1 Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655; and 2 Department of Physiology, University of Melbourne, Victoria 3010, Australia
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ABSTRACT |
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To determine
the mechanism of fatty acid modulation of rabbit pulmonary artery
large-conductance Ca2+-activated K+
(BKCa) channel activity, we studied effects of fatty acids
and other lipids on channel activity in excised patches with
patch-clamp techniques. The structural features of the fatty acid
required to increase BKCa channel activity (or average
number of open channels, NPo) were identified to
be the negatively charged head group and a sufficiently long (C > 8) carbon chain. Positively charged lipids like sphingosine, which have
a sufficiently long alkyl chain (C 8), produced a decrease in
NPo. Neutral and short-chain lipids did not
alter NPo. Screening of membrane surface charge
with high-ionic-strength bathing solutions (330 mM K+ or
130 mM K+, 300 mM Na+) did not alter the
modulation of the BKCa channel NPo
by fatty acids and other charged lipids, indicating that channel
modulation is unlikely to be due to an alteration of the membrane
electric field or the attraction of local counterions to the channel.
Fatty acids and other negatively charged lipids were able to modulate BKCa channel activity in bathing solutions containing 0 mM
Ca2+, 20 mM EGTA, suggesting that calcium is not required
for this modulation. Together, these results indicate that modulation
of BKCa channels by fatty acids and other charged lipids
most likely occurs by their direct interaction with the channel protein
itself or with some other channel-associated component.
arachidonic acid; sphingosine; calcium-activated potassium channel
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INTRODUCTION |
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IT IS WELL ESTABLISHED that fatty acids are able to modulate the activity of a wide variety of ion channels including K+, Na+, and Ca2+ channels as well as channels activated by N-methyl-D-aspartate (NMDA) and GABA (19, 36, 40, 47, 48, 51, 52, 55, 58, 63, 66, 70). Previously, Kirber et al. (32) characterized large-conductance Ca2+-activated K+ (BKCa) channels from rabbit pulmonary artery (RPA) smooth muscle cells and demonstrated that they are activated by two fatty acids, the polyunsaturated 20-carbon arachidonic acid and the fully saturated 14-carbon myristic acid. Because myristic acid is not metabolized via the lipoxygenase, cycloxygenase, or cytochrome P-450 oxygenase pathways to produce bioactive metabolites (22, 50, 55, 61, 68), modulation of BKCa channel activity was determined to be a direct consequence of the fatty acid molecule itself.
Although the study by Kirber et al. (32) suggested that two fatty acids, which vary in chain length and conformation, are capable of activating the BKCa channel, a comprehensive understanding of the structural features required for this modulation as well as the mechanism of fatty acid action were not determined. For example, do all fatty acids activate this BKCa channel or are only certain fatty acids effective? In addition, is modulation of this channel limited to fatty acids or are other lipids effective? Answers to these questions would also provide information regarding the mechanism of action of fatty acids on the RPA smooth muscle BKCa channel. To date, the fatty acid modulation of a number of BKCa channels has been studied, and these channels show variation in effective lipids as well as the mechanisms of modulation (1, 5, 7, 11, 17, 20, 38, 71, 76, 78).
One possible mechanism of action is direct interaction of fatty acids with the channel protein or some other protein closely associated with the channel (1, 29, 57). Alternatively, fatty acids may be acting through a mechanism that involves alterations of the bulk lipid properties of the membrane, for instance, by acting as detergents to perturb the lipid membrane (47, 73), by altering membrane fluidity, bilayer stiffness and/or membrane curvature (2, 31, 39, 44, 65), or by changing the "protein-lipid interface" (6). In addition, it is possible that fatty acids affect channel behavior by altering membrane surface charge. Changes in membrane surface charge may cause alterations in the local concentration of counterions in the vicinity of the channel and may change the electric field in the membrane, even when the membrane potential is unchanged (23, 66, 72). Because both membrane potential and ions, particularly Ca2+ (32), affect channel behavior, it is possible that the changes in membrane surface charge brought about by fatty acids and other charged lipids are responsible for the changes observed in BKCa channel activity. Fatty acid modulation has also been shown to occur via channel blockade (28, 60), through other modulatory proteins like protein kinase C (PKC) (3, 8, 62) and protein phosphatases (56), and indirectly by bioactive fatty acid metabolites (for example, see Refs. 4 and 7).
In an attempt to understand the mechanism of fatty acid modulation of BKCa channel activity, we determined the structural features of the fatty acid molecule required for channel modulation by studying the effects of a variety of fatty acids and other charged and uncharged amphiphiles on this channel. Four structural features of the fatty acid molecule were considered in this study: 1) the carboxylate head group, 2) the negative charge on the carboxylate head group, 3) the length of the acyl chain, and 4) the structural conformation of the acyl chain. In addition, experiments were performed to address whether mechanisms of action that involve alterations in membrane surface charge or changes in the Ca2+ concentration in the vicinity of the channel are responsible for the modulation of channel activity by fatty acids. A brief account of some of this work has been reported elsewhere in abstract form (14).
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MATERIALS AND METHODS |
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Recording conditions.
New Zealand White rabbits weighing between 3 and 5 lb were anesthetized
with a lethal dose of pentobarbital sodium (0.14 mg/g), and the
pulmonary artery was then dissected away from the heart and
lungs. Freshly isolated smooth muscle cells from the rabbit main pulmonary artery were obtained with the procedures of Clapp and
Gurney (13). Single-channel currents were recorded
from excised inside-out (I-O) and excised outside-out (O-O) patches with standard patch-clamp techniques (25). Single-channel
recordings were usually carried out in symmetric solutions that were
composed of (in mM) 130 K+, 1 Mg2+, 5 EGTA,
114.5 Cl, and 10 HEPES-HCl at pH 7.4 (ionic strength
I = 0.132; Ref. 74). Occasionally, recordings
were carried out in nonsymmetric solutions where the external solution
was composed of either (in mM) 127 Na+, 3 K+, 1 Mg2+, 5 EGTA, 114.5 Cl
, and 10 HEPES-HCl at
pH 7.4 or 120 Na+, 20 K+, 1 Mg2+, 5 EGTA, 122.5 Cl
, and 10 HEPES-HCl at pH 7.4. Solutions
containing 5 mM EGTA, zero calcium, and zero nucleotides were used to
avoid changes in channel activity that could occur because of the
involvement of calcium and other second messengers. In experiments in
which we wished to shield membrane surface charge, recordings were
carried out in symmetric solutions composed of (in mM) 130 K+, 300 Na+, 5 EGTA, 10 HEPES-HCl, 1 Mg2+, and 414.5 Cl
at pH 7.4 (I = 0.43)
or 330 K+, 5 EGTA, 10 HEPES-HCl, 1 Mg2+,
and 314.5 Cl
at pH 7.4 (I = 0.315).
High-EGTA-containing solutions were composed of (in mM) 130 K+, 48 Cl
, 20 EGTA, 10 HEPES-HCl, and 1 Mg2+ at pH 7.4.
Preparation and application of compounds.
The solubility of lipids used in this study varied. For example,
tetradecanesulfonate (TDS) could be easily dissolved in water, whereas
the water solubility of myristic acid was poor and it needed to be
first dissolved in dimethyl sulfoxide (DMSO; Fluka). In general, all
lipids were treated the same and were first dissolved in DMSO and then
diluted (1:1,000 dilution) in bathing solution. Compounds were applied
to the extracellular side of O-O patches and the cytosolic side of I-O
patches by pressure ejection (Picospritzer II; General Value,
Fairfield, NJ) from micropipettes ("puffer" pipette, 1- to 2-µm
tip diameter) placed 50-100 µm from the patch electrode
(35). We have defined the structural requirements with
concentrations of 10-50 µM (typically 50 µM) that were
determined previously to be effective at altering channel activity in
this (32) and other (17, 47, 57) preparations
and that did not greatly alter the resistance or integrity of the
patch. Because compounds are diluted with the bathing solution as they
exit the puffer pipette, their final concentration at the membrane
surface is difficult to estimate but is likely to be less than that in the puffer pipette. The concentration given is, therefore, the maximum
possible concentration. Bathing solutions containing DMSO were applied
to patches and had no obvious effect on BKCa channel behavior (Table 1).
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Data analysis and display. Recordings were made with a conventional patch-clamp amplifier (EPC 5; List). To obtain a similar background level of channel activity we recorded at a range of membrane potentials (usually +20 to +60 mV). However, channel activity varied greatly from patch to patch. The potential across the patch as well as other stimulus protocols were controlled by the software package pCLAMP 5 (Axon Instruments) and the laboratory interface TL1 (Axon Instruments). Data were filtered at 3 or 10 kHz and then digitally recorded onto videotape with a Sony PCM digital audio processor with a sampling frequency of 44 kHz. Data for figures were played back through the PCM to be converted back into an analog signal, filtered at 100 Hz, and then sampled at 300 Hz. Data for analysis were filtered at 300 Hz or 1 kHz and sampled at 1 or 3 kHz, respectively.
Channel activity in our case was defined as NPo (the average number of open channels), where N is the number of channels in the patch (unknown) and Po is the probability that an ion channel is in the open state. Qualitative changes in NPo were determined by visual inspection of the current record. Some records were analyzed quantitatively with the analysis packages described below. Patches chosen for analysis 1) showed low noise levels, 2) had very few or no other channel types evident in the patch that would have a major effect on the analysis, 3) could also show activation by a fatty acid control (when used), usually oleic acid, and 4) were representative of each compound. If many patches could fit these criteria, then patches used for analysis were chosen randomly from this group. The duration of the time period used for analysis before and during the application of a lipid was determined by visual inspection of the channel trace. Mean open times (To) were also determined over the same time periods as those used to determine NPo. Single-channel current amplitudes (i) were estimated by inspection with a custom software package, Erwin, kindly supplied by Michel Vivaudou (CEA Grenoble, Grenoble, France), or by the commercially available software package pCLAMP 5 or 6. NPo was determined by dividing the average current by the unitary current amplitude with Erwin. Alternatively, the software package pCLAMP 6, which idealized the real channel records, was used to determine NPo and To as described previously (67). Data for Tables 1 and 2 were analyzed with pCLAMP 6. NPo and To were usually calculated for a period of time (10-120 s) before the application of the lipid and for 10-120 s during the time that the lipid exerted an effect. Because the activity of the channels in the patch was not always in a steady state (during application), the values of NPo and To represent an average over the time period used.
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RESULTS |
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A range of fatty acids activate BKCa channels.
The fatty acids oleic acid and myristic acid, which are not substrates
for the arachidonic acid metabolic pathways that yield bioactive
compounds, as well as arachidonic acid increased channel activity of
RPA BKCa channels in both I-O and O-O membrane patches (Fig. 1, Table 1). Oleic acid increased
channel activity in 15 of 17 I-O patches and 1 of 1 O-O patches,
myristic acid increased activity in 10 of 12 I-O and 6 of 6 O-O
patches, and arachidonic acid increased channel activity in 3 of 3 I-O
and 2 of 2 O-O patches. Analysis of representative traces showed that
the increases in NPo produced by these fatty
acids was significant (Table 1). To, however,
was not significantly altered, and i appeared unaffected by
these fatty acids.
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The carboxylate group is not required for BKCa channel
activation.
To determine whether the carboxylate head group of the fatty acid was
required for increasing NPo, the fatty acid
analog TDS was applied to excised membrane patches. TDS, a 14-carbon,
fully saturated lipid that is similar in structure to fatty acids but possesses a negatively charged sulfonate head group, increased channel
activity in 16 of 16 I-O patches and 3 of 3 O-O patches. Analysis of
representative traces showed that this increase was significant (Fig.
2, Table 1). TDS did not significantly
alter To or i (Table 1). The
naturally occurring, negatively charged 16-carbon palmitoyl
lysophosphatidate (PLPA) also increased channel activity in 4 of 4 I-O
and 6 of 7 O-O patches. This increase in NPo was
also shown to be significant, but in this case,
To was also minimally but significantly
increased (Fig. 2, Table 1). However, the PLPA-induced changes in
To could not account for the observed changes in
NPo (i.e., NPo fold
change To fold change). Therefore, it
appears that the increase in NPo produced by
these negatively charged lipids is primarily the result of a decrease in Tc.
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Neutral lipids do not appear to alter NPo.
To determine whether the negative charge per se was an important
structural requirement for activation, uncharged or neutrally charged
lipids were applied to excised patches. The 18-carbon monounsaturated
neutral compound oleyl alcohol, the 10-carbon alcohol decanol, and the
12-carbon alcohol dodecanol had no effect on channel activity in 6 of 6 I-O and 2 of 2 O-O patches, 1 of 1 I-O patch, and 6 of 6 I-O and 5 of 5 O-O patches, respectively (Fig. 3). In
these same patches fatty acids were seen to increase channel activity.
Analysis of representative data for oleyl alcohol and dodecanol showed
that they had no significant effect on channel NPo (Table 1). In addition, the fully saturated
16-carbon palmitoyl lysophosphatidylcholine (PLPC), a zwitterion that
bears no net charge in the pH range used in this study, was also
without any apparent effect in 3 of 4 I-O patches. Analysis showed that
PLPC did not significantly change NPo and
To (Table 1). Application of PLPC and oleyl
alcohol did not alter i; however, dodecanol caused a
significant decrease in To.
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Positively charged lipids decrease NPo.
To determine whether positively charged head groups could also increase
NPo, compounds that are similar in structure to
fatty acids but instead have positively charged head groups were
applied to I-O and O-O patches. Two positively charged primary amines, the 14-carbon fully saturated compound tetradecylamine (TDA) and the
18-carbon monounsaturate oleylamine, both decreased channel activity,
showing a decrease in activity in 5 of 6 I-O and 4 of 4 I-O patches and
5 of 5 O-O patches, respectively. These decreases in
NPo were found to be significant (Table 1),
whereas no obvious effect on i was observed (Fig.
4). Unlike TDA, however, oleylamine significantly decreased To (Table 1). In the
case of oleylamine, the change in To alone could
only partially account for the change in NPo,
suggesting that the major reason for the observed change in
NPo was an increase in
Tc. The naturally occurring 14-carbon amino
alcohol sphingosine, which at the pH used in this study should bear a
positive charge, also decreased channel activity in 5 of 6 I-O and 2 of
2 O-O patches. This suppression of NPo was
significant (Table 1). Sphingosine did not significantly alter
To or have an apparent effect on i
(Fig. 4, Table 1).
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Short-chain lipids do not appear to alter NPo. Because a variety of negatively and positively charged lipids could effectively alter BKCa channel activity, we also wished to determine what structural features of the acyl and alkyl chains were required. As indicated in Figs. 1 and 4, saturated and unsaturated charged lipids could effectively alter BKCa channel NPo; however, a minimal chain length appears to be required. Octanesulfonate (4 of 4 I-O patches) and caprylic acid (4 of 4 I-O and 1 of 1 O-O patches) did not obviously change channel activity. Although small changes in channel activity were observed after analysis, the eight-carbon fully saturated fatty acid caprylic acid did not significantly alter NPo (Table 1). To and i were unaffected by its application (Fig. 1C, Table 1). Similarly, there was a lack of effect with the eight-carbon fully saturated alkyl sulfonate octanesulfonate (Fig. 2C, Table 1).
Octylamine appeared to produce no obvious effect on channel activity in 6 of 6 I-O and 1 of 1 O-O patches (Fig. 4C); however, on analysis, a small but significant decrease in NPo was observed (Table 1). Octylamine was much less effective than its longer-chain positively charged counterparts at decreasing NPo (Fig. 4C, Table 1). In addition, application of octylamine caused a decrease in i and a large and significant decrease in To in both patch configurations (Fig. 4, Table 1). Thus the decrease in channel NPo produced by octylamine, unlike most other lipids tested, is likely to result primarily from changes in To. In summary, short-chain negatively charged lipids do not increase NPo at the concentrations used here. The eight-carbon octylamine, although capable of decreasing BKCa channel NPo, was not as effective as the longer-chain, positively charged lipids. Octylamine, in fact, appears to alter channel NPo through a mechanism unlike that of the longer-chain positively charged lipids. Its action, which decreases i, is consistent with it acting as a fast open channel blocker, although it may also have an allosteric effect on channel gating (21). Thus, to effectively change NPo through a mechanism that appears to involve an alteration of Tc, negatively charged and positively charged lipids appear to require a chain length of greater than eight carbons.Fatty acids do not appear to affect NPo by altering the voltage dependence of channel activation. The activity of the RPA BKCa channel is strongly voltage dependent, showing, at low Po, an e-fold change in NPo for each 9-mV change in membrane potential (32). Therefore, the activation of BKCa channels by negatively charged lipids might be explained if we assume that fatty acids preferentially insert into the outer leaflet rather than into the inner leaflet of the cell membrane, thereby altering membrane surface charge. The addition of negatively charged lipids to the outer membrane leaflet would alter the electric field within the membrane in the direction expected for membrane depolarization, resulting in an increase in NPo. Similarly, preferential insertion of positively charged lipids into the outer leaflet would steepen the electric field within the membrane, producing an effective membrane hyperpolarization and a decrease in NPo (24).
To determine whether alterations in surface charge could explain the above results, fatty acids and other charged lipids were applied to I-O and O-O patches in the presence of high-ionic-strength bathing and pipette solutions (330 mM K+ or 130 mM K+, 300 mM Na+). High-ionic-strength solutions were previously used to shield membrane surface charge (41, 42, 49). It should also be pointed out, however, that shielding surface charge could also render these charged lipids less effective if they interact with a charged site on the channel protein that is exposed, or partially exposed, to the surrounding solutions. High-ionic-strength solutions failed to alter the effects of fatty acids and other charged lipids on BKCa channel activity seen under normal ionic strength conditions. TDS (6 of 6 I-O and 3 of 3 O-O patches) and oleic acid (5 of 6 I-O and 8 of 8 O-O patches) were still able to increase channel activity in 330 mM K+-containing solutions. A similar result was found for solutions containing 130 mM K+, 300 mM Na+, where TDS increased NPo in 4 of 4 I-O and 2 of 2 O-O patches and myristic acid increased NPo in 3 of 3 I-O and 1 of 1 O-O patch. Data analysis showed that these negatively charged lipids significantly increased NPo in high-ionic-strength solutions (Table 2, Fig. 5). Similarly, positively charged lipids were also effective in high-ionic-strength solutions. TDA (2 of 2 I-O and 2 of 2 O-O patches) and sphingosine (4 of 4 I-O and 2 of 2 O-O patches) decreased NPo when they were applied to membrane patches in 330 mM K+-containing solutions. Data analysis showed that these positively charged lipids significantly decreased NPo in high-ionic-strength solutions (Table 2).
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Fatty acids do not appear to affect NPo by altering concentration of calcium and other ions in the vicinity of the channel. Insertion of charged lipids into the inner leaflet of the membrane bilayer could attract or repel counterions in the vicinity of the channel (27). An increase in the internal concentration of the counterions Ca2+ and H+ near the channel could alter the activity of the BKCa channel in opposite ways. If the insertion of fatty acids into the inner membrane leaflet attracted Ca2+ to the channel, channel activity would be increased (54), whereas the attraction of H+ would cause a decrease in channel activity (33). Moreover, insertion of positively charged lipids would repel Ca2+ and H+, with the former causing a decrease in activity and the latter an increase. Experiments performed in high-ionic-strength solutions suggest that it is unlikely that channel activation by fatty acids and other charged lipids results from a change in the concentration of Ca2+ or H+ in the vicinity of the channel due to the alteration of membrane surface charge because responses were essentially unchanged in high-ionic-strength solutions.
However, calcium may still be involved in the charged lipid modulation of BKCa channel activity through a mechanism that does not involve an alteration of membrane surface charge. Because it has been suggested that calcium stores may exist in excised membrane patches (77) and that fatty acids can mobilize calcium from internal stores (12, 75), we further investigated the involvement of Ca2+. In this scheme we would have to assume that the positively and negatively charged lipids are acting to alter channel activity through two different mechanisms. The ability of TDS to activate the BKCa channel was tested in different concentrations of the calcium chelator EGTA (5 and 20 mM). If fatty acids and other negatively charged lipids were acting to increase NPo through a Ca2+-dependent mechanism, it would be expected that they would cause a much smaller increase in NPo in the presence of 20 mM EGTA than in the presence of 5 mM EGTA. Oleic acid was able to increase NPo in the presence of 20 mM EGTA (2 patches; not shown). TDS was also able to significantly increase NPo and To in solutions containing 20 mM EGTA (Table 2, Fig. 6A). In one I-O patch in which the experiment was carried out, the increase in NPo produced by TDS in a 20 mM EGTA-containing solution was similar to that seen when the solution was changed to one with 5 mM EGTA (Fig. 7A), suggesting that this increase was not due to a calcium-dependent mechanism. In addition, the increases in NPo produced by TDS were similar in different patches, whether these experiments were carried out in 5 or 20 mM EGTA (compare Fig. 6, A and B). In fact, the mean fold change in NPo seen in 20 mM EGTA was not significantly different from that seen in 5 mM EGTA [i.e., 13.45 ± 7.80 (6 patches, Table 2) vs. 12.70 ± 9.90 (5 patches, Table 1)], suggesting that fatty acids and fatty acid analogs do not increase NPo by increasing the Ca2+ concentration ([Ca2+]) in the vicinity of the channel. Moreover, these results show that fatty acids can affect BKCa channel activity independently of the levels of internal [Ca2+]. BAPTA was also used in experiments to chelate calcium, and in these cases the results were similar to those seen with EGTA. TDS could still increase NPo in high concentrations of BAPTA (20 mM, 4 patches; not shown). Interestingly, in one I-O patch in which it was examined, the voltage dependence of NPo was essentially the same in 5 and 20 mM EGTA (Fig. 7B). In addition to the above, the fact that fatty acids and other lipids could repetitively alter channel activity in solutions containing no ATP also argues against a mechanism involving Ca2+ release from stores. In these high-EGTA, zero-calcium solutions, the stores would not be refilled after emptying by fatty acids and a rundown in the response would be predicted.
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Membrane-bound protein kinases and phosphatases. Because the modulation of BKCa channel activity from the RPA by fatty acids and other charged lipids was studied in excised patches, in the absence of calcium and nucleotides, it is unlikely that they altered channel behavior by affecting the activity of protein kinases or protein phosphatases. Furthermore, the effects of fatty acids and other charged lipids could be obtained repeatedly in the same patch, and high concentrations of the kinase inhibitor staurosporin (70 nM) and the phosphatase inhibitor okadaic acid (2 mM) did not prevent fatty acid increases in NPo (4 patches; not shown). In addition, experiments that show that it is likely that charged lipids are acting from the external membrane surface (15) suggest that these lipids are not interacting directly with these enzymes to bring about an alteration in channel behavior.
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DISCUSSION |
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To elucidate the mechanism of fatty acid modulation of BKCa channel activity in RPA smooth muscle cells, we have studied the regulation of this channel by a variety of single-chain lipids. We identified certain structural features of the fatty acid molecule that are required for modulation of channel activity. While helping to address the mechanism of fatty acid action on this channel, these structural features should also provide clues as to the nature of the site with which the fatty acids interact. In addition, experiments were performed to address the mechanism by which fatty acids and other charged lipids modulate the activity of BKCa channels.
Structural requirements. The features of fatty acid compounds that were required for BKCa channel activation, at the concentrations used here, were found to be the negatively charged head group and a sufficiently long (C > 8) hydrophobic carbon chain. Thus not only could fatty acids increase NPo, but other negatively charged lipids could also do so. Interestingly, positively charged lipids were also able to affect the activity of the channel, but in this case NPo was decreased. Neutral and short-chain (C8) negatively charged lipids were apparently ineffective at the concentrations used. The short-chain, positively charged lipid octylamine produced a significant decrease in channel NPo; however, this decrease was much smaller than that produced by the longer-chain, positively charged lipids and appeared to be the result of a different mechanism. This suggests that chain length is also an important structural feature governing the effectiveness of positively charged lipids.
Chain conformation did not appear to be important, because saturates, monounsaturates, and polyunsaturates, as long as they were charged, could modulate channel activity. Fatty acids and other charged lipids, except octylamine, did not alter i and usually did not affect To. Therefore, all charged long-chain lipids affect NPo predominantly through an alteration in Tc. Changes in NPo produced by oleylamine are likely to result from changes in both Tc and To, whereas octylamine is likely to decrease NPo predominantly through changes in To. Fatty acids and charged lipids could effectively modulate channel activity when applied to both I-O and O-O patches, as would be expected for compounds that can traverse the lipid bilayer (26). The finding that fatty acid and lipid modulation of BKCa activity is not dependent on chain conformation is in agreement with the results from rabbit coronary artery smooth muscle cells obtained by Ahn et al. (1).Fatty acids and charged lipids do not appear to alter NPo through a lipid mechanism. The fact that the longer-chain lipids were more effective at altering channel activity than the shorter-chain compounds supports the contention that lipids affecting BKCa channel activity associate with the membrane or, alternatively, with a hydrophobic binding pocket in the channel protein, to exert their effects on BKCa channel activity. If lipids must partition into the membrane to be effective, they could be altering channel activity by affecting the bulk lipid properties of the membrane. Insertion of lipids into the membrane may have a number of affects on membrane properties. First, these inserted lipids may act as detergents and disrupt the membrane; second, they may change membrane fluidity; and, finally, they may alter the organization of the lipid bilayer to affect membrane curvature.
Because there are specific structural features of the fatty acid molecule that appear to be required for modulating channel activity, it is unlikely that these lipids affect channel activity by acting on membrane fluidity or by acting as detergents. The effects of charged lipids on channel behavior are correlated with the length of the carbon chain and the charge of the head group and not with their ability to alter properties of the membrane. For example, negatively charged cis-unsaturates such as arachidonic acid and oleic acid, which are more likely to disrupt membrane order and, therefore, increase membrane fluidity, had the same effect on channel activity as the fully saturated, and hence membrane-ordering, myristic acid (9). PLPA and PLPC are both excellent detergents; however, the former increases BKCa channel activity whereas the latter produces no obvious effect. Moreover, the effects of charged lipids were reversible, making them unlikely to result from the disruption of the cell membrane, i.e., detergent effects (47). Because this BKCa channel is mechanosensitive (32), intercalation of lipids into the membrane may affect membrane curvature, according to the "bilayer couple theory" (65), and thus produce stretch-induced alterations of channel activity, as suggested by Martinac et al. (44). According to the bilayer couple theory, charged amphipaths accumulate preferentially in one-half of the lipid bilayer, positively charged amphipaths in the inner layer and negatively charged amphipaths in the outer layer, and induce membrane curvature in opposite directions (concave or negative curvature and convex or positive curvature, respectively). Martinac et al. (44) first used the bilayer couple theory to explain the effects of amphipathic compounds on the stretch-sensitive channels of bacteria. In their study, they found that cationic and anionic amphipaths mimicked the effect of stretch and activated the channels. Thus, to explain their results according to the bilayer couple theory, they had to assume that a convex or a concave curvature of the membrane creates a mechanical stress on the channels equivalent to stretch. Our results suggest that such a mechanism is unlikely, because all lipids (irrespective of the charge on their head group) should produce the same effect, channel activation. This, however, was not the case. Our results could be consistent with the bilayer couple theory if we assume that the BKCa channels can differentiate between opposite curvatures, convex and concave. Thus, for lipids that can easily flip across the bilayer when applied from one side, preferential insertion of negatively charged lipids into the outer membrane leaflet would cause membrane "stretch" or positive bilayer curvature and thus channel activation, whereas preferential insertion of positively charged lipids into the inner leaflet would cause membrane "compression" or negative bilayer curvature and thus inhibit channel activity. However, this explanation is unlikely because in experiments in which we limited the application of a negatively charged lipid to one side of the membrane [palmitoyl coenzyme A, which does not flip across the bilayer (26)] under surface charge shielding conditions, channel activation was seen only when the compound was applied to the extracellular side. There was essentially no effect when the same lipid was applied to the intracellular surface (15). The bilayer couple hypothesis (where there was specific curvature dependence) would predict that for a lipid that only inserts into one side of the bilayer, insertion into one leaflet would cause channel activation whereas insertion into the other leaflet would cause channel inhibition. Another mechanism by which lipids could affect channel activity is by shape-dependent membrane deformation (see Refs. 10 and 39 and references therein). Casado and Ascher (10) ascribed the effect of a number of lipid compounds (similar to those used in this study) on the stretch-sensitive NMDA receptor to the shape of the compound. Lysophospholipids, whose head group structures are larger than their tails (cones), were found, like membrane compression, to inhibit the NMDA receptor when applied to the outside membrane surface, whereas application of arachidonic acid, a compound whose head group structure is smaller than its tail (inverted cone), produced the same effect as membrane stretch when applied to the outside membrane surface, channel activation. Our findings are not consistent with such a hypothesis because there was no correlation between the shape of the compound and its effect on channel activity. Negatively charged cones (palmitoyl lysophosphatidic acid) and inverted cones (arachidonic acid) activated the BKCa channel, whereas cones of differing charge [palmitoyl lysophosphatidic acid (negative) and PLPC (neutral)] had different affects on channel activity. These results highlight the importance of the charge on the head group of the lipid compound, and not its shape, and raise the question of how positively charged cones and inverted cones would affect the NMDA channel, which was not addressed by Casado and Ascher (10).Possible mechanisms of action of fatty acids and charged lipids. Our results show that single-chain charged lipids do not alter BKCa channel activity by altering membrane surface charge or by means of second messenger molecules like calcium or by altering the activity of protein kinases and phosphatases. Instead, our results are consistent with single-chain lipids having an effect on the channel protein complex itself or a closely associated membrane component. If the only role of the carbon chain is to attach the charged head group to the lipid bilayer or to a hydrophobic pocket in the channel, then this charged group must be in some way responsible for the alteration in channel activity. It is likely that the charged head group alters NPo by interacting with residues either on the channel itself or on some closely associated channel protein.
One possible way that this could happen is that the charged head groups interact with one, or some, of the positively charged residues found in the transmembrane spanning voltage sensor S4 (24, 34, 43). Kang and Leaf (30) previously proposed the involvement of a sequence within the voltage sensor in the fatty acid modulation of voltage-gated Na+, K+, and Ca2+ channels. Alternatively, these charged lipids may interact with other residues in the channel protein or in other membrane-bound proteins, and these residues may not be the same for positively charged and negatively charged lipids. Interestingly, the lipid modulation of the activity of a number of other proteins, PKC (37, 46, 62, 64), the Na+/Ca2+ exchanger (59), and a small-conductance K+ channel (53, 57), show structural requirements similar to those required for the BKCa channel studied here. However, these structural requirements are different from those described for the BK channel of GH3 cells (a channel that is Ca2+ activated via phospholipase A2; Ref. 18), in which it was found that there was a significant correlation between the degree of fatty acid unsaturation and channel activation (and not with the compounds' ability to affect membrane fluidity) (17). Saturated and trans-unsaturated fatty acids were found to be ineffective. It is clear that such a correlation does not apply to the RPA smooth muscle BKCa channel because fatty acids and other lipids without double bonds in the carbon chains, e.g., myristic acid and TDS, were very effective at increasing channel activity. Although our study identifies lipids that effectively modulate channel activity, limitations brought about by the method of application do not allow us to definitively compare the relative effectiveness of these lipids. Future studies that compare steady-state concentration-response curves for these lipids will enable such a comparison. In summary, we have identified the structural features required for single-chain lipids to both activate and inhibit BKCa channel activity. BKCa channel activation requires a negatively charged head group with a chain length of greater than eight carbons. These negatively charged lipids increase NPo, primarily by decreasing Tc. Longer-chain positively charged lipids decrease NPo primarily by increasing Tc. Together with the evidence indicating that these compounds do not appear to act by altering the properties of the lipid bilayer or membrane surface charge or the activity of second messenger molecules, this study suggests that it is likely that fatty acids and other charged lipids modulate BKCa channel activity by interacting with the channel protein itself, with some other channel-associated protein (e.g., ![]() |
ACKNOWLEDGEMENTS |
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We thank Paul Tilander, Rebecca McKinney, and Brian Packard for excellent technical assistance and Alejandro M. Dopico for helpful discussions.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-31620 and HL-61297.
Address for reprint requests and other correspondence: A. L. Clarke, Dept. of Physiology, Univ. of Melbourne, VIC 3010, Australia (E-mail: alisonlc{at}unimelb.edu.au).
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.
July 3, 2002;10.1152/ajpcell.00035.2002
Received 22 January 2002; accepted in final form 24 June 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ahn, DS,
Kim YH,
Lee BS,
and
Kang DH.
Fatty acids directly increase the activity of Ca2+-activated K+ channels in rabbit coronary smooth muscle cells.
Yonsei Med J
35:
10-24,
1994[Medline].
2.
Anel, A,
Richieri GV,
and
Kleinfeld AM.
Membrane partition of fatty acids and inhibition of T cell function.
Biochemistry
2:
530-536,
1993.
3.
Asaoka, Y,
Yoshida K,
Oka M,
Shinomura T,
Ogita K,
Kikkawa U,
and
Nishizuka Y.
The family of protein kinase C in transmembrane signalling for cellular regulation.
J Nutr Sci Vitaminol (Tokyo)
SpecNo:
7-12,
1992.
4.
Barlow, RS,
El-Mowafy AM,
and
White RE.
H2O2 opens BKCa channels via the PLA2-arachidonic acid signaling cascade in coronary artery smooth muscle.
Am J Physiol Heart Circ Physiol
279:
H475-H483,
2000
5.
Baron, A,
Frieden M,
and
Beny JL.
Epoxyeicosatrienoic acids activate a high-conductance, Ca2+-dependent K+ channel on pig coronary artery endothelial cells.
J Physiol
504:
537-543,
1997[Abstract].
6.
Barrantes, FJ.
Structural-functional correlates of the nicotinic acetylcholine receptor and its lipid microenvironment.
FASEB J
7:
1460-1467,
1993
7.
Benoit, C,
Renaudon B,
Salvail D,
and
Rousseau E.
EETs relax airway smooth muscle via an EpDHF effect: BKCa channel activation and hyperpolarization.
Am J Physiol Lung Cell Mol Physiol
280:
L965-L973,
2001
8.
Blobe, GC,
Khan WA,
and
Hannun YA.
Protein kinase C: cellular target of the second messenger arachidonic acid?
Prostaglandins Leukot Essent Fatty Acids
52:
129-135,
1995[ISI][Medline].
9.
Carruthers, A,
and
Melchior DL.
Role of bilayer lipids in governing membrane transport processes.
In: Lipid Domains and the Relationship to Membrane Function, edited by Aloia RC,
Curtain CC,
and Gordon LM.. New York: Liss, 1988, p. 201-225.
10.
Casado, M,
and
Ascher P.
Opposite modulation of NMDA receptors by lysophospholipids and arachidonic acid: common features with mechanosensitivity.
J Physiol
513:
317-330,
1998
11.
Chang, HM,
Reitstetter R,
and
Gruener R.
Lipid-ion channel interactions: increasing phospholipid headgroup size but not ordering acyl chains alters reconstituted channel behavior.
J Membr Biol
145:
9-13,
1995.
12.
Chow, SC,
and
Jondal M.
Polyunsaturated fatty acids stimulate an increase in cytosolic Ca2+ by mobilizing the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool in T cells through a mechanism independent of phosphoinositide turnover.
J Biol Chem
265:
902-907,
1990
13.
Clapp, LH,
and
Gurney AM.
Outward currents in rabbit pulmonary artery cells dissociated with a new technique.
Exp Physiol
76:
677-693,
1991[Abstract].
14.
Clarke, AL,
Petrou S,
Walsh JV, Jr,
and
Singer JJ.
Modulation of large conductance Ca2+-activated K+ channel activity by charged lipids: structural requirements (Abstract).
Soc Neurosci Abstr
21:
1326,
1995.
15.
Clarke AL, Walsh JV Jr, and Singer JJ. Site of
action of charged lipids on Ca2+-activated K+
(CAK) channels from rabbit pulmonary artery (Abstract). Abstract
Book of the Fourth Annual Neuropharmacology ConferencePotassium
Channels, 1996, p. 11.
16.
Cui, J,
Cox DH,
and
Aldrich RW.
Intrinsic voltage dependence and Ca2+ regulation of mslo large conductance Ca-activated K+ channels.
J Gen Physiol
109:
647-673,
1997
17.
Denson, DD,
Wang X,
Worrell RT,
and
Eaton DC.
Effects of fatty acids on BK channels in GH3 cells.
Am J Physiol Cell Physiol
279:
C1211-C1219,
2000
18.
Denson, DD,
Worrell RT,
Middleton P,
and
Eaton DC.
Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2.
Am J Physiol Cell Physiol
276:
C201-C209,
1999
19.
Devor, DC,
and
Frizzell RA.
Modulation of K+ channels by arachidonic acid in T84 cells. II. Activation of a Ca2+-independent K+ channel.
Am J Physiol Cell Physiol
274:
C149-C160,
1998
20.
Dumoulin, M,
Salvvail D,
Gaudreault SB,
Cadieux A,
and
Rousseau E.
Epoxyeicosatrienoic acids relax airway smooth muscles and directly activate reconstituted KCa channels.
Am J Physiol Lung Cell Mol Physiol
275:
L423-L431,
1998
21.
Eghbali, M,
Curmi JP,
Birnir B,
and
Gage PW.
Hippocampal GABAA channel conductance increased by diazepam.
Nature
388:
71-75,
1997[ISI][Medline].
22.
Fitzpatrick, FA,
and
Murphy RC.
Cytochrome P450 metabolism of arachidonic acid: formation and biological actions of "epoxygenase"-derived eicosanoids.
Pharmacol Rev
40:
229-241,
1994[ISI][Medline].
23.
Fraser, DD,
Hoehn K,
Weiss S,
and
MacVicar BA.
Arachidonic acid inhibits sodium currents and synaptic transmission in cultured striatal neurones.
Neuron
11:
633-644,
1997.
24.
French, RJ,
Prusak-Sochaczewski E,
Zamponi GW,
Becker S,
Kularatna AS,
and
Horn R.
Interactions between a pore-blocking peptide and the voltage sensor of the sodium channel: an electrostatic approach to channel gating.
Neuron
16:
407-413,
1996[ISI][Medline].
25.
Hamill, OP,
Marty A,
Neher E,
Sakmann B,
and
Sigworth FJ.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch
391:
85-100,
1981[ISI][Medline].
26.
Hamilton, JA,
and
Cistola DP.
Transfer of oleic acid between albumin and phospholipid vesicles.
Proc Natl Acad Sci USA
83:
82-86,
1986[Abstract].
27.
Hille, B.
Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer, 1992.
28.
Honore, E,
Barhanin J,
Attali B,
Lesage F,
and
Lazdunski M.
External blockade of the major cardiac delayed-rectifier K+ channel (Kv1.5) by polyunsaturated fatty acids.
Proc Natl Acad Sci USA
91:
1937-1941,
1994[Abstract].
29.
Kang, JX,
and
Leaf A.
Evidence that free polyunsaturated fatty acids modify Na+ channels by directly binding to the channel proteins.
Proc Natl Acad Sci USA
93:
3542-3546,
1996
30.
Kang, JX,
and
Leaf AL.
Antiarrhythmic effects of polyunsaturated fatty acids.
Circulation
94:
1774-1780,
1996
31.
Karnovsky, MJ,
Kleinfeld AM,
Hoover RL,
and
Klausner RD.
The concept of lipid domains in membranes.
J Cell Biol
94:
1-6,
1982[ISI][Medline].
32.
Kirber, MT,
Ordway RW,
Clapp LH,
Walsh JV, Jr,
and
Singer JJ.
Both membrane stretch and fatty acids directly activate large conductance Ca2+-activated K+ channels in vascular smooth muscle cells.
FEBS Lett
297:
24-28,
1992[ISI][Medline].
33.
Kume, H,
Takagi K,
Satake T,
Tokuno H,
and
Tomita T.
Effects of intracellular pH on calcium-activated potassium channels in rabbit tracheal smooth muscle.
J Physiol
424:
445-457,
1990[Abstract].
34.
Larsson, HP,
Baker OS,
Dhillon DS,
and
Isacoff EY.
Transmembrane movement of the Shaker K+ channel S4.
Neuron
16:
387-397,
1996[ISI][Medline].
35.
Lassignal, NL,
Singer JJ,
and
Walsh JV, Jr.
Multiple neuropeptides exert a direct effect on the same isolated single smooth muscle cell.
Am J Physiol Cell Physiol
250:
C792-C798,
1986
36.
Lesage, F,
and
Lazdunski M.
Molecular and functional properties of two-pore-domain potassium channels.
Am J Physiol Renal Physiol
279:
F593-F801,
2000
37.
Limatola, C,
Schaap D,
Moolenaar WH,
and
van Blitterswijk WJ.
Phosphatidic acid activation of protein kinase C- overexpressed in COS cells: comparison with other protein kinase C isotypes and other acidic lipids.
Biochem J
304:
1001-1008,
1994[ISI][Medline].
38.
Lu, T,
Katakam PV,
Van Rollins M,
Weintraub NL,
Spector AA,
and
Lee HC.
Dihydroxyeicosatrienoic acids are potent activators of Ca2+-activated K+ channels in isolated rat coronary arterial myocytes.
J Physiol
534:
651-667,
2001
39.
Lundbaek, JA,
Birn P,
Girshman J,
Hansen AJ,
and
Andersen OS.
Membrane stiffness and channel function.
Biochemistry
35:
3825-3830,
1996[ISI][Medline].
40.
Macica, CM,
Yang Y,
Hebert SC,
and
Wang WH.
Arachidonic acid inhibition of cloned renal K+ channel, ROMK1.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F588-F594,
1996
41.
MacKinnon, R,
Latorre R,
and
Miller C.
Role of surface electrostatics in the operation of a high-conductance Ca2+-activated K+ channel.
Biochemistry
28:
8092-8099,
1989[ISI][Medline].
42.
MacKinnon, R,
and
Miller C.
Functional modification of a Ca2+-activated K+ channel by trimethyloxonium.
Biochemistry
28:
8087-8092,
1989[ISI][Medline].
43.
Mannuzzu, LM,
Moronne MM,
and
Isacoff EY.
Direct physical measure of conformational rearrangement underlying potassium channel gating.
Science
271:
213-216,
1996[Abstract].
44.
Martinac, B,
Adler J,
and
Kung C.
Mechanosensitive ion channels of E. coli activated by amphipaths.
Nature
348:
261-263,
1990[ISI][Medline].
45.
Meera, P,
Wallner M,
Jiang Z,
and
Toro L.
A calcium switch for the functional coupling between (hslo) and
subunits (KV, Ca
) of maxi K channels.
FEBS Lett
382:
84-88,
1996[ISI][Medline].
46.
Merrill, AHJ,
Nimkar S,
Mendaldino D,
Hannun YA,
Loomis C,
Bell RM,
Tyagi SR,
Lambeth JD,
Stevens VL,
Hunter R,
and
Liotta DC.
Structural requirements for long-chain (sphingoid) base inhibition of protein kinase C in vitro and for the cellular effects of these compounds.
Biochemistry
28:
3138-3145,
1989[ISI][Medline].
47.
Meves, H.
Modulation of ion channels by arachidonic acid.
Prog Neurobiol
43:
175-186,
1994[ISI][Medline].
48.
Miller, B,
Sarantis M,
Traynelis SF,
and
Attwell D.
Potentiation of NMDA receptor currents by arachidonic acid.
Nature
355:
722-725,
1992[ISI][Medline].
49.
Moczydlowski, E,
Alvarez O,
Vergara C,
and
Latorre R.
Effect of phospholipid surface charge on the conductance and gating of a Ca2+-activated K+ channel in planar lipid bilayers.
J Membr Biol
83:
273-282,
1985[ISI][Medline].
50.
Needleman, P,
Turk J,
Jakschik BA,
Morrison AR,
and
Lefkowith JB.
Arachidonic acid metabolism.
Annu Rev Biochem
55:
69-102,
1986[ISI][Medline].
51.
Nishikawa, M,
Kimura S,
and
Akaike N.
Facilitatory effect of docosahexaenoic acid on N-methyl-D-aspartate response in pyramidal neurones of rat cerebral cortex.
J Physiol
475:
83-93,
1994[Abstract].
52.
Ordway, RW,
Singer JJ,
and
Walsh JV, Jr.
Direct regulation of ion channels by fatty acids.
Trends Neurosci
14:
96-100,
1991[ISI][Medline].
53.
Ordway, RW,
Walsh JV, Jr,
and
Singer JJ.
Arachidonic acid and other fatty acids directly activate potassium channels in smooth muscle cells.
Science
244:
1176-1179,
1989[ISI][Medline].
54.
Pallotta, BS,
Magleby KL,
and
Barrett JN.
Single channel recordings of Ca2+-activated K+ currents in rat muscle cell culture.
Nature
293:
471-474,
1981[ISI][Medline].
55.
Patel, AJ,
and
Honoré E.
Properties and modulation of mammalian 2P domain K+ channels.
Trends Neurosci
24:
339-346,
2001[ISI][Medline].
56.
Petit-Jacques, J,
and
Hartzell HC.
Effect of arachidonic acid on the L-type calcium current in frog cardiac myocytes.
J Physiol
493:
67-81,
1996[Abstract].
57.
Petrou, S,
Ordway RW,
Hamilton JA,
Walsh JV, Jr,
and
Singer JJ.
Structural requirements for charged lipid molecules to directly increase or suppress K+ channel activity in smooth muscle cells: effects of fatty acids, lysophosphatidate, acyl Coenzyme A, and sphingosine.
J Gen Physiol
103:
471-486,
1994[Abstract].
58.
Petrou, S,
Ordway RW,
Kirber MT,
Dopico AM,
Hamilton JA,
Walsh JV, Jr,
and
Singer JJ.
Direct effects of fatty acids and other charged lipids on ion channel activity in smooth muscle.
Prostaglandins Leukot Essent Fatty Acids
52:
173-178,
1995[ISI][Medline].
59.
Philipson, KD.
Interaction of charged amphiphiles with Na+-Ca2+ exchange in cardiac sarcolemmal vesicles.
J Biol Chem
259:
13999-14002,
1984
60.
Poling, JS,
Karanian JW,
Salem NJ,
and
Vicini S.
Time- and voltage-dependent block of delayed rectifier potassium channels by docosahexaenoic acid.
Am Soc Pharmacol Exp Therap
47:
381-390,
1995.
61.
Samuelsson, B,
Dahlen SE,
Lindgren JA,
Rouzer CA,
and
Serhan CN.
Leukotrienes and lipoxins: structures, biosynthesis, and biological effects.
Science
237:
1171-1176,
1987[ISI][Medline].
62.
Schmitt, H,
and
Meves H.
Protein kinase C as mediator of arachidonic acid-induced decrease of neuronal M-current.
Pflügers Arch
425:
134-139,
1993[ISI][Medline].
63.
Schwartz, RD,
and
Yu X.
Inhibition of GABA-gated chloride channel function by arachidonic acid.
Brain Res
585:
405-410,
1992[ISI][Medline].
64.
Sekiguchi, K,
Tsukuda M,
Ogita K,
Kikkawa U,
and
Nishizuka Y.
Three distinct forms of rat brain protein kinase C: differential response to unsaturated fatty acids.
Biochem Biophys Res Commun
145:
797-802,
1987[ISI][Medline].
65.
Sheetz, MP,
and
Singer SJ.
Biological membranes as bilayer couples. A molecular mechanism of drug-erythrocyte interactions.
Proc Natl Acad Sci USA
71:
4457-4461,
1974[Abstract].
66.
Shimada, T,
and
Somlyo AP.
Modulation of voltage-dependent Ca channel current by arachidonic acid and other long-chain fatty acids in rabbit intestinal smooth muscle.
J Gen Physiol
100:
27-44,
1992[Abstract].
67.
Singer, JJ,
and
Walsh JV, Jr.
Characterization of calcium-activated potassium channels in single smooth muscle cells using the patch-clamp technique.
Pflügers Arch
408:
98-111,
1987[ISI][Medline].
68.
Smith, WL.
The eicosanoids and their biochemical mechanisms of action.
Biochem J
259:
315-324,
1989[ISI][Medline].
69.
Stefani, E,
Ottolia M,
Olcese R,
Wallner M,
Latorre R,
and
Toro L.
Voltage-controlled gating in a large conductance Ca2+-sensitive K+ channel (hslo).
Proc Natl Acad Sci USA
13:
5427-5431,
1997.
70.
Sumida, C,
Graber R,
and
Nunez E.
Role of fatty acids in signal transduction: modulators and messengers.
Prostaglandins Leukot Essent Fatty Acids
48:
117-122,
1993[ISI][Medline].
71.
Twitchell, WA,
Penna TL,
and
Rane SG.
Ca2+-dependent K+ channels in bovine adrenal chromaffin cells are modulated by lipoxygenase metabolites of arachidonic acid.
J Membr Biol
158:
69-75,
1997[ISI][Medline].
72.
Vacher, P,
McKenzie J,
and
Dufy B.
Arachidonic acid affects membrane ionic conductances of GH3 pituitary cells.
Am J Physiol Endocrinol Metab
257:
E203-E211,
1989
73.
Wieland, SJ,
Fletcher JE,
and
Gong QH.
Differential modulation of a sodium conductance in skeletal muscle by intracellular and extracellular fatty acids.
Am J Physiol Cell Physiol
263:
C308-C312,
1992
74.
Williams, HB.
Basic Physical Chemistry for the Life Sciences (3rd ed.). San Francisco, CA: Freeman, 1978, p. 184-185.
75.
Wolf, BA,
Turk J,
Sherman WR,
and
McDaniel ML.
Intracellular Ca2+ mobilization by arachidonic acid. Comparison with myo-inositol 1,4,5-trisphosphate in isolated pancreatic islets.
J Biol Chem
261:
3501-3511,
1986
76.
Wu, SN,
Li HF,
and
Chiang HT.
Actions of epoxyeicosatrienoic acid on large-conductance Ca2+-activated K+ channels in pituitary GH3 cells.
Biochem Pharmacol
60:
251-262,
2000[ISI][Medline].
77.
Xiong, ZL,
Kitamura K,
and
Kuriyama H.
Evidence for contribution of Ca2+ storage sites on unitary K+ channel currents in inside-out membrane of rabbit portal vein.
Pflügers Arch
420:
112-114,
1992[ISI][Medline].
78.
Zhang, Y,
Oltman CL,
Lu T,
Lee HC,
Dellsperger KC,
and
Vanrollins M.
EET homologs potently dilate coronary microvessels and activate BKCa channels.
Am J Physiol Heart Circ Physiol
280:
H2430-H2440,
2001