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Vol. 57, Issue 6, 1256-1261, June 2000
University Laboratory of Physiology, Oxford, United Kingdom
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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ATP-sensitive K+ channels are the target for K+
channel openers such as pinacidil. These channels are formed from
pore-forming Kir6.2 and regulatory sulfonylurea receptor (SUR)
subunits. Pinacidil activates channels containing SUR2A (heart,
skeletal muscle), but not those containing SUR1 (
cells).
Surprisingly, binding of the pinacidil analog [3H]P1075
is dependent on added nucleotides, yet in electrophysiological studies,
pinacidil is effective in the absence of intracellular nucleotides. To
determine the reason for this anomaly, we examined the functional
interactions between pinacidil (or P1075) and nucleotides by expressing
cloned Kir6.2/SUR2A channels in Xenopus laevis oocytes. Both pinacidil and P1075 activated macroscopic Kir6.2/SUR2A currents in
the absence of added nucleotide, but the presence of intracellular ATP
or ADP slowed the off-rate of the response. Mutation of the Walker A
lysine in a single nucleotide binding domain (NBD) of SUR2A (K707A in
NBD1, K1348A in NBD2), abolished this action of nucleotide. The K1348A
mutation prevented stimulation by MgADP but had little effect on the
amplitude of the pinacidil response. In contrast, Kir6.2/SUR2A-K707A
currents were activated by MgADP, but only responded to pinacidil in
the presence of Mg-nucleotide. Off-rates in the absence (or presence)
of nucleotide were slower for the pinacidil analog P1075 than for
pinacidil, consistent with the higher affinity of P1075. We suggest
that slowing of P1075 dissociation by nucleotide enables binding to be detected.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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ATP-sensitive
K+ channels (KATP channels)
are found in a wide variety of tissues, including pancreatic cells,
cardiac, skeletal, and smooth muscles, and some neurons, where they
couple the metabolic state of a cell to its electrical activity
(Ashcroft and Ashcroft, 1990
; Aguilar-Bryan and Bryan, 1999). This is
considered to be mediated by changes in adenine nucleotide levels,
particularly ATP (which blocks the channel) and MgADP (which activates
the channel). KATP channels are also activated by
KATP-channel openers, a structurally diverse
group of drugs with a wide range of potential therapeutic applications,
and they are inhibited by sulfonylurea drugs that are used to treat
type 2 diabetes.
The KATP channel is a heteromeric complex of four
Kir6.x and four sulfonylurea receptor (SUR) subunits: Kir6.2 subunits
form the ATP-sensitive channel pore, and SUR acts as a regulatory
subunit, endowing the KATP channel with
sensitivity to the stimulatory effects of MgADP and
KATP-channel openers and to the inhibitory actions of sulfonylureas (Aguilar-Bryan et al., 1995; Inagaki et al.,
1995, 1996; Sakura et al., 1995; Isomoto et al., 1996; Clement et al.,
1997; Tucker et al., 1997). Like other members of the ATP-binding
cassette transporter family, SUR has two nucleotide-binding domains
(NBDs). Mutagenesis studies have revealed that Mg-nucleotides promote
channel activity by interacting with these NBDs (Nichols et al., 1996;
Gribble et al., 1997b, 1998; Shyng et al., 1997).
Two SUR genes have been identified; one encodes the cell isoform
(SUR1), and the other encodes the cardiac/skeletal muscle (SUR2A) and
smooth muscle (SUR2B) isoforms (Aguilar-Bryan et al., 1995, Inagaki et
al., 1996; Isomoto et al., 1996). KATP channels containing different SURs show distinct sensitivities to
KATP-channel openers. Thus, Kir6.2/SUR1 currents
are activated by diazoxide but not by pinacidil (Gribble et al.,
1997a), Kir6.2/SUR2A currents are strongly activated by pinacidil but
only weakly responsive to diazoxide (Inagaki et al., 1996), and
Kir6.2/SUR2B currents are activated by both drugs (Schwanstecher et
al., 1998).
There is currently a discrepancy between the effects of pinacidil in
electrophysiological experiments and that observed for the pinacidil
analog P1075 in binding studies. Provided that the drug is tested soon
after patch excision, pinacidil enhances cardiac KATP currents in the absence of added nucleotides
(Fan et al., 1990; Terzic et al., 1995), although it is ineffective
once channel activity has completely run down (Shen et al., 1991; Tung
and Kurachi, 1991; Allard and Lazdunski, 1992; Terzic et al., 1995). In
contrast, both nucleotide and Mg2+ ions are
required for detection of [3H]P1075 binding,
both to native skeletal and cardiac muscle KATP channels (Dickinson et al., 1997, Löffler-Walz and Quast, 1998), and to cloned SUR2A and SUR2B subunits (Schwanstecher et al., 1998;
Hambrock et al., 1998, 1999). It has not yet been determined whether
this difference in nucleotide dependence is caused by differences in
the mechanism of action of P1075 and pinacidil or has another explanation.
In this article, we explore the mechanism underlying the nucleotide modulation of pinacidil activity on Kir6.2/SUR2A currents and show that MgATP (and MgADP) can slow the off-rate of pinacidil through an interaction with the NBDs of SUR2. This may underlie the requirement for Mg-nucleotide in binding studies.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Molecular Biology.
Mouse Kir6.2 (GenBank accession no.
D50581; Sakura et al., 1995) and rat SUR2A (GenBank accession no.
D83598; Inagaki et al., 1996) cDNAs were subcloned into the pBF vector.
Mutagenesis of individual amino acids was performed using the altered
sites II System (Promega, Madison, WI). Synthesis of mRNA for oocyte expression was carried out using the mMessage mMachine large-scale in
vitro transcription kit (Ambion, Austin, TX).
Electrophysiology.
Oocytes were prepared from female
Xenopus laevis and coinjected with ~0.1 ng Kir6.2 mRNA and
~2 ng of mRNA encoding wild-type or mutated SUR2A (Gribble et al.,
1997a). The final injection volume was 50 nl/oocyte. Isolated oocytes
were maintained in Barth's solution and studied 1 to 4 days after
injection. Macroscopic currents were recorded from giant, excised,
inside-out patches at a holding potential of 0 mV and at 20 to 24°C
(Gribble et al., 1997a). 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 110 mM KCl, 1.4 mM
MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.2 with KOH;
final [K+] ~140 mM). The Mg-free solution
contained 107 mM KCl, 2.6 mM CaCl2, 10 mM EDTA,
10 mM HEPES, pH 7.2 with KOH; final [K+] ~140
mM). Stock solutions of pinacidil (Sigma, Poole, UK) and P1075 (Leo
Pharmaceuticals) were prepared in ethanol and dimethyl sulfoxide, respectively. Nucleotides were dissolved directly in the
bath solution and the pH was then readjusted as necessary. 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.
Data Analysis.
In some experiments, currents were recorded
in response to repetitive 3-sec voltage ramps from 
110 mV to +100 mV.
They were filtered at 0.5 kHz, digitised at 1 kHz using a Digidata 1200 Interface, and analyzed using pClamp software (Axon Instruments, Foster
City, CA). The slope conductance was measured by fitting a straight
line to the current-voltage relation between 20 mV and 100 mV: the
average response to five consecutive ramps was calculated in each solution.
50 mV, and macroscopic currents were recorded in
response to repeated drug applications. Currents were sampled at 20 Hz
and analyzed using Microcal Origin (Microcal Software, Northampton,
MA). The decay in current after drug removal was best fit with a sum of
three exponentials, one of which (
r) is
attributable to channel rundown. This was subtracted in further analysis. The remaining current, representing the drug-activated component, was fit by a sum of two exponentials, with fast
(f) and slow (s) time
constants. The relative contribution of the slow component was
calculated from [As/(As + Af)] × 100%, where As is
the amplitude of the slow component and Af that
of the fast component. The results obtained from repeated drug
applications on a single patch were averaged for each test condition.
Exponentials faster than ~1 s could not be resolved because of the
time taken to change solutions, and exponentials slower than 300 s
could not be resolved accurately because other processes, such as
rundown, start to influence the time course of the current. Statistical significance was tested using Student's t test. Data are
presented as mean ± 1 S.E.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Large currents were recorded immediately after excision of
inside-out patches from oocytes coinjected with mRNAs encoding Kir6.2
and SUR2A. These subsequently declined with time (rundown) at a
variable rate (Fig. 1A). Addition of 100 µM pinacidil in the absence of nucleotides to the intracellular
(bath) solution increased the Kir6.2/SUR2A current (Fig. 1A). The
extent of activation ranged from 2- to 9-fold, even within a single
patch (mean 4.7 ± 0.4-fold; n = 7 patches).
Responses were dependent on the time of drug application: they were
small immediately after patch excision, increased after partial channel
rundown, and then declined slowly over 10 to 15 min (Fig. 1A). The
maximal extent of activation never exceeded the current amplitude
measured immediately after patch excision. Channel activation did not
require intracellular Mg2+ ions, because
pinacidil enhanced Kir6.2/SUR2A currents even after ~10 min in
Mg2+-free solution (mean
activation = 4.1 ± 0.6-fold; n = 4; Fig. 1B). These results contrast with the requirement for both ATP and Mg2+ for [3H]P1075
binding to SUR2A (Schwanstecher et al., 1998; Hambrock et al., 1999).
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We next examined the effect of pinacidil on Kir6.2/SUR2A currents in
the presence of intracellular nucleotides. Figure 1B shows that
application of 100 µM MgATP inhibited channel activity, and that
subsequent addition of pinacidil (in the presence of ATP) restored the
current to the control level. In the presence of MgATP, the mean
activation by pinacidil was 8.1 ± 2.9-fold (n = 7; Fig. 2). Repeated drug applications
produced stable responses (Fig. 3),
consistent with the ability of MgATP to prevent channel rundown
(Ohno-Shosaku et al., 1987). In contrast to ATP, 100 µM MgADP
enhanced Kir6.2/SUR2A currents. The magnitude of this activation, like
that produced by pinacidil, varied with the extent of channel rundown,
and it was smallest immediately after patch excision. The subsequent
addition of pinacidil to the MgADP-containing solution produced a
further 1.5 ± 0.1-fold (n = 5) increase in
channel activity (Figs. 1B and 2).
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To investigate the role of the NBDs of SUR2A in Kir6.2/SUR2A channel
activation by nucleotides and pinacidil, we introduced mutations into
the Walker A (WA) motifs of the NBDs. In other ATP-binding cassette transporters, a lysine residue in the
WA motif forms part of the nucleotide-binding
site and is essential for ATP hydrolysis (Azzaria et al., 1989; Carson
et al., 1995; Ko and Pedersen, 1995). Furthermore, mutation of a single
WA lysine in either NBD of SUR1 abolishes channel
activation by Mg-nucleotides (Gribble et al., 1997b, 1998; Shyng et
al., 1997). We therefore mutated each of the WA
lysines in SUR2A individually to alanine (K707A in NBD1 and K1348A in NBD2).
Fig. 2 shows that mutation K707A in NBD1 of SUR2A to alanine had no
effect on channel activation by MgADP, in contrast to mutation of the
equivalent residue in SUR1 (Gribble et al., 1997b; Shyng et al., 1997).
Mutation of K1348A in NBD2 to alanine, however, abolished stimulation
by MgADP and unmasked an inhibitory effect of the nucleotide (mediated
by Kir6.2; Tucker et al., 1997). Figure 2 also shows the effects of the
WA mutations on the amplitude of the pinacidil
response. Only the K707A mutation (in NBD1) prevented the stimulatory
effect of pinacidil in the absence of Mg-nucleotide. Pinacidil was able
to activate all mutant channels in the presence of MgATP, although the
amount of activation of Kir6.2/SUR2A-K707A currents was very small
(Fig. 2). In the presence of MgADP, pinacidil caused clear activation
of wild-type, Kir6.2/SUR2A-K1348A and Kir6.2/SUR2A-K707A currents.
As is the case for pinacidil, P1075 stimulated Kir6.2/SUR2A currents in
both the absence and presence of nucleotides (Fig. 3). The mean
activation at 50 mV was 2.2 ± 0.3-fold (n = 6)
in the absence of nucleotide, when expressed as a fraction of the mean
current before and after drug addition (compared with 4.7-fold for
pinacidil). In the presence of 100 µM MgATP, the off-rate of P1075
was very slow and we have expressed the extent of drug activation
relative to the current recorded in MgATP solution before drug
addition. This was 13 ± 4-fold (n = 6) (for
comparison, pinacidil produced a 16 ± 4-fold increase in current
when calculated in this way). Thus, despite the fact that binding has
not been observed, P1075 is able to stimulate
KATP currents in the absence of nucleotide.
Rate of Reversal of Pinacidil Activation.
The marked increase
in KATP current produced by pinacidil and P1075
reversed rapidly on removal of the drug. The presence of Mg-nucleotide
seemed to slow the rate of reversal (off-rate; Figs. 1B and 3),
suggesting that nucleotides might either slow the dissociation of the
drug from its binding site or the rate at which activated channels
relax back to the resting state. To explore this phenomenon more fully,
we analyzed the off-rate of each drug by recording macroscopic currents
at a fixed membrane potential of 50 mV (Fig. 3).
r, 51 ± 8 s;
n = 5) was caused by the rundown of channel activity,
as it was observed even in the absence of drug. Its contribution
declined with time, in parallel with the rundown of the background
current, and it was subtracted in subsequent analysis. The decay of the
remaining current, which represents the pinacidil-activated component,
was best fit by the sum of a fast and a slow exponential, with mean
time constants of 1.3 ± 0.2 s (f,
n = 4) and 8.3 ± 1.4 s
(s, n = 4). The relative contributions of these two exponentials altered with time, although their time constants did not vary. Soon after patch excision (at ~75
s), the slow exponential comprised 54 ± 11% (n = 5) of the pinacidil-activated current, but after 3 min, its
contribution had fallen to 26 ± 8% (n = 5). When
KATP currents were refreshed by the application
and subsequent removal of MgATP (0.1 to 1 mM), the amplitude of the
slow component increased again, comprising 63 ± 7%
(n = 4) of the pinacidil response ~80 s after MgATP removal.
When pinacidil was tested in the presence of MgATP or MgADP (100 µM),
the off-rate of the drug followed a single exponential with a time
constant of 12 ± 2 s (n = 11) in MgATP and
9.7 ± 0.6 s in MgADP (n = 5) (Fig.
4), rates which resemble the slow time constant (s) observed in nucleotide-free
solution. The presence of nucleotide thus seemed to enhance the
proportion of the pinacidil-activated current that decayed slowly. The
effect of MgATP required Mg2+ ions, because in
Mg-free solution containing 100 µM ATP, the pinacidil response
decayed rapidly, with a time constant of 1.3 ± 4 s
(n = 5). In contrast to ATP and ADP, 200 µM AMP-PCP did not slow the off-rate of pinacidil in the presence of
Mg2+ (f = 1.6 ± 0.2 s, s = 9.9 ± 0.7 s,
amplitude s = 10 ± 6%, n = 7; Fig. 4).
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= 5.7 ± 0.6 s (n = 6) in control solution and 108 ± 17 s (n = 6) in the presence of 100 µM MgATP.
We next examined the off-rate of pinacidil for channels carrying
mutations in the NBDs of SUR2A (Fig. 4). In the absence of nucleotide,
most (>90%) of the pinacidil response of Kir6.2/SUR2A-K1348A currents
reversed with a fast time constant of ~1.0 s (Fig. 4). Neither MgADP
nor MgATP had a significant effect on this time constant. Thus, when
100 µM MgADP was present, the off-rate of the drug exhibited a single
fast time constant of 1.7 ± 0.3 s (n = 5),
whereas in the presence of 100 µM ATP, 83 ± 6% of the current
decayed with a time constant of 1.3 ± 0.2 sec, and the remaining
current declined with a time constant of 11 ± 2 s
(n = 5). The off-rate of pinacidil on
Kir6.2/SUR2A-K707A currents in the presence of MgADP followed a single
exponential with a mean time constant of 1.1 ± 0.3 s
(n = 5); in MgATP solution, the activation was too
small to measure the off-rate (Fig. 2).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Our results show that both pinacidil and its analog P1075 can enhance Kir6.2/SUR2A currents in the absence of nucleotides. However, the off-rate of both drugs is slowed in the presence of MgATP or MgADP, which interact with the NBDs of SUR2A. The ability of Mg-nucleotide to slow the drug dissociation may account for the fact that [3H]P1075 binding can only be detected in the presence of Mg-nucleotide.
Channel Activation by MgADP.
Mutation of K707 in NBD1 of SUR2A
did not prevent channel stimulation by MgADP, although the equivalent
mutation in SUR1 abolishes MgADP activation (Gribble et al., 1997b).
The amino acid sequence of NBD1 is not identical in SUR1 and SUR2A, so
this difference may simply be because the mutation disrupts MgADP
binding to NBD1 in SUR1 but not SUR2A. An alternative possibility is
that NBD2 is essential for MgADP-activation in both SURs, whereas NBD1
plays a facilitatory role that differs between SUR1 and SUR2A. Indeed, binding studies (using SUR1) suggest NBD1 constitutes a high-affinity binding site for ATP, that is modulated by MgADP binding at NBD2 (Ueda
et al., 1997, 1999).
Pinacidil Responses of Wild Type and Mutant KATP
Channels.
Consistent with studies on native cardiac and skeletal
muscle KATP channels (Fan et al., 1990; Shen et
al., 1991; Tung and Kurachi, 1991), pinacidil activated Kir6.2/SUR2A
currents in both the absence and presence of added nucleotide, and the
magnitude of the response varied with the degree of channel rundown.
, 1999). The
slow dissociation of nucleotide from NBD1 might also account for the
gradual reduction in the effect of pinacidil after patch excision.
Because the rate of dissociation of prebound nucleotide from SUR will
depend on the experimental conditions, this hypothesis may explain why
ATP-independent pinacidil responses have not always been reported for
native skeletal muscle KATP channels or
Kir6.2/SUR2B channels (Allard and Lazdunski, 1992; Schwanstecher et
al., 1998). The idea that pinacidil sensitivity requires nucleotide
binding to SUR is also consistent with reports that drug sensitivity
can be restored after rundown of native cardiac and skeletal muscle
KATP channels by the addition of MgADP or MgATP
(Fan et al., 1990; Shen et al., 1991; Tung and Kurachi, 1991; Allard
and Lazdunski, 1992; Terzic et al., 1995).
Addition of MgATP or MgADP to the intracellular solution partially
restored pinacidil sensitivity to Kir6.2/SUR2A-K707A currents. This
suggests that either the mutation does not completely abolish nucleotide binding (and/or hydrolysis) at NBD1, or that the interaction of nucleotide with NBD2 alone is sufficient to support the action of
the drug.
Effects of Nucleotide on the Pinacidil Off-Rate. In the case of wild-type SUR2A, addition of MgATP or MgADP markedly slowed the off-rate of pinacidil. Mutation of the WA lysine in a single NBD of SUR2A (either K707A or K1348A) largely abolished this effect. This suggests that binding and/or hydrolysis of nucleotide at both NBDs is necessary to slow the off-rate of the drug.
When pinacidil was tested on Kir6.2/SUR2A currents soon after the patch was excised into nucleotide-free solution, ~50% of the activated current reversed with a slow exponential whose time constant (s) was similar to that observed in the
presence of MgATP or MgADP. The magnitude of this component decreased
with time, but was restored after refreshment of channel activity by MgATP. It is thus possible that after exposure to high concentrations of MgATP (either intracellular or in the bath solution), some SUR2A
subunits have nucleotide bound at both NBDs, causing a proportion of
the pinacidil response to reverse with a slow time constant. The
time-dependent decrease in the magnitude of this exponential might then
reflect the dissociation of nucleotide from a single NBD.
When K1348 was mutated in NBD2 of SUR2A, the slow component of
pinacidil reversal was not completely eliminated. This component accounted for <10% of the response in the absence of nucleotide, but
increased to ~20% in 100 µM MgATP. If the slow component is only
observed when both NBDs are functional, as suggested above, the effect
of the K1348A mutation may be explained by a reduction, but not
complete abolition, of nucleotide binding and/or hydrolysis at NBD2.
Comparison of [3H]P1075 Binding and Channel
Activation.
The ability of P1075 to activate Kir6.2/SUR2A currents
in the absence of nucleotide contrasts with the MgATP-dependence of [3H]P1075 binding to skeletal muscle membranes
and to heterologously expressed SUR2A or SUR2B (Dickinson et al., 1997;
Schwanstecher et al., 1998; Hambrock et al., 1998, 1999). One
explanation for this anomaly might be that detection of
[3H]P1075 binding, and the functional response
to P1075 (or pinacidil), require MgATP binding/hydrolysis by SUR.
Association of nucleotide with SUR for 10 to 15 min after patch
excision might support pinacidil activation in electrophysiological
experiments but would play an insignificant role in binding studies
that are performed much later after membrane isolation. One problem
with this hypothesis, however, is that mutating the
WA lysine in NBD2 of SUR2B abolished binding of
[3H]P1075 (Schwanstecher et al., 1998), whereas
the corresponding mutation in SUR2A did not prevent channel activation
by pinacidil. An alternative idea, therefore, is that detection of
[3H]P1075 binding is only possible under
conditions in which drug dissociation from SUR is slow. This idea is
favored by the observation that maneuvers that prevent
[3H]P1075 binding to SUR2A or SUR2B (e.g.,
Mg2+ or ATP removal, mutation of the
WA lysine in NBD1 or NBD2 of SUR2B) also
abolished the slow reversal of pinacidil activation of Kir6.2/SUR2A
currents (Schwanstecher et al., 1998; Hambrock et al., 1998, 1999).
Although we have not attempted to interpret the on-rates for pinacidil
or P1075, we speculate that the slow off-rate in the presence of
Mg-nucleotides may be associated with an increase in the affinity of
drug binding. This could account for the inability to detect P1075
binding in the absence of nucleotide, because
[3H]P1075 binding studies are performed at
lower drug concentrations (1 to 10 nM) than that used in the current
study (10 µM).
=108 s) was ~10 times slower than that of pinacidil ( =12 s). This
difference is similar to the ~10-fold higher affinity of P1075 (17 nM) in binding studies, compared with that of pinacidil (~150 nM: 78 nM for the active enantiomer ()pinacidil, Hambrock et al., 1999). The
off-rate of P1075 is also similar to the rate of dissociation of
[3H]P1075 from SUR2A in binding studies
( = 95 s) (Hambrock et al., 1999). Although the binding
studies were performed at 37°C and the electrophysiology at 20 to
22°C, the similarity between these measurements suggests that the
time course of the current decline is determined by drug unbinding
rather than by the rate at which the activated channel relaxes back to
the resting state.
Although it is widely recognized that the activity of many
KATP-channel openers is dependent on the
nucleotide environment, the mechanisms behind these interactions have
remained elusive. It has been proposed, for example, that ATP
hydrolysis is essential for binding of
[3H]P1075 to SUR2B (Schwanstecher et al.,
1998). The roles of the individual NBDs of SUR have not, however, been
addressed. Our results suggest that nucleotides may modulate pinacidil
efficacy by interacting with the NBDs and that when a single NBD of
SUR2A is mutated, pinacidil activation reverses rapidly in the presence of nucleotide. The cooperative effect of nucleotide binding at both
NBDs, however, seems to stabilize a pinacidil-activated state, resulting in a slow reversal of the drug response.
| |
Acknowledgments |
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We thank Dr S Seino (Chiba University, Japan) for SUR2A and Leo Pharmaceuticals for the gift of P1075.
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Footnotes |
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Received December 6, 1999; Accepted February 14, 2000
This work was supported by the Wellcome Trust and the British Diabetic Association. FMG holds a Wellcome Trust Advanced Fellowship for Medical Graduates.
Send reprint requests to: Prof. Frances M. Ashcroft, University Laboratory of Physiology, Parks Rd., Oxford OX1 3PT, UK. E-mail: frances.ashcroft{at}physiol.ox.ac.uk
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Abbreviations |
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KATP channel, ATP-sensitive K+ channels; SUR, sulfonylurea receptor; NBD, nucleotide-binding domain; WA, Walker A.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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 F. Reimann, F. M. Gribble, and F. M. Ashcroft Differential Response of KATP Channels Containing SUR2A or SUR2B Subunits to Nucleotides and Pinacidil Mol. Pharmacol., April 13, 2001; 58(6): 1318 - 1325. [Abstract] [Full Text]
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M. Matsuo, K. Tanabe, N. Kioka, T. Amachi, and K. Ueda Different Binding Properties and Affinities for ATP and ADP among Sulfonylurea Receptor Subtypes, SUR1, SUR2A, and SUR2B J. Biol. Chem., September 8, 2000; 275(37): 28757 - 28763. [Abstract] [Full Text] [PDF]
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D.-K. Song and F. M. Ashcroft ATP Modulation of ATP-sensitive Potassium Channel ATP Sensitivity Varies with the Type of SUR Subunit J. Biol. Chem., March 2, 2001; 276(10): 7143 - 7149. [Abstract] [Full Text] [PDF]
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J. C. Koster, A. Knopp, T. P. Flagg, K. P. Markova, Q. Sha, D. Enkvetchakul, T. Betsuyaku, K. A. Yamada, and C. G. Nichols Tolerance for ATP-Insensitive KATP Channels in Transgenic Mice Circ. Res., November 23, 2001; 89(11): 1022 - 1029. [Abstract] [Full Text] [PDF]
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, 100 µM pinacidil; 
, 100 µM MgATP; 
, 100 µM MgATP + 100 µM
pinacidil; 
, 100 µM MgADP; 
, 100 µM MgADP + 100 µM
pinacidil.
