the hyperpolarizing shift in
voltage-dependent activation. b, effects of cAMP
on R823Q K+ current. Current-voltage (left
graph) and voltage-activation curves (right graph) show
that there was a 15% current reduction and a left shift in voltage
dependence of activation after cAMP treatment. Inset, data
normalized to the maximum current in the same curve.
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We have previously shown that cAMP decreased whole cell HERG current
amplitude with minimal alteration in voltage-dependent activation (12). This current inhibition was primarily achieved via
PKA-mediated phosphorylation of the channel protein. To assess the
effect of cAMP on mutant R823Q, a membrane-permeable cAMP analog
CPT-cAMP was applied to the extracellular solution at a concentration
of 500 µM. HERG K+ current was recorded 3 min
after CPT-cAMP addition. Fig. 2b shows that cAMP caused a
current reduction of ~15% and a small negative shift in
voltage-dependent activation
(V1/2 (baseline) =
20.44 ± 0.39 mV; V1/2(cAMP) =
26.86 ± 0.16 mV, n = 18).
Analysis of CNBD Mutant HERG Protein Expression--
Several LQT2
mutations are known to cause reduced Ikr by defective
transport of mutant proteins to the plasma membrane or through enhanced
protein degradation (24, 25, 27, 28). When misfolded mutant HERG
subunits assemble with wild-type protein, a dominant effect can be
exerted where both proteins are subjected to early and rapid
degradation (24). To assess whether the dysfunction of CNBD mutations
was also caused by abnormal protein biosynthesis or trafficking, we
examined the abundance and the membrane expression of c-Myc-tagged HERG
and CNBD mutants. Anti-Myc immunoblot analysis showed that the CNBD
mutants were expressed in abundance less than but comparable with
wild-type HERG (Fig. 3a).
There was no evidence of retention of CNBD mutant protein in
endoplasmic reticulum or Golgi compartments when fractions enriched for
microsomal membrane proteins were isolated from plasma membrane and
subjected to immunoblotting (Fig. 3b).

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Fig. 3.
Protein expression of WT and CNBD mutant
HERG. a, Western blot analysis of whole cell lysates. Cells
were harvested 24 h after transfection. GFP was used as a
transfection efficiency indicator and a negative control. b,
Western blot analysis of membrane (left panels) and
cytosolic proteins (right panels). Both wild-type and CNBD
mutant HERG are enriched in the plasma membrane fraction. As a control
for transfection efficiency Myc-tagged connexin43 was co-transfected
and appears in the lower left gel panel. c,
effect of temperature on the expression and maturation of wild-type and
CNBD mutations. 16 h after transfection, HEK293 cells were grown
for another 36 h either at 37 °C or 30 °C. Wild-type HERG
and CNBD mutations showed a mature glycosylated upper protein band
around 155 kDa. d, reducing temperature to 30 °C
can partially rescue mutant R823W function. Upper panel
shows examples of whole cell currents elicited by depolarizing steps
from cells transfected with wild-type HERG (left) or R823W
(right) after 36 h at 27 °C. Lower panel
shows summary data of the tail current density of R823W HERG grown
at 30 °C was ~7% of wild-type HERG.
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Recent reports have shown that loss of function associated with
misfolded and mistrafficked mutant HERG may be rescued by incubation at
low temperatures (24, 27, 28). We examined the effect of decreasing
culture temperature on the expression of CNBD mutations. Previous work
demonstrated that HERG existed as two bands on SDS-PAGE analysis when
expressed in HEK293 cells. A band migrating near 155 kDa represents the
mature fully glycosylated channel protein, and a band near 135-kDa
represents a core-glycosylated immature form of protein (29). As shown
in Fig. 3c, both wild-type HERG and the CNBD mutants were
expressed as two bands on Western blot. In all groups, both bands
migrated slightly slower when grown at 30 °C. No enhancement of
mutant protein abundance occurred in cells that were grown at the lower
temperature. Moreover, we were not able to detect any HERG current in
CHO cells transfected with the V822M or
NBD mutants after incubation
at 30 °C for 36-72 h. The R823W mutant, however, occasionally
produced a very small amount of current when the incubation temperature
was decreased to 27-30 °C for 24-48 h. Among 12 examined cells, 6 exhibited small HERG currents (Fig. 3d). The average tail
current density for wild-type and R823W at 30 °C was 117.6 ± 14.5 pA/pF and 8.25 ± 2.7 pA/pF, respectively (p < 0.01). Despite comparable protein abundance, low temperatures
resulted in a current that was expressed in only half of the cells with
an amplitude that was ~7% of the wild-type current. This argues
against misfolding and mistrafficking as a primary mechanism in the
loss of function for R823W.
To further examine whether the CNBD mutant proteins are expressed on
the plasma membrane, we visualized the location of HERG protein in
living cells using N-terminal GFP fusion proteins for both wild-type
and mutant HERG. GFP-tagged wild-type HERG produces K+
current similar to untagged wild-type HERG (data not shown). GFP-tagged
-adrenergic receptor (
-AR) was used as a control for plasma
membrane location (30). CNBD mutations exhibited a similar fluorescence
pattern as wild-type HERG and
-AR (Fig. 4). As frequently seen with transient
forced expression, a signal was seen in endoplasmic reticulum-Golgi
locations in all cells in addition to cell surface locations. These
results confirm that defective biosynthetic processing and protein
transport are not the major causes of the channel dysfunction in CNBD
mutations.

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Fig. 4.
Localization of WT and CNBD mutant HERG in
living cells by GFP fluorescence. CHO cells were transiently
transfected with cDNA of N-terminal-tagged GFP wild-type and CNBD
mutants for 48 h. Pairs of images for each cDNA show phase
contrast (left panel) and GFP fluorescence images of the
same cell (right panel). GFP· -AR was used as a positive
control for plasma membrane fluorescence. Wild-type and CNBD
mutants showed a similar fluorescent location to that of
GFP· -AR.
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Co-expression of CNBD Mutants with Wild-type HERG Produces
Functional Heterotetrameric Channels--
To assess the interactions
between wild-type and CNBD mutant proteins, we co-expressed equal
amounts of wild-type and mutant HERG in CHO cells and performed whole
cell patch clamp recordings. Compared with wild-type HERG homotetramer,
co-expression of wild-type with mutant HERG did not significantly alter
current amplitudes (Table I and
Fig. 5a). Alterations in
V1/2 observed in currents from cells co-expressing
wild-type HERG and CNBD mutants are evidence that support
heterotetrameric channel assembly (Table I). When cAMP was applied
during whole cell patch clamp measurements, the degree of current
amplitude suppression seen in cells transfected with wild-type HERG
alone was comparable with cells expressing both wild-type and CNBD
mutants (Fig. 5a). This is consistent with our finding that
cAMP-dependent current suppression is because of
PKA-mediated phosphorylation of HERG (12).

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Fig. 5.
Functional co-expression of CNBD mutants and
wild-type HERG. a, summary tail current density data
from cells expressing wild-type HERG (WT) or wild-type HERG
with CNBD mutants in a 1:1 molar ratio. Open bars represent
the current amplitude at baseline and filled bars show the amplitude
after cAMP treatment. b, similar to a, but for
cells also co-expressing KCNE2 plasmid. *, p 0.05 by
t test versus WT before treatment with cAMP.
c, immunoblot analysis of HERG and KCNE2 protein expression.
Cells were co-transfected with 1 µg of untagged wild-type HERG
cDNA, 1 µg of Myc-tagged wild-type or CNBD mutant HERG cDNA
and 1 µg of HA-tagged KCNE2 cDNA. The membrane was probed with
HERG-specific polyclonal antiserum to demonstrate the total HEG protein
abundance (top gel). Re-probing with anti-Myc antibody shows
the expression of the Myc-tagged subunits (middle gel) and
re-probing with anti-HA antibody shows the expression of KCNE2
(bottom gel).
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Association of KCNE2 Uncovers an LQT2 Phenotype for CNBD HERG
Mutants--
The current belief is that HERG is complexed to small
subunits of the KCNE family to form native Ikr channels
in vivo (4, 5). Furthermore, we reported that both KCNE1 and
KCNE2 altered the effects of cAMP regulation of the HERG channel (12).
To examine the effects of CNBD mutations on Ikr, we
co-expressed wild-type HERG and CNBD mutants with KCNE2. KCNE2 failed
to induce currents from the functionally defective CNBD mutants in the
absence of co-expressed wild-type HERG. The presence of KCNE2, however, did confer a suppressive effect on CNBD mutants when co-expressed with
wild-type HERG. The exception to this was the R823Q mutant that can
sustain current as a homotetramer (Table I and Fig. 5b).
Compared with expression of wild type alone, current density for
co-transfection with V822M, R823W, and
NBD were decreased 35.3%,
49.6, and 44.6%, respectively. Whereas this is a significant suppression, it does not represent a completely dominant-negative effect as seen with several other LQT2 mutants (24, 31). To exclude the
possibility that co-expression KCNE2 with wild-type and mutant HERG
would lower the expression of channel protein, we examined the
abundance of total HERG protein by immunoblot. As seen in Fig.
5c, the total HERG protein abundance was comparable in the
wild-type alone and mutant co-expression groups when KCNE2 was present.
Assuming random assembly of an equal amount of wild-type and mutant
subunits a binomial distribution would predict that a completely
dominant mutation should reduce the current amplitude by >90% whereas
the CNBD mutants range from ~35-50% current reduction. The cAMP
effects on wild-type and mutant heteromultimers are altered when KCNE2
is co-expressed (Table I and Fig. 5b). In cells expressing WT-HERG-KCNE2, cAMP induced a leftward shift in
voltage-dependent activation and did not significantly
alter current amplitudes, which is consistent with our previous report
(12). Addition of cAMP, however, causes an augmentation of current
density for WT-V822M-KCNE2 channel and a suppression of the others. The
V1/2 for WT-CNBD mutant-KCNE2 were all shifted to
more negative voltages by cAMP.
Fig. 6 shows a more detailed analysis of
the effects of the LQT2 mutant V822M on WT HERG current. CHO cells were
transfected with constant amount (5 µg) of wild-type HERG cDNA
with varying ratios of V822M. Expression of V822M alone produced no
current (Fig. 1b), whereas co-transfection of equal amounts
of wild-type and V822M cDNA (50% wild type) yielded a current
similar in magnitude to that of wild-type HERG (Fig. 5a and
Table I). Significant current suppression was only noted when the
fraction of transfected mutant cDNA to wild-type cDNA was
greater than 0.5. When current amplitudes were plotted against the
fraction of wild-type HERG cDNA (WT/[WT + V822M]), the data best
fit a model where mutant subunits exert no dominant effect over
co-assembled wild-type subunits. This is described by the binomial
expression for tetramer association, I = IWT(1
(fmut)4), where I is the
macroscopic current amplitude, IWT is the
current amplitude when all subunits are wild type and
fmut is the fraction of subunits that are mutant
(as shown by the solid curve on Fig. 6c). This
equation is reduced from the general expression, I = (fwt)n
Z0 IWT + n(fWT)(n
1)
fmut Zx
IWT + ... + (fmut)n
Zn IWT, where
fWT is the fraction of subunits that are wild
type, fmut is the fraction of mutant subunits,
and Zx is the fraction of wild-type current
passed by channels with x mutant subunits. For a completely
dominant-negative suppression, Zx will be 0 whenever x
1, and the binomial expression would be
reduced to I = IWT × (fWT)4 (Fig.
6c, dotted curve). When co-expressed with KCNE2,
however, mutant HERG subunit showed partial dominant-negative
suppression of current (Fig. 5b). Fig. 6c also
showed the current density at varying ratios of V822M HERG associated
with KCNE2 with a current expression intermediate between a completely
dominant and non-dominant phenotype. These data points were not well
fit to simple binomial expressions where the Zx
was varied to values between 0 and 1. If we assume that through the
association of KCNE2, mutant HERG subunits become dominant-negative,
then the predicted current density will be best described by a function
where a fraction of channels that are assembled with KCNE2 shows a
dominant-negative effect, and the remaining channels behave in a
completely non-dominant manner. Such a model may be expressed as
I = IWT[P × (fWT)4 + (1
P) × (1
(1
fWT)4)], where P is the
fraction of mutant HERG associated with KCNE2. Our experimental data
are best fit to this equation with P = 0.36 (Fig.
6c, dashed curve) corresponding to 36% of HERG channels associated with KCNE2.

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Fig. 6.
Requirement of KCNE2 for dominant phenotype
of V822M HERG. a, representative whole cell current of
CHO cells co-transfected with wild-type (WT) and V822M HERG
in different molar ratios as indicated above. b, summary
current-voltage (left graph) and voltage-activation curves
(right graph). Insets, same data as b
normalized to unity. c, tail current density in cells
transfected with varying ratios of wild type and V822M. Dotted
line describes a binomial function for a completely dominant
mutant in a tetrameric assembly. Solid line represents a
binomial function where one wild-type subunit in a tetramer confers
full functionality. Dashed line represents partial dominant
suppression of mutant subunit associated with KCNE2.
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cAMP Binding Capacity of Wild-type HERG Protein and CNBD
Mutations--
Our previous work demonstrated that cAMP binds to HERG
protein with a dissociation constant of ~ 40 µM
(12). We measured the specific [3H]cAMP binding of
wild-type HERG and the CNBD mutants in the presence and absence of
excess unlabeled cAMP. As shown in Fig.
7, in the presence of 400 µM unlabeled cAMP, only the wild-type HERG protein demonstrated a significant decrease in cAMP binding (p < 0.05), indicating a specific affinity for cAMP. The LQT2 mutant
V822M and CNBD deletion mutant
NBD did not demonstrate specific
binding to cAMP. The binding capacity for R823Q and R823W were
significantly reduced compared with wild-type HERG. The loss of
specific [3H]cAMP binding capacity of HERG CNBD mutants
suggests that the ability to bind cAMP is essential for normal channel
function.

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Fig. 7.
Comparison of cAMP binding capacity of
wild-type and CNBD mutant HERG. Binding capacity is expressed as
the ratio values relative to [3H]cAMP bound to
immunoprecipitated wild-type HERG in the absence of excess (400 µM) unlabeled cAMP. Data were obtained from four
independent experiments and normalized to 100% for comparison.
Asterisk represents p 0.05 by
t test versus wild-type control in the absence of
unlabeled cAMP.
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DISCUSSION |
In this study we have examined the function of CNBD in HERG
K+ channels in a heterologous mammalian expression system.
We selectively introduced mutations at the key sites in the cyclic
nucleotide binding domain, which we predicted would disrupt cAMP
binding. With the exclusion of R823Q, we found that the CNBD mutants do not form functional channels as homotetramers despite normal protein expression. Immunoblot analyses and microscopy of GFP fusion proteins demonstrated that CNBD mutant HERG surface expression was similar to
wild-type HERG at physiological temperature. Although reduced incubation temperatures have been shown to rescue several other LQT2
mutants (24, 28), this failed to restore function to our CNBD mutants.
Thus, defective protein transport to the plasma membrane does not
appear to be the major reason for the functional defect in CNBD
mutants. Furthermore, we show that CNBD mutant proteins have a
decreased or absent capability to bind cAMP when compared with
wild-type HERG. This suggests that the capacity to bind cAMP may be
essential for normal HERG channel function.
Although CNBD mutant homotetramers did not express voltage-gated
K+ currents, when co-expressed with wild-type HERG,
functional heterotetrameric channels were detected. Unlike several
other LQT2 mutations, the CNBD mutants that we examined demonstrated no
dominant-negative suppression of wild-type current. This finding is
consistent with previous reports of other C-terminal mutations (26,
28). The resulting current density values in our experiments
co-expressing V822M with wild-type HERG at varying molar ratios is best
fit to a model where mutant subunits are entirely non-dominant. We speculate that only one subunit capable of binding cAMP is needed for
full channel function. Whether cAMP must actually bind to the channel
for it to be active, however, is still not known.
Our model of a single cAMP binding subunit resulting in a functional
channel has similarities to the activation of PKA and cyclic
nucleotide-gated channels. The regulatory subunit of PKA has two cAMP
binding sites, and mutational analysis demonstrates that activation of
type I PKA requires only one of the cAMP binding sites to be capable of
binding cAMP for preservation of function (14). Cyclic nucleotide-gated
ion channels also contain a CNBD at the C terminus, analogous to HERG.
Binding of a cyclic nucleotide to a single subunit is sufficient to
produce significant activation of the channel. All four channel
subunits, however, must bind to cyclic nucleotide for full current
activation (32).
How the CNBD affects channel activity is still unknown. One
possibility is that CNBD binding of cAMP is required for activation of
the channel through a conformational change in the channel protein.
Kupershmidt et al. (33) reported that upon expressing a
recombinant HERG that entirely lacked the CNBD, a Ikr-like
current was produced. Deletion of the entire CNBD however, is more
likely to cause ancillary structural changes and channel perturbations in addition to loss of cAMP binding compared with point mutations that
leave the CNBD intact but unable to bind cAMP. Liu et al. (32) have proposed a model where the CNBDs within a tetramer of cyclic
nucleotide-gated channels act as ligand-bound dimers. Such a situation
seems unlikely for HERG given the absence of dominant current
suppression when CNBD mutants were co-expressed with wild-type
HERG. For HERG CNBD to act as ligand-bound dimers we would have
expected that increasing ratios of V822M/wild-type HERG would have
suppressed the current in a fashion consistent with a 2-fold stoichiometry.
Another role that CNBD may play is to modulate HERG current by
interacting with KCNE2. The interaction between HERG and a small
integral membrane subunit such as KCNE2 can alter HERG channel current
to more resemble native IKr (5). Moreover, we have shown
that the co-expression of either minK or KCNE2 can significantly alter
the cAMP-dependent response of HERG activity to favor
direct effects as opposed to PKA-mediated effects (12). Although
co-expression of CNBD mutant with WT HERG does not suppress current in
a dominant-negative fashion, the addition of KCNE2 results in a
mutant-dependent decrease in peak current density of about
40%. These results support a role for the CNBD of HERG in KCNE2
control of IKr. Clearly, further experiments are necessary
to define the functional and structural interaction between the CNBD
and KCNE2 in modulation of channel activity. Furthermore, it now
appears that KCNE2 may play a pivotal role in mechanisms that create
the LQTS phenotype for some LQT2 mutations. That the CNBD mutants were
only partially dominant over wild-type function may be consistent with
reports of another CNBD LQT2 mutant (26), and the observation that the
clinical presentations of C-terminal mutations are less severe than
those involving transmembrane and pore regions of HERG (7). Our present data however, suggest that association of KCNE2 with the HERG channel
uncovers the dominant phenotype of CNBD LQT2 mutants. The degree of
suppression we observed with differing ratios of mutant and wild-type
HERG are best explained by multiple channel populations, some complexed
with KCNE2 and some not. The stoichiometry of the KCNEs complexed to
their respective
-subunits is not absolutely known; therefore, our
results may alternatively represent variable degrees of mutant
suppression resulting from different combinations of subunits.