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 me
Trang 1channel complex
Lauren J Manderfield1and Alfred L George Jr1,2
1 Department of Pharmacology, Vanderbilt University, Nashville, TN, USA
2 Department of Medicine, Vanderbilt University, Nashville, TN, USA
Voltage-gated potassium (KV) channels are essential
for a variety of physiological processes, including the
control of membrane potential, electrical excitability
and solute transport Many KV 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 KV accessory subunit
have been identified, including KVb [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
KV 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 (KV) 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 KV channels, including KCNQ1 (KV7.1) that, together with KCNE1, generates the slow-delayed rectifier current (IKs) important for cardiac repolarization In particular, KCNE4 exerts a strong inhibitory effect on KCNQ1 and other KVchannels, 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 KVchannel 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; KVchannel, voltage-gated potassium channel.
Trang 2peptides encoded by human genes KCNE2, KCNE3,
KCNE4 and KCNE5 were identified [13,14] Although
different KCNE proteins functionally interact with a
variety of KV channels, all KCNE proteins have been
shown to modulate heterologously expressed KCNQ1
(KV7.1) with distinct effects [15–20] The
co-expres-sion of KCNE1 with KCNQ1 reconstitutes IKs, 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
KV channels, including KV1.1 and KV1.3, are also
inhibited by KCNE4 [23] KCNE4 can also exert
func-tional inhibition on KV channels even in the presence
of other accessory subunits For example, KCNE4 can
inhibit IKs stably expressed in Chinese hamster ovary
cells [24], as well as the transient outward current (Ito)
reconstituted in heterologous systems by the
co-expres-sion of KV4.3 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 KVchannel Finally,
we demonstrated that KCNE1 and KCNE4 can
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
KVchannels
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 with the highest KCNE4 mRNA expression, had low or no KCNE4 protein expression [22] We inferred from these data that post-transcriptional mechanisms contribute to the
Trang 3steady-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 IKs 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
HA IB:
+
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 Colon Heart 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.
Trang 4first examined by testing whether KCNE1 remained
associated with KCNQ1 even in the presence of
KCNE4 Cells were transiently transfected with
KCNE4, KCNE13FLAG 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
IKs [24] KCNE4 and an epitope-tagged KCNE1 (KCNE13FLAG) 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 IKs The multiple molecular mass bands ranging from approximately 15 to 25 kDa observed in the immunoblots probed for KCNE13FLAG 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 IKsexpression 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
75 kDa
IB:KCNQ1
25
IP:KCNQ1 IB:KCNQ1
75 kDa
50
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 anti-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.
Trang 5KCNE4 does not inhibit KCNQ1 trafficking
One potential mechanism by which KCNE4 could
sup-press IKs 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 IKs Total protein, non-biotinylated and biotinylated fractions from cells expressing KCNQ1 only, KCNQ1 with KCNE13FLAG and KCNQ1 with KCNE43HA 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 KCNE13FLAG
75 kDa
75 kDa
IB:KCNQ1
25
30 kDa
IB:KCNE4
25 kDa
50 kDa 30 25
IP:KCNQ1 IB:KCNE4
30 kDa 25 15
IP:KCNQ1 IB:FLAG
8 7 6 5 4 3 2
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 KCNE13FLAGonly 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 KCNE13FLAGtransfected cells immunoprecipitated with goat pre-immune serum Lane 9, cells expressing KCNQ1 only Lane 10, cells expressing KCNE13FLAGonly 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.
Trang 6(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 IKsby 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 bionon-bio-tinylated 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-associate with 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
E1
NT E1+E4
IB:FLAG
30
25
NT E4 E1+E4
+ – + + + + + –
– + + + + + + –
KCNE13FLAG 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 2 3 4 5 6 7
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 KCNE13FLAG 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, KCNE13FLAGand KCNE4 transfected cells
immuno-precipitated with Protein-G Sepharose Lane 6, KCNE13FLAG 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 KCNE13FLAG, E4
for KCNE4 and E1 + E4 for KCNE13FLAG+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.
Trang 7expression 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 KCNE13FLAG
8 7 6 5 4 3 2
30 kDa
30
50 kDa
25
IP:FLAG IB:KCNE4
25 kDa
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 with the KCNE4 antibody The sixth row of immunoblots shows the initial lysates confirming KCNE4 expression.
35 kDa
75 kDa
50
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
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.
Trang 8Diversity 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 KV1.1 and KV1.3 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 IKsand may be critical for the regulation of cardiac repolarization The fact that IKshas 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
IKs, 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 IKs-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 can co-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
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.
Trang 9be 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 (KV7.2 and KV7.3) [37], or by
the association of channels with different accessory
subunits Conceivably, the variety of channel
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 (IKs cells)
suppressed IKs [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 KV 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 KVb) have been shown
to increase membrane expression of KVchannel 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 KV1.1 trafficking and found that KCNE4 did not diminish the cell surface expression of either KV 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 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 KV channel gating modulation by accessory subunits, and this provides another potential mechanism for KCNE4 effects For example, heterologous expression
of KV1.5 generates a non-inactivating current when expressed alone, but becomes a rapidly inactivating outward current when co-expressed with KVb1 [45] The ability of KVb1 to dramatically alter the KV1.5 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 KV channels [1,46] This structure allows for rapid inacti-vation of the channel 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 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 KV 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
Trang 10mechanism 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 KVchannel 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
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-CMV-13 (Sigma-Aldrich, St
Louis, MO, USA) Similarly, a triple HA epitope
(YP-YDVPDYAGYPYDVPDYAGSYPYDVPDYA) was
intro-duced into the KCNE4 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 37C in 5% CO2 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 unitsÆmL)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 Na2HPO4, 2 mm KH2PO4, 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 4C, 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 Sepharose 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 4C 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 lL of borate buffer (200 mm sodium tetraborate decahydrate,
pH 9.0), added to 50 lL of Protein-G Sepharose 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