Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322-3030
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ABSTRACT |
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Ca-activated Cl currents are widely expressed in many cell types and play diverse and important physiological roles. The Xenopus oocyte is a good model system for studying the regulation of these currents. We previously showed that inositol 1,4,5-trisphosphate (IP3) injection into Xenopus oocytes rapidly elicits a noninactivating outward Cl current (ICl1-S) followed several minutes later by the development of slow inward (ICl2) and transient outward (ICl1-T) Cl currents. In this paper, we investigate whether these three currents are mediated by the same or different Cl channels. Outward Cl currents were more sensitive to Ca than inward Cl currents, as shown by injection of different amounts of Ca or by Ca influx through a heterologously expressed ligand-gated Ca channel, the ionotropic glutamate receptor iGluR3. These data could be explained by two channels with different Ca affinities or one channel with a higher Ca affinity at depolarized potentials. To distinguish between these possibilities, we determined the anion selectivity of the three currents. The anion selectivity sequences for the three currents were the same (I > Br > Cl), but ICl1-S had an I-to-Cl permeability ratio more than twofold smaller than the other two currents. The different anion selectivities and instantaneous current-voltage relationships were consistent with at least two different channels mediating these currents. However, after consideration of possible errors, the hypothesis that a single type of Cl channel underlies the complex waveforms of the three different macroscopic Ca-activated Cl currents in Xenopus oocytes remains a viable alternative.
inositol trisphosphate; glutamate receptor; voltage clamp; A-23187; store-operated calcium entry
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INTRODUCTION |
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INCREASES IN CYTOSOLIC Ca can occur by Ca influx from the extracellular space or by release of Ca from subcellular compartments (3, 40). For example, many G protein- and tyrosine kinase-associated receptors stimulate phospholipase C and the production of inositol 1,4,5-trisphosphate (IP3), resulting in a transient release of Ca from endoplasmic reticulum (ER) stores and then a long-lasting influx of extracellular Ca. This Ca influx, which is stimulated by decreases in the Ca content of internal stores, has been termed store-operated Ca entry (SOCE), formerly known as capacitative Ca entry (41, 42).
Xenopus oocytes have been used extensively as a model system for studying Ca signaling and have provided valuable information about spatial and temporal aspects of Ca signals and the mechanisms of regulation of SOCE. Xenopus oocytes are well suited for studies on Ca signaling, because they are easily voltage clamped with two microelectrodes (10), their large size permits imaging Ca waves with Ca-sensitive fluorescent dyes (23, 29, 33, 50), and Ca signaling proteins are easily expressed heterologously (7). Another important reason that Xenopus oocytes have been a popular system is that they have endogenous Ca-activated Cl channels, which can be used as a real-time assay for subplasmalemmal Ca (10, 25). For example, Ca-activated Cl channels have been used as an indirect measure of SOCE and for the evaluation of factors thought to regulate store-operated Ca channels (SOCs) (12, 21, 37-39).
The number of different types of Ca-activated Cl channels in the oocyte remains an open question. The pioneering studies of Miledi, Parker, Dascal, and their colleagues as well as other investigators (2, 11, 25, 30, 31, 34, 35, 44) show that responses to IP3 usually consist of two or more components. An initial transient component and subsequent oscillatory components are independent of extracellular Ca and are caused by Ca release from intracellular stores. These components are followed by a sustained component, which depends on Ca influx. Yao and Parker (49) suggested that all three components are mediated by the same population of Cl channels, which are activated with different kinetics in response to Ca released from stores and by Ca influx. In contrast, Boton et al. (4) concluded that there are two different Ca-activated Cl currents because of their differential sensitivities to Ca, EGTA, and anthracene-9-carboxylic acid. We suggested that the two different Cl currents activated by Ca released from stores and Ca influx were mediated by different channels, because the currents exhibited different instantaneous current-voltage (I-V) and activation curves (15). Unfortunately, there are no definitive single-channel data to support the existence of two Ca-activated Cl channels. Takahashi et al. (46) reported that activation of heterologously expressed 5HT1C receptors (which activated phospholipase C) activated 3-pS Cl channels in cell-attached patches and that Ca activated the same channels in excised patches. Although another type of channel was also sometimes observed, the predominance of the 3-pS channel suggested that there was only one species of Ca-activated Cl channel in the oocyte.
Whether the different currents activated by IP3 are mediated by the same or different channels, the fact that IP3 stimulates currents that differ significantly in their kinetics, voltage sensitivity, sensitivity to store-released and influxed Ca, and biophysical properties is extremely interesting. If these are mediated by different channels, their differing sensitivity to the source of Ca (influx from extracellular space vs. release from internal stores) suggests that differences in the amplitude or spatiotemporal features of the Ca signal from these two sources may be important in determining which channel is activated. Alternatively, if only one channel is responsible, the behavior of the Cl current must be dictated by the features of the Ca signal in intriguing ways. Because Ca-activated Cl channels have played such a prominent role in the study of Ca signaling in Xenopus oocytes, it is important to understand their mechanisms of regulation. The goal of the present study was to obtain additional evidence for the identity of these currents and to investigate the mechanisms of their regulation by Ca.
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METHODS |
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Isolation of Xenopus Oocytes
Stage V-VI oocytes were harvested from adult Xenopus laevis females (Xenopus I, Ann Arbor, MI) as described by Dascal (10). Animals were anesthetized by immersion in tricaine (1.5 g/l). Ovarian follicles were removed, cut into small pieces, and digested in normal Ringer solution with no added Ca containing 2 mg/ml collagenase type IA (Sigma Chemical, St. Louis, MO) for 2 h at room temperature. The oocytes were extensively rinsed with normal Ringer solution, placed in L-15 medium (GIBCO BRL, Gaithersburg, MD), and stored at 18°C. Oocytes were used 1-6 days after isolation.Electrophysiological Methods
Oocytes were voltage clamped with two microelectrodes with use of a GeneClamp 500 (Axon Instruments, Foster City, CA). Current was always recorded at maximal gain (10,000×) with a minimal stability setting (<200 µs) to achieve the fastest possible voltage clamp. Electrodes were usually filled with 3 M KCl and had resistances of 0.5-2 MOocyte Injection
Oocytes were injected with various substances with use of a Nanoject Automatic Oocyte Injector (Drummond Scientific, Broomall, PA). The injection pipette was pulled from glass capillary tubing in a manner similar to that for the recording electrodes and then broken so that it had a <20-µm-OD beveled tip. Typically, 23 nl of 1 mM IP3 solution in Chelex resin-treated H2O were injected to give a calculated oocyte concentration of ~50 µM.Solutions
Normal Ringer solution consisted of (in mM) 123 NaCl, 2.5 KCl, 2 CaCl2, 1.8 MgCl2, and 10 HEPES, with pH adjusted to 7.4 with NaOH. Zero-Ca Ringer solution was the same as normal Ringer solution, except CaCl2 was omitted, MgCl2 was increased to 5 mM, and 0.1 mM EGTA was added. NMDG Ringer solution consisted of (in mM) 116 NMDG chloride, 2 CaCl2, 2 MgCl2, and 10 HEPES, pH 7.4. In experiments on anion permeability, 123 mM NaCl in normal Ringer solution was replaced with 123 mM NaI or NaBr. For measuring ISOC, the oocytes were incubated overnight in a solution containing (in mM) 108 sodium aspartate, 1.6 potassium aspartate, 2 Ca(OH)2, 2 MgSO4, and 10 sodium HEPES, pH 7.4, before the experiment, which was conducted in Na- and Cl-free Ringer solution composed of (in mM) 113 aspartic acid, 5 calcium aspartate, and 5 HEPES, with pH adjusted to 7.4 with NMDG. Stock solutions of IP3 (Sigma Chemical) were made at 10 mM in H2O, stored atiGluR3 Expression
The rat flop form of iGluR3 cRNA was synthesized in vitro using Ambion mMessage mMachine capped RNA synthesis kit with the iGluR3 plasmid as template (accession number M85036, provided by Dr. Jim Boulter, University of California, Los Angeles). cRNA (10-23 ng) in water was injected near the equator of the oocyte 2-4 days before recording. Injection was performed as described above for IP3, and the oocytes were stored at 18°C in L-15 medium until they were used. ![]() |
RESULTS |
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Time Course of Development of ICl1 and ICl2
Injection of Xenopus oocytes with IP3 stimulates Cl currents composed of several different kinetic components (e.g., Refs. 4, 15, 30, 31, 44). Figure 1 recapitulates how we measure these currents, ICl1-S, ICl1-T, and ICl2 (15). The oocyte was voltage clamped with two microelectrodes, and the membrane potential was stepped from a holding potential of
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Relationship of Cl Channel Activation to ISOC Activation
In Fig. 1A, ICl2 developed more slowly than ICl1-T. This observation suggested that these two currents were activated differently and raised the question of how the development of these currents related to the development of SOCE. To examine this question, we measured ISOC directly, as we previously described (15). The oocytes were incubated in Cl-free solution overnight to deplete cytosolic Cl, and the experiments were performed in Cl-free solutions to minimize Cl currents. In addition, ISOC was isolated by blocking Ca-activated Cl currents by injection of BAPTA (5 mM oocyte concentration). Under these conditions, injection of IP3 or treatment with thapsigargin resulted in the development of an inwardly rectifying current that was blocked by La or removal of extracellular Ca (Fig. 2). On average, ISOC developed with a half time of ~100 s.
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The time course of development of
ISOC
is compared with the development of
ICl1-T
and
ICl2
measured by our standard +40-mV, 140-mV pulse,
35-mV
holding potential protocol in Fig.
3A.
Surprisingly, the time course of development of
ICl1-T
and
ICl2
lagged significantly behind the development of
ISOC.
We hypothesized that the lag between the development of
ISOC
and Cl currents might be related to the difference in conditions for
measuring
ISOC
(high intracellular BAPTA) and for measuring Cl currents. By reducing
the level of cytosolic Ca, BAPTA could accelerate the development of
ISOC
by reducing its inactivation by Ca (18, 53) and by reducing
deactivation of
ISOC
due to partial refilling of Ca stores. We tested this idea by changing
Ca influx by holding the oocyte at different potentials (Fig.
3B). The development of
ICl1-T
and
ICl2
was strongly affected by holding potential. With a +40-mV holding
potential, both currents developed more quickly and became larger than
with the
35-mV holding potential. Because one would expect that
Ca influx during the holding period would be less at +40 mV than at
35 mV, this supports the suggestion that the development of ISOC
varies with the availability of cytosolic Ca. With the +40-mV holding
potential the development of
ISOC
corresponded almost precisely with the development of
ICl1-T
(Fig. 3C). In contrast, the
development of
ICl2
remained considerably slower. The correspondence of
ISOC
and
ICl1-T
development in Fig. 3C might be
coincidental, but the important point is that the time course of
development of
ICl1-T
and
ICl2
differs significantly at both holding potentials tested. This
difference suggests that these two currents are regulated differently.
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These observations raised several questions. Are ICl1-S, ICl1-T, and ICl2 mediated by the same or by different channels? Do the different currents have the same or different sensitivity to Ca? Why is ICl2 apparently not activated in response to Ca released from stores? Do the waveforms of ICl2 and ICl1-T simply reflect subplasmalemmal Ca concentration, or is there a more complex relationship between Ca and channel activation? Why does ICl1-S turn off within several minutes after IP3 injection?
Activation of ICl2 by Ca Injection and Influx
To test whether the slow development of ICl2 involved a time-dependent activation of some metabolic process, we examined whether it was possible to activate ICl2 by Ca influx through other types of Ca channels. We predicted that if ICl2 activation required some time-dependent process subsequent to Ca influx, this process should occur with similar kinetics regardless of the method of elevation of subplasmalemmal Ca.A-23187. Initially, we tried to produce Ca influx directly via Ca ionophores such as A-23187 or ionomycin, but the interpretation was complicated, because A-23187 and ionomycin produced massive and immediate release of Ca from internal stores, as reported previously (4, 26, 48, 51). A-23187 stimulated ICl1-S in the absence of extracellular Ca in the same way that IP3 injection did (Fig. 4A), showing that it released Ca from intracellular stores. In the presence of extracellular Ca, ICl1-T developed in a distinctively biphasic manner (Fig. 4B). A-23187 resulted in a rapid initial increase in ICl1-T (the "hump" in the curve between 200 and 500 s) followed by a slower sigmoidal increase (approximated by the dashed line between 200 and 500 s). The hump was due to Ca influx through A-23187 channels in the plasma membrane, because it was dependent on extracellular Ca and because it was never seen with IP3 injection. Although ICl1-T was activated by Ca influx through A-23187 channels, ICl2 was not activated appreciably during this time period. ICl2 did activate later (>500 s), however, as SOCE developed as a consequence of depletion of Ca stores by A-23187. The observation that ICl2 was not activated by Ca influx through A-23187 channels suggested that ICl1-T and ICl2 were regulated differently by Ca.
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Ca injection.
We also activated Ca-activated Cl currents by direct injection of Ca
into the oocyte. The amount of Ca required to activate the Cl currents
depended critically on the depth and hemispheric location of the
injection pipette, as reported previously (25). Figure
5 shows a typical result. Injection of
~230 pmol of Ca into the oocyte elicited an outward current that
activated slowly on depolarization and exhibited a time-dependent
deactivating tail current on hyperpolarization (Fig. 5,
A and
B; 230 and 320 pmol). Very little
inward current was present at the end of the 1-s pulse at 140 mV
with these small Ca injections. In contrast, larger injections of Ca
(460-690 pmol) activated outward currents that exhibited little or
no time-dependent activation (Fig. 5, B and
C; cf. time course of currents
stimulated by 320 and 690 pmol Ca) and stimulated large inward currents
(Fig. 5, A and
B; 460 and 690 pmol). Figure
5D shows that the relationship between inward and outward current induced by Ca injection is nonlinear. Outward currents up to ~2 µA in amplitude were associated with only
small inward currents. These data suggest that inward currents may be
less sensitive to Ca than outward currents. The different Ca
sensitivity of inward and outward currents is confirmed in Fig. 5,
E and
F, which shows the
I-V relationships in response to
different Ca injections. In this experiment, multiple 23-nl injections
of 10 mM Ca were made as in Fig. 5A.
The I-V relationships were determined
by 5-s-duration linear ramps from
140 to +60 mV. It should be
emphasized that these I-V
relationships are neither instantaneous nor steady state, and their
shapes are influenced by the waveform of time-dependent currents.
Nevertheless, we chose to use a ramp protocol, rather than a step
protocol, so that we could obtain an approximation of the steady-state
I-V relationship in a short period of
time while the Ca concentration was (presumably) not changing
significantly. A single bolus of 23 nl stimulated only outward current
and no measurable inward current (Fig. 5E, trace
1). Two boluses (Fig. 5E, trace
2) injected in quick succession stimulated more
outward current but also stimulated significant inward current. Three
boluses (Fig. 5E, trace 3) increased
the outward current only a small amount over that evoked by two boluses but stimulated inward current twofold. The amount of inward current relative to the amount of outward current became greater with increasing amounts of Ca injected. This is shown in Fig.
5F, where the traces are normalized to
the same amount of outward current at +60 mV. As increasing amounts of
Ca were injected, significantly more inward current was recorded and
the curves become less outwardly rectifying.
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Expressed iGluR3 Ca channels.
The experiments in Fig. 5 suggested that the inward Cl current
stimulated by Ca injection was less sensitive to Ca than outward current. Is this inward current the same as
ICl2?
The waveform of the inward current induced by Ca injection was very
different from that of ICl2, but it is possible
that the waveform of
ICl2 is determined by the dynamics of the Ca signal rather than by some
intrinsic property of the Cl channel itself. If
ICl2
is mediated by the same pathway as the inward current induced by Ca
injection, we would expect that
ICl2
would have a lower Ca sensitivity than ICl1-T.
To test whether
ICl2
and
ICl1-T
have different Ca sensitivities, we examined Cl currents stimulated by
Ca influx through the ionotropic glutamate receptor iGluR3, a
ligand-gated ion channel that exhibits a high Ca permeability (5). cRNA
for iGluR3 was injected into the oocyte several days before the
experiment. The oocyte was bathed in a solution in which the only
permeant cation present was Ca (Na was replaced with NMDG). In the
absence of a glutamate receptor agonist, the currents in iGluR3 oocytes
were essentially identical to those in uninjected oocytes. However,
addition of the iGluR3 agonist kainic acid activated Cl currents (Fig.
6A). Although kainic acid did not stimulate
ICl1-S
(Fig. 6A and +40-mV[1] pulse in Fig. 6B), it did stimulate
ICl1-T
and
ICl2
(Fig. 6A and 140-mV and
+40-mV[2] pulses in Fig.
6B). Low concentrations of kainic
acid activated
ICl1-T
preferentially, whereas higher concentrations activated
ICl1-T
and
ICl2.
Another important observation was that ICl1-T
and
ICl2
increased maximally within 10 s after application of kainic acid.
Although this experiment did not exclude the involvement of a metabolic
step in activation of these Cl currents, it did demonstrate that the
~10 min typically required for
ICl2
activation (Fig. 3B) were unlikely
to be due to a slow metabolic step.
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Mechanism of Turnoff of ICl1-S
Figures 5 and 6 show that outward Cl current is generally more sensitive to Ca than is inward Cl current. If this is true, the following questions arise: Why does ICl1-S inactivate so quickly after IP3 injection (Fig. 1A)? Is ICl1-S a unique Cl current that has intrinsic inactivating properties, or does the turnoff reflect the decline in the Ca signal? The data in Fig. 8 show that the inactivation of ICl1-S is due to depletion of Ca from internal stores and is not intrinsic inactivation of the Cl channel. Several minutes after IP3 injection when ICl1-S has turned off, bath application of ionomycin (not shown) or A-23187 (Fig. 8, A and B) does not stimulate ICl1-S. However, injection of Ca does significantly stimulate outward current, which resembles ICl1-S (Fig. 8, C and D). Because Ca is able to activate ICl1-S, the Cl channel clearly is not inactivated and can be stimulated when Ca is provided. The inability of A-23187 to stimulate this current, however, suggests that the stores do not contain sufficient Ca to activate the Cl channels. We have shown in Fig. 4 that A-23187 does stimulate a large ICl1-S when applied before IP3 injection, showing that A-23187 is capable of releasing enough Ca from stores to activate the current, provided the stores are filled with Ca. These results show that ICl1-S turns off after IP3 injection, because Ca release from stores has waned, but SOCE has not yet completely developed. When SOCE has developed, the waveform of the current will be dependent on the voltage dependence of Ca influx and efflux.
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Are ICl1 and ICl2 Due to One or Multiple Channel Types?
The data presented so far show that the inward Ca-activated Cl current is less sensitive to Ca than is the outward current. This could be explained 1) if there were two different Cl channels having different sensitivities to Ca and different biophysical properties or 2) if the Ca sensitivity and biophysical properties of a single Cl channel were voltage dependent. For example, if hyperpolarization decreased the Ca sensitivity of the channel, more Ca would be required to activate it.To gain additional information about whether ICl1-S, ICl-1T, and ICl2 were mediated by different channels, we examined their ionic selectivity. The reversal potentials of the instantaneous I-V relationships for ICl1-S, ICl2, and ICl1-T were measured as described previously (15) with Cl, I, or Br as the charge-carrying species in the extracellular solution. Figure 9 shows typical current traces for 133.5 mM Cl and 123 mM I for ICl1-S (A and B), ICl2 (D and E), and ICl1-T (G and H). The instantaneous I-V relationships in Cl and I are shown. Table 1 summarizes the measured reversal potentials and calculated anion-to-Cl permeability ratios. For all three currents, the order of ion selectivity was the same: I > Br > Cl. However, there were quantitative differences between the currents. The I-to-Cl permeability ratio (PI/PCl) for ICl1-S was only about one-half of that of ICl1-T and ICl2. The differences between ICl2 and ICl1-T, however, were insignificant: the Br-to-Cl permeability ratios were the same, and PI/PCl values were statistically different only at the 0.04 level. These data suggest that two different channels may exist.
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Ca-Induced Ca Release and ICl1-T
Other investigators who believe that ICl1-T and ICl2 are the same current (36, 50) argue that the current we call ICl1-T is actually due to reactivation of ICl2 resulting from Ca release from the ER induced by Ca influx during the previous hyperpolarizing pulse. To test this hypothesis, we injected oocytes with a large concentration of heparin to block Ca release from the ER after ICl2 was fully activated to determine whether ICl1-T could be explained by Ca-induced Ca release (Fig. 10). We first tested whether heparin could block IP3-induced Ca release. In Fig. 10A, a concentration of IP3 was injected that released Ca from stores and activated SOCE and ICl2. After ~20 min, presumably as the IP3 was hydrolyzed, the Cl currents returned to baseline levels as Ca stores became refilled (16). Heparin was then injected. A second injection of IP3 after the heparin injection produced no effect as a result of blockage of IP3 receptors by heparin. Heparin was also able to block Ca oscillations very quickly (Fig. 10B). Low concentrations of IP3 stimulated ICl1-S oscillations (16), which were blocked within 1 min after heparin injection. In contrast (Fig. 10C), injection of heparin after ICl1-T and ICl2 had fully developed usually increased (n = 5) but never decreased ICl1-T and ICl2. We have not investigated the mechanism of the stimulatory effect of heparin. Nevertheless, these data show that ICl1-T does not require Ca-induced Ca release.
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DISCUSSION |
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We showed previously (15, 24) that Xenopus oocytes develop three different Ca-activated Cl currents after IP3 injection. ICl1-S is activated by Ca released from stores, and ICl1-T and ICl2 are activated in a transient manner by Ca influx. The present studies provide additional insights into the mechanisms of regulation of these currents. We show here that ICl2 is less sensitive to Ca than is ICl1-S or ICl1-T. This difference in sensitivity of these currents to Ca provides a plausible explanation for why Ca release from stores does not activate ICl2: the level of Ca released from stores may be below threshold for ICl2 activation. If we assume that ICl1-S and ICl1-T are due to the same channels (see below), we can estimate the relative subplasmalemmal Ca in response to release from stores and by SOCE by comparing the amplitude of ICl1-S immediately after IP3 injection with the amplitude of ICl1-T when SOCE is fully activated (Fig. 1, A and B). The fact that ICl1-T is usually twice as large as ICl1-S is consistent with the idea that subplasmalemmal Ca levels are lower in response to Ca release than they are to SOCE. The observations that injection of low concentrations of Ca into the oocyte or Ca influx through A-23187 channels selectively activates ICl1-T (Figs. 4 and 5) can be explained by these modes of Ca delivery being insufficient to provide enough Ca to activate ICl2.
The time course of activation of ICl2 is considerably slower than that of ICl1-T (Fig. 3C). This can be explained if ICl2 is less sensitive to Ca than ICl1-T. As SOCE develops, ICl1-T increases sooner than ICl2, simply because ICl2 requires higher levels of Ca to be activated. The alternative explanation that ICl2 activation requires intermediate steps between Ca influx and Cl channel activation is disfavored by the observation that Ca influx via heterologously expressed iGluR3 activates ICl2 rapidly (<10 s). Although this does not exclude the possibility that intermediates exist between Ca influx and Cl channel activation, this experiment shows that Ca influx can activate ICl2 much more quickly than the activation that occurs in response to IP3 injection.
How Many Types of Ca-Activated Cl Channels?
It is clear that Xenopus oocytes have several different Ca-activated Cl currents, which have different Ca sensitivities (4; present study), but whether these different currents are mediated by different channels remains to be established. These two currents could be due to two different Cl channels with different Ca affinities or one Cl channel with a Ca affinity that is dependent on voltage (Fig. 11). Any hypothesis needs to be able to explain the different waveforms of ICl1-S, ICl1-T, and ICl2 and also the differently shaped I-V relationships and ionic selectivities of these currents.
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Single-channel hypothesis.
The single-channel hypothesis suggests that
ICl1-S,
ICl1-T,
and
ICl2
are mediated by one Cl channel with voltage-dependent Ca affinity. The
Ca affinity is greater at depolarized potentials. In response to
IP3 injection,
ICl1-S
is stimulated as Ca is released from internal stores. The
time-dependent activation of the current in response to a depolarizing
voltage step could be attributed to a true voltage-dependent gating of
the channel or a voltage-dependent increase in channel Ca affinity. The
fact that the time-dependent component of the current disappears when
large amounts of Ca are injected (Fig.
5C) is consistent with the idea that
the voltage-dependent activation is due to an increase in Ca affinity.
When Ca concentration is saturating, the time dependence is absent,
because the channel is already maximally occupied with Ca. The
deactivating tail current on hyperpolarization could be explained by a
decrease in Ca affinity and/or voltage-dependent gating. The
fact that one sees a large tail current even when large concentrations
of Ca are injected to give a time-independent outward current is
consistent with a change in Ca affinity. After Ca has been released
from stores, ICl1-S
turns off as the stores become depleted of Ca.
ICl2
is not activated in response to Ca release from stores, because at negative potentials the affinity of the channel for Ca is low and the
subplasmalemmal Ca concentration in response to Ca release from stores
is relatively low. After SOCE becomes activated in response to store
depletion, Ca influx at hyperpolarized potentials activates
ICl2.
The time course of
ICl2
activation on stepping to 140 mV is most likely explained by the
time course of subplasmalemmal Ca accumulation during the pulse,
because similar
ICl2
waveforms are seen with Ca influx through SOCs and through iGluR3.
Repolarization to positive potentials evokes
ICl1-T
as a result of the voltage-dependent increase in Ca affinity of the
channel. The different time course of stimulation of
ICl2
and
ICl1-T
after an IP3 injection can be explained simply by the lower Ca affinity of the channel at
hyperpolarized potentials.
Multiple-channel hypothesis. Much of the data we have presented can also be explained by assuming that there are several Cl channels with different Ca affinities. Outward currents are mediated by channels with high affinity, and inward currents are mediated by channels with low affinity. The multiple-channel hypothesis has the advantage that it can more easily explain the differences in the instantaneous I-V relationships and the ionic selectivities of the currents. However, one problem with the hypothesis that there are just two channels is that the ionic selectivity data suggest that ICl1-T and ICl2 could be the same current, whereas the instantaneous I-V relationships suggest that they are not. Thus, if more than one channel is involved in mediating these currents, it seems that one must propose that there are three different kinds of channels.
Ca-Induced Ca Release
The idea that there is only one type of Cl channel would agree with the views of Parker and co-workers (30, 32, 35, 36, 49, 50) and Gomez-Hernandez et al. (13). However, we do not agree with the suggestion of Parker and co-workers that ICl1-T is due to Ca-induced Ca release. Parker has shown that, after release of Ca from stores stimulated by injection of IP3 into Xenopus oocytes, cytosolic Ca continues to increase after a hyperpolarizing voltage step has been terminated, presumably as a result of Ca-induced Ca release. He suggests that the outward current we call ICl1-T is actually ICl2 being reactivated by Ca-induced Ca release. However, we find that heparin does not diminish the size of ICl1-T or ICl2, as would be expected if Ca-induced Ca release were contributing to the cytosolic Ca under these conditions. Also we do not observe continued increase in cytosolic Ca after terminating the hyperpolarizing step (24a). We believe that the difference between the results of Parker and co-workers and our results is the quantity of IP3 that was injected. Parker and co-workers injected a much smaller amount of IP3, which resulted in Ca waves that were influenced by Ca influx. In contrast, we injected large amounts of IP3, which rapidly depleted the stores completely so that there is little effect of Ca influx on release. Thus, although we agree with Parker and co-workers that the multiple currents may be explained by a single conductance, we believe that the currents are explained by a voltage-dependent change in Ca affinity, whereas Parker and co-workers believe that the different currents are explained by differences in Ca dynamics. Resolution of these questions will require single-channel analysis.Physiological Significance of Cl Channels in Xenopus Oocytes
In the oocytes of many species, including Xenopus, sperm entry stimulates phosphatidylinositol 4,5bisphosphate hydrolysis, production of IP3 (28, 43, 45), and release of Ca from internal stores. As the Ca wave spreads from the sperm entry site to encompass the entire egg, it activates Ca-activated Cl channels (15, 22), which depolarize the membrane to produce the fertilization potential (20, 47). Because amphibian eggs in the wild are fertilized in fresh water with relatively low Cl concentration, ECl is positive, and activation of Cl currents will depolarize the egg. The fertilization potential is responsible for the rapidly developing, transient block to polyspermy ("fast electrical block") (8, 14), which lasts ~15 min. The electrical block to polyspermy, which is found in many (but not all) species, is caused by a voltage dependence of sperm-egg fusion, with positive membrane potentials being inhibitory. This has been demonstrated by voltage-clamp experiments and by altering extracellular ionic composition to alter the polarity of the fertilization potential (9, 19, 27, 47). To prevent polyspermy, it is important that the depolarization develop rapidly. The voltage-dependent Ca sensitivity of Cl currents would provide a strong positive-feedback mechanism to accelerate the depolarization. Inasmuch as the egg depolarizes as the result of activation of Ca-activated Cl channels, the depolarization will increase the affinity of the channels for Ca, which will increase the depolarization. This feedback might be important in facilitating the rate of depolarization to block polyspermy. ![]() |
ACKNOWLEDGEMENTS |
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We thank Alyson Ellingson and Elizabeth Lytle for excellent technical assistance, Dr. Khaled Machaca for comments on the manuscript, Dr. Jim Boulter for the iGluR3 plasmid, Dr. Raymond Dingledine for the iGluR3 cRNA, and Dr. Seiko Kawano for helpful discussion and for performing the experiment shown in Fig. 6 while she was visiting the author's laboratory.
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FOOTNOTES |
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This study was supported by National Institutes of Health Grants HL-21195 and GM-55276.
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: H. C. Hartzell, Dept. of Cell Biology, 1648 Pierce Dr., Emory University School of Medicine, Atlanta, GA 30322-3030. E-mail: criss{at}cellbio.emory.edu.
Received 15 May 1998; accepted in final form 6 October 1998.
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