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INTRODUCTION |
Voltage-dependent and Ca2+-activated
K+ (MaxiK, BK) channels are expressed in a variety of
tissues, including brain and smooth muscles (1, 2). Their functional
properties and relative abundance vary from tissue to tissue and within
the same tissue (1-5). The physiological role of MaxiK channels in
different tissues is influenced not only by their single channel
properties, but also by their expression level. In line with this view,
they are strikingly abundant in smooth muscle, where they play a
critical role in the control of vascular tone (6), and their expression level is exquisitely modulated during pregnancy (7). In brain, differential surface expression has been detected, suggesting a
critical role of MaxiK channels in shaping the electrical activity in
mammalian brain (8).
The elucidation of molecular mechanisms underlying MaxiK channel
diversity has received much attention in recent years; however, those
that determine channel expression levels in vertebrates are less
understood. Vertebrate MaxiK channels are composed of the pore-forming
and modulatory transmembrane
subunits (9-11). The
subunit
has seven transmembrane domains (S0-S6) (12) and seems to be the
product of a single gene (13). In contrast, modulatory
subunits are
products of several genes and contribute largely to the functional
diversity of MaxiK channels. For instance, the
1 subunit can
profoundly modulate the apparent voltage/Ca2+ sensitivity
and activation/deactivation kinetics of the
subunit (14-16).
Functional diversity of MaxiK channels is further enhanced by
alternative splicing of its
(11) and
(17) subunits. Ten splice
sites have been reported for the
subunit; eight of them are located
in the intracellular carboxyl terminus. However, only one splice insert
has been reported to have profound functional effects on activation and
deactivation kinetics and voltage/Ca2+ sensitivities of the
vertebrate MaxiK channel (11, 18). Thus, at least two mechanisms can
contribute to the functional diversity of MaxiK channels: association
with various
subunits and splice variation. Recently, a
Drosophila protein (DSLIP1) that reduces the number of
functional channels in the plasma membrane has been isolated. This
protein interacts with both human and Drosophila MaxiK
channels (19). However, a mammalian DSLIP1 counterpart has not been described.
We now report a novel mammalian MaxiK channel
subunit splice
variant (SV1)1 that can
regulate surface expression of both
(hSLO) and
1 subunits of the
human MaxiK channel when expressed in HEK293T cells. SV1 retains either
or
1 subunits in the endoplasmic reticulum (ER). These findings
suggest that SV1 may act as a dominant-negative expression regulator by
inhibiting MaxiK
and
1 subunit surface expression and show
splice variation as a possible mechanism for regulation of MaxiK
channel expression in mammalian tissues.
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EXPERIMENTAL PROCEDURES |
Molecular Biology
SV1 Isolation by RT-PCR--
Total RNA was extracted using
0.5~1.0 g of pregnant rat myometrium (Harlan Sprague-Dawley, 17 days
pregnant) and the Totally RNA isolation kit (Ambion Inc.). Poly(A) RNA
was purified using an Oligotex mRNA minikit (QIAGEN Inc.) according
to the manufacturer's instructions. Approximately 100 ng of poly(A)
RNA was converted to single-stranded cDNA by priming with an
oligo(dT) primer, followed by a 20-µl reverse transcription reaction
with 20 units of avian myeloblastosis virus reverse transcriptase
(Roche Molecular Biochemicals).
Single-stranded cDNA was mixed with MaxiK gene-specific primers
spanning a region between the linker of transmembrane segments S0 and
S1 and transmembrane segment S2 (see Fig. 1A,
arrows) and amplified with 3.5 units of Expand High Fidelity
DNA polymerase (Roche Molecular Biochemicals) in a 100-µl reaction.
The upstream primer anneals to nucleotides 419-437
(5'-AGGAGGTGGTGGCAGCCGA-3'), and the downstream primer anneals to
nucleotides 652-672 (5'-AAACCGCAAGCCAAAGTAGAG-3') according to
GenBankTM/EBI accession number U55995. PCR was
performed by denaturing at 94 °C for 5 min, followed by 35 cycles of
94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min, with a
final extension at 72 °C for 7 min. Amplification products were
separated on a 2% agarose gel and visualized by ethidium bromide
staining. Sequences were determined using automated fluorescent
sequencing (PerkinElmer Life Sciences).
Two amplification products were obtained: one with the expected size
corresponding to the reported mammalian SLO sequences (253 base pairs)
and another of larger size (352 base pairs). Sequence analysis showed
that this product had an amino acid sequence identical to mammalian
(human, rat, mice, bovine, and canine) SLO sequences, but contained an
in-frame 33-amino acid segment. As expected for a splice site, the
flanking nucleotides were Gs, which occur 60-80% of the time at
splice sites (20). For functional analysis, the 33-amino acid segment
was inserted into the human MaxiK
subunit (hSLO); this construct
was named splice variant 1 (SV1). Note that human and rat MaxiK channel
subunits have an overall 99% amino acid identity.
RT-PCR Analysis--
RT-PCR was performed as described above
using poly(A) RNA from rat brain and/or uterus (non-pregnant). The
primers used to check the presence of the SV1 insert in rSLO were as
follows (see Fig. 1C): Reaction 1 (Box B1),
5'-AAGATGGATGCGCTCATCATC-3' (forward primer,
nucleotides 166-186, GenBankTM/EBI accession number
U55995), which includes the start codon Met3 (21), and
5'-CATGAGGAGTCTAGGCATG-3' (reverse primer, SV1-specific; see Fig.
1B); and Reaction 2 (Box B2), 5'-GCATTTGAAAGATCCTCATTG-3' (forward primer, SV1-specific; see Fig. 1B) and
5'-CTCTATGATTTCAGGGACGTA-3' (reverse primer, nucleotides
1120-1140, GenBankTM/EBI accession number U55995).
Detection of SV1 in brain and uterus was performed using the same
SV1-specific primers. All products were confirmed by sequencing.
RNase Protection Assay (RPA)--
Probe DNAs were obtained by
RT-PCR amplification using gene-specific primers for rSLO and
SV1. To detect rSLO (generic sequence), we amplified a highly conserved
region that includes the domain DQDDDD, known as the "Ca-bowl"
(22), where no splicing sites have been reported. The upstream primer
anneals to nucleotides 2842-2861 (5'-GGACCAAGACGATGATGATG-3'), and the
downstream primer anneals to nucleotides 3060-3077
(5'-GCTAGTGTCTGCGGAGTG-3'). For SV1, the same specific primers used for
RT-PCR analysis were used. Both amplification products were subcloned
into the pCR2.1 vector (Invitrogen) and analyzed by nucleotide
sequencing. Clones with the correct inserts were digested with
EcoRV and KpnI and cloned into similarly digested
pBluescript II KS vector (Stratagene). Plasmids were linearized with
Acc65I, and digoxigenin-labeled antisense riboprobes were
synthesized using the digoxigenin RNA labeling kit (Roche Molecular Biochemicals).
The RPA was performed using RNA isolated from non-pregnant rat
myometrium and brain with the RNase protection kit (Roche Molecular Biochemicals). Each sample (100 µg of total RNA) was individually hybridized with 1 ng of rSLO probe or SV1 probe overnight at 45 °C.
The unprotected single-stranded RNAs were digested with RNase A/T1. For
each sample, total SV1-protected fragments and 1% of the
rSLO-protected fragments were mixed, separated on an 8 M
urea and 5% polyacrylamide gel, and transferred to positively charged nylon membranes (Roche Molecular Biochemicals) using contact blotting. Signals were detected by immunological and chemiluminescent detection with the chemiluminescent substrate CSPD® using the
digoxigenin luminescent detection kit (Roche Molecular Biochemicals).
Constructs--
hSLO (12) and SV1 were tagged with the c-Myc
epitope AEEQKLISEEDL at the N terminus. Tagged and untagged SV1 and
hSLO behaved similarly. For simplicity, we refer to the c-Myc-tagged
constructs as hSLO and SV1 throughout this work. hSLO-GFP and SV1-GFP
were constructed by fusing GFP (pEGFP-N2, CLONTECH)
to the C terminus of the human MaxiK
subunit. N-terminal GFP
tagging was avoided since it is known that the tag diminishes the
voltage and Ca2+ sensitivities of the hSLO fusion protein
(23). All constructs were subcloned into the pcDNA3 vector
(Invitrogen). The GenBankTM/EBI accession numbers are
U11058 for the human MaxiK pore-forming
subunit and U25138 for the
human MaxiK modulatory
1 subunit (KCNMB1).
Transfection
The calcium phosphate transfection protocol was used to
transiently transfect HEK293T cells (24). Cells were grown to ~10% confluency and incubated for ~22 h with the transfection mixture. The
transfection mixture was made by mixing (in a 1:1 ratio) a solution
containing 250 mM CaCl2 and 20 ng/µl
endotoxin-free plasmid with a buffer containing 42 mM
HEPES, 274 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4, and 11 mM
dextrose, pH 7.05. This mixture was incubated for 20 min at room
temperature prior to cell treatment. After transfection, cells were
washed with phosphate-buffered saline (10 mM
Na2HPO4, 2.3 mM
NaH2PO4, 138 mM NaCl, and 2.7 mM KCl, pH 7.4) and fed with complete medium (Dulbecco's
modified Eagle's medium, L-glutamine, 100 units/ml
penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal
bovine serum; Life Technologies, Inc.). The day before
immunocytochemistry experiments, cells were mechanically dissociated
and transferred to chamber slides (Lab-Tek) that were precoated with
0.1 mg/ml poly-D-lysine and 0.1 mg/ml collagen. In
experiments involving cotransfection of cDNAs, we used a 1:1
cDNA molar ratio.
Immunocytochemistry
Non-permeabilized Labeling (Live Cell Labeling)--
Three days
after transfection, cells were incubated for 1 h at 37 °C with
a 1:50 dilution of anti-c-Myc monoclonal antibody. Cells were washed
and fixed for 30 min with 4% paraformaldehyde. After washing with
phosphate-buffered saline, nonspecific binding was blocked by
preincubating the cells with 5% donkey serum at room temperature. The
excess serum was removed, and the secondary antibody (1:5000 dilution;
Alexa 594, Molecular Probes, Inc.) was added for 1 h at room
temperature. Cells were washed with phosphate-buffered saline, dried
for 5 min, and mounted (Molecular Probe Prolong) for imaging.
Permeabilized Labeling--
Three days after transfection, cells
were washed with phosphate-buffered saline and fixed for 30 min with
4% paraformaldehyde at room temperature. To permeabilize cells,
subsequent solutions contained 0.2% Triton X-100 (Sigma). Nonspecific
binding was blocked for 30 min at room temperature using 5% donkey
serum. Primary antibodies were then added and incubated overnight at
4 °C. The next day, cells were washed and incubated for 1 h
with the appropriate secondary antibodies at room temperature. The
secondary antibodies were rhodamine red-X succinimidyl ester-conjugated
donkey anti-rabbit or anti-mouse antibodies (1:200 dilution; Jackson
ImmunoResearch Laboratories, Inc.). Cells were washed again, and
mounting was performed as described for non-permeabilized cells. Images
were acquired by optically sectioning tissues every 0.5-1 µm with a confocal microscope (Leica) and analyzed using the Image-Pro Plus (Media Cybernetics) and Adobe Photoshop programs.
Antibodies--
Affinity-purified polyclonal antibodies were
used against hSLO, Kv4.3 (Alomone Labs), and ERp72 (endoplasmic
reticulum marker, Calbiochem) at 1:500, 1:500, and 1:1000 dilutions,
respectively. These antibodies were raised against purified peptides
corresponding to residues 883-896 (VNDTNVQFLDQDDD) of hSLO (7, 25),
residues 451-467 ((Y)NEALELTGTPEEEH-Nle-GK) of Kv4.3 (26), and
residues 623-638 ((C)FIDEHATKRSRTKEEL) of ERp72 protein (27, 28). To label the human
1 subunit, we used a polyclonal antibody (1:1000 dilution) raised against residues 85-102 (GRWAMLYHTEDTRDQNQQ) of
KCNMB1 (29). Anti-c-Myc was a monoclonal antibody (1:50 dilution) raised against sequence AEEQKLISEEDLLRKRREQLKHKLEQLRNSCA (clone 9E10,
Oncogene Science Inc.).
The lowest concentration of antibody that gives the highest signal was
used in these studies. In all experiments, a negative control was
performed by transfecting cells with no DNA and by preadsorbing each
antibody with the corresponding antigenic peptide.
Electrophysiolgy
hSLO, hSLO-GFP, SV1, and SV1-GFP cDNAs were transiently
expressed in HEK293T cells. One day after transfection, cells were plated on coverslips precoated with Cell-Tak (Collaborative Biomedical Products). Transfected cells were identified using a fluorescence microscope (for GFP constructs; CK40-RFL, Olympus) or by the CD8 method
(30). In this method, CD8 cDNA is cotransfected with the cDNA
of interest. Transfected cells are identified by light microscopy with
the aid of anti-CD8 antibody-coated magnetic beads (Dynabeads®, Dynal, Inc.). In our experience, this method
is highly successful when Shaker or hSLO K+ channels are
cotransfected. Currents were measured 1-5 days after transfection
using the cell-attached, inside-out, and whole-cell patch-clamp modes.
Recording pipettes had resistances of ~2-5 megaohms and were filled
with 105 mM potassium methanesulfonate, 5 mM
KCl, 10 mM HEPES, 5 mM HEDTA, and 58 nM free Ca2+, pH 7.0. The bath solution was 105 mM potassium methanesulfonate, 5 mM KCl, 10 mM HEPES, and 10 µM free Ca2+, pH
7.0. A Ca2+ electrode was used to measure pCa
(World Precision Instruments, Inc.) as previously described (31). Data
was filtered at one-fourth the sampling frequency and acquired and
analyzed with pCLAMP (Axon Instruments, Inc.). Normalized voltage
activation curves of macroscopic currents were calculated as described
previously (15). Values are expressed as means ± S.D.
(n = number of experiments).
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RESULTS |
A Novel MaxiK Splice Variant--
A new splice variant of the
MaxiK
subunit (SV1) (Fig.
1A, gray portion)
was isolated from rat myometrium by RT-PCR using MaxiK gene-specific
primers spanning the S0-S1 linker and the middle of transmembrane
segment S2 (arrows). Two PCR products were obtained (see
"Experimental Procedures"): one with identical amino acid sequence
to mammalian (human, rat, mice, bovine, and canine) SLO sequences and
another containing an in-frame insert of 33 amino acids in the S1
transmembrane domain. Fig. 1B shows the nucleotide and amino
acid sequences of the new insert and its position in transmembrane
segment S1 (arrowhead). This insert contains a homologous
sequence for the cAMP-dependent phosphorylation site
(RXS*) (32). To discard the possibility of a cloning
artifact, we amplified 447 bases of the 5'-end (including 3 bases
upstream to the Met3 start codon) (21) and 708 bases of the
3'-end using pairs of SV1- and rSLO-specific primers. Using this
strategy, only cDNAs containing SV1 sequences should be amplified.
The diagram in Fig. 1C depicts the sizes of the expected
products (1, 2) and the positions of the primers (arrows)
and SV1 (gray boxes). The RT-PCRs yielded single bands (Fig.
1C) of the expected sizes (447 (lane 1) and 708 (lane 2) nucleotides) and sequences, indicating that indeed
the SV1 insert is present in rat myometrium SLO cDNAs. As expected,
when water (lane W) was added instead of cDNA, no bands
were detected. RT-PCR using human myometrium cDNAs and the same
primers also generated products with the expected sizes and sequences
(data not shown).

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Fig. 1.
A new splice variant (SV1) at the N terminus
of mammalian SLO. A, proposed SV1 topology shows that
the insert elongates the linker between the S0 and S1 regions
(gray dash line) in addition to substituting some of the S1
amino acids (gray portion of S1). Arrows mark the
positions of RT-PCR primers used for initial isolation. B,
shown are the nucleotide and amino acid sequences of the 33-amino acid
splice insert. The arrowhead marks the location of the
insert in the S1 transmembrane region (solid line) of rat or
human SLO. The asterisk marks a potential
cAMP-dependent phosphorylation site. C, shown is
a bar representation of the SV1 5'- and 3'-amplified regions
(upper panel). Arrows mark the positions and
orientations of the primers. White boxes represent rSLO
sequences, and gray boxes correspond to SV1 sequence. RT-PCR
shows products of the expected sizes (lower panel).
Lane 1, ~447 nucleotides (nt); lane
2, ~708 nucleotides; lane W, water used instead of
cDNA as a control. D, RT-PCR (upper panel)
and RPA (lower panel) show the presence of SV1 transcripts
in uterus (lane U) and brain (lane B). RT-PCR
with SV1 site-specific primers shows products of the expected sizes in
uterus and brain, but no product in the control (lane W).
RPA was carried out using an SV1-specific probe (99 nucleotides) and a
rSLO-(2842-3078) generic probe (237 nucleotides) of a highly conserved
sequence at the carboxyl terminus (see "Experimental Procedures").
Protected fragments of the expected sizes were present in both uterus
(lane U) and brain (lane B). Only 1% of the
generic protected fragment was loaded on the gel. E,
hydrophobicity plots span the N terminus to the end of the S2 domain.
The Genetics Computer Group program PEPPlOT, which uses Kyte
and Doolittle hydropathy analysis (47), was used (averaging nine amino
acids). The filled regions mark hydrophobic residues in
stretches of 20 amino acids that correspond to transmembrane regions
S0-S2. The solid bar marks the position of the insert
(lower panel). F, the S1 hydrophobicity values of
SV1 (dotted line) and hSLO (solid line) are
almost identical.
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The mRNA of the novel splice variant was detected in the uterus
(Fig. 1D, lane U) and brain (lane B)
using both RT-PCR (upper panel) and RPA (lower
panel). RT-PCR was performed using SV1-specific primers that
detected the expected 99-base pair product. SV1 transcripts were also
detected by RPA. This method was used because of its much higher
specificity and sensitivity compared with RT-PCR and Northern blotting.
RPA analysis clearly demonstrated SV1 transcripts in uterus and also in
brain (Fig. 1D). SV1-specific and rSLO-(2842-3078) generic
probes distinguished two bands of the expected sizes. Signals for SV1
accounted for 1.2 ± 0.1% (n = 5) of the total rSLO message (note that only 1% of the total rSLO reaction was loaded;
see "Experimental Procedures"). This low expression, together with
the small size of the SV1-specific probe, likely contributed to the
difficulty in detecting transcripts using Northern blotting (data not
shown). Nevertheless, both the RT-PCR experiments (Fig. 1, C
and D) and the RPA results confirm the authenticity of the SV1 splice variant and the existence of its mRNA in rat uterus and brain.
Because the SV1 insert is located within the hydrophobic S1 domain of
SLO, we investigated if its insertion could impact the hydrophobic
properties of S1. Fig. 1E illustrates the hydrophobicity plots of SLO (upper panel) and SV1 (lower panel)
from the amino terminus to the end of S2; the location of the insert is
marked with a solid bar. Note that rSLO and hSLO sequences
are 100% conserved in this region and have 99% overall sequence
identity. Striking is the similar hydrophobic pattern of S1 in both SLO
and SV1 constructs. This similarity is further illustrated in Fig.
1F by overlapping S1 hydrophobicity values from SLO
(solid line) and SV1 (dotted line). The high
degree of overlap demonstrates that the overall hydrophobicity pattern
of SLO is not disrupted by the splice insert in SV1, but rather it
elongates the linker between S0 and S1.
SV1 Does Not Produce Detectable Ionic Currents in HEK293T
Cells--
To investigate the functional impact of the SV1 splice
insert, HEK293T cells were transfected with SV1 and hSLO constructs (Fig. 2; see "Experimental
Procedures"). Currents were measured in cell-attached or inside-out
configurations (Fig. 2, A-D, middle and
right panels). Fig. 2A shows that cells
transfected with SV1 (as assessed by coexpression of CD8; see
"Experimental Procedures") had no detectable currents when measured
in the cell-attached (n = 4) or inside-out
(n = 4; 10 µM Ca2+)
configurations. Changes in internal Ca2+ concentrations
from 1.5 µM to 1.4 mM did not produce
detectable currents. To ensure that patched cells were transfected with
SV1, GFP was fused at the C terminus of SV1 (Fig. 2B,
left panel). Similarly, SV1-GFP-transfected cells (as
assessed by GFP fluorescence) showed no detectable currents in
cell-attached (n = 12) or inside-out (n = 6) recordings in the presence of 10 µM intracellular
Ca2+ (Fig. 2B). Furthermore, in whole-cell
recordings, SV1- and SV1-GFP-transfected cells failed to generate any
current (data not shown).

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Fig. 2.
SV1- or SV1-GFP-transfected HEK293T cells
show no detectable current. Transfected cells were identified
using the CD8 method (A and C) or the GFP
fluorophore (B and D). Currents were elicited by
holding cells at 0 mV and pulsing from 60 to +145 mV every 5 mV. No
detectable current was measured from SV1 (A) or SV1-GFP
(B) in cell-attached or inside-out recordings. In contrast,
currents were detected from hSLO (C)- or hSLO-GFP
(D)-transfected cells in cell-attached or inside-out
configurations. In E, the fractional open probabilities
(FPo) of hSLO ( ) and hSLO-GFP ( ) from
inside-out recordings were plotted and fitted using a Boltzmann
distribution. The calculated V0.5 values (the
voltage at 50% FPo) in 10 µM internal
Ca2+ were 30 ± 4 mV (n = 4)
for hSLO and 35 ± 7 mV (n = 7) for
hSLO-GFP. In F, V0.5 was plotted as a
function of internal Ca2+ concentration. The
Ca2+ sensitivities of hSLO ( ) and hSLO-GFP ( ) are
practically the same. Values are the means of three to four
experiments.
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The lack of measurable currents from SV1 constructs was not caused by
the addition of the c-Myc tag at the SV1 N terminus or fusion of
GFP at its C terminus. SV1 constructs with (n = 8) or
without (n = 12) c-Myc yielded no measurable currents.
In addition, previous studies demonstrated that N-terminal c-Myc
addition does not change the overall properties of the wild-type hSLO
channels (12). We now demonstrate that the carboxyl-terminal GFP fusion does not change the overall voltage/Ca2+ sensitivities of
hSLO compared with c-Myc-tagged hSLO. Fig. 2 (C and
D) shows examples of currents in cell-attached or inside-out configurations. Voltage activation curves demonstrate that the voltage
dependence of c-Myc-tagged hSLO (Fig. 2E,
) and that of
GFP-tagged hSLO (
) were practically identical. Furthermore, current
measurements at different Ca2+ concentrations indicated
that the Ca2+ sensitivity of the hSLO-GFP channel was also
similar (Fig. 2F). Because the fusion of GFP to the C
terminus of hSLO does not interfere with its functional expression, it
is likely that the addition of GFP to SV1 also keeps its intrinsic
characteristics. Thus, our electrophysiological results using SV1-GFP
demonstrate the inability of SV1 to produce detectable currents in the
plasma membrane of HEK293T cells.
SV1 Displays No Surface Labeling--
To determine whether SV1
reaches the plasma membrane, we directly measured the cellular
localization of SV1 and compared it with the hSLO expression pattern
using fluorescent confocal microscopy. Since the N terminus of the
MaxiK channel is positioned extracellularly (12), we used N-terminal
c-Myc-tagged hSLO (Fig. 2C) or SV1 (Fig. 2A) and
anti-c-Myc antibody to detect their surface expression in
non-permeabilized cells (live cells). Fig.
3A clearly shows that hSLO was
expressed in the plasma membrane (arrowheads) of HEK293T
cells. The overlap of the fluorescent confocal image taken near the
middle of the cells and the differential interference contrast (DIC)
image show that the labeling coincided with the borders of the cells.
Because cells were not permeabilized, no signal was detected inside the
cells.

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Fig. 3.
Immunocytochemistry studies demonstrate the
inability of SV1 to reach the surface membrane of HEK293T cells.
A-C, confocal images of HEK293T cells transfected
with c-Myc-tagged hSLO (A) or SV1 (B and
C) and immunolabeled under non-permeabilized (A
and B) or permeabilized (C) conditions.
Anti-c-Myc antibody and Alexa 594 secondary antibody (red)
were used. A, the overlap of DIC and hSLO (single confocal
image) labeled cells shows the ability of hSLO to reach the plasma
membrane (arrowheads). B, under identical
conditions, SV1-transfected cells failed to show positive labeling
(eight confocal images). C, after permeabilization,
SV1-transfected cells display a perinuclear labeling pattern.
D, counting the number of labeled cells/0.02 mm2
demonstrates that, under non-permeabilized conditions, SV1 could not be
labeled because of its retention in the perinuclear region. Scale
bars (A-C) = 20 µm.
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In contrast to hSLO and under identical conditions, SV1-transfected
cells showed no labeling even when the whole depth of the cells was
scanned (Fig. 3B). This lack of labeling was observed in all
studied cells in four different experiments. However, when cells were
permeabilized, the anti-c-Myc antibody was able to reach its target
inside the cell, and labeling of SV1 could be observed. The overlap of
DIC and fluorescence images indicates that SV1 expression was
concentrated in the perinuclear region of the cells (Fig.
3C).
Fig. 3D is the quantification of the number of labeled
cells/field (0.02 mm2) in experiments as in Fig. 3
(A-C). In contrast to hSLO, SV1 showed no surface labeling;
note that 40 different fields were examined. On the other hand, under
permeabilized conditions, SV1 showed perinuclear labeling in every
field examined, consistent with accumulation of protein in the ER.
MaxiK Splice Variant SV1 Is Localized to the Endoplasmic
Reticulum--
The distribution of SV1 or SV1-GFP, seen at high
magnification, showed a strong network expression pattern that spread
within the cell bodies typical of ER localization. The overlap of DIC and fluorescent images (Fig. 4,
A and B) illustrates the strong labeling around
the nucleus (arrows) that extended outward in a network
manner (arrowheads) without reaching the plasma membrane (asterisks).

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Fig. 4.
SV1 is localized to the ER.
A and B, perinuclear distribution of both SV1 and
SV1-GFP. Asterisks mark the plasma membrane as seen by
DIC. Arrows mark the strong perinuclear staining, and
arrowheads mark the mesh pattern typical of ER labeling.
C, double-labeling experiments visualizing SV1 with GFP
(green) and the ER with anti-ERp72 antibody
(red). The high extent of colocalization was observed by
overlapping all images (yellow). In C, 13 optical
sections were overlapped to show the entire thickness of the cell.
These images are representative of 47 (A), 37 (B), and 24 (C) cells examined in four to seven
separate experiments. In this and the following figures,
double-labeling experiments were visualized with rhodamine
(red) and GFP (green) fluorophores. Scale
bars = 10 µm.
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To determine if the SV1 splice variant localizes in the ER, cells were
transfected with SV1-GFP and double-labeled with an antibody against a
resident ER protein, ERp72. SV1-GFP was used because no primary or
secondary antibodies that can introduce background signals are needed;
and thus, a more sensitive labeling is achieved. Anti-ERp72 antibody
was selected because of ERp72 abundance and localization in the ER (27,
28). Fig. 4C illustrates SV1-GFP
(green)-transfected cells that were permeabilized and labeled with anti-ERp72 antibody (red). The overlap of these
images shows a clear colocalization of SV1-GFP and ERp72 proteins
(yellow). These results indicate that SV1-GFP protein is
synthesized, but is unable to leave the ER. Similar results were
obtained in 24 cells in four separate experiments.
MaxiK Splice Variant SV1 Traps Coexpressed hSLO or
1
Subunits--
Tetramerization of Shaker K+ channels and
assembly with the cytoplasmic Kv
2 subunit take place in the ER
(33). In addition, increasing evidence indicates that
subunits play
important roles in regulating ion channel surface expression. For
example, cytoplasmic
subunits enhance plasma membrane expression of
voltage-dependent K+ (34) and Ca2+
(35) channels, and the transmembrane
subunit SUR1, when properly assembled with its corresponding
subunits (Kir6.1/2), masks retention signals located in both subunits, allowing surface expression of KATP channel complexes (36). Therefore, we investigated
whether SV1 could interact with the
(hSLO) or
1 subunits of
MaxiK channels and serve as a dominant-negative regulator of channel
expression, or if rescue of SV1 from the ER could occur.
The interaction of SV1-GFP with its counterpart hSLO or its
transmembrane
1 subunit was studied in cotransfected cells under permeabilized conditions. We found that neither hSLO nor
1 could rescue SV1 from the ER; but instead, SV1 prevented the normal expression of both subunits in the plasma membrane. Fig.
5 compares the normal expression patterns
of hSLO (A) and
1 (B) subunits when
transfected alone with their expression when SV1-GFP was also expressed
(C and D). Transfection with hSLO (43 cells,
seven experiments) or
1 (56 cells, seven experiments) alone showed almost a uniform expression that covered the entire plasma membrane (Fig. 5, A and B, arrowheads).
Perinuclear regions (arrows) had relatively higher
expression in these cells, which can be explained by normal cell
trafficking. This is in contrast to SV1 and SV1-GFP labeling, which
concentrates near the nucleus, with no expression in the plasma
membrane (Fig. 4, A and B). When SV1-GFP was
coexpressed with hSLO, both proteins were found colocalized. Fig.
5C shows images of the same cell coexpressing SV1-GFP
(assessed by GFP fluorescence; second panel) and hSLO
(assessed by rhodamine labeling; third panel). Similarly,
Fig. 5D shows that cotransfection of SV1-GFP with
1
induced its retention around the nucleus, consistent with ER retention.
SV1-GFP expression was assessed by GFP fluorescence (green,
second panel), and
1 expression in the same cell was assessed with rhodamine-conjugated secondary antibodies
(red, third panel). The overlap images in Fig. 5
(C and D, fourth panels) show the high
degree of SV1-GFP colocalization with hSLO or
1 subunits
(yellow). Similar results were observed in 80 cells
cotransfected with hSLO and 48 cells cotransfected with
1 (three
experiments).

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Fig. 5.
SV1 traps coexpressed hSLO or
1 subunits in HEK293T cells. A and
B show the normal expression patterns of MaxiK pore-forming
(hSLO) and modulatory 1 subunits in permeabilized cells. Note
that both subunits could express independently and readily reached the
plasma membrane (arrowheads). The slightly higher labeling
of the perinuclear region is likely due to normal trafficking
(arrows). C shows that when SV1-GFP
(green) and hSLO (red) were cotransfected, only a
perinuclear labeling was observed for both proteins. hSLO was labeled
with anti-c-Myc antibody. The overlap shows a high degree of
colocalization in the perinuclear region (yellow), which did
not extend to the plasma membrane (arrowheads). D
shows a similar retention of the 1 subunit (red) by
SV1-GFP and a high degree of colocalization (yellow).
Arrowheads mark the outer boundaries of the cells. These
results are representative of 43 (A), 56 (B), 80 (C), and 48 (D) cells examined in three to seven
experiments. A and B are single 0.5-1-µm
confocal sections; C and D are overlaps of eight
confocal sections. Scale bars = 10 µm.
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Our observations suggest that SV1-GFP may act as a dominant-negative
expression regulator by trapping the MaxiK
(hSLO) or
1 subunits
in the ER. In addition, these results are consistent with the early
coassembly of hSLO or
1 subunits with SV1-GFP in the ER.
MaxiK Splice Variant SV1 Allows the Coexpressed Kv4.3 Channel to
Leave the ER and Does Not Interact with Cytosolic GFP--
To
determine the specificity of the interaction of SV1 with hSLO or its
1 subunit, we cotransfected SV1 with Kv4.3 K+ channel or
GFP cDNAs. Fig. 6A
displays images of cells that were cotransfected with SV1-GFP and
Kv4.3. Cells expressing SV1 were identified with GFP fluorescence
(green), whereas cells expressing Kv4.3 were labeled with
rhodamine-conjugated secondary antibodies (red). Note that
not all cells in the field were expressing SV1-GFP (asterisks). Arrowheads in Fig. 6A
point to the location of the outer cell membrane of a cell expressing
both SV1-GFP and Kv4.3, as visualized by DIC. Note that SV1-GFP
remained in the perinuclear region (arrows), whereas Kv4.3
was able to reach the plasma membrane (arrowheads). It is
possible that Kv4.3 has a nonspecific interaction with SV1 since the
level of labeling in the perinuclear region of cells expressing both
Kv4.3 and SV1-GFP (arrows) was somewhat higher than in cells
expressing Kv4.3 alone (asterisks). However, this possible
interaction does not prevent Kv4.3 from reaching the plasma
membrane.

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Fig. 6.
SV1 does not trap Kv4.3 in the ER and does
not interact with GFP. A, HEK293T cells cotransfected
with SV1-GFP and the Kv4.3 K+ channel. The image of SV1-GFP
shows a single cell transfected with perinuclear distribution of SV1
(arrow). The Kv4.3 image shows, in the same field, several
cells expressing Kv4.3 in their plasma membrane that were not
cotransfected with SV1-GFP (asterisk). Arrows
mark the perinuclear region of the cell coexpressing SV1-GFP and Kv4.3;
note that Kv4.3 was able to reach the plasma membrane
(arrowheads) (see overlap image). B, cells
cotransfected with SV1 and GFP protein. SV1 labeling shows a clear
perinuclear localization in two transfected cells (arrows).
GFP fluorescence shows the expression of GFP alone in one cell
(asterisk) and that coexpression with SV1
(arrows) did not prevent its diffuse expression away from
the perinuclear region (arrowhead) (see overlap image).
These images (eight confocal sections) are representative of 9 (A) and 12 (B) cells examined in three separate
experiments. C, image analysis of experiments similar to
those shown in A and B and in Fig. 5. The plot
shows the ratio of signal intensity in the plasma membrane with respect
to the perinuclear region in cells that were (black bars) or
were not (gray bars; control) cotransfected with SV1 or
SV1-GFP. Coexpression of SV1 with hSLO or 1 significantly (*)
diminished expression of these MaxiK subunits in the plasma membrane.
In contrast, expression of Kv4.3 or GFP was not altered in a
significant manner. The numbers of cells examined were as follows:
hSLO, 43; hSLO + SV1 or SV1-GFP, 80; 1, 56; 1 + SV1 or SV1-GFP,
48; Kv4.3, 4; Kv4.3 + SV1 or SV1-GFP, 9; GFP, 11; and GFP + SV1 or
SV1-GFP, 12. Values are means ± S.E. Scale bars
(A and B) = 10 µm.
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Similar to Kv4.3, SV1 did not modify the GFP expression pattern (Fig.
6B). The asterisks mark a cell that was
transfected only with GFP (no label in the SV1 image), whose expression
was homogeneous throughout the cell. On the other hand, the
arrows mark two cells that were cotransfected with SV1 and
GFP; note that the expression pattern of GFP was unaltered in these
cells. Moreover, we observed no significant interaction of GFP protein with SV1 in the perinuclear region.
Fig. 6C plots the intensity ratio of membrane to perinuclear
labeling and demonstrates that cotransfection with SV1 (black bars) trapped both hSLO and
1 (*, p < 0.001),
but had no significant effect on Kv4.3 and GFP expression. In
summary, our results demonstrate that the MaxiK SV1 splice variant has
dominant-negative properties when coexpressed with hSLO and its
1
subunit and that their interaction is specific.
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DISCUSSION |
We isolated a novel MaxiK splice variant (SV1) that contains a
33-amino acid insert between the S0 and S1 transmembrane domains of the
subunit (Fig. 1, A and B). About six amino
acids of the insert contributed to the S1 segment without affecting its
hydrophobicity profile (Fig. 1, A and F). Even
though the overall transmembrane hydrophobicity of hSLO did not change,
the insert restrained the ability of SV1 to reach the plasma membrane
of HEK293T cells. Our electrophysiological experiments (Fig. 2,
A and B) and live cell labeling (Fig.
3B) demonstrate the inability of SV1 to produce any current
or surface labeling, contrary to hSLO (Figs. 2, C and
D; and 3A). In permeabilized cells, SV1 or
SV1-GFP showed high expression around the nucleus and a weaker network
expression within the cell bodies (Fig. 4, A and
B), which corresponded to ER retention (Fig. 4C).
In contrast, hSLO and
1 showed expression patterns that more
uniformly covered the entire plasma membrane (Fig. 5, A and
B). Taken together, our results demonstrate the inability of
SV1 or SV1-GFP to reach the plasma membrane because of their retention
in ER.
We also studied the interaction of the splice variant SV1 with hSLO,
1, Kv4.3, and GFP proteins. Our results demonstrate that the MaxiK
splice variant trapped coexpressed hSLO and
1 in the ER (Fig. 5),
but showed no effect on coexpressed GFP protein (Fig. 6). SV1-GFP
seemed to have a limited nonspecific interaction with
coexpressed Kv4.3 without inhibiting its surface expression (Fig.
6A, arrows). This interaction may be similar to
the interactions seen between
subunits of different classes of Kv
potassium channels (37). In addition, the coexpression studies provide
important insight into MaxiK subunit assembly and suggest the early
assembly of hSLO and
1 subunits in the ER organelle. This is
consistent with the early assembly of the
subunit of K+
channels with cytosolic
subunits in the ER (33). It is also relevant to note that hSLO and
1 subunits could be expressed independently (Fig. 5) and that the
1 subunit did not seem to increase the functional expression of hSLO (data not shown), as is the
case for the Kv
2 subunit and Shaker K+ channels
(33).
SV1 and hSLO have identical amino acid sequences except for the
33-amino acid insert; however, one is retained in the ER (SV1), and the
other is readily expressed in the plasma membrane (hSLO). The retention
of SV1-GFP was not due to fragmented translation of SV1 since the
C-terminal GFP domain of the SV1-GFP fusion protein was expressed.
Also, it is unlikely that a thiol-mediated retention mechanism (38)
could contribute to ER retention of SV1 since the insert is located in
the cytosol and not in the lumen of the ER. Therefore, the two cysteine
residues of the insert (Fig. 1B) are unable to interact with
protein-disulfide isomerase located in ER lumen, which is involved in
ER retention (38).
Another possible mechanism of ER retention may be caused by the
inability of transport vesicles to form that would carry SV1 protein
from ER to Golgi organelles. The cytosolic expression of the insert may
require special assembly particles that would recognize the insert and
include it in specific transport vesicles to deliver them to the Golgi
and subsequently to the plasma membrane (39, 40). It is possible that,
in HEK293T cells, this assembly particle(s) may be missing. This
possibility was addressed by expressing SV1 in oocytes and polarized
Madin-Darby canine kidney cells. SV1 produced no measurable currents in
oocytes and no surface labeling in Madin-Darby canine kidney cells
(data not shown).
Alternatively, SV1 may contain or unmask a novel ER retention signal.
Typical retention signals are luminal KDEL and cytoplasmic KKXX motifs located proximal to the C terminus (41). None of these motifs are present in the SV1 insert, which is far away from the
C terminus. However, other acidic retention signals containing arginines have been located in the N terminus of alternatively spliced
variants of syntaxin-5 and the human invariant (Ii) chain of major
histocompatibility complex class II (42, 43). Moreover, a new ER
retention/retrieval signal (RKR) distant to both N and C termini has
been identified that controls surface expression of fully assembled
KATP channel complexes (36). Thus, it is possible that SV1
modulates expression of MaxiK channel complexes in a variety of
tissues, allowing molecular plasticity and fulfillment of specific
functions. It will be interesting to determine if SV1 can modulate the
expression levels of MaxiK channels to different degrees, as is the
case for Kv1.1 modulation of Kv1.2 and Kv1.4 (44), KvLQT1 isoform 2 of
isoform 1 (45), or the HERG-A561V Long QT syndrome
mutant of HERG K+ channel surface expression (46). Future
studies will investigate SV1 effects on MaxiK channel expression in
myometrium and brain cells.
In summary, we found a novel MaxiK channel splice variant (SV1) that
acts as a dominant-negative regulator of MaxiK channel
and
1
subunit expression. It is likely that SV1 induces differential surface
expression of MaxiK channels and thus may contribute to plastic changes
necessary for the proper function of excitable and non-excitable
tissues. The role of SV1 as a dominant-negative regulator of MaxiK
channel expression under physiological or pathological conditions needs
to be determined. At present, we know that SV1 transcripts are present
in myometrium and brain, and it is possible that their levels may
change during the estrous cycle, pregnancy, or neurodevelopment or
neurodegenerative conditions modifying MaxiK channel expression.