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J Biol Chem, Vol. 274, Issue 7, 3931-3933, February 12, 1999
,,
,, and**
From the Laboratory of Biochemistry, Division of
Applied Life Sciences, Kyoto University Graduate School of Agriculture,
Kyoto 606-8502, Japan, the § University Laboratory of
Physiology, Oxford OX1 3PT, United Kingdom, and the Department
of Molecular Medicine, Chiba University Graduate School of Medicine,
Chuo-ku, Chiba 260-8670, Japan
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ABSTRACT |
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 Top
 Abstract
 Introduction
References
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ATP-sensitive potassium channels are under
complex regulation by intracellular ATP and ADP. The potentiating
effect of MgADP is conferred by the sulfonylurea receptor subunit of
the channel, SUR, whereas the inhibitory effect of ATP appears to be
mediated via the pore-forming subunit, Kir6.2. We determined whether
ATP directly interacts with a binding site on the Kir6.2 subunit to mediate channel inhibition by analyzing binding of a photoaffinity analog of ATP (8-azido-[ ATP-sensitive potassium
(KATP)1 channels
play important roles in many tissues by linking the metabolic status of
the cell to its membrane potential (1, 2). In pancreatic Transfection and Preparation of Membranes--
COS-7 cells were
cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine
serum in a humidified atmosphere of 95% air, 5% CO2 at
37 °C. COS-7 cells were transfected with expression vectors encoding
full-length wild-type or mutant mouse Kir6.2, tagged with the Flag
epitope at the NH2 terminus and with a hexahistidine tag at
the COOH terminus (Flag-Kir6.2), or tagged with the Flag epitope at the
COOH terminus (Kir6.2-Flag), using LipofectAMINEplus (Life
Technologies, Inc.) according to the manufacturer's directions. Rat
Kir4.1 was tagged with the Flag epitope at the COOH terminus (Kir4.1-Flag). Addition of these epitopes did not significantly affect
the functional properties of the channel (data not shown). Two days
after transfection, cellular membranes were prepared as described
previously (16). Immunoblotting was carried out with an anti-Flag M2
monoclonal antibody (Eastman Kodak).
Photoaffinity Labeling of Flag-Kir6.2 with
8-Azido-[ Electrophysiological Studies--
Macroscopic currents were
recorded from giant inside-out patches excised from Xenopus
oocytes expressing Kir6.2 8-Azido-ATP Inhibition of Kir6.2 8-Azido-ATP Labeling of Kir6.2--
We next investigated the
direct interaction of Kir6.2 with ATP using the photoaffinity ATP
analog, 8-azido-[ Concentration Dependence--
Membranes from Flag-Kir6.2
transfected cells were incubated with different concentrations of
8-azido-[ Competition of Photoaffinity Labeling by ATP--
To determine
whether photoaffinity labeling of Flag-Kir6.2 was specific, competition
by ATP was examined (Fig. 4). Membranes were preincubated with 100 µM, 1 mM, and 2 mM ATP on ice for 10 min, and then 50 µM
8-azido-[ Effects of Kir6.2 Mutations on 8-Azido-ATP Labeling--
We have
previously identified two mutations that reduce the apparent ATP
sensitivity of Kir6.2 The results we present here provide the first biochemical evidence
that ATP binds directly to Kir6.2. We demonstrate that Kir6.2 can be
specifically labeled by the ATP photoaffinity analog 8-azido-[ We have previously reported that SUR1 binds 8-azido-ATP with high
affinity (16). SUR1 was efficiently photoaffinity-labeled with
8-azido-[32P]ATP by UV irradiation even after the removal
of unbound 8-azido-ATP. In contrast, Kir6.2 was not
photoaffinity-labeled with 8-azido-[32P]ATP by UV
irradiation after the removal of unbound 8-azido-ATP (data not shown).
Photoaffinity labeling with 8-azido-[ Mutations in both the NH2- and COOH-terminal intracellular
domains have been identified that significantly reduce ATP inhibition of Kir6.2
-32P]ATP) to membranes
from COS-7 cells transiently expressing Kir6.2. We demonstrate that
Kir6.2 can be directly labeled by 8-azido-[-32P]ATP
but that the related subunit Kir4.1, which is not inhibited by ATP, is
not labeled. Photoaffinity labeling of Kir6.2 is reduced by
approximately 50% with 100 µM ATP. In addition,
mutations in the NH2 terminus (R50G) and the COOH terminus
(K185Q) of Kir6.2, which have both been shown to reduce the inhibitory
effect of ATP upon Kir6.2 channel activity, reduced photoaffinity
labeling by >50%. These results demonstrate that ATP binds directly
to Kir6.2 and that both the NH2- and COOH-terminal
intracellular domains may influence ATP binding.
INTRODUCTION
Top
Abstract
Introduction
References
-cells,
KATP channels are critical for the regulation of
glucose-induced insulin secretion (3, 4) and have recently been shown
to be an octameric complex of two subunits, which coassemble with a 4:4
stoichiometry (5-9). The pore-forming subunit, Kir6.2, is a member of
the inwardly rectifying K+ channel family (10, 11), whereas
the other subunit, the sulfonylurea receptor (SUR1), is a member of the
ATP-binding cassette transporter superfamily (12, 13). Unlike most
other Kir channels, expression of Kir6.2 alone does not produce
functional channel activity; instead, it requires coexpression with
SUR1. However, an isoform of Kir6.2 in which the last 26 amino acids
have been removed (Kir6.2
C26) is capable of expressing functional
K+ channel activity in the absence of SUR1. Kir6.2C26
retains sensitivity to inhibition by ATP, and mutations in this subunit
can significantly reduce the inhibitory effect of ATP (14, 15). This
has been taken as evidence that the primary site at which ATP acts to
cause KATP channel closure resides on Kir6.2. However,
controversy still remains as to whether ATP binds directly to Kir6.2,
whether truncation of Kir6.2 exposes a cryptic blocking site for
nucleotides, or whether ATP inhibition is mediated indirectly by
binding of the nucleotide to an endogenous subunit that modulates the
activity of Kir6.2 (9, 14). In the present study, we show that Kir6.2 directly binds the photoaffinity analog of ATP, 8-azido-ATP, and that
this labeling can be reduced by 50% with 100 µM ATP. We
also demonstrate that the related inwardly rectifying K+
channel subunit Kir4.1, which is not inhibited by ATP, exhibits no
significant photoaffinity labeling by
8-azido-[-32P]ATP. Furthermore, we show that mutations
in Kir6.2 that reduce the inhibitory effect of ATP on channel activity
also reduce photoaffinity labeling. This provides strong evidence that
ATP binds directly to Kir6.2.
MATERIALS AND METHODS
-32P]ATP--
8-Azido-[-32P]ATP
(500-600 GBq/mmol) was purchased from ICN Biomedicals. Membranes were
incubated with 50 µM 8-azido-[-32P]ATP,
2 mM ouabain, 0.1 mM EGTA, 4 mM
MgSO4, and 40 mM Tris-Cl (pH 7.5) in a total
volume of 6 µl for 10 min on ice. After UV irradiation (at 254 nm,
4.4-8.2 milliwatts/cm2) for 15 s to 3 min, 500 µl
of TE buffer (40 mM Tris-HCl (pH 7.5), 0.1 mM
EGTA) was added to the mixture, and free
8-azido-[-32P]ATP was removed by centrifugation
(15,000 × g, 10 min, 4 °C). The pellet was
solubilized with 100 µl of RIPA buffer (20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 0.15 M NaCl, 10 µg/ml leupeptin, 100 µg/ml
(p-amidinophenyl)-methane-sulfonyl fluoride). The lysate
was kept on ice for 15 min and centrifuged to remove insoluble
material. Flag-Kir6.2 was immunoprecipitated from the supernatant with
the anti-Flag M2 antibody. Samples were electrophoresed on a 10%
SDS-polyacrylamide gel and autoradiographed. Bound
8-azido-[-32P]ATP to Kir6.2 was measured by scanning
with a radioimaging analyzer (BAS2000, Fuji Photo Film Co.).
Experiments were carried out in duplicate.
C26, as described previously (17). Currents
were recorded at a holding potential of 0 mV in response to repetitive
voltage ramps from 
110mV to +100 mV, at 20-24 °C. The pipette
solution contained 140 mM KCl, 1.2 mM
MgCl2, 2.6 mM CaCl2, and 10 mM HEPES (pH 7.4 with KOH), and the internal (bath)
solution contained 110 mM KCl, 2 mM
MgCl2, 1 mM CaCl2, 30 mM KOH, 10 mM EGTA, and 10 mM HEPES
(pH 7.2 with KOH) and 8-azido-ATP as indicated. ATP dose-response
relationships were measured by alternating the control solution with a
test ATP solution, and the extent of inhibition by ATP was expressed as
a fraction of the mean of the value obtained in the control solution
before and after ATP application (17). ATP dose-response curves were
fit to the Hill equation G/Gc = 1/(1 + ([ATP]/Ki)h), where [ATP] is the
ATP concentration, Ki is the ATP concentration at
which inhibition is half-maximal, and h is the slope factor
(Hill coefficient).
RESULTS
C26 Currents--
We first
examined the ability of 8-azido-ATP to inhibit Kir6.2C26 currents.
Fig. 1 shows that this nucleotide blocks
Kir6.2C26 currents rather less potently than ATP, half-maximal
inhibition (Ki) occurring at 2.8 ± 0.4 mM (n = 6) compared with 172 ± 7 µM for ATP (n = 6). The Hill coefficients
were 0.9 ± 0.2 for 8-azido-ATP and 1.3 ± 0.1 for ATP.
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Fig. 1.
Inhibition of
Kir6.2 C26 by 8-azido-ATP. A,
macroscopic currents recorded from a giant inside-out patch on an
oocyte injected with mRNA encoding Kir6.2C26. Currents were
elicited in response to a series of voltage ramps from 110 mV to +100
mV. ATP or 8-Azido-ATP was added to the internal solution as indicated
by the bar. B, mean dose-response relationship
for ATP (n = 6) or 8-azido-ATP (n = 6).
Test solutions were alternated with control solutions, and the slope
conductance (G) is expressed as a fraction of the mean
(Gc) of that obtained in control solution before
and after exposure to ATP. Conductance was measured between 20 and
100 mV and is the mean of five voltage ramps. The solid
lines are the best fit of the data to the Hill equation (see
"Materials and Methods") using the mean values for
Ki and h given in the text.
-32P]ATP. Flag-Kir6.2 and Kir4.1-Flag
were transiently expressed in COS-7 cells, and expression levels were
monitored by immunoblot analysis of membrane fraction preparations
(Fig. 2A). Membranes were
incubated with 50 µM 8-azido-[-32P]ATP
for 10 min on ice and irradiated with UV light. Flag-Kir6.2 and
Kir4.1-Flag were immunoprecipitated with an anti-Flag M2 antibody and
subjected to electrophoresis. The autoradiogram in Fig. 2B shows an approximately 43-kDa photoaffinity-labeled protein to be
immunoprecipitated from Flag-Kir6.2 transfected cells (Fig. 2B, lanes 3 and
4) but not from untransfected cells (Fig. 3A, lanes 3 and 4). The molecular mass of this
photoaffinity-labeled membrane protein is identical to that of
Flag-Kir6.2 identified by Western blotting (Fig. 2A).
Neither of the two bands observed in the Western blot of Kir4.1-Flag
transfected cells (Fig. 2A) exhibited any significant
photoaffinity labeling (Fig. 2B, lanes 1 and
2). Attachment of the Flag epitope at the COOH terminus of
Kir4.1 is unlikely to hinder immunoprecipitation because
photoaffinity-labeled Kir6.2-Flag (COOH-terminal tag) was precipitated
as efficiently as the NH2-terminal fusion, Flag-Kir6.2
(data not shown). Flag-Kir6.2 could also be photoaffinity-labeled with
8-azido-[
-32P]ATP (data not shown).
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Fig. 2.
Photoaffinity labeling of Flag-Kir6.2 with
8-azido-[ -32P] ATP.
A, membranes (10 µg) from COS-7 cells expressing
Kir4.1-Flag and Flag-Kir6.2 were separated by 10% SDS-polyacrylamide
gel electrophoresis and detected by immunoblotting with anti-Flag
monoclonal antibody M2. Lane 1, untransfected cells;
lane 2, Kir4.1-Flag transfected cells; lane 3,
Flag-Kir6.2 transfected cells. B, membranes (80 µg) were
incubated with 50 µM 8-azido-[-32P] ATP
for 10 min on ice. Proteins were photoaffinity-labeled by UV
irradiation (at 254 nm, 8.2 milliwatts/cm2) for 15 s
(lanes 1 and 3) and 30 s (lanes 2 and 4). Kir4.1-Flag and Flag-Kir6.2 were immunoprecipitated
with antibody M2 after solubilization and analyzed as described under
"Materials and Methods."
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Fig. 3.
Concentration dependence of photoaffinity
labeling of Flag-Kir6.2 with
8-azido-[ -32P] ATP.
Membranes (100 µg) from untransfected COS-7 cells (A) or
COS-7 cells expressing Flag-Kir6.2 (B) were incubated with
8-azido-[-32P] ATP at 10 µM (lane
1), 50 µM (lane 2), 100 µM
(lane 3), and 200 µM (lane 4).
Proteins were photoaffinity-labeled with UV irradiation (at 254 nm, 4.4 milliwatts/cm2) for 3 min. Flag-Kir6.2 was
immunoprecipitated with antibody M2 after solubilization as described
under "Materials and Methods."
-32P]ATP and photoaffinity-labeled (Fig.
3B). Photoaffinity labeling increased with increasing
concentrations of 8-azido-[-32P]ATP, and no saturation
was observed at the highest concentration tested (200 µM). No protein smaller than 50-kDa showed specific photoaffinity labeling in membranes from untransfected COS-7 cells, even with 200 µM 8-azido-[-32P]ATP (Fig.
3A).
-32P]ATP was added. Photoaffinity labeling of
Flag-Kir6.2 was reduced as the concentration of ATP was increased.
Quantitation by radioimaging analysis revealed that photoaffinity
labeling was reduced by approximately 50% in the presence of 100 µM ATP.
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Fig. 4.
Inhibition of
8-azido-[ -32P] ATP photoaffinity
labeling of Flag-Kir6.2 by cold ATP. Membranes (100 µg) from
COS-7 cells expressing Flag-Kir6.2 were incubated without (lane
1) or with 100 µM (lane 2), 1 mM (lane 3), and 2 mM (lane
4) cold ATP for 10 min on ice, and 50 µM
8-azido-[-32P]ATP was added to the mixture. The
mixture was incubated for 10 min on ice, and proteins were
photoaffinity-labeled with UV irradiation (at 254 nm, 4.4 milliwatts/cm2) for 3 min. Flag-Kir6.2 was
immunoprecipitated after solubilization as described under "Materials
and Methods."
C26 from a Ki of ~100 µM to ~4 mM. These are R50G in the
NH2 terminus and K185Q in the COOH terminus (14, 15).
Neither mutation affects the level of channel expression as examined by
electrophysiological methods and immunoblotting. The effect of these
mutations on 8-azido-[-32P]ATP binding was examined.
Membranes prepared from cells expressing equivalent amounts of the
wild-type and mutant forms of Flag-Kir6.2 (Fig.
5A) were labeled using 100 µM 8-azido-[-32P]ATP (Fig.
5B). Quantitation of the labeling by radioimaging analysis
revealed that photoaffinity labeling of the mutants R50G and K185Q was
reduced by 50 and 65%, respectively, as compared with wild-type
Flag-Kir6.2.
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Fig. 5.
Mutations in Flag-Kir6.2 reduce photoaffinity
labeling with
8-azido-[ -32P]ATP.
Lanes 1, wild type; lanes 2, R50G; lanes
3, K185Q. A, membranes (10-20 µg) from COS-7 cells
expressing equivalent amounts of wild-type or mutant Flag-Kir6.2 were
separated by 10% SDS-polyacrylamide gel electrophoresis and detected
by immunoblotting with anti-Flag monoclonal antibody M2. B,
membranes (50-100 µg) from COS-7 cells expressing equivalent amounts
of wild-type and mutant Flag-Kir6.2 were photoaffinity-labeled with 100 µM 8-azido-[-32P]ATP. C,
relative photoaffinity labeling of wild-type and mutant Flag-Kir6.2,
expressed as percentages of that observed for wild-type
Flag-Kir6.2.
DISCUSSION
-32P]ATP in the absence of SUR and that this
labeling can be significantly reduced by competition with ATP.
Furthermore, the related subunit Kir4.1, which is not inhibited by ATP,
exhibits no photoaffinity labeling by
8-azido-[-32P]ATP. Further evidence of the direct and
specific interaction of ATP with Kir6.2 is provided by the observation
that mutations in Kir6.2 that reduce the inhibitory effect of ATP on
channel activity also reduce the photoaffinity labeling.
-32P]ATP did not
appear to saturate even at a concentration 200 µM (Fig.
3). These results indicate that Kir6.2 has much lower affinity for
8-azido-ATP than SUR1. This is consistent with the result of the lower
affinity of Kir6.2 for 8-azido-ATP (Ki = 2.8 mM) as compared with ATP itself (Ki = ~100 µM). Introduction of the reactive azido group at
the 8' position may account for this reduced affinity because Kir6.2
demonstrates high specificity toward the adenine moiety of ATP (15).
Labeling with 8-azido-[-32P]ATP required 0.1 mM ATP for approximately 50% displacement, a value that is
consistent with that found for half-maximal inhibition of Kir6.2C26
currents (~0.1 mM).
C26 currents (14, 15). The mutations R50G in the NH2 terminus and K185Q in the COOH terminus both reduce the
Ki for inhibition of Kir6.2C26 from ~100
µM to ~4 mM (14, 15). In support of the
fact that channel inhibition is mediated by a direct interaction of ATP
with Kir6.2, we found that both of these mutations also exhibit
significantly reduced photoaffinity labeling with
8-azido-[-32P]ATP (Fig. 5). Neither mutation affected
the channel gating kinetics (15) and so are predicted to influence ATP
sensitivity by effects on ATP binding and/or the link between binding
and gating. However, although we show that mutations in both the
NH2- and COOH-terminal intracellular domains influence
labeling, it remains unclear whether the effect of these mutations
reflects a direct interaction of these residues with ATP or whether
their effects on ATP binding are mediated indirectly.
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FOOTNOTES |
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* This work was supported by Grants-in-Aid for Scientific Research on Priority Areas "Channel-Transporter Correlation" 07276101 and "ATP-binding Cassette Proteins" 10217205 and by a Grant-in-Aid for Creative Basic Research from the Ministry of Education, Science, Sports, and Culture of Japan. Work in the Oxford group was supported by the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Wellcome Trust Research Career Development Fellow.
** To whom correspondence should be addressed.
The abbreviation used is: KATP, ATP-sensitive potassium.
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REFERENCES |
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Top
Abstract
Introduction
References
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