Phosphatidic acid stimulates cardiac KATP
channels like phosphatidylinositols, but with novel gating
kinetics
Zheng
Fan,
Lizhi
Gao, and
Wenxia
Wang
Department of Physiology, University of Tennessee
Health Science Center, College of Medicine, Memphis, Tennessee
38163
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ABSTRACT |
Membrane-bound anionic phospholipids such
as phosphatidylinositols have the capacity to modulate ATP-sensitive
potassium (KATP) channels through a mechanism involving
long-range electrostatic interaction between the lipid headgroup and
channel. However, it has not yet been determined whether the multiple
effects of phosphatidylinositols reported in the literature all result
from this general electrostatic interaction or require a specific
headgroup structure. The present study investigated whether
phosphatidic acid (PA), an anionic phospholipid substantially different
in structure from phosphatidylinositols, evokes effects similar to phosphatidylinositols on native KATP channels of rat heart
and heterogeneous Kir6.2/SUR2A channels. Channels treated with PA (0.2-1 mg/ml applied to the cytoplasmic side of the
membrane) exhibited higher activity, lower sensitivity to ATP
inhibition, less Mg2+-dependent nucleotide stimulation, and
poor sulfonylurea inhibition. These effects match the spectrum of
phosphatidylinositols' effects, but, in addition, PA also induced
a novel pattern in gating kinetics, represented by a decreased mean
open time (from 12.2 ± 2.0 to 3.3 ± 0.7 ms). This impact on
gating kinetics clearly distinguishes PA's effects from those of
phosphatidylinositols. Results indicate that multiple effects of
anionic phospholipids on KATP channels are related
phenomena and can likely be attributed to a common mechanism, but
additional specific effects due to other mechanisms may also coincide.
adenosine 5'-triphosphate-sensitive potassium channel; phosphatidic acid; sulfonylurea
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INTRODUCTION |
MEMBRANE-BOUND
ANIONIC PHOSPHOLIPIDS such as phosphatidylinositols modulate
gating function of various inward rectifier K+ channels,
including ATP-sensitive K+ (KATP) channels
(8, 12, 14). Because anionic phospholipids are abundant in
the inner leaflet of plasma membrane, this modulation may serve as a
control mechanism or even as a requirement for channel gating.
Available evidence has been consistent with the hypothesis that anionic
phospholipids modulate channels by interacting directly with
channel-forming subunits (8, 14, 22, 31), but little is
known about the nature of this interaction (24). By using
KATP channels as a model system, we previously reported that the potency of channel stimulation was proportional to the number
of negative charges in the phospholipid (8). This
correlation led us to propose that electrostatic interaction (that is,
long-range electrostatic interaction) may be a major mediator of the
modulation of channel gating by phospholipids. This idea was further
supported by site-directed mutagenesis analyses in which positively
charged residues of the channels were replaced with negatively charged residues (6, 8). Beyond electrostatic interactions,
specific interactions determined by structural details may also
play a role in channel modulation. Indeed, studies on Kir2.1 channels showed that different phosphatidylinositol isomers had different potencies in activating the channels (29).
The relative role of electrostatic vs. specific structural interaction
in the modulation of channels by lipids is still not well understood.
So far, most studies have focused on phosphatidylinositols, notably
phosphatidylinositol 4,5-disphosphate (PIP2), a significant signaling molecule. Such studies provide limited information on the
generalizability of the interaction, and to address this issue it is
necessary to examine the effects of anionic phospholipids whose
headgroups are less structurally related to phosphatidylinositols. Information from studies on phosphatidylinositols is also restricted by
the complexity of their effects. In KATP channels, it has
been shown that phosphatidylinositols produce a complex array of
modulatory effects: they stimulate the channel regardless of the
presence of intracellular nucleotides, reduce sensitivity to nucleotide inhibition and nucleotide stimulation in the presence of nucleotides, and retard sulfonylurea inhibition (2, 9, 17, 18, 28). It
has not yet been elucidated whether these effects result from a common
mechanism of interaction or several discrete mechanisms.
In this paper we focus on the effects of phosphatidic acid (PA), a
nonphosphatidylinositol anionic phospholipid, on the cardiac isoform of
KATP channel. The chemical structures of PA and
phosphatidylinositols are shown in Fig.
1A. One of the aims of the
present study is to test whether PA confers the same spectrum of
effects as do phosphatidylinositols. If so, this might be an indication
that these effects are all related phenomena in which a ubiquitous mechanism such as electrostatic interaction may play a key role, because the structure of the headgroups of PA and phosphatidylinositols differs substantially but both carry negative charges. Results of these
investigations may help understanding of the molecular nature whereby
membrane lipids modulate KATP channel functions, as well as
the intrinsic mechanisms governing the gating of these channels.
Physiologically, PA can act as a second messenger mediated by
phospholipase D (PLD) and other signaling molecules (5, 13), and the present study may implicate PA as a potential
regulator of KATP channels in cardiac myocytes.

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Fig. 1.
Stimulation of ATP-sensitive potassium (KATP)
channels by phosphatidic acid (PA). A: chemical structures
of PA and phosphatidylinositols (PPI). In the structure of PPI, "R"
can be either a hydrogen atom or a phosphate group (as shown at
right); a PPI can be denoted as PIPn, with "n"
indicating the number of phosphate groups. B:
KATP channel currents recorded at 0 mV in an inside-out
patch excised from a rat ventricular myocyte. The patch contained at
least 5 active channels. PA was applied at 0.5 mg/ml as indicated.
C: traces a-c are current recordings at the
corresponding time indicated in B shown on an expanded time
scale. Dotted lines indicate the level at which all channels were
closed. Open channel events deflect upward. No ATP was included at the
intracellular side of the patch.
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MATERIALS AND METHODS |
Isolated cardiac myocytes.
Single ventricular myocytes were isolated from rat hearts by enzymatic
(0.1% collagenase) dissociation. This method is well established in
our laboratory and produces plentiful, viable Ca2+-tolerant
myocytes for study.
Heterologous expression and cell culture.
A COS-1 cell line has been found optimal for transient transfection of
KATP channels as described previously (9).
Briefly, cells were transfected with cDNA clones using a LipofectAMINE PLUS kit (Invitrogen, Carlsbad, CA). The transfected cells were cultured 64-72 h before use in electrophysiological experiments. A
green fluorescent protein mutant (rsGFP, Clontech, Palo Alto, CA) was
cotransfected as a reporter to identify the transfected cells.
Patch-clamp recordings.
Single- or multiple-channel currents were recorded in the inside-out or
cell-attached configuration. The patch-clamp recording and data
acquisition used an Axopatch 200B amplifier (Axon Instruments, Union
City, CA) and a Digital 1200 interface controlled by pCLAMP 6.0 software. The extracellular solution contained (in mM) 10 KCl, 140 NaCl, 1.8 CaCl2, 0.53 MgCl2, and 5 HEPES (pH
7.4). In some extracellular solutions, 140 mM KCl was substituted for
the NaCl. The cytoplasmic solution contained (in mM) 140 KCl, 2 EGTA, and 5 HEPES (pH 7.2). ATP and MgCl2 were added to the
cytoplasmic solution to yield 1 µM ATP and 0.2 mM Mg2+,
unless specifically indicated otherwise. A computer program (7) was used to calculate the amount of MgCl2
to be added to the intracellular solution to maintain a free
Mg2+ concentration of 0.2 mM. PA from two sources was used
in this study. A PA sodium salt was prepared from naturally occurring phosphatidylcholine by hydrolysis with cabbage PLD (Sigma Chemical, St.
Louis, MO), and synthetic PA, 1-palmitoyl-2-oleoyl-PA (POPA), was
purchased from Avanti Polar Lipids (Alabaster, AL).
Phosphoinositides (PPIs, an extract from bovine brain containing
triphosphoinositide and diphosphoinositide, each at least 5 to
~10%, and phosphatidylinositol and phosphatidylserine) were
purchased from Sigma. The lipids were dispersed with sonication on ice.
PPIs have been shown to have a spectrum of effect similar to
PIP2 (8, 9, 11). The dispersed lipids formed
micelles as judged by the transparency of the solutions and were used
shortly after preparation. Use of the chemicals is described along with
the specific experimental results. The perfusion solutions were
switched within 100 ms by a computer-controlled fast solution-switching
system (DAD12, ALA Scientific Instruments, Westbury, NY). All
experiments were done at room temperature.
Data analysis.
Open channel activity was assessed as overall open probability
(NPo) which was calculated from an average
current in a 10- or 30-s time window divided by the single-channel
current amplitude. The gating kinetics of single-channel current were
assessed by open and closed time histograms, which were plotted on a
logarithmic time scale with event duration log-binned at a resolution
of 20 bins per log unit and a minimum resolution of
tmin = 150 µs, as described previously
(9). The histograms were fitted by probability distribution
|
(1)
|
where Sf,n is a scale factor of the
proportional contribution for component "f,n";
f,n is a time constant; subscript
f denotes channel state; and n = 1, 2, 3, 4 identifies the individual component. The total number of components was
determined by an F-test. A burst was defined as any series
of openings interrupted only by gaps shorter than a specified critical
time of 3 ms. Mean open times were calculated and corrected for missed
closings by
|
(2)
|
where an is the area of the exponential
component n.
Statistical analysis.
Mean data are reported with their standard errors. The Student's
t-test was used to determine statistical significance
between two data groups, and a one-way ANOVA test followed by a post
hoc Student-Newman-Keuls test was used to assess the statistical
differences among more than two data groups.
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RESULTS |
Effects of PA in the absence of ATP.
The effect of PA on KATP channels was examined by applying
PA to the cytoplasmic surface of patch membranes excised from
ventricular cells of rat heart. In the absence of inhibitory
concentrations of ATP (denoted as [ATP]), PA increased the average
channel current soon after application (<10 s). Like phosphoinositides
(8), channel stimulation by PA is significant only after
channels run down. Therefore, channels were allowed to run down for at
least 5 min after patch excision in the ATP-free cytoplasmic solution (9). Figure 1 shows typical results. In this experiment,
the patch contained multiple channels, but the single-channel opening events can be identified clearly. The increase of the channel current
was evidently due to significant increases in open channel activity and
in the number of active channels; no perceptible change in the
single-channel current amplitude was observed (Fig. 1). It is also
noteworthy that, after ~30 s, the open channel events became shorter
so that the current record appeared to flicker, although the general
level of activation (judged by the number of active channels) was not
further increased. The effect of PA, therefore, appears to have two
phases: 1) initial channel stimulation and 2)
later induction of current flickering. Both effects persisted after
removal of the PA from the perfusate, indicating that they were not
readily reversible. PA exerted similar effects on Kir6.2/SUR2A recombinant channels expressed in COS-1 cells. When measured within 30 s after application, 0.5 mg/ml PA initially increased channel activity by 1,235 ± 269% (n = 15; both native
KATP channels and Kir6.2/SUR2A channels are pooled). It
should be noted that these numbers vary with the basal activity of the
channels before the application of PA. POPA had qualitatively similar
effects. Similar effects of PA were also observed when ATP and
Mg2+ were not included in the cytoplasmic solution.
Effects of PA on channel interaction with nucleotides.
In the presence of normally inhibitory [ATP] (1 mM), PA was
able to open KATP channels, suggesting an antagonistic
effect against ATP inhibition. This effect becomes more clearly evident when concentration-dependent inhibition of channel activity by ATP was
compared before and after treatment with PA. In the example shown in
Fig. 2, treating a patch containing
Kir6.2/SUR2A channels with 0.5 mg/ml PA significantly increased the
channel activity in the presence of ATP concentrations that were
sufficient to inhibit the channel before PA treatment. Treatment for
70 s reduced the Ki of the inhibitory curve
from 20 to 152 µM (Fig. 2B). Figure 2C gives a
statistical summary of similar experiments. It should be noted that the
Ki before PA treatment is lower than reported averages for Kir6.2A/SUR2A channels (see Ref. 3); this is
most likely due to the channel rundown at the time of measurement. High
sensitivity to ATP inhibition has been reported previously in rundown
channels (25). Under this condition, however, the effect
of PA on ATP sensitivity was observed more profoundly. Besides this
inhibitory effect, ATP and some other nucleotides can also stimulate
KATP channels in the presence of Mg2+. Figure
3, A and B, illustrates an example in which
application of ATP, ADP, and Mg2+ transiently increased
channel activity under optimal concentration combinations. This
stimulatory effect, however, was not observed after the channels were
treated with PA. These results were repeated in a total of three
patches and were statistically significant (Fig.
3C).

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Fig. 2.
Decrease in ATP sensitivity after treatment with PA.
A: response of KATP channel currents to ATP
concentration steps before and after treatment with PA. The current
traces were recorded sequentially from the same inside-out patch
excised from a COS-1 cell expressing Kir6.2/SUR2A channels. PA (0.5 mg/ml) was applied to the intracellular side of the patch as indicated
in the middle trace. PA was completely removed from the
perfusion solution when the bottom trace was recorded.
B: corresponding concentration-inhibition curves measured
before ( ) and after ( ) PA treatment.
C: statistical summary of the effect of PA on ATP
inhibition. Ki, half-inhibitory
concentration; S, slope constant. These parameters were obtained by
fitting the measured relative open probability
(NPo; in the presence of ATP/control
NPo) to a Hill equation (e.g., those shown by
lines in B). Data from native and Kir6.2/SUR2A
KATP channels treated with either PA or
1-palmitoyl-2-oleoyl-PA (POPA) are pooled together.
* P < 0.05; ** P < 0.001. The
numbers of pooled experiments are indicated as bracketed.
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Fig. 3.
Absence of channel stimulation by nucleotides in channels treated
with PA. A: response of KATP channel currents to
ADP, ATP, and Mg2+ before and after treatment with PA.
KATP channel current traces shown in the figure were
sequentially recorded in the same patch isolated from a rat ventricular
myocyte. As indicated, 1 mg/ml PA, 0.1 mM ADP, ATP (concentrations in
mM given above the bars), and 0.2 mM Mg2+ were included in
the intracellular solution. The stimulatory effect of ADP/ATP before PA
treatment and absence of this effect after PA treatment are indicated
by arrows. B: corresponding concentration-effect curves
measured before (open symbols) and after (filled symbols) PA treatment.
Circles indicate measurements in the absence of ADP-Mg2+,
whereas triangles indicate measurements in the presence of
ADP-Mg2+. The lines were plots of the Hill equations fitted
to the relative NPo in the absence of
ADP-Mg2+. The dashed and solid lines are before and after
PA treatment, respectively. C: statistical summary of the
effect of PA on nucleotide stimulation. The relative
NPo was measured in the presence of 0.1 mM ADP,
10 µM ATP, and 0.2 mM Mg2+ before and after PA treatment.
* P < 0.05. The numbers of experiments are
indicated in brackets.
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Effect of PA on channel inhibition by sulfonylureas.
The effect of PA on the pharmacological characteristics of cardiac
KATP channels was also examined. As shown in Fig.
4A, treatment with PA
attenuated the inhibitory effect of tolbutamide on Kir6.2/SUR2A channels. A previous study showed that PIP2, a
phosphatidylinositol, could also reduce the inhibitory effect of
sulfonylureas on Kir6.2/SUR1 channel activity (17).
Because this effect has not been previously reported for Kir6.2/SUR2A
channels, we confirmed it by using PPIs (Fig. 4A).
Statistical analysis of these results is given in Fig. 4B,
which also includes data obtained from native KATP channels of rat heart.

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Fig. 4.
Attenuation of tolbutamide block by treatment with PA.
A: KATP channel currents recorded from an
untreated patch (top trace) and patches treated with PA
(middle trace) or PPIs (bottom trace). The
patches were isolated from COS-1 cells expressing Kir6.2/SUR2A
channels. Tolbutamide (0.5 mM), 1 mg/ml PA, or 1 mg/ml PPIs were
applied as indicated by bars. B: statistical summary of the
effect of PA and PPIs on tolbutamide block. Relative
NPo, NPo in the presence
of tolbutamide/control NPo. Both PA and PPIs
significantly (*P < 0.001) reduced block by
tolbutamide.
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Changes in kinetics of single-channel current in response to PA.
As noted above, PA causes a dramatic "flickering" in the gating
pattern of channel opening. Figure 5
shows the current record from an inside-out patch that contained only
one active Kir6.2/SUR2A channel throughout the experiment. Examination
of the single-channel records confirmed that the kinetics of
single-channel current gating were apparently more rapid after
treatment with 0.2 mg/ml PA. Interestingly, this change of channel
gating kinetics appeared with latency after the initial increase in
channel open probability. In this example, the latency was ~2 min.
After the latency, the mean open time decreased dramatically from 37 (15) to 8.8 (5.1) ms (parenthetical values are corrected
mean open times). As summarized in Table
1, in three experiments using patches
containing only a single channel, PA treatment initially stimulated the
channel open activity, and this effect was consistently followed by a reduction in channel open times. On the other hand, PA consistently reduced mean closed time during the initial and later periods of
effect. As a result of these changes in gating kinetics, the open
probability (Po) after the application of PA
underwent a biphasic change (Table 1). Po was
increased during the initial 2 min of PA application and decreased
thereafter, but still remained greater than in controls. Figure
6 presents a more detailed kinetic analysis of the channel shown in Fig. 5. The single-channel events were
recorded before and after 2 min of treatment with 0.2 mg/ml PA. Table
2 lists all parameters of the
distribution functions. This analysis reveals that the gating change
results from drastic shortening of the slowest component of open time
distributions, plus an increase of the fractional probability of an
intermediate component with a time constant of ~23 ms in closed time
distribution.

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Fig. 5.
Time-dependent effect of PA on single KATP
channel current. A: single-channel Kir6.2/SUR2A current
recorded continuously in an inside-out patch at 0 mV. PA (0.2 mg/ml)
was applied at arrowhead. B: selected current traces
displayed in an expanded time scale. From left to
right, the current traces were recorded before, during the
initial 15 s, and 120 s after application of PA,
respectively.
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Fig. 6.
Histograms of open and closed time distributions of the
single-channel events before and after treatment with PA. The
histograms shown in the figure were constructed from the current events
shown in Fig. 5 recorded before (A), during the initial 2 min (B), and 2 min after application of PA (C).
Solid lines are drawn according to the probability density function of
multiple exponential components as described in MATERIALS AND
METHODS. The parameters of the probability density functions
obtained from the fitting are listed in Table 2.
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Cardiac KATP channels open in bursts, and several bursts
can further form cluster activities (10). Recently,
significant work has revealed features of the relationship between
intraburst and interburst gating involving the open state of the
KATP channel and nucleotide inhibition (19,
20). Under our criterion for burst detection, PA considerably
changed the burst behavior, as indicated by the significant shortening
in mean burst duration after the initial channel stimulation. In
contrast, simple examination of the current recordings indicates that
the cluster behavior was maintained during and after the latency.
Further characterization of the burst and cluster behaviors was not
attempted in the present study (see DISCUSSION).
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DISCUSSION |
In this report, we demonstrate that PA applied to the
intracellular side of patch membranes causes multiple changes in the function of cardiac KATP channels. We previously
hypothesized and showed indirect evidence that PA might modulate
channel activity in a way similar to phosphatidylinositols
(8), but, to our knowledge, systematic experimentation
specifically designed to examine the effect of PA on KATP
channels has not yet been reported. PA exhibits a complex spectrum of
effects that not only completely overlaps with but also is broader than
that of phosphatidylinositols. We present evidence that PA induces
novel gating kinetics in cardiac KATP channels, an effect
that has not been found with other anionic phospholipids. These
findings add to the growing body of evidence that anionic phospholipids
can exert a diverse range of effects on KATP channels.
These results allow further inferences to be made concerning the
molecular mechanism underlying KATP channel gating and the
role of embedded lipids in controlling the gating process and provide
potentially new functional information on the properties of cardiac
KATP channels and their regulation.
Complex effects of PA and comparison with phosphatidylinositols.
Specifically, treatment with PA 1) increases channel
activity independent of ATP, 2) reduces sensitivity to ATP
inhibition, 3) attenuates pharmacological sensitivity as
represented by a reduction in sulfonylurea inhibition, 4)
abolishes Mg2+-dependent channel stimulation by
nucleotides, and 5) switches the intrinsic gating of
KATP channels to one with faster kinetics. All these
effects, except the effect on intrinsic gating, have also been reported
to be induced by phosphatidylinositols (2, 8, 12, 17, 28).
In general, however, the effects of PA are relatively weaker than those
of polyphosphatidylinositols such as PIP2. For example, 0.5 mg/ml PA reduces the Ki of ATP inhibition by
~6-fold, whereas 1 mg/ml PPIs left-shifts Ki
by ~400-fold (9). Effects on kinetics represent the most
significant difference between PA and phosphatidylinositols. Under the
same conditions, treatment of cardiac KATP channels with
phosphatidylinositols results in longer mean open and bursting times
but shorter mean closed time (9). In the present study, we
found that PA induced more complex kinetic changes. During the initial
period after application of PA, the mean open time was slightly
prolonged with an unchanged mean bursting duration. The mean open and
bursting times then decreased and became much shorter than in controls. Interestingly, the mean closed time was reduced consistently after application of PA. It should be noted that, because the effect of PA
during the initial period of application is a time-dependent process,
the statistical parameters measured during this period contain a
time-dependent error component so that they must be interpreted with
caution. We used these parameters only to qualitatively reveal the
different effects caused by PA during the initial and later period of
PA treatment. We were unable to characterize burst behavior
quantitatively and describe cluster behavior even qualitatively because
of the small number of these events during the short initial period.
Possible mechanisms whereby PA acts on KATP channels.
Overlap in their spectrum of effects indicates that PA and
phosphatidylinositols share, at least partly, a common mechanism of
interaction with KATP channels. Previous studies using
mutagenesis analysis have revealed that negative charges on the
headgroups of phosphatidylinositols play a critical role, and it has
been postulated that phosphatidylinositols' effects may be mediated by
direct interaction between lipid headgroups and the channel-forming subunit of KATP channels (8, 16, 27).
Phosphatidylinositols also modulate activity of other Kir channels
(14, 26), probably by a similar mechanism
(8). Biochemical binding assays conducted in Kir2.1 and
Kir1.1 channels confirmed that phosphatidylinositols can directly
interact with charged residues of the cytoplasmic domain of these
channels (14, 29). However, some uncharged residues also
seem to be involved in the interaction (29), and it has
also been shown that the strength of the effect of channel activation
varies among PIP2 isomers (31). In spite of
these studies, so far it remains unclear whether the proposed
lipid-channel interaction requires a specific lipid head structure or
whether it is primarily determined by the charges the headgroup
carries. Moreover, because phosphatidylinositols appear to induce
several changes in KATP functions, the previously published
data are not sufficient to indicate whether these effects share a
common mechanism of interaction. The present study demonstrates that,
although the headgroup structure of PA differs substantially from that of a phosphatidylinositol, both lipids induce overlapping effects on
KATP channels. Together with the previous studies, these
results indicate that a specific headgroup structure such as an
inositol is not essential for anionic phospholipids to induce complex
changes in KATP channel gating. Rather, as we have argued
previously (8), electrostatic interaction between the
lipid headgroup and the channel dominates the modulatory effects.
Noticeably, the present data imply that the complex spectrum of effects
reported for PIP2 on KATP channels may result
entirely from this electrostatic interaction. Alternatively, because it
is known that PA can regulate phosphatidylinositol-4-phosphate 5-kinase, an enzyme that synthesizes PIP2
(15), PA may also act on KATP channels
indirectly by stimulating PPI synthesis.
The delayed change in intrinsic gating kinetics after PA treatment is
an interesting phenomenon that deserves special attention. This effect
contrasts the effect of phosphatidylinositols on gating kinetics, i.e.,
prolongation of open times and burst durations (9).
Because they may directly interact with the gating structure, phosphatidylinositols have been utilized as probes to investigate the
gating mechanism in recent studies (6, 9). Similarly, elucidation of the mechanism underlying the change in kinetics induced
by PA may also provide insights into how the channel gates. Several
lines of information can be gained from the current data, which could
help us understand the nature of this effect. First, the different time
courses distinguish this effect from those effects that are also
observed with phosphatidylinositols; the difference can be interpreted
as indicating that different mechanisms are involved. Secondly, the
major effect is the shortening of mean open times, especially the
slower components of open times (Tables 1 and 2). It is generally
agreed that the fast component is determined by a gating process
separate from the process(es) that cause the slower components (for
example, see Refs. 1, 6, 9,
10, and 30). Therefore, PA appears to affect
the slower process(es) but not the fast process. So far, agents that can promote channel activity have been linked to these slower process(es). Interestingly, all of them induce longer mean opening times and shorter mean closed times [for example, potassium channel openers such as pinacidil (10), phosphatidylinositols
(6, 9), and even fatty acid metabolites,
epoxyeicosatrienoic acids, that activate the channel
(21)]. To our knowledge, PA is the only agent that
shortens the open times while concurrently stimulating channel
activity. One possibility is that the exposed phosphorous group of
membrane-incorporated PA specifically interacts with the channel and
closes it via an allosteric mechanism. The short headgroup may have
more physical interaction with the end of transmembrane segments and
thus may interfere with the movement of these segments. Another
possibility is that PA may act directly at or within the channel pore.
Presumably, removal of PA from the perfusate removes it from the
channel pore, which is inconsistent with the persistent flickery gating
kinetics observed in our experiments. It remains a possibility,
however, that the persistent flickery gating might also arise from
direct blocking effect of PA remaining in the membrane by the channel
even after its removal from the perfusate.
Possible physiological implications.
PA is an intermediate product of the de novo synthesis of membrane
phospholipids, but it is also a potential signal molecule in the
myocardium. Although the de novo synthesis and turnover of PA occur
mostly in the endoplasmic reticulum, PA in plasma membrane is formed
primarily through the hydrolysis of phosphatidylcholine by PLD. Under
normal physiological conditions, PA is present in the plasma membrane
only in trace amounts, but studies have shown that
ischemia-reperfusion potentiates PLD activity in
heart (4, 23), and PA concentration may be expected to be
elevated accordingly. To date, a relationship between KATP
channel activity and the PLD signal cascade has not been investigated.
Although our knowledge of the amount of active PA in membranes is still
limited due to technical difficulties, the results of this study
suggest that PA is a potential mediator linking KATP
channel activity and the PLD signal cascade and may provide novel
perspectives for studying the regulation of cardiac KATP
channels under pathological conditions.
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ACKNOWLEDGEMENTS |
We thank Dr. P. Hoffman and associates for providing rat heart
cells and Dr. Y. Cui for conducting some experiments.
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FOOTNOTES |
This work was supported by National Institutes of Health Grants
HL-58133 and GM-61943 and a Grant-in-Aid from the American Heart
Association Southeast Affiliation.
Address for reprint requests and other correspondence: Z. Fan, Dept. of Physiology, Univ. of Tennessee Health Science Center, College of Medicine, Memphis, Tennessee 38163 (E-mail:
zfan{at}physiol.utmem.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published September 18, 2002;10.1152/ajpcell.00255.2002
Received 31 May 2002; accepted in final form 9 September 2002.
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