Laboratory of Cellular and Molecular Neurobiology, Department of Neurobiology and Behavior, University of California Irvine, Irvine, California 92697-4550
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ABSTRACT |
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Ca2+-activated
Cl currents (ICl,Ca) were
examined using fluorescence confocal microscopy to monitor
intracellular Ca2+ liberation evoked by flash photolysis of
caged inositol 1,4,5-trisphosphate (InsP3) in
voltage-clamped Xenopus oocytes. Currents at +40 mV exhibited a
steep dependence on InsP3 concentration
([InsP3]), whereas currents at
140 mV exhibited a higher threshold and more graded relationship
with [InsP3]. Ca2+ levels
required to half-maximally activate ICl,Ca were
about 50% larger at
140 mV than at +40 mV, and currents evoked
by small Ca2+ elevations were reduced >25-fold. The
half-decay time of Ca2+ signals shortened at increasingly
positive potentials, whereas the decay of ICl,Ca
lengthened. The steady-state current-voltage (I-V) relationship
for ICl,Ca exhibited outward rectification with
weak photolysis flashes but became more linear with stronger stimuli.
Instantaneous I-V relationships were linear with both strong
and weak stimuli. Current relaxations following voltage steps during
activation of ICl,Ca decayed with half-times that shortened from about 100 ms at +10 mV to 20 ms at
160 mV. We conclude that InsP3-mediated Ca2+
liberation activates a single population of Cl
channels, which exhibit voltage-dependent Ca2+ activation
and voltage-independent instantaneous conductance.
calcium-activated chloride current; voltage dependence; inositol 1,4,5-trisphosphate
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INTRODUCTION |
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NUMEROUS Ca2+-activated
Cl channels are present in the plasma membrane of
Xenopus oocytes (13) and serve to generate the fertilization potential that provides a fast electrical block to polyspermy in the
egg (7). Voltage-clamp recordings of Cl
current provide a convenient reporter of subplasmalemmal
Ca2+ concentration ([Ca2+]) and are
widely used to study both endogenous Ca2+ signaling
pathways in the oocyte and to monitor the expression of exogenous
Ca2+-mobilizing receptors (8, 14).
Ca2+-activated Cl
currents
(ICl,Ca), however, show complex time- and
dose-dependent characteristics that complicate interpretation of the
underlying Ca2+ signals (11, 21). In particular, it remains
unclear whether they are generated through a single population of
Cl
channels or through two or more channel types
that have different sensitivities to Ca2+ (3, 11).
This problem is particularly vexing in the case of responses mediated
by the inositol 1,4,5-trisphosphate (InsP3)
messenger pathway. Ca2+ signals evoked by
InsP3 involve a rapid, transient liberation of
Ca2+ from endoplasmic reticulum stores, followed by influx
of extracellular Ca2+ via a plasma membrane pathway
activated by store depletion (2, 23). These two sources of
Ca2+ can be discriminated by recording
ICl,Ca evoked by hyperpolarizing voltage steps
because entry of extracellular Ca2+ depends on the
electrochemical gradient across the cell membrane, whereas
intracellular Ca2+ mobilization is largely independent of
membrane potential (11, 19, 28). We had previously interpreted the
resulting Cl currents as arising through a single
class of Cl
channels (28), but a more detailed
electrophysiological analysis led Hartzell (11) to propose the
involvement of two channel types: one generating an outwardly
rectifying current activated by both intracellular Ca2+
liberation and Ca2+ influx and a second channel with lower
affinity for Ca2+ that is activated selectively by the high
subplasmalemmal Ca2+ levels resulting from Ca2+
influx during hyperpolarizing voltage steps. An alternative hypothesis, however, is that only a single class of ICl,Ca
channels is present in the oocyte, but that the sensitivity of these
channels for Ca2+ is voltage dependent (12).
To study the influence of membrane potential on the Ca2+
sensitivity of Cl channels, we recorded
Cl
currents and cytosolic Ca2+ signals
generated by Ca2+ arising from transient liberation of
Ca2+ from intracellular stores induced by flash photolysis
of caged InsP3. Currents recorded at
positive holding potentials exhibited a lower threshold and steeper
dose dependence on InsP3 concentration ([InsP3]) than corresponding currents
at hyperpolarized potentials, even though intracellular
Ca2+ signals monitored by confocal microfluorimetry were
similar at both voltages. Furthermore, the steady-state current-voltage
(I-V) relationship of the Cl
current changed
progressively from strongly outward rectifying to linear with
increasing intracellular [Ca2+]. These
results are consistent with the presence of a single class of
Cl
channels that show increasing sensitivity
to Ca2+ at more positive potentials.
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METHODS |
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Immature (stage V and VI) oocytes of Xenopus laevis were obtained as previously described (25). Frogs were anesthetized by immersion in a 0.17% aqueous solution of MS-222 (3-aminobenzoic acid ethyl ester) for 15 min, and a small portion of the ovary was removed surgically through an abdominal incision, after which the wound was sutured and animals were allowed to recover. After manual removal of epithelial layers, oocytes were loaded 30-60 min before recording with caged InsP3 [myo-inositol 1,4,5-trisphosphate, P4(5)-1-(2-nitrophenyl) ethyl ester] together with the low-affinity Ca2+ indicator Oregon Green 488 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-5N (OG-5N) to final intracellular concentrations of about 5 and 50 µM, respectively (26).
Recordings were made at room temperature, with oocytes bathed in normal
Ringer solution (in mM, 120 NaCl, 2 KCl, 1.8 CaCl2, 5 HEPES, at pH about 7.2). Ca2+-free solution contained no
added Ca2+, and additionally 1 mM EGTA and 5 mM
Mg2+. Measurements of membrane currents were obtained using
a two-electrode voltage clamp (GeneClamp 500; Axon Instruments; Foster
City, CA), with KCl-filled microelectrodes broken to resistances of
1-3 M. Simultaneously, measurements were made of intracellular
Ca2+ signals (monitored by OG-5N fluorescence) evoked by
photostimulation, using a linescan confocal microscope and flash
photolysis system as described previously (4, 17). In brief, a
custom-built confocal scanner assembly was interfaced to an Olympus IX
70 inverted microscope fitted with a ×40 oil-immersion objective
(NA 1.35). Fluorescence excited in the oocyte by a diffraction-limited
spot of 488 nm light from an argon ion laser was monitored through a
confocal aperture at
> 510 nm while the spot was repeatedly scanned along a 50-µm line. The microscope was focused at the depth
of the pigment granules within the oocyte, which lie about 2 µm
inward from the cell surface. Measurements are presented as a ratio
relative to the resting fluorescence before stimulation (
F/F) and
represent an average across the scan line. Scans were obtained at a
rate of 125 Hz, and current records were low-pass filtered at 50 Hz.
The low-affinity indicator OG-5N was used to avoid saturation during
strong InsP3-evoked Ca2+ signals and
provide a fluorescence signal linearly proportional to
[Ca2+] over the range of interest. The
dissociation constant of OG-5N is about 31 µM [measured using
Molecular Probes buffer kit 3 in the presence of 1 mM Mg2+
(24)], and we observed a maximal fluorescence change of 12.6 following injections of saturating amounts of Ca2+ into
oocytes (n = 3) through a micropipette filled with 100 mM CaCl2. Thus for small Ca2+ signals, the dye
provides a fluorescence change of ~0.20
F/F per micromole of free
Ca2+.
Flashes of ultraviolet (UV; 340-400 nm) light, derived from a continuous mercury arc lamp, were used to photorelease InsP3 from the caged precursor loaded into the oocyte. The relative amount of InsP3 formed was varied using an electronic shutter to regulate the duration of light flashes and neutral density filters to control intensity. The photolysis light was introduced through the epifluorescence port of the microscope and was focused uniformly as a 200-µm diameter spot on the cell, centered around and parafocal with the laser scan line. For experiments with Ca2+ injections, oocytes were not loaded with caged InsP3 or indicator, and injections were made by applying pneumatic pressure pulses to a third micropipette filled with 0.5 M CaCl2 while the cell was voltage clamped (13, 18). All experiments were done in the animal hemisphere of oocytes, because this contains a higher density of Ca2+-activated channels (10, 13) and InsP3-sensitive Ca2+ release sites (6).
Fluorescence signals and currents were quantified by measuring the
amplitudes of their respective peaks. We did not attempt to correlate
simultaneous measurements of current and fluorescence, because of
uncertainties in their kinetic relationship (e.g., delays due to
Ca2+ diffusion and Cl channel
activation). However, the peak current was usually attained shortly (ca
100 ms) following the peak fluorescence, at a time when the
fluorescence had declined only slightly (<10%). To compare responses
at different membrane potentials, measurements of currents were
expressed as membrane conductance, so as to normalize for ohmic changes
in current and better reveal the voltage dependence of activation of
ICl,Ca. Conductances were calculated using measured values of reversal potentials for ICl,Ca, which
were close to
20 mV, corresponding to the Cl
equilibrium potential in the oocyte (13). Outward currents at voltages
positive to this potential are thus carried by an influx of
Cl
, and inward currents at more negative potentials
by efflux of Cl
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In addition to Cl currents evoked by
InsP3-mediated Ca2+ liberation, oocytes
from some donor frogs display Cl
currents on
depolarization due to activation of voltage-dependent membrane
Ca2+ channels (13). These latter currents were absent or
negligible (<50 nA) in the oocytes used for the present experiments.
Furthermore, they would not have complicated measurements of
InsP3-evoked signals, because the
Cl
currents generated by Ca2+ entry
through endogenous voltage-gated channels decay within about 2 s (13),
whereas photolysis flashes were delivered after the oocytes had been
held at a given potential for >10 s.
OG-5N and caged InsP3 were obtained from Molecular Probes (Eugene, OR). All other reagents were from Sigma Chemical (St. Louis, MO).
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RESULTS |
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Cl currents and
Ca2+ fluorescence signals evoked by
photoreleased InsP3. Figure
1 shows records of Ca2+
fluorescence signals (top traces) and currents (bottom
traces) obtained at clamp potentials of
160 mV (Fig.
1A) and +40 mV (Fig. 1B) in a single oocyte in response
to photorelease of InsP3 by UV light flashes of
increasing durations. These data are representative of results obtained
in a total of 12 oocytes. As we have described previously,
Ca2+ waves (22) and Ca2+-activated currents
(18) were evoked only by flashes exceeding a certain threshold
duration. With suprathreshold stimuli, the amplitudes of the
Ca2+ signals then increased progressively with increasing
flash duration and were similar at the two holding potentials. In
contrast, the corresponding membrane currents differed markedly at
negative and positive potentials. Inward currents at
160 mV were
brief and varied progressively in magnitude with the size of the
underlying Ca2+ transient. Outward currents at +40 mV, on
the other hand, were slower and exhibited a steeper dose-response
characteristic.
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Measurements of peak fluorescence signals and currents are shown
plotted as a function of flash duration in Figs. 1, C and D. At 160 mV the current amplitude increased in a graded
manner as the flashes were progressively lengthened. In contrast,
currents at +40 mV exhibited a lower threshold and increased more
steeply with increasing flash duration so as to more rapidly attain a maximum, saturating value. Maximal currents at +40 mV were smaller than
at
160 mV, but this could be attributed simply to the different electrical driving force for Cl
flux through the
channels. To eliminate the ohmic voltage dependence of the current, the
data are expressed as membrane conductance in Fig. 1E. This
displays more clearly the differing dose dependence of activation of
ICl,Ca at positive and negative potentials and shows that the maximal conductances at high
[InsP3] were similar at positive and
negative potentials. The different dose dependence was not due to
differences in [Ca2+] transients, because the
corresponding fluorescence signals showed closely similar peak
amplitudes at positive and negative holding potentials (Fig.
1C). Instead, the differences in dose dependence likely arise
through voltage-dependent changes in Ca2+ activation of the
Cl
channels.
A possible concern, however, was whether Ca2+ influx across
the plasma membrane may have locally activated Cl
channels without producing appreciable fluorescence signals. This is
unlikely because similar data (not shown) were obtained in four oocytes
bathed in Ca2+-free Ringer solution. Furthermore,
InsP3-mediated activation of store-operated
Ca2+ influx develops much more slowly than the transient
responses evoked here (28) and, in any case, is not expected to
contribute to the increased sensitivity of ICl,Ca
to Ca2+ at positive potentials because the electrochemical
gradient for Ca2+ entry is reduced at these voltages.
Density of Ca2+-activated
Cl channels. Figure
2, inset, shows the mean values of currents
evoked by supramaximal photolysis flashes (intensities >50times
threshold) in oocytes from five different donor frogs.
Measurements were made at a holding potential of +40 mV, using a
200-µm-diameter photolysis spot positioned near the animal pole. The
maximal currents were relatively consistent within oocytes obtained
from a given donor frog but varied more widely between oocytes from
different frogs. Among the donors examined, mean currents ranged from
~300-1,200 nA. The overall current was 843 ± 66 (SE) nA (35 oocytes), which corresponds to a conductance of 14.05 µS (assuming a
Cl
equilibrium potential of
20 mV). Given the
restricted spatial photorelease of InsP3
(200-µm-diam spot), the maximal conductance was ~600 pS per square
micrometer of cell surface (neglecting membrane infoldings) and the
maximal whole cell conductance from the entire oocyte is predicted to
be about 1,200 µS, assuming a diameter of 1.2 mm and a twofold lower
density of Cl
channels in the vegetal hemisphere
(6). The single channel conductance of Ca2+-activated
Cl
channels in the oocyte membrane measured by
patch-clamp studies is about 3 pS (27), indicating that the channel
density in the animal hemisphere is roughly 200/µm2
considering the oocyte as a smooth sphere, a value that may be reduced
as much as 10-fold in terms of density per square micrometer of actual
membrane area because of the numerous microvilli (8).
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Relationship between ICl,Ca and
cytosolic [Ca2+]. The
relationship between the cytosolic Ca2+ signal and
activation of ICl,Ca was determined from
experiments such as that in Fig. 1, using a range of flash strengths to
evoke responses of varying magnitudes while fluorescence transients were imaged with the confocal scan line focused within 2 µm of the
cell surface. Figure 2A shows pooled measurements from 12 oocytes, plotting the peak conductance change at holding potentials of
+40 and 150 mV as a function of OG-5N fluorescence ratio. The
maximal conductance at high [Ca2+] was similar
at both voltages, but the relationship at
150 mV was shifted to
the right compared with that at +40 mV so that intermediate
Ca2+ levels resulted in smaller conductance changes at the
hyperpolarized potential. The fluorescence signal associated with
half-maximal activation of ICl,Ca increased by
about 46% on polarization from +40 to
150 mV, but a much more
pronounced effect was evident at low Ca2+ levels. For
example, the conductance change associated with a fluorescence signal
of 0.25
F/F was nearly 20-fold greater at +40 mV than at
150 mV.
Measurements of fluorescence signals corresponding to half-maximal
Cl currents at various voltages are shown in Fig.
2B and provide an estimate of how the apparent affinity for
Ca2+ activation of the Cl
conductance
varies with voltage. Little difference was apparent between +40 mV and
60 mV, but the Ca2+ levels associated with
half-maximal activation then increased progressively at more negative
potentials and were about 57% greater at
150 mV compared with
+40 mV. From the calibration factor given in METHODS, the
fluorescence ratio values in Fig. 2B correspond to free
Ca2+ concentrations of about 2.1 µM at +40 and
60
mV, 3 µM at
120 mV, and 3.5 µM at
150 mV.
Voltage dependence of InsP3-evoked
currents and Ca2+ signals. To explore
further the interaction between voltage and
[Ca2+] in the activation of
ICl,Ca, we recorded fluorescence signals and
membrane currents evoked by photorelease of InsP3
while the membrane potential was clamped over a range of holding
voltages. Figure 3 shows representative
records from an oocyte stimulated by photolysis flashes that were
relatively strong (135 ms; Fig. 3A) or weak (35 ms; Fig.
3B) in relation to the threshold flash duration required to
evoke ICl,Ca at +40 mV (about 25 ms). The InsP3-evoked Ca2+ signals changed
relatively little with voltage, other than a slight acceleration of
their decay at more positive potentials. In contrast, the amplitudes
and kinetics of ICl,Ca changed markedly with
voltage. Currents evoked by both strong and weak flashes reversed
direction at about 18 mV in this oocyte, and at increasingly positive potentials, the currents became slower and exhibited a
prolonged plateau. The peak amplitudes of outward currents evoked by
both weak and strong flashes increased progressively as the membrane
potential was clamped at voltages increasingly positive to the reversal
potential. At more negative potentials, however, responses to strong
flashes differed markedly from those evoked by weak flashes. Inward
currents evoked by the 35-ms flashes increased only slightly or even
declined with hyperpolarization beyond about
60 mV, despite the
increased electrical driving force for Cl
efflux
(Fig. 3B). In contrast, the peak amplitudes of currents evoked
by 135-ms flashes continued to increase progressively with hyperpolarization to at least
140 mV (Fig. 3A).
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I-V relationships (Fig. 3C) derived from these data
showed a nearly linear voltage dependence for
ICl,Ca evoked by the 135-ms flash, whereas currents
evoked by the 35-ms flash showed a marked outward rectification. The
ratio of the current amplitudes evoked by the strong and weak flashes
varied from about 1.5 at a potential of +50 mV to about 4 at 180
mV. This shift from an outwardly rectifying to a linear I-V
relationship with increasing photorelease of InsP3
was consistently observed in all oocytes examined (12 cells; 5 donor frogs).
The outward rectification of currents evoked by weak flashes was not
due to decreased Ca2+ liberation at more negative
potentials, because the corresponding fluorescence signals changed
little or not at all at voltages between 180 and +60 mV (Fig.
3D). Instead, we interpret the differing shapes of the
I-V relationships with weak and strong stimuli to arise through
the voltage-dependent decrease in sensitivity of the
Cl
conductance to Ca2+ at more negative
potentials. Thus intracellular free [Ca2+]
levels resulting from the strong flash were sufficient to maximally activate the conductance at all voltages between approximately +60 and
140 mV, whereas currents evoked by the weak flashes diminished at increasingly hyperpolarized potentials as the sensitivity of the
Cl
conductance became progressively lower in
relation to the smaller cytosolic [Ca2+] transient.
In accordance with this interpretation, the shape of the I-V
relationship depended on the current density (current per unit area of
membrane) rather than on the absolute whole cell current. For example,
when the photolysis light was focused sharply as a 200-µm diameter
spot, a flash of 60-ms duration evoked currents with a linear voltage
dependence. After deliberately defocusing the light to illuminate a
broader area of the cell, currents evoked by flashes of the same
duration then exhibited a pronounced outward rectification, so that the
current at 130 mV was only 55% of that with the sharply focused
spot, even though the currents at +30 mV were similar (2 oocytes examined).
Voltage-dependent kinetics of Ca2+
transients and ICl,Ca. As illustrated in Fig.
3, A and B, the kinetics of ICl,Ca varied strongly with holding potential, although the time course of the
corresponding Ca2+ signals exhibited only slight voltage
dependence. To quantify these effects, we measured the time to fall to
one-half the peak value (t1/2) of currents and
fluorescence signals evoked by supramaximal photolysis flashes over a
range of holding potentials (Fig.
4A). The decay of the currents
shortened markedly at increasingly negative potentials, with
t1/2 decreasing as an exponential function of voltage from about 1.6 s at +60 mV to 350 ms at 175 mV. In
contrast, the voltage dependence of decay of the fluorescence signals
was more shallow and in the opposite direction; the
t1/2 lengthened from about 1.5 s at +60 mV to about
1.75 s at strongly negative potentials.
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The increasing discrepancy in time course between the
Cl currents and Ca2+ signals at more
negative potentials was not due simply to a reduction in
Ca2+ sensitivity, such that the current terminated more
rapidly because the Ca2+ level, as indicated by the
fluorescence signal, declined below the higher threshold level needed
for activation of ICl,Ca. This is illustrated in
Fig. 4B, which compares fluorescence and current signals evoked
at
160 mV by photolysis flashes with durations of 22 and 50 ms.
The current evoked by the stronger flash terminated rapidly, and no
current was evident during an appreciable time for which the
fluorescence signal remained higher than the peak level evoked by the
weaker flash, to which there was a clear current response.
Graded rectification of ICl,Ca as a
function of stimulus strength. Figure
5A plots pooled data from five
oocytes (3 donor frogs), showing that the form of the I-V
relationship of ICl,Ca varied progressively from
almost complete outward rectification with just suprathreshold
photolysis flashes to near perfect linearity with very strong flashes.
Normalized Cl conductance changes derived from these
data are shown in Fig. 5B, to remove the ohmic voltage
dependence of the current and display more clearly the effect of
voltage on activation of ICl,Ca. The strongest
flash was >100 times stronger than the threshold required to evoke a
current at +40 mV and produced a conductance that remained constant
between +40 and
160 mV, suggesting that cytosolic
[Ca2+] was sufficiently high to maximally
activate the channels even at strongly hyperpolarized potentials. In
contrast, the conductance change evoked by the weakest flash (~1.2
times threshold) declined rapidly at potentials negative to +40 mV and
decreased to ~5% at
120 mV. Flashes of intermediate strengths
gave intermediate conductance-voltage relationships.
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Instantaneous and steady-state I-V relationships. The results
presented above are consistent with ICl,Ca arising
through channels, the gating of which is determined by
[Ca2+] and modulated by voltage and which
exhibit an ohmic open channel I-V relationship (as demonstrated
by the linear voltage dependence with strong stimuli). To confirm this
latter point, we measured instantaneous I-V relationships
(reflecting current flow through open channels) as well as steady-state
relationships (reflecting both probability of channel opening and open
channel current). Figure 6 shows data from
an oocyte stimulated by relatively weak (1.5 times threshold at +20 mV)
and stronger (7 times threshold) photolysis flashes. Steady-state
current measurements were obtained, as in Fig. 2, by delivering
photolysis flashes after clamping at a given potential for several
seconds. To determine instantaneous currents, flashes were delivered
with the oocyte clamped at +20 mV and the potential was stepped to more
negative levels at about the time of the peak current (800 ms following
the flash). Measurements of instantaneous currents were made about 8 ms
after the voltage step, to allow time for capacitative currents to
settle, and are presented after subtraction of passive currents evoked
by equivalent voltage steps in the absence of photolysis flashes. The
steady-state I-V relationship for the weak flash exhibited
characteristic outward rectification, whereas the corresponding
instantaneous I-V relationship was close to linear. With the
strong flash, on the other hand, both the instantaneous and
steady-state relationships were linear and closely similar.
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Kinetics of Ca2+-sensitivity
changes. To examine the rapidity with which the Ca2+
sensitivity of the Cl conductance changed with
potential, we measured the relaxation times of tail currents following
voltage steps. These measurements were not readily obtained from
responses evoked by photoreleased InsP3, due to
their rapid decay (e.g., Fig. 1). Instead, we applied voltage pulses
during submaximal currents evoked by microinjecting Ca2+
into the oocyte through a pipette filled with 0.5 M CaCl2,
which persist for a few seconds (13). Hyperpolarizing steps applied from a holding potential of
50 mV evoked large instantaneous inward currents that decayed rapidly, whereas positive-going steps evoked smaller instantaneous currents followed by a more slowly increasing outward current (Fig.
7A). Similar to the I-V
relationships obtained with InsP3-evoked responses,
the steady-state currents measured following Ca2+
injections exhibited outward rectification (Fig. 7B), whereas instantaneous currents were more closely linear. The half-times of the
current relaxations lengthened from ~20 ms at
160 mV to nearly
100 ms at +10 mV (Fig. 7C).
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DISCUSSION |
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We found that Cl currents in Xenopus oocytes
activated by InsP3-mediated liberation of
intracellular Ca2+ exhibited a marked increase in
sensitivity to [Ca2+] at increasingly positive
membrane potentials. Consequently, the shape of the I-V
relationship varied with [Ca2+]. Currents
evoked by small Ca2+ elevations exhibited outward
rectification, with little or no response at strongly negative
potentials, whereas a linear I-V relationship was obtained with
Ca2+ elevations sufficiently large to cause maximal
activation over a wide voltage range. These findings are most easily
explained by the presence of a single class of
Ca2+-activated Cl
channels that exhibit
a voltage-dependent activation by cytosolic Ca2+ and a
voltage-independent open-channel conductance. The idea that there are
two or more classes of Cl
channels with differing
Ca2+ affinities (11) appears less plausible, because our
data indicate that this would require both outwardly rectifying
channels with high Ca2+ affinity and inwardly rectifying
channels with low affinity, present at an appropriate relative density
in the oocyte membrane so as to result in an overall linear I-V
relationship with strong Ca2+ activation. Our results and
conclusion are similar, however, to a recent study by Kuruma and
Hartzell (12), who employed Ca2+ injections and
Ca2+ influx through heterologously expressed
Ca2+ channels to show that outward Cl
currents in the oocyte are more sensitive to Ca2+ than
inward currents. In that study they also described apparent differences
in anion selectivity and instantaneous I-V relationships between Cl
currents activated by different
voltage-clamp protocols but concluded that these may arise through
experimental limitations and that the hypothesis that a single type of
Cl
channel remained valid (12).
In agreement with Kuruma and Hartzell (12), we believe a likely
explanation for the voltage-dependent sensitivity of the Cl current to cytosolic Ca2+ is that the
apparent affinity of the channel for Ca2+ is voltage
dependent, a property that is common among Ca2+-activated
membrane channels. For example, Ca2+-activated
Cl
channels in rat parotid acinar cells (1) and in
pulmonary artery endothelial cells (15) exhibit increased activation at more positive potentials, as do large-conductance
Ca2+-activated K+ channels (9). The increasing
sensitivity of the Cl
current at more positive
potentials may be due to Ca2+ remaining bound to the
channel for a longer time, and in agreement relaxations of
ICl,Ca following voltage steps occur with a
half-time that lengthens from about 20 ms at
160 mV to about 100 ms at +10 mV. These values are closely similar to those obtained for half-times of deactivation of ICl,Ca in excised
patches of oocyte membrane following removal of Ca2+ (10),
suggesting that the voltage-dependent changes in affinity of the
Cl
channel arise largely through changes in off-rate
for Ca2+ dissociation rather than in the rate of
Ca2+ binding. Our measurements, however, represent
population behavior of the underlying channels and do not allow a
precise molecular interpretation. It therefore remains possible that
processes other than changes in affinity may account for the
voltage-dependent sensitivity of the Cl
current
(e.g., changes in channel gating subsequent to Ca2+
binding; modulation of local cytosolic [Ca2+]
gradients near the plasma membrane). Detailed kinetic studies employing
patch-clamp recording are required to unambiguously resolve these issues.
Fluorescence measurements of the intracellular free Ca2+
transients underlying the Cl currents indicated that
half-maximal activation of the Cl
conductance
required ~3.5 µM Ca2+ at
150 mV and about 2 µM
Ca2+ at +40 mV. These values are within the range of
cytosolic Ca2+ concentrations attained during
InsP3-mediated signaling (22), and even at strongly
negative potentials sufficient Ca2+ could be liberated in
most oocytes by strong photorelease of InsP3 to
maximally activate the conductance. However, the voltage-dependent shift in the Ca2+ activation of ICl,Ca
(Fig. 2B) results in much more prominent effects at low
[Ca2+] than suggested by the relatively modest
change in concentrations required for half-maximal activation. For
example, conductances evoked by just suprathreshold photorelease of
InsP3 decreased by almost 95% with polarization
from +40 to
140 mV. This large change may account for
observations that intracellular Ca2+ elevations
preferentially activate outward Cl
currents (12) and
that Ca2+ influx (which is expected to produce a high,
local [Ca2+] near the plasma membrane) readily
activates inward currents at hyperpolarized potentials, whereas
Ca2+ liberation from more distant intracellular stores is
relatively ineffective (11, 12).
Gomez-Hernandez et al. (10) reported that a concentration of ~27 µM
Ca2+ was required for 40% activation of
Cl current in excised patches of Xenopus
oocyte membrane. One explanation for the apparent discrepancy between
this value and our results may be that the sensitivity of the channels
is higher in the intact oocyte compared with an excised patch.
Alternatively, high Ca2+ levels may be sensed by the
Cl
channels if they are located close to
InsP3-sensitive Ca2+ release sites but
would not be accurately reflected in the fluorescence measurements. If
this is the case, such local Ca2+ gradients must be very
narrowly delineated, as our fluorescence measurements were obtained
from a confocal section <1 µm thick (17) focused within about 2 µm of the plasma membrane, and we did not observe larger signals
closer to the plasma membrane (5).
A further unresolved question concerns the kinetic relationship between
Ca2+ signals and ICl,Ca. The decay of
InsP3-evoked fluorescence signals exhibited a
slight slowing with polarization to more positive potentials, possibly
because the mechanisms extruding Ca2+ across the plasma
membrane functioned more effectively because of the reduced
electrochemical gradient for Ca2+ across the membrane. In
contrast, the decay of Cl currents lengthened
considerably at increasingly positive potentials, following an
exponential relationship with an e-fold change in t1/2 per 210 mV. The increasing discrepancy between
time courses of the fluorescence signal and ICl,Ca
with hyperpolarization is not readily explained as a simple consequence
of the reduced sensitivity to Ca2+ but suggests that the
kinetics of termination of the Cl
conductance are
voltage sensitive. The reason why ICl,Ca declines more rapidly than the Ca2+ signal at negative voltages,
however, remains unclear. We had previously proposed that the
Cl
channels exhibit an adaptive behavior to
Ca2+ (21), but subsequent findings of sustained
Cl
currents evoked by Ca2+ application
to excised membrane patches (10) and by photolysis of caged
Ca2+ in intact oocytes (unpublished data) make this idea
less tenable. Instead, a close coupling between Ca2+
release sites and Cl
channels may help explain the
dissociation in time courses of the confocal fluorescence signal and
ICl,Ca.
The high sensitivity of the Cl conductance to
[Ca2+] at positive membrane potentials may
assist in experiments where voltage-clamp recording of
Cl
current is used as a convenient, endogenous
monitor of intracellular Ca2+. Furthermore, it is likely to
be of physiological importance in the generation of the fertilization
potential that results from an InsP3-dependent wave
of intracellular Ca2+ spreading from the sperm entry site
(16). The Cl
equilibrium potential for eggs in pond
water is positive, so that opening of Cl
channels
results in a depolarization. This is likely to be reinforced robustly
by the regenerative characteristic imparted by the increasing sensitivity to intracellular Ca2+ at increasingly positive potentials.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jennifer Kahle for editorial assistance. This work was supported by the National Institute of General Medical Sciences Grant GM-48071.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: I. Parker, Laboratory of Cellular and Molecular Neurobiology, Dept. of Neurobiology and Behavior, Univ. of California, Irvine, CA 92697 (E-mail: iparker{at}uci.edu).
Received 16 August 1999; accepted in final form 26 October 1999.
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REFERENCES |
---|
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---|
1.
Arreola, J,
Melvin JE,
and
Begenisich T.
Activation of calcium-dependent chloride channels in rat parotid acinar cells.
J Gen Physiol
108:
35-47,
1996[Abstract].
2.
Berridge, MJ.
Inositol trisphosphate and calcium signaling.
Nature
361:
315-325,
1993[ISI][Medline].
3.
Boton, R,
Dascal N,
Gillo B,
and
Lass Y.
Two calcium-activated chloride conductances in Xenopus laevis oocytes permeabilized with the ionophore A23187.
J Physiol (Lond)
408:
511-534,
1987[Abstract].
4.
Callamaras, N,
and
Parker I.
Caged inositol 1,4,5-trisphosphate for studying release of Ca2+ from intracellular stores.
Methods Enzymol
291:
497-499,
1998.
5.
Callamaras, N,
and
Parker I.
Radial localization of InsP3-sensitive Ca2+ release sites in Xenopus oocytes resolved by axial confocal linescan imaging.
J Gen Physiol
113:
199-213,
1999
6.
Callamaras, N,
Sun X-P,
Ivorra I,
and
Parker I.
Hemispheric asymmetry of macroscopic and elementary calcium signals mediated by InsP3 in Xenopus oocytes.
J Physiol (Lond)
511:
395-405,
1998
7.
Cross, NL,
and
Elinson RP.
A fast block to polyspermy in frogs mediated by changes in the membrane potential.
Dev Biol
75:
187-198,
1980[ISI][Medline].
8.
Dascal, N.
The use of Xenopus oocytes for the study of ion channels.
Crit Rev Biochem
22:
317-387,
1987[ISI][Medline].
9.
DiChiara, TJ,
and
Reinhart PH.
Distinct effects of Ca2+ and voltage on the activation and deactivation of cloned Ca2+-activated K+ channels.
J Physiol (Lond)
489:
403-418,
1995[Abstract].
10.
Gomez-Hernandez, J-M,
Stuhmer W,
and
Parekh AB.
Calcium-dependence and distribution of calcium-activated chloride channels in Xenopus oocytes.
J Physiol (Lond)
502:
569-574,
1997[Abstract].
11.
Hartzell, HC.
Activation of different Cl currents in Xenopus oocytes by Ca liberated from stores and by capacitative Ca influx.
J Gen Physiol
108:
157-175,
1996[Abstract].
12.
Kuruma, A,
and
Hartzell HC.
Dynamics of calcium regulation of chloride currents in Xenopus oocytes.
Am J Physiol Cell Physiol
276:
C161-C175,
1999
13.
Miledi, R,
and
Parker I.
Chloride current induced by injection of calcium into Xenopus oocytes.
J Physiol (Lond)
357:
173-183,
1984[Abstract].
14.
Miledi, R,
Parker I,
and
Sumikawa K.
Transplanting receptors from brains into oocytes.
In: Fidia Research Foundation Neuroscience Award Lectures. New York: Raven, 1989, p. 57-90.
15.
Nilius, B,
Prenen J,
Szucs G,
Wei L,
Tanzi F,
Voets T,
and
Droogmans G.
Calcium-activated chloride channels in bovine pulmonary artery endothelial cells.
J Physiol (Lond)
498:
381-396,
1997[Abstract].
16.
Nuccitelli, R,
Yim DL,
and
Smart T.
The sperm-induced Ca2+ wave following fertilization of the Xenopus egg requires the production of Ins(1,4,5)P3.
Dev Biol
158:
200-212,
1993[ISI][Medline].
17.
Parker, I,
Callamaras N,
and
Wier WG.
A high-resolution, confocal laser-scanning microscope and flash photolysis system for physiological studies.
Cell Calcium
21:
441-452,
1997[ISI][Medline].
18.
Parker, I,
and
Ivorra I.
Characteristics of membrane currents evoked by photoreleased inositol trisphosphate in Xenopus oocytes.
Am J Physiol Cell Physiol
263:
C154-C165,
1992
19.
Parker, I,
and
Miledi R.
Inositol trisphosphate activates a voltage-dependent calcium influx in Xenopus oocytes.
Proc R Soc Lond B Biol Sci
231:
27-36,
1987[ISI][Medline].
20.
Parker, I,
and
Miledi R.
Nonlinearity and facilitation in phosphoinositide signaling studied by the use of caged inositol trisphosphate in Xenopus oocytes.
J Neurosci
9:
4068-4077,
1989[Abstract].
21.
Parker, I,
and
Yao Y.
Relation between intracellular Ca2+ signals and Ca2+-activated Cl current in Xenopus oocytes.
Cell Calcium
15:
276-288,
1994[ISI][Medline].
22.
Parker, I,
Yao Y,
and
Ilyin V.
Fast kinetics of calcium liberation induced in Xenopus oocytes by photoreleased inositol trisphosphate.
Biophys J
70:
222-237,
1996[Abstract].
23.
Putney, JW.
Inositol phosphates and calcium entry.
Adv Second Messenger Phosphoprotein Res
26:
143-160,
1992[Medline].
24.
Song, LS,
Sham JS,
Stern MD,
Lakatta EG,
and
Cheng H.
Direct measurement of SR release flux by tracking "Ca2+ spikes' in rat cardiac myocytes.
J Physiol (Lond)
512:
677-691,
1998
25.
Sumikawa, K,
Parker I,
and
Miledi R.
Expression of neurotransmitter receptors and voltage-activated channels from brain mRNA in Xenopus oocytes.
Methods Neurosci
1:
30-45,
1989.
26.
Sun, X-P,
Callamaras N,
and
Parker I.
A continuum of InsP3-mediated elementary Ca2+ signaling events in Xenopus oocytes.
J Physiol (Lond)
509:
67-80,
1998
27.
Takahashi, T,
Neher E,
and
Sakmann B.
Rat brain serotonin receptors in Xenopus oocytes are coupled by intracellular calcium to endogenous channels.
Proc Natl Acad Sci USA
84:
5063-5067,
1986[ISI].
28.
Yao, Y,
and
Parker I.
Inositol trisphosphate-mediated Ca2+ influx into Xenopus oocytes triggers Ca2+ liberation from intracellular stores.
J Physiol (Lond)
468:
275-295,
1993[Abstract].