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Vol. 58, Issue 6, 1318-1325, December 2000
Oxford University Laboratory of Physiology, Oxford, UK
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
Conclusions
References
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ATP-sensitive K-channels (KATP channels) are the target for KATP-channel openers (KCOs), such as pinacidil and P1075. These channels are formed from pore-forming Kir6.2 and regulatory sulfonylurea receptors (SUR2A in heart and skeletal muscle; SUR2B in smooth muscle). The two isoforms of SUR2 differ only in their final 42 amino acids, a region that includes neither the Walker A and B nucleotide binding motifs nor the proposed KCO binding site, yet channels containing SUR2A or SUR2B respond differently to both nucleotides and KCOs. We explored the basis for this difference by expressing Kir6.2/SUR2A and Kir6.2/SUR2B currents in Xenopus laevis oocytes. Kir6.2/SUR2B but not Kir6.2/SUR2A currents were activated by the Mg-nucleoside triphosphates MgATP and MgGTP, whereas both channel types responded to the diphosphates MgADP and MgGDP. This activation of Kir6.2/SUR2B currents by MgATP explains how the ATP concentration-response curve is shifted to the right in the presence of Mg2+. In the absence of nucleotide, pinacidil and P1075 activated Kir6.2/SUR2B and Kir6.2/SUR2A currents, but the presence of nucleotide slowed the drug off-rates. In the presence of MgATP, the response to pinacidil reversed ~14 times more slowly with SUR2B than SUR2A. The EC50 for ATP, measured by its ability to slow the pinacidil off-rate, was also ~20 times higher for channels containing SUR2A than SUR2B. Our findings suggest that nucleotide binding and/or hydrolysis is enhanced in SUR2B compared with SUR2A, and that the greater KCO-affinities of SUR2B compared with SUR2A may be a consequence of this altered nucleotide handling.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusions
References
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KATP
channels couple the metabolic state of a cell to its electrical
activity. They are found in a variety of cell types, including smooth,
cardiac, and skeletal muscle, pancreatic 
-cells and some neurons
(Ashcroft and Gribble, 1999
). In cardiac muscle, they are involved in
the response to ischemia and ischemic preconditioning (Nichols and
Lederer, 1991), in vascular smooth muscle they regulate vessel tone
(Quayle et al., 1997), in skeletal muscle they may be activated during
fatigue (Davis et al., 1991), and in pancreatic -cells, they play a
key role in glucose-stimulated insulin secretion (Ashcroft and Gribble,
1999). KATP channels are the target for a range
of therapeutic drugs, including the sulfonylureas used in the treatment
of type 2 diabetes, and the KCOs such as diazoxide and pinacidil
(Edwards and Weston, 1993; Ashcroft and Gribble, 1999).
The pore of the KATP channel consists of a
tetramer of Kir6.2 subunits, each of which is associated with a
regulatory SUR (Aguilar-Bryan et al., 1995; Sakura et al., 1995;
Clement et al., 1997). The SUR subunit endows the channel with
sensitivity to sulfonylureas and to the stimulatory actions of MgADP
and the KATP-channel openers (Tucker et al.,
1997). SUR is a member of the ABC transporter family, and like other
members of this group, has multiple transmembrane domains and two
intracellular NBDs, each containing a Walker A and a Walker B motif. It
is believed that a conserved aspartate in the Walker B motif
coordinates the Mg2+ ion of MgATP and that a
conserved lysine residue in the Walker A motif is involved in the
binding and/or hydrolysis of ATP (Azzaria et al., 1989; Carson et al.,
1995; Ko and Pedersen, 1995; Ueda et al., 1997, 1999). The sulfonylurea
receptors found in cardiac (SUR2A) and smooth (SUR2B) muscles are
splice variants of a single gene, differing in their final exon
(Inagaki et al., 1996; Isomoto et al., 1996). The splice site is distal
to the Walker A and B motifs in NBD2, and alters only the final 42 amino acids of the C terminus.
Several drug and nucleotide binding sites have been localized on the
sulfonylurea receptor. Sulfonylurea binding involves the C-terminal
group of transmembrane helices of SUR1 (Ashfield et al., 1999), and a
neighboring, but not exactly overlapping, region in the homogous domain
of SUR2 has been implicated in binding of the
KATP-channel openers pinacidil, P1075, and
cromakalim (D'hanan et al., 1999a; Uhde et al., 1999; Babenko et al.,
2000). A question that has remained unanswered, however, is why many
KCOs bind with 3- to 4-fold higher affinity to SUR2B than to SUR2A even
though the splice variation does not alter the sequence of the proposed KCO-binding site (Schwanstecher et al., 1998; Shindo et al., 1998; Hambrock et al., 1999). Based on earlier data, we suggested that the
potency of pinacidil on Kir6.2/SUR2A channels is modified by the
presence of Mg-nucleotides (Gribble et al., 2000). One possibility,
therefore, is that the greater KCO-binding affinity of SUR2B compared
with SUR2A is related to a difference in Mg-nucleotide handling between
the two SUR2 isoforms. In favor of this idea, lower concentrations of
ATP are required for [3H]P1075 binding to SUR2B
than to SUR2A (Hambrock et al., 1999). In addition, Kir6.2/SUR2B
channels respond to diazoxide in the presence of MgATP, whereas
Kir6.2/SUR2A channels require the additional presence of ADP (D'hanan
et al., 1999b).
In this article, we explore this idea by comparing the functional responses of Kir6.2/SUR2A and Kir6.2/SUR2B channels with Mg-nucleotides and with KATP channel openers. We show that channels containing SUR2B, but not those containing SUR2A, show marked activation by nucleoside triphosphates even in the absence of KCOs. In the presence of MgATP, both channel types are activated by pinacidil and P1075 but the drugs reverse more slowly with SUR2B than with SUR2A. The results indicate that the C-terminus of SUR2 influences how the channels respond to Mg-nucleotide, and suggest that this difference may itself determine the binding affinity for KCOs.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusions
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) and SUR2B (Genbank accession no. D86038;
Isomoto 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 SUR2 (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 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 either 110 mM KCl, 1.4 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.2, with KOH (final [K+] ~140
mM) or 110 mM KCl, 2 mM MgCl2, 1 mM
CaCl2, 10 mM EGTA, 10 mM HEPES, pH 7.2 with KOH
(final [K+] ~140 mM). Results were similar
using the two solutions, and data were therefore combined. 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, Ballerup, Denmark)
were prepared in ethanol and dimethyl sulfoxide, respectively.
Nucleotides were dissolved directly in the bath solution and the pH
then readjusted as necessary. MgCl2 was added to
maintain the free Mg2+ concentration. 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-s voltage ramps from 
110 mV to +100 mV,
with a holding potential of 0 mV. They were filtered at 0.5 kHz,
digitized at 1 kHz using a Digidata 1200 Interface, and analyzed using
pClamp software (Axon Instruments, Burlingame, 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. ATP
concentration-response curves were fit to the Hill equation:
G/Gc = 1 / (1 + ([ATP] / Ki)h), where [ATP]
is the ATP concentration, Ki is the ATP
concentration at which inhibition is half-maximal, and h is the slope
factor (Hill coefficient).
50 mV and macroscopic currents were recorded in
response to repeated drug applications. Currents were sampled at 20 Hz
and analyzed using Microcal Origin software (Microcal Software,
Northampton, MA). The decay in current after drug removal was best fit
with the 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 the sum of two exponentials,
with fast (f) and slow
(s) time constants. Exponentials with time
constants <1 s could not be detected because of the time required to
exchange solutions. 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.
Statistical significance was tested using Student's t test.
Data are presented as mean ± S.E.M.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusions
References
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Macroscopic currents were recorded from X. laevis oocytes expressing either Kir6.2/SUR2A or Kir6.2/SUR2B channels. In both cases, the currents were small in the cell-attached patch, but increased on patch excision. Current amplitudes were similar for Kir6.2/SUR2A and Kir6.2/SUR2B channels. We first examined the effects of nucleotides alone, and then in conjunction with pinacidil, on the macroscopic KATP current in inside-out membrane patches.
Effects of Nucleotides.
Application of ATP in the presence of
Mg2+ to the intracellular membrane surface
inhibited both Kir6.2/SUR2A and Kir6.2/SUR2B currents. Inhibition of
Kir6.2/SUR2B currents became weaker over the course of 10 to 20 s
but remained stable in the case of Kir6.2/SUR2A currents (Fig.
1A). Concentration-response relationships
for ATP inhibition of Kir6.2/SUR2B currents were constructed by
measuring the steady-state extent of block. Half-maximal inhibition
(KI) was produced by 29 ± 3 µM ATP
(n = 13) for Kir6.2/SUR2A currents and by 117 ± 22 µM ATP (n = 11) for Kir6.2/SUR2B currents (Fig. 1B).
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-cell type of KATP
channel, Kir6.2/SUR1, and that the current amplitude in the presence of Mg-nucleotides reflects the balance between these two opposing actions
(Gribble et al., 1998). Inhibition is mediated by a direct interaction
of the nucleotide with Kir6.2, and is independent of
Mg2+ ions, whereas activation occurs when
Mg-nucleotides interact with the NBDs of SUR1 (Tucker et al., 1997;
Gribble et al., 1998). A further complication is that the presence of
SUR1 enhances the sensitivity of Kir6.2/SUR1 currents to the inhibitory
effect of ATP (Tucker et al., 1997). Thus, although the different
responses of Kir6.2/SUR2A and Kir6.2/SUR2B currents to MgATP must be
conferred by the sulfonylurea receptor, it is not clear whether it
results from a difference in the degree of inhibition or of activation.
To distinguish between these possibilities, we tested the effect of ATP
in the absence of Mg2+, a procedure that
selectively abolishes the stimulatory action of nucleotides. In
contrast to what we observed in the presence of
Mg2+, ATP (100 µM) blocked Kir6.2/SUR2A and
Kir6.2/SUR2B currents to similar degrees in Mg-free solution (Fig. 1C).
The extent of block was 90 ± 4% (n = 4) for
Kir6.2/SUR2A channels and 94 ± 2% (n = 11) for
Kir6.2/SUR2B channels. This indicates that Kir6.2/SUR2A and
Kir6.2/SUR2B are blocked by ATP to similar extents and suggests that
the different ATP sensitivities observed in the presence of
Mg2+ are the consequence of an additional
stimulatory action of MgATP on Kir6.2/SUR2B currents. If this
activation takes time to develop, it could also account for the slow
increase in Kir6.2/SUR2B currents in the maintained presence of MgATP.
Our results are consistent with previous reports that the ATP
concentration-response curve of vascular smooth muscle
KATP channels and cloned Kir6.2/SUR2B currents is
shifted to the right in the presence of Mg2+
(Kamouchi and Kitamura 1994; Isomoto et al., 1996), unlike that of
cardiac KATP channels and cloned Kir6.2/SUR2A
currents (Lederer and Nichols 1989; Okuyama et al., 1998).
In contrast to MgATP, 100 µM MgADP activated Kir6.2/SUR2A and
Kir6.2/SUR2B currents to similar extents (Fig. 1C) [by 1.8 ± 0.2-fold (n = 11) and 1.7 ± 0.1-fold
(n = 9), respectively]. This suggests that the
stimulatory effect of MgADP is similar for both types of channel,
whereas that of MgATP differs.
To determine whether this difference between nucleoside di- and
tri-phosphates also holds for other nucleotides, we tested the effects
of MgGTP and MgGDP. These nucleotides have been shown to activate
Kir6.2/SUR1 currents via the SUR subunit (Trapp et al., 1997) but have
very little inhibitory effect on Kir6.2 (Trapp et al., 1997; Tucker et
al., 1998). Because the degree of activation is greater when the
channel open probability has first been reduced by nucleotide
inhibition, currents were blocked with 200 µM AMP-PCP. Figure
2, A and C, shows that MgGDP activated
both Kir6.2/SUR2A and Kir6.2/SUR2B currents. In contrast, MgGTP
activated Kir6.2/SUR2B currents but produced a small inhibition of
Kir6.2/SUR2A currents (Fig. 2, A and B). The diphosphate was more
effective than the triphosphate at activating Kir6.2/SUR2B, as
previously reported for SUR1 (Trapp et al., 1997; Gribble et al.,
1998).
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Effect of the Walker A Mutations on Nucleotide Activation.
To
investigate the roles of the individual NBDs of SUR2A and SUR2B, we
mutated the lysine residue in the Walker A motif of either NBD1 or NBD2
to alanine (K707A and K1348A, respectively). In other ABC transporters,
these mutations have been shown to abolish ATP binding and/or
hydrolysis (Azzaria et al., 1989; Carson et al., 1995; Ko and Pedersen,
1995; Ueda et al., 1997, 1999), and in SUR1 they prevent activation by
Mg-nucleotides (Gribble et al., 1997b, 1998; Shyng et al., 1997).
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).
Effects of KATP-Channel Openers.
Both pinacidil
(100 µM) and P1075 (10 µM) activated Kir6.2/SUR2A and Kir6.2/SUR2B
currents in the absence of nucleotide (Table 1; Fig. 4,
5A). Mutation of the Walker A lysine in NBD1 (K707A), but not in NBD2
(K1348A), of either SUR2A or SUR2B abolished the nucleotide-independent
action of pinacidil. Addition of 100 µM MgATP fully restored the
response of Kir6.2/SUR2B-K707A currents to pinacidil (Table 1), but
only partially restored the response of Kir6.2/SUR2A-K707A currents.
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Rates of Reversal.
As reported previously (Gribble et al.,
2000), the activation of Kir6.2/SUR2A by pinacidil or P1075 reversed
rapidly in the absence of added nucleotide. This was also found to be
the case for Kir6.2/SUR2B currents (Fig. 4, 5A). The off-rate of
pinacidil could be fit by a single exponential with a mean time
constant of 1.8 ± 0.2 s (n = 5) for
Kir6.2/SUR2A currents and of 3.3 ± 0.5 s (n = 6) for Kir6.2/SUR2B currents. The off-rates of P1075 were slightly
slower: = 5.7 ± 0.6 s (n = 6) for
Kir6.2/SUR2A currents and = 5.7 ± 0.9 s
(n = 7) for Kir6.2/SUR2B currents. The C-terminus does
not, therefore, appear to have a marked effect on the off-rate in the
absence of added nucleotide.
) and
the activation was effectively irreversible for Kir6.2/SUR2B currents
(Fig. 4).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusions
References
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Different Responses of SUR2A and SUR2B to Nucleotides.
In the
absence of Mg2+, Kir6.2/SUR2A and Kir6.2/SUR2B
currents were inhibited to similar degrees (~90%) by 100 µM ATP,
suggesting that there is little difference in the ability of the two
SUR2 isoforms to enhance the sensitivity of the ATP inhibitory site on
Kir6.2. Thus, although the C-terminal 42 amino acids of SUR have been
implicated in conferring the different ATP sensitivities of
KATP channels containing SUR1 and SUR2A (Babenko
et al., 1999), our results suggest that the splice variation between
SUR2A and SUR2B does not have a marked effect on Mg-independent ATP
inhibition. In the presence of Mg2+, however, the
inhibitory effect of ATP on Kir6.2/SUR2B currents was greatly reduced.
Taken together with the effects of GTP, our results suggest that
Mg-nucleoside triphosphates have a much greater stimulatory action on
Kir6.2/SUR2B currents than on Kir6.2/SUR2A currents. This additional
stimulation accounts for the apparent lower ATP sensitivity of
Kir6.2/SUR2B currents when measured in the presence of
Mg2+.
), and recent azido-ATP binding studies on SUR1 have
suggested that the second NBD may be able to hydrolyze ATP (Matsuo et
al., 1999). An alternative explanation, however, is that the different
effects of Mg-nucleoside triphosphates on Kir6.2/SUR2A and Kir6.2/SUR2B
currents are caused by a lower nucleotide potency on SUR2A compared
with SUR2B. In support of this idea, it has been shown previously that
the sensitivity of Kir6.2/SUR2A channels to activation by UDP is lower
than that of Kir6.2/SUR2B channels [Ki
values of ~240 µM and ~70 µM, respectively (Isomoto et al.,
1996; Okuyama et al., 1998)].
Responses to KATP Channel Openers. Pinacidil and P1075 activated Kir6.2/SUR2A and Kir6.2/SUR2B currents in the absence of added nucleotide, and under these conditions the effect of both drugs was rapidly reversible. The off-rate of P1075 was similar for channels containing SUR2A and SUR2B, suggesting that the C terminus does not influence the rate of reversal in the absence of nucleotide.
In the presence of MgATP, however, the activation of Kir6.2/SUR2B currents by pinacidil and P1075 reversed more slowly, as shown previously for Kir6.2/SUR2A currents (Gribble et al., 2000). In fact,
the off-rate of pinacidil was ~14 times slower for Kir6.2/SUR2B currents than for Kir6.2/SUR2A currents when measured in the presence of 10 µM MgATP ( = ~110 s for SUR2B and ~8 s for SUR2A).
A similar difference was also observed with P1075: in the presence of
100 µM MgATP, activation of Kir6.2/SUR2A currents reversed with a time constant of ~110 s, whereas the rate of reversal of Kir6.2/SUR2B currents was too slow to be measured.
Binding studies have shown half-lives for the dissociation of
[3H]P1075 that are comparable with the
off-rates we measured electrophysiologically (0.5 of ~10 min for SUR2B and ~1.1 min for
SUR2A, in 3 mM MgATP at 37°C: Hambrock et al., 1999). Our data
suggest that the greater potency of pinacidil and P1075 on Kir6.2/SUR2B
currents compared with Kir6.2/SUR2A currents is caused by differences
in the ability of MgATP to slow the off-rate of these drugs. This may
explain why the potencies of the drugs on channels containing SUR2A and SUR2B seem to differ even though the SUR splice variation does not form
part of the proposed KCO-binding site (D'hanan et al., 1999a; Uhde et
al., 1999; Babenko et al., 2000). Indeed, because Kir6.2/SUR2A and
Kir6.2/SUR2B currents behaved differently even in the absence of
pinacidil, it is most likely that the principal difference between
Kir6.2/SUR2A and Kir6.2/SUR2B channels is in either the binding and/or
hydrolysis of nucleotide at the NBDs or in coupling these events to the
opening of Kir6.2.
At intermediate MgATP concentrations, the pinacidil off-rate was best
fit by the sum of two exponentials, one fast and one slow. The
concentration of ATP at which half the current decayed rapidly
(IC50) was lower for Kir6.2/SUR2B (0.1 µM) than
Kir6.2/SUR2A (1.4 µM) currents. The time constant of the slow
exponential found for Kir6.2/SUR2B currents was also ATP-dependent, and
increased with ATP concentration. There are at least two possible
explanations for these results. First, there may be a real difference
in the ATP affinity of SUR2A and SUR2B, and the ATP-dependence of the slow exponential for Kir6.2/SUR2B currents might be caused by enhanced
ATP binding at higher concentrations. In support of this idea, we
measured different ATP IC50 values for
Kir6.2/SUR2A and Kir6.2/SUR2B currents in the presence of pinacidil
(1.4 µM versus 0.1 µM, respectively), and differences in ATP
sensitivity have also been reported from
[3H]P1075 binding studies [110 µM versus 3 µM, for SUR2A and SUR2B, respectively (Hambrock et al., 1999)].
Secondly, nucleotide hydrolysis might slow the pinacidil off-rate more
than nucleotide binding alone. If more hydrolysis occurs with SUR2B
than with SUR2A, this might cause the longer time constants found for
Kir6.2/SUR2B currents. The enhancement of hydrolysis at higher ATP
concentrations might account for the ATP-dependence of the slow exponential.
Effect of the Walker A Mutations.
Mutation of the Walker A
lysine in NBD2 abolished the stimulatory effect of MgADP on both
Kir6.2/SUR2B and Kir6.2/SUR2A currents. Similar results have been
described previously for SUR1 and SUR2A (Gribble et al., 1997b, 2000;
Shyng et al., 1997; D'hanan et al., 1999b). Our data are consistent
with the idea that the K1348A mutation reduces MgADP binding to NBD2 of
SUR2, and thereby impairs Mg-nucleotide stimulation of channel activity.
)], whereas the identical
mutation in SUR2A did not prevent stimulation by MgADP (Gribble et al.,
2000). This finding is slightly curious because the sequence of NBD1 is
identical in SUR2A and SUR2B. The results suggest either that the
mutation affects nucleotide binding at NBD1 differently in SUR2A and
SUR2B, or that nucleotide binding at NBD1 is critical for MgADP
activation of Kir6.2/SUR2B but not Kir6.2/SUR2A channels. In either
case, our data suggest that the alternatively spliced C terminus of
SUR2 modifies the properties of NBD1. Interactions between the NBDs of
SUR1 have been demonstrated in binding studies using azido-ATP (Ueda et al., 1997, 1999) but have not yet been studied biochemically for SUR2.
The finding that SUR2B behaves more like SUR1 when NBD1 is mutated is
interesting because the alternatively-spliced final exon of SUR2B is
more homologous to that of SUR1 (75% identity) than to that of SUR2A
(36% identity) (Isomoto et al., 1996).
Effect of the Walker Mutations on Pinacidil Activation.
We
suggested previously that pinacidil may only be effective on wild-type
Kir6.2/SUR2A channels when Mg-nucleotide is bound at NBD1 and that the
activation observed in nucleotide-free solutions is caused by the
presence of prebound ATP or ADP, which takes several minutes to
dissociate from its binding site (Gribble et al., 2000). In agreement
with this idea, mutation of lysine 707 in SUR2B, as in SUR2A, abolished
the effect of pinacidil in the absence of added nucleotide. Unlike what
was found for Kir6.2/SUR2A-K707A currents, however, addition of MgATP
fully restored pinacidil activation of Kir6.2/SUR2B-K707A currents. One
possible interpretation of this result is that the C terminus of SUR2
influences the nucleotide binding site at NBD1, consistent with the
idea that the tail of SUR is involved in mediating interactions between
the NBDs. Alternatively, the result might be caused by a difference in
the nucleotide handling of SUR2B compared with SUR2A.
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Conclusions |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusions
References
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Our results demonstrate two principal differences between
SUR2A and SUR2B. First, Mg-nucleoside triphosphates stimulated
Kir6.2/SUR2B but not Kir6.2/SUR2A currents. Secondly, MgATP was more
effective at slowing the off-rate of KATP channel
openers on Kir6.2/SUR2B compared with Kir6.2/SUR2A currents. It thus
seems that SUR2B can make use of nucleoside triphosphates where SUR2A
cannot, consistent with the report that Mg-ATP is sufficient for the
activation of Kir6.2/SUR2B currents by diazoxide, whereas Kir6.2/SUR2A
currents only responded to the drug in the presence of Mg-ADP (D'hanan et al., 1999b). The different effects of nucleoside triphosphates on
channels containing SUR2A or SUR2B suggest the possibility that MgATP
and MgGTP may stimulate channel activity because of their hydrolysis to
the corresponding nucleoside diphosphate and that the rate of this
hydrolysis may be faster for SUR2B than SUR2A. Our results are
consistent with a model in which the C terminus of SUR influences
interactions between the two NBDs and so accounts for the different
nucleotide sensitivities and KCO off-rates of Kir6.2/SUR2A and
Kir6.2/SUR2B channels. The marked difference in pinacidil off-rates for
Kir6.2/SUR2A and Kir6.2/SUR2B currents in the presence of MgATP
probably accounts for the different binding affinities of SUR2A and
SUR2B to this KCO. A similar effect may occur with other
KATP channel openers.
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Acknowledgments |
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We thank Dr S Seino (Chiba University, Tokyo, Japan) for SUR2A and Leo Pharmaceuticals for the gift of P1075.
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Note Added in Proof |
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As this article was being readied for publication, studies of
nucleotide binding to the NBDS of SUR2A and SUR2B have been reported
(Matsuo et al., 2000).
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Footnotes |
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Received March 28, 2000; Accepted September 7, 2000
This work was supported by the Wellcome Trust and the British Diabetic Association. F.M.G. holds a Wellcome Trust Clinician Scientist Fellowship.
F.R. and F.M.G. contributed equally to this article.
Send reprint requests to: Prof. Frances M. Ashcroft, University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK. E-mail: frances.ashcroft{at}physiol.ox.ac.uk
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Abbreviations |
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KATP, ATP-sensitive potassium (channel);
KCO, KATP channel opener;
SUR, sulphonylurea
receptor;
Kir, inwardly rectifying potassium (channel);
ABC, ATP
binding cassette (transporter);
NBD, nucleotide binding domain;
AMP-PCP, -
-methylene-ATP.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusions
References
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  A. Haupt, C. Kausch, D. Dahl, O. Bachmann, M. Stumvoll, H.-U. Haring, and S. Matthaei Effect of Glimepiride on Insulin-Stimulated Glycogen Synthesis in Cultured Human Skeletal Muscle Cells: A comparison to glibenclamide Diabetes Care, December 1, 2002; 25(12): 2129 - 2132. [Abstract] [Full Text] [PDF]
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 M. Dabrowski, F. M. Ashcroft, R. Ashfield, P. Lebrun, B. Pirotte, J. Egebjerg, J. Bondo Hansen, and P. Wahl The Novel Diazoxide Analog 3-Isopropylamino-7-Methoxy-4H-1,2,4-Benzothiadiazine 1,1-Dioxide Is a Selective Kir6.2/SUR1 Channel Opener Diabetes, June 1, 2002; 51(6): 1896 - 1906. [Abstract] [Full Text] [PDF]
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 C. Loffler-Walz, A. Hambrock, and U. Quast Interaction of KATP Channel Modulators with Sulfonylurea Receptor SUR2B: Implication for Tetramer Formation and Allosteric Coupling of Subunits Mol. Pharmacol., February 1, 2002; 61(2): 407 - 414. [Abstract] [Full Text] [PDF]
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F. Reimann, F. M. Ashcroft, and F. M. Gribble Structural Basis for the Interference Between Nicorandil and Sulfonylurea Action Diabetes, October 1, 2001; 50(10): 2253 - 2259. [Abstract] [Full Text]
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 K. Matsushita, K. Kinoshita, T. Matsuoka, A. Fujita, T. Fujikado, Y. Tano, H. Nakamura, and Y. Kurachi Intramolecular Interaction of SUR2 Subtypes for Intracellular ADP-Induced Differential Control of KATP Channels Circ. Res., March 22, 2002; 90(5): 554 - 561. [Abstract] [Full Text] [PDF]
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, n = 13) and
Kir6.2/SUR2B (
, n = 11) currents, in the
presence of Mg2+. The slope conductance (G) is expressed as
a fraction of the mean (Gc) of that obtained in control
solution before and after exposure to ATP. The lines are the best fit
of the data to the Hill equation. For Kir6.2/SUR2A currents,
Ki = 29 and h = 1.5. For
Kir6.2/SUR2B currents, Ki = 117 and
h = 1.5. C. Macroscopic currents recorded from oocytes
coexpressing Kir6.2 and either SUR2A or SUR2B in the presence of ATP,
ADP, and Mg2+ as indicated. Mean conductance in the
presence of these agent(s) (G) is expressed relative to the mean of the
conductance in the control solution before and after their addition
(Gc). The dashed line indicates the control conductance.
The number of patches is given above each bar. Statistical significance
is indicated by: n.s., not significant; ***P < .001.
) currents.
The lines drawn through the points are fitted with the equation y = 100/[1 + ([ATP]/IC50)], with IC50 = 1.4 µM for Kir6.2/SUR2A currents and 0.09 µM for Kir6.2/SUR2B
currents. The numbers adjacent to the symbols indicate the number of
patches. C, ATP-dependence of the time constants of the fast (above)
and slow (below) exponentials for the data shown in B.