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Vol. 56, Issue 2, 308-315, August 1999
Laboratoire de Biophysique Moleculaire et Celluraire, Département de Biologie Moléculaire et Structurale, Commissariat à l'Energie Atomique, Grenoble, France
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Summary |
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 Top
 Summary
 Introduction
Materials and Methods
Results
Discussion
References
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ATP-sensitive K+ (KATP) channels are a
complex of an ATP-binding cassette transporter, the sulfonylurea
receptor (SUR), and an inward rectifier K+ channel subunit,
Kir6.2. The diverse pharmacological responsiveness of KATP
channels from various tissues are thought to arise from distinct SUR
isoforms. Thus, when assembled with Kir6.2, the pancreatic 
cell
isoform SUR1 is activated by the hyperglycemic drug diazoxide but not
by hypotensive drugs like cromakalim, whereas the cardiac muscle
isoform SUR2A is activated by cromakalim and not by diazoxide. We
exploited these differences between SUR1 and SUR2A to pursue a chimeric
approach designed to identify the structural determinants of SUR
involved in the pharmacological activation of KATP
channels. Wild-type and chimeric SUR were coexpressed with Kir6.2 in
Xenopus oocytes, and we studied the resulting channels
with the patch-clamp technique in the excised inside-out configuration.
The third transmembrane domain of SUR is found to be an important
determinant of the response to cromakalim, which possibly harbors at
least part of its binding site. Contrary to expectations, diazoxide
sensitivity could not be linked specifically to the carboxyl-terminal
end (nucleotide-binding domain 2) of SUR but appeared to involve
complex allosteric interactions between transmembrane and
nucleotide-binding domains. In addition to providing direct evidence
for the structure-function relationship governing KATP
channel activation by potassium channel-opening drugs, a family of
drugs of the highest therapeutic interest, these findings delineate the
determinants of ligand specificity within the modular ATP-binding
cassette-transporter architecture of SUR.
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Introduction |
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Summary
Introduction
Materials and Methods
Results
Discussion
References
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ATP-sensitive
K+ (KATP) channels are
present in the plasma membrane of muscle cells, neurons, and most other
excitable cells, where they serve to adjust the resting membrane
potential to the metabolic state of the cell (Ashcroft and Ashcroft,
1990
; Isomoto and Kurachi, 1997). These channels are the targets of a
number of drugs that can block them, like the sulfonylureas, or open them, like cromakalim, pinacidil, or diazoxide (Gopalakrishnan et al.,
1993). These drugs are of great therapeutic interest because they
provide a way to pharmacologically adjust the excitability of cells,
raising it with blockers and lowering it with openers. Widespread use
of potassium channel openers (KCOs) is impaired, however, by their poor
tissue specificity and somewhat low affinity (Lawson, 1996). In the
search for better drugs, specially in terms of specificity, it appears
necessary to obtain a better understanding of the mechanisms of action
of these pharmacological agents.
The KATP channel is a complex of two proteins:
the sulfonylurea receptor (SUR; Aguilar-Bryan et al., 1995), which is a
member of the ATP-binding cassette (ABC) transporter family, and a
smaller protein, Kir6.2 (Inagaki et al., 1995), which belongs to the
inward rectifier K+ channel family. Four Kir6.2
subunits assemble to form a K+-selective pore
that is rendered functional by 4 auxiliary SUR subunits (Clement et
al., 1997; Inagaki et al., 1997; Shyng and Nichols, 1997) through
direct physical association (Lorenz et al., 1998).
Several isoforms of SUR have been identified. When the isoforms
SUR1, SUR2A, or SUR2B are coexpressed with Kir6.2, channels are
reconstituted that resemble native KATP channels
from pancreatic cells, cardiac muscle, and smooth muscle (Inagaki
et al., 1995, 1996; Isomoto et al., 1996). The variable tissue-specific
properties, particularly the response to openers, of
KATP channels would then arise from the identity
of the SUR isoform expressed in that tissue. This observation, as well
as more direct functional (Tucker et al., 1997) and biochemical
(Schwanstecher et al., 1998) evidence, designates SUR as the primary
target of KCOs. Presently, scarce structural data are available on the
underlying mechanism of action of KCOs. It is well established that the
nucleotide-binding domains of SUR are tightly linked to the sites of
action of KCOs because the binding and effect of KCOs require
hydrolyzable nucleotides and are compromised by mutations that, in
other ABC transporters, impair the ability of these domains to bind and
hydrolyze nucleotides (Gribble et al., 1997; Shyng et al., 1997;
Schwanstecher et al., 1998). It has also been suggested that the
carboxyl terminus of SUR could play an important role in this mechanism
because SUR2A and SUR2B differ only in their last 42 amino acids but
give rise to channels with very distinct apparent KCO affinity (Isomoto et al., 1996; Schwanstecher et al., 1998).
As an ABC transporter (Croop, 1998), SUR is organized as a modular
protein with clearly identifiable transmembrane domains and cytoplasmic
nucleotide-binding domains (Aguilar-Bryan et al., 1998). Taking
advantage of the differential sensitivity to the openers diazoxide and
cromakalim of two SUR isoforms, the pancreatic isoform SUR1
(diazoxide-sensitive, cromakalim-insensitive) and the cardiac isoform
SUR2A (diazoxide-insensitive, cromakalim-sensitive), we have followed a
chimeric approach to examine whether specific domains of SUR are
involved in the activation of KATP channels by
these KCOs.
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Materials and Methods |
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Summary
Introduction
Materials and Methods
Results
Discussion
References
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Molecular Biology.
Mouse Kir6.2 (GenBank accession no.
D50581; Inagaki et al., 1995), hamster SUR1 (GenBank accession no.
L40623; Aguilar-Bryan et al., 1995), and rat SUR2A (GenBank accession
no. D83598; Inagaki et al., 1996) were subcloned in the
Xenopus oocyte expression vector pGEMHE (Liman et al., 1992)
or its modified versions with enhanced polylinkers, pGH2 and pGH3
(kindly provided by Dr. F. Pagès, Grenoble). Three nonsilent
differences were found between the hamster SUR1 cDNA sequence and the
published sequence: G3601C,
T3751C, and A4099G, which
produce the amino acid changes V1201L,
C1251R, and T1367A. Because
these residues are also Leu, Arg, and Ala in all other rat, mouse, and
human SUR1 and SUR2 isoforms, it is likely that these differences were
due to errors in the original sequence.
). Hamster SUR1 and rat SUR2A were used as templates for making all
of the chimeras listed in Fig. 4. Successive PCRs with Vent DNA
polymerase (New England Biolabs, Beverly, MA) were performed for 25 cycles to produce the junctional cDNA fragments, which had a length of
2 kilobase pairs or less. These fragments were ligated to the
flanking 5' and 3' remaining sequence elements to produce the final
constructs. Plasmid DNAs were amplified, confirmed by restriction
analysis and by sequencing of the PCR-derived region, linearized, and
transcribed in vitro with the T7 mMessage mMachine kit (Ambion, Austin,
TX). cRNAs were electrophoresed on formaldehyde gels, and
concentrations were estimated from two dilutions with RNA marker as a standard.
Electrophysiology. Xenopus laevis were anesthetized with 3-aminobenzoic acid ethyl ester (1 g/liter water). Part of one ovary was removed, the incision was sutured, and the animal was allowed to recover. Stage V or VI oocytes were defoliculated by an ~60-min incubation at 19°C with 2 mg/ml type A collagenase (Sigma Chemical Co., St. Louis, MO). Selected oocytes were injected the next day with 50 nl of water containing ~2 ng of Kir6.2 cRNA and with ~6 ng of cRNA encoding wild-type or chimeric SURs. They were stored at 19°C in a modified Barth's solution with 1 mM KCl, 0.82 mM MgSO4, 88 mM NaCl, 2.4 mM NaHCO3, 0.41 mM CaCl2, 0.3 mM Ca(NO3)2, and 16 mM HEPES (pH 7.4) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml gentamycin.
Two to 15 days after injection, oocytes were devitellinized, and exogenous KATP channels were characterized by the patch-clamp technique in the excised inside-out configuration (Hamill et al., 1981). Experimental methods were similar to those used in our laboratory to record native frog skeletal muscle
KATP channels (Vivaudou and Forestier, 1995;
Forestier et al., 1996).
Conditions were designed to optimize recording of
KATP currents and to minimize contributions by
endogenous oocyte Cl
currents. Patch pipettes
(2-10 M
) contained 154 mM K+, 146 mM
Cl, 5 mM Mg2+, and 10 mM
piperazine-N,N'-bis(2-ethanesulfonic acid) (pH
7.1). The cytoplasmic face of the patch was bathed in solutions, all of
which contained 174 mM K+, 40 mM
Cl, 1 mM EGTA, 10 mM
piperazine-N,N'-bis(2-ethanesulfonic acid) (pH
7.1), and methanesulfonate as the
remaining anions. When present, Mg2+ was 1 mM.
ATP (potassium salt; Sigma Chemical Co.), diazoxide [100 mM stock in
dimethyl sulfoxide (DMSO); Sigma Chemical Co.], and SR47063 (20 mM
stock in DMSO; Sanofi Recherche, Montpellier, France) were added as
specified. The concentration of contaminant Mg2+
in nominally Mg2+-free solutions was less than 10 µM (Forestier and Vivaudou, 1993). The membrane potential was
maintained at 
50 mV. Experiments were conducted at room temperature
(22-24°C).
Applications of the various solutions to the intracellular face of the
patch were performed with a RSC-100 rapid solution changer (Bio-Logic,
Claix, France) controlled by in-house software Perf 2.10. Analog
signals were filtered at 300 Hz and sampled at 1 kHz. Slow fluctuations
of the no-channel-open baseline of the signal were removed by
interactive fitting of the baseline with a spline curve and subtraction
of this fit from the signal. Acquisition, analysis, and presentation
were performed with in-house software Erwin 3.2. ATP dose-response
curves were obtained and processed as in Vivaudou and Forestier (1995).
Results are displayed as mean ± S.E.M.
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Results |
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Summary
Introduction
Materials and Methods
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Discussion
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Expression of SUR1 and SUR2A/Kir6.2 KATP Channels in
Oocytes.
Oocytes coinjected with equimolar quantities of mRNAs
encoding Kir6.2 (2 ng) and either SUR1 or SUR2A (6 ng) were found to express a high density of KATP channels within 2 days. In excised patches with small patch pipettes of ~5-M
resistance, macroscopic ATP-inhibitable currents of 100 pA or more at a
membrane potential of 50 mV with 150 mM intracellular and
extracellular K+ could be recorded routinely.
; Vivaudou and Forestier, 1995).
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Pharmacological Properties of Wild-Type KATP
Channels.
SUR1/Kir6.2 and SUR2A/Kir6.2 channels were clearly
distinguishable on the basis of their pharmacological profiles. In
agreement with previous reports (Inagaki et al., 1995, 1996),
SUR1/Kir6.2 channels were activated by diazoxide but not by SR47063, a
cromakalim analog, whereas the opposite was true for SUR2A/Kir6.2
channels (Fig. 2).
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; Larsson et al., 1993). Taking into
account only the result of the first application in every patch
(usually performed in the first 5 min after patch excision), diazoxide
caused on average a more than 5-fold increase in current.
In oocytes expressing SUR2A and Kir6.2, diazoxide caused a much
smaller, 56 ± 15% (n = 17) increase in current,
but this could be attributed to the drug vehicle, DMSO, which alone
increased current by 48 ± 12% (n = 5).
In contrast, SR47063 (100 µM) consistently activated SUR2A/Kir6.2
channels, causing on average a 19-fold increase in current, but had no
significant effect on SUR1/Kir6.2 channels because it produced the same
increase (12 ± 10%; n = 17) as its vehicle alone
(13 ± 5%; n = 4). Higher concentrations of this
hydrophobic drug were not tested because they produced cloudy
solutions, indicative of solubility problems.
Magnesium Dependence of Opener Effects.
In an attempt to
clarify the role of nucleotide hydrolysis on opener action, experiments
were conducted on wild-type channels to determine whether the presence
of Mg2+ affected activation by diazoxide or
SR47063. In the presence of 100 µM ATP, channel activity was higher
when Mg2+ was present, which is consistent with
recent data on the activatory role of MgATP (Gribble et al., 1998a).
Figure 3 shows that diazoxide becomes
ineffective when Mg2+ is removed, an observation
already reported for native (e.g., Dunne et al., 1987) and recombinant
(Shyng et al., 1997) channels. This was not the case for SR47063, which
retained part of its activatory potential in the nominal absence of
Mg2+, although its effect was much weaker than
that with Mg2+ and rapidly reversed on washout of
the drug (Fig. 2). To verify that this effect was not a residual effect
due to contaminant Mg2+, the experiments
represented in Fig. 3B were repeated in the added presence of 5 mM
EDTA. Under those conditions, contaminant Mg2+
estimated at 8 µM (Forestier and Vivaudou, 1993) would be mostly chelated by EDTA, leaving ~7 nM free Mg2+ and
~5 nM MgATP. Still, SR47063 augmented SUR2A/Kir6.2 currents by
~3-fold (2.87 ± 0.61; n = 5), whereas its
vehicle alone, 0.5% DMSO, had a negligible effect in those experiments
(test/control = 0.92 ± 0.06; n = 4; data not
shown).
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Design and Expression of Chimeric SUR Receptors. The high sequence homology between SUR1 and SUR2A (67% identity) suggests that they have very similar structures. The clear dichotomy in terms of pharmacological properties of these two highly homologous proteins suggested that a chimeric approach could constitute a viable strategy to pinpoint the regions of SUR responsible for its pharmacological phenotype.
On the basis of hydrophobicity profiles and comparison with other ABC transporters (Tusnády et al., 1997), SUR may be divided in five
domains, as shown in Fig. 4: three
transmembrane domains (TMDs; TMD0, TMD1, and TMD2) and two cytoplasmic
nucleotide-binding domains (NBD1 and NBD2). These last two domains,
which incorporate Walker A and Walker B motifs capable of forming an
ATP-binding pocket (Walker et al., 1982), are the most conserved
regions of ABC transporters and the most homologous regions of SUR1 and
SUR2A. In some ABC transporters, distinct proteins form each of these domains (Croop, 1998), suggesting that each domain is a structurally and functionally independent module. Therefore, we designed chimeras along the logic of this modular architecture.
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Effects of KCOs on Chimeric KATP Channels.
The
responses to diazoxide and SR47063 of the chimeric channels were
tested. Results are illustrated in Fig. 6
and summarized in Fig. 7. Unlike either
SUR1 or SUR2A, Chim1 associated with Kir6.2 was activated by both
openers. Although a lack of response would have been difficult to
interpret, this gain-of-function phenotype suggests that Chim1 acquired
diazoxide sensitivity from its SUR1 regions (TMD0, TMD1, and
NBD1) and SR47063 sensitivity from its SUR2A regions (TMD2 and
NBD2). The response of this chimera to diazoxide was intermediate
between that of SUR2A (mainly due to vehicle as discussed above) and
that of SUR1, suggesting that domains in both halves of SUR participate
in that response. In contrast, the robust response to SR47063 suggests
a tight link between this opener and the second half of SUR (TMD2 + NBD2).
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Discussion |
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Summary
Introduction
Materials and Methods
Results
Discussion
References
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Identical ATP Sensitivity of SUR1 and SUR2A/Kir6.2 KATP
Channels.
In agreement with recently published data (Gribble et
al., 1998b), we found that SUR1 and SUR2A/Kir6.2 channels had the same sensitivity to block by intracellular ATP. For both, the concentration of ATP for 50% inhibition (K1/2) was 16 µM
and the Hill coefficient was slightly above 1, suggesting at least two
coupled nucleotide-binding sites. In agreement with this observation,
all SUR1/SUR2A chimeras displayed K1/2 values
that were not significantly different.
; Aguilar-Bryan et al., 1998; Okuyama et
al., 1998). We do not know the reason for this discrepancy. Even in
well-controlled conditions, ATP sensitivity is unstable (Findlay and
Faivre, 1991) and could well change with the expression system where
the presence and amount of certain channel-regulating factors, such as
membrane phospholipids (Fan and Makielski, 1997), remain unpredictable.
Therefore, constituent SUR isoforms cannot be identified dependably on
the responses to ATP of KATP channels. As far as
we can tell, in Xenopus oocytes, SUR1/Kir6.2 and
SUR2A/Kir6.2 KATP channels can be separated with
confidence only on the basis of their pharmacological properties.
Essential Role of TMD2 Domain in SR47063 Action.
The high
sequence homology and clear pharmacological divergence of SUR1 and
SUR2A make these proteins perfect candidates for a chimeric approach.
The two SUR isoforms used, hamster SUR1 and rat SUR2A, indeed have very
homologous primary sequences. Overall, 79% of their amino acids are
conserved, with identical amino acids amounting to 67% of the total.
Alignment between the two sequences is straightforward over most of
their length and provides an ideal basis for the design of chimeric
proteins. ABC transporters are organized in characteristic structural
domains (Croop, 1998), and chimeras were constructed along the lines of
this modular architecture to identify the respective role of domains
TMD2 and NBD2.
.
The TMD2 region that makes up a little less than one third of the SUR
receptor is the region least conserved between SUR1 and SUR2, with 60%
identities compared with more than 80% for the NBD1 and NBD2 domains.
It contains four highly hydrophobic stretches of amino acids compatible
with at least four and possibly six transmembrane helices
(Tusnády et al., 1997). A direct involvement of such a region in
the binding of KCOs would be consistent with the hydrophobic nature of
KCOs, which could reach their target via the lipid bilayer as has been
postulated for the substrates of another ABC transporters, the
P-glycoprotein (Sharom, 1997).
Our data indicate that in the absence of Mg2+,
SR47063 was still able to produce activation of SUR2A/kir6.2 channels.
Although the subunits of the skeletal muscle KATP
channels have not been formally identified, these data are in agreement
with our previous observations on native KATP
channels from skeletal muscle showing little effect of
Mg2+ on KCO activation (Forestier et al., 1996)
and suggesting that KCOs can bind in the absence of both
Mg2+ and nucleotides (Forestier et al., 1993).
Without Mg2+, SUR2A/kir6.2 activation was weaker
and reversed much more rapidly on washout of the drug. This could
possibly explain why binding of a tritiated KCO to native (Dickinson et
al., 1997) or recombinant (Schwanstecher et al., 1998) channels could
only be measured when hydrolyzable nucleotides and
Mg2+ were present. Thus, nucleotide binding and
hydrolysis by the NBDs would serve to modulate interaction of KCOs with
the TMD2 region.
Role of Carboxyl-Terminal End in Diazoxide Action.
The role of
the carboxyl-terminal extremity of SUR in diazoxide activation was
anticipated in view of the functional differences between the two
splice variants SUR2A and SUR2B, which, in the mouse, have only 28 nonidentical amino acids all within the last 42 amino acids. Because
diazoxide activates SUR2B/Kir6.2 channels (Isomoto et al., 1996) but
not SUR2A/Kir6.2 channels, elementary logic dictates that diazoxide
sensitivity resides in the carboxyl-terminal end of SUR. Unfortunately,
experimental evidence is at odds with this simple reasoning. The two
chimeras Chim1 and Chim4 that have the carboxyl-terminal extremity of
the diazoxide-insensitive SUR2A isoform were activated by diazoxide
although not to the same degree as SUR1, and the chimera Chim6, which
retained the SUR1 carboxyl terminus, was nearly insensitive to
diazoxide. Therefore, it does appear that the carboxyl-terminal
extremity is one among several determinants of diazoxide action.
Indirect estimates of diazoxide binding affinity have shown that
despite their functional differences, SUR1 and SUR2A have comparable
affinities for diazoxide (Schwanstecher et al., 1998). The site of
action of diazoxide would therefore be located within conserved
sequence elements, and functional differences among isoforms would be
due to variations in efficacy (i.e., in the structures linking channel
opening to binding). The present observations do not permit an
association of these structures to a specific domain but imply that
they are distributed over the whole protein and that their functional
role is governed by complex allosteric interactions. On the other hand,
the nucleotide binding domains, the most conserved regions of the SUR
isoforms, of which the integrity is indispensable for diazoxide
activation (Gribble et al., 1997; Shyng et al., 1997), are more likely
to be directly involved with the binding than with the efficacy of the opener.
Conclusions.
Despite the therapeutic potential of KCOs, the
molecular details of their mechanisms of action remain mysterious. We
have demonstrated here by a chimeric approach that distinct structures of SUR mediate KATP channel activation by the
openers diazoxide and cromakalim. If a single domain, the last
transmembrane spanning region of SUR, was found to be fundamental for
cromakalim action, functional determinants of diazoxide action appeared
distributed over several domains, and not only over the
carboxyl-terminal extremity as previously postulated (Isomoto et al.,
1996). This constitutes a first step toward the precise determination
of KCO interaction sites that should lead to a better understanding of the structure and mechanisms of SUR and other ABC transporters.
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Acknowledgments |
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We are grateful to Dr. D. Logothetis (Mount Sinai Hospital, New York, NY) for providing vector pGEMHE, Dr. F. Pagès (Commissariat à l'Energie Atomique, Grenoble, France) for vectors pGH2 and pGH3, Dr. S. Seino (Chiba University School of Medicine, Chiba, Japan) for mouse Kir6.2 and rat SUR2A, Dr. J. Bryan (Baylor College of Medicine, Houston, TX) for hamster SUR1, and Dr. P. Gautier (Sanofi Recherche, Montpellier, France) for SR 47063.
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Footnotes |
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Received February 4, 1999; Accepted May 2, 1999
This work was supported by grants from Association Francaise contre les Myopathies and Association Francaise de Lutte contre la Mucoviscidose. Additional support was provided by Commissariat à l'Energie Atomique and Centre National de la Recherche Scientifique. N.D., H.J., and C.M. were supported by a fellowship from La Société des Amis des Sciences, a studentship from Association pour la Recherche contre le Cancer, and a studentship from La Ligue contre le Cancer, respectively. A preliminary account of this work has been published in abstract form [Jacquet H, D'hahan N, Moreau C and Vivaudou M (1999) A transmembrane domain of the sulfonylurea receptor mediates activation of K-ATP channels by K-channel-openers. Biophys J 76:A413].
Send reprint requests to: Dr. Michel Vivaudou, Commissariat à l'Energie Atomique, Département de Biologie Moléculaire et Structurale, Biophysique Moleculaire et Celluraire, 17 rue des Martyrs, 38054, Grenoble, France. E-mail: vivaudou{at}cea.fr
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Abbreviations |
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SUR, sulfonylurea receptor; KCO, potassium channel opener; KATP channel, ATP-sensitive potassium channel; TMD, transmembrane domain; NBD, nucleotide-binding domain; DMSO, dimethyl sulfoxide; PCR, polymerase chain reaction; ABC, ATP-binding cassette.
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Summary
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Materials and Methods
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Discussion
References
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on nucleotide dependent potassium channels in an insulin-secreting cell line.
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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. BIENENGRAEBER, A. E. ALEKSEEV, M. R. ABRAHAM, A. J. CARRASCO, C. MOREAU, M. VIVAUDOU, P. P. DZEJA, and A. TERZIC ATPase activity of the sulfonylurea receptor: a catalytic function for the KATP channel complex FASEB J, October 1, 2000; 14(13): 1943 - 1952. [Abstract] [Full Text]
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A. P. Babenko, G. Gonzalez, and J. Bryan Pharmaco-topology of Sulfonylurea Receptors. SEPARATE DOMAINS OF THE REGULATORY SUBUNITS OF KATP CHANNEL ISOFORMS ARE REQUIRED FOR SELECTIVE INTERACTION WITH K+ CHANNEL OPENERS J. Biol. Chem., January 14, 2000; 275(2): 717 - 720. [Abstract] [Full Text] [PDF]
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 N. D'hahan, C. Moreau, A.-L. Prost, H. Jacquet, A. E. Alekseev, A. Terzic, and M. Vivaudou Pharmacological plasticity of cardiac ATP-sensitive potassium channels toward diazoxide revealed by ADP PNAS, October 12, 1999; 96(21): 12162 - 12167. [Abstract] [Full Text] [PDF]
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H. Malhi, A. N. Irani, P. Rajvanshi, S. O. Suadicani, D. C. Spray, T. V. McDonald, and S. Gupta KATP Channels Regulate Mitogenically Induced Proliferation in Primary Rat Hepatocytes and Human Liver Cell Lines. IMPLICATIONS FOR LIVER GROWTH CONTROL AND POTENTIAL THERAPEUTIC TARGETING J. Biol. Chem., August 18, 2000; 275(34): 26050 - 26057. [Abstract] [Full Text] [PDF]
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) and
SUR2A/Kir6.2 (
) channels versus ATP concentration. Each symbol and
bar represent the average and S.E.M. of measurements obtained in 12 SUR1 and 9 SUR2A patches using the protocols of A and B. Lines are best
fits of the data points to the Hill equation with values of
K1/2 and h of 15.8 µM and 1.16 (SUR1) and 15.9 µM and 1.24 (SUR2A).
) and Chim4/Kir6.2 (
) channels
versus ATP concentration. Each symbol and bar represent the average and
S.E.M. of 2 to 13 measurements obtained using the protocols of A and B. Lines are best fits of the data points to the Hill equation with values
of K1/2 and h of 11.4 µM and 1.14 (Chim1) and 20.0 µM and 1.22 (Chim4).
