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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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REFERENCES

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
f=<LIM><OP>∑</OP></LIM> S<SUB><IT>f,n</IT></SUB> [exp(<IT>−</IT>10<SUP>(<IT>x−&tgr;<SUB>f,n</SUB></IT>)</SUP>)]10<SUP>(<IT>x−&tgr;<SUB>f,n</SUB></IT>)</SUP> (1)
where Sf,n is a scale factor of the proportional contribution for component "f,n"; tau 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
t<SUB>o</SUB><IT>=t′</IT><SUB>o</SUB> <FENCE><LIM><OP>∑</OP></LIM> a<SUB><IT>n</IT></SUB> exp(<IT>−t</IT><SUB>min</SUB><IT>/&tgr;<SUB>c,n</SUB></IT>)</FENCE> (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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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 (open circle ) 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.

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.

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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Table 1.   Effect of phosphatidic acid on single-channel kinetics of KATP channels



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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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Table 2.   Parameters of the closed and open time distribution functions fit to the histograms in Fig. 6

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).


    DISCUSSION
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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.


    ACKNOWLEDGEMENTS

We thank Dr. P. Hoffman and associates for providing rat heart cells and Dr. Y. Cui for conducting some experiments.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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