Correspondence to: Donghee Kim, Department of Physiology & Biophysics, Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064. Fax:847-578-3265 E-mail:kimd{at}mis.finchcms.edu.
Released online: 28 February 2000
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Abstract |
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This study reports the identification of an endogenous inhibitor of the G proteingated (KACh) channel and its effect on the KACh channel kinetics. In the presence of acetylcholine in the pipette, KACh channels in inside-out atrial patches were activated by applying GTP to the cytoplasmic side of the membrane. In these patches, addition of physiological concentration of intracellular ATP (4 mM) upregulated KACh channel activity approximately fivefold and induced long-lived openings. However, such ATP-dependent gating is normally not observed in cell-attached patches, indicating that an endogenous substance that inhibits the ATP effect is present in the cell. We searched for such an inhibitor in the cell. ATP-dependent gating of the KACh channel was inhibited by the addition of the cytosolic fraction of rat atrial or brain tissues. The lipid component of the cytosolic fraction was found to contain the inhibitory activity. To identify the lipid inhibitor, we tested the effect of ~40 different lipid molecules. Among the lipids tested, only unsaturated free fatty acids such as oleic, linoleic, and arachidonic acids (0.22 µM) reversibly inhibited the ATP-dependent gating of native KACh channels in atrial cells and hippocampal neurons, and of recombinant KACh channels (GIRK1/4 and GIRK1/2) expressed in oocytes. Unsaturated free fatty acids also inhibited phosphatidylinositol-4,5-bisphosphate (PIP2)-induced changes in KACh channel kinetics but were ineffective against ATP-activated background K1 channels and PIP2-activated KATP channels. These results show that during agonist-induced activation, unsaturated free fatty acids in the cytoplasm help to keep the cardiac and neuronal KACh channels downregulated by antagonizing their ATP-dependent gating. The opposing effects of ATP and free fatty acids represent a novel regulatory mechanism for the G proteingated K+ channel.
Key Words: acetylcholine, long-chain fatty acid, phosphatidylinositol-4,5-bisphosphate, atria, arachidonic acid
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INTRODUCTION |
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In the heart and brain, binding of neurotransmitters and hormones to their specific GTP binding protein (G protein)coupled receptors activates an inwardly rectifying K+ channel (KACh channel)1 via the ß subunit of Gi/o proteins (
have been described in many earlier studies. Thus, in cell-attached patches of mammalian atrial cells, KACh channels activated by acetylcholine (ACh) typically show one open state with an open time constant of ~1 ms and a single-channel conductance of 3540 pS in symmetrical 140-mM KCl solution (
The absence of ATP-induced modification of KACh channels (i.e., long-lived openings and high open probability state) in cell-attached patches has previously led us to hypothesize that an inhibitor of ATP-dependent mechanism exists in the cell (
This study reports the successful identification of the endogenous inhibitor and its effect on KACh channel kinetics. The inhibitor was determined to be a lipid molecule in the cytoplasm. Tests of various lipid molecules on the KACh channel function showed that only unsaturated free fatty acids such as oleic, linoleic, and arachidonic acids were potent inhibitors of ATP-dependent gating. These findings indicate that downregulation of the KACh current by cytoplasmic unsaturated free fatty acids, achieved by interfering with ATP-mediated changes in KACh channel gating, is an important mechanism for the control of heart rate and synaptic transmission in the brain.
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MATERIALS AND METHODS |
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Cell Preparation
Cultured atrial cells from 1-d-old newborn rats were prepared as described previously (
Electrophysiology
Gigaseals were formed using Sylgard-coated, thin-walled borosilicate pipettes (Kimax) with ~4 M resistances. Channel currents were recorded with an Axopatch 200 patch-clamp amplifier (Axon Instruments), digitized with a PCM adapter (VR10; Instrutech Corp.), and stored on video tape using a video tape recorder. The recorded signal was filtered at 3 kHz using an eight-pole Bessel filter (-3 dB; Frequency Devices Inc.) and transferred to a Dell computer using the Digidata 1200 interface (Axon Instruments) at a sampling rate of 20 kHz. The filter dead time was ~100 µs (0.3/cutoff frequency, and therefore events <50 µs will be missed in our analysis. Continuous single-channel currents were then analyzed with the pClamp program without further filtering (Version 6.0.3). Data were analyzed to obtain amplitude histogram and channel activity (nPo). n is the number of channels in the patch, and Po is the probability of a channel being open. nPo was determined from 12 min of channel recording. Current tracings shown in figures were filtered at 100 Hz except for expanded tracings, which were filtered at 1 kHz. Data are represented as mean ± SD. Student's t test was used to test for significance between two values at the level of 0.05.
Single Channel Analysis
As every patch contained multiple openings, a maximum likelihood algorithm was used to determine single channel kinetic parameters from idealized patch clamp data (
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Preparation of Cytosol
Tissues (~2 g) were homogenized for ~30 s in ice using a polytron in 5 ml HEPES-buffer containing 50 mM HEPES, 5 mM EDTA, pH 7.4, 0.5 mM dithiothreitol, and 0.1 mM PMSF, and then centrifuged at 3,300 g for 20 min at 4°C (J2-21M, rotor JA-21; Beckman Instruments, Inc.). The supernatant was collected and mixed with same volume of cytoplasmic extract buffer containing 140 mM KCl, 3.0 mM MgCl2, and 30 mM HEPES, pH 7.9. This mixture was centrifuged at 100,000 g for 1 h using an L-60 ultracentrifuge (rotor, 70-TI; Beckman Instruments, Inc.). The supernatant was collected and placed in a dialysis tubing (molecular weight cutoff, 10 kD; Spectrum Medical Industries, Inc.) and dialyzed in 4 liters of solution containing 140 mM KCl, 2 mM MgCl2, 5 mM EGTA, 0.5 mM dithiothreitol, 0.1 mM PMSF, 1 mg/ml leupeptin and 1 mg/ml pepstatin A, and 10 mM HEPES, pH 7.2, for 12 h at 4°C. This cytosolic fraction (20 mg protein/ml) was then stored at -70°C. Protein concentration was determined by Bradford assay. The lipid component of the cytosolic fraction was prepared by vigorous mixing with the same volume of chloroform for ~1 min. After centrifugation, organic phase was transferred and evaporated completely under nitrogen. Perfusion solution was then added and the mixture sonicated for 5 min before applying to the patch.
Solutions and Materials
The pipette and bath solutions contained 140 mM KCl, 0.5 mM MgCl2, 10 mM HEPES, and 5 mM EGTA, pH 7.2. To change solutions perfusing the cytosolic surface of the inside-out patches, the pipette with the attached membrane was brought to the mouth of the polypropylene tubing through which the desired solution flowed at a rate of ~1 ml/min. For studies using ATP, amounts of MgCl2 and ATP were determined to produce desired concentrations of free Mg2+ and MgATP using EQCAL software (Biosoft). The free Mg2+ concentration in solutions was always kept constant at 0.5 mM. All experiments were performed at 2426°C. Free fatty acids were purchased from Sigma Chemical Co., dissolved in chloroform to form a 50-mM stock solution, and stored at -70°C under nitrogen. For free fatty acids supplied in dried powdered form, they were dissolved in either ethanol or DMSO, and subsequently sonicated in the perfusion solution at desired concentrations. ACh, GTP, and ATP were purchased from Boehringer Mannheim Biochemicals. Trypsin (porcine pancreas, Type 2), chymotrypsin, papain, and fatty acid-free albumin were purchased from Sigma Chemical Co. Purified bovine ß subunit and GTP
S were purchased from Calbiochem Corp. Phosphatidylinositol-4,5-bisphosphate (PIP2), phosphatidylcholine, and phosphatidylethanolamine were purchased from Sigma Chemical Co. Mono- and diacylglycerols were purchased from Biomol and Sigma Chemical Co., and dissolved in chloroform similar to free fatty acids. All other lipids were first dissolved in chloroform and kept at -80°C freezer. The solvent (chloroform) was evaporated and free fatty acids were dissolved by sonication (W-380; Heat Systems-Ultrasonics, Inc.) on ice for ~10 min in bath recording solution at a desired concentration. The final ethanol or DMSO concentration in the perfusion solution used was <0.1% and had no effect on the KACh channel function.
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RESULTS |
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Presence of an Endogenous Inhibitor of KACh Channel in the Cytoplasm
We first confirmed the marked difference in KACh channel gating kinetics observed in the presence and absence of intracellular ATP using both outside-out and inside-out patches. The potential role of intracellular ATP in agonist-induced KACh current has not been previously studied using outside-out patches. Fig 1 A shows current recordings from outside-out patches excised from atrial cells. Pipette (intracellular) solution contained GTP (100 µM), or both GTP and ATP (4 mM). These nucleotide concentrations are within the physiological levels found in mammalian heart cells. Extracellular application of 10 µM ACh resulted in activation of KACh channels in both cases as expected, but the channel activity (nPo) was approximately fivefold higher with ATP in the pipette solution than without it. In the presence of GTP alone, only a single open state with an open time constant of 1.0 ± 0.1 ms was sufficient to describe the channel openings. In the presence of GTP and ATP, an additional open state with longer-lived openings was detected (see Fig 2 B and 3). These results clearly confirm the profound effect of ATP on KACh channel gating that we described in earlier studies. To show that an inhibitor of ATP-dependent gating is present in the cytoplasm of atrial cells, cytosolic fraction (5 mg protein/ml) prepared from rat atrial tissues was included in the pipette along with GTP and ATP. In the presence of the cytosolic fraction, long-lived channel openings were no longer present, indicating that atrial cytosolic fraction contains a substance that inhibits ATP-dependent gating of the KACh channel, and thus reduces the magnitude of KACh channel activation by ACh.
As reported previously, the mean open time of the KACh channel in cell-attached patches was 1.0 ± 0.1 ms, and very few or no long-lived openings were observed, presumably due to the intact cytosol, which contains the substance that inhibits the ATP-dependent gating (Fig 1 B). In inside-out patches with ACh in the pipette, GTP applied to the bath solution activated KACh channels, and further addition of ATP (4 mM) produced an approximately fivefold increase in nPo, similar to that observed in outside-out patches. The [ATP]nPo relationship showed that K1/2 was 32 ± 8 µM (n = 4) and the Hill coefficient was 1.9 ± 0.2 (n = 3). The effect of ATP did not reverse after its washout for at least 10 min, provided that the patch gigaseals were formed with a mild suction pressure of less than ~3 mmHg. When the cytosolic fraction (5 mg protein/ml) was added together with GTP and ATP to the bath solution, ATP-induced long-lived openings were no longer observed and the channel activity decreased significantly (Fig 1 B). In agreement with this result, a rapid inhibition of the ATP effect was observed when the patch containing ATP-modified KACh channels was crammed through the plasma membrane and into the cytosol of a spherically shaped cultured atrial cell (Fig 1 B). The combined results from outside-out and inside-out patches are summarized in Fig 2A and Fig B, which shows changes in channel activity and open time durations, respectively. These results, particularly those obtained using outside-out patches, show that an inhibitor of ATP-dependent gating is present in atrial cytoplasm and that it plays a crucial role in keeping the KACh channel activity downregulated in the presence of an agonist.
As nearly all patches contained multiple openings, we were unable to determine open time durations using the pClamp programs. Therefore, the kinetic scheme shown in Fig 2 B was used to fit the channel data, from which transition rates between states and open time constants could be extracted (
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The Endogenous Inhibitor Is a Lipid Molecule
Our previous study suggested that the inhibitor might be a protein as treatment with proteases (with trypsin or -chymotrypsin for ~30 min, 37°C) abolished its effect on the ATP-induced changes in KACh channel kinetics. However, we now acknowledge that this suggestion was premature, as incubation of the cytosolic fraction in room air at 37°C for a prolonged period of time (~30 min) was also found to markedly reduce the modulatory activity of certain lipids on the KACh channel (
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Using an experimental protocol identical to that described in Fig 1 A, we tested whether the agonist-induced activation of KACh channels is also dependent on the presence of the lipid fraction. In outside-out patches, ACh-elicited KACh channel activity was markedly reduced after addition of the lipid extracted from atrial cytosolic fraction to the pipette, and this was again reversed by 50 µg/ml albumin (Fig 4 C). Similar results were obtained when the lipid fraction was prepared from brain cytosolic fraction. The combined results obtained from inside-out and outside-out patches are summarized in Fig 5. For kinetic analysis, we used patches containing two to four channel openings. As described above, the two kinetic schemes were used to obtain open time constants when ATP-induced gating was present. In the presence of cytosolic fraction or its lipid component, ATP-induced long-lived openings were absent and a single open state was sufficient to describe the channel kinetics. The currentvoltage relationship of the GTP-activated KACh channel was unaffected by ATP, cytosolic fraction, or the lipid component (Fig 4 B, inset). These results show that the endogenous factor that downregulates the KACh channel activity by interfering with the ATP-dependent modulation is a lipid substance that binds albumin.
Unsaturated Free Fatty Acids Inhibit ATP-dependent Gating
To identify the lipid molecule that inhibits ATP-dependent gating, we tested many different types of lipid molecules that are normally found in the cell (Table 1). A lipid molecule (0.150 µM) was applied to the cytoplasmic side of inside-out patches containing ATP-modified KACh channels and concentration-dependent changes in channel activity and open time constants were determined. From these experiments, we found that unsaturated free fatty acids such as oleic, linoleic, arachidonic (AA), eicosapentaenoic, and docosahexaenoic acids were good inhibitors of ATP-dependent gating and thus the channel activity (Fig 6). In each case, fatty acid-free albumin (50 µg/ml) was able to completely remove the inhibitory effect of free fatty acids. Linoleic acid produced a similar inhibitory effect when KACh channels were activated with purified bovine brain ß subunit (20 nM; Fig 6 D), showing that Gi
and their downstream signaling molecules were not involved. Fig 7 A shows concentration-dependent effects of four free fatty acids, a diacylglycerol, and the lipid derived from brain cytosol. The four unsaturated free fatty acids (oleic, linoleic, arachidonic, and docosahexaenoic acids) inhibited KACh channel activity with a K1/2 of ~1.5 µM. The Hill coefficients were ~2 (1.82.2), similar to that obtained with the cytosolic lipid, indicating that at least two fatty acid molecules bind the channel or an associated regulatory molecule. The effect of free fatty acids on open time constants calculated from transition rates are shown in Fig 7 B. Free fatty acids completely inhibited the appearance of the long-open state even in the presence of 4 mM ATP. As expected from the results shown in Fig 6, when ß
-activated KACh channels were exposed to linoleic or AA (2 µM) first, further addition of 4 mM ATP produced only a small increase in channel activity (1.3 ± 0.1-fold; n = 5; not shown). AA, oleic acid, and linoleic acid (5 µM) added to the pipette solution (extracellular) did not inhibit ATP-dependent gating even after 5 min (n = 4 each). However, when a higher concentration (50 µM) of oleic acid was used, a partial inhibition (65 ± 6%; n = 6) of ATP-dependent gating was present after 5 min, indicating that enough free fatty acids probably crossed the lipid bilayer to reach the intracellular site to inhibit the KACh channel.
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AA produced the same inhibitory effect in the presence of indomethacin (50 µM; cyclooxygenase inhibitor) or nordihydroguaiaretic acid (10 µM; lipoxygenase inhibitor), indicating that AA itself is the inhibitor (n = 5 each). This is supported by the results that fatty acids that are not substrates for cyclooxygenases or lipoxygenases (i.e., oleic acid) are also potent inhibitors of ATP-dependent gating. Therefore, free fatty acids probably acted directly rather than via their metabolites. Treatment of patches with calphostin-C (protein kinase C inhibitor, 1 µM, 10 min), neomycin (phospholipase C inhibitor, 50 µM, 15 min), or pirenzepine (M1 receptor antagonist, 0.1 µM) also did not affect the inhibition by AA (n = 5 each). These results show that the two enzymes that can be activated by free fatty acids (
Interestingly, synthetic diacylglycerols and several monoacylglycerols were also effective in inhibiting the ATP-dependent gating, albeit at higher concentrations (Table 1). Their inhibitory effect, however, could not be easily removed by washout or fatty acidfree albumin (100 µg/ml). The inhibitory effect of diacylglycerols and monoacylglycerols could be due to long-chain fatty acids present in these molecules. The naturally occurring diacylglycerols (steroyl-arachidonoyl-glycerol and steroyl-linoleoyl-glycerol), however, showed no inhibitory effect even at 100 µM. Methyl ester and alcohol derivatives of arachidonic acid also caused inhibition of KACh channel activity at relatively high concentrations, indicating that the free carboxyl group is not necessary for inhibition of ATP-dependent gating (Table 1). Polyamines (
Unsaturated Free Fatty Acids Inhibit PIP2- and Phospholipid-dependent Gating
It has been reported that the ATP-induced change in K+ channel function may occur via generation of PIP2 in the membrane as a result of phosphorylation of PI and PIP by lipid kinases (o1, 23 ms) and KACh channel activity (
S to inside-out patches, 30 µM PIP2 was further added. This resulted in approximately twofold increase in channel activity and the appearance of a second open state with an open time constant of 2.6 ± 0.3 ms (Fig 8). The effect of PIP2 on the KACh channel was always small compared with that produced by ATP, which produces an approximately fivefold increase in channel activity and
o1 of 78 ms. Nevertheless, when AA (2 µM) was applied together with PIP2, AA fully reversed the effect of PIP2 (Fig 8B and Fig C). Although not shown, oleic (2 µM) and linoleic (2 µM) acids also inhibited PIP2-dependent gating of the KACh channel (n = 3 each).
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As phosphatidylcholine (100 µM) and phosphatidylethanolamine (100 µM) have been shown to produce effects similar to PIP2 on KACh channel kinetics (o1 values were 2.5 ± 0.6 and 2.8 ± 0.5 ms, respectively (n = 3). After application of 2 µM AA, only one open state with a time constant of 0.9 ± 0.1 ms was present for both phospholipids. Application of 2 µM linoleic acid resulted in a similar inhibition such that the open time constant was 0.8 ± 0.1 ms (n = 3). Therefore, our results show that unsaturated free fatty acids inhibit the appearance of long-lived openings; i.e., abolish the second open state, regardless of which substance was used to induce it.
Concentration-dependent Effect of Arachidonic Acid on KACh Channel Kinetics
To understand in more detail the effect of a free fatty acid on the KACh channel function, we determined the concentration-dependent effect of AA on transition rates between all connected states in our kinetic scheme. As shown in Fig 9, KACh channel openings in the cell-attached or in the inside-out state with GTP alone could be described by a linear kinetic scheme with only one open state. The averaged transition rates determined using the QuB analysis from three cells with similar channel activities are shown on the right. Under the experimental conditions of our study, the channel activity in the inside-out patch with 100 µM GTP alone was always lower than that in the cell-attached patch from the same cell. However, the open time constant remained unchanged from that in the cell-attached state (0.91.0 ms). Application of 4 mM ATP produced an additional open state with long-lived openings (O1) and also increased the transition rate to the longest closed state (C1). The latter accounts for the many long closings observed in the presence of ATP in inside-out patches, with a mean closed time of 62 ms. At 0.5 µM AA, the primary effect was a decrease in the open time constant (O1; Fig 10 A), without a change in the transition rate to the longest closed state (C1). At higher AA concentrations, the transition rate to C1 was greatly reduced, in addition to a further decrease in the open time constant (O1). Transition rates between all connected states were affected by AA, indicating that AA acts on both closed and open states. The results show clearly that 4 mM ATP and 2 µM AA together can reestablish the channel kinetics observed in the cell-attached state. A mixture containing 0.5 µM each of oleic acid, linoleic acid, AA, and docosahexaenoic was as effective as 2 µM AA. The effect of AA was voltage-insensitive (Fig 10 B). Furthermore, the K1/2 values for inhibition by linoleic acid with pipette solution containing 70, 140, and 210 mM K+ were 1.6 ± 0.4, 1.5 ± 0.3, and 1.7 ± 0.4 µM, respectively (n = 3 each). Therefore, the fatty acid effect is unlikely to involve sites within the pore of the channel.
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Inhibition of KACh Channel Activity by Arachidonic Acid in the Absence of ATP
The inhibition of the ATP-induced increase in mean open time and activity of the KACh channel by unsaturated free fatty acids could be due to a selective block of the ATP-induced effect on the KACh channel. It could also be due to a nonselective effect, affecting the KACh channel regardless of whether the channel is in the ATP-modified state or not. To address this issue, we studied the effect of AA on GTP-activated KACh channels in inside-out patches with ACh in the pipette in the absence of ATP. As described previously, application of 100 µM GTP to the cytoplasmic side of the membrane caused an immediate activation of the KACh channels (Fig 11 A). AA was then applied to the membrane starting at 0.1 µM and the concentration was increased progressively to 10 µM. Channel activity and mean open times were determined and plotted as a function of AA concentration (Fig 11C and Fig D). The concentration at which channel activity decreased by half was 1.6 ± 0.4 µM and the Hill coefficient was 1.7. These values are very close to those obtained from patches in which the KACh channels were studied in the presence of ATP (K1/2 = 1.7 µM, Hill coefficient = 1.9). AA also caused a significant shortening of the open time duration in the absence of ATP, but only at high concentrations of the free fatty acid. Similar inhibitory effects were observed when purified bovine ß (100 nM) was used to activate the KACh channel (K1/2 = 1.5 µM, Hill coefficient = 1.9; mean of two values; data not shown), suggesting that AA may be interfering with ß
-KACh channel interaction to reduce channel activity. The approximately eightfold decrease in the open time constant (
o1) of the ATP-modified KACh channel produced by AA cannot easily be explained by such a mechanism, as 1 µM AA has a negligible effect on the open time constant in the absence of ATP. Changes in [GTP] and, by inference, the amount of ß
available for interaction with the KACh channel, does not affect the open time constant significantly in atrial cells. Therefore, these results suggest that free fatty acids produce two separate effects: a decrease in frequency of opening and inhibition of ATP-induced long-lived openings. Both of these mechanisms are probably involved in AA-induced decrease in channel activity.
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Effect of Free Fatty Acids on the KACh Channel in Hippocampal Neuron and in Oocytes Expressing GIRK
To determine whether free fatty acids also downregulate KACh channels in neurons and cloned KACh channels expressed in oocytes, we tested their effects on KACh channels in hippocampal pyramidal neurons and in oocytes expressing GIRK1/4 and GIRK1/2. In neurons, 5-hydroxytryptamine (5-HT) was used as agonist to activate the KACh channel as we have done previously (
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When expressed in Xenopus oocytes, heteromeric GIRK1/4 that represents the atrial KACh channel also undergoes ATP-dependent gating (
Free Fatty Acids Do Not Inhibit Background (K1) and ATP-sensitive K+ Channels
Other members of the Kir family of K+ channels including the classical background (IRK, K1) and ATP-sensitive K+ channels (Kir6/SUR and ROMK) have also been shown to be activated by ATP or PIP2 (
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To test the effect of AA on KATP channels, we used inside-out patches formed from adult rat ventricular cells that usually show many KATP channels in each patch. In Fig 13 B, six KATP channels were activated upon formation of the inside-out state. In this patch, KATP channel rundown was slow and allowed us to test the effect of AA. AA (5 µM) caused a marked inhibition of the KATP channel activity, and this was irreversible. Although 2 µM AA caused a significant inhibition (62 ± 12% inhibition, n = 3), we used 5 µM AA to obtain a near-maximal inhibition. Linoleic acid (5 µM) also caused a near maximal inhibition. These results show that AA is an effective inhibitor of the KATP channel, as we have described previously (
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DISCUSSION |
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Since 1991, when intracellular ATP was first shown to cause marked upregulation of the KACh channel activity in inside-out patches and to increase the number of long-lived openings (
Are Unsaturated Free Fatty Acids True In Vivo Inhibitors of ATP-dependent Gating?
It would be important to know whether free fatty acids in the cytoplasm are true in vivo inhibitors of the KACh channel. The following points support the role of endogenous free fatty acids in the control of the KACh channel function. (a) Studies using the cytosolic fraction and its lipid component clearly show that an endogenous lipid substance is inhibiting the ATP-dependent gating of the KACh channel. (b) Of the ~40 different lipophilic compounds tested so far, only unsaturated free fatty acids were found to be potent inhibitors of ATP-dependent gating of the KACh channel with inhibitory properties indistinguishable from that of the cytosolic fraction or its lipid fraction. In earlier studies, intracellular molecules such as cAMP, cGMP, Ca2+, and inositol trisphosphate at physiological concentrations were found to have no significant effect on the KACh channel activity (
The results of this study suggest that the cytoplasmic concentration of a free fatty acid that inhibits ATP-dependent gating is 12 µM. The actual concentration of free fatty acids that inhibits the ATP effect may be much lower, as oxidation of fatty acids probably occurs during preparation and experiment. In support of this, it was found that the effect of AA decreased significantly (K1/2 = 5.5 ± 1.1 µM, n = 4) after ~10 min exposure of the AA solution to air and, after 1 hr, K1/2 was 12.2 ± 3.4 µM (n = 3), although all solutions were kept in ice. We also found that incubation of oleic, linoleic, and linolenic acids at room temperature or at 37°C for 30 min also reduced their inhibitory potency significantly. Different types of fatty acid binding proteins exist at high levels in the cell (~1 mM), and bind fatty acids with high affinity (Kd, ~110 µM;
Structural Requirements for Modulation of the KACh Channel by Free Fatty Acids
The inhibitory profile of free fatty acids shows that a long-chain fatty acid with at least one double bond is necessary for blocking ATP-dependent gating. Trans isomers (eladic and linoelaidic acids) were less potent than their cis counterparts, suggesting that some structural elements within the hydrophobic region may be important. Uncharged alcohol and methyl ester forms of AA inhibited ATP-dependent gating irreversibly, suggesting that the carboxyl group of free fatty acids is important for reversibility but is not necessary for inhibition. This supports the idea that the hydrophobic region of free fatty acids may be important for causing inhibition. Mono- and diacylglycerols were also found to be effective inhibitors of ATP-dependent gating, albeit at higher concentrations. The inhibition by mono- and diacylglycerols may be due to the fatty acids in these molecules and their low affinity may be explained by the reduced accessibility due to steric hindrance. Although these synthetic diacylglycerols showed inhibitory effect, naturally occurring diacylglycerols (steroyl-arachidonoyl glycerol and steroyl-linoleoyl-glycerol) were found to have no effect, presumably due to greater steric hindrance. Therefore, these results suggest that all unsaturated free fatty acids may be able to downregulate the KACh channel by inhibiting the ATP-dependent gating. It seems likely, however, that commonly found free fatty acids with high inhibitory potency, such as oleic, linoleic, linolenic, and arachidonic acids, are probably the major players in downregulating the KACh channel activity. The relative lack of effect of free fatty acids from the extracellular side is interesting, considering that fatty acids are thought to cross the membrane rapidly. Therefore, free fatty acids in the plasma are not expected to regulate KACh channel function to any significant degree. Fatty acids in plasma are also mostly bound to albumin (
Mechanism of Inhibition of KACh Channel Activity by Free Fatty Acids
Our results show that the observed channel activity in cell-attached patches at steady state when ACh is present in the pipette is the sum of the effects produced by GTP (ß)-induced activation, ATP-induced stimulation, and free fatty acidinduced inhibition. The inhibitory effect of free fatty acid on ATP-dependent gating seems to be the crucial factor that keeps the KACh channel in the short-lived, single open state and maintains low channel activity despite the presence of ATP in the cell. To understand how free fatty acids inhibit ATP-dependent gating, it is first necessary to identify the signaling pathway by which ATP modifies the KACh channel. The evidence from recent studies indicate that PIP2 formed in the membrane via lipid kinases mediates the effect of intracellular ATP on different ion transporters and channels (
If PIP2 promotes the active state for all inward-rectifier K+ channels mentioned above by a common mechanism, a blocker of the PIP2 effect would be expected to reduce the active state of these K+ channels. Therefore, we predicted that if unsaturated free fatty acids inhibit ATP- and PIP2-dependent gating of KACh channels, they would also inhibit ATP- or PIP2-induced modification of KATP and K1 channels. Our results in Fig 13 show, however, that AA (25 µM) does not inhibit PIP2-modified KATP channels or ATP-activated background K1 channels. This suggests that the mechanism of ATP- or PIP2-induced effect on the KACh channel may be different from that on K1 and KATP channels. However, recent studies measuring the kinetics of K+ current inhibition by PIP2 antibody show that PIP2channel interaction is strong for IRK1 and ROMK1, and weak for GIRK ( causes stabilization of the PIP2-KACh channel interaction that may be directly responsible for channel activation. Thus, the level of PIP2 in the membrane is now believed to be the critical factor that determines the degree of channel activation and open time kinetics when ß
is applied. At a low basal level of PIP2 (no applied ATP), activation by ß
is small and shows only short-lived openings, whereas at high PIP2 levels (applied ATP), channel activity is increased and long-lived openings are induced. Therefore, a likely mechanism by which free fatty acids inhibit the KACh channel activity and shorten the open time duration either in the presence or absence of ATP may involve a reduction of the PIP2channel interaction.
Interestingly, the KACh channels in bullfrog atrial cells show many long-lived openings (bursts) even in the absence of ATP ( subunit (
on the open time duration of KACh channels in mammalian atrial cells show that the ATP effect is far greater than that of ß
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Physiological Significance of the Free Fatty Acid Effect on the KACh Channel
The existence of the dual and opposite effect of intracellular ATP and certain unsaturated free fatty acids on the KACh channel is interesting and curious. Since these molecules provide energy to the cell, it is plausible that the KACh channel activity is influenced by the metabolic state, particularly by the fatty acid metabolism. Small changes in total free fatty acid concentration near 12 µM could in principle have a significant effect on KACh channel activity. It is also possible that free fatty acid concentrations are kept at a constant level and do not modulate the KACh channel. Nevertheless, it is clear that without the inhibitory action of free fatty acids, the KACh channels would be in the "high open probability" state due to the ATP-induced increase in channel activity and induction of long-lived openings. Single-channel studies show that in oocytes expressing GIRK1/4, the channels in the cell-attached state are already in the ATP-modified gating mode and show long-lived openings (
In previous studies from our laboratory, it was found that long-lived openings can be observed during the initial several seconds upon formation of cell-attached patches with ACh in the pipette (
It has been reported recently that endothelin causes inhibition of GIRK when endothelin receptor and GIRK channels are expressed in oocytes (
Arachidonic Acid, Free Fatty Acids and Ion Channel Function
In 1987, AA and its lipoxygenase metabolites were reported to act as second messengers in the modulation of K+ channels in Aplysia neurons (S activation of the KACh current while decreasing the ACh-activated current (
The role of free fatty acids in ion channel regulation is increasingly recognized as many ion channels are affected by free fatty acids. AA and other long-chain free fatty acids have been shown to affect Na+, Ca2+, and K+ channels in various cell types in different ways (
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Footnotes |
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Dr. Pleumsamran's current address is Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand.
1 Abbreviations used in this paper: 5-HT, 5-hydroxytryptamine; AA, arachidonic acid; ACh, acetylcholine; KACh channel, G proteingated channel; PIP2, phosphatidylinositol-4,5-bisphosphate.
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Acknowledgements |
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The authors thank Drs. L.Y. Jan, H.A. Lester, and D.E. Clapham for providing GIRK1, GIRK2, and GIRK4 clones, respectively.
This work was supported by grants from the National Institutes of Health and the American Heart Association.
Submitted: 8 October 1999
Revised: 24 January 2000
Accepted: 25 January 2000
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References |
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