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(Received for publication, December 3, 1996, and in revised form, December 27, 1996)
From the Department of Medicine, University of Wisconsin, Madison,
Wisconsin 53792 and University of Chicago, Chicago, Illinois 60637
ABSTRACT The ATP-sensitive potassium channel (KATP)
controls insulin release in pancreatic INTRODUCTION The ATP-sensitive potassium channel
(KATP)1 is a highly regulated channel type
important in the physiology and pathophysiology of pancreas, heart,
vascular smooth muscle, and perhaps other tissues as well (1-5). KATP
activity can be reconstituted by co-expression of an inwardly
rectifying channel (Kir6.2) (6, 7), and a sulfonylurea receptor (SUR)
(8, 9). Major regulatory mechanisms are postulated to reside in the
nucleotide binding domains on SUR (8-10). Activation of KATP generally
requires a reduced ATP concentration. This regulation, including
inhibition of KATP activity by other nucleotides, does not require
phosphorylation. Activation of KATP, however, also depends on another
less well characterized regulatory process, because KATP gradually
inactivates when the integrity of the cell is disrupted by, for
example, the excised patch method of voltage clamp (11-13). This
property is shared not only by most KATP channels from different
tissues, but also by other channels of the inward rectifier potassium
(Kir) superfamily (for reviews, see Refs. 14 and 15). Despite intense investigation, the mechanisms for maintaining activation of KATP have
not been fully identified and characterized, although previous work has
focused on protein phosphorylation processes on the channel or related
subunit (for a review, see Ref. 16). We found that anionic
phospholipids, especially phosphoinositides, are necessary and
sufficient to activate and maintain KATP activity in native and cloned
KATP and in two other Kir channels. We also propose and test a novel
hypothesis involving the cytoplasmic tail of the pore-forming subunit
to account for this regulation. Modulation of channel activity through
the composition and phosphorylation of membrane phospholipids may
represent an important regulation for KATP in particular, and for
channels in the Kir superfamily in general.
EXPERIMENTAL PROCEDURES Single pancreatic The pipette solution (extracellular
side, 140-K+-pipette) contained (in mM) KCl
(140.0), CaCl2 (1.8), MgCl2 (0.5), HEPES (5.0), and glucose (5.5), pH 7.4. The pipette solution for membrane vesicles from skeletal muscle cells and cloned KATP (SUR/Kir6.2) contained (in
mM) NaCl (140.0), CaCl2 (1.8),
MgCl2 (0.5), KCl (10.0), HEPES (5.0), and glucose (5.5).
The bath solution (cytoplasmic side, 140-K+-bath) contained
(in mM) KCl (142.0), HEPES (5.0), glucose (5.5), and EGTA
(2.0), pH 7.4. EGTA was included unless indicated otherwise. For whole
cell clamp experiments, the pipette solution (140-K+ with
Mg-ATP) contained (in mM) KCl (140.0), EGTA (2.0), HEPES (5.0), glucose (5.5), and Mg-ATP (5.0), pH 7.3. The bath solution (5.4-Tyrode solution) contained (in mM) KCl (5.4), NaCl
(137.0), CaCl2 (1.8), MgCl2 (0.5), and HEPES
(5.0), and the pH was adjusted to 7.4. K2-ATP
(Sigma) was dissolved into the bath solution just before use, and the pH of the solution was readjusted. DNase I and
cytochalasin B were products of Sigma. Polyvalent
cations were prepared in stock solutions and added to the solution
prior to use. The concentration of free cations was calculated (19). A
2-ml bath was perfused with a solution of dispersed lipids at concentrations of 0.005-1 mg/ml. PPIs (a mixture of
PI(4,5)P2, PI(4)P (15-20%), PI, PS, and PC, from bovine
brain; Sigma) were used. Other lipids were as follows:
PI(4,5)P2 (Boehringer Mannheim), PI(4)P (sodium salt, from
bovine brain; Sigma), PI (from bovine liver; Avanti
Polar Lipids), PS (from brain; Avanti Polar Lipids), PC, PE (from
bovine heart; Avanti Polar Lipids), IP3 (potassium salt,
from bovine brain; Sigma), DOG (8:0 DG), and DPG (16:0
DG; Sigma). Anionic phospholipids were dispersed in
the solution with 30-min sonication (half setting) on ice. The
following uncharged lipids were dissolved first in
dimethylsulfoxide and then sonicated for 30 min: PLC-PI (from
Bacillus cereus; Sigma), PLC (Type IV, from
Bacillus cereus; Sigma), PLA2
(Sigma), and PLD (Sigma). All enzymes
were dissolved directly into reaction solution. Experiments were done
at room temperature (21-24 °C).
The wild type
recombinant complementary DNAs encoding the rat SUR (rSUR), and rat
Kir6.2 (rKir6.2) were cloned from a cDNA library of insulinoma beta
cell tumor line, and a rat islet cDNA library (7), respectively. A
polymerase chain reaction-based site-directed mutagenesis kit (ExSite,
Stratagene) was used to generate the site-directed point mutations,
which were confirmed by sequencing. The cDNA of SUR (in a
pcDNA3 vector, Invitrogen) and wild type Kir6.2 or its mutants (in
a pCR3 vector, Invitrogen) were co-transfected and heterologously
expressed in mammalian cells either transiently (tsA201 cell line (20))
or stably (a human embryonic kidney cell line, HEK) using a
calcium-phosphatide transfection kit (Life Technologies, Inc.). The
transfected cells were cultured in Dulbecco's modified Eagle's medium
for 12 h with the precipitated cDNA. The transiently
transfected cells were kept in culture for another 24-48 h before
being used in electrophysiological experiments. Stably transfected
cells were selected by growth in media containing 400-600 µg/ml
active G418 (Life Technologies, Inc.) for at least 1 week, and single
colonies surviving were isolated and grown to harvest in the continuous
pressure of selection with 200-300 µg/ml active G418.
Currents
were recorded using inside-out patch clamp (cytoplasmic membrane
exposed to the bath) or whole cell clamp (external membrane exposed to
bath) through a patch clamp amplifier (Axopatch 1-D or Axopatch 200A,
Axon Instruments) and filtered through a built-in low-pass filter at 1 kHz, unless indicated otherwise. Leak current was compensated
electronically on-line or subtracted off-line. Data were acquired by
digitizing at 2 kHz and analyzed by a pClamp 6.0 (Axon Instruments) and
graphic plotting software running on a PC-compatible computer (Gateway
2000).
RESULTS Currents of native KATPs were recorded from inside-out patches of
pancreatic
The preparation of PPIs was a mixture of PI(4,5)P2, PI(4)P,
PI, PS, and PC; to evaluate the contribution of the individual components to the activation effect, we individually tested purified PI(4,5)P2 (5 negative charges), PI(4)P (3 negative
charges), PI (1 negative charge), PS (1 negative charge), PC (neutral),
and another neutral phospholipid, PE, using the same
protocol. All negatively charged phospholipids tested
activated KATP; Fig. 3A shows an example for
PI(4,5)P2, and results for all lipids tested are summarized
in Fig. 3D. Similar to PPIs, the activation effect also
remained after washing the lipids from the bath solution. The most
effective anionic phospholipid was PI(4,5)P2, and others were less effective. The effectiveness sequence correlated with the
number of negative charges in the head groups, suggesting a role for
electrostatic force. Uncharged PC (1 mg/ml) had no effect on KATP
activity, while uncharged PE (1 mg/ml) induced a slight depression of
KATP activity. In intact cells, phospholipase C hydrolyzes
phospholipids such as PI(4,5)P2 into the cascade products,
soluble IP3 and membrane-delimited diacylglycerol, which as
important second messengers, may affect channel function. Although IP3 has 6 negative charges/molecule, it had no effect on
KATP activity (0.1 mg/ml) (Fig. 3B). The diacylglycerols
(DPG and DOG) actually reduced activity of KATP (results summarized in
Fig. 3D). This indicates that an intact unit of a
hydrophobic tail and a charged hydrophilic head are a basic structural
requirement for the anionic phospholipids to activate KATPs. Other
cascade products of phospholipids, arachidonates, had inconsistent
effects on KATPs in cardiac cells (21) and insulinoma cells (22). The
effect of intact phospholipids to activate KATP was also tested by
using phospholipase-treated lipids (Fig. 3C). PLC-PI, PLC, or PLA2 was incubated with PI(4,5)P2, PI(4)P,
and PS. The enzyme-treated lipids were much less potent than the
untreated lipids (Fig. 3E). Interestingly, PLD treatment of
PC increased KATP activity (Fig. 3E). PLD adds an anion to
the head group of neutral PC, making this observation consistent with
the effectiveness sequence depending upon net negative charge. Finally,
exogenous PLA2 decreased KATP activity in inside-out
patches, consistent with a recent report (23). This effect of
PLA2 was reversed by subsequent application of PPIs.
We further tested the hypothesis that the negative charges in the
anionic heads of the anionic phospholipids are the functional group
critical to the activation of KATP by screening those charges with
polyvalent cations. As an example (Fig. 4A),
the polyvalent cation antibiotic neomycin decreased KATP activity, and
PPIs could reverse and antagonize this effect. Divalent cations such as
Ca2+ and Mg2+ are already known to accelerate
the decrease of KATP activity (24, 25) by an unknown mechanism. We
found that trivalent cations such as Gd3+ and
La3+ (ED50 = 70 nM and 0.3 µM, respectively; ED50 defined as 50%
inhibition at 1 min) were even more potent inhibitors of KATPs than
divalent cations (Fig. 4B). Other polyvalent cations
including aminoglycoside antibiotics and polyamines used as negative
charge chelators for phospholipids also have the similar effect.
Neomycin (ED50 = 20 µM), gentamicin
(ED50 = 60 µM), and spermine
(ED50 = 0.94 mM) inhibited KATP activity
irreversibly (Fig. 4B). A decreased potency for the bigger
molecules may reflect the influence of the molecular sizes in chelating
negative charges of the phospholipids for these polyvalent cations.
Application of exogenous PPIs activated KATPs previously inhibited by
polyvalent cations, and application of polyvalent cations with PPIs
reduced the effect of PPIs.
The effects of phospholipids on KATP were rapid, sustained, and general
to all anionic phospholipids and all types of KATPs tested. The
mechanism for the effect of these lipids appeared less likely to
involve a very specific or complex cellular process requiring many
steps. We hypothesized that PPIs directly interact with KATPs or one of
its subunits, mediated by electrostatic force, to maintain the channel
in a functional state. We noted a segment of Kir6.2 composed of a high
concentration of positively charged residues at the beginning of the
cytoplasmic C terminus (Fig. 5A). This
positioning of a cluster of positive charges has been shown to be an
important determinant of protein topology in other membrane proteins
(26, 27). To test our hypothesis, two mutants were made in this region:
1) adjacent positively arginine residues at positions 176 and 177 were
mutated to neutral alanines (R176A,R177A), and 2) the arginine at 176 was mutated to the negatively charged glutamic acid (R176E). Wild type
Kir6.2 co-transfected with SUR expressed a peak KATP current of
1.59 ± 0.3 nA (n = 8) recorded by whole cell
clamp at 0 mV with no ATP in the pipette. The current was blocked by
glibenclamide and also exhibited the usual loss of activity with time.
The R176A,R177A mutant co-transfected with rSUR also expressed a
glibenclamide-sensitive K+ current, but the current was
much smaller, with a mean value of 0.32 ± 0.07 nA
(n = 4, p = 0.008, compared with wild
type) at 0 mV at maximum. Loss of channel activity was much faster than wild type, and the current showed more fluctuation. Examples of whole
cell wild type and R176A,R177A currents are shown in Fig. 6, panels A and B, respectively.
In inside-out patches, exogenous application of PPIs also activated the
channels expressed from the R176A, R177A mutant, but activation was
less effective than for wild type, as demonstrated by a lower open
probability and by the transient nature of the activation. Single
channel recordings (Fig. 7A) from excised
patches showed that the lower open probability in the mutant was caused
by a decrease in open times (Fig. 7B) with a possible
increase in the closed times. No glibenclamide-sensitive current could
be recorded from the cells co-transfected with the R176E mutant and
rSUR, suggesting that either this mutant did not express or that the
mutation rendered it incapable of opening.
DISCUSSION Gradual inactivation of KATP
with time was recognized with the first description of KATP (1) using
the inside-out patch clamp technique, a method that severely disrupts
the cytoplasmic environment. This inactivation, sometimes called
run-down, has been reported or addressed in nearly all research on
KATP. Loss of activity is a consistent feature of all native and cloned
KATP (Fig. 1, and Refs. 11-13 and 16). Despite much study, the
mechanism required to maintain KATP activity is still unclear. Mg-ATP
(28, 29) was found to transiently and partially activate and maintain KATP in cardiac, pancreatic, and skeletal muscle cells. Some anions (F Activation of KATP in cardiac cells by
PI(4,5)P2 and Mg-ATP/PI has been recently reported (33,
34). All negatively charged intact phospholipids we tested possess the
ability to activate KATP, including PS, PI, PI(4)P, and
PI(4,5)P2, directly, and in the absence of magnesium and
ATP. Mg-ATP and PI, therefore, are not critical requirements to
activate the channel. Although Mg-ATP is not required to mediate
activation by anionic phospholipids, the anionic lipid hypothesis can
account for the well known Mg-ATP activation effect on KATPs. It has
previously been reported that nonphosphorylating analogs of ATP,
AMP-PNP and AMP-PCP, are incapable of increasing KATP activity,
suggesting that Mg-ATP acts as a phosphoryl group donor in
kinase-mediated phosphorylation reaction, although such kinases have
not yet been identified (29). Our effectiveness sequence of
PI(4,5)P2 > PI(4)P > PI suggests the possibility that in vivo, Mg-ATP potentiates the activity of
KATPs by serving as a substrate for the phosphoinositide kinases (35) and aminophospholipid translocases that maintain sufficient
membrane concentrations of PPIs to maintain KATP by increasing net
negative membrane charge. PI(4,5)2 and phosphorylation of
PI may perhaps be the important physiological regulators of KATP
activity in this mechanism, a subject that requires further study.
Do
anionic phospholipids activate KATPs through an intermediate structure
or process or through a direct structural interaction with the channel?
Some components of PPIs, especially PI(4,5)P2, are known to
directly regulate various cellular proteins. One of the most important
proteins interacting with the anionic phospholipids is protein kinase
C, which uses Ca2+ as part of its signaling pathway. In our
cell-free patch clamp experiments, however, ATP was not included in the
perfusion solution to the cytoplasmic side of the membrane, and
Ca2+ was chelated by EGTA to less than 5 × 10 The apparent importance of electrostatic interactions suggested by our
data lead us to propose a molecular/physical model involving the
channel protein (Fig. 5B). Kir channels that are not thought
to associate with SUR (mIRK1, but hROMK1) were also activated by
anionic phospholipids. This caused us to focus on the channel structure
(Kir6.2) rather than SUR for the mechanisms of the effect. In our
model, the positively charged residuals at the beginning of the C
terminus of Kir6.2 are anchored or tethered by the electrostatic force
of the anionic phospholipid head groups at the cytoplasmic face of the
membrane. A decrease in the concentration of anionic phospholipids
causes a release or conformation change of the tethered portion of the
C terminus, triggering the formation of a gate that closes off the
inner vestibule of the channel pore. This model is highly speculative,
but protein-membrane electrostatic interactions at this portion of
membrane proteins have been proposed previously (26, 27). The model
also has value because it can account for the known experimental data
such as the inhibitory effects of pH and di- and trivalent ions (by
charge screening or neutralization), the anionic charge effectiveness
sequence, and suggests possible explanations for such effects as
trypsin (cleaving the gate) and Mg-ATP (increasing net negative
membrane charge by phosphorylation of membrane phospholipids). This
model was tested by the Kir6.2 mutant R176A,R177A, which reduced
positive charge on the cytoplasmic tail. As the model predicted, the
mutant channel activity was reduced and less sensitive to PPIs.
How widely might this protein-lipid interaction mechanism apply to ion
channel activity in general? To date, KATP has been cloned from
pancreatic cells (6, 7) and cardiac cells (9, 37); both require a
sulfonylurea receptor and Kir6.2 to function. Kir6.2 is one member of
the Kir family with functional and structural similarities, including a
concentration of positive charges at the beginning section of the C
terminus (Fig. 5A). Loss of channel activity has been
generally found in most members of the Kir family, in both native
channels (Refs. 38-40, for example) and expressed channels (41, 42).
Our demonstration that PPIs activated native non-KATP inward rectifier
(Fig. 1B) and also two cloned non-KATP inward rectifiers
(Fig. 2) indicates that this mechanism may be general to all channels
of this family. The higher effectiveness for more highly charged
phospholipids suggests a role for lipid phosphorylation in this
regulation. Thus, the PPIs, especially PI(4,5)P2 and
phosphatidylinositol 3,4,5-triphosphate, along with enzymes maintaining
them such as phosphoinositide kinases (35), may be important cellular
regulators by maintaining a channel protein-anionic lipid interaction
required to activate channels.
FOOTNOTES Acknowledgment We thank Dr. Y. Tokuyama for providing the rat
pancreatic REFERENCES
Volume 272, Number 9,
Issue of February 28, 1997
pp. 5388-5395
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
and
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES
-cells and also modulates
important functions in other cell types. In this study we report that
anionic phospholipids activated KATP in pancreatic -cells, cardiac
myocytes, skeletal muscle cells, and a cloned KATP composed of two
subunits (SUR/Kir6.2) stably expressed in a mammalian cell line. The
effectiveness was proportional to the number of negative charges on the
head group of the anionic phospholipid. Screening negative charges with
polyvalent cations antagonized the effect. Enzymatic treatment with
phospholipases that reduced charge on the lipids also reduced or
eliminated the effect. These results suggest that intact phospholipids
with negative charges are the critical requirement for activation of
KATP, in distinction from the usual cell signaling pathway through
phospholipids that requires cleavage. Mutations of two positively
charged amino acid residues at the C terminus of Kir6.2 accelerated
loss of channel activity and reduced the activating effects of
phospholipids, suggesting involvement of this region in the activation.
Metabolism of anionic phospholipids in plasmalemmal membrane may be a
novel and general mechanism for regulation of KATP and perhaps other ion channels in the family of inward rectifiers.
-cells were
enzymatically (0.05% trypsin) dispersed from isolated islets of Wistar
rats collected by centrifugation through a discontinuous gradient of
Ficoll (17). Single ventricular myocytes were isolated from rabbit
hearts by an enzymatic (0.1% collagenase, Williton) dissociation
method (17, 18). Single skeletal muscle cells were dissociated
enzymatically (0.3% collagenase, Williton) from the flexor digitorum
brevis muscles of the hind legs of Wistar rats (13). Isolated single
skeletal muscle cells were suspended in Dulbecco's modified Eagle's
medium until they were used. Membrane vesicles were formed by washing
the isolated cells with high K+ solution.
-cells (Fig. 1A), cardiac
ventricular myocytes (Fig. 1B), and sarcomeric membrane
vesicles from skeletal muscle cells (Fig. 1C). KATP current
expressed in HEK cells stably transfected by cloned cDNA of rSUR
and rKir6.2 was also studied (Fig. 1D). Open channel
activity was increased within 1-2 s to a maximal value after excising
the patch membrane into an ATP-free solution; this was followed by
decreased channel activity for pancreatic cells, cardiac cells, and
transfected HEK cells. In the vesicles from skeletal muscle cells, the
patch was initially devoid of KATP activity in most cases (Fig.
1C), presumably because these channels had inactivated
during the vesicle formation process. An anionic phospholipid-rich
tissue extraction containing PPIs (0.05-1 mg/ml) consistently
activated KATP in cells from all three tissues and also the cloned
KATP. Maximal effects were reached within 30 s to 2 min and did
not change with time if the perfusion of the lipid-containing solution
was maintained. After wash-out of PPIs from the bath solution, the
activation effect remained for a time but diminished slowly with a time
course that depended upon the previous concentration of PPIs. PPIs had
no effects on single channel current amplitudes or ATP sensitivity. A
non-KATP inwardly rectifying K+ channel, presumably the
cardiac inward rectifier, or IRK1, noted coincidentally in Fig.
1B (small conductance channel observed before excision) also
lost activity and was then activated by PPIs (small conductance
apparent after ATP suppressed KATP). In separate experiments, PPIs
activated currents expressed from clones of mouse inwardly rectifying
K+ channels mIRK1 and the ATP-regulated inward
rectifier hROMK1 (Fig. 2).
Fig. 1.
PPIs activated KATP from diverse
tissues. Activity was studied in inside-out patch membranes
containing between 10 and 100 channels from a pancreatic beta cell
(A), a cardiac cell (B), an isolated membrane
vesicle from skeletal muscle cells (C), and KATP
(rSUR/rKir6.2) channels stably expressed in HEK cells (D).
The membranes were clamped at 
50 mV (A and B),
0 mV (C), or 80 mV (D), and the inside-out
patch was formed at the time indicated by the arrow labeled
i-o. The pipette solution contained 140 mM
K+ for A, B, and D and 10 mM K+ for C, with 140 mM
K+ in the bath for all. Open channel currents were inward
for A, B, and D and outward for
C. For clarity of illustration, all currents are shown as a
downward deflection from the zero current (dotted line). The
bath solution contained EGTA for A, C, and
D but not for B. Omitting EGTA from the bath
solution potentiated loss of KATP activity by increasing the residual
Ca2+ concentration in the bath solution. PPIs were
effective in cardiac KATP with or without EGTA. The cytoplasmic surface
of the patch membrane was perfused with PPIs (0.25 mg/ml) or ATP (2 mM) as indicated by bars. PPIs activated KATPs
in the examples shown and in n = 6, 5, 3, and 3 for
-cells, cardiac cells, membrane vesicles of skeletal muscle cells,
and rSUR/rKir6.2, respectively.
[View Larger Version of this Image (29K GIF file)]
Fig. 2.
PPIs activated currents in cloned non-KATP
Kir channels expressed in cultured cells. Methods used were the
same as for Fig. 1. A, activity of a cloned mouse heart Kir
(mIRK1, Kir2.1) was inhibited by Ca2+ and then activated by
the addition of PPIs to the cytoplasmic surface. B, activity
of an ATP-regulated Kir cloned from human kidney (hROMK1, Kir1.1) was
also activated by application of PPIs.
[View Larger Version of this Image (25K GIF file)]
Fig. 3.
Intact anionic phospholipids, but not their
breakdown products, activated KATP. Effects of
PI(4,5)P2- (25 µg/ml) (A), IP3-
(0.1 mg/ml) (B), and phosphatidylinositol-specific PLC-PI- (C) treated versus untreated
PI(4,5)P2 (25 µg/ml in both cases) on KATPs are shown.
KATPs in A and C were recorded from cardiac cells, while those in B were recorded from pancreatic
-cells. The same results were obtained with both tissue types, and
thus summary data were pooled. D, summaries of the effects
of different phospholipids and their cascade products on KATP activity.
The quantities of the lipids indicated in the figure are per ml volume of the bath solutions. E, summaries of the effects of
phospholipase on the phospholipid activation of KATP. Phospholipids
were incubated with PLC-PI (1 unit/ml), PLC (5 units/ml),
PLA2 (1 unit/ml), and PLD (20 units/ml) (indicated in the
figure by a plus sign) for 2 h or overnight at
37 °C. In the case of PLC, 1 mM Ca2+ was
added to the incubating solution, and the reaction was stopped by
adding 5 mM EGTA to the Ca2+-containing
solution. To determine relative activity for D and E, the open probability (NPopen) was
first calculated as the average current in a 5-s time window divided by
the known single channel current amplitude under the same recording
conditions. Relative activity was then obtained by normalizing
NPopen to the NPopen value measured within the initial 20 s (Initial) after
the formation of the inside-out patch. NPopen
measured after the inactivation and just before the application of
lipids was taken as a control. Control mean value is indicated by a
dotted line in both D and E. Most data
are from cardiac cells, with some data from pancreatic -cells
included in the effects of PPIs, PI(4,5)P2, PI, PS, and PE.
Data are expressed as mean ± S.D., with numbers from 3 to 5 for
each point except for the control (n = 67), and for
DOG, PI(4)P plus PLC-PI, and PC plus PLD (n = 1).
Experimental conditions were the same as described in the Fig. 1
legend, except in some cardiac cells EGTA was included. The lipids were
sonicated immediately before the experiment. The lipids were sonicated
again after incubation with phospholipase and before the
experiments.
[View Larger Version of this Image (33K GIF file)]
Fig. 4.
Polyvalent cations inhibited KATP activity,
and inhibition was reversed by application of PPIs. A,
neomycin in combination with PPIs on KATP from an inside-out patches
from a cardiac cell. Neomycin and PPIs were perfused on the membrane
cytoplasmic side during the period indicated (bars), at the
concentrations noted. The bath solution contained 2 mM EGTA
to slow down inactivation. Inhibitory effects of polyvalent cations on
KATP of cardiac cells at different concentrations are summarized in
B. Relative activity was calculated by normalizing the
NPopen (as described in the legend to Fig. 3) at
the end of a 1-min application of the polyvalent cations to the
membrane surface, to the NPopen just prior to
application. Symbols represent the mean, and bars
show the S.D., with n = 3-5 for each point. The
lines are fits of the data for each polyvalent cation to an
apparent binding isotherm y = 1/(1 + C/IC50), where C is the polyvalent
cation concentration. The fits are presented for descriptive purposes
only, and no implication for binding versus shielding
mechanism is intended.
[View Larger Version of this Image (25K GIF file)]
Fig. 5.
Schematic diagrams for structure and amino
acid sequence of Kir channels to account for electrostatic
channel-membrane lipid interactions. A, block diagram of the
linear amino acid sequence of Kir channels indicating the two
transmembrane (M1 and M2) and pore-forming (H5) domains. The amino acid
sequence for the initial C terminus (shaded area) for six
representatives of the Kir family are shown. B, a diagram of
two Kir subunits indicating how positively charged residues might
interact with anionic membrane phospholipids to "tether" a
"gate" away from the pore, allowing the channel to be active.
[View Larger Version of this Image (45K GIF file)]
Fig. 6.
A double mutation in the proximal C terminus
of Kir6.2 accelerated loss of channel activity. Shown is the
inactivation of KATP current recorded from a tsA201 cell transiently
co-expressed with wild type rKir6.2 and rSUR (A) and from a
tsA201 cell transiently co-expressed with R176A,R177A
(RR176AA) mutant and rSUR (B). The time label
indicates the elapsed time after the rupture of the cell membrane. No
ATP was included in the pipette solution. Currents were elicited by the
voltage steps from a holding potential of 20 mV to 100 mV and +60
mV with a 20-mV increment.
[View Larger Version of this Image (30K GIF file)]
Fig. 7.
PPIs were less effective in maintaining
channel activity in the mutant and resulted in shorter channel open
times. A, effects of PPIs on the single channel currents of
KATP expressed from the co-transfection of rKir6.2 and rSUR and KATP
expressed from the co-transfection of R176A,R177A (RR176AA)
and rSUR. The outward current is downward. The K+
concentrations were as follows: [K+]o = 10 mM; [K+]i = 144 mM. The
holding potential was 0 mV. The currents were low-pass filtered at 100 Hz. In the patch with wild type rKir6.2/rSUR (left), a
single channel was present. In the patch with R176A,R177A/rSUR
(RR176AA/rSUR) (right), two channels were present. 
indicates the time when the excised patch formed, and indicates the time when PPIs (1 mg/ml) were applied to the bath.
B, histogram analysis for the open times of the single
channel events in the presence of PPI shown in A. The open
times were fitted to two components of the exponential. The time
constants were 4.3 and 121 ms for Kir6.2/SUR, and were 3.8 and 32 ms
for R176A,R177A/rSUR, respectively.
[View Larger Version of this Image (40K GIF file)]
, VO) (30, 31) were also shown to have
similar effects. Trypsin treatment was found to dramatically maintain
KATP activation (18, 32). Mg2+ was found to be necessary
for ATP activation of KATP; therefore, many researchers speculated that
phosphorylation/dephosphorylation was the key to maintenance of KATP
channel activity (16). Mg-ATP, however, was often ineffective in
activating KATP in rat skeletal muscles (13). In addition, intensive
searches for protein kinases and the protein phosphorylation targets
have not been successful (32). These results suggest that although
phosphorylation may be a part of the regulatory process, additional key
elements are involved. In rat skeletal muscle cells, KATP was found to
be activated by high concentrations (>50 mM) of gluconate,
with the effect proportional to the length of the carbon chain (13).
This finding suggested to us the hypothesis that native anionic
phospholipids might be an endogenous mediator of this effect, because
like gluconate they have an anionic head with a hydrophobic tail.
10 M. Indeed, regardless of the presence of
ATP, raising the cytoplasmic Ca2+ concentration decreased,
rather than increased, the effect of PPIs. Thus, ATP and
Ca2+ are not required for the phospholipid effect on KATP,
making it unlikely that the protein kinase C signaling pathway is
involved in these effects of anionic lipids. Our observation that the
protein kinase C activator diacylglycerol was ineffective in activating KATPs is consistent with noninvolvement of protein kinase C. Likewise, the immediate requirement of other phosphorylation processes in maintaining KATP activity is unlikely because of the ATP-free conditions of our study. Another important protein group known to
interact with PI(4,5)P2 is the cytoskeletal regulating
proteins such as gelsolin. The possibility of involvement of
cytoskeletal structure in the KATP activity has been suggested (33).
The effects of PPIs remained even after application of high
concentrations of the cytoskeleton disrupters DNase I and cytochalasin
B (data not shown). Also, PS, which does not interact with most
cytoskeletal proteins (36), was effective in activating KATPs in our
experiments. Participation of the cytoskeleton network is evidently not
a requirement for the effect, although a modulatory role has not been
excluded.
To whom correspondence should be addressed: Dept. of Medicine,
Room 1683, MSC Bldg., 1300 University Ave., Madison, WI 53706. Tel.:
608-263-2250; Fax: 608-262-4761; E-mail: zfan{at}facstaff.wisc.edu.
1
The abbreviations used are: KATP,
ATP-sensitive potassium channel; PPI, phosphoinositide;
PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI(4)P,
phosphatidylinositol 4-phosphate; PI, phosphatidylinositol; PS,
phosphatidylserine; PC, phosphatidylcholine; PE,
phosphatidylethanolamine; IP3, inositol
1,4,5-triphosphate; DOG, 1,2-dioctanoyl glycerol (8:0 DG); DPG,
1,2-dipalmitoyl glycerol (16:0 DG); PLC-PI,
phosphatidylinositol-specific phospholipase C; PLC, phospholipase C;
PLA2, phospholipase A2; PLD, phospholipase D;
AMP-PNP, adenosine 5
-(,
-imino)triphosphate; AMP-PCP, adenosine 5-(,-methylenetriphosphate); HEK, human embryonic kidney.
-cells; Drs. Y. Tokuyama and H. Yano in Dr. G. Bell's
lab for rKir6.2, rSUR, and hROMK1 clones; Drs. J. Kyle and L. Phillipson for the mIRK1 clone, B. Ye for technical assistance, Dr. C. T. January for reading and commenting on the manuscript, and D. Pittz
for secretarial help.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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N. D'hahan, H. Jacquet, C. Moreau, P. Catty, and M. Vivaudou A Transmembrane Domain of the Sulfonylurea Receptor Mediates Activation of ATP-Sensitive K+ Channels by K+ Channel Openers Mol. Pharmacol., August 1, 1999; 56(2): 308 - 315. [Abstract] [Full Text]
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D. Kim and H. Bang Modulation of rat atrial G protein-coupled K+ channel function by phospholipids J. Physiol., May 15, 1999; 517(1): 59 - 74. [Abstract] [Full Text] [PDF]
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S. Misra, P. Ujhazy, L. Varticovski, and I. M. Arias Phosphoinositide 3-kinase lipid products regulate ATP-dependent transport by sister of P-glycoprotein and multidrug resistance associated protein 2 in bile canalicular membrane vesicles PNAS, May 11, 1999; 96(10): 5814 - 5819. [Abstract] [Full Text] [PDF]
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H.-H. Liou, S.-S. Zhou, and C.-L. Huang Regulation of ROMK1 channel by protein kinase A via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism PNAS, May 11, 1999; 96(10): 5820 - 5825. [Abstract] [Full Text] [PDF]
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W. A. Chutkow, J. C. Makielski, D. J. Nelson, C. F. Burant, and Z. Fan Alternative Splicing of sur2 Exon 17 Regulates Nucleotide Sensitivity of the ATP-sensitive Potassium Channel J. Biol. Chem., May 7, 1999; 274(19): 13656 - 13665. [Abstract] [Full Text] [PDF]
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A. B. Zhainazarov and B. W. Ache Effects of Phosphatidylinositol 4,5-Bisphosphate and Phosphatidylinositol 4-Phosphate on a Na+-Gated Nonselective Cation Channel J. Neurosci., April 15, 1999; 19(8): 2929 - 2937. [Abstract] [Full Text] [PDF]
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 L. Aguilar-Bryan and J. Bryan Molecular Biology of Adenosine Triphosphate-Sensitive Potassium Channels Endocr. Rev., April 1, 1999; 20(2): 101 - 135. [Abstract] [Full Text]
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 I. Heilmann, I. Y. Perera, W. Gross, and W. F. Boss Changes in Phosphoinositide Metabolism with Days in Culture Affect Signal Transduction Pathways in Galdieria sulphuraria Plant Physiology, April 1, 1999; 119(4): 1331 - 1340. [Abstract] [Full Text]
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I. H. M. Ho and R. D. Murrell-Lagnado Molecular Determinants for Sodium-dependent Activation of G Protein-gated K+ Channels J. Biol. Chem., March 26, 1999; 274(13): 8639 - 8648. [Abstract] [Full Text] [PDF]
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L.-H. Xie, M. Takano, M. Kakei, M. Okamura, and A. Noma Wortmannin, an inhibitor of phosphatidylinositol kinases, blocks the MgATP-dependent recovery of Kir6.2/SUR2A channels J. Physiol., February 1, 1999; 514(3): 655 - 665. [Abstract] [Full Text] [PDF]
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 Y Sai, A. Nies, and I. Arias Bile acid secretion and direct targeting of mdr1-green fluorescent protein from Golgi to the canalicular membrane in polarized WIF-B cells J. Cell Sci., January 12, 1999; 112(24): 4535 - 4545. [Abstract] [PDF]
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 S. Shyng and C. G. Nichols Membrane Phospholipid Control of Nucleotide Sensitivity of KATP Channels Science, November 6, 1998; 282(5391): 1138 - 1141. [Abstract] [Full Text]
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T. Baukrowitz, U. Schulte, D. Oliver, S. Herlitze, T. Krauter, S. J. Tucker, J. P. Ruppersberg, and B. Fakler PIP2 and PIP as Determinants for ATP Inhibition of KATP Channels Science, November 6, 1998; 282(5391): 1141 - 1144. [Abstract] [Full Text]
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F. M. Gribble, P. Proks, B. E. Corkey, and F. M. Ashcroft Mechanism of Cloned ATP-sensitive Potassium Channel Activation by Oleoyl-CoA J. Biol. Chem., October 9, 1998; 273(41): 26383 - 26387. [Abstract] [Full Text] [PDF]
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 G. G. MacGregor, J. Z. Xu, C. M. McNicholas, G. Giebisch, and S. C. Hebert Partially active channels produced by PKA site mutation of the cloned renal K+ channel, ROMK2 (kir1.2) Am J Physiol Renal Physiol, September 1, 1998; 275(3): F415 - F422. [Abstract] [Full Text] [PDF]
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X W Niu and R W Meech The effect of polyamines on KATP channels in guinea-pig ventricular myocytes J. Physiol., April 15, 1998; 508(2): 401 - 411. [Abstract] [Full Text] [PDF]
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S. Gudi, J. P. Nolan, and J. A. Frangos Modulation of GTPase activity of G proteins by fluid shear stress and phospholipid composition PNAS, March 3, 1998; 95(5): 2515 - 2519. [Abstract] [Full Text] [PDF]
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J. L. Sui, J. Petit-Jacques, and D. E. Logothetis Activation of the atrial KACh channel by the beta gamma subunits of G proteins or intracellular Na+ ions depends on the presence of phosphatidylinositol phosphates PNAS, February 3, 1998; 95(3): 1307 - 1312. [Abstract] [Full Text] [PDF]
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 L. AGUILAR-BRYAN, J. P. CLEMENT IV, G. GONZALEZ, K. KUNJILWAR, A. BABENKO, and J. BRYAN Toward Understanding the Assembly and Structure of KATP Channels Physiol Rev, January 1, 1998; 78(1): 227 - 245. [Abstract] [Full Text] [PDF]
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F. M. Gribble, G. Loussouarn, S. J. Tucker, C. Zhao, C. G. Nichols, and F. M. Ashcroft A Novel Method for Measurement of Submembrane ATP Concentration J. Biol. Chem., September 22, 2000; 275(39): 30046 - 30049. [Abstract] [Full Text] [PDF]
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H. Cho, G.-B. Nam, S. H. Lee, Y. E. Earm, and W.-K. Ho Phosphatidylinositol 4,5-Bisphosphate Is Acting as a Signal Molecule in alpha 1-Adrenergic Pathway via the Modulation of Acetylcholine-activated K+ Channels in Mouse Atrial Myocytes J. Biol. Chem., January 5, 2001; 276(1): 159 - 164. [Abstract] [Full Text] [PDF]
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H. Xu, N. Cui, Z. Yang, J. Wu, L. R. Giwa, L. Abdulkadir, P. Sharma, and C. Jiang Direct Activation of Cloned KATP Channels by Intracellular Acidosis J. Biol. Chem., April 13, 2001; 276(16): 12898 - 12902. [Abstract] [Full Text] [PDF]
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D.-K. Song and F. M. Ashcroft ATP Modulation of ATP-sensitive Potassium Channel ATP Sensitivity Varies with the Type of SUR Subunit J. Biol. Chem., March 2, 2001; 276(10): 7143 - 7149. [Abstract] [Full Text] [PDF]
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G. Loussouarn, L. J. Pike, F. M. Ashcroft, E. N. Makhina, and C. G. Nichols Dynamic Sensitivity of ATP-sensitive K+ Channels to ATP J. Biol. Chem., July 27, 2001; 276(31): 29098 - 29103. [Abstract] [Full Text] [PDF]
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