Ca2+ Selectivity and Fatty Acid Specificity of the Noncapacitative, Arachidonate-regulated Ca2+ (ARC) Channels*

Olivier MignenDagger, Jill L. Thompson, and Trevor J. Shuttleworth§

From the Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642

Received for publication, December 9, 2002, and in revised form, January 8, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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The arachidonate-regulated, Ca2+-selective ARC channels represent a novel receptor-activated pathway for the entry of Ca2+ in nonexcitable cells that is entirely separate from the widely studied store-operated, Ca2+ release-activated Ca2+ channels. Activation of ARC channels occurs specifically at the low agonist concentrations typically associated with oscillatory Ca2+ signals and appears to provide the predominant mode of Ca2+ entry under these conditions (Mignen, O., Thompson, J. L., and Shuttleworth, T. J. (2001) J. Biol. Chem. 276, 35676-35683). In this study we demonstrate that ARC channels are present in a variety of different cell types including both cell lines and primary cells. Examination of their pharmacology revealed that currents through these channels are significantly inhibited by low concentrations (< 5 µM) of Gd3+, are unaffected by 100 µM 2-aminoethyoxydiphenyl borane, and are not activated by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (100 µM). Their selectivity for Ca2+ was assessed by determining the EC50 for external Ca2+ block of the monovalent currents observed in the absence of external divalent cations. The value obtained (150 nM) indicates that the Ca2+ selectivity of ARC channels is extremely high. Examination of the ability of various fatty acids, including arachidonic acid, to activate the ARC channels demonstrated that activation does not reflect any nonspecific membrane fluidity or detergent effects, shows a high degree of specificity for arachidonic acid over other fatty acids (especially monounsaturated and saturated fatty acids), and is independent of any arachidonic acid metabolite. Moreover, studies using the charged analogue arachidonyl coenzyme A demonstrate that activation of the ARC channels reflects an action of the fatty acid specifically at the internal face of the plasma membrane. Whether this involves a direct action of arachidonic acid on the channel protein itself or an action on some intermediary molecule is, at present, unclear.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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The generation and shaping of [Ca2+]i signals in nonexcitable cells are profoundly influenced by the receptor-activated entry of Ca2+. At high agonist concentrations, this entry determines the sustained elevated level of [Ca2+]i achieved after the inositol 1,4,5-trisphosphate-dependent discharge of the internal agonist-sensitive Ca2+ stores. It is also responsible for the refilling of those stores on termination of the signal (1). At lower agonist concentrations the induced entry of Ca2+ acts, along with generated inositol 1,4,5-trisphosphate, to initiate and/or drive the characteristic oscillatory changes in [Ca2+]i generally observed and to modulate their frequency (2-6). Until recently, the entry of Ca2+ under both these conditions was believed to occur via a single pathway whose activation was entirely dependent on the emptying of the internal Ca2+ stores (1, 7). Although several distinct conductances may be responsible for this store-operated or capacitative pathway in different cell types, the most thoroughly characterized are the so-called Ca2+ release-activated Ca2+ (CRAC)1 channels first described in mast cells and in Jurkat cells (8, 9). These are identified as very low conductance, highly Ca2+-selective, channels whose gating is independent of voltage but entirely dependent on depletion of internal Ca2+ stores (10-12). Such CRAC channels, or at least conductances displaying very similar properties, have since been described in a wide variety of different cell types. Their molecular identity and precise mechanism of activation, however, remain unclear.

Despite the apparent ubiquitous nature of store-operated Ca2+ entry, recent evidence has indicated that other, noncapacitative pathways for the receptor-activated entry of Ca2+ exist in various cells. In particular, a Ca2+ entry pathway that is independent of store-depletion and is regulated by receptor-activated increases in arachidonic acid has been identified in a wide variety of different cell types (13-16). The conductance underlying this novel noncapacitative Ca2+ entry in HEK293 cells has been described recently (17) and defined as the arachidonate-regulated Ca2+ (ARC) channel. Patch clamp studies have shown that ARC channels and CRAC channels share many features including selectivity for Ca2+, a small macroscopic conductance, and voltage-independent gating (17). Despite this superficial similarity, biophysical analysis reveals that ARC and CRAC channels represent entirely distinct conductances with several unique characteristics, the most important of which is that activation of the ARC channels is entirely independent of store depletion (17-19). These findings have lead to the conclusion that ARC and CRAC channels represent co-existing, but independent, Ca2+ influx pathways.

Although pathways for the store-operated entry of Ca2+ have focused on the role of the CRAC channels, such entry in certain cell types appears to involve nonselective cation conductances that are clearly distinct from the Ca2+-selective CRAC channels. This raises the question whether the various arachidonic acid-dependent noncapacitative Ca2+ entry pathways that have been described may also reflect the activity of different conductances. This is of particular importance as it has become increasingly common for the properties of such a pathway in one cell type to be used as definitive identifiers of the presumed same pathway in another cell type. In the present study, we have first examined the presence of ARC channels in a range of cell types and have demonstrated that it appears to be a widely distributed conductance. Examination of certain pharmacological approaches commonly used to distinguish between store-operated and noncapacitative Ca2+ entry pathways in various cells revealed that they are of questionable validity and of only limited use in distinguishing between the relative contributions of ARC and CRAC channels in overall Ca2+ entry. Given this, and as the molecular identity of the ARC channels is as yet unknown, we considered it important to establish and characterize, wherever possible, those features and properties that define this novel conductance and distinguish it from other channels that may be present, either in the same cell or in different cell types. To this end, we have focused on the key defining features of ARC channels, namely their selectivity for Ca2+ and their specificity for arachidonic acid for activation.

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EXPERIMENTAL PROCEDURES
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Cell Culture-- Cells from the human embryonic kidney cell line HEK293 stably transfected with the human m3 muscarinic receptor (m3-HEK cells; generous gift from Dr. Craig Logsdon, University of Michigan) were cultured in Dulbecco's modified Eagle's medium with 10% calf serum and antibiotics in a 5% CO2 incubator at 37 °C as reported previously (15). Rat basophilic leukemia (RBL-1) cells, COS cells, and HeLa cells were all obtained from the American Type Culture Collection. COS cells were cultured in the same way as the m3-HEK cells except 10% fetal bovine serum was substituted for the calf serum. HeLa and RBL-1 cells were cultured in Eagle's medium with 10% fetal bovine serum and antibiotics. Cells from the chicken B-lymphocyte DT40 cell line (gift from Dr. David Yule, University of Rochester) were cultured in RPMI 1640 medium supplemented with 2 mM glutamine, 10% fetal bovine serum, 1% chicken serum, 50 µM beta -mercaptoethanol, and antibiotics. Mouse parotid acinar cells were isolated from freshly dissected parotid glands by sequential digestion with trypsin and collagenase P as described by Bruce et al. (20).

Whole-cell Patch Clamp Recording-- All cells were plated on glass coverslips that formed the bottom of a patch clamp chamber (Warner Instruments, Hamden, CT) at least 4 h before experimentation. Patch clamp recordings of macroscopic whole-cell currents were performed using an Axopatch-1C patch clamp amplifier (Axon Instruments, Foster City, CA) as described previously (17, 19). Patch pipettes had resistances of 3-6 megohms when filled with standard internal solution. Whole-cell currents were recorded using 250-ms voltage steps to -80 mV from a holding potential of 0 mV delivered every 2 s. Current-voltage relationships were recorded either by using 150-ms voltage ramps from -100 to +30 mV or by pulsing to a series of potentials between -100 mV and +30 mV at 10- or 20 mV-intervals. Data were sampled at 20 kHz during the voltage steps and at 5.5 kHz during the voltage ramps and digitally filtered off-line at 1 kHz. Initial current-voltage relationships obtained immediately upon going whole cell (i.e. before activation of IARC) were averaged and used for leak subtraction of subsequent current recordings. Changes in the external (bath) solution were by perfusion of solution through the patch clamp chamber (rate of ~1.5 ml/min). All experiments were carried out at room temperature (20-22 °C). The standard pipette (internal) solution contained the following (in mM): cesium acetate, 140; NaCl, 10; MgCl2, 1.22; CaCl2, 1.89; EGTA, 5; HEPES, 10; pH 7.2, unless otherwise specified. The free Ca2+ concentration of this solution was calculated to be 100 nM as computed with Maxchelator (21). The standard extracellular solution contained the following (in mM): NaCl, 140; MgCl2, 1.2; CaCl2, 10; CsCl, 5; D-glucose, 10; HEPES, 10; pH 7.4, unless otherwise specified. For the experiments examining the inhibition of monovalent currents by external Ca2+ and Mg2+, the extracellular solution used was as follows (in mM): NaCl, 140; CsCl, 10; HEDTA, 2; D-glucose, 20; HEPES, 10, pH 7.4. Ca2+ or Mg2+ was added to this as appropriate to obtain a series of concentrations ranging from 0.1 to 100 µM (Ca2+) or 1.2 mM (Mg2+) as calculated using Maxchelator. For Ca2+ concentrations below 0.5 µM the external solution was supplemented with 2 mM EGTA. In these same experiments, the pipette solution used contained the following (in mM): cesium acetate, 140; MgCl2, 8; CaCl2, 1.6; EGTA, 5; HEPES, 10; pH 7.2) so as to eliminate any contribution from the MagNuM (or MIC) channels (see "Results" for details). The free Ca2+ concentration of this solution was calculated to be 100 nM as computed with Maxchelator. Analysis of the Ca2+ and Mg2+ inhibition curves and determination of EC50 values was performed using Origin software (Microcal, Northampton, MA). Where applicable, data are given as means ± S.E.

[Ca2+]i Determinations-- Single cell measurements of changes in [Ca2+]i were carried out as described previously (13).

Materials-- Arachidonic, linolenic, linoleic, eicosatetraynoic acids, and 1-oleoyl-2-acetyl-sn-glycerol were purchased from BioMol (Plymouth Meeting, PA). 2-APB was obtained from Calbiochem (San Diego, CA). All other chemicals and drugs were purchased from Sigma.

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INTRODUCTION
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Cell Distribution-- Although an arachidonic acid-dependent noncapacitative entry of Ca2+ has been described in a wide variety of different cells, to date the detailed characterization of the ARC channels has only been reported for HEK293 cells stably transfected with the human m3 muscarinic receptor (m3-HEK cells) (17). It therefore remains uncertain whether these channels are also likely to provide the route for this mode of entry of Ca2+ in other cell types. To address this, we examined a range of different cells for the presence of arachidonic acid-activated Ca2+-selective conductances displaying the specific characteristics of IARC as defined previously. We were able to successfully identify such a conductance in all the cell types examined, including HeLa cells, RBL-1 cells, COS cells, DT40 cells, and freshly isolated mouse parotid acinar cells (Fig. 1). In all these cells, currents activated promptly on addition of 8 µM arachidonic acid to the bath. The resulting whole-cell Ca2+ current densities measured under standard conditions at -80 mV ranged from 0.52 pA/pF in RBL-1 cells to 1.39 pA/pF in HeLa cells. These currents displayed all the key features associated with ARC channels (17) including marked inward rectification, very positive (>+30 mV) reversal potential, inhibition by La3+ (50 µM), absence of any fast inactivation, and, most critically, an activation that was specifically dependent on low concentrations of arachidonic acid and entirely independent of store depletion.


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Fig. 1.   IARC in different cell types. A, mean values of the arachidonic acid-activated (8 µM) currents measured at -80 mV in different cells types. Currents were measured in the standard extracellular solution. B, representative current-voltage relationships of the currents activated by addition of arachidonic acid (8 µM) in HeLa cells (), DT40 cells (open circle ), and freshly isolated mouse parotid acinar cells (black-square). Currents were measured in the standard extracellular solution, and steady-state currents were determined during pulses to the indicated voltages.

Pharmacology of ARC Channels: Sensitivity to Gd3+, 2-APB, and Diacylglycerols-- Superficially, macroscopic currents through the noncapacitative ARC channels share many of the biophysical features displayed by the store-operated CRAC channels, and discriminating between the two conductances requires more detailed analysis of such features as the presence or absence of fast inactivation etc. (17, 22). Of course, physiologically, currents through ARC channels can be distinguished by their critical dependence on arachidonic acid for activation and their independence from store depletion. However, it is often much more convenient to use pharmacological approaches to identify specific Ca2+ entry pathways. In this context, several recent studies have made extensive use of the lanthanide gadolinium and the drug 2-APB as a means to distinguished between putative noncapacitative Ca2+ entry pathways and store-operated pathways. Thus, Gd3+ at 1 µM and 2-APB at 100 µM are typically regarded as selectively inhibiting the capacitative entry of Ca2+ in a variety of different cell types (16, 23), with the implicit assumption that these agents do not inhibit noncapacitative Ca2+ entry pathways. However, such distinctions are largely based on studies of Ca2+ entry using fluorescent methods, and their effects on the noncapacitative ARC channels has never been reported. We therefore examined the effects of Gd3+ and 2-APB on the macroscopic Ca2+ currents through ARC channels activated by 8 µM arachidonic acid. The data show that 1 µM Gd3+ induces a significant, but incomplete, inhibition of Ca2+ currents through the ARC channels (Fig. 2A). The magnitude of this inhibition measured at voltages between -100 mV and -20 mV averaged 47.5% (range, 30 to 70%). At potentials more positive than -20 mV, the currents were too small to reliably estimate any differences. At a concentration of 5 µM, Gd3+ completely inhibited all currents through the ARC channels (Fig. 2A). The inhibitory effect of 1 µM Gd3+ on Ca2+ entry through the ARC channels was confirmed in fluorescence measurements of arachidonic acid-induced increases in cytosolic Ca2+ (Fig. 2B). As for 2-APB, concentrations >30 µM have been shown to block currents through CRAC channels in various cell types (24-26). In contrast, application of 100 µM 2-APB had no significant effect on macroscopic ARC currents that had been activated by addition of arachidonic acid (8 µM) (Fig. 2C). Prior addition of 2-APB also failed to influence the ability of arachidonic acid to activate the ARC currents. We conclude that 2-APB, at a concentration that has been shown to completely inhibit currents through CRAC channels in several cell types, has no significant effect on the Ca2+ currents through the noncapacitative ARC channels.


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Fig. 2.   Effect of Gd3+, 2-APB, and OAG on currents through ARC channels. A, the effect of Gd3+ on IARC. Average currents activated by 8 µM arachidonic acid under normal conditions in the absence of Gd3+ () or in the presence of 1 µM Gd3+ (open circle ) or 5 µM Gd3+ () (n = 6, 8, and 3, respectively). Steady-state currents were measured in the standard extracellular solution and determined during pulses to the indicated voltages. B, representative trace showing the effect of Gd3+ (1 µM) on the increase in cytosolic Ca2+ induced by exogenous addition of 8 µM arachidonic acid (indicated by arrow). Changes in cytosolic Ca2+ were measured as the 405/485 fluorescence ratio of intracellular indo-1. C, the effect of 2-APB on IARC. Average currents activated by 8 µM arachidonic acid in the presence (open circle ) and absence () of 100 µM 2-APB are shown (n = 5 and 8, respectively). Experimental conditions were as in A. D, the effect of OAG on IARC. Mean steady-state currents recorded during 250-ms pulses to -80 mV following addition of 100 µM OAG to the bath (n = 7; filled bar) and after the subsequent addition of 8 µM arachidonic acid (n = 4; hatched bar) are shown.

Although the molecular identity of both ARC channels and CRAC channels is unknown, it has been widely suggested that likely candidates for the latter are the TRPC family of channels. Interestingly, three closely related members of the TRPC family of ion channels, TRPC3, TRPC6, and TRPC7, have been shown to be activated in a manner that is independent of store depletion (i.e. noncapacitative). This noncapacitative mechanism apparently involves an action of diacylglycerol that is independent of the activation of PKC and can be mimicked by diacylglycerol analogues such as OAG (27, 28). This raises the question of whether a similar mechanism might underlie activation of the noncapacitative ARC channels. We therefore examined whether OAG was able to activate ARC channels. Addition of OAG (100 µM) to cells consistently failed to activate significant IARC (mean inward current at -80 mV was 0.09 ± 0.02 pA/pF, n = 7) (Fig. 2D). This lack of a significant effect was not because of any problem with the channels themselves, as the subsequent addition of arachidonic acid (8 µM) produced a normal activation of IARC (mean inward current at -80 mV = 0.54 ± 0.08 pA/pF, n = 4). The data obtained clearly demonstrate that ARC channels are insensitive to OAG.

Ca2+ Ion Selectivity-- Like the CRAC channels of T-lymphocytes and of mast cells, ARC channels are defined as highly Ca2+-selective conductances. Consistent with this, currents through the ARC channels are dependent on the presence of external Ca2+; they show a marked inward rectification at negative membrane potentials and a reversal potential significantly more positive than +30 mV (17). Moreover, complete substitution of extracellular Na+ with NMDG+ has no significant effect on the observed current-voltage relationship (Fig. 3A), indicating a negligible permeability for Na+ under normal conditions. Similar to other highly Ca2+-selective conductances, including both voltage-gated Ca2+ channels and CRAC channels, complete removal of extracellular divalent cations reveals a significant permeability to monovalent cations through ARC channels (19). Based largely on extensive study of this phenomenon in voltage-gated Ca2+ channels, this is believed to reflect the effects of a Ca2+-binding site within the channel pore (29, 30). Occupation of this site by Ca2+ precludes the permeation of monovalent ions, and such permeation only becomes possible when this site is vacant.


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Fig. 3.   Ca2+ and Mg2+ selectivity of ARC channels. A, the effect of complete replacement of external Na+ with NMDG+ on IARC. Average currents activated by 8 µM arachidonic acid measured in the standard extracellular solution (; n = 8), compared with those measured in a modified extracellular solution in which Na+ had been replaced with equimolar NMDG+ (open circle ; n = 3), are shown. Steady-state currents were determined during pulses to the indicated voltages. B, average currents (n = 6) activated by 8 µM arachidonic acid in a divalent-free external solution containing 2 mM HEDTA and 2 mM EGTA (see "Experimental Procedures" for details). Steady-state currents were determined during pulses to the indicated voltages. Under these conditions, currents through the ARC channels are carried by monovalent cations. C, the effect of increasing external Ca2+ () and Mg2+ (open circle ) concentrations on monovalent currents through ARC channels measured at -80 mV (n = 3-6) in each case. The dashed lines represent logistic fits of the data as determined using Origin software (Microcal).

Although these common features indicate a significant Ca2+ selectivity for these different channel types, they provide little information on their selectivity relative to each other. One way in which the relative selectivity of these channels for Ca2+ can be compared is by determining the affinity of the putative Ca2+ site responsible for the inhibition of monovalent permeation through the channel. We therefore examined this for ARC channels by determining the magnitude of the macroscopic monovalent currents in m3-HEK cells at different external Ca2+ concentrations. It should be noted that recent data have indicated that similar studies on the monovalent permeation through CRAC channels were probably significantly distorted by the presence of an additional conductance that is activated in the absence of internal Mg2+ (via the so-called MagNuM or MIC channels (31, 32). In our previously published data, we could detect no significant contribution from such contaminating conductances as the magnitude of the monovalent currents through ARC channels was essentially unaffected by removal of internal Mg2+ (19) or if the measurements were made using the perforated patch technique where normal physiological internal Mg2+ (and ATP) levels are maintained (33). Nevertheless, to be certain that our determinations of monovalent currents through ARC channels in the present study were not subject to contamination from MIC channels the Mg2+ concentration of the internal (pipette) solution was raised to 8 mM for these series of experiments. Under these conditions, the inward monovalent current recorded at negative internal potentials in the absence of external divalent cations is carried by Na+ and amounted to 20.7 ± 1.7 pA/pF (mean ± S.E., n = 6) at -80 mV (Fig. 3B). This is 40 times the inward Ca2+ current measured at the same potential in the presence of normal external divalent cations. This Na+:Ca2+ current ratio is essentially identical to that reported previously (19, 22) for the ARC channels and differs markedly from the corresponding ratio of around 5-10 reported for currents through CRAC channels in various cell types (19, 34, 35). Using these same conditions, the magnitude of the arachidonic acid-activated monovalent current was examined at a range of different external Ca2+ concentrations (with zero Mg2+ externally). The data for the macroscopic monovalent currents measured at -80 mV are shown in Fig. 3C. Because it proved difficult to obtain measurements at all the different Ca2+ concentrations in each individual experiment on a single cell, the current values obtained could not be normalized to any maximal value (e.g. at 0.01 µM external Ca2+). Data were therefore pooled from different experiments without normalization. Although this resulted in somewhat increased standard errors, the essential relationship remained clear. Increasing external Ca2+ inhibited the monovalent ARC current in a concentration-dependent manner with a calculated EC50 of 147 ± 35 nM. Based on the hypothesis that this reflects the binding of Ca2+ to a monovalent blocking site in the channel, the data indicate a Ca2+ affinity for this site of ~150 nM at an internal potential -80 mV. As a comparison, we also performed a parallel series of experiments examining the ability of external Mg2+ to block the monovalent current through ARC channels in the absence of external Ca2+ (Fig. 3C). As with Ca2+, external Mg2+ inhibited the monovalent current in a concentration-dependent manner but with a calculated EC50 at -80 mV of 32.8 ± 10.1 µM, more than 200 times that seen with Ca2+. It must be emphasized that it would be incorrect to assume that this necessarily reflects the relative affinity for Ca2+ and Mg2+ of the same site in the channel as it is far from certain that Ca2+ and Mg2+ bind to the same site or even whether the underlying mechanism of block of monovalent permeation is the same for Ca2+ and Mg2+.

Fatty Acid Specificity-- Of course, the truly unique feature of ARC channels that distinguishes them from the other highly Ca2+-selective channels is that their activation is entirely dependent on either the agonist-activated generation, or bath application, of arachidonic acid. To characterize this dependence more thoroughly, the ability of arachidonic acid to activate the Ca2+ current carried through the ARC channels was examined at different concentrations of the fatty acid. The data obtained (Fig. 4A) showed that detectible inward currents measured at -80 mV could be obtained with concentrations of exogenous arachidonic acid as low as 2 µM. Despite the small magnitude of the macroscopic currents measured at such low concentrations, the characteristic ARC channel features of inward rectification, a reversal potential of >+30 mV, complete inhibition by 50 µM La3+, and, most importantly, the absence of any fast inactivation were all apparent. Concentrations of arachidonic acid above 8 µM were not examined as it is known that the fatty acid will tend to form micelles at such concentrations (36), raising the possibility of detergent-like effects on the membrane. In addition, the possibility of nonspecific effects on membrane fluidity, etc. are greatly increased at elevated concentrations. For example, we have found that concentrations of arachidonic acid greater than 25 µM routinely induce large, highly nonselective leak conductances capable of passing NMDG+. Such currents presumably reflect the result of a significant perturbation of the phospholipid properties of the cell membrane.2


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Fig. 4.   Effect of fatty acids on currents through ARC channels. A, the effect of different concentrations of exogenous arachidonic acid on the magnitude of IARC. Average currents (n = 4-6) activated by arachidonic acid added to the external bath at the final concentrations indicated are shown. Steady-state currents were measured during 250-ms pulses to -80 mV in the standard extracellular solution. B, the saturated fatty acid, palmitic acid (16:0), or the monounsaturated fatty acid oleic acid (18:1, cis-9) did not activate IARC. Average steady-state currents activated by addition of 8 µM fatty acids were determined during 250-ms pulses to -80 mV using the standard extracellular solution (n = 6 and 7, respectively). Subsequently, arachidonic acid (8 µM) was added, and the magnitude of the resulting IARC was determined at -80 mV (n = 3 and 4, respectively). C, the ability of various polyunsaturated fatty acids to activate IARC. Mean steady-state currents were recorded during 250-ms pulses to -80 mV following addition of 8 µM of the following polyunsaturated fatty acids: a, linolelaidic acid (18:2, trans-9,12) (n = 7); b, linolenic acid (18:3, cis-9,12,15) (n = 6); c, linoleic acid (18:2, cis-9,12) (n = 5); d, eicosatetraynoic acid (ETYA, 20:4, yne-5,8,11,14) (n = 4); e, arachidonic acid (20:4, cis-5,8,11,14) (n = 11).

Arachidonic acid is a cis-polyunsaturated fatty acid (20:4, cis-5,8,11,14), and like many other highly hydrophobic molecules, can exert its effects on membrane transport properties either directly by interacting with specific proteins or indirectly by inducing perturbations of the lipid bilayer of the membrane in which the proteins are incorporated. Many of these effects on membrane lipids are induced by a variety of amphiphilic molecules, including different fatty acids, and are therefore essentially nonspecific in nature. To examine the fatty acid specificity of the activation of ARC channels we tested the ability of a range of different fatty acids (all at 8 µM) to activate IARC at -80 mV. Both the saturated fatty acid, palmitic acid (16:0), and the monounsaturated fatty acid, oleic acid (18:1, cis-9), failed to induce significant current (Fig. 4B). This was not because of any problem with the ARC channels themselves, as in each case, the subsequent addition of 8 µM arachidonic acid resulted in the appearance of a normal Ca2+-selective IARC of 0.62 ± 0.04 pA/pF (n = 4) and 0.52 ± 0.05 pA/pF (n = 3), respectively (Fig. 4B). This suggests that the activation of IARC is a property limited to the polyunsaturated fatty acids. To examine this further, a range of different polyunsaturated fatty acids including both cis and trans varieties, and with different chain lengths and numbers of double bonds, was examined for their ability to activate IARC. (Fig. 4C). For comparative purposes, each fatty acid was examined at a concentration of 8 µM. The trans-polyunsaturated fatty acid linolelaidic acid (18:2, trans-9,12) produced only a very modest activation of IARC as measured at -80 mV to a maximum value of 0.2 ± 0.02 pA/pF (n = 7), or ~35% of the corresponding value for arachidonic acid-activated IARC. Among the cis-polyunsaturated fatty acids, linolenic acid (18:3, cis-9,12,15) produced a similar modest stimulation to a value of 0.21 ± 0.03 pA/pF (n = 6), or 38% of the normal arachidonic acid-activated value of IARC, whereas linoleic acid (18:2, cis-9,12) was significantly more effective, resulting in a maximal current of 0.42 ± 0.03 pA/pF (n = 5), or ~75% of the normal arachidonic acid-stimulated current.

Another way in which arachidonic acid can exert its effects on cell function is as a result of its rapid metabolism inside the cell into a variety of eicosanoid products, many of which are known to have important actions in cells. However, previous studies using cyclooxygenase and lipoxygenase inhibitors have indicated that the effects of exogenous arachidonic acid on the noncapacitative entry of Ca2+ reflect the actions of the fatty acid itself and not one of its many bioactive metabolites (15). To confirm that this is true for the activation of the ARC channels themselves, the ability of ETYA (20:4, yne-5,8,11,14), to activate IARC was examined. ETYA is a triple-bond non-metabolizable analog of arachidonic acid that also acts as an effective blocker of lipoxygenase, cyclooxygenase and P450 pathways for the metabolism of the fatty acid (37). Exogenous addition of 8 µM ETYA produced a significant, although submaximal, stimulation of IARC to a value of 0.28 ± 0.04 pA/pF (n = 4), or 50% of that seen with the same concentration of arachidonic acid (Fig. 4C). Despite the reduced magnitude, the overall characteristics of the currents activated by ETYA were indistinguishable from those activated by exogenous arachidonic acid.

Site of Arachidonic Acid Action-- In the m3-HEK cells, we have demonstrated previously (33, 38) that the muscarinic agonist carbachol, at low concentrations, specifically activates the ARC channels in a manner dependent on the intracellular generation of arachidonic acid via the action of a cytosolic phospholipase A2. It therefore seems most likely that the activation of the channel is dependent on intracellular levels of arachidonic acid. However, our method for activating the ARC channels with arachidonic acid in the above experiments involves the exogenous addition of the fatty acid to the external surface of the cell. Nevertheless, because arachidonic acid is able to readily traverse the plasma membranes (39), we assume that the arachidonic acid rapidly gains access to its putative cytosolic site of action.

To test this assumption, we made use of the arachidonic acid analog ACoA. This molecule contains a large negatively charged head group that results in the molecule being confined to the side of the membrane to which it is applied (40). We therefore examined the relative ability of externally applied and internally applied ACoA to activate IARC. Inclusion of 8 µM ACoA in the standard pipette solution resulted in the prompt activation of inward current measured at -80 mV that began within ~20 s of achieving the whole-cell configuration (Fig. 5A). The mean value of this ACoA-induced inward current was 0.39 ± 0.02 pA/pF at -80 mV (n = 7), somewhat less that the value for ARC currents activated under identical conditions by the exogenous addition of the same concentration (8 µM) of arachidonic acid (0.60 ± 0.07 pA/pF at -80 mV, n = 5) (Fig. 5B). More importantly, the current/voltage relationship of the ACoA-induced current showed marked inward rectification and a reversal potential significantly greater than +30 mV (Fig. 5C) and, as such, was essentially identical to the normal ARC current activated by exogenous addition of arachidonic acid. In marked contrast, addition of the same concentration of ACoA to the bath solution produced only very small currents (0.07 ± 0.05 pA/pF at -80 mV, n = 5) that were indistinguishable from background noise (Fig. 5, B and C). It should be noted that the experiments involving the internal application of ACoA required that it be included in the pipette solution, with the result that exposure of the cell to this drug would begin immediately on achieving the whole-cell configuration. Because we were concerned that such exposure might effect internal stores of Ca2+, possibly resulting in an activation of CRAC channels, we repeated the measurements of the current induced by internal ACoA in the presence of 2-APB (100 µM) in the bath. As demonstrated above, under the strict conditions of the experiments reported here, application of 2-APB provides a convenient method of avoiding any possible contamination from the activity of CRAC channels in our current measurements. The results of such experiments, however, showed that 2-APB (100 µM) was completely without effect on the magnitude of current produced by internal ACoA (0.35 ± 0.04 pA/pF at -80 mV, n = 4) (Fig. 5B), demonstrating that activation of CRAC channels had not occurred.


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Fig. 5.   Effect of internally and externally applied arachidonyl coenzyme A on currents through ARC channels. A, representative trace showing that the inclusion of the charged arachidonic acid analogue arachidonyl-coenzyme A (8 µM) in the standard pipette solution results in the prompt activation of an inward current, measured during pulses to -80 mV. Recording was begun immediately on achieving the whole-cell condition (at zero time). B, comparison of the average currents activated by arachidonic acid added to the external bath (AA-OUT; n = 5), arachidonyl coenzyme A added to the pipette solution (ACoA-IN; n = 6), and arachidonyl coenzyme A added to the external bath (ACoA-OUT; n = 5). Also shown is the average current activated by arachidonyl coenzyme A added to the pipette solution measured in the presence of 100 µM 2-APB in the bath (ACoA-IN + 2-APB; n = 4). Concentrations of added fatty acids and analogues were 8 µM in each case. Steady-state currents were measured during 250-ms pulses to -80 mV in the standard extracellular solution. C, average current-voltage relationship of the currents activated by application of arachidonyl coenzyme A (8 µM) added in the pipette solution (; n = 6) or in the external bath (open circle ; n = 5).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Distribution-- As noted previously, arachidonic acid-dependent noncapacitative Ca2+ entry has been described in a wide variety of different cells, including avian exocrine nasal gland cells (13), Balb-c 3T3 fibroblasts (14), HEK293 cells (15), and A7r5 smooth muscle cells (16). However, it is unclear whether ARC channels, as characterized and defined in our earlier studies on HEK293 cells (17, 19), are specifically responsible for the observed Ca2+ entry in all these cases. Our examination of a series of different cell types, including cell lines from both mammalian and avian sources, as well as primary cells from freshly dissociated tissues, indicated the presence of ARC channels in all cases. Similarly, Yoo et al. (41) have described an arachidonic acid-activated conductance in Chinese hamster ovary cells that appears to be consistent with the activity of ARC channels. Given this, it seems likely that ARC channels are indeed widely distributed. The observed current densities were uniformly small (~0.5 pA/pF to 1.5 pA/pF at -80 mV), but this is essentially consistent with reported values of the other highly Ca2+-selective entry channel pathway of nonexcitable cells, namely CRAC channels (11).

Pharmacology of ARC Channels-- As discussed above, our reason for examining the effects of Gd3+ (1 µM) and 2-APB (100 µM) on Ca2+ currents through the ARC channels was that these agents have been increasingly used as presumed definitive discriminators between capacitative and noncapacitative Ca2+ entry pathways in cells (23). However, this assumption is based almost exclusively on studies in which Ca2+ entry has been assessed from changes in cytosolic fluorescence of Ca2+-sensitive probes (e.g. fura-2), either on addition of extracellular Ca2+ after prior treatment of the cells in a Ca2+-free medium or induced by external addition of Mn2+, Sr2+, or Ba2+ as surrogates for Ca2+. Both of these approaches have their limitations, such as the inability to control the membrane potential. However, perhaps more importantly, it would seem premature to rely on the assumption that the characteristics and pharmacology of Ca2+ entry pathways in one cell type can be automatically applied to other cell types. For example, the assumption that 1 µM Gd3+ selectively inhibits capacitative entry without affecting noncapacitative entry derives largely from the studies of Broad et al. (16) on A7r5 smooth muscle cells. However, there is increasing evidence that receptor-activated Ca2+ entry in A7r5 cells, and probably other smooth muscle cells, occurs via nonselective cation channels, rather than the highly Ca2+-selective channels (e.g. CRAC and/or ARC) seen in many other cell types (42-44). Our direct examination of Ca2+ entry through the Ca2+-selective noncapacitative ARC channels clearly shows that 1 µM Gd3+ is capable of significantly inhibiting this entry, a fact supported by fluorescence measurements of arachidonic acid-induced increases in cytosolic Ca2+. It should be noted that Luo et al. (23) reported that 1 µM Gd3+ failed to significantly effect arachidonic acid-induced increases in Ca2+ in HEK293 cells using fluorescence measurements. However, their data do indicate that increasing the concentration of Gd3+ only slightly (to 3 µM) induced an approximately 50% inhibition. Consistent with this, we found that 5 µM Gd3+ completely inhibited Ca2+ currents through the ARC channels. These data demonstrate that the use of Gd3+, even at a concentration of only 1 µM, cannot be considered a reliable means to accurately discriminate between Ca2+ entry through ARC and CRAC channels.

With regards to the use of 2-APB, our data demonstrate that this agent, at a concentration that profoundly inhibits CRAC channels (100 µM) (24-26), has no significant effect on the ARC channels. However, the usefulness of 2-APB in definitively discriminating between these two channels remains questionable as recent reports have shown that it also blocks the MagNuM channels (31), and it actually slightly potentiates currents through CaT1 (ECaC2/TRPV6) (45). Moreover, although originally used as a cell-permeant inhibitor of inositol 1,4,5-trisphosphate receptors, 2-APB has been shown to have diverse actions on a variety of other processes involved in overall Ca2+ regulation in cells (46). Such effects will likely severely impact the interpretation of its effects, particularly when used in intact cells and under conditions where signaling pathways involving multiple steps are activated (e.g. during stimulation with receptor agonists).

Finally, our data show that the cell-permeable diacylglycerol analogue OAG, even at high concentrations, failed to induce any significant activation of the ARC channels. This is in marked contrast with the members of the TRPC family of channels (TRPC3, TRPC6, and TRPC7) that have been shown to be activated in a noncapacitative manner via a diacylglycerol-dependent mechanism. Consistent with this, we have found that overexpression of human TRPC6 in the m3-HEK cells fails to effect the magnitude of arachidonic acid-activated Ca2+ entry.3 It should also be noted that none of the currently identified TRPC channels display biophysical characteristics consistent with ARC channels.

Ca2+ Ion Selectivity-- Although the biophysical evidence indicates that the ARC channels are highly Ca2+-selective, no direct comparison of the Ca2+ selectivity of these channels relative to other Ca2+-selective conductances has been reported. To obtain such a comparison, we determined the Ca2+ affinity for the block of monovalent ion permeation through the channel. The particular usefulness of this parameter is that similar studies have been reported for the store-operated Ca2+ CRAC channels (34), as well as for voltage-gated Ca2+ channels (47), and the Ca2+ channels of Ca2+-transporting epithelia ECaC1 (= TRPV5, CaT2) (48) and ECaC2 (= TRPV6, CaT1) (49). As already discussed, internal Mg2+ concentration in these experiments was raised to 8 mM throughout to avoid any possible contamination from MagNuM or MIC channels. Under these conditions, the data obtained indicated an affinity for the putative Ca2+-blocking site of ~150 nM. This is much higher than the corresponding estimated value of ~10 µM (at -80 mV) reported for the CRAC channels of RBL cells (35) and ~5 µM for the CRAC channels of Jurkat lymphocytes (34). Studies of other highly Ca2+-selective channels in which similar measurements have been made include voltage-gated Ca2+ channels (0.7 µM; see Ref. 29) and the Ca2+ channels of Ca2+-transporting epithelia ECaC1 (TRPV5) and ECaC2 (TRPV6) at 200 nM and 150 nM, respectively (48, 49), values that are much closer to those reported here.

As expected for a highly Ca2+-selective conductance, the block of monovalent currents through ARC channels showed an ~200-fold lower affinity for external Mg2+ than for Ca2+. The EC50 value obtained for external Mg2+ block of the ARC currents (~30 µM at -80 mV) can be compared with corresponding values determined under similar conditions of 62 ± 9 µM for ECaC1/TRPV5 (48) and 200 ± 14 µM for ECaC2/TRPV6 (49).

Fatty Acid Specificity and Site of Action-- The unique feature of ARC channels among the various Ca2+ entry channels of nonexcitable cells is their specific dependence on arachidonic acid for activation. We have now demonstrated that this activation occurs at concentrations of arachidonic acid between 2 and 10 µM. Such concentrations are considered physiologically relevant as they lie within the range of reported values for the KD of the cytosolic enzymes responsible for the metabolism of the fatty acid (cyclooxygenases and lipoxygenases) in cells. This is important, because although several fatty acids including arachidonic acid have been shown to influence the activity and behavior of different ion channels in various tissues (see Refs. 50 and 51), many of these effects are only seen at relatively high concentrations. At such concentrations, a variety of essentially nonspecific effects on the physical properties or structural integrity of the cell membrane are known to occur. For example, the hydrophobic nature of long chain fatty acids results in their rapid incorporation into cell membranes often with consequent effects on membrane fluidity, etc. Moreover, the amphiphilic nature of fatty acids can produce detergent-like effects on membranes. These generally result from the formation of micelles, which can even create lipidic pores through which ions can move (52).

The data presented here indicate that such nonspecific effects are not involved in the observed ability of arachidonic acid to activate ARC channels. For example, the concentrations of arachidonic acid shown to activate IARC are significantly below the critical micellar concentration for this fatty acid in saline (36). Moreover, nonspecific detergent-like effects should also be seen with saturated fatty acids, yet palmitic acid (16:0) clearly failed to activate IARC. With regard to possible changes in membrane fluidity, it is generally considered that such changes result from an increase the overall unsaturation (increase in the number of cis double bonds) of the membrane lipids. However, the precise nature of this relationship is complex and is influenced by other factors including the position of the double bonds and the total chain length, etc. (53). In the experiments presented here we found that linoleic acid (18:2, cis-9,12) was significantly more potent as an activator of IARC than was linolenic acid (18:3, cis-9,12,15), yet simple predictions would suggest that the latter would have a greater influence on membrane fluidity (because of the presence of an additional double bond). In addition, Brown et al. (54) report that ETYA is even more potent than arachidonic acid in increasing membrane fluidity, yet our data indicate that it was only ~50% as effective in activating IARC. Together then, the data indicate that the action of arachidonic acid on the ARC channels is not a result of any nonspecific modification of the physical properties of the lipid membrane, including changes in membrane fluidity, in which the channel is located.

Moreover, the demonstrated ability of non-metabolizable arachidonic acid analog ETYA to, at least partially, activate IARC shows that the activation of the ARC channels is not dependent on the generation of any eicosanoid metabolites of arachidonic acid. Clearly then, the activation of the ARC channels results from a specific action of the fatty acid itself and not of any metabolite. The data further suggest that the activation of the ARC channels is a property restricted to the polyunsaturated fatty acids. Among these, there appeared to be no clear relationship between either the chain length or the number of double bonds and the ability to activate IARC. For example, linolelaidic acid is the trans-isomer of the cis-polyunsaturated fatty acid linoleic acid; they are the same chain length and have the same number of double bonds. Yet, linolelaidic acid produced only a very modest stimulation of IARC, whereas linoleic acid produced a fairly robust stimulation equivalent to 75% of that seen with arachidonic acid. Moreover, the ability of ETYA to act as an effective inhibitor of the various enzymes that metabolize arachidonic acid (see above) is a reflection of its close structural analogy to arachidonic acid. However, it is significantly less effective in its ability to activate IARC than arachidonic acid itself. It would seem therefore, that the activation of ARC channels displays a high degree of specificity for arachidonic acid.

Finally, the data obtained from the studies using the membrane-impermeable arachidonic acid analog ACoA revealed that activation of IARC uniquely involves an action of the fatty acid either in, or at the cytosolic face of, the inner leaflet of the membrane. Whether this involves a direct action of the fatty acid on the channel protein itself or an action on some intermediary molecule is, at present, uncertain. Whatever its precise nature, it would seem that any such interaction must display a fairly high degree of specificity. In this context, it is known that non-esterified arachidonic acid takes the form of a highly curved, hairpin-like conformation with one hydrocarbon arm of the hairpin having a hydrophilic end and the other having a hydrophobic end. As pointed out by Rich (55), this highly flexible hairpin-like structure of arachidonic acid makes it more ideally suited to interact directly with concave protein surfaces than the essentially linear conformation displayed by saturated, monounsaturated, and trans-polyunsaturated fatty acids molecules. This is supported by the data obtained using ACoA. In this molecule, the COOH terminus of the arachidonic acid molecule is replaced by the coenzyme A group, but the essential hairpin structure of the carbon chain is retained.

In conclusion, we have shown that ARC channels represent a novel member of the group of highly selective Ca2+ entry channels that includes CRAC channels, voltage-gated Ca2+ channels, and the epithelial ECaC channels. Based on the ability of external Ca2+ ions to block monovalent cation permeation through the channels, we would conclude that the ARC channels are among the most highly Ca2+-selective of all these channel types. Our evidence also suggests that current pharmacological approaches widely used to dissect the relative contributions of CRAC and ARC channels are not reliable. Of course, the particular unique feature of the ARC channels is that their activation is dependent on arachidonic acid and is entirely independent of store depletion. However, ARC channels are not the only Ca2+ channels whose activity is influenced by arachidonic acid. For example, L-type Ca2+ channels in cardiac myocytes (56) and both L-type and N-type Ca2+ channels in various neuronal preparations (57) are inhibited by low concentrations of arachidonic acid, although the actual mechanisms involved appear to vary with the different channel types. Based on the data presented here, it would seem that the activation of ARC channels shows a high degree of specificity for arachidonic acid over other fatty acids (especially monounsaturated and saturated fatty acids). Moreover, this activation involves action of the fatty acid at an intracellular site, either directly on the channel itself or on some intermediary protein. Finally, our demonstration that ARC channels are widely distributed in different cell types indicates that these channels are likely candidates for providing the route for the receptor-activated, arachidonic acid-dependent, noncapacitative entry of Ca2+ that has been described in a wide variety of different cells. An important caveat to this conclusion is that this would only apply where such entry is occurring through a highly Ca2+-selective pathway. Their demonstrated high selectivity for Ca2+ suggests that where such entry is taking place through a non-selective cation pathway (e.g. in smooth muscle cells), ARC channels are unlikely to be involved.

    ACKNOWLEDGEMENTS

We thank Dr. Ted Begenisich for helpful comments and suggestions and Pauline Leakey for excellent technical assistance.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM40457 (to T. J. S.).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.

Dagger Present address: UMR 8078, Laboratoire de Physiologie Cellulaire, bât.442 bis, Université de Paris-Sud, 91405 Orsay Cedex, France.

§ To whom correspondence should be addressed: Dept. of Pharmacology and Physiology, Box 711, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-275-2076; Fax: 585-273-2652; E-mail: trevor_shuttleworth@urmc.rochester.edu.

Published, JBC Papers in Press, January 9, 2003, DOI 10.1074/jbc.M212536200

2 J. L. Thompson and T. J. Shuttleworth, unpublished data.

3 T. J. Shuttleworth, unpublished data.

    ABBREVIATIONS

The abbreviations used are: CRAC, Ca2+ release-activated Ca2+; 2-APB, 2-aminoethyoxydiphenyl borate; OAG, 1-oleoyl-2-acetyl-sn-glycerol; IARC, current through ARC channels; NMDG+, N-methyl-D-glucamine; MagNuM, magnesium nucleotide-regulated metal; MIC, Mg2+-inhibited cation; ETYA, eicosatetraynoic acid; ACoA, arachidonyl coenzyme A; HEK, human embryonic kidney; ARC, arachidonate-regulated Ca2+; RBL, rat basophilic leukemia; HEDTA, N-(2-hydroxyethyl)ethylenediaminetriacetic acid; pF, picofarad.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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