From the University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, United Kingdom
Received for publication, November 1, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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ATP-sensitive potassium (KATP)
channels comprise Kir and SUR subunits. Using recombinant
KATP channels expressed in Xenopus oocytes, we
observed that MgATP (100 µM) block of Kir6.2/SUR2A currents gradually declined with time, whereas inhibition of
Kir6.2/SUR1 or Kir6.2 ATP-sensitive potassium
(KATP)1 channels
are widely distributed, being found in pancreatic B-cells, cardiac,
smooth, and skeletal muscles, and neurones (1). They play important
functional roles in all these tissues by linking cellular metabolism to
electrical activity. Opening of the KATP channel produces a
voltage-independent K+ current that hyperpolarizes the cell
and reduces its electrical excitability; conversely, KATP
channel closure usually decreases membrane excitability.
The KATP channel is an octamer, formed by the physical
association of four inwardly rectifying potassium channel subunits (Kir6.2) and four regulatory sulfonylurea receptor subunits (2). KATP channels in different tissues are composed of
different Kir and SUR subunits. In most tissues, Kir6.2 acts as the
pore-forming subunit (3, 4). Two different sulfonylurea receptor genes (SUR1 and SUR2) have been identified, and further diversity is created
by alternative splicing of SUR2 (5-9). In this paper, the major
isoform of SUR2 (6) is called SUR2A. It shares 67% sequence identity
with SUR1. There is evidence that SUR1 serves as the regulatory subunit
of the KATP channel in pancreatic B-cells and some types of
neurone (5, 10), whereas a similar role is played by SUR2A in cardiac
and skeletal muscle (6).
Binding sites for drugs and modulatory agents are found on both Kir6.2
and SUR subunits. The nucleotides ATP and ADP bind to an intracellular
site on Kir6.2 and bring about KATP channel closure (11,
12), whereas a range of magnesium nucleotides (including ATP and ADP)
interact with the NBDs of SUR and thereby increase channel
activity (13-16). The balance between these inhibitory and stimulatory
effects results in channel inhibition by most concentrations of MgATP
and high concentrations of MgADP (10 mM) and in channel
activation by low micromolar concentrations of MgADP. It is thought
that metabolic regulation of KATP channel activity is
mediated, in part, by changes in the intracellular concentrations of
these nucleotides.
Recent studies indicate that membrane phospholipids also play an
important role in modulating KATP channel activity. Thus, phosphatidylinositol 4,5-bisphosphate (PIP2) increases the
activity and reduces the ATP sensitivity of both native and cloned
KATP channels (17-21). Overexpression of PI5-kinase, which
enhances PIP2 levels, reduces the ATP sensitivity of the
channel in membrane patches (21), whereas breakdown of PIP2
by phospholipase C enhances the KATP channel ATP
sensitivity (22). This is in agreement with data indicating that
PIP2 binds directly to Kir channels (23). Interestingly,
application of PIP3 and PI 3-P also enhances KATP channel activity (19, 20, 24).
The rundown of both native and cloned KATP channels that
occurs in excised membrane patches is reversed by application of MgATP
(25-27), and PIP2 has also been implicated in this effect (25). It is postulated that PIP2 is produced in plasma
membrane by serial phosphorylation of PI and that this process is
sequentially catalyzed by PI 4-kinase and PI 5-kinase. MgATP
generation of phospholipids, therefore, has multiple but related
effects on KATP channel activity. It decreases the rate of
channel rundown, it causes reactivation of the channel after removal of
ATP, and it reduces the channel ATP sensitivity.
In this paper, we demonstrate that the time course of KATP
channel inhibition by ATP is markedly different for Kir6.2/SUR1 and
Kir6.2/SUR2A. Whereas inhibition of Kir6.2/SUR1 currents by 100 µM ATP does not change with time, that of Kir6.2/SUR2A
decreases during prolonged nucleotide application. This
ATP-dependent activation of Kir6.2/SUR2A currents by MgATP
can be prevented by 10 µM LY 294002, a specific inhibitor
of PI 3-kinase (28), suggesting that it results from
MgATP-dependent production of PIP3 or
PI(3,4)P2 rather than PI(4,5)P2
(PIP2). Both PIP3 and PIP2, however,
are able to promote channel activity in excised patches. Studies with chimeric SUR further suggest that the different responses of
Kir6.2/SUR1 and Kir6.2/SUR2A channels are conferred by the first set of
transmembrane domains of the sulfonylurea receptor.
Molecular Biology--
Mouse Kir6.2 (GenBankTM
accession number D50581; Refs. 3 and 4), rat SUR1
(GenBankTM accession number L40624; Ref. 5), and SUR2A
(GenBankTM accession number D83598; Ref. 6) cDNAs were
cloned in the pBF vector. A truncated form of Kir6.2 (Kir6.2 Oocyte Collection--
Female Xenopus laevis were
anesthetized with MS222 (2 g/liter added to the water). One ovary was
removed via a mini-laparotomy, the incision was sutured, and the animal
was allowed to recover. Immature stage V and VI oocytes were incubated
for 60 min with 1.0 mg/ml collagenase (Sigma, type V) and manually
defolliculated. Oocytes were either injected with ~1 ng of Kir6.2 Electrophysiology--
Patch pipettes were pulled from
borosilicate glass and had resistances of 250-500 k
In most experiments, currents were recorded in response to repetitive
3-s voltage ramps from Macroscopic currents were recorded in inside-out patches from
Xenopus oocytes expressing either Kir6.2/SUR1 or
Kir6.2/SUR2A. Current amplitudes were similar for both types of
KATP channel.
Time Course of ATP Inhibition--
Fig.
1A shows that application of
100 µM ATP to the intracellular membrane surface
initially inhibited both Kir6.2/SUR1 and Kir6.2/SUR2A currents by
~90%. Inhibition of Kir6.2/SUR1 currents did not change, or even
slightly increased, over the course of a 10-min exposure to ATP. In
contrast, there was a gradual decline in the ATP sensitivity of
Kir6.2/SUR2A currents with time; despite the continued presence of
nucleotide, a slow increase in current was observed that began about 2 min after the onset of ATP application and stabilized 8-10 min later
at around 75% block. This decrease in ATP sensitivity was not reversed
by a 1-min exposure to nucleotide-free solution (Fig. 1A).
Mean data are shown in Fig. 1B.
The extent of initial block of both types of KATP current
by 100 µM ATP is in agreement with that previously
published for Kir6.2/SUR1 and Kir6.2/SUR2A channels. When expressed in
oocytes and measured immediately after patch excision, half-maximal
inhibition (Ki) of Kir6.2/SUR1 and Kir6.2/SUR2A
channels is produced by 28 and 29 µM ATP, respectively
(31). The Ki value for Kir6.2/SUR1 is in good
agreement with that found when the channel is expressed in mammalian
cells (8-47 µM; Refs. 21 and 32) and with what is found
for the native
To confirm that the difference in nucleotide response between
Kir6.2/SUR1 and Kir6.2/SUR2A currents was conferred by the SUR subunit,
we also measured the effect of continued exposure to ATP on a truncated
form of Kir6.2 (Kir6.2 Effect of Walker A Mutation on the ATP Sensitivity of SUR
2A--
It is possible that the time-dependent
stimulatory effect of MgATP is mediated via the NBDs of SUR2A, either
directly or via hydrolysis of MgATP to MgADP. It is known that MgADP is
able to stimulate both Kir6.2/SUR1 (13-16, 31) and Kir6.2/SUR2A (31). Mutation of the lysine residue in the Walker A motif of NBD2 (but not
NBD1) of SUR2A abolishes this stimulatory effect (35). We therefore
examined the effect of MgATP on channels containing this mutation
(Kir6.2/SUR2A-K2A).
Fig. 2 shows that initial ATP sensitivity
of Kir6.2/SUR2A-K2A currents is slightly greater than that found for
the wild type channel; the mean block by 100 µM ATP was
97.7 ± 0.6% (n = 6) compared with 87.6 ± 4.3% (n = 7) for Kir6.2/SUR2A-K2A and Kir6.2/SUR2A, respectively. This is not unexpected because a similar increase in ATP
sensitivity is found when the equivalent residue is mutated in SUR1
(14). As in the case of SUR1, therefore, it may result from loss of
MgATP activation mediated via the NBDs of SUR. Despite the enhanced ATP
sensitivity, however, Kir6.2/SUR2A-K2A currents showed a
time-dependent activation in the presence of MgATP that resembled that found for wild type channels; it began 1-2 min after
exposure to ATP, and the current amplitude increased 3-fold within 5 min. This decrease in ATP sensitivity did not occur in the presence of
100 µM LY 294002, an inhibitor of PI 3-kinase (see
below); rather the currents declined with time (as was also observed
for Kir6.2/SUR2A; Fig. 3B).
These results therefore suggest that the MgATP-dependent
decline in ATP sensitivity is not mediated via nucleotide interaction
with the NBDs of SUR.
Mechanism of ATP-dependent Activation--
Another
mechanism by which the ATP sensitivity of the KATP channel
might be reduced is by the ATP-dependent generation of PIP2 in the patch membrane caused by the action of
endogenous lipid kinases (21). To test this hypothesis, we examined the effect of the lipid kinase inhibitor LY 294002 on the ATP sensitivity of Kir6.2/SUR2A currents (Fig. 3). LY 294002 is a potent and relatively specific inhibitor of PI 3-kinase with an IC50 of 1.5-4
µM (27, 36). Thus, at a concentration of 10 µM, PI 3-kinase is totally inhibited, but there is little
effect on PI 4-kinase, whereas at a concentration of 100 µM, PI 4-kinase is also completely blocked (36). As shown
in Fig. 3, 10 µM LY 294002 blocked the
time-dependent decline in ATP sensitivity observed for
Kir6.2/SUR2A currents and prevented the reduction in ATP sensitivity
produced by preincubation with ATP (compare Figs. 1 and 3). It also
slightly enhanced the ATP sensitivity of the channel. This suggests
that production of PI(3)P, PI(3,4)P2, or PIP3
is required for the response. 100 µM LY 294002 also
blocked the ATP-dependent activation of Kir6.2/SUR2A currents and further increased the extent of inhibition by 100 µM ATP. Wortmannin (100 µM,
n = 3), which also blocks PI 3-kinase (37), and
neomycin (100 µM, n = 3), which chelates
phospholipids, produced an immediate decline in Kir6.2/SUR2A channel
activity and prevented the MgATP-dependent loss of ATP
sensitivity (data not shown). As expected if a lipid kinase is
involved, ATP was not effective at stimulating Kir6.2/SUR2A currents in
the absence of Mg2+ (data not shown).
Taken together, these results suggest that MgATP is used as a substrate
by PI 3-kinase in the oocyte membrane to generate PIP3 or
other membrane lipids and that the gradual accumulation of
PIP3 causes a slow decline in the ATP sensitivity of
Kir6.2/SUR2A currents. Because PI 4-kinase is not blocked by 10 µM LY 294002, this activation cannot be mediated by
PI(4,5)P2 (Fig. 3C). Earlier studies have shown
that KATP channel is very sensitive to the level of PI
5-kinase activity (21), which favors the possibility that the effects
of MgATP are mediated via production of PIP3 rather than
PI(3)P or PI(3,4)P2. Although we cannot formally exclude a
role for the latter two phospholipids, for simplicity, we will simply
refer to PIP3 (rather than PIP3, PI(3)P, or
PI(3,4)P2) in the rest of this paper.
The time lag observed before ATP induces activation of Kir6.2/SUR2A
currents may therefore reflect the time taken for sufficient PIP3 to accumulate within the membrane to cause a
measurable reduction in ATP sensitivity. Accumulation of
PIP3 within the membrane may also explain why the ATP
sensitivity of Kir6.2/SUR2A currents is not immediately restored on
return to control solution. Clearly, if the lipid is not rapidly
removed from the membrane, then subsequent ATP application will produce
a smaller inhibitory response.
Mechanism of Action of PIP3--
Although exogenously
applied PIP3 is known to modulate
The effect of cytochalasin might be mediated subsequent to
PIP3 production, or it might affect generation of the
phospholipid. To distinguish between these possibilities, we examined
whether LY 294002 was able to inhibit the response to cytochalasin; if cytochalasin influences PIP3 production, then LY 294002 should block the response whereas, conversely, if cytochalasin has an effect downstream of the phospholipid it should still be effective in
the presence of LY 294002. As shown in Fig. 4 (A and
B), both 10 and 100 µM LY 294002 completely
abolished the response to cytochalasin. The simplest explanation of
this result is that changes in the cell cytoskeleton do not modify the
ATP sensitivity of Kir6.2/SUR2A currents directly. Rather, disruption
of the cytoskeleton enhances MgATP-dependent production of
PIP3 (or its metabolites) or facilitates PIP3
targeting to the KATP channel and thereby enhances the loss of ATP sensitivity.
In contrast to Kir6.2/SUR2A channels, KATP channels
containing the SUR1 subunit were not activated by cytochalasin in the presence of ATP (Fig. 4C). Thus, like the
ATP-dependent activation itself, this response is specific
to SUR2A.
Differential Sensitivity of Kir6.2/SUR1 and Kir6.2/SUR2A
Currents to PIP3--
It is striking that whereas
Kir6.2/SUR2A currents show an ATP-dependent activation that
appears to be mediated by PIP3 production, this is not the
case for Kir6.2/SUR1 currents. In some patches, however, we observed a
slow, time-dependent activation of Kir6.2/SUR1 currents
when exposed to 1 mM ATP (data not shown). This suggests that Kir6.2/SUR1 channels may simply be less sensitive to
PIP3 than Kir6.2/SUR2A channels.
We therefore tested the effect of direct application of
PIP2 or PIP3 on Kir6.2/SUR1 and Kir6.2/SUR2A
currents in the presence of 100 µM ATP. Fig.
5 (A and B) shows
that (in the absence of Mg2+) PIP3 produces a
time-dependent decline in the ATP sensitivity of both types
of channel but that this effect was more rapid and pronounced in the
case of Kir6.2/SUR2A than Kir6.2/SUR1. The magnitude and time course of
the PIP3 activation of Kir6.2
Fig. 5C shows that neither LY 294002 nor phalloidin is able
to block the effect of PIP3 on Kir6.2/SUR2A currents.
Similar results were found with PIP2 (data not shown). The
lack of effect of phalloidin is consistent with the idea that
stabilization of the cell cytoskeleton is involved in the
MgATP-dependent generation of phospholipids rather than
directly affecting the KATP channel. The data also indicate
that PIP2 does not have to be metabolized to
PIP3 to mediate its effect, because its effect was not
blocked by LY 294002.
Molecular Basis of the Differential Sensitivity of Kir6.2/SUR1 and
Kir6.2/SUR2A Currents to Phospholipids--
To determine the molecular
basis of the differential sensitivity of Kir6.2/SUR1 and Kir6.2/SUR2A
currents to phospholipids, we made chimeras between SUR1 and SUR2A and
investigated the ability of 100 µM MgATP to produce a
time-dependent activation of KATP channels
containing chimeric SUR. These results are summarized in Fig.
6. SUR1 containing both NBDs, or just the
last 42 amino acids of SUR2A, behaved like SUR1. Likewise, SUR2A
containing the last 42 amino acids, or NBD2, of SUR1 behaved like the
parent channel. When the first six TMs of SUR1 were transferred
into SUR2A, however, the ability of MgATP to stimulate KATP
channel activity was abolished (Fig. 6A). Thus, it appears
that this region of SUR is critical for the different response of
Kir6.2/SUR1 and Kir6.2/SUR2A channels to MgATP.
Our results suggest that the gradual loss of ATP sensitivity of
Kir6.2/SUR2A currents is due to a MgATP-dependent synthesis of membrane phospholipids, which causes a secondary decrease in the
channel ATP sensitivity. This hypothesis is supported by the facts that
LY 294002, wortmannin, neomycin, and Mg2+-free solutions
prevent this effect and that the effect of MgATP is mimicked by the
phospholipids PIP2 and PIP3.
In the cell membrane, PI 4-kinase phosphorylates PI to give
PI(4)P, which is subsequently phosphorylated by PI
5-kinase to PIP2 (PI(4,5)P2). All three
phospholipids are also phosphorylated by PI 3-kinase to produce PI(3)P,
PI(3,4)P2, and PIP3 respectively (Fig.
3C). Biochemical studies have shown that 10 µM
LY 294002 specifically inhibits PI 3-kinase and is without effect on PI 4-kinase but that at a concentration of 100 µM, LY 294002 also totally blocks PI 4-kinase (36). Because 10 µM LY
294002 abolished the magnesium-dependent loss of ATP
sensitivity, we conclude that either PI(3)P, PI(3,4)P2,
and/or PIP3 is involved the response. At this
concentration, LY 294002 does not prevent PI(4)P or
PI(4,5)P2 formation, suggesting that neither of these
lipids is responsible for the magnesium-dependent decrease
in ATP sensitivity.
When applied directly to the membrane patch, both PIP2 and
PIP3 were able to reduce the ATP sensitivity of
Kir6.2/SUR2A, although no MgATP-dependent activation of the
channel was observed in the presence of 10 µM LY 294002. This suggests that PIP3 is a more potent regulator of the
channel and that, in the presence of LY 294002, PIP2
generated by addition of MgATP does not accumulate to a concentration
sufficient to reduce the channel ATP sensitivity. This may be due to
the action of endogenous phospholipase C or lipid phosphatases. It is
also worth pointing out that because many proteins bind PIPs, they may
influence KATP channel activity simply by sequestering the
amount of PIPs available for interaction with the channel.
Mechanism of Action of PIP3--
Our results suggest
that the effect of PIP3 on the ATP sensitivity of the
KATP channel is not mediated via the cell cytoskeleton, because it was not blocked by phalloidin. We did observe, however, that
agents that perturb the cytoskeleton have a marked effect on the
MgATP-dependent decline in ATP sensitivity. Notably,
disruption of actin microfilaments by cytochalasin enhanced the
response, whereas stabilization of microfilaments by phalloidin
decreased the response. It is possible that the effect of cytochalasin
may be mediated upstream of PI 3-kinase because it can be blocked by 10 µM LY 294002. This might suggest that disruption of the cytoskeleton enhances PIP3 levels (or targeting to the
KATP channel), whereas, conversely, stabilization of the
cytoskeleton by phalloidin reduces PIP3 levels.
Furthermore, the ability of phalloidin to inhibit the
ATP-dependent loss of ATP sensitivity suggests that MgATP
may mediate its effect by inducing cytoskeletal disruption. This idea
is supported by the fact that phalloidin did not prevent the loss of
ATP sensitivity produced by direct application of PIP3 to
the intracellular membrane surface. In contrast, the stimulatory action
of PIP3 on native
Interestingly, cytochalasin reduced the activity of native cardiac
KATP channels in the absence of ATP (42) but enhanced it
when ATP was present (43). It seems plausible that the latter effect is
due to an increase in MgATP-mediated PIP3 generation, as in
the case for the cloned channel Kir6.2/SUR2A. A different mechanism may
underlie the action of cytochalasin in the absence of ATP, which
promotes KATP channel rundown (42).
Differential Sensitivity of Kir6.2/SUR1 and Kir6.2/SUR2A
Channels--
In contrast to Kir6.2/SUR2A currents, Kir6.2/SUR1
currents were not activated by 100 µM MgATP. Moreover,
when PIP3 was added directly to the patch in the presence
of 100 µM MgATP, the phospholipid produced a much faster
and more marked activation of Kir6.2/SUR2A than of Kir6.2/SUR1. Our
results indicate that these different responses are mediated by the
first set of transmembrane domains of SUR (TMs 1-6). It is
noteworthy that this region of SUR confers the different gating
kinetics of Kir6.2/SUR1 and Kir6.2/SUR2A channels (46, 47). This region
of SUR may therefore be involved in transducing conformational changes
in SUR into gating of the channel pore.
Both Kir6.2/SUR1 and Kir6.2
It is therefore possible that the different responses of Kir6.2/SUR2A
and Kir6.2/SUR1 are due to their different single-channel kinetics; the
higher Po of Kir6.2/SUR2A channels means that a further increase in Po produced by
PIP2 is more likely to reduce the channel ATP sensitivity
than in the case of Kir6.2/SUR1 channels. An alternative interpretation
of our results, however, is that PIP3 is able to interact
directly with the SUR2A subunit of the KATP channel to
decrease the channel ATP sensitivity (in addition to its effect on
Kir6.2) and that the functional effect of this interaction involves the
first six TMs of SUR.
C36 currents did not change. The decline in
Kir6.2/SUR2A ATP sensitivity was not observed in Mg2+ free
solution and was blocked by the phosphatidylinositol (PI) 3-kinase inhibitors LY 294002 (10 µM) and wortmannin (100 µM), and by neomycin (100 µM). These
results suggest that a MgATP-dependent synthesis of
membrane phospholipids produces a secondary decrease in the ATP
sensitivity of Kir6.2/SUR2A. Direct application of the phospholipids
PI 4,5-bisphosphate and PI 3,4,5-trisphosphate in the presence
of 100 µM MgATP activated all three types of channel, but
the response was faster for Kir6.2/SUR2A. Chimeric studies indicate
that the different responses of Kir6.2/SUR2A and Kir6.2/SUR1 are
mediated by the first six transmembrane domains of SUR. The MgATP-dependent loss of ATP sensitivity of Kir6.2/SUR2A was
enhanced by the actin filament disrupter cytochalasin and blocked by
phalloidin (which stabilizes the cytoskeleton). Phalloidin did not
block the effect of PI 3,4,5-trisphosphate. This suggests that MgATP may cause disruption of the cytoskeleton, leading to enhanced membrane
phospholipid levels (or better targeting to the KATP channel) and thus to decreased channel ATP sensitivity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C),
which lacks the C-terminal 36 amino acids and forms functional channels
in the absence of SUR, was prepared as described previously (11). Chimeras between SUR1 and SUR2A were constructed as described previously (29). Capped mRNA was prepared using the mMESSAGE mMACHINE large scale in vitro transcription kit (Ambion,
Austin, TX), as previously described (30).
C
mRNA or coinjected with ~0.1 ng of Kir6.2 mRNA and ~2 ng of
mRNA encoding either SUR1 or SUR2A. The final injection volume was
50 nl/oocyte. Isolated oocytes were maintained in Barth's solution and
studied 1-4 days after injection (30).
when filled
with pipette solution. Macroscopic currents were recorded from giant
excised inside-out patches at a holding potential of 0 mV and at
20-24 °C using an EPC7 patch-clamp amplifier (List Electronik,
Darmstadt, Germany; Ref. 30). The pipette (external) solution contained
140 mM KCl, 1.2 mM MgCl2, 2.6 mM CaCl2, 10 mM HEPES (pH 7.4 with
KOH). The intracellular (bath) solution contained 107 mM
KCl, 2 mM MgCl2, 1 mM
CaCl2, 10 mM EGTA, 10 mM HEPES (pH
7.2 with KOH; final [K+], ~140 mM). The
magnesium-free intracellular solution contained 140 mM KCl,
1 mM EGTA, 10 mM HEPES (pH 7.2 with KOH). LY
294002 (CalBiochem), phalloidin, and cytochalasin (CalBiochem) were
dissolved in Me2SO to make 10 mM stock
solutions. Stock solutions (1 mM) of PIP2 and
PIP3 were made in magnesium-free intracellular solution and
diluted to the desired concentration and sonicated (30 min on ice)
immediately before use. Rapid exchange of solutions was achieved by
positioning the patch in the mouth of one of a series of adjacent
inflow pipes placed in the bath. Test solutions were applied in random
order unless otherwise stated.
110 mV to +100 mV. They were filtered at 10 kHz, digitized at 0.4 kHz using a Digidata 1200 Interface, and analyzed
using pClamp software (Axon Instruments, Foster City, CA). Records were
stored on videotape and resampled at 20 Hz for presentation in the
figures. The slope conductance was measured by fitting a straight line
to the current-voltage relation between
20 and
100 mV; the average
response to five consecutive ramps was calculated in each solution. In
other experiments, macroscopic currents were recorded at a fixed
holding potential of
50 mV in response to nucleotide or drug
applications. Currents were sampled at 20 Hz and analyzed using
Microcal Origin software (Microcal Software, Northampton, MA). Data are
presented as the means ± S.E.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Time course of current response in the
presence of 100 µM MgATP.
A, macroscopic currents recorded from oocytes coexpressing
Kir6.2 and either SUR1 or SUR2A in response to a series of voltage
ramps from 110 to +100 mV. ATP was added to the intracellular
solution as indicated by the bars. All solutions contained
Mg2+. B, mean KATP conductance
recorded for Kir6.2
C36 (
, n = 3), Kir6.2/SUR1
(
, n = 6), or Kir6.2/SUR2A (
, n = 7) currents at different times after the addition of 100 µM MgATP to the intracellular solution. The slope
conductance (G) is expressed as a fraction of the mean
(Gc) of that obtained in control solution before exposure to
ATP.
-cell KATP channel (26 µM;
Ref. 33). Reported values for half-maximal inhibition of cloned
Kir6.2/SUR2A channels and for native cardiac KATP channels vary widely, from 17 to 100 µM (6, 31, 34). It seems
possible that this may reflect, at least in part, the time at which the measurements were made during exposure to ATP. It may also reflect differences in the concentration of endogenous membrane phospholipids.
C), expressed in absence of SUR. Fig.
1B shows that Kir6.2
C currents resemble Kir6.2/SUR1 currents and show a slow decline with time in the presence of ATP,
despite the lower ATP sensitivity. These data argue that the
time-dependent activation of Kir6.2/SUR2A currents is
conferred by the SUR2A subunit.
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Fig. 2.
Effects of an NBD mutation on the response to
MgATP. Mean conductance recorded for Kir6.2/SUR2A-K2A currents at
different times after the addition of 100 µM MgATP to the
intracellular solution in the absence ( , n = 6) or
presence (
, n = 3) of 100 µM LY
294002. The slope conductance (G) is expressed as a fraction
of the mean (Gc) of that obtained in control solution before
exposure to ATP.
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Fig. 3.
Effects of LY 294002 on Kir6.2/SUR2A
currents. A, macroscopic Kir6.2/SUR2A currents recorded
in response to a series of voltage ramps from 110 to +100 mV. ATP and
LY 294002 (10 µM, above; 100 µM,
below) were added to the intracellular solution as indicated
by the bars. All solutions contained Mg2+.
B, mean conductance recorded for Kir6.2/SUR2A currents at
different times after the addition of 100 µM MgATP to the
intracellular solution in the presence of 10 µM (
,
n = 3) or 100 µM (
, n = 3) LY 294002. The dashed line indicates the data obtained
in control solution (as shown in Fig. 1B). The slope
conductance (G) is expressed as a fraction of the mean
(Gc) of that obtained in control solution before exposure to
ATP. C, schematic illustrating the metabolism of
phospholipids. The pathways inhibited by 10 and 100 µM LY
294002 are indicated.
-cell KATP
channel activity (20, 38), it need not necessarily interact directly
with the channel. It might also exert its effect indirectly, by
modulating a protein that regulates the KATP channel. It is
well established that PIP2 and other membrane lipids
influence the cell cytoskeleton by inhibiting the activity of
actin-binding proteins that sever and cap actin filaments (39).
Likewise, PIP3 associates with the Rho family of GTPases
(40), which are key regulators of actin filament structure (41). Agents
that modulate the cytoskeleton also influence the activity of native KATP channels in cardiac membranes in both the presence and
absence of ATP (42, 43). We therefore examined the effects of
phalloidin, which stabilizes the cytoskeleton, and of cytochalasin,
which disrupts the cytoskeleton, on the ATP-dependent
activation of Kir6.2/SUR2A currents. Cytochalasin (10 µM)
increased the time-dependent current activation produced by
100 µM ATP, whereas phalloidin (20 µM)
diminished the extent of activation (Fig.
4A). These results argue that
the ATP-dependent activation of Kir6.2/SUR2A currents involves the cell cytoskeleton and that disruption of the cytoskeleton enhances the ATP-dependent decline in the channel ATP
sensitivity.
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Fig. 4.
Effects of cytoskeletal agents.
A, mean KATP conductance recorded for
Kir6.2/SUR2A currents at different times after the addition of 100 µM MgATP to the intracellular solution in the presence of
10 µM cytochalasin ( , n = 4) or 20 µM phalloidin (
, n = 3) or of 10 µM cytochalasin plus 10 µM LY 294002 (
,
n = 3). The slope conductance (G) is
expressed as a fraction of the mean (Gc) of that obtained in
control solution before exposure to ATP. The dashed line
indicates the control response in the absence of either agent.
B, mean conductance recorded for Kir6.2/SUR2A currents at
50 mV at different times after the addition of 100 µM
MgATP (
, n = 5), 100 µM MgATP plus 10 µM cytochalasin (
, n = 4), 100 µM MgATP and 10 µM cytochalasin plus 10 µM (
, n = 5), or 100 µM
LY 294002 (
, n = 5). The dashed line
indicates the initial current level. The conductance (G) is
expressed as a fraction of the mean (Gc) of that obtained at
50 mV in control solution before exposure to ATP. The patch was held
at
50 mV throughout the experiment. C, mean conductance
recorded for Kir6.2/SUR1 currents at different times after the addition
of 100 µM MgATP to the intracellular solution in presence
of 10 µM cytochalasin (n = 3). The
dashed line indicates the response in the absence of
cytochalasin. The slope conductance (G) was measured using a
ramp protocol and is expressed as a fraction of the mean
(Gc) of that obtained in control solution before exposure to
ATP.
C36 was similar to that
observed for Kir6.2/SUR1 (Fig. 5B), providing additional support for the idea that the differential action of the phospholipid on Kir6.2/SUR1 and Kir6.2/SUR2A is mediated by the SUR2A subunit. Similar results were found with PIP2 (n = 2-3 in each case, date not shown). Because the effects of
PIP3 were tested in the absence of Mg2+, we can
be certain that current activation is the result of the phospholipid
rather than ATP itself. It has previously been reported that the ATP
sensitivity of both types of channel is reduced by PIP2 in
excised patches (18-20, 22, 44), but the difference in sensitivity has
not been remarked.
View larger version (53K):
[in a new window]
Fig. 5.
Time course of current response in the
presence of 5 µM PIP3
and 100 µM
MgATP. A, macroscopic currents recorded from oocytes
coexpressing Kir6.2 and either SUR2A (above) or SUR1
(below) in response to a series of voltage ramps from 110
to +100 mV. ATP and PIP3 were added to the intracellular
solution as indicated by the bars. All solutions were
Mg2+-free. B, mean KATP conductance
recorded for Kir6.2
C36 (
, n = 3); Kir6.2/SUR1
(
, n = 3) or Kir6.2/SUR2A (
, n = 3) currents at different times after the addition of 5 µM
PIP3 and 100 µM ATP to the intracellular
solution. The slope conductance (G) is expressed as a
fraction of the mean (Gc) of that obtained in control
solution before exposure to ATP. All solutions were
Mg2+-free. C, mean conductance recorded for
Kir6.2/SUR2A currents at different times after the addition of 5 µM PIP3 and 100 µM ATP to the
intracellular solution in the presence of 20 µM
phalloidin (
, n = 3) or 10 µM LY
294002 (
, n = 3). The dashed line
indicates the response in the absence of either agent. The slope
conductance (G) is expressed as a fraction of the mean
(Gc) of that obtained in control solution before exposure to
ATP.
View larger version (29K):
[in a new window]
Fig. 6.
Effects of chimeras. A, mean
conductance recorded from an inside-out patch excised from an oocyte
coexpressing Kir6.2 and SUR-215, at different times after the addition
of 100 µM MgATP to the intracellular solution. The slope
conductance (G) is expressed as a fraction of the mean
(Gc) of that obtained in control solution before exposure to
ATP. The dashed lines indicate the response of Kir6.2/SUR1
or Kir6.2/SUR2A currents. B, macroscopic conductance
recorded from patches excised from oocytes coexpressing Kir6.2 and
either SUR1, SUR2A, or the SUR chimera indicated, after 5 min of
exposure to MgATP. Mean conductance in the presence of the test
solution (G) is expressed relative to the mean conductance
in the control (nucleotide-free) solution before nucleotide addition
(Gc). The number of patches is given by each
bar.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell KATP channels is
prevented by phalloidin (24, 45). Presumably, this difference reflects the different cell types, which may metabolize PIP3 differently.
C channels have shorter bursts of
openings and a lower open probability than Kir6.2/SUR2A channels. Loussouarn and colleagues (48) have shown that there is an exponential correlation between the burst duration (or open probability) of the
KATP channel and its Ki for ATP
inhibition, and it has been suggested that PIP2 mediates
its effect on KATP channel ATP sensitivity, at least in
part, via changes in the channel open probability (49). Whether or not
a given increase in open probability (Po)
results in a change in ATP sensitivity will therefore depend on the
initial Po. Because this is lower for
Kir6.2/SUR1 and Kir6.2
C currents, the likelihood of observing a
change in ATP sensitivity will be less.
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ACKNOWLEDGEMENTS |
---|
We thank Phillippa Jones for expert technical assistance and Colin Nichols and Cathy Cukras for advice on the use of phospholipids.
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FOOTNOTES |
---|
* This work 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.
Permanent address: Dept. of Physiology, Keimyung University School
of Medicine, 194 Dongsang Dong, Choong Gu, Taegu, 700-712 Korea.
§ To whom correspondence should be addressed. E-mail: frances.ashcroft@physiol.ox.ac.uk.
Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M009959200
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ABBREVIATIONS |
---|
The abbreviations used are: KATP, ATP-sensitive potassium; NBD, nucleotide-binding domain; PIP2, and PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PI, phosphatidylinositol; PI(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PI(4)P, phosphatidylinositol 4-phosphate; TM, transmembrane domain; PI(3)P, phosphatidylinositol 3-phosphate.
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