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INTRODUCTION |
Large conductance, voltage-dependent, and
Ca2+-activated K+ (MaxiK, or
BK)1 channels are widely
distributed in neurons (1, 2), smooth muscle (3-5), and several
nephron segments (6-9), where they play roles in regulating the
neurotransmitter release, cell contractility, and volume decrease under
hypotonic stress (10-14). The expression level of MaxiK channels
varies from tissue to tissue and even within the same tissue (15).
The MaxiK channels are composed of the pore-forming
-subunit and
modulatory
-subunit (16-20). A single gene encodes the
-subunit with the S1-S6 region homologous to the tetrameric voltage-gated K+ channels, especially in the pore-forming domain,
S5-P-S6, and the voltage-sensing element, S4 (21, 22). Structures
unique to the MaxiK channel
-subunit include an additional
N-terminal transmembrane domain, S0, and a large C terminus. There are
four hydrophobic segments (S7-S10) in the C-terminal region of the channel protein (21, 23). These segments were previously thought to be
additional transmembrane domains, but now it is agreed that they are
located inside the cell (21). The MaxiK C-terminal region has
Ca2+ sensors and also affects the channel's gating
(24-27).
MaxiK channels play a fundamental role in many physiological processes,
including action potential repolarization, modulation of calcium
signals, hormone release, and regulation of vascular tone (28-31).
MaxiK channels from different cell types vary in their calcium
sensitivity, unitary conductance, and gating kinetics (5). The
interaction of the MaxiK
-subunit with the modulatory
-subunits
or other accessory proteins partially explains their overall functional
diversity. More importantly, the function variation of MaxiK channels
comes from the alternative splicing of its
-subunit (17, 22, 32). So
far, ten splice sites have been reported with most of them localized in
the intracellular C terminus.
The renal MaxiK channel is distributed in glomeruli, thin limbs of
Henle's loop, cortical and medullary ascending limbs of Henle's loop,
cortical collecting tubules, and outer and inner medullary collecting
ducts (9). Renal MaxiK channels function in cell volume regulation and
flow-mediated K+ secretion (12). Previously, we reported
the cloning of two isoforms of the MaxiK channel
-subunit from a
rabbit medullary thick ascending limb (mTAL) cell line (9). The longer
one, rbslo1 (NCBI accession number AF201702), is highly homologous to
MaxiK channels from other species such as mslo, hslo, and cslo and has
an additional 58-amino acid (aa) exon insert between the S8 and S9
regions. The other isoform, rbslo2, is truncated at position
Gly781 with a unique 15-aa sequence appended at the end
(see Fig. 1 below). Using electrophysiological techniques, we have
shown that rbslo1 functions in a voltage- and
Ca2+-dependent way, whereas rbslo2 cannot be
activated at all.
The objectives of the present study were to determine at the protein
level why rbslo2 is functionally silent. To this end we examined the
electrophysiology, assembly, and intracellular localization of rbslo1,
rbslo2, and several deletion mutants in cultured mammalian cell lines.
Using whole cell current recordings, confocal microscopy, and cell
surface biotinylation, we report here that the cytoplasmic tail present
in rbslo1 but missing in rbslo2 is critically involved in apical cell
membrane expression of the MaxiK channel in renal cells.
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EXPERIMENTAL PROCEDURES |
DNA Constructs of rbslo1, rbslo2, and MaxiK
Subunits--
The MaxiK channel has seven transmembrane domains
(S0-S6, Fig. 1) with an
extracellular N terminus and a large cytoplasmic tail. The wild type
rbslo1 and rbslo2 cDNAs, i.e. wt-rbslo1 and wt-rbslo2
(see Fig. 1), were prepared as described before (9). All the myc-tagged
constructs were created by PCR amplification using the wild type rbslo1
or 2 as the template. The PCR primers were synthesized to flank the
desired DNA sequence with a BamHI (for forward primer) or
SalI (for reverse primer) enzyme digestion site at the end.
The PCR products were purified, double-digested by restriction enzymes
BamHI and SalI, and ligated into pCMV-TAG3 (Stratagene) vector. To make the GFP-tagged rbslo2 construct, the
coding region of wt-rbslo2 was isolated by double digestion using
restriction enzymes HindIII and XbaI. The
fragment was subcloned into pEGFP-C3 (Clontech)
vector. All the rbslo1 and 2 constructs were sequenced to verify that
the correct insert and the coding sequences were in-frame with the tag.
All vectors contained the cytomegalovirus (CMV) promoter to obtain
better transfection rates and higher expression level in mammalian
cells.

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Fig. 1.
rbslo1 and rbslo2. Wild type
rbslo1 has seven predicted transmembrane domains (TMD),
S0-S6; four hydrophobic domains, S7-S10; and a 58-aa insertion
between S8 and S9. myc-rbslo1 contains a myc tag at the N terminus.
myc-rbslo1-Del, rbslo1 with N-terminal myc tag and truncated
at Gly839. wt-rbslo2 is missing 332 aa of the C terminus
and has a unique 15-aa C terminus. GFP-rbslo2 has a GFP tag at the N
terminus, myc-rbslo2 has an myc tag at the N terminus, and
myc-rbslo2-Del has an N-terminal myc tag and is truncated at
Gly781. The myc-rbslo1-Del and myc-rbslo2-Del have the same
C terminus. The unique 15-aa C terminus in wt-rbslo2, GFP-rbslo2, and
myc-rbslo2 is shown as a closed circle.
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The wt-rbslo1 was investigated by a rabbit polyclonal antibody against
its C terminus. For other experiments, we chose to add myc or GFP tag
in-frame at the N terminus of the signal sequence of rbslo1 and 2. The
fusion proteins were investigated by using antibodies against the
corresponding tag peptide. To determine whether the 58-aa insertion in
rbslo1 and the unique C terminus of 15 aa in rbslo2 may have some
function to the channel's property, two truncated DNA constructs
designated myc-rbslo1-Del and myc-rbslo2-Del were generated from their
corresponding DNA myc-rbslo1 and myc-rbslo2, respectively. Both of the
deletion constructs stop at a Gly residue (Gly839 in
wt-rbslo1 or Gly781 in wt-rbslo2, respectively).
The MaxiK
3 and
4 subunits were cloned from human kidney library
(Clontech) using PCR technique according to their
published sequences (NCBI accession numbers AF170916 and AF160967 for
3 and
4, respectively). The PCR products were then subcloned into
pEGFP-C3 vector and sequenced as described above.
Cell Culture and Transfection--
Both HEK293 and mTAL cells
were grown in 1:1 Dulbecco's modified Eagle's medium/Ham's F-12
nutrient mixture supplemented with 10% fetal bovine serum, 50 units/ml
penicillin, and 50 µg/ml streptomycin. Cells were grown in a 37 °C
incubator with 5% CO2 and passaged twice weekly through
exposure to 0.05% trypsin, 0.5 mM EDTA. For transfection,
HEK293 cells were transferred to poly-L-lysine-coated glass
coverslips and transiently transfected using LipofectAMINE 2000 (Invitrogen) when 90% confluent. For electrophysiology, GFP plasmid
was cotransfected with either rbslo1 or 2 DNA. To increase the
transfection rates in polarized mTAL cells, the DNA/LipofectAMINE mixture was added immediately after the cells were seeded on the 12-mm
Transwell (0.4-µm pore size, Corning Inc.). Remaining transfection steps were carried out according to the manufacturer's instructions. Forty eight to seventy two hours after transfection, the cells were
used for electrophysiological studies or fixed with 4%
paraformaldehyde for 10 min and, if desired, permeabilized with 0.5%
Triton X-100 for 3 min for immunofluorescent experiments.
Current Recordings--
Currents were measured using a
conventional tight seal whole cell recording at room temperature. The
GFP plasmid was used to identify green fluorescent cells, which were
likely cotransfected with the rbslo1 or 2 constructs, using a Nikon
Eclipse TE200 inverted fluorescence microscope intrinsic to the current
recording system. GFP-positive cells were superfused with an
extracellular solution containing (in mM): 135 sodium
aspartate, 10 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2,
0.1 niflumic acid, and 10 Hepes (pH 7.4 with Tris). Aspartate-rich solution and niflumic acid were used to prevent the activation of
endogenous currents. The pipette solution contained (in
mM): 135 potassium aspartate, 15 KCl, 1 MgCl2,
10 Hepes (pH 7.4 with Tris). Extracellular solution containing 10 or 30 µM ionomycin was puff-applied using a nearby pipette of
30- to 35-µm diameter. pClamp8 software (Axon Instruments) was used
to sample and analyze data. The access conductance was monitored in the
trace of capacitate surge. To minimize the voltage errors due to the
lower access conductance, results that showed the amplitude of the
current above 1 nA were obtained from the experiments showing the
access conductance over 70 nanosiemens. Current-voltage (I-V)
relationships were obtained at 100 ms of each voltage step from a
holding potential of
80 mV.
Antibodies and Immunofluorescent Staining--
Rabbit polyclonal
antibody against C terminus of rbslo1 was from Alomone Laboratories;
mouse monoclonal antibody (9E10) for myc and rabbit polyclonal antibody
for ZO-1 were from Zymed Laboratories Inc.; antibody
for GFP (rabbit) was from Clontech; antibody for CD4 (mouse) was from Calbiochem; chicken anti-myc antibody was from
Aves Laboratories Inc.; mouse anti-KDEL antibody was from StressGen;
mouse anti-
-adaptin antibody was from Transduction; mouse
anti-mannose 6-phosphate receptor antibody was from Affinity Bioreagents Inc. The lowest concentration of antibody giving the highest signal was used in these studies. In all experiments, negative controls were performed by either: 1) transfecting
cells with DNA vectors or no DNA or 2) pre-adsorbing each antibody with the corresponding antigenic peptide or both as appropriate. For double-labeling experiments, single-stained specimens were examined under the other fluorescent channel to assure minimized bleed-through. Moreover, the intensity of the two dyes in double-stained specimens was
balanced to reduce artifacts. No antibodies from the same species were
used in any of the double-stained specimens.
For immunostaining, the fixed cells were blocked with 5% nonfat dry
milk in PBS for 1 h. The cells were then incubated with the
primary antibody for 1 h, washed with PBS three times, and incubated with the appropriate secondary antibody conjugated to fluorescein isothiocyanate or Cy3 fluorescent dye (Sigma) for 1 h. After staining, the coverslips (for HEK293) or Transwells (for mTAL) were washed in PBS and mounted using Prolong Antifade kit
(Molecular Probes).
Western Blotting Analysis--
Cells transiently transfected
with DNA constructs encoding rbslo1 or rbslo2 fusion proteins were
lysed in 20 mM Hepes, pH 7.5, 120 mM NaCl, 5.0 mM EDTA, 1.0% Triton X-100, 0.5 mM
dithiothreitol, and 1.0 mM phenylmethylsulfonyl fluoride
containing Complete protease inhibitor (Roche Molecular Biochemicals, 1 tablet per 50-ml solution). The lysates were spun at 6000 × g for 5 min to pellet the insoluble material, and the
proteins from supernatant were quantified using a Bio-Rad Protein Assay
kit. After mixing in Laemmli buffer (Bio-Rad) and incubating at
37 °C for 15 min, the protein sample was separated by SDS-PAGE and
electrophoretically transferred to polyvinylidene difluoride membranes
(Amersham Biosciences). The probing with specific antibodies and
subsequent detection with ECL plus system (Amersham Biosciences) was
performed according to standard procedure as described previously
(33).
Confocal Laser Microscopy--
The fluorescence label was
examined with UltraView confocal imaging system (PerkinElmer Life
Sciences). Images were generated using the manufacturer's software
suite. Contrast and brightness settings were chosen to ensure that all
pixels were within the linear range. To obtain three-dimensional images
of the cell, each XY plane of the sample with a depth of 0.4 µm in Z
direction was scanned by the confocal laser, and the picture serials
along the Z-axis were combined, reconstructed, and presented as XZ and YZ cross-section images using the Volocity software package
(Improvision Ltd.) Images were prepared for publication with Adobe Photoshop.
Cell Surface Biotinylation--
HEK293 cells were grown on
six-well plates for 48-72 h after DNA transfection. Cell surface
biotinylation was carried out by overlaying 1.0 ml of
Sulfo-N-hydroxysuccinimidobiotin (Pierce) (1.0 mg/ml in
PBS buffer) on the cells. The sample was incubated for 45 min on ice
with gentle shaking. Cold PBS buffer (1 volume, 1.0 ml) containing 100 mM glycine was then added to stop the reaction. After an
additional 20-min incubation, the cells were collected and lysed as
described above. The biotinylated proteins from the cell surface were
recovered from lysates by incubation with 100 µl of immobilized
streptavidin-agarose beads (Pierce). Bound proteins were eluted from
the beads in Laemmli buffer, and the unbound samples were also saved.
All the protein samples were quantified and then analyzed by SDS-PAGE
and Western blotting as described above.
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RESULTS |
Electrophysiology of rbslo1 and -2--
HEK293 cells were
cotransfected with the wt-rbslo1 and GFP plasmid. Outward currents were
observed in cells selected for positive GFP expression (Fig.
2A). These outward currents
were also time-dependent. Five seconds after treatment of
the same cell with ionomycin, a Ca2+ ionophore, outward
currents were observed at lower voltage steps (Fig. 2, A and
B). A prolonged treatment with ionomycin (> 15 s)
resulted in the activation of an inward current at
100 mV and an
almost linear I-V relation (data not shown) with a reversal potential
of
70 mV, suggesting the high sensitivity of wt-rbslo1 channel to
Ca2+ (34). Similar results were observed in eight of eight
cells tested. These features in cells expressing wt-rbslo1 are similar to previous measurements in rbslo1-expressing Xenopus
oocytes and Chinese hamster ovary cells (9). As expected,
cells expressing GFP only did not show any similar current activity
(five cells, data not shown).

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Fig. 2.
Electrophysiology of rbslo1 and -2 channels. Time-, voltage-, and Ca2+-dependent
currents in HEK293 cells transfected with GFP and wt-rbslo1
(A and B), wt-rbslo2 (C and
D), myc-rbslo1 (E and F),
myc-rbslo1-Del (G and H), or myc-rbslo2
(I and J). Typical recordings measured before
(left panels) and after (middle panels)
application of ionomycin (10 µM). The relationship of the
steady-state current to membrane voltage is shown in the right
panels. The cell was held at 80 mV, and 100-ms voltage steps
were applied (from 100 through +100 mV at increments of 20 mV).
Open circles indicate basal currents, and closed
circles indicate ionomycin-stimulated currents.
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When wt-rbslo1 was tagged with myc at the N terminus (myc-rbslo1) and
cotransfected with GFP into HEK293 cells, current activities similar to
those with wt-rbslo1 were observed in 9 cells tested (Fig. 2,
E and F). These data suggested that the myc tag
at the N terminus does not affect the channel function of rbslo1. A
G839X truncation mutant, myc-rbslo1-Del, which lacks the cytoplasmic tail of rbslo1, did not generate any time-, voltage-, and
Ca2+-dependent currents when coexpressed with
GFP (Fig. 2, G and H).
In contrast to wt-rbslo1 channel, when cells were transfected with
wt-rbslo2, no time-, voltage-, and
Ca2+-dependent current activities were observed
(10 cells tested, Fig. 2, C and D). Similar
results were obtained using myc-rbslo2 (five cells tested, Fig. 2,
I and J), GFP-rbslo2 (eight cells tested, data
not shown), or the G781X mutant, myc-rbslo2-Del (four cells tested,
data not shown), which lacks the C-terminal unique tail of 15 aa in
rbslo2. These data suggest the cytoplasmic tail of the rbslo1 is
essential for the generation of plasma membrane currents.
The rbslo1 and -2 Exist as Oligomers--
Like the other potassium
channels, functional MaxiK channels formed by the tetramerization of
monomers (35, 36). Therefore, we asked whether both rbslo1 and 2 exist
as oligomers using non-reduced electrophoresis and immunoblotting
techniques. HEK293 cell lysates containing the exogenously expressed
rbslo1 or 2 proteins were pretreated with non-mercaptoethanol
containing Laemmli buffer. An immunoblot with mouse anti-myc antibody
(Fig. 3A) shows that the
monomer, as well as various oligomers, were present in all samples,
i.e. myc-rbslo1, myc-rbslo1-Del, myc-rbslo2, and
myc-rbslo2-Del. The stoichiometry of the oligomers was initially
determined by comparing the apparent molecular weight of each band to
its respective theoretical value. For example, bands labeled with
one to four stars for myc-slo1 have calculated
apparent molecular masses of about 146, 262, 314, and 431 kDa,
respectively. They were designated monomer, dimer, trimer, and
tetramer, respectively. Because membrane and soluble proteins have
different hydrodynamic properties, soluble molecular weight markers may
not provide an accurate estimate of the molecular mass of a hydrophobic
protein (37). Therefore, we further tested our proposed multimeric
state of each band by plotting the relative migration (Rf)
of each band against the putative number of subunits they represent
(Fig. 3B). The linear relationship in each of the four plots
indicates that the relative mobility of each band corresponds to its
assigned subunit stoichiometry, suggesting that rbslo1 and 2 monomer,
dimer, trimer, and tetramer existed (38, 39). These results in Fig. 3
indicate that both rbslo1 and 2 proteins retain the ability to assemble
as oligomers and that the cytoplasmic tail of rbslo1 is not necessary
for rbslo1 or 2 channel assembly.

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Fig. 3.
Both rbslo1 and -2 assemble as
oligomers. A, immunoblot showing transient expression
of myc-rbslo1 (lane 2), myc-rbslo1-Del (lane 3),
myc-rbslo2 (lane 4), or myc-rbslo2-Del (lane 5)
in HEK293 cells. Mock transfected cells were run as lane 1.
SDS-PAGE was run on 5% gel with no mercaptoethanol in the system.
Western blotting was performed with mouse anti myc-antibody (1:200) and
horseradish peroxidase-linked sheep anti-mouse Ig (1:1000) as primary
and secondary antibody, respectively. The monomer, dimer, trimer, and
tetramer forms of the protein are here labeled as *, **, ***, and ****,
respectively. B, relative mobility (Rf) of
myc-rbslo1 ( ), myc-rbslo1-Del ( ), myc-rbslo2 (×), and
myc-rbslo2-Del ( ) through SDS-PAGE as a function of the logarithm of
the number of subunit.
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Comparing the Distribution of rbslo1 and 2 in HEK293 Cells--
To
see if the lack of rbslo2 channel currents was due to its
misdistribution in the cell, we transiently transfected HEK293 cells
with various rbslo1 or 2 constructs. After 48 h, the cells were
examined under indirect immunofluorescent confocal microscopy using
rabbit polyclonal anti-rbslo1 antibody for wt-rbslo1 and mouse
monoclonal anti-myc antibody for myc-rbslo1 (Fig.
4, A and B). Both
wt-rbslo1 and myc-rbslo1 displayed a clear cell surface distribution.
Therefore, as suggested by our electrophysiological studies, rbslo1
protein traffics to the cell surface and the N terminus myc-tag does
not interfere with the localization of the channel proteins. We also
compared the effects of myc and GFP tags by cotransfecting both
myc-rbslo2 and GFP-rbslo2 into the HEK293 cells (Fig. 4,
C-E). We found that when coexpressed in one cell,
myc-rbslo2 (C, Cy3-labeled secondary antibody) and
GFP-rbslo2 (D) proteins have the same distribution as
indicated by the yellow in the merged image
(E). Thus neither N-terminal myc nor GFP tag disturbs the
characteristics of rbslo channels.

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Fig. 4.
Comparison of wild type, myc-, and GFP-tagged
rbslo1 and -2 in HEK293 cells by immunofluorescent microscopy.
Shown are the confocal microscopic images of wt-rbslo1 (A),
myc-rbslo1 (B), myc-rbslo2 (C), and GFP-rbslo2
(D). Rabbit anti-rbslo1 (1:500) and mouse anti-myc (1:1000)
antibodies were used for wt-rbslo1, myc-rbslo1, and myc-rbslo2
staining, respectively. GFP-rbslo2 was examined using the intrinsic
fluorescence of GFP. The plasma membrane localization of wt-rbslo1 and
myc-rbslo1 in A and B is indicated by white
arrows. E is the merged image of
C and D. All experiments were performed at least
three times separately, and only representative images (from about 40)
are shown.
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rbslo2 Does Not Traffic to the Cell Surface--
Fig. 4 suggests
that the majority of rbslo2 is localized inside the cell. To see if
some of the rbslo2 proteins are able to reach the cell surface, we
compared the expression of rbslo2 constructs with the endogenous or
transfected cell membrane markers ZO-1 (40) and CD4 (41), respectively.
Fig. 5 shows either membrane marker, ZO-1
(A) or CD4 (D), in red and rbslo2 in
green (B and E). A merge of the images
(C and F) shows no regions of overlap (yellow) at the cell surface, indicating that rbslo2 was
almost exclusively confined to the cytoplasmic region of the cell.

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Fig. 5.
Immunocytochemistry studies show rbslo2 is
not able to reach the HEK293 cell surface. A-C,
confocal images of HEK293 cells transfected with myc-rbslo2 and
immunolabeled under permeabilized conditions. ZO-1 labeling, using
rabbit anti-ZO-1 antibody (1:1000), is shown in red
(A) as the cell surface marker. myc-rbslo2 stained with
mouse anti-myc antibody (1:1000) is shown in green
(B). C, is the overlap of images A and
B. D-F, confocal images of HEK293
cells cotransfected with CD4 and GFP-rbslo2, and immunolabeled under
non-permeabilized condition. The CD4 labeling, using mouse anti-CD4
antibody (1:500), is shown in red (D) as the cell
membrane marker and GFP-rbslo2 in green (E).
F is the overlap of images D and E. At
least four separate experiments were examined, and the representative
images out of 30 were displayed.
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In a separate experiment to verify the surface presentation of rbslo1
and rbslo2 proteins, we conducted cell surface biotinylation experiments (Fig. 6). The data showed
that a large amount of rbslo1 proteins was biotinylated, indicating its
strong cell surface expression. On the contrary, for rbslo2, only a
negligible amount of the protein was biotinylated. The Western blotting
results for rbslo2 were similar to GFP, a control protein that does not express at the cell surface. Taken together with our immunofluorescent data, these results confirm that the majority of rbslo1, but not rbslo2, is delivered to the cell surface region.

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Fig. 6.
Surface biotinylation study of rbslo1 and -2 channel proteins. A, DNA construct of wt-rbslo1
(top), GFP-rbslo2 (middle), or GFP
(bottom) was transfected into HEK293 cells, and the
exogenously expressed proteins were examined in Western blot using
rabbit anti-rbslo1 (1:1000) and rabbit anti-GFP (1:1000) antibodies,
respectively. Surface biotinylation studies were performed as described
under "Experimental Procedures." For all three samples, cell
lysates containing 30 µg of protein were loaded in the first
lane. The unbound (second lane) and biotinylated
(third lane) samples come from cell lysates originally
containing 30 and 50 µg of protein, respectively. B,
densitometry measurements of the three gels. Bars depict the
percentage of total cell lysate at the cell surface (ratio of the
amount of surface-biotinylated protein/the total protein in the cell
lysate × 100%). The star shows a significant
difference between rbslo1 and rbslo2 surface biotinylation.
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Intracellular Distribution of rbslo1 and rbslo2--
To further
pursue the intracellular localization of rbslo1 and 2 proteins, we
double-labeled rbslo1- or rbslo2-expressing cells with antibodies
against channel proteins and markers of intracellular organelles. Both
rbslo1 and 2 appear (Fig. 7, A and D, respectively) in green, and the ER, as
detected by an anti-KDEL antibody, appears in red. Regions
of overlap appear in yellow. Whereas rbslo1 is expressed
primarily at the plasma membrane, some rbslo1 did colocalize with the
ER marker. In contrast, rbslo2 resides predominately in the ER region.
Using the anti-
-adaptin antibody (Cy3-labeled secondary) to label
the Golgi apparatus, it can be seen that only rbslo2 colocalized
with Golgi marker, whereas the colocalization signal of rbslo1 and
Golgi marker was very weak (Fig. 7, B and E).
Endosomal compartments were labeled by anti-mannose 6-phosphate
receptor antibody (Fig. 7, C and F). Only a
negligible amount of rbslo1 or rbslo2 localized within the endosomal
compartments of the cell. Importantly, both myc-rbslo1-Del and
myc-rbslo2-Del have intracellular ER and Golgi localization similar as
rbslo2, again pointing out the role of the C-terminal domain in the
trafficking of rbslo1 (Fig. 7, G-L).

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Fig. 7.
Intracellular localization of rbslo1 and -2 in HEK293 cells. Immunofluorescence images of wt-rbslo1
(green in A-C, stained with rabbit anti-rbslo1
antibody (1:500)), GFP-rbslo2 (green in D-F,
using GFP fluorescence directly), myc-rbslo1-Del (green in
G-I, using chicken anti-myc antibody (1:100)), or
myc-slo2-Del (green in J-L, using chicken
anti-myc antibody (1:100)) were transiently expressed in HEK293 cells.
The first column (A, D, G,
and J), ER was revealed with mouse anti-KDEL antibody
(1:500) (red). The second column (B,
E, H, and K), Golgi apparatus was
indicated with anti- -adaptin antibody (1:500) (red). The
third column (C, F, I, and
L), endosomal compartments were labeled with anti-mannose
6-phosphate receptor antibody (1:300) (red). The
yellow appearing in A, D,
G, and J as well as in E,
H, and K denotes colocalized regions of the
rbslo1 or 2 protein with the respective cell organelle markers. At
least four images were taken for each sample and shown are
representative ones.
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Coexpression of MaxiK
Subunits Does Not Change the Subcellular
Distribution of Either rbslo1 or rbslo2--
To study whether the
MaxiK
subunits play a role in the intracellular trafficking of
rbslo1 and 2, we cotransfected either rbslo1 or rbslo2 with the MaxiK
3 or
4 subunit (Fig. 8). In the
presence of the
subunit in HEK293 cells, rbslo1 was still located
at the cell surface (Fig. 8, A-C and G-I),
whereas rbslo2 remained mostly in the cytoplasm (Fig. 8,
D-F and J-L). Comparing these
results with those in Figs. 5 and 7, it can be seen that the
subcellular distribution of rbslo2 did not change after coexpression with MaxiK
subunits.

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Fig. 8.
MaxiK subunits do
not change the subcellular localization of rbslo1 or rbslo2.
A-C, coexpression of rbslo1 (A) and 3
(B) in HEK293 cells. The merged image was shown
in C; D-F, coexpression of rbslo2
(D) and 3 (E) in HEK293 cells. The
merged image was shown in F; G-I,
coexpression of rbslo1 (G) and 4 (H) in HEK293
cells. The merged image was shown in I;
J-L, coexpression of rbslo2 (J) and 4
(K) in HEK293 cells. The merged image was shown
in L. At least four images were taken for each sample, and
representative images are shown.
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rbslo1 Does Not Facilitate the Surface Expression of
rbslo2--
To study the possible effect of rbslo1 on the
intracellular trafficking of rbslo2, we cotransfected both rbslo1 and 2 into HEK293 cells. When coexpressed, rbslo1 and 2 colocalized most intensely within the cytoplasmic region of the cell (Fig.
9A, panels A-F).
Similar to what was observed with rbslo1 alone, only the red
but not the yellow color can be seen at the cell surface, indicating the presence of only rbslo1 but not rbslo2. This is more
clearly seen under non-permeabilized conditions. In a merged image of
wt-rbslo1 (in red) and GFP-rbslo2 (in green), no
yellow color can be detected (Fig. 9A,
panels G-I). Thus rbslo1 did not promote the surface
expression of rbslo2. In addition, rbslo2 did not exert a strong
dominant-negative effect on the surface expression of rbslo1.

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Fig. 9.
Immunocytochemistry and surface biotinylation
studies on HEK293 cells coexpression of rbslo1 and -2. A,
panels A-C, HEK293 cells cotransfected with wt-rbslo1 and
myc-rbslo2 were immunolabeled under permeabilized conditions. Shown are
confocal images of wt-rbslo1 (A, using rabbit anti-rbslo1
antibody (1:500)), myc-rbslo2 (B, using mouse anti-myc
antibody (1:1000)), and the overlaid image with wt-rbslo1 in
red and myc-rbslo2 in green (C).
D-I, HEK293 cells cotransfected with wt-rbslo1
and GFP-rbslo2 were immunolabeled under permeabilized
(D-F) or non-permeabilized (G-I)
conditions. Shown are confocal images of wt-rbslo1 (D and
G) and GFP-rbslo2 (E and H) stained
with rabbit anti-rbslo1 antibody or examined using intrinsic GFP
fluorescence, respectively. F and I, the overlay
images with wt-rbslo1 in red and GFP-rbslo2 in
green. B, surface biotinylation study on HEK293
cells transfected with wt-rbslo1 (left two lanes),
GFP-rbslo2 (middle two lanes), or cotransfected with both
wt-rbslo1 and GFP-rbslo2 (right two lanes). Upper
gel, rabbit anti-rbslo1 antibody (1:1000) was used to show the
signal of rbslo1 as indicated by the arrow; lower
gel, rabbit anti-GFP antibody (1:1000) was used to show the signal
of rbslo2 as indicated by the arrow. L, cell
lysates samples. 15 µg of protein was loaded for rbslo1 sample
(lane 1), 30 µg of protein for rbslo2 (lane 3),
and cotransfection sample (lane 5); B,
surface-biotinylated samples. All samples (lanes 2,
4, and 6) from cell lysates containing 150 µg
of protein. C, densitometry study on gels in B. Bars depict the percentage of total cell lysate at the cell
surface (ratio of the amount of surface-biotinylated protein/the total
protein in the cell lysate × 100%). The left three
bars come from data in the upper gel; the right
three bars come from data in the lower gel. The
star shows the significant difference in protein amount for
surface-biotinylated rbslo1 before and after coexpression with
rbslo2.
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Fig. 9B confirms these results with surface biotinylation
experiments. As shown in the top panel, rbslo1 protein was
biotinylated when either expressed alone or coexpressed with rbslo2. In
contrast, rbslo2 was not biotinylated when expressed alone in HEK293
cells (bottom panel). Coexpression with rbslo1 did not
result in the biotinylation of rbslo2, indicating that rbslo2 remained
primarily inside the cell. Interestingly, after coexpression with
rbslo2, surface biotinylation of rbslo1 was weaker, implying that less rbslo1 reaches the cell surface (Fig. 9C). When transfected
together, rbslo2 may reduce surface expression of rbslo1 somewhat,
perhaps by competing with the same intracellular trafficking machinery.
Distribution of rbslo1 and rbslo2 in Polarized Renal
Epithelia--
Endogenous MaxiK channel proteins are expressed in
renal epithelia (9, 12). In our Western blot experiments we also
detected a specific band in cultured medullary thick ascending limb
cell (mTAL) lysates using anti-rbslo1 antibody (data not shown). The band is very weak and has an apparent molecular mass of about 70 kDa,
corresponding to another isoform of the MaxiK channel (42). To examine
how rbslo1 and 2 channels are trafficked in polarized epithelial cells,
we transfected myc-rbslo1 and myc-rbslo2 constructs into cultured mTAL
cells. Use of the myc-tagged fusion construct allowed detection of only
exogenously expressed rbslo1 and 2. At 72 h post-transfection,
polarized mTAL cells were fixed and permeabilized, and antibodies were
added at both the apical and basolateral sides of the cell. After the
immunostaining procedure, the sample was examined by confocal
microscopy with ZO-1, the tight junction protein, as apical
membrane marker. Pictures in Fig. 10
(A and B) are a reconstruction of
serial sections of immunofluorescent images along the Z direction (see
"Experimental Procedures"). The en face pictures depict
signals from all confocal sections. The X-Z (top) or Y-Z
cross-section (left) pictures show that myc-rbslo1 was
almost exclusively localized at the apical surface of mTAL cells (Fig.
10A). In contrast, myc-rbslo2 did not achieve a polarized distribution. It spread diffusely in the cytoplasm of the cell (Fig.
10B). The location of rbslo2 was further determined by
cotransfection of rbslo1 and 2 in mTAL cells. In Fig. 10C,
rbslo1 was shown in red and rbslo2 in green.
Although some small amount of apical membrane location of rbslo2 may
occur, the results indicate that the majority of rbslo2 did not reach
the plasma membrane of polarized mTAL cells. Both truncation mutants,
i.e. myc-rbslo1-Del myc-rbslo2-Del, showed intracellular
distributions similar to myc-rbslo2 (data not shown).

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Fig. 10.
Localization of rbslo1 and -2 in polarized
mTAL cells. Shown are reconstructed immunofluorescent confocal
pictures of myc-rbslo1 (A) and myc-rbslo2 (B)
expressed in polarized mTAL cells. The cell-cell tight junction protein
ZO-1 was stained with rabbit anti-ZO-1 antibody (1:1000) and is shown
in green. The rbslo1 or 2 proteins were stained with mouse
anti-myc antibody (1:500) and are shown in red. The en
face images are the collapsed serials of XY plane confocal images
along the Z direction. At the top are the XZ cross-section
images with the apical side facing upward and the
basolateral side facing downward. To the left of
each en face image are YZ cross-section images with the apical side on the left and the basolateral side
on the right. C, immunofluorescent confocal
images of myc-rbslo1 (in red) and GFP-rbslo2 (in
green) coexpressed in polarized mTAL cells. At the
top is the XZ cross-section image with the apical side
facing upward. The bottom 12 images are XY plane
confocal images along the Z direction. From left to
right, and then from top to bottom,
the focus plane moved from the apical to the basolateral side of the
cell. All experiments were performed at least three times separately,
and only representative images (out of 10) are shown.
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DISCUSSION |
MaxiK channels exist in several nephron segments (6-9). Although
their exact physiological roles in renal epithelia are unclear, the
existing literature suggests that they may function in cell volume
regulation and flow-mediated K+ secretion (12). We
previously cloned two MaxiK channel splicing isoforms, rbslo1 and
rbslo2, from rabbit mTAL cells. Although rbslo1 expression leads to
functional currents in either mammalian cell lines or
Xenopus oocytes, rbslo2 cannot be activated in
any of the systems. Therefore, we asked whether the problem arises from
a failure of assembly or one of cell surface delivery. In addition,
because complex renal function relies upon the correct sorting,
targeting, and restriction of ion channel proteins to specific regions
of the nephronal epithelial cells (43, 44), it is important to know the
detailed intracellular distribution of renal MaxiK channels at the
protein level.
We used myc- and GFP-tagged fusion protein in some of our experiments.
To verify that the tags did not have an effect, the channel's behavior
was carefully checked in the experiments shown in Figs. 2 and 4.
Bravo-Zehnder et al. (45) also reported that myc-tagged
MaxiK channel has characteristics similar to those of wild type. N
terminus GFP tagging of hslo changes the channel's electrophysiological properties (46). In our case, the GFP-tagged rbslo2 did not generate currents, like the wild type, and its intracellular distribution was not changed. Therefore, we used these
constructs for our further immunofluorescence and Western blotting experiments.
In the present study we demonstrated, using Western blotting methods,
that both rbslo1 and rbslo2 have the ability to form oligomers,
including tetramers. The missing part in the C-terminal region of
rbslo2 does not play an important role in the assembly of the channel.
There is an additional 58-aa insertion in rbslo1 and a unique C
terminus of 15 aa long in rbslo2. Because the MaxiK channel can still
be assembled as oligomers without either of the two domains, we further
conclude that neither of these two domains is important for channel
assembly. For voltage-gated KV channels, which are
homologous to MaxiK channels in the N-terminal region, the structural
element facilitating subunit assembly is localized in the hydrophilic N
terminus (47). Recently, Quirk et al. (36) showed that a
region between S6 and S7 in hslo1 is capable of self-association and
required for functional channel expression. They concluded that this
region mediates the tetramerization of hslo1. Elucidation of the
detailed sequence that is required for MaxiK channel assembly needs
further investigation.
The mechanism underlying the renal MaxiK channel sorting remains
unknown. We demonstrate here, using two separate methods, i.e. immunofluorescent microscopy and surface biotinylation,
that the cytoplasmic domain of the MaxiK channel is necessary for its surface delivery. The rbslo2, which lacks this cytoplasmic domain, remained predominately within an intracellular pool and did not appear
abundantly at the cell surface in either HEK293 or mTAL cells.
Using anti-KDEL and other antibodies as cell organelle markers, we
showed localization of rbslo2 within the ER and Golgi apparatus. Little
rbslo2 proteins localized to endosomes. Thus, the majority of rbslo2 is
unable to process further beyond the ER or Golgi. Interestingly, the
unique 15-aa C terminus of rbslo2 is a potential PSD-95, Dlg, zo-1
(PDZ) binding domain that has been reported to be involved in
ion channel trafficking (33, 48, 49). However, the similar behavior of
myc-rbslo2-Del, which has this unique domain deleted, compared with
myc-rbslo2 suggests that this domain does not inhibit the function or
surface expression of the channel.
At present, four MaxiK
subunits have been found in human and other
mammals (19, 20, 50-52). Results in Fig. 8 showed that, when
coexpressed either with the MaxiK
3 or
4 subunit, neither rbslo1
nor rbslo2 acquired a different subcellular distribution compared with
that observed in the absence of the MaxiK
subunits.
There are three differences between rbslo1 and rbslo2: 1) rbslo1 has a
58-aa insertion in the cytoplasmic domain between S8 and S9, 2) rbslo2
is truncated at Gly781, missing a 332-aa cytoplasmic tail,
and 3) has a unique 15-aa C terminus. Our experiments here and
previously (34) showed that neither the 58-aa insertion in rbslo1 nor
the unique 15-aa C terminus in rbslo2 is likely to play a major role in
plasma membrane trafficking. On the other hand, we present compelling data to show that the cytoplasmic tail of rbslo1 does play a role.
Many other MaxiK splice variants exist in different tissues. At least
one of these can act as a dominant negative one that, when coexpressed
with other subunits, inhibits the surface expression of the other
subunits (53). We also examined whether rbslo2 can influence the
surface expression of coexpressed rbslo1. Our experiments in Fig. 9
showed that both isoforms colocalized in the cytoplasm but only rbslo1
presented at the cell surface. Although rbslo2 still did not reach the
cell surface, the amount of rbslo1 on the cell surface was partially
reduced. These results, together with our previous electrophysiological
data (9), indicate that rbslo2 has some effect on the trafficking and
function of rbslo1, but it does not fully prevent the surface
expression of rblso1. However, this effect may also result from
transfection of more than one plasmid and may not reflect an actual
physiological reduction of rbslo1 by rbslo2.
Localization of ion channels and pumps to the correct membrane of
polarized epithelia is important for vectorial ion movement (54, 55).
Several prior studies have shown the distribution of MaxiK channel in
polarized epithelial cells. Using single channel patch recording,
currents from MaxiK channels were recorded at apical or basolateral
membranes of different cell types (23, 56-63). Bravo-Zehnder et
al. (45) reported that when transfected into Madin-Darby canine
kidney cells, hslo protein traffics to the apical membrane. Endogenous
rabbit MaxiK channel localizes at the apical membranes of collapsed
thick ascending limb of the loop of Henle, as shown using indirect
immunofluorescence microscopy (12). Because the rbslo1 and rbslo2 are
different MaxiK splice variants having distinct molecular weights and
sequences, we examined using confocal microscopy the reconstructed
three-dimensional pictures of rbslo1 and 2 expressed in mTAL cells. The
representative images in Fig. 10 clearly indicate that rbslo1 localized
abundantly at the apical membrane of the renal epithelia. In contrast,
very little rbslo2 colocalized with rbslo1 at the plasma membrane (Fig. 10C), suggesting that most rbslo2 proteins remain in the
cytoplasm. The results from polarized epithelial cells are
fundamentally consistent with those from non-polarized HEK293 cells.
That rbslo1 proteins exist at the apical membrane of renal epithelial
cells is consistent with a role for these channels in K+
secretion in kidney.
In summary, we demonstrated that rbslo1 is localized at the cell
surface in non-polarized cells and at the apical surface of polarized
renal epithelial cells. Whereas rbslo2 retains the ability to assemble
into oligomers, the majority does not reach the cell surface, remaining
trapped in the ER and Golgi. Considering the high degree of similarity
between the two isoforms, we conclude that the cytoplasmic tail of
rbslo1, missing in rbslo2, is critically involved in the cell surface
expression of MaxiK channels.