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KCNE4canco-associatewiththe I
Ks
(KCNQ1–KCNE1)
channel complex
Lauren J. Manderfield
1
and Alfred L. George Jr
1,2
1 Department of Pharmacology, Vanderbilt University, Nashville, TN, USA
2 Department of Medicine, Vanderbilt University, Nashville, TN, USA
Voltage-gated potassium (K
V
) channels are essential
for a variety of physiological processes, including the
control of membrane potential, electrical excitability
and solute transport. Many K
V
channels are hetero-
multimeric protein complexes consisting of pore-form-
ing subunits, encoded by a large number of distinct
potassium channel gene subfamilies, and accessory
proteins. At least four classes of K
V
accessory subunit
have been identified, including K
V
b [1–4], KChIP [5,6],
KChAP [7] and the KCNE proteins [8]. Accessory
proteins provide an important mechanism for achiev-
ing functional diversity amongst potassium channels.
KCNE proteins are small, single transmembrane
domain subunits that function to control or modulate
K
V
channels in the heart, cochlea, small intestine and
other tissues. KCNE1, originally named minK, was
the first identified member of this family [9], and its
expression has been demonstrated in several tissues,
including the kidney, heart and uterus [10–12]. More
than a decade later, the paralogous minK-related
Keywords
accessory subunits; KCNE4; KCNQ1; K
V
7.1;
potassium channel
Correspondence
A. L. George Jr, 529 Light Hall,
2215 Garland Avenue, Nashville,
TN 37232-0275, USA
Fax: +1 615 936 2661
Tel: +1 615 936 2660
E-mail: al.george@vanderbilt.edu
(Received 24 August 2007, revised 11
December 2007, accepted 15 January 2008)
doi:10.1111/j.1742-4658.2008.06294.x
Voltage-gated potassium (K
V
) channels can form heteromultimeric com-
plexes with a variety of accessory subunits, including KCNE proteins. Het-
erologous expression studies have demonstrated diverse functional effects
of KCNE subunits on several K
V
channels, including KCNQ1 (K
V
7.1)
that, together with KCNE1, generates the slow-delayed rectifier current
(I
Ks
) important for cardiac repolarization. In particular, KCNE4 exerts a
strong inhibitory effect on KCNQ1 and other K
V
channels, raising the pos-
sibility that this accessory subunit is an important potassium current modu-
lator. A polyclonal KCNE4 antibody was developed to determine the
human tissue expression pattern and to investigate the biochemical associa-
tions of this protein with KCNQ1. We found that KCNE4 is widely and
variably expressed in several human tissues, with greatest abundance in
brain, liver and testis. In heterologous expression experiments, immunopre-
cipitation followed by immunoblotting was used to establish that KCNE4
directly associates with KCNQ1, and canco-associate together with
KCNE1 in the same KCNQ1 complex to form a ‘triple subunit’ complex
(KCNE1–KCNQ1–KCNE4). We also used cell surface biotinylation to
demonstrate that KCNE4 does not impair plasma membrane expression of
either KCNQ1 or the triple subunit complex, indicating that biophysical
mechanisms probably underlie the inhibitory effects of KCNE4. The obser-
vation that multiple KCNE proteins canco-associatewith and modulate
KCNQ1 channels to produce biochemically diverse channel complexes has
important implications for understanding K
V
channel regulation in human
physiology.
Abbreviations
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, haemagglutinin; I
Ks
, cardiac slow-delayed rectifier current; I
to
, cardiac transient
outward current; K
V
channel, voltage-gated potassium channel.
1336 FEBS Journal 275 (2008) 1336–1349 ª 2008 The Authors Journal compilation ª 2008 FEBS
peptides encoded by human genes KCNE2, KCNE3,
KCNE4 and KCNE5 were identified [13,14]. Although
different KCNE proteins functionally interact with a
variety of K
V
channels, all KCNE proteins have been
shown to modulate heterologously expressed KCNQ1
(K
V
7.1) with distinct effects [15–20]. The co-expres-
sion of KCNE1 with KCNQ1 reconstitutes I
Ks
,a
potassium current important for myocardial repo-
larization and the most well-studied physiological
phenomenon mediated by a KCNE subunit [15,16].
Biophysical and biochemical experiments have demon-
strated that two KCNE1 subunits associate with each
tetramer of KCNQ1 [21]. All other KCNE proteins
exert functional effects on KCNQ1 ranging from
potentiation (KCNE3) [18] to suppression (KCNE4,
KCNE5) [19,20] of channel activity. Given the varied
KCNQ1 phenotypes generated by different KCNE
proteins, and the overlapping expression patterns of
these subunits [22], there may be multiple and diverse
KCNE–KCNQ1 interactions within the same cells or
tissues.
One of the least characterized, but biophysically
potent, members of this family is KCNE4. When
expressed in heterologous systems, KCNE4 exerts dra-
matic functional effects on KCNQ1 channels. Grunnet
et al. [19] first demonstrated complete suppression of
KCNQ1 activity by KCNE4 in both oocytes and Chi-
nese hamster ovary cells. In addition to KCNQ1, other
K
V
channels, including K
V
1.1 and K
V
1.3, are also
inhibited by KCNE4 [23]. KCNE4can also exert func-
tional inhibition on K
V
channels even in the presence
of other accessory subunits. For example, KCNE4 can
inhibit I
Ks
stably expressed in Chinese hamster ovary
cells [24], as well as the transient outward current (I
to
)
reconstituted in heterologous systems by the co-expres-
sion of K
V
4.3 with KChIP2 [25].
KCNE4 inhibition of heterologously expressed
KCNQ1 in the presence or absence of KCNE1, the
overlapping mRNA expression patterns of KCNE and
KCNQ1 genes, and the observation that the KCNQ1
tetramer can accommodate at least two KCNE sub-
units has raised the possibility that multiple accessory
subunits can interact simultaneously with KCNQ1
channels. In this study, the expression of KCNE4 pro-
tein was demonstrated in human tissues. We further
show that KCNE4 physically interacts with KCNQ1,
but does not suppress channel activity by impairing
the cell surface expression of this K
V
channel. Finally,
we demonstrated that KCNE1 and KCNE4can simul-
taneously associate with KCNQ1 to form KCNE1–
KCNQ1–KCNE4 channel complexes expressed at the
plasma membrane. Together, our findings contribute
to the understanding of the role of KCNE4 as a
potentially important regulator of KCNQ1 and other
K
V
channels.
Results
Characterization of KCNE4 antibody
A rabbit polyclonal antibody raised against a C-termi-
nal epitope of human KCNE4 was characterized. The
antibody (anti-KCNE4) recognized a single band of
approximately 28 kDa on immunoblots of proteins
from cells transfected with an epitope (haemagglutinin,
HA)-tagged KCNE4 cDNA, but did not recognize spe-
cific bands in non-transfected cells or when excess anti-
genic peptide was present to block immunodetection
(Fig. 1A). An identical band was observed when the
immunoblots were probed with anti-HA, but not when
the immunoblots were probed with pre-immune rabbit
serum. In separate experiments designed to demon-
strate specificity, anti-KCNE4 recognized a band of
approximately 25 kDa only in cells transfected with
untagged KCNE4, and did not exhibit cross-reactivity
with other human KCNE proteins (Fig. 1B). The
observed mass of the native KCNE4 protein
( 25 kDa) is slightly larger than that predicted from
the ORF ( 18 kDa), and we speculate that this dis-
crepancy may be the result of anomalous electropho-
retic migration of KCNE4 on SDS-PAGE, as observed
with other small, highly acidic proteins [26,27]. The
molecular mass difference between tagged and untag-
ged KCNE4 ( 28 versus 25 kDa) is very consistent
with the predicted mass of the epitope tag ( 3 kDa).
All subsequent biochemical experiments utilized untag-
ged KCNE4 unless otherwise stated.
Expression of KCNE4 in human tissues
Anti-KCNE4 was utilized to probe immunoblots pre-
pared with a panel of 16 human tissues to determine
the expression pattern of this protein (Fig. 1C).
KCNE4 exhibited the highest levels of expression in
the brain, liver and testis. By contrast, colon, lung,
placenta and prostate had little or no KCNE4 expres-
sion. Many of the tissues examined had been studied
previously by real-time quantitative RT-PCR [22] and,
in most tissues, mRNA levels were concordant with
protein levels. Interestingly, brain and liver, two of the
tissues with high levels of KCNE4 protein expression,
had low KCNE4 mRNA expression [22]. Conversely,
placenta and spleen, two of the tissues withthe highest
KCNE4 mRNA expression, had low or no KCNE4
protein expression [22]. We inferred from these data
that post-transcriptional mechanisms contribute to the
L. J. Manderfield and A. L. George Jr KCNE4 co-association with KCNQ1–KCNE1 channels
FEBS Journal 275 (2008) 1336–1349 ª 2008 The Authors Journal compilation ª 2008 FEBS 1337
steady-state level of KCNE4 protein in certain tissues.
A similar lack of correlation between mRNA levels
and protein expression has also been observed for
KCNE1 throughout regions of the heart [28].
KCNE4 interacts with KCNQ1
We and others have demonstrated that KCNE4 inhib-
its KCNQ1 function in vitro [19,24]. We hypothesized
that this effect is a result of a direct interaction of
KCNE4 with KCNQ1. This hypothesis was tested by
examining whether KCNE4 forms protein complexes
with KCNQ1. KCNE4 and KCNQ1 were transiently
co-expressed in COS-M6 cells, the protein complexes
were immunoprecipitated from cellular lysates using a
KCNQ1 antibody (anti-KCNQ1), and the immuno-
blots were probed with anti-KCNE4. The results indi-
cated that KCNE4 interacts with KCNQ1 (Fig. 2).
The specificity of this interaction was demonstrated by
several control experiments. Pre-incubation of anti-
KCNQ1 with antigenic peptide prevented the immuno-
precipitation of KCNQ1 or KCNE4 (Fig. 2, lane 3).
When cell lysates from cells expressing only KCNQ1
or KCNE4 were mixed, interaction was not observed,
thus excluding a post-lysis artefact (Fig. 2, lane 4).
Neither KCNE4 nor KCNQ1 was immunoprecipitated
with Protein-G Sepharose beads alone (Fig. 2, lane 5)
or pre-immune serum matched to the species origin of
anti-KCNQ1 (Fig. 2, lane 6). When KCNQ1 and
KCNE4 were expressed alone (Fig. 2, lanes 7 and 8),
no cross-reactivity was observed between the respective
antibodies. These experiments offer conclusive evidence
that KCNE4 forms channel complexes with KCNQ1
in vitro.
The suppression of I
Ks
by KCNE4 could poten-
tially be explained by displacement or sequestra-
tion of KCNE1 by KCNE4. The possibility that
KCNE4 can displace KCNE1 from KCNQ1 was
+
KCNE4
+
KCNE4 +
antigenic
peptide
+
Rabbit pre-
immune
serum
HAIB:
+
––
50 kDa
30
25
37 kDa
IB:GAPDH
50 kDa
30
25
IB:KCNE4
NT
KCNE1
KCNE2
KCNE3
IB:KCNE4
IB:GAPDH
50 kDa
30
25
35 kDa
KCNE4
KCNE5
–
–
Brain
Heart
Colon
Ileum
Kidney
Liver
Lung
Ovary
Pancreas
Palcenta
Prostae
Muscle
Spleen
Testicle
Thymus
Uterus
A
B
C
Fig. 1. Specificity of anti-KCNE4. (A) Whole cell lysates from COS-M6 cells transfected with HA epitope-tagged KCNE4 (+) or non-transfect-
ed cells ()) were subjected to SDS-PAGE and western blotting withthe indicated immunoreagent. A specific protein with a molecular mass
of approximately 28 kDa was identified by immunoblotting with either anti-HA or anti-KCNE4. (B) Western blot of lysates derived from non-
transfected cells (NT) or cells expressing each individual KCNE protein probed for KCNE4. All lysates were also probed for GAPDH in order
to demonstrate protein expression. (C) Western blot of lysates derived from specified human tissues probed for KCNE4. Brain lysates were
derived from the cerebellum. Colon lysates were derived from the descending colon. Heart lysates were derived from the left ventricle.
Muscle lysates were derived from skeletal muscle (quadriceps). Supplementary Table S1 provides age and sex information for the tissue
donors. All lysates were also probed for GAPDH in order to demonstrate protein expression.
KCNE4 co-association with KCNQ1–KCNE1 channels L. J. Manderfield and A. L. George Jr
1338 FEBS Journal 275 (2008) 1336–1349 ª 2008 The Authors Journal compilation ª 2008 FEBS
first examined by testing whether KCNE1 remained
associated with KCNQ1 even in the presence of
KCNE4. Cells were transiently transfected with
KCNE4, KCNE1
3FLAG
and KCNQ1, and the cell
lysates were subjected to immunoprecipitation with
anti-KCNQ1. In these experiments, KCNE4 and
KCNE1 interactions with KCNQ1 were detected
by immunoblot using anti-KCNE4 or anti-FLAG.
Figure 3 illustrates that, in cells transfected with all
three channel subunits, anti-KCNQ1 immunoprecipi-
tates both KCNE1 and KCNE4. This interaction
was specific for the KCNQ1 antibody, did not occur
during processing of the cell lysates, and could not
be attributed to antibody cross-reactivity or non-spe-
cific interactions with Protein-G Sepharose. In the
immunoblots in Fig. 3, KCNE4 appears as a dou-
blet, which may be a result of post-translational pro-
cessing. These experiments demonstrate that KCNQ1
can associate with both KCNE1 and KCNE4 in the
same population of cells, providing evidence that dis-
tinct KCNQ1–KCNE1 and KCNQ1–KCNE4 com-
plexes are formed.
We next examined the hypothesis that KCNE4
directly binds and sequesters KCNE1 was examined as
an explanation of why KCNE4 functionally suppresses
I
Ks
[24]. KCNE4 and an epitope-tagged KCNE1
(KCNE1
3FLAG
) were co-expressed and immunoprecipi-
tated with anti-FLAG, followed by immunoblotting
using anti-KCNE4. There was no evidence of
KCNE1–KCNE4 interaction when both subunits were
co-expressed (Fig. 4A). Furthermore, both KCNE
subunits were expressed at the plasma membrane
(Fig. 4B), and this observation rules out intracellular
degradation as an explanation for a lack of KCNE1–
KCNE4 interaction [29]. The apparent decrease in
KCNE1 at the plasma membrane in the presence of
KCNE4 is not sufficient to explain the dominant effect
of KCNE4 on I
Ks
. The multiple molecular mass bands
ranging from approximately 15 to 25 kDa observed in
the immunoblots probed for KCNE1
3FLAG
represent
differentially glycosylated forms of this protein that
have been described previously [30].
KCNE1 and KCNE4 co-assemble with KCNQ1
The existence of KCNQ1–KCNE1 complexes in the
experiment described above would be expected to con-
tribute some level of I
Ks
expression. However, this was
not observed in previous electrophysiological studies
when KCNQ1, KCNE1 and KCNE4 were co-
expressed [24]. One possible explanation is that all
three subunits form a triple subunit complex (i.e.
KCNE1–KCNQ1–KCNE4) in which KCNE4 exerts a
dominant inhibitory effect. To probe for the existence
of KCNE1–KCNQ1–KCNE4, we examined whether
both KCNE1 and KCNE4 could be incorporated into
the same KCNQ1 complex. As stated above, we estab-
lished that these two different KCNE subtypes did not
interact with each other in the absence of KCNQ1
(Fig. 3) and that both subtypes bound KCNQ1 when
co-expressed in the same cell population (Fig. 4). To
detect KCNE1–KCNQ1–KCNE4 complexes, cells were
transfected with all three channel subunits, the cell
lysates were immunoprecipitated using anti-FLAG
(recognizes KCNE1), and KCNE4 was immunodetect-
ed. Figure 5 illustrates that anti-FLAG was indeed
able to co-immunoprecipitate both KCNQ1 and
KCNE4, thus providing evidence for the existence of
KCNE1–KCNQ1–KCNE4 complexes. These interac-
tions were specific, as demonstrated by the absence of
co-immunoprecipitation of KCNE4 and KCNQ1 in
any of the control conditions. Therefore, these data
represent biochemical evidence for a KCNQ1 chan-
nel complex incorporating two different KCNE
subunits.
+–+++++–
–++++++–
KCNQ1
KCNE4
30
50 kDa
25
IP:KCNQ1
IB:KCNE4
75 kDa
IB:KCNQ1
25
30 kDa
IB:KCNE4
IP:KCNQ1
IB:KCNQ1
75 kDa
50
12 456783
Fig. 2. KCNE4 interacts with KCNQ1. Whole cell lysates were
immunoprecipitated with anti-KCNQ1 and then subjected to SDS-
PAGE and western blot analysis. Lane 1, non-transfected COS-M6
cells. Lane 2, cells transfected with KCNQ1 and KCNE4. Lane 3,
cells transfected with KCNQ1 and KCNE4, but anti-KCNQ1 used for
immunoprecipitation was pre-incubated with antigenic peptide.
Lane 4, mixture of lysates from cells expressing either KCNQ1 or
KCNE4 only combined prior to immunoprecipitation. Lane 5,
KCNQ1 and KCNE4 transfected cells immunoprecipitated with Pro-
tein-G Sepharose. Lane 6, KCNQ1 and KCNE4 transfected cells
immunoprecipitated with goat pre-immune serum. Lane 7, cells
expressing KCNQ1 only. Lane 8, cells expressing KCNE4 only. The
first immunoblot shows samples immunoprecipitated with anti-
KCNQ1 and immunoblotted for KCNQ1. The second image shows
a KCNQ1 immunoblot of the initial lysates demonstrating KCNQ1
expression. The third immunoblot shows the anti-KCNQ1 immuno-
precipitated samples which were probed with anti-KCNE4. The final
image shows a KCNE4 immunoblot of the initial lysates demon-
strating KCNE4 expression.
L. J. Manderfield and A. L. George Jr KCNE4 co-association with KCNQ1–KCNE1 channels
FEBS Journal 275 (2008) 1336–1349 ª 2008 The Authors Journal compilation ª 2008 FEBS 1339
KCNE4 does not inhibit KCNQ1 trafficking
One potential mechanism by which KCNE4 could sup-
press I
Ks
is by impairing KCNQ1 cell surface expres-
sion. This possibility was examined previously by
Grunnet et al. [19], where it was demonstrated that
KCNE4 did not decrease KCNQ1 plasma membrane
expression in Xenopus oocytes assayed by cell surface
biotinylation. Here we investigated whether KCNE4
expression affected KCNQ1 plasma membrane expres-
sion in mammalian cells, and whether KCNQ1,
KCNE1 and KCNE4 reached the cell surface when
co-expressed.
We examined KCNQ1 trafficking when KCNQ1
was either expressed alone, with KCNE1 or with
KCNE4. KCNQ1 co-expression with KCNE1 served
as a control for KCNQ1 trafficking, as it is presumed
that KCNE1–KCNQ1 complexes reach the plasma
membrane to enable functional I
Ks
. Total protein,
non-biotinylated and biotinylated fractions from cells
expressing KCNQ1 only, KCNQ1 with KCNE1
3FLAG
and KCNQ1 with KCNE4
3HA
were collected and
probed with anti-KCNQ1. Figure 6 illustrates that
KCNQ1 cell surface expression was not inhibited by
the expression of either KCNE1 or KCNE4. KCNQ1
was specifically detected in all protein fractions under
all three conditions. KCNQ1 was not immunodetected
in any fraction from non-transfected cells (data not
shown). Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) protein was immunodetected in only the
total protein and non-biotinylated fractions, demon-
strating clean separation of plasma membrane
+++++++–
+++++++–
+++++++–
–+–
+––
––+
KCNQ1
KCNE4
KCNE1
3FLAG
75 kDa
50
IP:KCNQ1
IB:KCNQ1
75 kDa
IB:KCNQ1
25
30 kDa
IB:KCNE4
25 kDa
15
IB:FLAG
50 kDa
30
25
IP:KCNQ1
IB:KCNE4
30 kDa
25
15
IP:KCNQ1
IB:FLAG
87654321
91011
Fig. 3. KCNE4 interacts with KCNQ1 in the presence of KCNE1. Whole cell lysates were immunoprecipitated with anti-KCNQ1 and then
subjected to SDS-PAGE and western blot analysis. Lane 1, non-transfected COS-M6 cells. Lane 2, cells transfected with KCNQ1, KCNE4
and KCNE1
3FLAG
. Lane 3, cells transfected with KCNQ1, KCNE4 and KCNE1
3FLAG
, but anti-KCNQ1 used for immunoprecipitation was pre-
incubated with an antigenic peptide. Lane 4, mixture of lysates from cells expressing KCNQ1, KCNE4 or KCNE1
3FLAG
only were combined
prior to immunoprecipitation. Lane 5, mixture of lysates from cells expressing either KCNQ1 and KCNE4 or KCNE1
3FLAG
only combined prior
to immunoprecipitation. Lane 6, mixture of lysates from cells expressing either KCNQ1 and KCNE1
3FLAG
or KCNE4 only combined prior to
immunoprecipitation. Lane 7, KCNQ1, KCNE4 and KCNE1
3FLAG
transfected cells immunoprecipitated with Protein-G Sepharose. Lane 8,
KCNQ1, KCNE4 and KCNE1
3FLAG
transfected cells immunoprecipitated with goat pre-immune serum. Lane 9, cells expressing KCNQ1 only.
Lane 10, cells expressing KCNE1
3FLAG
only. Lane 11, cells expressing KCNE4 only. The first row of immunoblots shows samples immuno-
precipitated with anti-KCNQ1 and immunoblotted for KCNQ1. The second row of immunoblots shows the initial lysates confirming KCNQ1
expression. The third row of immunoblots shows the anti-KCNQ1 immunoprecipitated samples that were probed withtheKCNE4 antibody.
The fourth row of immunoblots shows the initial lysates confirming KCNE4 expression. The fifth row of immunoblots shows the anti-KCNQ1
immunoprecipitated samples which were probed withthe FLAG antibody. The sixth row of immunoblots shows the initial lysates confirming
KCNE1 expression.
KCNE4 co-association with KCNQ1–KCNE1 channels L. J. Manderfield and A. L. George Jr
1340 FEBS Journal 275 (2008) 1336–1349 ª 2008 The Authors Journal compilation ª 2008 FEBS
(biotinylated fraction) and cytosolic proteins (non-bio-
tinylated fraction). Similarly, calnexin was only immu-
nodetected in the total protein and non-biotinylated
fractions (data not shown). The percentage of KCNQ1
protein present at the cell surface was not significantly
different between the three conditions (KCNQ1 alone,
49.8 ± 8.4%; KCNQ1 plus KCNE1, 40.1 ± 7.6%;
KCNQ1 plus KCNE4, 31.0 ± 3.4%; mean ± SEM;
n = 3 each; Fig. 6), indicating that impaired KCNQ1
cell surface expression cannot explain the suppression
of I
Ks
by KCNE4.
Next, we examined the ability of KCNE proteins to
traffic to the plasma membrane in the presence of
KCNQ1. Total protein, non-biotinylated and biotiny-
lated fractions from cells transfected with KCNQ1
plus KCNE1 and KCNQ1 plus KCNE4 were collected
and probed for KCNE1 (anti-FLAG) or KCNE4
(anti-HA). Figure 7A illustrates that KCNE1 reaches
the cell surface in the presence of KCNQ1. Multiple
bands representing the differentially glycosylated forms
of KCNE1 were detected, indicating normal matura-
tion of the protein. Figure 7B illustrates that KCNE4
also traffics to the cell surface in the presence of
KCNQ1.
Finally, we examined if KCNE1–KCNQ1–KCNE4
complexes exist at the cell surface. The three channel
subunits were co-expressed, and total protein, non-bio-
tinylated and biotinylated fractions were collected.
Figure 7C illustrates qualitatively that KCNQ1,
KCNE1 and KCNE4 were all detected in the biotiny-
lated fraction, suggesting expression of the KCNE1–
KCNQ1–KCNE4 complex at the surface plasma mem-
brane (Fig. 7C).
Discussion
In this study, we demonstrated the expression pattern
of KCNE4 protein in human tissues, and provided
in vitro biochemical evidence that KCNE4 interacts
with KCNQ1. We also determined that KCNE1 and
KCNE4 can simultaneously co-associatewith KCNQ1
to form KCNE1–KCNQ1–KCNE4 ‘triple’ subunit
complexes, and that the inhibitory effect of KCNE4
cannot be explained by impaired cell surface
30 kDa
75 kDa
IB:Transferrin
75 kDa
IB:Transferrin
50 kDa
IB:KCNE4
Biotinylated Fractions
25
15
E1NT
E1+E4
IB:FLAG
30
25
NT E4 E1+E4
+ – + + + + + –
– + + + + + + –
KCNE1
3FLAG
KCNE4
IB:FLAG
IP:FLAG
IB:KCNE4
IB:KCNE4
IP:FLAG
IB:KCNE1
30 kDa
25
15
25 kDa
15
30
50 kDa
25
30 kDa
25
1 8 7 6 5 4 3 2
A
B
Fig. 4. KCNE4 does not interact with KCNE1. (A) Whole cell lysates
were immunoprecipitated with anti-FLAG and then subjected to
SDS-PAGE and western blot analysis. Lane 1, non-transfected
COS-M6 cells. Lane 2, cells transfected with KCNE1
3FLAG
and
KCNE4. Lane 3, cells transfected with KCNE1
3FLAG
and KCNE4, but
anti-FLAG used for immunoprecipitation was pre-incubated with an
antigenic peptide. Lane 4, mixture of lysates from cells expressing
either KCNE4 or KCNE1
3FLAG
only combined prior to immunoprecipi-
tation. Lane 5, KCNE1
3FLAG
and KCNE4 transfected cells immuno-
precipitated with Protein-G Sepharose. Lane 6, KCNE1
3FLAG
and
KCNE4 transfected cells immunoprecipitated with mouse pre-
immune serum. Lane 7, cells expressing KCNE1
3FLAG
only. Lane 8,
cells expressing KCNE4 only. The first immunoblot shows samples
immunoprecipitated with anti-FLAG and immunoblotted for KCNE1.
The second blot shows a FLAG immunoblot of the initial lysates
confirming KCNE1 expression. The third immunoblot shows the
anti-FLAG immunoprecipitated samples which were probed with
the KCNE4 antibody. The fourth image shows a KCNE4 immunoblot
of the initial lysates confirming KCNE4 expression. (B) Representa-
tive western blots examining KCNE1 and KCNE4 protein trafficking
to the plasma membrane. The protein lysate composition of each
lane is denoted as NT for non-transfected, E1 for KCNE1
3FLAG
,E4
for KCNE4 and E1 + E4 for KCNE1
3FLAG
+KCNE4. Only the bio-
tinylated fractions are illustrated. Lysates were probed with anti-
FLAG to demonstrate the presence of KCNE1, or anti-KCNE4 to
demonstrate the presence of KCNE4. All lysates were also probed
with an antibody against transferrin to demonstrate complete sepa-
ration of biotinylated proteins.
L. J. Manderfield and A. L. George Jr KCNE4 co-association with KCNQ1–KCNE1 channels
FEBS Journal 275 (2008) 1336–1349 ª 2008 The Authors Journal compilation ª 2008 FEBS 1341
expression. The observation that multiple KCNE pro-
teins can associate with and modulate KCNQ1 chan-
nels at the plasma membrane to produce biochemically
diverse channel complexes has important implications
for understanding physiologically relevant channel reg-
ulation.
+++++++–
+++++++–
+++++++–
+––
––+
–+–
KCNQ1
KCNE4
KCNE1
3FLAG
87654321
9
10 11
30 kDa
25
IB:KCNE4
30
50 kDa
25
IP:FLAG
IB:KCNE4
25 kDa
15
IB:FLAG
30 kDa
25
15
IP:FLAG
IB:KCNE1
75 kDa
50
IP:FLAG
IB:KCNQ1
IB:KCNQ1
75 kDa
Fig. 5. KCNE1 and KCNE4 co-assemble with KCNQ1. Whole cell lysates were immunoprecipitated with anti-FLAG and then subjected to
SDS-PAGE and western blot analysis. All lane compositions are the same as defined in Fig. 3. The first row of immunoblots shows samples
immunoprecipitated with anti-FLAG and immunoblotted for KCNE1. The second row shows FLAG immunoblots of the initial lysates confirm-
ing KCNE1 expression. The third row of immunoblots shows the anti-FLAG immunoprecipitated samples which were probed with KCNQ1
antibody. The fourth row of immunoblots shows the initial lysates confirming KCNQ1 expression. The fifth row of immunoblots shows the
anti-FLAG immunoprecipitated samples that were probed withtheKCNE4 antibody. The sixth row of immunoblots shows the initial lysates
confirming KCNE4 expression.
35 kDa
75 kDa
50
Total protein
KCNQ1 alone
KCNQ1 + KCNE1
IB:KCNQ1
IB:GAPDH
KCNQ1 + KCNE4
0
10
20
30
40
50
60
70
NS
Non-biotinylated
Biotinylated
Total protein
Non-biotinylated
Biotinylated
Total protein
Non-biotinylated
Biotinylated
% Surface expression
KCNQ1 + KCNE4
KCNQ1 + KCNE1
KCNQ1 alone
Fig. 6. KCNE4 does not inhibit KCNQ1 cell surface expression. Representative western blots examining KCNQ1 trafficking to the
plasma membrane when expressed alone, with KCNE1 or with KCNE4. The protein lysate composition of each lane is denoted as total
protein, non-biotinylated or biotinylated for the three conditions examined. A bar graph illustrates the relative proportions of surface-
expressed KCNQ1 as a percentage of total protein for the three conditions (NS, non-significant). All lysates were probed with
anti-KCNQ1 to demonstrate KCNQ1 expression and with anti-GAPDH in order to demonstrate complete separation of biotinylated and
non-biotinylated proteins.
KCNE4 co-association with KCNQ1–KCNE1 channels L. J. Manderfield and A. L. George Jr
1342 FEBS Journal 275 (2008) 1336–1349 ª 2008 The Authors Journal compilation ª 2008 FEBS
Diversity of KCNE4 expression
KCNE4 protein is expressed widely in both excitable
and non-excitable human tissues, suggesting that this
subunit could impact a wide array of cell types and
physiological functions. Excitable tissues that express
KCNE4 include the brain, heart and skeletal muscle.
The expression of KCNE4 in brain, coupled with the
previously demonstrated inhibitory effect of this sub-
unit on K
V
1.1 and K
V
1.3 channels, raises the possibil-
ity of important physiological effects on neuronal
excitability, synaptic neurotransmission and impulse
conduction [23]. In the heart, we speculate that
KCNE4 exerts a suppressive effect on I
Ks
and may be
critical for the regulation of cardiac repolarization.
The fact that I
Ks
has been detected in cardiac myocytes
suggests that KCNE4 does not associate with all avail-
able KCNQ1 channels, possibly because of excess
KCNE1, the most highly expressed KCNE mRNA in
heart [24]. We previously showed significant changes in
KCNE4 mRNA expression in the setting of end-stage
cardiomyopathy [24] and there have been recent sug-
gestions of an influence of KCNE4 polymorphisms on
the susceptibility to atrial arrhythmias [31]. There are
no data available on the role of KCNE4 in skeletal
muscle.
KCNE4 also exhibits robust expression in epithelial
tissues, including the pancreas and kidney. Several
studies have indicated that pancreatic acinar cells gen-
erate a slowly activating potassium current resembling
I
Ks
, and that this current promotes a driving force
for efficient chloride secretion [32,33]. Conceivably,
KCNE4 could modulate pancreatic exocrine secretion
through attenuation of the I
Ks
-like current. In the kid-
ney, KCNE4 may interact with KCNQ1, which is
localized to the lumenal membranes of the mid to late
proximal tubule [15,34,35]. Evidence from studies of
knockout mice has revealed that KCNQ1 is important
for proximal tubule repolarization and the mainte-
nance of the electrical driving force for Na
+
reabsorp-
tion under conditions of enhanced electrogenic
reabsorption [36]. We speculate that renal expression
of KCNE4 may modulate this channel activity and
affect reabsorption, but additional studies are needed
to demonstrate the subcellular location of this protein
in the proximal tubule.
Multiple KCNE subunits canco-associate with
KCNQ1
There is substantial evidence for the functional diver-
sity of potassium channels as a result of the differential
assembly of channel subunits. Functional diversity can
B
35 kDa
IB:GAPDH
50 kDa
T
NB
B
KCNQ1 + KCNE4
30
25
IB:HA
C
IB:KCNQ1
IB:GAPDH
IB:FLAG
KCNQ1 + KCNE1 + KCNE4
B
75 kDa
50
35 kDa
30 kDa
25
15
30
25
50 kDa
IB:HA
A
IB:GAPDH
35 kDa
KCNQ1 + KCNE1
IB:FLAG
NB
B
30 kDa
25
15
T
T
NB
Fig. 7. KCNE trafficking in the presence of KCNQ1, and KCNQ1
trafficking in the presence of multiple KCNE proteins. Representa-
tive western blots examining KCNE protein trafficking to the
plasma membrane when expressed with KCNQ1. The protein
lysate composition of each lane is denoted as T for total protein,
NB for non-biotinylated proteins and B for biotinylated proteins. All
lysates were probed with anti-GAPDH in order to demonstrate
complete separation of biotinylated and non-biotinylated proteins.
(A) KCNE1 traffics to the plasma membrane in the presence of
KCNQ1. All lysates were probed with anti-FLAG to demonstrate
the presence of KCNE1. (B) KCNE4 traffics to the plasma mem-
brane in the presence of KCNQ1. All lysates were probed with anti-
HA to demonstrate the presence of KCNE4. (C) KCNQ1 traffics to
the plasma membrane in the presence of KCNE1 and KCNE4. The
top immunoblot was probed with anti-KCNQ1 to examine KCNQ1
expression. The second immunoblot was probed with anti-FLAG to
examine KCNE1 expression. The third immunoblot was probed
with anti-HA to examine KCNE4 expression.
L. J. Manderfield and A. L. George Jr KCNE4 co-association with KCNQ1–KCNE1 channels
FEBS Journal 275 (2008) 1336–1349 ª 2008 The Authors Journal compilation ª 2008 FEBS 1343
be achieved through either the assembly of different
pore-forming subunits, as illustrated by the generation
of the neuronal M-current through the co-assembly of
KCNQ2 and KCNQ3 (K
V
7.2 and K
V
7.3) [37], or by
the association of channels with different accessory
subunits. Conceivably, the variety of channel com-
plexes can be expanded further by mechanisms com-
bining pore-forming subunits with multiple different
types of accessory subunits. In considering this possi-
bility with regard to the KCNE family, we were
inspired by the well-established heteromultimeric nat-
ure of neuronal voltage-gated sodium channels which
comprise a single a-subunit combined with two distinct
accessory b-subunits. This precedent led us to investi-
gate the possibility that more than one type of
KCNE protein could simultaneously co-associate with
KCNQ1.
We first proposed that KCNQ1 could associate
with two different KCNE proteins based on our find-
ing that the transient expression of KCNE4 in a cell
line stably expressing KCNQ1 and KCNE1 (I
Ks
cells)
suppressed I
Ks
[24]. This observation suggested that
either KCNE1 was displaced from KCNQ1 com-
plexes, or that KCNE4 and KCNE1 associated jointly
with KCNQ1, but the inhibitory effect of KCNE4
was dominant. In our study, biochemical strategies
were applied to determine that the latter explanation
was most likely. Since our initial biophysical study,
other groups have examined KCNQ1 co-association
with KCNE1 and KCNE2 [38,39]. One of these stud-
ies used a similar biochemical strategy to demonstrate
the presence of a KCNE1–KCNQ1–KCNE2 channel
complex [39]; however, these data are somewhat
difficult to interpret because of the evidence for
KCNQ1 protein aggregation (aberrant molecular mass
of KCNQ1 monomer) and the absence of control
experiments to exclude artefactual subunit interac-
tions. In Caenorhabditis elegans, an A-type K
+
chan-
nel, KVS-1, has been shown to biophysically associate
with MPS-2 and MPS-3, two KCNE-related subunits
[40]. The association of both MPS proteins with
KVS-1 generates potassium currents which are distinct
from those generated when KVS-1 is expressed with
either MPS-2 or MPS-3 alone [40]. We speculate that
other K
V
channel complexes can be modulated dis-
tinctly by the incorporation of multiple KCNE
subunits.
KCNE4 does not impair KCNQ1 membrane
expression
Finally, it was tested whether impairment of cell
surface expression might explain the inhibition of
KCNQ1 by KCNE4. Certain classes of potassium
channel accessory subunit (i.e. K
V
b) have been shown
to increase membrane expression of K
V
channel a-sub-
units [41,42], and it was hypothesized that other types
of accessory subunit could have the opposite effect.
Indeed, a missense KCNE1 mutant (L51H) associated
with congenital long-QT syndrome causes retention of
both KCNE1 and KCNQ1 in the endoplasmic reticu-
lum [43,44]. One previous study examined KCNQ1
and K
V
1.1 trafficking and found that KCNE4 did not
diminish the cell surface expression of either K
V
chan-
nel [19,23]. We confirmed this finding related to
KCNQ1, but also demonstrated cell surface expression
of KCNE4 protein and the triple KCNE1–KCNQ1–
KCNE4 complex.
Mechanisms other than impaired plasma membrane
expression must explain the impaired KCNQ1 func-
tion in the presence of KCNE4. For example,
KCNE4 may cause a strong shift in the voltage
dependence of activation, or lock thechannel in a
closed state by immobilizing the activation gate or
voltage sensor. A strong depolarizing shift in activa-
tion appears to explain the suppression of KCNQ1
by KCNE5 [20]. There are many examples of K
V
channel gating modulation by accessory subunits, and
this provides another potential mechanism for
KCNE4 effects. For example, heterologous expression
of K
V
1.5 generates a non-inactivating current when
expressed alone, but becomes a rapidly inactivating
outward current when co-expressed with K
V
b1 [45].
The ability of K
V
b1 to dramatically alter the K
V
1.5
current has been attributed to a specific structure
within the N-terminus of the protein that is similar to
the inactivating N-terminal peptide in A-type K
V
channels [1,46]. This structure allows for rapid inacti-
vation of thechannel through blocking of the internal
pore following depolarization of the membrane. The
KCNE4 protein possesses a large cytoplasmic C-ter-
minal tail. We speculate that this structure may func-
tion in a similar, albeit voltage-independent, manner
to block the internal pore of KCNQ1. The C-termi-
nus of KCNE4 might also stabilize thechannel in
another non-conducting state. There have been no
investigations into the structural determinants of
KCNE4 inhibition.
Conclusions
KCNE4 is a widely expressed K
V
accessory subunit
implicated in the assembly of biophysically diverse
channel complexes in both excitable and non-excitable
tissues. The inhibitory actions of KCNE4 are exerted
at the plasma membrane, but the precise functional
KCNE4 co-association with KCNQ1–KCNE1 channels L. J. Manderfield and A. L. George Jr
1344 FEBS Journal 275 (2008) 1336–1349 ª 2008 The Authors Journal compilation ª 2008 FEBS
mechanism remains unknown. KCNE4can co-associ-
ate with KCNE1 and KCNQ1 to form a heteromulti-
meric complex that is non-functional at the cell
membrane. These findings indicate that KCNE4 is a
physiologically relevant K
V
channel modulator.
Experimental procedures
Generation of KCNE4 polyclonal antibody
A polyclonal rabbit antibody, targetted to a unique
sequence in theKCNE4 C-terminus (residues 73–94,
YKDEERLWGEAMKPLPVVSGLR), was generated by
Proteintech Group, Inc. (Chicago, IL, USA). A cysteine
residue was added to the N-terminus of the peptide to facil-
itate KLH conjugation. Sera were screened using ELISA
and the final antibody preparations were affinity purified
against the antigenic peptide.
Construction of epitope-tagged KCNE1 and
KCNE4
KCNE1 and KCNE4 were subcloned into pIRES2-EGFP
(BD Biosciences-Clontech, Mountain View, CA, USA), as
described previously [24]. A triple FLAG epitope (DY-
KDHDGDYKDHDIDYKDDDDK) was introduced by
recombinant PCR into the KCNE1 cDNA, immediately
upstream of the stop codon. The FLAG sequence was PCR
amplified from p3XFLAG-CMVÔ-13 (Sigma-Aldrich, St
Louis, MO, USA). Similarly, a triple HA epitope (YP-
YDVPDYAGYPYDVPDYAGSYPYDVPDYA) was intro-
duced into theKCNE4 cDNA, immediately upstream of
the stop codon. The HA sequence was PCR amplified from
a plasmid provided by Sabina Kuperschmidt (Vanderbilt
University, Nashville, TN, USA). All constructs were veri-
fied by complete sequencing of the coding regions. The
addition of epitope tags did not affect the electrophysiologi-
cal effects of KCNE1 or KCNE4 (supplementary Figs S1
and S2).
Cell culture and transfection
COS-M6 cells were grown at 37 °Cin5%CO
2
in Dul-
becco’s modified Eagle’s medium (DMEM; Life Technolo-
gies, Grand Island, NY, USA) supplemented with 10%
fetal bovine serum (ATLANTA Biologicals, Norcross, GA,
USA), penicillin (50 unitsÆ mL
)1
), streptomycin (50 lgÆmL
)1
)
(Life Technologies) and 20 mm HEPES. COS-M6 cells were
transiently transfected using FuGene-6 (Roche Applied
Science, Indianapolis, IN, USA). Full-length KCNQ1 was
expressed from the pcDNA5 ⁄ FRT vector (Invitrogen, San
Diego, CA, USA), whereas all KCNE cDNAs were
constructed in pIRES2-EGFP. Cells were harvested 48 h
post-transfection.
Preparation of cell lysates
Two 100 mm dishes of COS-M6 cells were transfected per
condition, and two dishes of non-transfected COS-M6 cells
were used in parallel as a control. Forty-eight hours post-
transfection, cells were placed on ice and washed twice with
ice-cold phosphate buffered saline (PBS) (137 mm NaCl,
2.7 mm KCl, 10 mm Na
2
HPO
4
,2mm KH
2
PO
4
, pH 7.4).
The cells from one dish were lysed with 1 mL of ice-cold
NP-40 buffer (1% NP-40, 150 mm NaCl, 50 mm Tris,
pH 8.0) supplemented with a Complete miniprotease inhibi-
tor tablet (Roche Applied Science) for 3 min. Cells were
then scraped and incubated on ice for another 3 min. The
lysate was transferred to a 1.5 mL microfuge tube and
rocked for 15 min at 4 °C, followed by centrifugation at
14 000 g for 10 min. The supernatant was collected and
centrifuged again under the same conditions. Prior to
immunoprecipitation experiments, aliquots of the final
supernatant were incubated with Protein-G SepharoseÔ 4
Fast Flow (GE Healthcare Life Sciences, Piscataway, NJ,
USA) to pre-clear non-specific protein binding to the
Sepharose beads. Final pre-cleared lysates were quantified
using the Bradford reagent (Bio-Rad Laboratories, Hercu-
les, CA, USA), and equal amounts of proteins were used in
the immunoprecipitation experiments.
Preparation of tissue lysates
Human autopsy tissues were obtained from the NICHD
Brain and Tissue Bank for Developmental Disorders under
contracts N01-HD-4-3368 and N01-HD-4-3383. All tissues
had previously been frozen and stored at )80 °C. Prior to
homogenization, tissues were ground with a mortar and
pestle. Three millilitres of ice-cold NP-40 buffer with pro-
tease inhibitors was added to 1 g of ground tissue and
homogenized for 30 s using a mechanical homogenizer
(Tekmar Company, Cincinnati, OH, USA). Homogenates
were rocked at 4 °C for 1 h, and then centrifuged at
14 000 g for 10 min. The supernatant was collected and
centrifuged again under the same conditions. The Bradford
reagent was used to quantify the protein concentration in
the final lysates.
Cross-linking of antibodies to Protein-G
Sepharose
Ten micrograms of antibody were combined with 750 lLof
borate buffer (200 mm sodium tetraborate decahydrate,
pH 9.0), added to 50 lL of Protein-G SepharoseÔ 4 Fast
Flow and rocked at room temperature for 1 h. The beads
were then washed twice with borate buffer. After the sec-
ond wash, the beads were resuspended in 1 mL of borate
buffer supplemented with 20 mm dimethyl pimelimidate di-
hydrochloride, and rocked at room temperature for 30 min.
L. J. Manderfield and A. L. George Jr KCNE4 co-association with KCNQ1–KCNE1 channels
FEBS Journal 275 (2008) 1336–1349 ª 2008 The Authors Journal compilation ª 2008 FEBS 1345
[...]... washing twice with DPBS with 100 mm glycine The cells were then incubated in the same DPBS with glycine solution for 10 min and then washed twice with DPBS Cellular lysates were then prepared as described above, except that the cells were lysed with ice-cold RIPA buffer (150 mm NaCl, 50 mm Tris-Base, pH 7.5, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with a Complete miniprotease inhibitor... part of the online article from http://www.blackwell-synergy.com KCNE4 co-association with KCNQ1–KCNE1 channels Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 1336–1349 ª 2008 The Authors... at 4 °C The supernatant fraction was collected and an aliquot was retained as the total protein fraction The remaining supernatant was incubated with ImmunoPure Immobilized Streptavidin beads (Pierce Chemical Co.) overnight at 4 °C The samples were centrifuged for 1 min at 14 000 g, and the supernatant fraction was collected and retained as the non-biotinylated fraction The beads were washed with RIPA... three times for 5 min at 4 °C Then, the biotinylated proteins were eluted with Laemmli sample buffer (Bio-Rad Laboratories) for 30 min at room temperature, and this final elution was retained as the biotinylated fraction All fractions were then subjected to western blot analysis as described above, withthe exception that all membranes were incubated overnight at 4 °C withthe appropriate primary antibody... transfection, the cells were washed twice with ice-cold Dulbecco’s PBS (DPBS) supplemented with CaCl2 and MgCl2 (GIBCO ⁄ Invitrogen, Grand Island, NY, USA) After washing, the cells were incubated in DPBS plus 1.5 mgÆmL)1 sulfo-NHS-biotin (Pierce Chemical Co., Rockford, IL, USA) for 1 h on ice with shaking The biotinylation solution was removed and the reaction was quenched by washing twice with DPBS with 100.. .KCNE4 co-association with KCNQ1–KCNE1 channels L J Manderfield and A L George Jr The cross-linking reaction was quenched by the addition of ethanolamine buffer (200 mm ethanolamine, pH 8.0) at room temperature for 5 min The Sepharose beads were centrifuged and resuspended in 1 mL of ethanolamine buffer and rocked at room temperature for 1 h The beads were washed twice in PBS and then resuspended... temperature with a mouse transferrin monoclonal antibody (1 : 500, Zymed Laboratories ⁄ Invitrogen Corporation, Carlsbad, CA, USA) Proteins were quantified by densitometry using imagej software (National Institutes of Health, Bethesda, MD, USA) The percentage of protein at the membrane was calculated by dividing the biotinylated fraction value by the total protein fraction value, and is reported as the mean... research fellowship FEBS Journal 275 (2008) 1336–1349 ª 2008 The Authors Journal compilation ª 2008 FEBS L J Manderfield and A L George Jr award from the American Heart Association Southeast Affiliate We wish to thank Dr Carlos Vanoye for valuable comments on the manuscript We also acknowledge the Brain and Tissue Bank for Developmental Disorders at the University of Maryland for providing human tissue samples... resuspended in PBS and stored at 4 °C until use Co-immunoprecipitations Cellular lysates pre-cleared with Protein-G Sepharose were rocked with 50 lL of cross-linked antibody at 4 °C for 4 h Following incubation, the samples were washed three times with ice-cold NP-40 for 5 min at 4 °C Immune complexes were then eluted with SDS-PAGE sample buffer (1% SDS, 50 mm Tris, 1% glycerol, 100 mm dithiothreitol) at 50... proteins to form cardiac IKs potassium channel Nature 384, 80–83 16 Barhanin J, Lesage F, Guillemare E, Fink M, Lazdunski M & Romey G (1996) KvLQT1 and IsK (minK) proteins associate to form theIKs cardiac potassium current Nature 384, 78–80 17 Tinel N, Diochot S, Borsotto M, Lazdunski M & Barhanin J (2000) KCNE2 confers background current characteristics to the cardiac KCNQ1 potassium channel EMBO J 19, . establish that KCNE4
directly associates with KCNQ1, and can co-associate together with
KCNE1 in the same KCNQ1 complex to form a ‘triple subunit’ complex
(KCNE1–KCNQ1 KCNE4) and
KCNE4 can simultaneously co-associate with KCNQ1
to form KCNE1–KCNQ1 KCNE4 ‘triple’ subunit
complexes, and that the inhibitory effect of KCNE4
cannot