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
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ABSTRACT |
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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.
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.
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 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 [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.
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 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
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 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.
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 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
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
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 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 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
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 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 (
), and freshly isolated mouse
parotid acinar cells (
). Currents were measured in the standard
extracellular solution, and steady-state currents were determined
during pulses to the indicated voltages.
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+ (
) 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 (
) 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.
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.
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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+
(
; 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+ (
) 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).
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+.
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).
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.
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 (
; n = 5).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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).
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.
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).
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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.
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.
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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.
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