KCNE4 can co-associate with the 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

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 can co-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 can co-associate with and modulate
KCNQ1 channels to produce biochemically diverse channel complexes has
important implications for understanding K
V
channel regulation in human
physiology.

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]. KCNE4 can 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

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-

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

–
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 with the 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

(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-

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-

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

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 with the KCNE4 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 with the 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

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

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-

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 with the KCNE4 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

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

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

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-

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 the channel in a

to block the internal pore of KCNQ1. The C-termi-
nus of KCNE4 might also stabilize the channel 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. KCNE4 can 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 the KCNE4 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

), 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

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
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 buf-
fer and rocked at room temperature for 1 h. The beads
were washed twice in PBS and then 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 °C
for 5 min.
SDS-PAGE and western blotting
Immunoprecipitated samples were loaded on to 4–20% linear
gradient polyacrylamide gels (Bio-Rad Laboratories) and
run at 200 mV for 45 min at room temperature. The gels
were transferred to Hybond ECL nitrocellulose membranes
(GE Healthcare Life Sciences) at 4 °C, at 70 mV for 30 min

system (GE Healthcare Life Sciences), and exposed to
Kodak BioMax MS film (Kodak, New Haven, CT, USA).
Cell surface biotinylation
COS-M6 cells were grown in 35 mm tissue culture dishes to
70% confluence and transfected as described above. Forty-
eight hours after transfection, the cells were washed twice
with ice-cold Dulbecco’s PBS (DPBS) supplemented with
CaCl
2
and MgCl
2
(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 bioti-
nylation solution was removed and the reaction was
quenched by washing twice with DPBS with 100 mm gly-
cine. 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% IGE-
PAL, 0.5% sodium deoxycholate, 0.1% SDS, supplemented
with a Complete miniprotease inhibitor tablet), and were
centrifuged for 30 min at 4 °C. The supernatant fraction
was collected and an aliquot was retained as the total pro-
tein fraction. The remaining supernatant was incubated
with ImmunoPure Immobilized Streptavidin beads (Pierce

valuable comments on the manuscript. We also
acknowledge the Brain and Tissue Bank for Develop-
mental Disorders at the University of Maryland for
providing human tissue samples.
References
1 Rettig J, Heinemann SH, Wunder F, Lorra C, Parcej
DN, Dolly JO & Pongs O (1994) Inactivation properties
of voltage-gated K
+
channels altered by presence of
b-subunit. Nature 369, 289–294.
2 Martens JR, Kwak YG & Tamkun MM (1999) Modu-
lation of K
v
channel alpha ⁄ beta subunit interactions.
Trends Cardiovasc Med 9, 253–258.
3 Pongs O, Leicher T, Berger M, Roeper J, Bahring R,
Wray D, Giese KP, Silva AJ & Storm JF (1999) Func-
tional and molecular aspects of voltage-gated K
+
chan-
nel beta subunits. Ann NY Acad Sci 868, 344–355.
4 Trimmer JS (1998) Regulation of ion channel expres-
sion by cytoplasmic subunits. Curr Opin Neurobiol 8,
370–374.
5 Pourrier M, Schram G & Nattel S (2003) Properties,
expression and potential roles of cardiac K
+
channel
accessory subunits: MinK, MiRPs, KChIP, and

Biol 113, 39–47.
12 Demolombe S, Franco D, de Boer P, Kuperschmidt S,
Roden D, Pereon Y, Jarry A, Moorman AF & Escande
D (2001) Differential expression of K
v
LQT1 and its reg-
ulator I
sK
in mouse epithelia. Am J Physiol Cell Physiol
280, C359–C372.
13 Abbott GW, Sesti F, Splawski I, Buck ME, Lehmann
MH, Timothy KW, Keating MT & Goldstein SA
(1999) MiRP1 forms I
Kr
potassium channels with
HERG and is associated with cardiac arrhythmia. Cell
97, 175–187.
14 Piccini M, Vitelli F, Seri M, Galietta LJ, Moran O,
Bulfone A, Banfi S, Pober B & Renieri A (1999)
KCNE1-like gene is deleted in AMME contiguous
gene syndrome: identification and characterization of
the human and mouse homologs. Genomics 60, 251–
257.
15 Sanguinetti MC, Curran ME, Zou A, Shen J, Spector
PS, Atkinson DL & Keating MT (1996) Coassembly of
K
v
LQT1 and minK (I
sK
) proteins to form cardiac I

Ks
pore demon-
strates two MinK subunits in each channel complex.
Neuron 40, 15–23.
22 Lundquist AL, Turner CL, Ballester LY & George AL
Jr (2006) Expression and transcriptional control of
human KCNE genes. Genomics 87, 119–128.
23 Grunnet M, Rasmussen HB, Hay-Schmidt A, Rosensti-
erne M & Klaerke DA (2003) KCNE4 is an inhibitory
subunit to Kv1.1 and Kv1.3 potassium channels.
Biophys J 85, 1525–1537.
24 Lundquist AL, Manderfield LJ, Vanoye CG, Rogers
CS, Donahue BS, Chang PA, Drinkwater DC,
Murray KT & George AL Jr (2005) Expression of
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 1347
multiple KCNE genes in human heart may enable
variable modulation of I
Ks
. J Mol Cell Cardiol 38,
277–287.
25 Radicke S, Cotella D, Graf EM, Banse U, Jost N, Var-
ro A, Tseng GN, Ravens U & Wettwer E (2006) Func-
tional modulation of the transient outward current I
to
by KCNE beta-subunits and regional distribution in
human non-failing and failing hearts. Cardiovasc Res
71, 695–703.
26 Alves VS, Pimenta DC, Sattlegger E & Castilho BA
(2004) Biophysical characterization of Gir2, a highly

fibrillation-associated KCNE4 (145E ⁄ D) gene polymor-
phism. Chin Med J (Engl) 120, 150–154.
32 Kim SJ & Greger R (1999) Voltage-dependent, slowly
activating K
+
current (I
Ks
) and its augmentation by
carbachol in rat pancreatic acini. Pflugers Arch 438,
604–611.
33 Kottgen M, Hoefer A, Kim SJ, Beschorner U, Schreiber
R, Hug MJ & Greger R (1999) Carbachol activates a
K
+
channel of very small conductance in the basolater-
al membrane of rat pancreatic acinar cells. Pflugers
Arch 438, 597–603.
34 Bleich M & Warth R (2000) The very small-conduc-
tance K
+
channel K
v
LQT1 and epithelial function.
Pflugers Arch 440, 202–206.
35 Vallon V, Grahammer F, Richter K, Bleich M, Lang F,
Barhanin J, Volkl H & Warth R (2001) Role of
KCNE1-dependent K
+
fluxes in mouse proximal
tubule. J Am Soc Nephrol 12, 2003–2011.

channel surface expression through effects early in bio-
synthesis. Neuron 16, 843–852.
42 Campomanes CR, Carroll KI, Manganas LN, Hersh-
berger ME, Gong B, Antonucci DE, Rhodes KJ &
Trimmer JS (2002) Kv beta subunit oxidoreductase
activity and Kv1 potassium channel trafficking. J Biol
Chem 277, 8298–8305.
43 Bianchi L, Shen Z, Dennis AT, Priori SG, Napolitano
C, Ronchetti E, Bryskin R, Schwartz PJ & Brown AM
(1999) Cellular dysfunction of LQT5-minK mutants:
abnormalities of I
Ks
,I
Kr
and trafficking in long QT syn-
drome. Hum Mol Genet 8, 1499–1507.
44 Krumerman A, Gao X, Bian JS, Melman YF, Kagan A
& Mcdonald TV (2004) An LQT mutant minK alters
K
v
LQT1 trafficking. Am J Physiol Cell Physiol 286,
C1453–C1463.
45 Sewing S, Roeper J & Pongs O (1996) Kv beta 1 sub-
unit binding specific for shaker-related potassium chan-
nel alpha subunits. Neuron 16, 455–463.
46 Heinemann S, Rettig J, Scott V, Parcej DN, Lorra C,
Dolly J & Pongs O (1994) The inactivation behaviour
of voltage-gated K-channels may be determined by
association of alpha- and beta-subunits. J Physiol Paris
88, 173–180.