From the University Laboratory of Physiology, Oxford OX1 3PT, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ATP-sensitive potassium (KATP) channels are reversibly inhibited by intracellular ATP. Agents that interact with sulfhydryl moieties produce an irreversible inhibition of KATP channel activity when applied to the intracellular membrane surface. ATP appears to protect against this effect, suggesting that the cysteine residue with
which thiol reagents interact may either lie within the ATP-binding site or be inaccessible when the channel is
closed. We have examined the interaction of the membrane-impermeant thiol-reactive agent p-chloromercuriphenylsulphonate (pCMPS) with the cloned cell KATP channel. This channel comprises the pore-forming Kir6.2 and regulatory SUR1 subunits. We show that the cysteine residue involved in channel inhibition by pCMPS resides on
the Kir6.2 subunit and is located at position 42, which lies within the NH2 terminus of the protein. Although ATP
protects against the effects of pCMPS, the ATP sensitivity of the KATP channel was unchanged by mutation of C42
to either valine (V) or alanine (A), suggesting that ATP does not interact directly with this residue. These results
are consistent with the idea that C42 is inaccessible to the intracellular solution, and thereby protected from interaction with pCMPS when the channel is closed by ATP. We also observed that the C42A mutation does not affect
the ability of SUR1 to endow Kir6.2 with diazoxide sensitivity, and reduces, but does not prevent, the effects of MgADP and tolbutamide, which are mediated via SUR1. The Kir6.2-C42A (or V) mutant channel may provide a
suitable background for cysteine-scanning mutagenesis studies.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ATP-sensitive K channels (KATP channels) couple electrical activity to the metabolic state of the cell in a variety of tissues, including muscle, nerve, and endocrine
cells (Ashcroft and Ashcroft, 1990; Nichols and Lederer, 1991
; Quayle et al., 1997
; Trapp and Ashcroft,
1997
). These channels are inhibited by ATP and activated by MgADP and it is currently thought that metabolic regulation of the KATP channel is achieved through
changes in the cytosolic levels of these nucleotides.
KATP channels are also blocked by sulfonylurea drugs,
which are used to treat non-insulin-dependent diabetes mellitus, and activated by a group of chemically unrelated drugs collectively known as K channel openers
(Ashcroft and Ashcroft, 1992
; Edwards and Weston,
1993
). Recent studies have demonstrated that the
cell
KATP channel is an octameric 4:4 complex of two structurally distinct proteins: an inwardly rectifying K channel subunit, Kir6.2, and a sulfonylurea receptor, SUR1
(Inagaki et al., 1995
, 1997
; Sakura et al., 1995
; Clement
et al., 1997
; Shyng and Nichols, 1997
). Both subunits
are required for functional expression. Kir6.2 acts as an
ATP-sensitive pore, while SUR1 is a regulatory subunit
that endows Kir6.2 with sensitivity to sulfonylureas, K-channel openers, and the potentiatory effects of Mg
nucleotides such as MgADP (Nichols et al., 1996
; Gribble et al., 1997a
; Shyng et al., 1997
; Trapp et al., 1997
;
Tucker et al., 1997
). Two different sulfonylurea receptor genes have been cloned, SUR1 and SUR2, that encode proteins with different pharmacological sensitivity
and show different tissue expression (Chutkow et al.,
1996
; Inagaki et al., 1996
; Isomoto et al., 1996
). KATP
channels in pancreatic
cells comprise Kir6.2 and
SUR1 subunits, while those of cardiac muscle appear to
be composed of Kir6.2 and a splice variant of SUR2,
SUR2A (Aguilar-Bryan et al., 1998
).
Agents that interact with sulfhydryl (SH) moieties
produce an irreversible inhibition of both
cell and
cardiac muscle KATP channel activity when applied to
the intracellular membrane surface (Weik and Neumcke,
1989
; Lee et al., 1994
; Coetzee et al., 1995
). Inhibition
can be reversed, however, by subsequent application of
the reducing agent dithiothreitol (DTT).1 These results
argue that a sulfhydryl group, and thus a cysteine residue, is associated with the normal function of the KATP
channel. ATP appears to protect against the effects of
thiol reagents, for if ATP is applied before the sulfhydryl-modifying agent is added, and removed after it is
washed out, the irreversible inhibition of channel activity is substantially reduced (Weik and Neumcke, 1989
;
Lee et al., 1994
). A similar protective effect was observed with the sulfonylurea tolbutamide, which also
blocks the KATP channel (Lee et al., 1994
). A possible
explanation for the ability of both ATP and tolbutamide to protect against the effects of sulfhydryl agents
is that the critical cysteine is not accessible when the
channel is closed.
In the present study, we examined the interaction of the
membrane-impermeant thiol-reactive agent p-chloromercuriphenylsulphonate (pCMPS) with the KATP channel.
We used a truncated isoform of Kir6.2 (Kir6.2C26),
which expresses ATP-sensitive channel activity in the absence of a sulfonylurea receptor (Tucker et al., 1997
), to
demonstrate that the cysteine residue involved in channel inhibition by pCMPS lies on Kir6.2. Using site-
directed mutagenesis, we show that this residue is located at position 42, which lies within the intracellular
NH2 terminus of the protein. The ATP sensitivity of the
channel was unchanged by mutation of C42 to either
valine or alanine, suggesting that ATP does not interact
directly with this residue. It remains possible, however,
that C42 lies within the ATP-binding motif, although it
does not contribute to the affinity for ATP. Our results indicate that C42 is inaccessible to the intracellular solution,
and thereby protected against inhibition by pCMPS, when
the channel is closed by ATP. They also suggest that the
inhibitory effect of pCMPS is state dependent.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molecular Biology
Mouse Kir6.2 (Genbank D50581; Inagaki et al., 1995; Sakura et al.,
1995
) and rat SUR1 (Genbank L40624; kindly provided by Dr. G. Bell, University of Chicago, Chicago, IL; Aguilar-Bryan et al.,
1995
) were used in this study. A 26 amino acid COOH-terminal deletion of mouse Kir6.2 (Kir6.2
C26) was also used (Tucker et al.,
1997
). Site-directed mutagenesis of Kir6.2
C26 was carried out
by subcloning the appropriate fragments into the pALTER vector (Promega, Madison, WI). The mutations are indicated by the single amino acid letter code. Synthesis of capped mRNA was carried out using the mMessage mMachine large scale in vitro transcription kit (Ambion Inc., Austin, TX).
Electrophysiology
Oocyte collection.
Female Xenopus laevis were anaesthetized with
MS222 (2 g/liter in water). One ovary was removed via a mini
laparotomy, the incision was sutured, and the animal was allowed
to recover. Once the wound had completely healed, the second
ovary was removed in a similar operation and the animal was
then killed by decapitation while under anaesthesia. Immature
stage V-VI Xenopus oocytes were incubated for 60 min with 1.0 mg/ml collagenase (type V; Sigma Chemical Co., St. Louis, MO)
and manually defolliculated. In most experiments, oocytes were
injected with ~2 ng of mRNA encoding Kir6.2C26 (either
wild type or mutant). For coexpression experiments, ~0.04 ng of
full-length Kir6.2 (wild type or mutant) was coinjected with ~2 ng
of SUR1 (giving a 1:50 ratio). The final injection volume was ~50
nl/oocyte. Control oocytes were injected with water. Isolated oocytes were maintained in tissue culture and studied 1-4 d after injection (Gribble et al., 1997b
).
Data Analysis
The slope conductance was measured by fitting a straight line to
the current-voltage relation between 20 and
100 mV. The average of five consecutive ramps was calculated in each solution.
ATP dose-response relationships were measured by alternating the control solution with a test ATP concentration. The extent of inhibition by ATP was then expressed as a fraction of the mean of the value obtained in the control solution before and after ATP application. Dose-response curves were fitted 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). The time course of the block by pCMPS was fit with a monoexponential function to obtain the time constant of block.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effects of pCMPS on the KATP Wild-Type Channel
We first investigated whether the effect of sulfhydryl-modifying reagents on the cloned cell KATP channel
(Kir6.2/SUR1) was the same as that reported for native
KATP channels (Weik and Neumcke, 1989
; Lee et al.,
1994
; Coetzee et al., 1995
). Fig.1 A shows that the sulfhydryl reagent pCMPS (50 µM) produced a complete block of Kir6.2/SUR1 currents when applied to the intracellular surface of an inside-out patch. Inhibition
was not reversible on removal of pCMPS, but application of the reducing agent DTT partially restored the
current. In seven patches, 5 mM DTT restored the current amplitude to 31 ± 4% of its control value. This
supports the idea that the inhibitory effect of pCMPS is
mediated by sulfhydryl modification of the channel.
The fact that the current amplitude did not recover to
its original value may be due to an incomplete action of
DTT, which acts only slowly at physiological pH. Alternatively, it may reflect the presence of rundown, a time-dependent decline in channel activity that is observed
for both native and cloned KATP channels in excised
patches.
ATP has been shown to protect against the effects of
thiol reagents on native KATP channels (Weik and
Neumcke, 1989; Lee et al., 1994
). This was also the case
for Kir6.2/SUR1 currents (Fig. 1 B). Provided that the
patch was exposed to pCMPS in the continuous presence of ATP, the sulfhydryl reagent did not induce an
irreversible block and the currents recovered on removal of ATP (to 40 ± 13% of the control value, n = 5). This suggests that the cysteine residue with which
the thiol reagent interacts is not accessible from the intracellular solution when the channel is inhibited by
ATP, either because it is not exposed when the channel
is closed by ATP, or because access to the cysteine residue is occluded by the presence of ATP in its binding
site.
|
Effects of pCMPS on Kir6.2C26 Currents
We next explored whether the site at which thiol reagents interact with the KATP channel lies on the pore-forming Kir6.2 subunit, or on SUR1, by examining the
effect of pCMPS on a truncated isoform of Kir6.2
(Kir6.2C26) that is able to form functional channels independently of SUR1. Fig. 2 shows that 50 µM pCMPS
completely inhibited Kir6.2
C26 currents and that
DTT was able to partially reverse this inhibition. This
result indicates that pCMPS block is not mediated by
SUR1, and further suggests that the thiol reagent interacts either directly with Kir6.2
C26 or (possibly) with a third protein, endogenously expressed in Xenopus
oocytes, that modulates the activity of Kir6.2
C26.
|
Cysteine Mutations Identify the Residue Involved in pCMPS Block
To determine whether pCMPS interacts directly with
Kir6.2, and to identify the cysteine residue involved, we
carried out site-directed mutagenesis of Kir6.2C26.
The critical cysteine is accessible to the intracellular solution and must therefore lie either on the NH2 or
COOH terminus of Kir6.2
C26, or within the inner
mouth of the channel pore itself. There are five cysteines within these regions of Kir6.2
C26, at positions
42, 81, 166, 197, and 344 (Fig. 3 A). We therefore
tested the effects of mutating each of these, in turn, to
serine.
|
Currents expressed by Kir6.2C26 containing either
the C81S or C344S mutations were of similar amplitude
to wtKir6.2
C26, while those containing the C166S mutation were approximately sevenfold larger (see accompanying paper, Trapp et al., 1998
). For all three mutant
channels, 50 µM pCMPS caused a complete block of
the current. Functional expression of Kir6.2
C26-C42S
and Kir6.2
C26-C197S channels was too low to be able
to study: only rare single-channel openings were observed in excised patches containing Kir6.2
C26-C42S, and none were detected in patches excised from oocytes injected with Kir6.2
C26-C197S mRNA. We therefore tested the effect of both alanine and valine substitutions at positions 42 and 197. When expressed alone,
these mutants only produced very small currents but,
when coexpressed with SUR1, significant functional expression was observed for Kir6.2
C26 containing the
C197A, C197V, C42A, or C42V mutations. This suggests
that C42 and C197 may be needed for correct targeting
of the truncated Kir6.2 isoform to the surface membrane, or for its functional activity.
Mutation of C197 did not prevent inhibition by
pCMPS. By contrast, pCMPS was without effect on
Kir6.2C26-C42V/SUR1 currents (Fig. 3 B), nor did it affect the KATP currents observed when the full length form
of Kir6.2 containing the C42A mutation was coexpressed
with SUR1 (Fig. 3 C). These results confirm that pCMPS inhibits the KATP channel by interaction with Kir6.2,
rather than with an endogenous oocyte protein. A single
cysteine residue in Kir6.2, at position 42, which lies in the
cytosolic NH2-terminal part of the protein, appears to be
the target for the inhibitory action of pCMPS. Since
SUR1 was present in these experiments, they also indicate that pCMPS does not interact with additional sites
on SUR1 to inhibit channel activity: in wild-type channels, the presence of such sites would be masked by the
inhibitory effect of the thiol agent on Kir6.2.
We next examined the rate of pCMPS block of the
mutant channels (with the exception of C42A and
C42V, which are not blocked). The rate of block at
100 mV was quantified by fitting a single exponential
to the decay of the current after pCMPS application, and the mean time constants for the different cysteine
mutations are plotted in Fig. 4 C. The mutant channels
were fully blocked by pCMPS and, with two exceptions,
the kinetics of the block were similar to those observed
for both Kir6.2
C26 and Kir6.2/SUR1 currents. The
rate of block of Kir6.2
C26-C197V currents was slightly increased, while that of Kir6.2
C26-C166S was markedly slowed. However, mutation of C166 to valine did
not alter the kinetics of inhibition by pCMPS: this may
reflect the fact that the C166V mutation, unlike C166S,
did not alter the single-channel kinetics (see Trapp et
al., 1998
). The rate of block of wild-type Kir6.2/SUR1 channels by pCMPS (100 µM) was unaffected by membrane potential, being 1.36 ± 0.32 s (n = 7) at
100
mV and 1.76 ± 0.20 s (n = 7) at +100 mV.
|
One possible explanation for the ability of ATP to protect against the effects of pCMPS is that the critical cysteine lies within, or close to, the ATP-binding site and is
thus inaccessible when ATP is bound. We therefore compared the ATP sensitivity of Kir6.2C26-C42V/SUR1
currents with that of Kir6.2
C26/SUR1 currents (Fig. 5,
A and B). Application of 1 mM ATP to the inner membrane surface markedly inhibited both currents. There
was no significant difference in the ATP sensitivity of the
channels, half-maximal inhibition being produced by
13 ± 1 µM ATP (n = 4) for Kir6.2
C26-C42V/SUR1 and by 18 ± 3 µM ATP (n = 5) for Kir6.2
C26/SUR1
currents. In both cases, the Hill coefficient was close to
unity: 0.91 ± 0.07 for Kir6.2
C26-C42V/SUR1 and
0.92 ± 0.14 for Kir6.2
C26/SUR1 currents. Thus, the
C42V mutation does not influence the block by ATP.
|
No change in ATP sensitivity was observed for the
Kir6.2C26 isoform containing either the C81S or
C344S mutation, nor for Kir6.2
C26-C197V/SUR1 currents or Kir6.2-C42A/SUR1 currents (Fig. 5). When
C166 was replaced by serine, however, the ATP sensitivity of the channel was markedly reduced (Fig. 5; see
Trapp et al., 1998
). It is worth noting that in addition
to removing the pCMPS sensitivity, the C42V mutation
also induces an increased rate of rundown of the current, and an accompanying increase in the refreshment
of the current after MgATP application (Fig. 5 A).
Coupling to SUR1
We next examined the effect of mutating C42 on the
ability of SUR1 to couple to Kir6.2, by coexpressing the
full length version of Kir6.2 containing the C42A mutation (Kir6.2-C42A) with SUR1. Since Kir6.2 does not express functional channels independently of SUR, this
procedure ensures that the KATP current we record only
reflects current flow through octameric channels comprising both Kir6.2-C42A and SUR1 subunits. We compared the ability of MgADP, diazoxide, and tolbutamide,
all of which interact with SUR1, to influence the activity
of wild-type and mutant KATP channels. The stimulatory
effect of diazoxide requires the presence of hydrolyzable nucleotides at the inner face of the membrane (Dunne,
1989; Kozlowski et al., 1989
) and we therefore tested the
ability of this drug to activate channels partially blocked
by 100 µM ATP. Fig. 6 shows that the stimulatory effect
of diazoxide was not significantly different for Kir6.2-C42A/SUR1 and wild-type KATP currents (unpaired t
test). By contrast, both MgADP and tolbutamide were
less effective when C42 was mutated. Thus, 100 µM
MgADP was without significant effect on Kir6.2-C42A/
SUR1 currents, in contrast to the potentiatory effect of
the nucleotide on Kir6.2/SUR1 currents (P = 0.007). In
addition to the potentiatory effect of MgADP mediated
by the nucleotide-binding domains of SUR1, ADP inhibits the KATP channel by interaction with Kir6.2 (Gribble
et al., 1997a
). Complete abolition of the stimulatory
effects of MgADP unmasks the inhibitory effect of ADP,
reducing the current to ~50% of its control value
(Gribble et al., 1997a
). Since inhibition of Kir6.2-C42A/
SUR1 currents by MgADP was not observed, the stimulatory effect of MgADP must be impaired but not abolished by the mutation. Tolbutamide was also less effective when C42 was mutated (P = 0.017; Fig. 6). Thus,
mutation of C42 to alanine reduces, but does not prevent, the ability of SUR1 to transduce binding of MgADP
and tolbutamide into changes in Kir6.2 gating.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Both native KATP channels and the cloned wild-type cell KATP channel (Kir6.2/SUR1) are blocked by the
sulfhydryl reagent pCMPS with a time constant of ~3 s.
Our results argue that this effect involves interaction of
the sulfhydryl reagent with the cysteine residue at position 42 of Kir6.2. Mutation of C42 to either alanine or
valine abolished the inhibitory effect of pCMPS, while
mutation of the cysteine residues at positions 81, 166, 197, and 344 had no effect on the magnitude of pCMPS
block. The ability of these mutant channels to express
functional currents suggests that none of the mutated
cysteines participate in disulfide bonds that are critical
for channel formation. Although the kinetics of pCMPS
block were altered when C166 was mutated to serine,
this was not observed for the C166V mutation, indicating that this cysteine is also not involved in inhibition
by thiol reagents. The ability of intracellular pCMPS to
interact with C42 also implies that the NH2 terminus of
Kir6.2 is intracellular, as suggested by hydropathy analysis and by analogy with other Kir channels. The block
by pCMPS is not obviously voltage dependent, which
implies that C42 lies outside the membrane voltage
field and is again consistent with an intracellular location for the NH2 terminus. It is noteworthy that one or
more cysteine residues in the NH2 terminus of the voltage-gated K+ channel Kv2.1 must also be targeted by
sulfhydryl reagents, since deletion of the NH2 terminus
(which contains six cysteines) largely abolishes the effect of these reagents (Pascaul et al., 1997
).
Effects of C166 Mutation
Mutation of C166S markedly enhanced the level of current expression. This effect can be entirely explained
by the fact that the mutation alters the single-channel
kinetics and enhances the channel open probability,
rather than affecting the single-channel conductance
or channel density (Trapp et al., 1998).
The simplest kinetic model of the KATP channel assumes the presence of a single open state and two
closed states, one short and one long (Trapp et al.,
1998). The slower time course of pCMPS block of
Kir6.2
C26-166S currents argues that the thiol reagent may not block the open state of the channel, which is
markedly increased in frequency by the C166S mutation (Trapp et al., 1998
). Consistent with this idea, the
rate of pCMPS block was unaffected in Kir6.2
C26-C166V channels, which had single-channel kinetics similar to those of wild-type channels. One possibility,
therefore, is that pCMPS reduces the probability of
channel reopening from the long closed state. Alternatively, the C166S mutation may independently affect
channel gating and slow access to the pCMPS binding
site.
Effects of C42 Mutation
Mutation of C42 to either valine or alanine did not alter the ATP sensitivity of Kir6.2C26, indicating that a
cysteine at this site is not essential for ATP inhibition.
We cannot exclude the possibility, however, that C42 is
located close to the ATP-binding site. If access of pCMPS
to this residue in wild-type channels is physically prevented by the presence of ATP in its binding site, this
would account for the ability of ATP to protect against irreversible pCMPS inhibition. The fact that tolbutamide, which primarily interacts with the SUR rather
than the Kir6.2 subunit, also protects against thiol reagent block of native KATP channels (Lee et al., 1994
) is
less easily explained by this hypothesis. However, we
were unable to demonstrate protection against pCMPS block of Kir6.2/SUR1 currents by tolbutamide under
our experimental conditions (not shown). Although
previous studies have implicated residues in the COOH
terminus in ATP inhibition of Kir6.2
C26 (Tucker et
al., 1997
), there is also evidence that residues in the
NH2 terminus may be involved: mutation of R50, for
example, may also markedly alter the ATP sensitivity
(Tucker et al., 1998
). It remains unclear, however,
whether the ATP-binding site involves residues in both
the NH2 and COOH terminus, whether mutations in
the NH2 terminus allosterically influence an ATP-binding site in the COOH-terminal part of the molecule or if
the NH2 and COOH termini cooperate in channel closure by ATP. A precedent for the latter possibility is
provided by the cyclic nucleotide-activated channels,
where the cyclic nucleotide binding site is located in
the COOH terminus, but channel activation is modified by physical interaction between the NH2 and
COOH termini (Gordon and Zagotta, 1995
; Varnum
and Zagotta, 1997
).
The mechanism by which SUR1 enables functional expression of full-length Kir6.2 was unaffected by the C42A mutation. Likewise, the ability of SUR1 to confer diazoxide sensitivity on Kir6.2 was unchanged. By contrast, both MgADP and tolbutamide were significantly less effective than this mutant. This suggests that C42 may be required for full functional coupling of SUR1 to Kir6.2: our data, however, do not allow us to say whether the cysteine residue physically interacts with SUR1, or whether the effect of its mutation on Kir6.2-SUR1 coupling is mediated indirectly.
Conclusion
Cysteine scanning mutagenesis is a well established
method for identifying key residues involved in forming the channel pore, in which individual residues are
mutated to cysteine and the ability of thiol reagents to
block the permeation pathway are investigated. Such
studies can only be carried out on a channel that has no intrinsic sensitivity to thiol reagents. This is not the case for either the wild-type KATP channel or the Kir6.2C26
channel. Our results demonstrate that the inhibitory effect of sulfhydryl reagents is mediated by interaction
with C42 of Kir6.2, and thus that cysteine scanning mutagenesis could be performed using a Kir6.2 subunit in
which C42 has been changed.
![]() |
FOOTNOTES |
---|
Address correspondence to Frances M. Ashcroft, University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK. Fax: 44-1865-272469; E-mail: frances.ashcroft{at}physiol.ox.ac.uk
Original version received 18 March 1998 and accepted version received 22 May 1998.
We thank Dr. G. Yellen for constructive advice, and Drs. F. Gribble and J. Röper for useful discussion.
This study was supported by the Medical Research Council, the Wellcome Trust, and the British Diabetic Association. S. Trapp was supported by a fellowship from the Deutsche Forschungsgemeinschaft. S.J. Tucker is a Wellcome Trust Research Fellow.
![]() |
Abbreviations used in this paper |
---|
DTT, dithiothreitol; pCMPS, p-chloromercuriphenylsulphonate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Aguilar-Bryan, L.,
C.G. Nichols,
S.W. Wechsler,
J.P. Clement IV,
A.E. Boyd,
G. González,
H. Herrera-Sosa,
K. Nguy,
J. Bryan, and
D.A. Nelson.
1995.
Cloning of the ![]() |
2. |
Aguilar-Bryan, L.,
J.P. Clement IV,
G. González,
K. Kunjilwar,
A. Babenko, and
J. Bryan.
1998.
Towards understanding the assembly
and structure of KATP channel.
Physiol. Rev.
78:
227-245
|
3. | Ashcroft, F.M., and S.J.H. Ashcroft. 1990. Properties and functions of ATP-sensitive K-channels. Cell. Signalling 2: 197-214 [Medline]. |
4. | Ashcroft, F.M., and S.J.H. Ashcroft. 1992. The sulphonylurea receptor. Biochim. Biophys. Acta. 1175: 45-59 [Medline]. |
5. | Chutkow, W.A., M.C. Simon, M.M. Le Beau, and C.F. Burant. 1996. Cloning, tissue expression, and chromosomal localization of SUR2, the putative drug-binding subunit of cardiac, skeletal muscle, and vascular KATP channels. Diabetes. 45: 1439-1445 [Abstract]. |
6. | Clement, J.P. IV, K. Kunjilwar, G. González, M. Schwanstecher, U. Panten, L. Aguilar-Bryan, and J. Bryan. 1997. Association and stoichiometry of KATP channel subunits. Neuron 18: 827-838 [Medline]. |
7. |
Coetzee, W.A.,
T.Y. Nakamura, and
J.-F. Faivre.
1995.
Effects of
thiol reagents on KATP channels in guinea-pig ventricular cells.
Am. J. Physiol.
269:
H1625-H1633
|
8. | Dunne, M.J.. 1989. Phosphorylation is required for diazoxide to open ATP-sensitive potassium channels in insulin-secreting cells. FEBS Lett 250: 262-266 [Medline]. |
9. | Edwards, G., and A.H. Weston. 1993. The pharmacology of ATP-sensitive potassium channels. Annu. Rev. Pharmacol. Toxicol. 33: 597-637 [Medline]. |
10. | Gordon, S.E., and W.N. Zagotta. 1995. Localization of regions affecting an allosteric transition in cyclic nucleotide-gated channels. Neuron. 14: 857-864 [Medline]. |
11. |
Gribble, F.M.,
S.J. Tucker, and
F.M. Ashcroft.
1997a.
The essential
role of the Walker A motifs of SUR1 in K-ATP channel activation
by MgADP and diazoxide.
EMBO (Eur. Mol. Biol. Organ.) J.
16:
1145-1152
|
12. | Gribble, F.M., R. Ashfield, C. Ämmälä, and F.M. Ashcroft. 1997b. Properties of cloned ATP-sensitive K-currents expressed in Xenopus oocytes. J. Physiol. (Camb.). 498: 87-98 [Abstract]. |
13. | Inagaki, N., T. Gonoi, J.P. Clement IV, N. Namba, J. Inazawa, G. Gonzalez, L. Aguilar-Bryan, S. Seino, and J. Bryan. 1995. Reconstitution of IKATP: an inward rectifier subunit plus the sulphonylurea receptor. Science 270: 1166-1169 [Abstract]. |
14. | Inagaki, N., T. Gonoi, J.P. Clement IV, C.Z. Wang, L. Aguilar-Bryan, J. Bryan, and S. Seino. 1996. A family of sulfonylurea receptors determines the properties of ATP-sensitive K+ channels. Neuron 16: 1011-1017 [Medline]. |
15. |
Inagaki, N.,
T. Gonoi, and
S. Seino.
1997.
Subunit stoichiometry of
the pancreatic ![]() |
16. |
Isomoto, S.,
C. Kondo,
M. Yamada,
S. Matsumoto,
O. Higashiguchi,
Y. Horio,
Y. Matsuzawa, and
Y. Kurachi.
1996.
A novel sulphonylurea receptor forms with BIR (Kir6.2) a smooth muscle type of
ATP-sensitive K+ channel.
J. Biol. Chem.
271:
24321-24325
|
17. | Kozlowski, R.J., C.N. Hales, and M.L.J. Ashford. 1989. Dual effects of diazoxide on ATP-K+ currents recorded from an insulin- secreting cell line. Br. J. Pharmacol. 97: 1039-1050 [Abstract]. |
18. | Lee, K., S.E. Ozanne, C.N. Hales, and M.L.J. Ashford. 1994. Effects of chemical modification of amino and sulfhydryl groups on KATP channel function and sulphonylurea binding in CRIG1 insulin secreting cells. J. Membr. Biol 139: 167-181 [Medline]. |
19. |
Nichols, C.G., and
W.J. Lederer.
1991.
Adenosine triphosphate-sensitive potassium channels in the cardiovascular system.
Am. J. Physiol.
261:
H1675-H1686
|
20. | Nichols, C.G., S.-L. Shyng, A. Nestorowicz, B. Glaser, J.P. Clement IV, G. Gonzalez, L. Aguilar-Bryan, M.A. Permutt, and J. Bryan. 1996. Adenosine diphosphate as an intracellular regulator of insulin secretion. Science 272: 1785-1787 [Abstract]. |
21. |
Pascaul, J.M.,
C.C. Shieh,
G.E. Kirsch, and
A.M. Brown.
1997.
Contribution of the NH2 terminus of Kv2.1 to channel activation.
Am.
J. Physiol.
273:
C1849-C1858
|
22. |
Quayle, J.M.,
M.T. Nelson, and
N.B. Standen.
1997.
ATP-sensitive
and inwardly-rectifying potassium channels in smooth muscle.
Physiol. Rev.
77:
1165-1232
|
23. |
Sakura, H.,
C. Ämmälä,
P.A. Smith,
F.M. Gribble, and
F.M. Ashcroft.
1995.
Cloning and functional expression of the cDNA encoding a novel ATP-sensitive potassium channel expressed in
pancreatic ![]() |
24. |
Shyng, S.L.,
T. Ferrigni, and
C.G. Nichols.
1997.
Regulation of KATP
channel activity by diazoxide and MgADP: distinct functions of
the two nucleotide binding folds of the sulphonylurea receptor.
J. Gen. Physiol
110:
643-654
|
25. |
Shyng, S.L., and
C.G. Nichols.
1997.
Octameric stoichiometry of
the KATP channel complex.
J. Gen. Physiol.
110:
655-664
|
26. |
Trapp, S., and
F.M. Ashcroft.
1997.
A metabolic sensor in action: news
from the ATP sensitive K+ channel.
News Physiol. Sci.
12:
255-263
.
|
27. |
Trapp, S.,
S.J. Tucker, and
F.M. Ashcroft.
1997.
Activation and inhibition of KATP currents by guanine nucleotides is mediated by different channel subunits.
Proc. Natl. Acad. Sci. USA
94:
8872-8877
|
28. |
Trapp, S.,
P. Proks,
S.J. Tucker, and
F.M. Ashcroft.
1998.
Molecular
analysis of KATP channel gating and implications for channel inhibition by ATP.
J. Gen. Physiol.
112:
333-349
|
29. | Tucker, S.J., F.M. Gribble, C. Zhao, S. Trapp, and F.M. Ashcroft. 1997. Truncation of Kir6.2 produces ATP-sensitive K-channels in the absence of the sulphonylurea receptor. Nature 387: 179-183 [Medline]. |
30. |
Tucker, S.J.,
F.M. Gribble,
P. Proks,
S. Trapp,
T.J. Ryder,
T. Haug,
F. Reimann, and
F.M. Ashcroft.
1998.
Molecular determinants of
KATP channel inhibition by ATP.
EMBO (Eur. Mol. Biol. Organ.) J.
17:
3290-3296
|
31. |
Varnum, M.D., and
W.N. Zagotta.
1997.
Interdomain interactions
underlying activation of cyclic nucleotide-gated channels.
Science.
278:
110-113
|
32. | Weik, R., and B. Neumcke. 1989. ATP-sensitive potassium channels in adult skeletal muscle: characterization of the ATP-binding site. J. Membr. Biol. 110: 217-226 [Medline]. |