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* Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905, USA; and
CEA, DBMS, Laboratoire de Biophysique Moléculaire et Cellulaire, 38054 Grenoble, France
1Correspondence: Guggenheim 7, Mayo Clinic, 200 First St. S.W., Rochester, MN 55905, USA. E-mail: terzic.andre{at}mayo.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: ATP-sensitive K+ channels • enzyme • ABC proteins • nucleotide binding domains • potassium channel openers • Kir6.2 • SUR
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. In this way, ATP-sensitive
K+ (KATP) channels are
formed by association of an inwardly rectifying
K+ channel, Kir6.2, with the ATP binding cassette
(ABC) protein, the sulfonylurea receptor SUR (5
, 6)
.
Originally discovered in the heart (7)
,
KATP channels transduce cellular metabolic
signals into membrane potential changes and regulate critical cellular
functions, including cytoprotection (8
9
10
11)
. Although SUR
confers metabolic sensing to the KATP channel
complex by serving as a binding site for adenine nucleotides (12
, 13)
, it is unknown whether intrinsic properties of this protein
promote nucleotide exchange and thereby contribute to channel gating.
SUR, including the cardiac SUR2A isoform, possess two nucleotide
binding domains, NBD1 and NBD2, located between the eleventh and
twelfth transmembrane regions and at the carboxyl terminus of the
protein (14
, 15)
. A common feature of ABC proteins is that
NBDs contain conserved Walker motifs that form nucleotide binding
pockets (16
17
18)
. Mutations in these domains cause
life-threatening diseases such as persistent hyperinsulinemic
hypoglycemia of infancy or Tangier disease, a disorder of lipid
metabolism (19
20
21)
. Mutations of key residues in SUR,
which preclude nucleotide binding and/or hydrolysis in other ABC
transporters (22
, 23)
, alter the responsiveness of
KATP channels to endogenous channel ligands, ATP
and ADP (24
, 25)
. These mutations also impede channel
activation by KATP channel opening drugs
(25
26
27)
. MgADP, interacting through NBDs, may stabilize
an ‘activated’ state of SUR associated with a reduced sensitivity of
the KATP channel complex to inhibition by ATP
(18
, 26
, 28)
. Although it has been proposed that this SUR
state could result from Mg ATP hydrolysis (18
, 26
, 28)
,
such a catalytic function for NBDs in KATP
channels has not been demonstrated.
We report that NBDs in SUR2A harbor an ATPase activity that determines the sensitivity of KATP channels to ATP and is modulated by potassium channel openers. This intrinsic property, reduced by mutations in Walker motifs, contributes to KATP channel activation by favoring catalytic conversion of nucleotides at the channel site. Assigning a catalytic activity to the sulfonylurea receptor subunit indicates that the cardiac KATP channel complex functions not only as a K+ conductance, but also as an enzyme regulating channel gating.
A preliminary account of this work has been published in abstract form
(29)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) NBD1
(Ser684-Ser884) and NBD2
(Gly1306-Thr1498) were
amplified by polymerase chain reaction from rat SUR2A cDNA
(15)
and cloned in-frame with the maltose binding protein
(MBP) coding sequence in pMal-c2 (New England Biolabs, Beverly, Mass.).
The identity of fusion constructs was confirmed by DNA sequencing. NBDs
were expressed in Escherichia coli TB1 induced with 0.1 mM
isopropyl-ß-D-thiogalactoside (IPTG) and fusion proteins were
purified by affinity chromatography on an amylose resin in (in mM) 200
NaCl, 1 EDTA, 0.2 PMSF and 20 Tris (pH 7.4). Mutated NBDs
(Lys1348 to alanine and/or
Asp1469 to asparagine) were constructed in
pMal-c2 by site-directed mutagenesis (QuickChange, Stratagene, San
Diego, Calif.). ATPase activity of NBDs (10 µg) was measured by
monitoring production of
[32P]Pi from 1 µCi
[
-32P]ATP (30)
in 34 mM KCl, 4
mM MgCl2, 50 mM HEPES (pH 7.4), and 4 mM ATP (4
h; 37°C). Using polyethylenimine cellulose thin-layer chromatography
plates (Sigma, St. Louis, Mo.), nucleotides were resolved by ascending
chromatography with 0.75 M
KH2PO4 and quantified with
a PhosphorImager and ImageQuant software (Molecular Dynamics,
Sunnyvale, Calif.). ATPase activity was confirmed by measuring ADP
formation using high-performance liquid chromatography (HPLC) and a
spectrophotometric coupled enzyme assay (31)
.
|
ATPase activity in KATP channel immunoprecipitates
KATP channels were immunoprecipitated from
guinea pig heart membranes with a Kir6.2 antibody (32)
.
Cardiac membranes (400 µg) were solubilized in an immunoprecipitation
buffer (IP in mM: 50 Tris-HCl, 150 NaCl, 5 EDTA, 50 NaF; pH 8.3) and
incubated with a Kir6.2 antibody (raised in rabbit against amino acids
N19–32C of rat Kir6.2). The resulting
antibody/KATP channel complex was precipitated
with protein A Sepharose. After washes in IP buffer with 1% Nonidet
P-40, 1 mM PMSF, 10 mg/ml leupeptin, and phosphate-buffered saline
(PBS), samples were centrifuged and resuspended in PBS buffer. The
amount of SUR2A protein was calculated assuming a density of 5
KATP channels/µm2 of
cardiac membrane (33)
and an immunoprecipitation
efficiency of 10% (34)
. To measure ATPase activity,
channel immunoprecipitates or corresponding controls were incubated
with 2 mM ATP and 2 mM MgCl2 for 20 h at
37°C while shaken at 170 rpm. The reaction was stopped by
HClO4 (2 mM), and kept on ice for 5 min. Proteins
were precipitated by centrifugation at 15,000 g (4°C, 5
min). Supernates were neutralized with 2 M
K2CO3; upon removal of
potassium perchlorate precipitate, adenine nucleotides were determined
by HPLC (31)
.
ATPase activity in cardiac membranes
Purified cardiac membranes were isolated as described
(35)
. Hearts from guinea pigs (0.2–0.3 kg), anesthetized
with pentobarbital (75 mg/kg), were homogenized in hypotonic buffer (in
mM: 10 HEPES, 1 EGTA, 1 DTT, 1 aprotinin, 0.2 phenylmethylsulfonyl
fluoride, and 1 µg/ml leupeptin; pH 7.4) and spun at 5000
g (4°C, 15 min). Supernatant was centrifuged at 100,000
g (4°C, 1 h) and membrane pellets were suspended by
sonication in (in mM) 20 HEPES (pH 7.4), 140 NaCl, 5 KCl, 2
MgCl2, 0.5 dithiothreitol, 1 aprotinin, 0.2
phenylmethylsulfonyl fluoride, and 2 µg/ml leupeptin. Sarcolemmal
fraction was purified by sucrose density gradient centrifugation, and
the degree of enrichment was determined based on Na, K-ATPase activity
(36)
. Nascent ADP produced by ATPase activity was detected
using a spectrophotometric coupled enzyme assay (31)
.
Reaction medium contained (in mM) 50 Tris-HCl (pH 7.5), 50 KCl, 2
MgCl2, 2 dithiothreitol, 2 phosphoenolpyruvate,
0.15 NADH, 0.2 ouabain, 10 levamisole, 10 U/ml pyruvate kinase, 10 U/ml
lactate dehydrogenase, and 10–20 µg membrane protein. Release of
inorganic phosphate (Pi), the other product of
ATPase activity, was measured by spectrophotometry using an EnzChek
Phosphate Assay kit (Molecular Probes, Eugene, Oreg.). Incubation
medium contained (in mM) 50 Tris-HCl (pH 7.5), 0.2 MESG substrate, 2
ATP, 2 MgCl2, 2 dithiothreitol, 0.2 ouabain, 10
levamisole, and 5 U/ml purine nucleoside phosphorylase.
Recording of cardiac KATP channels
Electrophysiological recordings were performed in ventricular
myocytes dissociated from guinea pig hearts (37)
. Pipettes
(
7–10 M
) were filled with (in mM) KCl 140,
CaCl2 1, MgCl2 1, HEPES-KOH
5 (pH 7.3). For the inside-out configuration, cells were superfused
with ‘internal solution’ (in mM): KCl 140,
MgCl2 1, EGTA 5, HEPES-KOH 5 (pH 7.3). For the
open cell-attached patch, ‘internal solution’ was supplemented with
glucose (1 g/l), malic acid (5 mM), and pyruvic acid (5 mM). After seal
formation, the open cell-attached configuration was obtained by
applying digitonin (8 µg/ml) through a second pipette (filled with 5
µg/ml propidium iodide and 0.5 µg/ml rhodamine). Solution flow was
visualized by rhodamine under ultraviolet light; staining the cell
nucleus with propidium iodide served as a criterion for sarcolemmal
permeabilization. For whole-cell recording, pipettes (5 M) were
filled with ‘internal solution’ plus 4 mM ATP, and cardiomyocytes
superfused with Tyrode (in mM): NaCl 136.5, KCl 5.4,
CaCl2 1.8, MgCl2 0.53,
glucose 5.5, HEPES-NaOH 5.5 (pH 7.4). Whole-cell currents were obtained
in response to 1 s rectangular pulses from a holding potential of
-50 mV to 0 mV. Channel activity was expressed as
NP0, where N represents the number of channels
and P0 the open channel probability.
Concentration-dependent relationships were expressed in relative terms
as NP0 values measured in the presence
vs. absence of a channel inhibitor and fitted with the Hill
equation (37)
. Single-channel analysis was performed as
described (37
, 38)
.
Recording of recombinant KATP channels
Kir6.2 (5)
and SUR2A (15)
, subcloned
into a pGEMHE vector (25)
, were amplified and transcribed
using the T7 mMessage mMachine kit (Ambion, Austin, Tex.). Mutagenesis
of Lys1348 to methionine or
Asp1469 to asparagine in SUR2A was done in the
pGEMHE-SUR2A plasmid (QuickChange, Stratagene). cRNAs coding Kir6.2 (2
ng) and SUR2A (6 ng) were coinjected into defoliculated Xenopus
laevis oocytes (25
, 39)
. Recombinant
KATP channel currents were subsequently recorded
in inside-out membrane patches (25
, 39)
using pipettes
(2–10 M) containing (in mM) 154 K+, 146
Cl-, 5 Mg2+, and 10
PIPES-KOH (pH 7.1). The cytoplasmic face of the patch was bathed in (in
mM) 174 K+, 40 Cl-, 1
Mg2+, 1 EGTA, 10 PIPES-KOH (pH 7.1), and
methanesulfonate-. Membrane potential was
maintained at -50 mV.
Statistical analysis
Results are expressed as mean ± SE;
n refers to the number of samples from different
preparations used in each analysis. Significant differences for
unpaired samples were assessed by Student’s t test.
Difference at P<0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. NBD1 and NBD2 of the SUR2A isoform were
engineered in-frame with the MBP to facilitate purification (Fig. 1A
). E. coli, transformed with respective
constructs and induced with IPTG, expressed a high yield of NBD1 and
NBD2 fusion proteins detected at corresponding molecular weights on
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE;
Fig. 1A
). NBDs purified by affinity chromatography were
exposed to -labeled [32P]ATP and ATPase
activity was measured by monitoring generation of
[32P]Pi (Fig. 1B
). The estimated ATPase activity was 9.1 ± 0.6 and
17.9 ± 1.0 nmol Pi/min/mg for NBD1
(n=5) and NBD2 (n=7) fusion constructs,
respectively (Fig. 1C
). These values were significantly
(P<0.01) different between each other and higher than
3.2 ± 0.9 nmol Pi/min/mg (n=7)
measured in samples containing MBP alone (Fig. 1C
). The
ATPase activity of NBD1 and NBD2 was further confirmed by following ADP
formation using HPLC and spectrophotometry (not illustrated). Either
approach consistently demonstrated a significantly higher ATPase
activity in NBD2 compared to NBD1 fusion proteins. The ATPase activity
in NBD-containing samples was insensitive to inhibitors of conventional
ATPases and phosphatases, ouabain (1 mM), oligomycin (10 µg/ml),
thapsigargin (0.1 mg/ml), and/or levamisole (10 mM). The ATPase
activity of NBDs was dependent on ATP concentration (Fig. 1D
). For the most active NBD2, the Michaelis-Menten plot
exhibited a Vmax of 35.4 nmol/min/mg
and an apparent Km of 4.4 mM (Fig. 1D
).
Removal of Mg2+, a cofactor in the ATPase
reaction, reduced the ATPase activity (Fig. 1E
).
Site-directed mutagenesis of the lysine residue (K1348A), in the
signature Walker A motif of NBD2, reduced ATPase activity (Fig. 1E
). This catalytic activity was further diminished in the
double NBD2 mutant in which the Walker A
lysine1348 moiety was mutated in combination with
the Walker B aspartate1469 (K1348A+D1469N). Thus,
nucleotide binding domains in the sulfonylurea receptor, primarily
NBD2, possess intrinsic ATPase activity.
ATPase activity of the KATP channel complex regulates
channel sensitivity to ATP
SUR2A associates with Kir6.2 to form cardiac
KATP channels (15
, 32)
. Accordingly,
a Kir6.2 antibody (11)
coimmunoprecipitates both Kir6.2
and SUR2A subunits from cardiac sarcolemma (Fig. 2A
, inset). In immunoprecipitates of
KATP channel proteins, ATPase activity was
assayed by HPLC (Fig. 2A
). Consistent with enzymatic
activity of KATP channels, immunoprecipitates of
the channel complex converted ATP into ADP, with an ATPase activity
estimated at 31 ± 8 nmol ADP/min/mg (n=7). Such ATPase
activity could catalyze local hydrolysis of ATP, and thereby promote
KATP channel opening. Indeed, 10 µM ATP failed
to inhibit native cardiac KATP channels
(40)
under conditions of unimpeded ATPase activity (Fig. 2B
). However, when the product of the ATPase reaction, ADP,
was continuously removed through creatine phosphate (CrP) -activated
creatine kinase (41)
, 10 µM ATP suppressed
KATP channel opening (Fig. 2B
). In
fact, in the presence of CrP, the IC50 for
channel inhibition was reduced by over threefold, from 25.1 ± 1.4
µM (n=9) to 7.4 ± 0.4 µM (n=4; Fig. 2C
). Moreover, mutations of the Walker A
lysine1348 and Walker B
aspartate1469, which reduce ATPase activity of
NBD2 (Fig. 1E
), produced recombinant
KATP channels with a higher sensitivity to ATP
(100 µM) compared to wild-type Kir6.2/SUR2A (Fig. 2D
).
Thus, the ATPase activity of KATP channels
regulates the channel responsiveness to ATP.
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Potassium channel opener-induced ATPase activity
Potassium channel openers bind to SUR and promote
KATP channel opening by reducing channel
sensitivity to ATP (39
, 42
43
44
45)
. In cardiac sarcolemma,
the structurally distinct potassium channel openers rilmakalim (10
µM), pinacidil (50 µM), cromakalim (100 µM), diazoxide (100
µM), and nicorandil (100 µM) promoted hydrolysis of ATP into ADP,
indicating activation of ATPase activity (Fig. 3A
). Depending on the potassium channel opener tested,
opener-induced ATPase activity ranged from 28 to 132 nmol ·
min-1 · mg protein-1
above the basal membrane ADP-generating capacity (Fig. 3A
).
Opener-induced ADP generation was associated with increased liberation
of inorganic phosphate, the other product in the ATPase reaction. In
the presence of a representative potassium channel opener, rilmakalim
(10 µM), the ATPase activity calculated by increased inorganic
phosphate (Pi) generation was 139 ± 8 nmol
Pi · min-1 · mg
protein-1 (n=6), a value similar to
that obtained from ADP-generation measurements (114±6 nmol ADP ·
min-1 · mg protein-1;
n=10). Opener-induced increase in ATPase activity required
Mg2+. However, it was not inhibited by ouabain
(200 µM), oligomycin (1 µg/ml), and/or levamisole (10 mM).
Corresponding rilmakalim-induced ATPase activities were 139 ± 10,
119 ± 8, and 118 ± 9 nmol · min-1 · mg
protein-1 with ouabain (n=4),
oligomycin (n=4) and levamisole (n=4) alone, and
111 ± 6 nmol · min-1 · mg protein-1
(n=4) in the presence of a mixture containing all three
conventional ATPase and phosphatase inhibitors. This suggests that
the effect of openers on ADP and Pi generation is
not due to activation of sarcolemmal Na,K-ATPase, mitochondrial
F1F0-ATPase, or alkaline phosphatase. In
contrast, 10 µM rilmakalim failed to promote ATPase activity in the
presence of 10 mM azide, an inhibitor of ATPase activity in purified
ABC proteins (46)
. The effect of openers on cardiac
membrane ATPase activity was concentration dependent; with rilmakalim,
maximal activation was observed between 10 and 20 µM (Fig. 3B
). Beyond this concentration the opener was less effective
(Fig. 3B
), and at 200 to 500 µM could inhibit membrane
ATPase activity by 15 to 35%. Within the concentration range from 0.1
to 100 µM, the bell-shaped dependence of rilmakalim-induced ATPase
activity closely correlated with the concentration dependence of
KATP channel activation (Fig. 3B
).
Mutations in SUR2A that suppressed ATPase activity (Fig. 1E
)
also reduced the ability of rilmakalim (10 µM) to activate
KATP channels inhibited by ATP (Fig. 3C
, D
). Rilmakalim vigorously activated wild-type
Kir6.2/SUR2A, and was four- to fivefold less effective after mutations
in Walker A lysine1348 and Walker B
aspartate1469, respectively (Fig. 3C
, D
). Thus, potassium channel openers modulate ATPase activity
and mutations that reduce such catalytic activity reduce
opener-mediated channel activation.
|
Openers mimic ADP-induced KATP channel behavior
At the single-channel level, the product of the ATPase
reaction, ADP, induces a readily recognizable pattern of
KATP channel transitions between open and closed
conformations (37)
. ADP (0.1 mM) had no effect on
intraburst transitions (C1
O; rates
k01 and k10), but slowed
burst closure (rate k02) and diminished lifetime
in long-lasting (C2 C3)
closed states (accelerated rates k20 and
k32, and reduced rate k23;
Fig. 4A
, B
). This pattern was associated with burst prolongation
(from 1.3 s in the absence to 3.9 s in the presence of ADP)
and shortened interburst events leading to increased open channel
probability (from 0.22 to 0.62; Fig. 4A
). Similarly to
MgADP, rilmakalim (10 µM) and pinacidil (50 µM) also did not affect
intraburst transitions (rates k01 and
k10; Fig. 4A
, B
). Like ADP,
both openers slowed k02 and
k23 while accelerating k20
and k32 (Fig. 4A
, B
). This
prolonged burst duration (from 0.17 to 0.46 s for rilmakalim and
from 0.05 to 0.17 s for pinacidil) shortened interburst events and
increased open channel probability (from 0.28 to 0.84 and from 0.11 to
0.54, respectively). Thus, openers and MgADP induce the same profile of
KATP channel conformational transitions
associated with channel activation.
|
Creatine kinase regulates opener-induced KATP channel
opening
Creatine kinase, which catalyzes ADP phosphorylation in the MgADP
+ CrP creatine + MgATP reaction, provides the major phosphotransfer
pathway in the heart (31)
. After sarcolemmal
permeabilization, which results in loss of cellular CrP and creatine
kinase-dependent ADP utilization, millimolar concentrations of ATP were
required to inhibit KATP channels (Fig. 5A
). Under such conditions, rilmakalim (10 µM; Fig. 5A
) and pinacidil (50 µM) readily reversed ATP-inhibited
channel activity. On average, KATP channel
activity in the presence of openers (NP0 =
4.9±0.6; n=35 and 4.5±0.9; n=15, respectively)
was similar to that obtained in the absence of ATP (4.9±0.4;
n=53). In fact, more than 10 mM ATP was required to abolish
rilmakalim (10 µM) or pinacidil (50 µM) -induced channel opening.
However, the effect of openers was abolished by supplying CrP (Fig. 5A
, B
). CrP antagonized the effect of distinct openers with
the same potency (Fig. 5C
), suggesting a common mechanism,
such as ADP removal, as responsible for inhibition of rilmakalim- and
pinacidil-induced channel activity. This effect of CrP was abolished by
0.1 mM 2–4-dinitrofluorobenzene (DNFB), an irreversible creatine
kinase inhibitor (Fig. 5D
, E
). Application of
exogenous creatine kinase (0.2 mg/ml) restored the ability of CrP to
suppress opener-induced KATP channel activity
(Fig. 5D
, E
). Thus, the efficacy of potassium
channel openers to activate KATP channels is
determined by the availability of a reaction system capable of
scavenging ADP, the product of the ATPase reaction.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In some ABC proteins, NBDs are known to contain ATPase activity
critical for protein function (47
48
49
50)
. The ATPase
intrinsic to ABC transporters has been proposed to serve as a switch
between ATP- and ADP-liganded conformations and the energy of ATP
hydrolysis implicated in supporting transport function and ion
conductance (17
, 51)
. The SUR subunit of the
KATP channel has been recognized for its role in
channel trafficking and biogenesis (52
, 53)
, as a receptor
for pharmacological modulators (39
, 43
, 44)
, and as a site
for nucleotide binding (12
, 13
, 18
, 28
, 54)
. The present
demonstration of ATPase activity in NBDs identifies a new property for
SUR, supporting previous suggestions that such catalytic activity could
serve as an underlying mechanism for the nucleotide regulation of
KATP channels (18
, 26
, 28
, 55)
.
The Vmax and Km for
the ATPase in NBDs of SUR2A were within the range of values reported
for ATPase activity of other ABC proteins (47
, 56)
. As in
other ABC transporters (57)
, the
KATP channel ATPase activity was insensitive
to inhibitors of F- or P-ATPase types, indicating that the SUR ATPase
is distinct from conventional ATPases. Nevertheless, it required
Mg2+, confirming the catalytic nature of ATP
hydrolysis. Although mutations in conserved Walker motifs did not
completely abolish ATPase activity, the rate of ATP hydrolysis was
significantly decreased, particularly for the double mutant
K1348A+D1469N where amino acids in both Walker A and B were
neutralized. Equivalent site-directed mutagenesis in Walker motifs of
other ABC transporters also reduce their respective ATPase activities
(47
, 58
, 59)
.
Suppression of ATPase activity by mutations in Walker motifs of SUR2A
increased the sensitivity of cardiac KATP
channels to ATP, as in the mutated SUR1 isoform (24)
.
Furthermore, neutralizing the product of ATP hydrolysis, by the
CrP/creatine kinase phosphotransfer system, revealed a threefold higher
KATP channel sensitivity to ATP
(60)
. Thus, the intrinsic ATPase activity of
KATP channels sets the apparent ATP-sensitivity
of the channel to a level lower than that expected in the absence of
ATP hydrolysis.
Potassium channel openers, which specifically bind to SUR (39
, 43
, 44)
, promoted ATPase activity. The concentration dependence
of opener-induced ATPase activity closely correlated with
opener-induced KATP channel activation. At higher
concentrations, potassium channel openers inhibited ATPase activity
while still producing KATP channel activation
(61)
. This apparent contradiction can be related to an
opener-induced stabilization of MgADP in the active ATPase site after
ATP hydrolysis (29)
. ADP trapping at the ATPase active
site observed with conventional ATPase inhibitors (48)
would keep the channel predominantly in the ADP-bound state, thereby
promoting channel opening. As openers mimicked MgADP-induced
KATP channel kinetic behavior, this would support
the proposed concept that activation of KATP
channels may be associated with ADP production and/or stabilization of
the ADP-bound state at the SUR subunit (26
, 28
, 55)
.
Mutations of conserved lysine to methionine (K1348M) and aspartate to
asparagine (D1469N) residues in Walker A and B motifs of NBD2, which
abolish channel activation by ADP (25)
, reduced
rilmakalim-induced channel opening. This may be associated with partial
inhibition of ATPase activity observed with such mutations or with the
possibility that openers could act through an alternative pathway,
including an ADP-independent mechanism. This is supported by the
observation that analogous mutations in the SUR1 isoform abolished the
effect of ADP, but only reduced the action of openers or metabolic
stress on channel activity (27)
.
Potassium channel opener-induced KATP channel
opening was inhibited by the CrP/creatine kinase system, which removes
ADP from the channel site. Under this condition, due to relief of
end-product inhibition, the ATPase reaction should proceed at an even
higher rate (41
, 62)
. Thus, the product of ATPase
catalysis, ADP, rather than ATP hydrolysis per se, appears
to be essential for channel activation. By scavenging the ATPase
product, creatine kinase would provide an efficient means of regulating
KATP channel behavior (41
, 60
, 63)
.
In the heart, creatine kinase is the major phosphotransfer system whose
flux is dramatically reduced under metabolic stress (31
, 62)
. Here, inhibition of creatine kinase promoted
KATP channel activation by openers. The reported
higher responsiveness of ischemic hearts to openers (64)
could be the consequence of reduced creatine kinase flux early in
ischemia, which would facilitate activation of
KATP channels and associated cardioprotective
processes (65)
. Thus, the balance between ATP hydrolysis,
through the opener-sensitive channel ATPase, and ADP removal, through
the creatine kinase system, provides an integral mechanism of
KATP channel regulation under different cellular
metabolic states.
It should be noted that removal of ADP by creatine kinase is associated
with ATP generation, which, if sufficient, could inhibit opener-induced
channel activation. Inhibition of opener-induced
KATP channel activity would require 10 mM of
ATP. Under our experimental conditions, such elevation of ATP is
unlikely since the source for ATP regeneration is ADP resulting from
intracellular ATP hydrolysis, and therefore the overall concentration
of synthesized ATP, cannot far exceed 1 mM of ATP applied in the bath
solution. Although activation of an ATP-regenerating system, which
removes ADP, may reduce opener binding to the cardiac SUR2A isoform
(66)
, this has not been observed at concentrations of
nucleotides used in our experiments. Thus, loss of opener-induced
channel activation in the presence of an ADP-scavenging system is
apparently not due to an increase in the local ATP concentration or
reduction of opener binding.
In summary, this study demonstrates that the cardiac KATP channel complex possesses an ATPase activity found in NBDs of the SUR2A subunit. Such intrinsic enzymatic activity defines KATP channels not only as passive targets responding to alterations in the cellular metabolic status, but also as active contributors to their nucleotide-dependent gating. Therefore, modulation of the channel ATPase activity and/or of metabolic systems that scavenge the product of the ATPase reaction provides a novel means of regulating vital cellular functions associated with KATP channel opening.
|
ACKNOWLEDGMENTS |
|---|
Received for publication January 12, 2000.
Revision received March 22, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 B. Ng, Y. Kang, H. Xie, H. Sun, and H. Y. Gaisano Syntaxin-1A inhibition of P-1075, cromakalim, and diazoxide actions on mouse cardiac ATP-sensitive potassium channel Cardiovasc Res, December 1, 2008; 80(3): 365 - 374. [Abstract] [Full Text] [PDF]
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 R. Masia and C. G. Nichols Functional Clustering of Mutations in the Dimer Interface of the Nucleotide Binding Folds of the Sulfonylurea Receptor J. Biol. Chem., October 31, 2008; 283(44): 30322 - 30329. [Abstract] [Full Text] [PDF]
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 D. Tricarico, A. Mele, G. M. Camerino, A. Laghezza, G. Carbonara, G. Fracchiolla, P. Tortorella, F. Loiodice, and D. C. Camerino Molecular Determinants for the Activating/Blocking Actions of the 2H-1,4-Benzoxazine Derivatives, a Class of Potassium Channel Modulators Targeting the Skeletal Muscle KATP Channels Mol. Pharmacol., July 1, 2008; 74(1): 50 - 58. [Abstract] [Full Text] [PDF]
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 A. I. Tarasov, T. J. Nicolson, J.-P. Riveline, T. K. Taneja, S. A. Baldwin, J. M. Baldwin, G. Charpentier, J.-F. Gautier, P. Froguel, M. Vaxillaire, et al. A Rare Mutation in ABCC8/SUR1 Leading to Altered ATP-Sensitive K+ Channel Activity and {beta}-Cell Glucose Sensing Is Associated With Type 2 Diabetes in Adults Diabetes, June 1, 2008; 57(6): 1595 - 1604. [Abstract] [Full Text] [PDF]
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Y. Shi, X. Chen, Z. Wu, W. Shi, Y. Yang, N. Cui, C. Jiang, and R. W. Harrison cAMP-dependent Protein Kinase Phosphorylation Produces Interdomain Movement in SUR2B Leading to Activation of the Vascular KATP Channel J. Biol. Chem., March 21, 2008; 283(12): 7523 - 7530. [Abstract] [Full Text] [PDF]
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 A. B. Karger, S. Park, S. Reyes, M. Bienengraeber, R. B. Dyer, A. Terzic, and A. E. Alekseev Role for SUR2A ED Domain in Allosteric Coupling within the KATP Channel Complex J. Gen. Physiol., February 25, 2008; 131(3): 185 - 196. [Abstract] [Full Text] [PDF]
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 L. V. Zingman, A. E. Alekseev, D. M. Hodgson-Zingman, and A. Terzic ATP-sensitive potassium channels: metabolic sensing and cardioprotection J Appl Physiol, November 1, 2007; 103(5): 1888 - 1893. [Abstract] [Full Text] [PDF]
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B. Ng, Y. Kang, C. L. Elias, Y. He, H. Xie, J. B. Hansen, P. Wahl, and H. Y. Gaisano The Actions of a Novel Potent Islet {beta}-Cell Specific ATP-Sensitive K+ Channel Opener Can Be Modulated by Syntaxin-1A Acting on Sulfonylurea Receptor 1 Diabetes, August 1, 2007; 56(8): 2124 - 2134. [Abstract] [Full Text] [PDF]
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 S. Yamada, G. C. Kane, A. Behfar, X.-K. Liu, R. B. Dyer, R. S. Faustino, T. Miki, S. Seino, and A. Terzic Protection conferred by myocardial ATP-sensitive K+ channels in pressure overload-induced congestive heart failure revealed in KCNJ11 Kir6.2-null mutant J. Physiol., December 15, 2006; 577(3): 1053 - 1065. [Abstract] [Full Text] [PDF]
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 G. C. Kane, A. Behfar, R. B. Dyer, D. F. O'Cochlain, X.-K. Liu, D. M. Hodgson, S. Reyes, T. Miki, S. Seino, and A. Terzic KCNJ11 gene knockout of the Kir6.2 KATP channel causes maladaptive remodeling and heart failure in hypertension Hum. Mol. Genet., August 1, 2006; 15(15): 2285 - 2297. [Abstract] [Full Text] [PDF]
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 T. Jons, D. Wittschieber, A. Beyer, C. Meier, A. Brune, A. Thomzig, G. Ahnert-Hilger, and R. W. Veh K+-ATP-channel-related protein complexes: potential transducers in the regulation of epithelial tight junction permeability J. Cell Sci., August 1, 2006; 119(15): 3087 - 3097. [Abstract] [Full Text] [PDF]
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 M. W. Bowler, M. G. Montgomery, A. G. W. Leslie, and J. E. Walker How azide inhibits ATP hydrolysis by the F-ATPases PNAS, June 6, 2006; 103(23): 8646 - 8649. [Abstract] [Full Text] [PDF]
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C. Moreau, F. Gally, H. Jacquet-Bouix, and M. Vivaudou The Size of a Single Residue of the Sulfonylurea Receptor Dictates the Effectiveness of KATP Channel Openers Mol. Pharmacol., April 1, 2005; 67(4): 1026 - 1033. [Abstract] [Full Text] [PDF]
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G. C. Kane, A. Behfar, S. Yamada, C. Perez-Terzic, F. O'Cochlain, S. Reyes, P. P. Dzeja, T. Miki, S. Seino, and A. Terzic ATP-Sensitive K+ Channel Knockout Compromises the Metabolic Benefit of Exercise Training, Resulting in Cardiac Deficits Diabetes, December 1, 2004; 53(suppl_3): S169 - S175. [Abstract] [Full Text] [PDF]
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M. Yamada and Y. Kurachi The Nucleotide-Binding Domains of Sulfonylurea Receptor 2A and 2B Play Different Functional Roles in Nicorandil-Induced Activation of ATP-Sensitive K+ Channels Mol. Pharmacol., May 1, 2004; 65(5): 1198 - 1207. [Abstract] [Full Text]
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 P. P. Dzeja, P. Bast, C. Ozcan, A. Valverde, E. L. Holmuhamedov, D. G. L. Van Wylen, and A. Terzic Targeting nucleotide-requiring enzymes: implications for diazoxide-induced cardioprotection Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1048 - H1056. [Abstract] [Full Text] [PDF]
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 M. Chachin, M. Yamada, A. Fujita, T. Matsuoka, K. Matsushita, and Y. Kurachi Nateglinide, a D-Phenylalanine Derivative Lacking Either a Sulfonylurea or Benzamido Moiety, Specifically Inhibits Pancreatic beta -Cell-Type KATP Channels J. Pharmacol. Exp. Ther., March 1, 2003; 304(3): 1025 - 1032. [Abstract] [Full Text] [PDF]
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U. Lange, C. Loffler-Walz, H. C. Englert, A. Hambrock, U. Russ, and U. Quast The Stereoenantiomers of a Pinacidil Analog Open or Close Cloned ATP-sensitive K+ Channels J. Biol. Chem., October 18, 2002; 277(43): 40196 - 40205. [Abstract] [Full Text] [PDF]
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M. Gopalakrishnan, S. A. Buckner, K. L. Whiteaker, C.-C. Shieh, E. J. Molinari, I. Milicic, A. V. Daza, R. Davis-Taber, V. E. Scott, D. Sellers, et al. (-)-(9S)-9-(3-Bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-8(4H)-one 1,1-Dioxide (A-278637): A Novel ATP-Sensitive Potassium Channel Opener Efficacious in Suppressing Urinary Bladder Contractions. I. In Vitro Characterization J. Pharmacol. Exp. Ther., October 1, 2002; 303(1): 379 - 386. [Abstract] [Full Text] [PDF]
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L. V. Zingman, D. M. Hodgson, P. H. Bast, G. C. Kane, C. Perez-Terzic, R. J. Gumina, D. Pucar, M. Bienengraeber, P. P. Dzeja, T. Miki, et al. Kir6.2 is required for adaptation to stress PNAS, October 1, 2002; 99(20): 13278 - 13283. [Abstract] [Full Text] [PDF]
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 C. G. Nichols and J. C. Koster Diabetes and insulin secretion: whither KATP? Am J Physiol Endocrinol Metab, September 1, 2002; 283(3): E403 - E412. [Abstract] [Full Text] [PDF]
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R. Weiss, M. Mevissen, D. S. Hauser, G. Scholtysik, C. J. Portier, B. Walter, U. E. Studer, and H. Danuser Inhibition of Human and Pig Ureter Motility in Vitro and in Vivo by the K+ Channel Openers PKF 217-744b and Nicorandil J. Pharmacol. Exp. Ther., August 1, 2002; 302(2): 651 - 658. [Abstract] [Full Text] [PDF]
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H. Huopio, S.-L. Shyng, T. Otonkoski, and C. G. Nichols KATP channels and insulin secretion disorders Am J Physiol Endocrinol Metab, August 1, 2002; 283(2): E207 - E216. [Abstract] [Full Text] [PDF]
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M. R. Abraham, V. A. Selivanov, D. M. Hodgson, D. Pucar, L. V. Zingman, B. Wieringa, P. P. Dzeja, A. E. Alekseev, and A. Terzic Coupling of Cell Energetics with Membrane Metabolic Sensing. INTEGRATIVE SIGNALING THROUGH CREATINE KINASE PHOSPHOTRANSFER DISRUPTED BY M-CK GENE KNOCK-OUT J. Biol. Chem., June 28, 2002; 277(27): 24427 - 24434. [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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G. Taschenberger, A. Mougey, S. Shen, L. B. Lester, S. LaFranchi, and S.-L. Shyng Identification of a Familial Hyperinsulinism-causing Mutation in the Sulfonylurea Receptor 1 That Prevents Normal Trafficking and Function of KATP Channels J. Biol. Chem., May 3, 2002; 277(19): 17139 - 17146. [Abstract] [Full Text] [PDF]
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L. V. Zingman, D. M. Hodgson, M. Bienengraeber, A. B. Karger, E. C. Kathmann, A. E. Alekseev, and A. Terzic Tandem Function of Nucleotide Binding Domains Confers Competence to Sulfonylurea Receptor in Gating ATP-sensitive K+ Channels J. Biol. Chem., April 12, 2002; 277(16): 14206 - 14210. [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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L. Li, X. Geng, and P. Drain Open State Destabilization by Atp Occupancy Is Mechanism Speeding Burst Exit Underlying KATP Channel Inhibition by Atp J. Gen. Physiol., January 1, 2002; 119(1): 105 - 116. [Abstract] [Full Text] [PDF]
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S. A. John, J. N. Weiss, and B. Ribalet Regulation of Cloned Atp-Sensitive K Channels by Adenine Nucleotides and Sulfonylureas: Interactions between Sur1 and Positively Charged Domains on Kir6.2 J. Gen. Physiol., October 1, 2001; 118(4): 391 - 406. [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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E. A. Cartier, L. R. Conti, C. A. Vandenberg, and S.-L. Shyng Defective trafficking and function of KATP channels caused by a sulfonylurea receptor 1 mutation associated with persistent hyperinsulinemic hypoglycemia of infancy PNAS, February 27, 2001; 98(5): 2882 - 2887. [Abstract] [Full Text] [PDF]
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D. Pucar, E. Janssen, P. P. Dzeja, N. Juranic, S. Macura, B. Wieringa, and A. Terzic Compromised Energetics in the Adenylate Kinase AK1 Gene Knockout Heart under Metabolic Stress J. Biol. Chem., December 22, 2000; 275(52): 41424 - 41429. [Abstract] [Full Text] [PDF]
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D. Pucar, P. P. Dzeja, P. Bast, N. Juranic, S. Macura, and A. Terzic Cellular Energetics in the Preconditioned State. PROTECTIVE ROLE FOR PHOSPHOTRANSFER REACTIONS CAPTURED BY 18O-ASSISTED 31P NMR J. Biol. Chem., November 21, 2001; 276(48): 44812 - 44819. [Abstract] [Full Text] [PDF]
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A. J. Carrasco, P. P. Dzeja, A. E. Alekseev, D. Pucar, L. V. Zingman, M. R. Abraham, D. Hodgson, M. Bienengraeber, M. Puceat, E. Janssen, et al. Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels PNAS, June 19, 2001; 98(13): 7623 - 7628. [Abstract] [Full Text] [PDF]
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) or MBP-NBD2 (
).
Fusion proteins with NBD1 or NBD2 were purified (purif.) from lysates
by affinity chromatography using an amylose resin. Purified MBP alone
migrated at the expected size (43,000 kDa). B) ATPase
activity in NBD1 and NBD2. The generation of
[32P]Pi from [
