From the Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
Received for publication, March 14, 2001, and in revised form, March 30, 2001
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ABSTRACT |
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In a manner similar to voltage-gated
Ca2+ channels and Ca2+ release-activated
Ca2+ (CRAC) channels, the recently identified
arachidonate-regulated Ca2+ (ARC) channels display a large
monovalent conductance upon removal of external divalent cations. Using
whole-cell patch-clamp recording, we have characterized the properties
of these monovalent currents in HEK293 cells stably transfected with
the m3 muscarinic receptor and compared them with the corresponding
currents through the endogenous store-operated Ca2+ (SOC)
channels in the same cells. Although the monovalent currents seen
through these two channels displayed certain similarities, several
marked differences were also apparent, including the magnitude of the
monovalent current/Ca2+ current ratio, the rate and nature
of the spontaneous decline in the currents, and the effects of external
monovalent cation substitutions and removal of internal
Mg2+. Moreover, monovalent ARC currents could be activated
after the complete spontaneous inactivation of the corresponding SOC
current in the same cell. We conclude that the non-capacitative ARC
channels share, with voltage-gated Ca2+ channels and
store-operated Ca2+ channels (e.g. SOC and CRAC
the general property of monovalent ion permeation in the nominal
absence of extracellular divalent ions. However, the clear differences
between the properties of these currents through ARC and SOC channels
in the same cell confirm that these represent distinct conductances.
Receptor-stimulated increases in Ca2+ entry in
non-excitable cells are known to play a pivotal role in the generation
and maintenance of the intracellular Ca2+ signals
responsible for the control of such diverse functions as secretion,
motility, growth, proliferation, and gene expression. However, despite
extensive study, the mechanisms underlying such receptor-stimulated
Ca2+ entry are currently far from clear. To date, most
studies have focused on the so-called capacitative or store-operated
model in which activation of Ca2+ entry occurs as a result
of the emptying of intracellular Ca2+ stores (1-3). Such
entry is described as occurring via store-operated Ca2+
(SOC)1 channels (4, 5). The
ionic conductances associated with this store-operated entry were first
characterized using patch-clamp techniques in mast cells and Jurkat
lymphocytes (6-8), where the resulting whole-cell currents were
identified as ICRAC (for calcium
release-activated calcium
currents). These CRAC channels therefore represent the archetypal
capacitative or store-operated channel. Although store-operated
Ca2+ entry appears to be an almost ubiquitous feature of
cells, the biophysical characterization of SOC channels from other cell
types is rather limited, and the mechanism of activation of the
channels is still unknown. Moreover, it seems unlikely that such
channels are the exclusive route for the receptor-stimulated entry of
Ca2+ in non-excitable cells (9). Recently, we have
identified a novel receptor-activated Ca2+ entry pathway
that appears to be specifically responsible for the Ca2+
entry associated with agonist stimulation at physiologically relevant
concentrations (10-12). This pathway is independent of store depletion
(i.e. non-capacitative) and is instead regulated by the
receptor-mediated generation of arachidonic acid (AA). Similar
AA-dependent non-capacitative Ca2+ entry
pathways have now been identified in a variety of different cell types,
including Balb/c 3T3 fibroblasts (13), A7r5 smooth muscle cells (14),
and mouse parotid cells2.
Subsequent characterization of the Ca2+-selective
conductance associated with this pathway (named
IARC, for
arachidonate-regulated
Ca2+ current) in HEK293 cells stably
transfected with the m3 muscarinic receptor (m3-HEK cells) revealed
that it showed certain features that clearly distinguished it from the
store-operated current (ISOC) in the same cells
(12).
Both ARC and CRAC channels, as well as the endogenous SOC channels of
m3-HEK cells (HEK-SOC channels), share the properties of being highly
Ca2+-selective with relatively small magnitude currents
(~0.5-1 pA/pF at Cell Culture--
Cells from the human embryonic kidney cell
line HEK293 that had been stably transfected with the human m3
muscarinic receptor were a generous gift from Dr. Craig Logsdon
(University of Michigan). The cells were cultured in Dulbecco's
modified Eagle's medium with 10% calf serum and antibiotics in a 5%
CO2 incubator at 37 °C as previously reported (11).
Cells were plated on glass coverslips that formed the bottom of a
patch-clamp chamber (Warner Instrument Corp., Hamden, CT) at least
24 h before experimentation.
Whole-cell Patch-clamp Recordings--
Patch-clamp recordings
using the standard whole-cell mode (17) were performed at room
temperature (20-22 °C) using an Axopatch-1C patch-clamp amplifier
(Axon Instruments, Inc., Foster City, CA). Patch pipettes were pulled
from borosilicate glass (GC150-F, Warner Instrument Corp.) and
fire-polished to a resistance of 3-6 megaohms when filled with
internal solution. Whole-cell currents were recorded using 250-ms
voltage steps from a holding potential of 0 to Solutions--
The standard pipette (internal) solution
contained 140 mM Cs+ acetate, 1.22 mM MgCl2, 1.89 mM
CaCl2, 5 mM EGTA, and 10 mM HEPES (pH 7.2 with CsOH). The free Ca2+ concentration of this
solution was calculated to be 100 nM as computed with
Maxchelator (18). CaCl2 or MgCl2 was omitted
for the experiments involving Ca2+- or
Mg2+-free internal solutions, respectively. Where
indicated, the Cs+ acetate was replaced with
Na+ acetate or NMDG+ acetate as appropriate.
The standard extracellular solution contained 140 mM NaCl,
1.2 mM MgCl2, 20 mM
CaCl2, 10 mM D-glucose, and 10 mM
HEPES (pH 7.4). Both MgCl2 and CaCl2 were
omitted from this solution for the experiments involving nominally
divalent ion-free external solutions, and the osmolarity (320 mosmol/liter) was maintained with additional D-glucose.
Measurements of the Ca2+ concentration in this nominally
divalent ion-free external solution were made spectrofluorometrically
using fura-2-free acid and compared against commercially prepared
standards (Molecular Probes, Inc., Eugene, OR). The values obtained
ranged from 0.12 to 0.6 µM. Where indicated, the NaCl was
replaced with CsCl, LiCl, or NMDG-Cl as appropriate.
Ca2+-selective ARC Channels Become Permeable to
Monovalent Cations upon Removal of External Divalent Cations--
In
normal external medium containing Ca2+ and Mg2+
in millimolar concentrations, addition of exogenous arachidonic acid (8 µM) to the bath solution resulted in the activation of a
small inward current at
Upon nominal removal of external divalent cations, a much larger inward
current (measured at
As already noted, both voltage-gated Ca2+ channels and the
CRAC channel of non-excitable cells have been shown to display a large
permeability to monovalent ions upon removal of extracellular divalent
cations (7, 15, 16). The features of the AA-dependent monovalent current described above are consistent with the existence of
a similar phenomenon in the ARC channels we have previously identified.
To examine this, we compared the magnitude of the observed
AA-dependent monovalent current measured at Endogenous Store-operated Channels of HEK293 Cells (HEK-SOC
Channels) Also Show a Permeability to Monovalent Cations upon Removal
of Extracellular Divalent Cations--
As noted, the aim of this study
was to further define the features of the non-capacitative
arachidonate-regulated conductance that distinguish it from
store-operated or capacitative conductances. The features of the
macroscopic monovalent conductance that develops upon reduction of
external divalent cations in the store-operated conductance
(ICRAC) of Jurkat lymphocytes have been
extensively characterized (15, 16). However, it appears likely that
store-operated channels represent a family of conductances of which
ICRAC is only one member. Therefore, it was
important to be able to compare the AA-activated conductance with the
endogenous SOC conductance in the same cell type. We therefore examined
the effect of removal of divalent cations from the extracellular medium
in cells in which the internal Ca2+ stores had been
passively depleted by dialyzing with a highly buffered
Ca2+-free pipette solution. In the normal bath solution
containing millimolar Ca2+ and Mg2+, a small
inward current measured at
I/V curves for the store-operated current in the nominal absence of
divalent cations were obtained by application of voltage ramps from
From the above results, it is clear that both the endogenous
store-operated or capacitative conductance
(ISOC) and the non-capacitative AA-regulated
Ca2+-selective conductance (IARC)
display a marked permeability to monovalent cations upon nominal
removal of extracellular divalent ions, a property shared with
voltage-gated Ca2+ channels and the classic store-operated
Ca2+ conductance (ICRAC). The most
immediately obvious difference between the macroscopic currents
displayed by ARC and SOC channels is the much larger monovalent
conductance shown by IARC relative to
ISOC (Fig.
3A). Thus, although the
whole-cell currents observed for the two conductances when in their
Ca2+-selective mode are similar in magnitude (~0.5-0.6
pA/pF at Monovalent Selectivity--
As noted above, both
ISOC and IARC in the
nominal absence of extracellular divalent ions displayed characteristic
tilde-shaped I/V curves with significant inward and outward monovalent
currents under the standard conditions (Na+ in the bath
solution and Cs+ in the pipette solution). Moreover, when
the Cs+ in the pipette solution was replaced with
NMDG+, neither ISOC nor
IARC displayed any significant outward currents. These data indicate that both SOC and ARC channels have a significant permeability to both Na+ and Cs+ ions under
these conditions. To examine the monovalent cation selectivity of the
ARC and SOC channels in more detail, experiments were performed
involving various monovalent cation substitutions in both the bath and
pipette solutions. The data obtained are illustrated (A-D)
and summarized (E and F) in Fig.
4. Estimates of the respective sequences
of monovalent conductance for the two channels were then determined
from the resulting macroscopic current magnitudes.
Substitution of Na+ in the bath solution with
Cs+ had a statistically insignificant (p = 0.07) effect on the inward current density of the ARC channels measured
at
A small but statistically significant (p = 0.05)
increase in the inward current density at
Substitutions were also made to the monovalent cations in the
internal (pipette) solutions. Substitution of internal Cs+
with Na+ markedly reduced the outward current density
measured at +30 mV through the ARC channels (from 22.6 ± 3.4 to
8.1 ± 1.0 pA/pF, n = 5) (Fig. 4, B and
F). A similar, although much less dramatic decrease in
outward current density at +30 mV was seen with SOC channels when
Na+ replaced internal Cs+ (from 10.8 ± 1.7 pA/pF (n = 8) to 5.2 ± 0.8 pA/pF
(n = 6)) (Fig. 4, D and F).
Importantly, this latter response could not be caused by an effect on
the rate of spontaneous inactivation, as this was much slower when
Na+ replaced Cs+ in the pipette solution (time
to attain 50% inactivation of 54.0 ± 11.4 versus
17.6 ± 4.2 s). As has already been noted, replacing internal
Cs+ with NMDG+ resulted in the complete absence
of any significant outward currents through either ARC or SOC channels
(Fig. 4, B and D). Based on these data for
outward current densities, a monovalent cation selectivity for both ARC
and SOC channels of Cs+ > Na+ Effects of Internal Mg2+--
The similarity of the
inward currents carried by Na+ and Cs+ reported
here for both ARC and HEK-SOC channels (Fig. 4E) is not seen
in the CRAC channels of Jurkat cells measured under the same conditions
(15, 16). However, it was reported that magnesium-free internal
solutions increase the magnitude of the inward Na+ currents
and, more particularly, dramatically increase the Cs+
permeability of the Jurkat CRAC channels such that the Na+
and Cs+ currents are essentially identical (16). It was
also noted that large outward currents develop in the absence of
internal Mg2+ that are not observed in the presence of
internal Mg2+ (16). We therefore examined the effects of
removal of internal Mg2+ on the monovalent currents
recorded for ARC and HEK-SOC channels.
Under the standard conditions of Na+ in the bath solution
and Cs+ in the pipette solution, removal of internal
(pipette) Mg2+ had no significant effect on the magnitude
of the inward (Na+) currents through ARC channels (Fig.
5A). Similarly, no significant change in the magnitude of the inward current was seen when the external cation was Cs+ (data not shown). However,
examination of the I/V relationship clearly indicated that removal of
internal Mg2+ reduced the degree of rectification in the
inward (Na+) current (Fig. 5B).
Mg2+-free internal solutions also markedly reduced the
outward current (measured at +30 mV) carried by Cs+ from
22.6 ± 3.4 to 13.4 ± 2.2 pA/pF (n = 5 and
4, respectively; p = 0.04) (Fig. 5, A and
B).
When monovalent SOC currents were examined, Mg2+-free
internal solutions again had no significant effect on inward currents, whether these were carried by Na+ (Fig. 5C) or
Cs+ (data not shown). Unlike the ARC currents, however,
removal of internal Mg2+ did not significantly influence
the degree of rectification of the inward current (Fig. 5D).
Moreover, in marked contrast to the response seen in ARC currents,
removal of internal Mg2+ significantly increased the
outward (Cs+) current measured at +30 mV from 10.8 ± 1.7 to 16.6 ± 2.1 pA/pF (n = 6 and 8, respectively; p = 0.03) (Fig. 5, C and
D). Finally, Kerschbaum and Cahalan (16) reported that
removal of internal Mg2+ eliminates the spontaneous
inactivation of the monovalent current through the CRAC channels in
Jurkat cells. However, no such effect was observed in the endogenous
SOC channels of HEK293 cells measured under the standard conditions
(Na+ in the bath solution and Cs+ in the
pipette solution). In these cells, the times required to achieve a 50%
decline in the measured current at Activation of ARC Currents after SOC Current
Inactivation--
As already noted, the monovalent HEK-SOC current
showed a rather rapid spontaneous decline in magnitude after activation
upon removal of extracellular divalent cations (see Fig.
3B). Following such inactivation, re-exposure of the cells
to normal divalent ion-containing medium (i.e. with
Ca2+) demonstrated that the Ca2+-selective
current was also now absent (Fig.
6A). However, continued exposure of the cells to external divalent ions induced a gradual reactivation of the normal Ca2+-selective current over a
1-2-min period (Fig. 6A, inset). Subsequent exposure to divalent ion-free medium showed that this reappearance of
the Ca2+-selective current was also associated with a
reactivation of the monovalent current. The data show that the sizes of
these two currents (monovalent and Ca2+-selective) were
directly related to each other during this reactivation process
(r2 = 0.94) (Fig. 6B), confirming
that the two currents reflect the activity of the same family of
channels. An essentially identical phenomenon was observed in the CRAC
channels of Jurkat cells (15). Without implying any specific molecular
mechanism for this spontaneous decline and in accordance with the
similar decline reported for monovalent CRAC currents (15, 16), we have
described this phenomenon as a process of inactivation. Use of such a
term is consistent with the demonstrated ability to reactivate the
current by exposure to extracellular divalent ions.
The rate of decline in the corresponding monovalent ARC current after
initial activation was always very much slower (typically 10-15 times)
than those seen with the HEK-SOC currents under identical conditions
(see Fig. 3B). In addition, after the decline in the monovalent ARC current was complete, re-exposing the cells to normal
external medium containing divalent cations for up to 5 min produced
only a minimal subsequent reactivation of the monovalent ARC current
(equal to 5.6 ± 0.9% of the original monovalent current, n = 5). These data suggest that the decline in the
monovalent ARC current likely involves a rather different process from
that seen with HEK-SOC currents. To distinguish between these two
apparently rather different phenomena and consistent with the failure
to reactivate ARC currents upon re-exposure to extracellular divalent ions, we will refer to this decline in the monovalent ARC current as a
"run-down" rather than an inactivation. Again, the use of this term
is not meant to signify the involvement of any specific molecular mechanism.
As noted above, the inactivated HEK-SOC current was absolutely
dependent on exposure to normal divalent ion-containing solutions in
the bath for its re-activation, and no "spontaneous" reactivation of the monovalent HEK-SOC current was ever seen without exposure of the
cell to divalent ion-containing external medium. However, addition of
exogenous AA (8 µM) to a cell in which the monovalent SOC
current had been completely inactivated resulted in the
development of a large inward monovalent current (Fig. 6C).
The appearance of this AA-dependent current was not due to
the activation of any residual SOC current resulting from any failure
to completely deplete the internal Ca2+ stores, as an
identical AA-dependent monovalent current could be
activated after spontaneous inactivation of SOC currents that had been
stimulated by incubation in thapsigargin or by inclusion of
adenophostin A (2 µM) in the pipette solution (data not
shown). The magnitude of the AA-activated inward current in cells whose stores had been completely depleted by either thapsigargin or adenophostin A was not significantly different from that of the normal
monovalent ARC current (26.2 ± 1.1 pA/pF (n = 6)
versus 28.3 ± 2.7 pA/pF (n = 5)) and
was consistently larger than the magnitude of the previously recorded
monovalent SOC current in the same cell (7.5 ± 0.7 pA/pF,
n = 6). Moreover, it displayed the characteristically
slow spontaneous decline previously described for the monovalent ARC
current. These data demonstrate that the ARC currents can be activated
in a cell whose SOC currents have been completely inactivated and that
the two currents therefore represent the activities of distinct conductances.
The data reported here show that ARC channels in HEK293 cells
along with the endogenous SOC channels share, with CRAC channels and
voltage-gated Ca2+ channels, the general property of
monovalent cation permeation in the presence of low external divalent
ion concentrations. This transition from high Ca2+
selectivity to monovalent cation permeation has been most extensively analyzed in voltage-gated Ca2+ channels (20-23). In these
channels, the normally high selectivity for Ca2+ is thought
to involve the binding of Ca2+ with high affinity to a site
(or sites) in the pore of the channel. This binding enables
Ca2+ to act as a blocking ion to reduce nonselective
monovalent currents. A similar analysis has more recently been applied
to the store-operated CRAC channels of Jurkat lymphocytes (15, 16). The
proposed binding of Ca2+ also plays a role in
Ca2+ permeation through these channels, as it explains the
saturation of the Ca2+ current with increasing external
Ca2+ concentrations. Specific models based on these
principles have been developed that can simulate the selectivity,
permeation, and block properties of voltage-gated Ca2+
channels (20, 21). However, it has recently been argued that such
binding models can take several different possible forms, so the
precise details are currently far from fully understood (24).
Nevertheless, the general principles are reasonably clear. Selectivity
in these channels is not a result of any molecular sieving process, but
instead relies on the binding of ions to sites within the channel.
These sites show a high affinity for Ca2+ compared with
Na+. In the presence of external divalent cations,
occupation of the sites by Ca2+ precludes the permeation of
Na+ and other monovalent cations. At the same time,
Ca2+ permeation is favored, despite the intrinsic high
affinity of the sites, possibly by repulsive effects (strong ion-ion
interactions) of ions in two closely positioned binding sites of
similar high affinity (20, 22) or by a "stepwise" series of sites
with graded binding affinities (24). At low concentrations of external divalent cations, Ca2+ is lost from the binding sites and
thus permits the permeation of Na+ (and other monovalent
cations), whose high permeation reflects their low affinity for the
channel binding sites.
As such, it would seem that the overall behavior of both the ARC
channels and the endogenous SOC channels of HEK293 cells is consistent
with these models. Both ARC and HEK-SOC channels are highly
Ca2+-selective in normal divalent cation-containing
external medium and display a saturating current magnitude with
increasing external Ca2+ concentrations. Despite this high
selectivity for Ca2+, these channels reveal relatively
large monovalent currents in the nominal absence of extracellular
divalent cations. These monovalent currents are rapidly and markedly
inhibited by extracellular Ca2+ and are blocked by the same
ions that block the Ca2+ conductance. Moreover, in both
cases, the magnitude of the measured monovalent current is directly
related to the magnitude of the corresponding Ca2+ current.
All these features argue that the Ca2+ currents measured in
the presence of extracellular divalent ions and the much larger
monovalent currents measured in the nominal absence of divalent ions
reflect the behavior of the same channels.
The characteristic small Ca2+ current and yet relatively
large monovalent current of CRAC channels is therefore a feature shared by both HEK-SOC and ARC channels. Nevertheless, certain striking differences are apparent in the details of this phenomenon between these different channels. We have summarized the key features of the
three channels (CRAC, HEK-SOC, and ARC) in Table
I. First, although both ARC and
HEK-SOC channels have broadly similar Ca2+-selective
current densities compared with the CRAC channels of Jurkat cells
(~0.5-0.6 pA/pF for ARC and HEK-SOC channels and 0.8 pA/pF for CRAC
channels) (8, 12), HEK-SOC and, more particularly, ARC channels have
much higher monovalent current densities than Jurkat CRAC channels.
Monovalent (Na+)/Ca2+ current density ratios
for CRAC channels range from 5 to 10 (mean = 7.5 ± 2.7)
(14). The corresponding value for HEK-SOC channels was ~20 and was
even larger for ARC channels (~50). In CRAC channels, this ratio was
profoundly influenced by removal of internal Mg2+, which
markedly increased inward Na+ currents. Under these
conditions, the Na+/Ca2+ current ratio for the
CRAC channels becomes equal to 24.6 ± 4.9 (16), a value similar
to that observed for HEK-SOC channels in the presence of internal
Mg2+, but still considerably less than that observed for
ARC channels. Moreover, unlike Jurkat CRAC currents, removal of
internal Mg2+ had no significant effect on the magnitude of
the inward Na+ currents for either HEK-SOC or ARC channels.
The underlying basis for the observed differences in the relative
magnitudes of the Ca2+ and Na+ currents between
the different channels remains unclear. As explained, estimates of the
monovalent (Na+) currents for both HEK-SOC and CRAC
channels may be underestimated due to the spontaneous inactivation
phenomenon seen in both these conductances. However, it seems unlikely
that this can account for all the differences seen, especially those
between HEK-SOC and CRAC channels, both of which show broadly similar
inactivation kinetics. Until these properties have been examined in
detail at the single channel level, we can only speculate on the basis for the observed differences. They may reflect, for example, distinct permeation properties of the individual channels or differences in the
respective channel kinetics (e.g. open probability) induced by divalent ion-free external medium.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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80 mV), voltage-independent gating, inward
rectification, and very positive reversal potentials (4, 5, 7, 12). The
maintenance of the high selectivity for Ca2+ shown by these
channels (as well as by voltage-gated Ca2+ channels) is
problematic given that, under normal conditions, Na+ ions
vastly outnumber Ca2+ ions in the extracellular medium.
Despite this high selectivity for Ca2+ over Na+
under normal conditions, a characteristic feature of both CRAC channels
and voltage-gated Ca2+ channels is that lowering external
divalent cation concentrations to the micromolar range results in the
appearance of large monovalent currents through these channels (7, 15,
16). Evidence from voltage-gated Ca2+ channels suggests
that the high selectivity for Ca2+ over monovalent ions
seen under normal conditions does not result from a process of
rejection as from a molecular "sieve," but rather from the binding
of Ca2+ ions to sites within the channel. In other words,
the Ca2+ selectivity of these channels is
Ca2+-dependent. A similar conclusion was
reached in an analysis of the monovalent permeability of CRAC channels
(15, 16). Interestingly, we observed a similar increase in monovalent
ion permeability in the nominal absence of extracellular divalent
cations in our earlier studies on the ARC channels (12). This raises
the possibility that, despite the diverse mechanisms of gating shown by
these different Ca2+-selective channels (depolarization
versus store depletion versus arachidonic acid),
they all appear to share similar mechanisms for Ca2+
selectivity. In this study, we examine this feature in more detail with
the overall aim of attempting to further develop a unique "fingerprint" for the ARC channels to provide the means of
distinguishing them from the store-operated channels in the same cells.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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80 mV delivered every
2 s. Alternatively, current-voltage relationships were recorded
using 150-ms voltage ramps from
100 to +30 mV. Ramps were terminated
at +30 mV to avoid any contribution from a depolarization-activated
Cl
current. 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 traces obtained upon going whole
cell (i.e. before activation of IARC
or ISOC) 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 ~0.5 ml/min). For the experiments investigating fast
inactivation, peak and steady-state currents were compared during
hyperpolarizing pulses to
80 mV. Examination of the capacitative
currents during pulses to
80 mV indicated a mean time constant for
the capacitative transient of 240 ± 7.6 µs (n = 159). To minimize contributions from these transients, peak currents
(average currents over a 1-ms period) were determined at a point 3 ms
after the start of the hyperpolarizing pulse. Steady-state currents
were similarly determined at a point 200 ms later.
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ABSTRACT
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80 mV. Consistent with our previous report
(12), this current displayed a marked inward rectification, a positive (greater than +30 mV) reversal potential, and an absence of any fast
inactivation and was inhibited by La3+ (50 µM). Substitution of external sodium with
NMDG+ had negligible effects on the current, confirming a
minimal permeability to Na+ under these conditions. All
these features are consistent with the characterization of this
arachidonate-induced current as a Ca2+-selective current
(IARC).
80 mV) was seen to develop (Fig. 1A). Under normal conditions
(i.e. Na+ in the bath solution and
Cs+ in the pipette solution), this current increased to
reach a maximum amplitude of 28.3 ± 2.7 pA/pF (n = 5) or ~50 times the Ca2+-selective current seen in
normal divalent ion-containing external medium. The observed current
was entirely dependent on the presence of AA, as no significant
currents were seen to develop upon nominal removal of external divalent
cations without prior addition of AA. In the continued absence of
extracellular divalent ions, the AA-dependent current
slowly declined in magnitude (50% reduction in 229 ± 23 s)
(Fig. 1A). In contrast, replacing the divalent ion-free bath
solution with normal divalent ion-containing medium after the current
had reached its maximum value resulted in a rapid inhibition of the
current to levels consistent with those seen in normal medium
(i.e. ~0.5 pA/pF) (data not shown). We used voltage ramps
from
100 to +30 mV to examine the current/voltage (I/V) relationship
of the AA-dependent current seen in the nominal absence of
divalent cations. As shown in Fig. 1B, the I/V relationship showed a reversal potential of ~0 mV with significant inward and outward currents (mean current at +30 mV equals 22.6 ± 3.4 pA/pF, n = 5). The I/V curve showed a characteristic tilde
(~) shape, with marked inward rectification at negative voltages and
outward rectification at positive voltages. The obvious nonlinear
nature of the I/V curve strongly suggested that the observed
macroscopic current was not a simple nonspecific "leak" current
caused by removal of extracellular divalent ions. This was further
confirmed by the demonstration that substitution of extracellular
Na+ with NMDG+ completely inhibited the inward
AA-dependent current, but did not affect the outward
current, whereas substitution of Cs+ in the pipette
solution with NMDG+ resulted in the complete absence of any
outward current, without affecting the magnitude of the observed inward
current (see Fig. 4B). Moreover, AA-dependent
inward currents were completely inhibited (mean inhibition of 95.6 ± 3.5%, n = 5) by extracellular La3+ (50 µM) and partially inhibited (50.8 ± 2.2%,
n = 4) by extracellular Cd2+ (50 µM). Both of these inhibitions were reversible. This
further supports the contention that the AA-dependent
current observed was not a nonspecific leak and indicates that the
large current seen upon nominal removal of extracellular divalent
cations represents the activity of an AA-dependent
monovalent cation-permeable conductance. Comparison of the
magnitudes of the inward current (at
80 mV) and the corresponding
outward current (at +30 mV) showed a direct linear relationship
(r2 = 0.89) (Fig. 1C), consistent
with both currents being carried through the same channels. As both
inward and outward currents were observed under these divalent ion-free
conditions, it is clear that the channels responsible are appreciably
permeable to both Na+ and Cs+ ions (see below);
however, they show negligible conductance to NMDG+.
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Fig. 1.
Development of arachidonic
acid-dependent monovalent currents in the nominal absence
of extracellular divalent cations. A, representative
trace showing the current magnitude measured during 250-ms voltage
steps to 80 mV from a holding potential of 0 mV applied every 2 s under standard conditions (Na+ as the principal cation in
the bath solution and Cs+ as the principal cation in the
pipette solution). In the presence of exogenous AA (8 µM), large inward currents developed promptly upon
nominal removal of extracellular divalent cations
(divalent-free) and then subsequently slowly declined.
B, current-voltage relationship of the
AA-dependent current in the nominal absence of
extracellular divalent cations. Currents were recorded during 150-ms
voltage ramps from
100 mV to +30 mV at the peak of the
AA-dependent current. C, relationship between
the magnitude of the inward current measured at
80 mV and the
simultaneous outward current measured at +30 mV. Each point represents
the value from a different cell. D, relationship between the
magnitude of the inward Ca2+-selective current measured at
80 mV immediately prior to removal of extracellular divalent ions and
the corresponding peak inward monovalent current (also measured at
80
mV) in the same cell.
80 mV with that of the corresponding AA-dependent
Ca2+-selective current measured in the same cell
immediately prior to removal of extracellular divalent cations. The
data obtained show a direct linear relationship between the magnitudes
of the two AA-dependent currents (r2 = 0.88) (Fig. 1D), supporting the contention that they
reflect the behavior of a common set of channels (i.e. ARC channels).
80 mV slowly developed upon achieving
whole-cell conditions. As previously reported, this current displayed
all the features consistent with its characterization as a
Ca2+-selective store-operated current
(ISOC), including marked inward rectification,
very positive reversal potential, fast inactivation, and inhibition by
La3+ (50 µM) (12). Essentially identical
currents were observed in cells in which intracellular Ca2+
stores were maximally depleted by preincubation in thapsigargin (1 µM for 15 min) (12) or by inclusion of the
non-metabolizable inositol 1,4,5-trisphosphate analog
adenophostin A (2 µM) in the pipette solution (data not
shown). Subsequent exposure to nominally divalent ion-free external
solution evoked an increase in the inward current measured at
80 mV
to reach a maximum amplitude of 11.7 ± 1.6 pA/pF
(n = 8) or some 20 times the current observed in normal
divalent ion-containing external medium (Fig.
2A). This store-operated
current subsequently declined fairly rapidly in the continued absence
of extracellular divalent ions, showing a 50% reduction in magnitude
in a mean time of 17.6 ± 4.2 s (n = 5).
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Fig. 2.
Development of the endogenous monovalent
store-operated currents in the nominal absence of extracellular
divalent cations. Internal Ca2+ stores were depleted
passively using a Ca2+-free pipette solution. A,
representative trace showing the inward current magnitude measured
during 250-ms voltage steps to 80 mV applied every 2 s from a
holding potential of 0 mV under standard conditions (Na+
and Cs+ as the principal cations in the bath and pipette
solutions, respectively). Large inward currents developed upon nominal
removal of external divalent cations and then spontaneously declined
back to resting levels. B, current-voltage relationship of
the store-operated current in the nominal absence of extracellular
divalent cations. Currents were recorded during 150-ms voltage ramps
from
100 to +30 mV at the peak of the monovalent store-operated
current. C, relationship between the magnitude of the inward
and the simultaneous outward store-operated currents (measured at
80
and +30 mV, respectively). Each point represents the value from a
different cell. D, relationship between the magnitude of the
inward Ca2+-selective store-operated current measured at
80 mV immediately prior to removal of extracellular divalent ions and
the corresponding peak inward monovalent current (also measured at
80
mV) in the same cell.
100 to +30 mV (Fig. 2B). Under standard conditions (Na+ in the bath solution and Cs+ in the
pipette solution), these showed a current that reversed at ~0 mV with
significant inward and outward currents (mean current at +30 mV of
10.8 ± 1.7 pA/pF, n = 6). Inward rectification at negative voltages and outward rectification at positive voltages were
also apparent, giving the I/V curve a tilde shape. The magnitudes of
the inward (at
80 mV) and outward (at +30 mV) currents in individual
cells were linearly related (r2 = 0.72) (Fig.
2C). Moreover, the inward store-operated currents were
reversibly inhibited by extracellular La3+ (50 µM), and both inward and outward currents were rapidly
inhibited upon re-exposure to normal divalent ion-containing medium in
the bath (data not shown). Substitution of extracellular
Na+ with NMDG+ completely inhibited the inward
currents, but did not affect the outward currents. Similarly,
substitution of cesium in the pipette solution with NMDG+
resulted in the complete absence of any outward current, without affecting the magnitude of the observed inward current (see Fig. 4D). Based on these data, we conclude that the observed
macroscopic currents are not the result of a simple nonspecific leak
and that the currents seen upon nominal removal of extracellular
divalent cations after depletion of the intracellular Ca2+
stores represent the activity of the endogenous store-operated conductance (ISOC). This was further confirmed
by comparison of the magnitude of the macroscopic SOC current in its
Ca2+-selective mode seen in the presence of divalent ions
in the bath solution with that of the corresponding current seen in the
same cell upon removal of external divalent ions. This showed that the
magnitudes of the two currents were linearly related to each other
(r2 = 0.64) (Fig. 2D).
80 mV), the monovalent ARC currents seen upon removal of
extracellular divalent ions are ~2.5 times the corresponding
monovalent SOC currents (28.3 versus 11.7 pA/pF at
80 mV).
Such a difference may reflect a different sensitivity of ARC and SOC
channels to the very low (submicromolar) Ca2+
concentrations in our nominally divalent ion-free external medium. However, such sensitivity would have to be much higher than that previously reported for CRAC channels (IC50 for
Ca2+ block of Na+ currents = 4 µM) (13). Moreover, as noted above, the monovalent SOC
currents displayed a fairly rapid spontaneous decline, whereas the
decline in the monovalent ARC current was much slower (Fig. 3,
B and C). As discussed further below, the decline
in the monovalent SOC current appears to reflect some kind of
"inactivation" process of unknown origin. Given this, it is
possible that the inactivation process shown by the SOC channels
is occurring simultaneously with the activation of the monovalent
permeability upon removal of extracellular divalent ions and could
therefore contribute to the relatively smaller monovalent current seen
with these channels. Despite these reservations, it is clear that, like
the store-operated CRAC channels (7, 15, 16), both the endogenous SOC
channels and the ARC channels of HEK293 cells are capable of conducting monovalent ions much more readily than Ca2+ ions, although
there may be some relative difference in this ability between the ARC
and SOC channels.
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Fig. 3.
Comparison of the mean current-voltage
relationships and time courses of inactivation of the monovalent ARC
and SOC conductances. A, mean current-voltage
relationships of the macroscopic monovalent currents through ARC ( )
and SOC (
) conductances. Curves represent mean values from five
(ARC) or seven (SOC) different cells. For clarity, error
bars are shown only for the values obtained at
100 and +30 mV.
B, comparison of the time courses of inactivation of the
monovalent currents through ARC (
) and SOC (
) conductances.
Traces represent responses from typical cells in which the decay in
macroscopic current is displayed by first normalizing to their
corresponding peak values measured at
80 mV. C, the times
required for a 50% decline in current magnitude (mean ± S.E.,
n = 5) for SOC (white bar) and ARC
(black bar) channels.
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Fig. 4.
Selectivity of ARC and SOC conductances for
monovalent cations. ARC currents were activated by addition of
exogenous arachidonic acid (8 µM), and SOC currents were
induced by passive depletion using a Ca2+-free pipette
solution. A-D, shown are mean current-voltage curves
illustrating the effects of substitution with different cations
compared with the currents seen under standard conditions with
Na+ as the principal cation in the bath solution and
Cs+ as the principal cation in the pipette solution ( ).
The effects of substitution of Na+ in the external (bath)
solution with either Cs+ (
) or Li+ (
) are
illustrated for the monovalent currents through ARC (A) and
SOC (C) channels. Similarly, the effects of substitution of
Cs+ in the internal (pipette) solution with either
Na+ (
) or NMDG+ (
) are illustrated for
monovalent currents through ARC (B) and SOC (D)
channels. E, summary data illustrating the effects of
different external cations on inward ARC and SOC currents measured at
80 mV (mean ± S.E., n = 4-11). The principal
external cation was Na+ (white bars),
Cs+ (black bars), or Li+
(shaded bars). F, summary data illustrating the
effects of different internal cations on outward ARC and SOC currents
measured at +30 mV (mean ± S.E., n = 4-8). The
principal internal cation was Cs+ (black bars)
or Na+ (white bars). As can be seen in the
corresponding I/V traces (B and D), there was no
detectable outward current when the principal internal cation was
NMDG+.
80 mV (Fig. 4, A and E). When external
Na+ ions were replaced by Li+, the inward
current density at
80 mV was markedly reduced (from 28.3 ± 2.7 pA/pF (n = 5) to 5.0 ± 2.1 pA/pF
(n = 6)) (Fig. 4, A and E).
Outward (Cs+) currents were also affected by substitutions
of the external cations (Fig. 4A). Substitution of external
Na+ with Cs+ reduced the outward
(Cs+) current measured at +30 mV by ~50% to 11.9 ± 3.0 pA/pF (n = 6; p = 0.02), whereas
substitution with Li+ further reduced the outward current
to 8.0 ± 3.4 pA/pF (n = 6). Clearly then, the
identity of the external ion affects the ability of the internal ion to
carry outward current through the ARC channels. We have not analyzed
the basis for these effects in detail, but they are indicative of an
interaction of the different ions within the channel, consistent with
the channel possessing a multi-ion conductance pathway (19). Based on
these values of relative inward current density, a selectivity sequence
for the monovalent conductance of the ARC channels of Cs+
Na+
Li+
NMDG+ is
indicated. True relative permeabilities are normally analyzed from
changes in reversal potentials. However, in the experiments reported
here, the small magnitudes of the overall currents and the fact that
the I/V curves obtained were generally rather flattened around the
reversal potential made such analysis quantitatively difficult.
Nevertheless, examination of the changes in reversal potentials
observed indicted a similar relative permeability sequence of
Cs+-Na+-Li+-NMDG+.
80 mV upon substitution of
external Na+ with Cs+ was seen with the SOC
current (Fig. 4, C and E). However, as noted
above, the magnitude of the observed currents may be significantly influenced by the simultaneous rate of the inactivation process. In
this case, the rate of the spontaneous inactivation of the SOC current
was noticeably reduced in the presence of external Cs+
(current magnitude reduced to 50% in a mean time of 49.3 ± 2.4 s (n = 3) versus 17.6 ± 4.2 s (n = 5)). Thus, the apparent increased inward SOC current density in the presence of external Cs+
may result, at least in part, from the slower rate of inactivation seen
under these conditions. Replacement of external Na+ with
Li+ resulted in an approximate 50% reduction of the inward
current density to 6.3 ± 3.0 pA/pF (n = 4) (Fig.
4C), a value very similar to that seen with ARC
currents (Fig. 4E). In marked contrast to the
response of ARC currents, outward (Cs+) currents through
SOC channels were not significantly affected by replacement of external
Na+ with either Cs+ or Li+. The
data obtained indicate a monovalent selectivity sequence for inward
currents in the HEK-SOC channels of Cs+
Na+ > Li+
NMDG+. Once again, despite the
difficulties in obtaining any precise quantitative assessment,
examination of the changes in reversal potential observed indicated an
identical relative permeability sequence of
Cs+-Na+-Li+-NMDG+.
NMDG+ is indicated. Again, despite the caveats already
mentioned, examination of the changes in reversal potential observed
indicated an identical relative permeability sequence of
Cs+-Na+-NMDG+.
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Fig. 5.
Effects of internal Mg2+
removal on monovalent ARC and SOC currents. Shown are the mean
(±S.E.) inward (at 80 mV) and outward (at +30 mV) monovalent
currents and overall current-voltage relationships for ARC
(A and B, respectively; n = 4-5)
and SOC (C and D, respectively; n = 6-9) channels in the presence (white bars and
) and
absence (black bars and
) of internal Mg2+
(1.22 mM).
80 mV were 17.6 ± 4.2 s
in the presence of internal Mg2+ and 23.6 ± 4.9 s in its absence (n = 5).
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Fig. 6.
A, representative trace showing that,
after spontaneous inactivation of the monovalent SOC current,
reactivation is possible, but only after exposure to external
Ca2+. The inset is an expanded portion of the
main graph for the period at 450-650 s showing the reactivation of the
Ca2+-selective SOC current upon re-exposure to normal
divalent ion-containing medium. B, the relationship between
the magnitude of the Ca2+-selective current observed
immediately prior to return to a nominally divalent ion-free external
medium (first arrow in A) and the subsequent
reactivated monovalent current (second arrow) in individual
cells. C, representative trace illustrating the sequential
activation of monovalent SOC and ARC currents in the same cell. After
spontaneous inactivation of the monovalent SOC current, addition of
exogenous AA (8 µM) induced the activation of a large
monovalent ARC current.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Comparison of the key features of monovalent currents through CRAC
channels of Jurkat lymphocytes, HEK-SOC channels, and ARC channels
With regard to the monovalent selectivity, the data on inward current
densities indicate a broadly similar conductance sequence for both ARC
and HEK-SOC channels of Cs+ Na+ > Li+
NMDG+, although, compared with
HEK-SOC channels, ARC channels did show a much lower conductance for
Li+ relative to that for Na+ or
Cs+. The corresponding sequence for the outward current
densities for both channels is Cs+ > Na+
NMDG+. It should be noted that these apparent conductance
sequences are based on the measured macroscopic current densities and
therefore do not allow for any possible effects of ion substitution on
the open probability of the channels. Such analysis would require information on the behavior of single channels, which is currently unavailable. The observed monovalent selectivity sequence for both ARC
and HEK-SOC channels stands in marked contrast to that reported for the
CRAC channels of Jurkat cells under the same conditions (15, 16). In
these cells, the measured Cs+ conductance through the CRAC
channels is much smaller than the corresponding Na+
conductance, with a reported ratio of inward Na+ to
Cs+ currents (measured at
80 mV) of 26 ± 3. The
corresponding ratios for ARC and HEK-SOC channels are 0.80 and 0.74, respectively (Fig. 4). Lepple-Wienhues and Cahalan (15) noted that they
sometimes observed a nonspecific leak conductance in Jurkat cells that
showed a Cs+ permeability similar to that of
Na+. However, this leak showed a linear I/V relationship
and lacked the characteristic inactivation seen with either CRAC or
HEK-SOC currents. Given these features, it seems unlikely that the high apparent Cs+ permeability we have observed in the HEK293
cells results from such a similar leak current. Clearly, CRAC channels
are highly selective for Na+ over Cs+ in the
nominal absence of extracellular divalent cations, a feature that is
not shared by either the ARC channels or the endogenous HEK-SOC
channels. Importantly, Kerschbaum and Cahalan (16) reported that the
very low conductance to Cs+ shown by CRAC channels
(relative to Na+) is another feature that is dramatically
affected by internal Mg2+. Although removal of internal
Mg2+ did significantly increase outward Cs+
currents through HEK-SOC channels (Fig. 5C), the effect was
quantitatively much smaller than that reported for CRAC channels (16).
Moreover, in marked contrast to the data from both CRAC and HEK-SOC
channels, removal of internal Mg2+ significantly
decreased outward Cs+ currents through the ARC
channels (Fig. 5A). Obviously, the conclusion reached by
Kerschbaum and Cahalan (16) that internal Mg2+ normally
blocks outward current through CRAC channels clearly does not apply to
ARC channels.
Another reported effect of internal Mg2+ removal on CRAC channels is the loss of the spontaneous inactivation of the monovalent current (16). Although this was not seen in the HEK-SOC currents, many of the other features of the inactivation of monovalent CRAC currents were seen in the HEK-SOC currents, including the ability to reactivate the monovalent currents only after a brief period of re-exposure to external Ca2+. This reactivation process involved both Ca2+ currents (measured in normal divalent ion-containing medium) and the monovalent currents (measured in divalent ion-free medium), which reactivated in synchrony, as was previously reported for CRAC channels (15). A rather different kind of decay was observed in the monovalent currents through the ARC channels. This decay was much slower than the inactivation seen in the HEK-SOC currents, and after decaying to resting levels, re-exposure to external Ca2+ was able to induce only a minimal reactivation of the monovalent ARC currents. Although we have no direct information on the basis for this phenomenon, such behavior is suggestive of a run-down of the currents rather than an inactivation. Precise characterization of these two processes and the mechanisms involved must await further studies. Nevertheless, we were able to utilize the absolute requirement of exposure to extracellular Ca2+ for the reactivation of the HEK-SOC currents after their spontaneous inactivation to demonstrate that ARC currents could be activated (by addition of extracellular AA) in cells in which the monovalent SOC currents had been previously fully inactivated. The subsequently activated AA-dependent monovalent currents displayed all the properties characteristic of ARC currents. Clearly then, ARC channels can be activated in a cell whose SOC channels have been completely inactivated, consistent with our earlier findings that the two conductances are entirely separate and distinct entities (12, 25).
Together, the data presented indicate that there are both similarities and marked differences in the behavior of the CRAC channels of Jurkat cells, the endogenous SOC channels of HEK293 cells, and non-capacitative ARC channels. We have already described the similarities and differences between the HEK-SOC and ARC channels in their normal Ca2+-conducting state (12). In this study, we have shown that, as highly Ca2+-selective conductances, these channels share the property of conversion to a monovalent cation-permeable mode upon removal of extracellular divalent cations. Comparing first the two store-operated conductances CRAC and HEK-SOC, it is clear that, although they are very similar in several respects, they also appear to display some marked differences. Examination of these differences reveals that they particularly revolve around the effects of internal Mg2+ ions on the channel properties. For example, two of the most obvious differences between the CRAC and HEK-SOC conductances are their respective Na+/Ca2+ current density ratios and their monovalent selectivity sequence. However, these differences largely disappear when CRAC currents are measured in the absence of internal Mg2+ (16). In other words, HEK-SOC channels under normal conditions behave much like CRAC channels in the absence of internal Mg2+, suggesting that the principal difference between these two store-operated conductances lies simply in the presence or absence of some Mg2+-binding site(s) or in its affinity for Mg2+. Whether this reflects the influence of differences in the cellular environment (membrane composition, presence of modulatory factors, etc.) or actual differences in the molecular identity of the channel proteins involved must await their characterization at the molecular level. However, it seems that these two store-operated conductances are probably closely related members of what is likely to be a family of similar channels.
More importantly, our ability to measure the endogenous SOC
channels and ARC channels in the same cells allows a much more direct
comparison between these two conductances. Here, several marked
differences are apparent. These include differences in the magnitude of
the ratio of monovalent to Ca2+ currents, in the rate and
nature of the spontaneous decline in monovalent current, and in the
effects of external monovalent cation substitutions and of internal
Mg2+ removal on outward currents. All these features
indicate that ARC and SOC currents reflect the activity of distinct
conductances. Moreover, we have demonstrated that monovalent ARC
currents can be activated after the complete spontaneous inactivation
of SOC currents in the same cell. As such, this confirms our previous findings based on the characteristics and additive nature of the Ca2+-selective currents for these two conductances (12) and
on their distinct relative abilities to activate
Ca2+-sensitive adenylyl cyclases (25). Of course, the most
important and fundamental distinction between these two conductances is that, unlike SOC channels, ARC channels are not activated by
store depletion (12). Importantly, the individually unique
characteristics displayed by these two conductances in their monovalent
cation-permeable modes described here, together with the much
larger macroscopic currents observed, are features that will
undoubtedly prove useful in the identification of the specific
activities of the respective channels under different experimental
conditions and, in addition, raise the possibility of further more
detailed analysis at the single channel level, as has already been
demonstrated for CRAC channels (26, 27).
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ACKNOWLEDGEMENTS |
---|
We thank Jill Thompson for excellent technical assistance and Drs. Ted Begenisich and Bob Dirksen for helpful discussions and suggestions and comments on an earlier version of the manuscript.
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FOOTNOTES |
---|
* This work was supported by NIGMS Grant GM 40457 from the National Institutes of Health (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.
To whom correspondence should be addressed: Dept. of Pharmacology
and Physiology, University of Rochester Medical Center, P. O. Box 711, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-2076; Fax:
716-273-2652; E-mail: trevor_shuttleworth@urmc.rochester.edu.
Published, JBC Papers in Press, April 2, 2001, DOI 10.1074/jbc.M102311200
2 O. Mignen, J. Bruce, and T. J. Shuttleworth, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: SOC, store-operated Ca2+; CRAC, Ca2+ release-activated Ca2+; ARC, arachidonate-regulated Ca2+; AA, arachidonic acid; pF, picofarad; NMDG, N-methyl-D-glucamine; I/V, current/voltage.
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