© 2000 American Heart Association, Inc.
Molecular Medicine |
C-Terminal Tails of Sulfonylurea Receptors Control ADP-Induced Activation and Diazoxide Modulation of ATP-Sensitive K+ Channels
From the Departments of Pharmacology II (T.M., K.M., Y.K., A.F., K.I., M.T., A.I., Y.K.) and Internal Medicine and Molecular Science (T.M., S.Y., Y.M.), Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.
Correspondence to Y. Kurachi, Department of Pharmacology II, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail ykurachi{at}pharma2.med.osaka-u.ac.jp
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Abstract |
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Abstract—The ATP-sensitive K+ (KATP) channels are composed of the pore-forming K+ channel Kir6.0 and different sulfonylurea receptors (SURs). SUR1, SUR2A, and SUR2B are sulfonylurea receptors that are characteristic for pancreatic, cardiac, and vascular smooth muscle–type KATP channels, respectively. The structural elements of SURs that are responsible for their different characteristics have not been entirely determined. Here we report that the 42 amino acid segment at the C-terminal tail of SURs plays a critical role in the differential activation of different SUR-KATP channels by ADP and diazoxide. In inside-out patches of human embryonic kidney 293T cells coexpressing distinct SURs and Kir6.2, much higher concentrations of ADP were needed to activate channels that contained SUR2A than SUR1 or SUR2B. In all types of KATP channels, diazoxide increased potency but not efficacy of ADP to evoke channel activation. Replacement of the C-terminal segment of SUR1 with that of SUR2A inhibited ADP-mediated channel activation and reduced diazoxide modulation. Point mutations of the second nucleotide-binding domains (NBD2) of SUR1 and SUR2B, which would prevent ADP binding or ATP hydrolysis, showed similar effects. It is therefore suggested that the C-terminal segment of SUR2A possesses an inhibitory effect on NBD2-mediated ADP-induced channel activation, which underlies the differential effects of ADP and diazoxide on KATP channels containing different SURs.
Key Words: KATP channel • sulfonylurea receptor • C-terminus • ADP • diazoxide
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Introduction |
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ATP-sensitive K+ (KATP) channels are inhibited by intracellular ATP and activated by nucleoside diphosphates (NDPs) and thus provide a link between the metabolic state and cellular excitability in various organs, including pancreas, heart, vasculature, and brain.1 2 These channels are associated with such cellular functions as insulin secretion, cardiac preconditioning, vasodilatation, and neurotransmitter release. KATP channels are hetero-octamers composed of an ATP-binding cassette protein known as the sulfonylurea receptor (SUR) and an inwardly rectifying K+ channel (Kir) subunit.3 4 5 Three kinds of SUR, SUR1, SUR2A, and SUR2B, have been isolated, and their roles in formation of functional K+ channels with Kir6.0 subunits have been examined.3 4 5 6 7 8 9 It is now widely accepted that SUR1, SUR2A, and SUR2B represent pancreatic, cardiac, and vascular smooth muscle–type SURs, respectively. When expressed with Kir6.2, all 3 types of SURs form KATP channels that exhibit the same single-channel characteristics; weak inward rectification and a unitary conductance of
80 pS in the inward direction with 150 mmol/L
extracellular K+. However, they show
distinct sensitivities to various vasorelaxant
K+ channel opener compounds (KCOs),
intracellular MgADP, and sulfonylurea drug derivatives. For instance,
SUR1/Kir6.2 channel is activated by diazoxide but not by
pinacidil, SUR2A/Kir6.2 channel is strongly activated by
pinacidil but only weakly responsive to diazoxide, and SUR2B/Kir6.2
channel is activated by both
agents.10 11 12 13
Babenko et al14 recently
indicated using chimeras between SUR1 and SUR2A that the transmembrane
domains (TMDs) 6-11 and the first nucleotide-binding domain
(NBD1) of SUR1 are necessary for channel activation by diazoxide. Uhde
et al 15 showed using
chimeras between SUR1 and SUR2B that TMD16-17 and the cytosolic linker
between TMD13 and 14 (CL13-14) of SUR2 are required for the action of
pinacidil, levocromakalin, and P1075, but they did not examine the
effect of diazoxide. Therefore, these studies do not provide any
mechanistic insight to explain why diazoxide can effectively
activate the KATP channels containing
SUR2B but not SUR2A.
It has been known for a long time that diazoxide causes
shortening of the action potential recorded from cardiac
tissue,16 17
although it has almost no effect on SUR2A/Kir6.2 channel heterologously
expressed in mammalian
cells.6 18
D’hahan et al19 recently
showed that diazoxide activation of the reconstituted SUR2A/Kir6.2 and
native cardiac KATP channels requires the
presence of high concentrations of intracellular ADP, which is
analogous to the effect of nicorandil on cardiac
KATP
channel.2 20 SUR2A
and SUR2B are generated from a single gene and differ only in their 42
amino acid residue C-terminal tails
(C42).7 The C42 of SUR2B
shares 30% homology with that of SUR2A, but it shares 70%
homology with that of SUR1. Therefore, we considered that the
C-terminal segment of SURs might be involved in the SUR
subtype–dependent activation of KATP channels
by diazoxide, although it has been
reported14 that the
replacement of the C-terminus of the diazoxide-insensitive SUR2A with
the C-terminus of the diazoxide-sensitive SUR1 did not confer diazoxide
sensitivity to the chimera.
To examine this possibility, we compared the effects of intracellular ADP and diazoxide on KATP channels containing wild-type, mutant, and chimeric SURs. We found that in the presence of ATP, much higher concentrations of ADP were needed to activate SUR2A/Kir6.2 channels than SUR1 or SUR2B/Kir6.2. In all SUR/Kir6.2 channels, diazoxide increased the potency of ADP for channel activation without significantly affecting its efficacy. The chimera of SUR2A whose C42 was replaced with that of SUR1 formed a KATP channel effectively responding to ADP and diazoxide. When the SUR2A-C42 was adopted to SUR1, the chimera SUR1-2A/Kir6.2 channel was hardly activated by ADP and diazoxide. Mutations in the second nucleotide-binding domain (NBD2) of SUR1 and SUR2B that are known to reduce the binding of ADP21 resulted in behavior similar to that of the SUR1-2A chimera. This result suggests that the C-terminal segment of SUR2A might interfere with ADP binding to NBD2 and high concentration of ADP would be required to counter this effect and thus facilitate the activation of SUR2A/Kir6.2 by diazoxide.
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Materials and Methods |
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Molecular Biology
The cDNAs of mouse Kir6.2 and SURs were used.7 22 23 The coding region of each cDNA was individually subcloned into an expression vector, pcDNA3 (Invitrogen). Mutant SURs (SUR1 [K1348A] and SUR2B [K1348M]) were constructed using the GeneEditorTM in vitro site-directed mutagenesis system (Promega Corp). Chimeric SURs (SUR1-2A, SUR1-2B, and SUR2-1) were made by exchanging the C-terminal 42 amino acids of SUR1 or SUR2A with those of SUR2A, SUR2B, or SUR1 by overlap extension PCR method, respectively. All mutated and chimeric SURs were confirmed by DNA sequencing.
Functional Coexpression of SURs and Kir6.2
cDNAs
The plasmid containing Kir6.2 was cotransfected with
one of the SURs into human embryonic kidney 293T cells using
LipofectAMINE (Life Technology, Inc). To monitor the efficiency of
transfection, pCA-GFP (S65A) was also cotransfected. The cells
expressing GFP were identified by fluorescence microscopy and
used for electrophysiology.
Electrophysiology
The channels expressed in the cotransfected human
embryonic kidney 293T cells were recorded in the inside-out
configuration of the patch-clamp technique with a patch-clamp amplifier
(Axopatch 200A, Axon Instruments Inc). Pipettes were pulled from
thin-walled glass tube. The tip of pipettes were coated with silgard
and heat-polished. The bath was perfused with a solution containing
(in mmol/L) KCl 145, EGTA 5, MgCl2 2, and
HEPES-KOH 5 (pH 7.3), in which the concentration of free
Mg2+ was adjusted to 1.4 mmol/L with
the presence of various nucleotides. Pipettes were filled
with a solution containing (in mmol/L) KCl 145,
MgCl2 1, CaCl2 1, and
HEPES-KOH 5 (pH 7.4). Single-channel ion currents were recorded in
excised membrane patches voltage-clamped at -60 mV. All experiments
were performed at room temperature (25°C).
The data were recorded on videocassette tapes with a PCM converter system (VR-10B, Instrutech Corp). They were reproduced, low pass-filtered at 1 kHz (-3 dB) by an 8-pole Bessel filter (Frequency Devices), sampled at 5 kHz, and analyzed offline on a computer (Macintosh G3, Apple Computer Inc) using a commercially available software (Patch Analyst Pro, MT Corporation). The channel activity was expressed as relative NPo(rNPo) with reference to the maximum NPo measured in the absence of intracellular nucleotides in each inside-out patch. All data were derived from at least 4 distinct patches and expressed as mean±SE.
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Results |
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Effects of Intracellular MgADP and Diazoxide on SUR2A/Kir6.2 and SUR2B/Kir6.2 Channels
The effects of intracellular MgADP on SUR2A/Kir6.2 and SUR2B/Kir6.2 channels and their modulation by diazoxide are compared in Figures 1A
and 1B. On formation of inside-out patches,
vigorous activity of KATP channels appeared. ATP
(1 mmol/L) added to the internal solution almost completely
suppressed the channel activity in both cases. Even in the presence of
Mg2+, the SUR2A/Kir6.2 channel activity was
only weakly enhanced by ADP
(Figure 1A). The channel activity evoked by ADP was
negligible up to 300 µmol/L, and even with 3 mmol/L ADP,
rNPo was 0.3. Diazoxide (300 µmol/L)
induced little channel activity in the absence of ADP but significantly
enhanced it at each concentration of ADP ([ADP]). As [ADP] was
increased, diazoxide induced more channel activity, which saturated at
0.3-0.4 in rNPo at [ADP] >300 µmol/L.
The saturated rNPo value was nearly equal to
that obtained by 3 mmol/L ADP alone
(Figure 1A, middle panel).
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) and presence of diazoxide (
, 30 µmol/L
and •, 300 µmol/L). Right, Relationship between the
concentration of ADP and relative NPo
(rNPo) in the absence (
, 1 µmol/L; 
, 10 µmol/L; and
, 100 µmol/L). Relative NPo is expressed
as
mean±SE.
Unlike the SUR2A/Kir6.2 channel, the SUR2B/Kir6.2 channel
was effectively activated by ADP in a concentration-dependent
fashion
(Figure 1B
); as the [ADP] was increased from 0 to 30, 300,
and 1000 µmol/L, rNPo increased from
0.03±0.03 to 0.26±0.07, 0.37±0.10, and 0.42±0.08 (n=5 for each),
respectively. Diazoxide enhanced the channel activity at each [ADP].
As the concentration of the agent was raised from 0 to 30 and 300
µmol/L, the sensitivity of channel to ADP increased in a
concentration-dependent fashion, but the maximum
rNPo remained at 0.4-0.5
(Figure 1B, middle panel). Thus, diazoxide mainly increased
the potency but not the efficacy of ADP to enhance the
KATP channel activity in both SUR2A/Kir6.2 and
SUR2B/Kir6.2.
In contrast, pinacidil activated both SUR2A and
SUR2B KATP channels even in the absence of ADP
in a similar concentration-dependent fashion
(Figures 1A and 1B, right panels). Also different from
diazoxide, at high [ADP], the rNPo evoked by
even 1 µmol/L pinacidil reached a value beyond that obtained by ADP
alone. Pinacidil (1 µmol/L) shifted the concentration-response
relationship between [ADP] and rNPo upward
almost in a parallel fashion by 0.2- 0.3 in rNPo
at each [ADP]. Pinacidil (10 µmol/L) increased the activity of both
SUR2A and SUR2B/Kir6.2 channels by 0.8-0.9 in
rNPo in the absence of ADP. In the presence of
various [ADP], the channel activity was enhanced by the drug
essentially in an additive manner, which saturated at 1.0
rNPo at [ADP] >300 µmol/L. When the
concentration of pinacidil was additionally increased to 100 µmol/L,
channel activity decreased to 0.6-0.7 in rNPo,
probably because of nonspecific inhibitory or desensitizing
effects of the drug as previously
reported.20 Thus, it seems
likely that pinacidil activates SUR2A and
SUR2B-KATP channels independent of [ADP].
Therefore, it is strongly suggested that diazoxide and pinacidil
enhance the SUR2-KATP channel activity by using
distinct mechanisms.
SUR2A and SUR2B are splicing isoforms generated from a
single gene that differ from each other only in the 42 amino acid
segment at their C-terminal end. The C-terminal segment may then be
responsible for the differential effects of ADP and diazoxide on the
KATP channels containing these SUR2-isoforms.
The C-terminal 42 amino acids (C42) of SUR2B possess only 30%
homology with those of SUR2A but 70% with those of SUR1. Therefore,
to clarify the role of C42, we examined the effect of ADP and diazoxide
on the chimera SUR2-1, whose main part was SUR2, and C42 was adopted
from SUR1.
The chimera SUR2-1 formed a fully functional
KATP channel with Kir6.2
(Figure 1C). The channel activity in the inside-out patches
was almost completely suppressed by ATP (1 mmol/L), and diazoxide
(300 µmol/L) enhanced it to 0.35 in rNPo in
the absence of ADP. The channel activity was enhanced by ADP in a
concentration-dependent fashion in a similar manner to the SUR2B/Kir6.2
channel. Diazoxide increased the channel activity at each [ADP] but
did not exceed the maximum level induced by ADP also as for
SUR2B/Kir6.2 channel. Therefore, the C42 of SUR1 seems to possess a
similar effect on the ADP-mediated activation of the
SUR2B-KATP channel as the SUR2B-C42, which might
have been suggested by the high homology between the
two.
C-Terminal Tail of SUR2A Suppresses
Intracellular ADP-Activation and the Diazoxide Effect on SUR1/Kir6.2
Channel
The above results suggest that the SUR-C42 may play a
specific functional role in the control of ADP-mediated activation of
KATP channels and the modulation by diazoxide.
To additionally examine this possibility, we next constructed chimera
SURs, whose main part was SUR1 with the C-terminal segment of either
SUR2A (SUR1-2A) or SUR2B (SUR1-2B) and compared the responses of
wild-type SUR1/Kir6.2 and chimeric channels (SUR1-2A/Kir6.2 and
SUR1-2B/Kir6.2) toward ADP and diazoxide.
Figure 2A shows the effects of ADP and diazoxide on the
wild-type SUR1/Kir6.2 channel. The wild-type SUR1/Kir6.2 channel was
effectively activated by ADP in a concentration-dependent
fashion; as [ADP] was increased from 0 to 30, 300, and 1000 µmol/L,
rNPo of the channel increased from 0.07±0.04 to
0.18±0.07, 0.37±0.07 and 0.42±0.08 (n=5 for each), respectively.
This concentration-response relationship was similar to that of the
SUR2B/Kir6.2 channel
(Figure 1B). Like SUR2B/Kir6.2, diazoxide (30 µmol/L)
enhanced the wild-type SUR1/Kir6.2 channel openings at each [ADP] and
even without additional ADP. When the concentration of the agent was
increased to 300 µmol/L, the Kd
value for the effect of ADP on channel activity decreased, but the
maximum rNPo remained the same at 0.6 as 30
µmol/L diazoxide. However, this value was higher by 0.2 than the
rNPo at the highest level of channel activity
induced by [ADP] alone
(Figure 2A, right panel).
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The chimera SUR1-2A/Kir6.2 channel was hardly
activated by even high [ADP]
(Figure 2B). Diazoxide (300 µmol/L) enhanced the
SUR1-2A/Kir6.2 channel activity. The magnitude of the increase was the
same, 0.2 rNPo, at each [ADP]. Thus, the
concentration-response relationship between [ADP] and
rNPo of the channel was shifted upward almost in
a parallel fashion by 0.2 in rNPo
(Figure 2B, right panel). On the other hand, the
SUR1-2B/Kir6.2 channel
(Figure 2C) was as responsive to ADP and diazoxide as
SUR1/Kir6.2
(Figure 2A), which confirms the functional similarity between
the C42 of SUR1 and SUR2B.
Because the activation level of SUR1-2A-KATP channel by diazoxide was nearly the same as the difference between the drug-induced maximum channel activity in the presence of ADP, the above results can be interpreted as indicating that the SUR1/Kir6.2 channel activity could be enhanced by diazoxide via two distinct mechanisms; one is ADP-dependent and the other is ADP-independent. It seems likely that the C42 of SUR2A, but not of SUR1 or SUR2B, inhibits the ADP-dependent mechanism for the activation of SUR1/Kir6.2 channel and the modulation by diazoxide.
Effects of Mutations in NBD2 of SUR2B and SUR1
on the ADP and Diazoxide Action
Like other ATP-binding cassette proteins, SURs
possess 2 nucleotide-binding domains (NBD1 and NBD2,
respectively).3 It was shown
that SUR1 binds ATP at NBD1 and ADP at
NBD2.24 The NBDs possess the
Walker A and B motifs to form a portion of a
nucleotide-binding
pocket.25 In various
ATP-binding proteins, Walker A motif in NBDs is responsible for binding
or hydrolysis of nucleotides, where the highly conserved
lysine residue plays a critical
role.26 27 28
The mutation of this lysine residue in NBD2 extinguishes the
ADP-mediated activation of SUR1 and SUR2A-KATP
channels.19 21 29
Therefore, we introduced mutations of the lysine residue in NBD2 of
SUR2B and SUR1 (K1348M in SUR2B and K1384A in SUR1) and examined the
effects of ADP and diazoxide on the mutant SUR/Kir6.2 channels
(Figure 3).
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The mutant SURs formed with Kir6.2 fully functional
KATP channels, whose activity was almost
completely suppressed by 1 mmol/L ATP.
Figure 3A shows that ADP up to 1 mmol/L had little
effect on SUR2B(K1348M/Kir6.2 channel and that the effect of diazoxide
was dramatically reduced. In contrast, pinacidil (100 µmol/L)
enhanced the SUR2B(K1348M)/Kir6.2 channel to 0.7 in
rNPo in the absence of ADP
(Figure 3A, right panel), which was the same level as the
wild-type SUR2B/Kir6.2
(Figure 1B). The mutant SUR1(K1384A)/Kir6.2 channel also
scarcely responded to ADP
(Figure 3B). Different from the SUR2B mutant, diazoxide (30
and 300 µmol/L) enhanced the SUR1(K1384A)/Kir6.2 channel to 0.1
and 0.2 in rNPo, respectively, irrespective
of [ADP]. As a result, the concentration-response relationship
between rNPo and [ADP] was shifted by
diazoxide upward almost in a parallel fashion
(Figure 3B), which was similar to the reaction of the chimera
SUR1-2A/Kir6.2 channel
(Figure 2B).
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In this study, we examined the effect of intracellular ADP and diazoxide on the reconstituted KATP channels composed of Kir6.2 and SUR1, SUR2A, or SUR2B. We found that the SUR2A/Kir6.2 channel is much less sensitive to ADP than SUR2B or SUR1/Kir6.2. In all types of SUR/Kir6.2 channels, diazoxide increased the potency of ADP for channel activation without affecting its efficacy. The experiments using chimera and mutant SURs strongly suggest that the 42 amino acids at the C-terminal end (C42) of SURs play critical roles in the ADP-mediated activation of KATP channels and that the C42 of SUR2A may reduce ADP-mediated channel activation at NBD2. Thus, high concentrations of ADP were required for diazoxide-activation of the SUR2A/Kir6.2 channel.
Diazoxide May Enhance SUR2/Kir6.2 Channel
Activity Exclusively by an ADP-Mediated Activation Mechanism
The modes of KCO action on native cardiac
KATP channels have been
electrophysiologically classified into 3
distinct types.2 The type 1
KCOs, such as pinacidil and lemakalim, enhance the maximum response of
KATP channels and decrease the channel
sensitivity to intracellular ATP (ATPi). The
type 2 KCOs, such as ER-001533 and HOE234, selectively decrease the
channel sensitivity to ATPi. The type 3 KCO,
nicorandil, requires the presence of intracellular ADP
(ADPi) to enhance the channel activity. Because
diazoxide activation of the reconstituted SUR2A/Kir6.2 as well as
native cardiac KATP channels was shown to
require the presence of high concentrations of intracellular
ADP,19 diazoxide can also be
classified as a type 3 KCO. The present study provides molecular
insights for the type 3 action of diazoxide on various types of
KATP channels.
Although the potency was different, ADP induced openings of
SUR2A and SUR2B/Kir6.2 channels in a concentration-dependent fashion.
The potency of ADP for channel activation was much lower in
SUR2A/Kir6.2 channel than in SUR2B/Kir6.2. Diazoxide (300 µmol/L)
enhanced both types of KATP channel activity at
each [ADP], but the SUR2-channel activity in the presence of the drug
did not exceed the maximum level of ADP-induced channel openings.
Furthermore, the effect of diazoxide was largely reduced when the
lysine residue at NBD2 of SUR2B was mutated. These results indicate
that in the case of SUR2s, diazoxide enhances the
KATP channel activity by acting on the
ADP-mediated mechanism located at NBD2
(Figure 4).
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Pinacidil, the type 1 KCO for SUR2-containing
KATP channels, induced channel activation
apparently in an additive manner to the channel openings induced by ADP
(Figures 1A and 1B). The drug also activated the
SUR2B(K1348M)/Kir6.2 channel, whose ADP-dependent activation was
largely attenuated
(Figure 3A, right panel). This is consistent with a
recent study by Uhde et al15
using chimeras between SUR1 and SUR2B that showed that the TMD 16-17
and the CL 13-14 of SUR2 but not of SUR1 confer the activation by
pinacidil, levocromakalin, and P1075
(Figure 4).
In the case of SUR1, the effect of diazoxide could be
divided apparently into 2 components. One is ADP-dependent, similar to
that of SUR2-KATP channels, and the other is
ADP-independent. In the chimera SUR1-2A/Kir6.2 and the mutant
SUR1(K1384A)/Kir6.2 channels, the ADP-dependent component was largely
attenuated, and diazoxide (300 µmol/L) increased the channel
rNPo by 0.2 irrespective of [ADP].
Consistently, in the concentration-response relationship
between ADP and wild-type SUR1/Kir6.2 channel activity, the channel
rNPo in the presence of diazoxide was higher by
0.2 than the highest activity induced by ADP alone. Babenko et
al14 recently showed using
chimeras between SUR1 and SUR2A that TMD 6-11 and NBD1 of SUR1 are
required for the activation by diazoxide and that the corresponding
regions of SUR2 do not have such function. The ADP-independent
component of diazoxide on the SUR1/Kir6.2 channel may be related to
these regions of SUR1. But because their experiments were performed in
the absence of ADP, they missed the ADP-dependent component shown in
this study. This study showed that the ADP-dependent component of
diazoxide activation may be conferred by the NBD2 of either SUR1 or
SUR2 and the C-terminal tails of SUR1 or SUR2B
(Figure 4). Therefore, diazoxide may have at least two target
regions in SUR1 but only one (ie, NBD2) in SUR2
(Figure 4).
The Functional Role of the 42 Amino Acids at
C-Terminal Tail End of SUR2
This study indicates that the difference in the 42
amino acids at the C-terminal tail (C42) of SURs plays critical roles
in the control of ADP-mediated activation of the
KATP channel and the modulation of it by a KCO,
diazoxide. SUR2A and SUR2B are splicing isoforms from a single gene and
divergent only in C42. Therefore, the ADP-mediated activation of
KATP channels might be either inhibited by the
SUR2A-tail or enhanced by the SUR2B-tail. The experiments using the
chimera of SUR1 whose C-terminal segment was replaced with that of
SUR2A strongly support the former possibility. Furthermore, the point
mutation in NBD2 of SUR2B and SUR1, which is shown to reduce the
binding or hydrolysis of nucleotides in other ABC proteins,
abolished ADP-mediated activation of these KATP
channels and also attenuated the modulation of it by diazoxide.
Consistent with these results, Matsuo et
al30 have recently reported
using photoaffinity-labeled nucleotides that the affinity
of NBD2 of SUR2A for ADP binding was significantly lower than that of
SUR2B and SUR1.
It is known that KATP channels in pancreatic ß cells are active under physiological conditions and regulate the resting membrane potential.1 As a result, an increase of the ratio between [ATP]i and [ADP]i induced by high blood glucose inhibits the resting KATP channel, causes depolarization of the ß cells, and generates Ca2+-dependent action potentials, which triggers insulin secretion. A mutation in NBD2 of SUR1 was identified in an individual diagnosed PPHI.10 The reconstituted KATP channels possessing the mutated SUR1 exhibited a decreased sensitivity to MgADP and diazoxide and could not be opened in response to metabolic inhibition.10 12 Thus, the high channel sensitivity to ADP at NBD2 of SUR1 may play an essential role in regulation of insulin secretion in pancreatic ß cells.
In some vascular smooth muscle cells, KATP channels were shown to be active under physiological conditions.31 Thus, high sensitivity to intracellular ADP allowed by the SUR2B-C42 may also be essential for the channels to play a role in physiological control of vascular tone. In contrast, the low sensitivity of SUR2A to ADP because of its particular C-terminus means that the physiological alteration of the [ATP]i/[ADP]i ratio may not significantly induce KATP channel activity in cardiomyocytes. But when [ADP]i increases under pathophysiological conditions, however, the cardiac KATP channels would be activated and may contribute to the shortening of action potential duration.
Therefore, it is indicated that the differences in the 42 amino acid segment at C-terminal end among the subtypes of SUR may play critical roles in the physiological and pathophysiological control of KATP channels in different tissues. Additional studies are needed to elucidate the molecular mechanisms to determine how the C-terminal segments control the interaction between ADP and NBD2, how the ADP binding to NBD2 induces the KATP channel openings, and how diazoxide enhances the ADP-mediated channel activation process.
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Acknowledgments |
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This work was supported by a Research Grant for Cardiovascular Disease (11C-1) from the Ministry of Health and Welfare of Japan, Grant-in-Aid for Scientific Research on Priority Areas (B) from the Ministry of Education, Culture, Sports and Science of Japan, and grants from Research for the Future Program (96L00302) of Japan Society of the Promotion of Science, Human Frontier Science Program (RG0158/1997-B), and The Vehicle Racing Commemorative Foundation. We thank Dr Ian Findlay (Tours, France) for critical reading of this manuscript. We also thank Kazue Takahashi and Miho Fukui for excellent technical assistance and Keiko Tsuji for secretarial support.
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Footnotes |
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1 Both authors contributed equally to this study.
Received July 10, 2000; revision received September 22, 2000; accepted October 5, 2000.
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References |
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