From the Department of Cell Biology and Physiology, Washington
University School of Medicine, St. Louis, Missouri 63110
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INTRODUCTION |
ATP-sensitive potassium (KATP) channels couple
cellular metabolism to electrical activity in multiple cell types.
Insulin secretion in pancreatic
cells provides one of the best
understood examples of such coupling (1). KATP channels are
generated by coexpression of a potassium channel subunit (Kir6.2) with
the sulfonylurea receptor (SUR1) (2). Neither sulfonylurea receptor nor
Kir6.2 subunits are normally capable of channel activity when expressed
alone in mammalian cells or in Xenopus oocytes (2-5). Experiments on functional reconstitution of characteristic
cell currents by coexpression of SUR1 receptor and Kir6.2 suggested their
association in a multidomain complex (2). It has been shown that Kir6.2
subunits form the channel pore (6), in a tetrameric complex, with each
Kir6.2 subunit associated with 1 SUR1 subunit to form a higher order
octamer (7-9), and physical association of the two subunits has been
demonstrated biochemically (8). Clement et al. (8) have
shown that SUR1 and Kir6.2 protein can be detected in independent
expression, but in this case, higher order glycosylation of SUR1 is not
seen, and glibenclamide labeling of Kir6.2 is not observed. Fusion
proteins in which SUR1 and Kir6.2 are linked in tandem generate
functional KATP channels (7, 8). Sedimentation experiments
with channel complexes formed either by monomeric SUR1 and Kir6.2
subunits or by fusion proteins (8) further suggest a molecular weight
that is consistent with an octameric model of the KATP
channel containing 4 channel and 4 receptor subunits.
Nothing is known, however, about the cell biology of complex formation.
The inability of Kir6.2 subunits to function as K+ channels
in the absence of SUR1 (Inagaki et al. (2)) may be due to
trafficking failure and raises the possibility that one role of SUR1 is
to act as a chaperone to permit Kir6.2 subunits to form into a tetramer
and/or to traffic to the surface membrane (5). Conversely, there is no
direct demonstration of the surface expression of SUR1 independent of
association with Kir6.2 in the KATP complex. Ozanne
et al. (10) showed that whereas a 170-kDa sulfonylurea
receptor (SUR)1 was present
in the plasma membrane, a 140-kDa form was only present on internal
membranes. Clement et al. (8) show that SUR1 is seen as a
core-glycosylated 140-kDa species seen when expressed in the absence of
Kir6.2 but that a higher order (170 kDa) species is seen when
coexpressed with Kir6.2. Together, these results might suggest that
SUR1 cannot reach the surface membrane in the absence of Kir6.2. In the
present study we have utilized green fluorescence protein (GFP)
labeling to visualize in vivo Kir6.2 and SUR1 targeting in
mammalian cells and to address the question of whether Kir6.2
trafficking to the cell membrane is dependent on association with SUR1
subunits. The results indicate that both SUR1 and Kir6.2 can
independently traffic toward the cell membrane.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction and Nomenclature--
GFP optimized for
maximal fluorescence (11) was fused to the N or C terminus of Kir6.2
(GFP-Kir6.2 and Kir6.2-GFP, respectively) or to the C terminus of SUR1
(SUR1-GFP) using an overlapping PCR technique. In the latter case, a
linker of six glycine residues was placed in between SUR1 and GFP. A
dimeric SUR1-Kir6.2 construct (7, 8) was tagged at the C terminus with
GFP by substituting an XhoI-EcoRI fragment
containing Kir6.2-GFP. Kir6.2 and its derivatives were expressed under
the control of the cytomegalovirus promoter in pCMV6 vector and
SUR1-containing constructs in pECE expression vector. GFP-tagged
-1,4-galactosyltransferase gene was a gift from Dr. Jennifer
Lippincott-Schwartz (12).
Cell Transfection and Cultivation--
COSm6, HEK293, and
BHK cells were transiently transfected using LipofectAMINE reagent
(Life Technologies, Inc.) according to the manufacturer's suggestions
for microscopy and electrophysiology or by a DEAE-dextran/chloroquine
method for rubidium flux measurements. In the latter case, cells were
plated on glass coverslips at a density of 0.5-2.5 × 105 cells/well (30-mm six-well dishes) and cultured in
Dulbecco's modified Eagle's medium plus 10 mM glucose
supplemented with fetal calf serum (10%). The following day, 5 µg of
total cDNA was transfected into COSm6 cells with diethylaminoethyl
dextran (0.5 mg/ml). Cells were incubated for 2 min in HEPES-buffered
salt solution containing Me2SO (10%) and then for 4 h
in Dulbecco's modified Eagle's medium plus 10 mM glucose
plus 2% fetal calf serum and chloroquine (100 µM) and
then returned to Dulbecco's modified Eagle's medium plus 10 mM glucose plus 10% fetal calf serum.
Rubidium Flux Experiments--
86RbCl (1 mCi/ml) was
added in fresh growth medium 24 h after transfection. Cells were
incubated for 12-24 h before measurement of Rb efflux. For efflux
measurements, cells were incubated for 30 min at 25 °C in
Krebs-Ringer solution with or without metabolic inhibitors (2.5 mg/ml
oligomycin plus 1 mM 2-deoxy-D-glucose). At
selected time points, the solution was aspirated from the cells and
replaced with fresh solution. The 86Rb+ in the
aspirated solution was counted.
Light and Laser Microscopy--
Cells were cultivated for 24-72
h after transfection and photographed in UV light under 1,000 × magnification in a Zeiss microscope equipped with a 515-nm emission
filter. Confocal analysis was performed using an argon-krypton laser
(Bio-Rad). Green fluorescence was detected at
= 515 nm after
excitation at
= 488 nm. Cells were observed on thin coverslips used
for their plating or after being treated with 0.1% trypsin for 30 s and resuspended in phosphate-buffered saline. Digitized images from
confocal experiments were prepared for presentation using Corel
Photopaint (Corel Inc.).
Patch-clamp Experiments--
Patch-clamp experiments were
made at room temperature in a chamber that allowed the solution bathing
the exposed surface of the isolated patch to be changed in less than 50 ms. Shards of glass were removed from the culture dishes and placed in
the experimental chamber. Micropipettes were pulled from thin-walled
glass (WPI Inc., New Haven, CT) on a horizontal puller (Sutter
Instrument, Co., Novato, CA). Membrane patches were voltage-clamped
with an Axopatch 1B patch-clamp (Axon Inc., Foster City, CA). PClamp
software and a Labmaster TL125 D/A converter (Axon Inc.) were used to
generate voltage pulses. Data were normally filtered at 0.5-3 kHz, and signals were digitized at 22 kHz (Neurocorder, Neurodata, NY, NY) and
stored on video tape. Experiments were replayed onto a chart recorder
or digitized into a microcomputer using Axotape software (Axon Inc.).
The standard bath (intracellular) and pipette (extracellular) solution
had the following composition: 140 mM KCl, 10 mM K-HEPES, 1 mM K-EGTA, pH 7.3, with additions
as described. Off-line analysis was performed using ClampFit and
Microsoft Excel programs. Wherever possible, data are presented as
mean ± S.E. (standard error). Microsoft Solver was used to fit
data by a least-square algorithm.
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RESULTS |
Tagged Constructs Form Essentially Normal KATP
Channels--
To follow KATP channel activity in living
cells, GFP was fused to the C terminus of SUR1 (SUR1-GFP) or to the N
or C terminus of Kir6.2 (GFP-Kir6.2 or Kir6.2-GFP). To examine
functional integrity of chimeric channels, SUR1-GFP was cotransfected
with Kir6.2, and Kir6.2-GFP or GFP-Kir6.2 was cotransfected with SUR1
into COSm6 cells. Rubidium efflux measurements performed on the
metabolically inhibited transfectants (Fig.
1A) show that functional
KATP channels are formed from each tagged construct when
coexpressed with the complementary subunit. Neither GFP-tagged
construct of Kir6.2 showed detectable channel activity in the absence
of coexpression with SUR1, based on rubidium efflux measurements (not
shown).

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Fig. 1.
86Rb+ efflux from
untransfected COSm6 cells and cells expressing SUR1 and Kir6.2
subunits. Graphs show percent of Rb+ released
into the medium as a function of time in the presence of metabolic
inhibitors for a typical experiment. B, representative currents recorded from inside-out membrane patches containing wild type
or mutant KATP channels (as indicated) at 50 mV. Patches were exposed to differing [ATP] as indicated. The calibration bars represent 1 nA (5 nA for SUR1-Kir6.2-GFP) and 10 s. C, steady-state dependence of membrane current (relative
to current in zero ATP) on [ATP] for wild type and mutant
KATP channels (as indicated). Data points represent the
mean ± S.E. (n = 3-8 patches). The fitted lines correspond to least squares fits of the Hill equation
(relative current = 100/(1+([ATP]/Ki)H), with
H = 1.0, and Ki = 11.1 µM (wild type), 8.6 µM (Kir6.2-GFP + SUR1),
13.5 µM (SUR1-GFP + Kir6.2), and 39.1 µM (SUR1-Kir6.2-GFP).
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Further characterization of GFP-tagged channels constructs was
made using patch-clamp experiments. As shown in Fig. 1, B
and C, each of the tagged constructs generated channels that
were inhibited by ATP. The tagged SUR1 and Kir6.2 monomers, when
coexpressed with the relevant other subunit, generated channels with
wild type ATP sensitivity ([ATP] causing half-maximal inhibition
(Ki) = 13.5 µM and 8.6 µM for SUR1-GFP + Kir6.2 and Kir6.2-GFP + SUR1, respectively, cf. 11.1 µM for wild type
channels). SUR1-Kir6.2-GFP triple fusions generated functional channels
in the absence of additional subunits. These channels were less
sensitive to ATP inhibition, the fitted Ki (39.1 µM) being very similar to that seen with expression of
untagged SUR1-Kir6.2 dimeric fusion proteins (45.3 µM,
Ref. 7; Ref. 8). The functional similarity of tagged channels to the
wild type enables us to use them as analogs of the authentic
KATP channel in studying channel biogenesis.
Tagged Constructs Can Be Visualized in Endoplasmic Reticulum,
Perinuclear Space, and Plasma Membrane--
When GFP is fused to
Kir6.2, a strongly fluorescent protein is produced in transfected cells
and allows us to follow the fate of the Kir6.2 subunit in
vivo. When coexpressed with SUR1 to generate functional
KATP channels, the intracellular distribution of GFP-tagged Kir6.2 clearly differs from the distribution of free GFP, as shown in
populations of transfected BHK cells (Fig.
2) and in individual COSm6 cells at
higher magnification in Fig. 3. A strong
signal associated with Kir6.2 is concentrated in the endoplasmic
reticulum (ER) and perinuclear space, which contains the Golgi complex
(Fig. 3C). Unfortunately, contrast plasma membrane
fluorescence is not obvious in adherent COSm6 cells. To reveal the
surface signal, we minimized the cell surface by mild trypsinization
immediately before confocal analysis. With visualization in a confocal
microscope, edge fluorescence is clearly visible in Kir6.2-GFP + SUR1-transfected COSm6 cells (Fig. 3D) but is absent in
control cells expressing GFP alone (Fig. 3B) or GFP-tagged
-1,4-galactosyltransferase, an integral membrane protein that
resides in the Golgi but not in the plasma membrane (Fig.
3F, Ref. 12).

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Fig. 2.
Fluorescence associated with GFP-tagged
Kir6.2 plus SUR1 in BHK cells. Fluorescent images of BHK cells
expressing GFP alone (A) or Kir6.2-GFP and SUR1
(B) are shown. The calibration bar represents 5 µm. In this and following figures, typical cells are shown from three
or more independent transfections. In BHK cells, >70% transfection
was apparent, as judged by the fraction of fluorescent cells. In COSm6
cells, transfection was typically >30%, and in all cases, similar
fluorescence was evident in essentially all transfected cells on a
given plate (20 to >1000 cells).
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Fig. 3.
Fluorescence associated with GFP-tagged
Kir6.2 plus SUR1 in COSm6 cells. Fluorescent images of COSm6 cells
expressing GFP alone (A), Kir6.2-GFP (C), or
GFP-tagged -1,4-galactosyltransferase (Tgal-GFP)
(E) and confocal fluorescent images of trypsinized COSm6
cells expressing GFP alone (B), Kir6.2-GFP and SUR1
(D), or Tgal-GFP (F) are shown. The
calibration bar represents 3 µm in A,
C, and E and 2 µm in B,
D, and F. In this figure and Figs. 4 and 5,
white arrows indicate the endoplasmic reticulum,
darts indicate the plasma membrane, and asterisks
indicate the Golgi apparatus. try, trypsinized.
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In contradiction to the hypothesis that SUR1 might act as a chaperone
to guide Kir6.2 through the secretory pathway (4, 5), nearly identical
fluorescence patterns were observed when GFP-tagged Kir6.2 constructs
were expressed with (Fig. 3, C and D) or without
SUR1 (Fig. 4, A and
B). Adherent transfected COSm6 cells show the Kir6.2-GFP
subunit entering the secretory pathway, but again, a visible plasma
membrane signal is not obvious (Fig. 4A). Again, however, a
clear edge fluorescence is apparent after mild trypsinization (Fig.
4B). Adherent HEK293 cells (Fig.
5) do not spread so thinly on coverslips,
and in this case, edge fluorescence is visible in Kir6.2-GFP single
transfectants (Fig. 5A). Since Kir6.2-GFP showed no
electrical activity when expressed alone, we could not confirm membrane
localization functionally. As a positive control of a homomeric,
integral membrane protein with assessable expression, we studied Kir2.3
(HRK1) inward rectifier channel (13) subunits fused at the C terminus
to GFP. Kir2.3-GFP chimeric channels conduct inward rectifier
K+ currents with wild type efficiency (14) and show
essentially identical fluorescence patterns to those seen in Kir6.2-GFP
cells (Figs. 4, C and D, and 5B).

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Fig. 4.
Fluorescence associated with GFP-tagged
inward rectifier subunits. Fluorescent images of COSm6 cells
expressing Kir6.2-GFP alone (A), Kir2.3-GFP (C),
or SUR-Kir6.2-GFP (WTF-GFP) (E) and confocal
fluorescent images of trypsinized (typ) COSm6 cells
expressing Kir6.2-GFP alone (B), Kir2.3-GFP (D),
or WTF-GFP (F) are shown. The calibration bar
represents 3 µm in A, C, and E and 2 µm in B, D, and F.
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Fig. 5.
Fluorescence associated with GFP-tagged
inward rectifier subunits in HEK293 cells. Fluorescent images of
HEK293 cells expressing Kir6.2-GFP alone (A), Kir2.3-GFP
(B), SUR-GFP alone (C), and SUR-Kir6.2-GFP
(WTF-GFP) (D) are shown. The calibration bar represents 3 µm in each case.
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Coexpression of Kir6.2-GFP with SUR1 (in ~1:1 cDNA ratio), which
results in characteristic KATP currents (Fig. 1), does not obviously affect the intracellular distribution of Kir6.2-GFP (Fig. 4,
A and B, cf. Fig. 3, C and
D). One possible reason is that in coexpression, the
predominant Kir6.2 signal that we observe is still actually from
non-SUR1-associated subunits. To examine whether the distribution of
SUR1-associated Kir6.2 differs from the distribution of Kir6.2 alone,
we transfected cells with a SUR1-Kir6.2 tandem (8) tagged at the
C-terminal end with GFP (SUR1-Kir6.2-GFP). This triple-fusion
construct, lacking free Kir6.2 subunits, shows ER, Golgi, and cell
surface fluorescence of a similar intensity to cells expressing
Kir6.2-GFP in both COSm6 cells (Fig. 4, E and F)
and in HEK293 cells (Fig. 5D).
Since Kir6.2-GFP can traffic toward the plasma membrane independently
of SUR1, we addressed the question of whether SUR1 is capable of
Kir6.2-independent trafficking to the plasma membrane? COSm6 cells were
transfected with SUR1-GFP with or without additional Kir subunits
(Figs. 6, 7). In COSm6 cells, SUR1-GFP
fluorescence also localizes to the endoplasmic reticulum, perinuclear
space (Fig. 6A), and plasma membrane (Fig. 5C) in
untreated cells, and plasma membrane fluorescence is also clearly
visible in trypsinized COSm6 cells (Fig. 6B). However, a
strikingly different fluorescence pattern was observed when SUR1-GFP
was cotransfected with Kir6.2 (Fig. 6C), indicating a
physical interaction. The perinuclear and reticular distribution was
substantially replaced by a diffuse fluorescence across the cell (Fig.
6C), although membrane fluorescence was still clearly
visible in trypsinized cells (Fig. 6D). As shown in Fig.
7, this effect of Kir6.2 coexpression is
specific. When SUR1-GFP is coexpressed with Kir1.1 (Fig. 7C)
or Kir2.3 (Fig. 7D), which do not interact with SUR1, the
fluorescence is not different from that seen with expression of
SUR1-GFP alone (Fig. 7A).

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Fig. 6.
Fluorescence associated with GFP-tagged SUR1
subunits in COSm6 cells indicates a physical interaction between SUR
and Kir6.2. Fluorescent images of COSm6 cells expressing SUR-GFP
alone (A) and SUR-GFP and Kir6.2 (C) and confocal
fluorescent images of trypsinized (try) COSm6 cells
expressing SUR-GFP alone (B) and SUR-GFP and Kir6.2
(D) are shown. The calibration bar represents 3 µm in A and C and 2 µm in B and
D.
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Fig. 7.
Fluorescence associated with GFP-tagged SUR1
subunits indicates a specific interaction with Kir6.2.
Fluorescent images of COSm6 cells expressing SUR-GFP alone
(A), SUR-GFP and Kir6.2 (B), SUR-GFP and Kir1.1
(C), and SUR-GFP and Kir2.3 (D) are shown. The
calibration bar represents 8 µm in each case.
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DISCUSSION |
Neither receptor (SUR1) nor pore-forming (Kir6.2) subunits of the
KATP channel normally demonstrate a plasma
membrane-associated phenotype, with the exception of sulfonylurea
binding to SUR1 in total membranes (3), when expressed in the absence
of the other subunit. SUR1 appears as a core-glycosylated 140-kDa
species in the absence of Kir6.2 (8), and this species does not appear to reach the plasma membrane in various insulin-secreting cell lines
(10). A C-terminal deletion construct (5) and even full-length
Kir6.22 may form some active
ATP-sensitive K+ channels in the absence of SUR1, but in
both cases a considerably higher level of channel expression is
observed when coexpressed with SUR1. Lack of channel function when wild
type Kir6.2 is expressed without SUR1 may be due to an inability to
express stably or to reach the cell surface and reside there for a
physiologically significant period of time. The general concept of
multimeric integral membrane protein biogenesis is that subunits
assemble in early steps and are co-transported through the Golgi
complex and the trans-Golgi network to the plasma membrane (reviewed in Ref. 15). Unincorporated subunits typically do not reach the plasma
membrane because of proteolytic degradation in the endoplasmic reticulum or in lysosomes. The T-cell antigen receptor, consisting of
seven transmembrane subunits, provides an example of both degradation pathways. Although the fully assembled, seven-membered complex is
transported to the plasma membrane, free
,
, and
chains rapidly degrade in the endoplasmic reticulum, and pentamers lacking the
chain pass the Golgi complex but are then redirected to lysosomes
(reviewed in Ref. 16). To explore the possibility that a similar
degradation of free KATP subunits underlies the lack of
functional channels in different expression conditions, we labeled them
with GFP and followed their fate in living cells. Both SUR1-GFP and
Kir6.2-GFP were observed in ER, Golgi, and cell surface compartments,
indicating that neither unincorporated subunit contains any specific
signal for rapid and effective proteolytic degradation in the ER or
rerouting to lysosomes.
With a few exceptions (e.g. connexin-43, gap-junction
channel, type 2 sodium channel) where oligomerization takes place after exit (17, 18), subunit assembly of integral membrane proteins generally
occurs in the endoplasmic reticulum, and proteins that fail to assemble
into requisite oligomeric complexes are degraded without release from
the endoplasmic reticulum (16). In contrast to Kv1.2 or Kv2.2 channels,
where auxiliary
subunits promote surface staining with antibodies
raised against
-subunits (19, 20), coexpression of KATP
channel subunits does not obviously change the expression pattern of
individual subunits. The stability of free KATP channel
subunits in the secretory pathway indicates that assembly is not
necessary for trafficking without necessarily implying that assembly
into the functional complex should normally occur after independently
reaching the plasma membrane. The formation of functional channels by
the SUR1-Kir6.2-GFP triple fusion construct together with the visible
intracellular distribution (Fig. 4, E and F)
shows that preassembled SUR1 plus Kir6.2 dimer is also not subject to
endoplasmic reticular or lysosomal degradation and is as viable outside
the plasma membrane as the individual subunits. The effect of
coexpression with Kir6.2 on the intracellular distribution of SUR1-GFP
(Figs. 6 and 7) indicates a specific physical interaction between SUR1
and Kir6.2, since coexpression with other noninteracting Kir
subunits (Kir1.1, Kir2.3) has no effect on the SUR1-GFP
fluorescence (Fig. 7).
A complex picture is beginning to emerge for KATP channel
formation. The demonstration that the channel requires two subunits (2)
and recent indications of an obligate stoichiometry (7-9) might
suggest a classical endoplasmic reticular oligomeric assembly (21) in
which each subunit is necessary for normal trafficking. On the other
hand, Ammala et al. (22) reported that coexpression with
SUR1 was capable of conferring sulfonylurea sensitivity on Kir1.1
channels, which can clearly function in the absence of SUR1 (23) and
suggested that SUR1 may "promiscuously" couple to different inward
rectifier subunits. In the present study, we find that SUR1-GFP
fluorescence patterns are affected specifically by expression with
Kir6.2 but not by expression with Kir1.1 or Kir2.3. Together with the
demonstration that azidoglibenclamide can label Kir6.2, but not Kir1.1,
in coexpression with SUR1 (8), these results indicate that SUR1 does
not in fact interact promiscuously with other Kir subunits and
interacts specifically with Kir6.x subunits. The present results
further indicate that independent trafficking of each subunit toward
the cell membrane can occur, but altered fluorescence of SUR1-GFP when
coexpressed with Kir6.2 indicates a specific interaction and suggests
the following as a working hypothesis. Independent synthesis of Kir6.2
tetramers (7) and SUR1, perhaps as monomers, can occur in the
endoplasmic reticulum, and these homomeric constructs can traffic to
the surface membrane. However, when both subunits are coexpressed, as
happens in native cells, a physical interaction between SUR1 and Kir6.2 occurs in the endoplasmic reticulum, possibly involving the C terminus
of SUR1, which either facilitates insertion of channel-containing vesicles into the plasma membrane or stabilizes channels once inserted
into the plasma membrane.
We are grateful to Dr. J. Lippincott-Schwartz for providing us with the
-1,4-galactosyltransferase-GFP construct, to Drs. S. Seino and J. Bryan for providing us with the original Kir6.2 and the SUR1-Kir6.2
fusion, respectively, and to Drs. S. John, J. R. Monck, J. N. Weiss, and B. Ribalet for unpublished data. We are grateful to the
Washington University Diabetes Research and Training Center for
continued molecular biology support.