Adenophostin A and Inositol 1,4,5-Trisphosphate Differentially Activate Cl− Currents in Xenopus Oocytes Because of Disparate Ca2+ Release Kinetics*

Depletion of endoplasmic reticulum Ca2+ stores induces Ca2+ entry from the extracellular space by a process termed “store-operated Ca2+ entry” (SOCE). It has been suggested that the novel fungal metabolite adenophostin-A may be able to stimulate Ca2+ entry without stimulating Ca2+ release from stores. To test this idea further, we compared Ca2+release, SOCE, and the stimulation of Ca2+-activated Cl− currents in Xenopus oocytes in response to inositol 1,4,5-trisphosphate (IP3) and adenophostin-A injection. IP3 stimulated an outward Cl−current, I Cl1-S, in response to Ca2+ release from stores followed by an inward current,I Cl2, in response to SOCE. In contrast, low concentrations of adenophostins (AdAs) activatedI Cl2 without activatingI Cl1-S, consistent with the suggestion that AdA can activate Ca2+ entry without stimulating Ca2+ release. However, when Ca2+ entry has been stimulated by AdA, Ca2+ stores are largely depleted of Ca2+, as assessed by the inability of ionomycin to release additional Ca2+. The Ca2+ release stimulated by AdA, however, was 7 times slower than the release stimulated by IP3, which could explain the minimal activation ofI Cl1-S; when Ca2+ is released slowly, the threshold level required for I Cl1-Sactivation is not attained.

Ca 2ϩ signals regulate many cellular processes including cell growth, fertilization, gene transcription, and apoptosis (1). Increases in cytosolic Ca 2ϩ levels are produced both by Ca 2ϩ released from internal stores and Ca 2ϩ influxed from the extracellular space. A major pathway for Ca 2ϩ mobilization from internal stores is through inositol 1,4,5-trisphosphate receptors (IP 3 R) 1 after stimulation of G-protein-or tyrosine kinase-coupled plasma membrane receptors linked to phospholipase C (2)(3)(4). The decrease in the Ca 2ϩ content of the internal store then stimulates Ca 2ϩ entry through plasma membrane storeoperated Ca 2ϩ channels (SOCs) by a process called store-operated Ca 2ϩ entry (SOCE) (5,6). The mechanism by which a reduction in the content of store Ca 2ϩ results in opening of SOCs remains unknown, but there are two major hypotheses. The conformational coupling hypothesis suggests that there is direct physical contact between IP 3 Rs and SOCs such that conformational changes in the IP 3 R occurring upon Ca 2ϩ de-pletion of the internal store can affect the opening of SOCs (7,8). The diffusible messenger hypothesis suggests that the Ca 2ϩ store (endoplasmic reticulum) produces a diffusible messenger that opens SOCs (9,10).
Recently, a novel family of compounds called adenophostins (AdAs), which are structurally distinct from IP 3 , have been isolated from cultures of the fungus Penicillium brevicompactum (11,12). The AdAs are 10 -100-fold more potent than IP 3 in opening IP 3 Rs (13) and are capable of activating all three IP 3 R subtypes (13)(14)(15). Recently, Hartzell et al. (16) and DeLisle et al. (17) have shown that in Xenopus oocytes, low concentrations of AdA stimulate Ca 2ϩ -activated Cl Ϫ currents that are activated by Ca 2ϩ influx more than Cl Ϫ currents that are activated by Ca 2ϩ released from stores. Based on these observations, DeLisle et al. (17) suggested that AdA may be capable of activating store-operated Ca 2ϩ entry without first stimulating Ca 2ϩ release from stores. This is significant because it suggests that AdA may share structural features with the putative diffusible Ca 2ϩ entry signal released by Ca 2ϩ -depleted endoplasmic reticulum.
Ca 2ϩ -activated Cl Ϫ currents have been used for many years as real time indicators of sub-plasmalemmal Ca 2ϩ in Xenopus oocytes (18 -24), but clearly Cl Ϫ currents are only indirect indicators of Ca 2ϩ concentration. Consequently, conclusions about cytosolic Ca 2ϩ concentration derived from these measurements are subject to different interpretations. We have recently found that there are two Ca 2ϩ -activated Cl Ϫ currents in the oocyte that are selectively activated by Ca 2ϩ released from stores and by Ca 2ϩ influx (24). The Ca 2ϩ release-activated Cl Ϫ current (I Cl1-S ) has an outwardly rectifying steady-state current-voltage relationship, whereas the Ca 2ϩ influx-activated Cl Ϫ current (I Cl2 ) has an inwardly rectifying steady-state current-voltage relationship (24). This means that Ca 2ϩ -activated Cl Ϫ currents measured at constant negative membrane potentials, as was done in the experiments of DeLisle et al. (17), are relatively insensitive indicators of Ca 2ϩ released from stores. In our experiments (16), we measured I Cl1-S as an outward current at positive membrane potentials and I Cl2 as an inward current at negative membrane potentials to more clearly differentiate between Ca 2ϩ influx and Ca 2ϩ release and to increase the sensitivity of detection of Ca 2ϩ release. Using this protocol, IP 3 activated both I Cl1-S ("Ca 2ϩ release") and I Cl2 ("Ca 2ϩ influx"), but low concentrations of AdA often activated only a tiny amount of I Cl1-S , even though I Cl2 was robustly activated. But, because we could not find a concentration of AdA which could activate I Cl2 without activating some small amount of I Cl1-S , we concluded that AdA did not activate SOCE independently of Ca 2ϩ release from stores. We hypothesized that AdA activated relatively little I Cl1-S either because AdA released Ca 2ϩ from stores very slowly or that AdA released Ca 2ϩ from a subpopulation of stores which was tightly coupled to SOCs.
The purpose of this paper was to examine further the mechanisms of AdA regulation of Ca 2ϩ -activated Cl Ϫ currents using confocal scanning microscopy of oocytes loaded with fluorescent Ca 2ϩ indicators and two-microelectrode voltage clamp. Here we show that activation of SOCE following injection of low concentrations of AdA depends upon depletion of intracellular Ca 2ϩ stores. However, at low AdA concentrations the kinetics of Ca 2ϩ release from stores was Ͼ7-fold slower than that observed with IP 3 . This slower mode of Ca 2ϩ release is apparently not effective in activating I Cl1-S . Therefore, different kinetics of Ca 2ϩ release can differentially affect Cl Ϫ current activation.

EXPERIMENTAL PROCEDURES
Isolation of Xenopus Oocytes-Stage V-VI oocytes were harvested from adult albino or normal Xenopus laevis females (Xenopus I) as described by Dascal (18). Xenopus were anesthetized by immersion in Tricaine (1.5 g/liter). Ovarian follicles were removed and digested in normal Ringer with no added calcium, containing 2 mg/ml collagenase type IA (Sigma Chemical Co., St. Louis, MO), for 2 h at room temperature. The oocytes were extensively rinsed with normal Ringer, placed in L-15 medium (Life Technologies, Inc., Gaithersburg, MD) and stored at 18°C. Oocytes were usually used within 1-5 days after isolation.
Imaging and Electrophysiological Methods-Xenopus oocytes were injected with 9 nl Ca-green-1 coupled to 70kd dextran (333 M) for a final calculated oocyte concentration of ϳ3 M, and voltage-clamped with two-microelectrodes using a GeneClamp 500 (Axon Instruments, Foster City, CA). Electrodes were filled with 3 M KCl and had resistances of 1-4M⍀. Oocyte resting potentials were between Ϫ20 mV and Ϫ50 mV. Typically, the membrane was held at 0 mV and stepped to ϩ40 mV for 1.5 s every 15 s to monitor I Cl1-S . Every 2.25 min, a 1.5-s duration pulse to Ϫ140 mV followed by a 1.5-s duration pulse to ϩ40 mV was given to monitor I Cl2 and I Cl1-T , respectively. Images (256 ϫ 256 pixels) were acquired 500 ms after the onset of each voltage pulse using a Zeiss LSM 410 confocal box fitted to a Zeiss Axiovert 100TV inverted microscope using a Zeiss 10ϫ objective (0.5 numerical aperature). The confocal aperture was set at the maximal opening, resulting in a focal section 1267 ϫ 1267 ϫ 35 m. Image data was analyzed using the LSM 410 software or NIH image 1.60 on a Mac IIfx. Current data was analyzed on a Pentium PC using Origin 5.0 (Microcal Software, Northampton, MA). For plots of Ca 2ϩ fluorescence, the fluorescence intensity of the entire confocal section was averaged and expressed as a ratio of the background fluorescence taken before IP 3 injection. Experiments were performed at room temperature (22-26°C). Normal Ringer solution contained 123 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl 2 , 1.8 mM MgCl 2 , 10 mM HEPES, pH 7.4; Ca 2ϩ -free Ringer solution was the same except that CaCl 2 was omitted and, MgCl 2 was increased to 5 mM.
Oocytes were injected with IP 3 using a Nanoject automatic oocyte injector (Drummond Scientific Co., Broomall, PA). The injection pipette was pulled from glass capillary tubing in a manner similar to the recording electrodes and then broken so that it had a beveled tip with an inside diameter Ͻ20 m. Solutions of IP 3 or AdA were prepared in Chelex resin-treated H 2 O. The Ca 2ϩ concentration in this solution was not buffered, but injection of H 2 O produced no change in Ca-green FIG. 1. Effect of large amounts of IP 3 on Cl ؊ currents and Ca 2؉ fluorescence in Xenopus oocyte. The voltage protocol was designed to minimize the amount of Ca 2ϩ influx while still allowing the visualization of the time-dependent activation of the Cl Ϫ channels after Ca 2ϩ influx. The cell was stepped to ϩ40 mV for 1.5 s from a holding potential of 0 mV every 15 s for 9 consecutive episodes. In the 10th episode, the cell was stepped to -140 mV then to ϩ40 mV for 1.5 s each. Therefore, every 10th pulse elicited I Cl2 and I Cl1-T , whereas the intervening pulses elicited I Cl1-S . Cells were bathed in normal Ringer. The oocyte was injected with Ca-green-1 coupled to 70-kDa dextran 30 min before the experiment. The oocyte was voltage-clamped with two microelectrodes, and 23 nl of 1 mM IP 3 was injected at the arrow. At the end of the experiment, the oocyte was exposed to Ca 2ϩ -free Ringer containing 14 M ionomycin (Ionom.) to assess the Ca 2ϩ content remaining in the stores. a, summary of Cl Ϫ current amplitudes before and after IP 3 injection. Filled squares, I Cl1-S ; open circles, I Cl2 ; and open triangles, I Cl1-T . b, current traces corresponding to the ϩ40 mV pulses labeled 1 and 2 in a. The voltage protocol used is shown at the top. I Cl1-S was measured as the outward current at the end of the ϩ40 mV pulse. c, current traces corresponding to the Ϫ140 mV/ϩ40 mV pulse combination labeled 3 and 4 in a. The voltage protocol used is shown at the top. I Cl2 was measured as the inward current at the end of the Ϫ140 mV pulse. I Cl1-T was measured as the peak time-dependent outward current during the ϩ40 mV pulse after the Ϫ140 mV pulse. d, Ca 2ϩ fluorescence measured simultaneously with the Cl Ϫ currents. Ca 2ϩ levels were measured by Ca-green-1 fluorescence at ϩ40 mV during the ϩ40 mV pulses from the 0-mV holding potential (F Caϩ40 (filled squares) and at -140 mV (F Ca-140 (open circles)) every 10th pulse. Ca 2ϩ fluorescence levels were measured from the entire focal section and normalized to Ca 2ϩ -dependent fluorescence before IP 3 injection. At the end of the experiment, the oocyte was exposed to 14 M ionomycin in calcium-free Ringer (Ionom./0 Ca 2ϩ ) to release any residual Ca 2ϩ from stores (see Fig. 6). This cell is representative of 13 cells.
fluorescence or membrane current. Levels of the IP 3 R were lowered by injection of 60 ng of the IP 3 R antisense primer (AACTAGACATCTT-GTCTGACATTGCTGCAG) one day before the experiment as described by Kume et al. (25). The reverse sense primer (CTGCAGCAATGTCA-GACAAGATGTCTAGTT) was injected at the same level as a control.

Ca 2ϩ Transient and Cl Ϫ Currents Activated by High Concentrations of IP 3 -
The protocol used to measure Ca 2ϩ -activated Cl Ϫ currents in Xenopus oocytes in response to IP 3 or AdA injection while simultaneously measuring cytosolic Ca 2ϩ with confocal microscopy and Ca-green dextran is shown in Fig. 1. About 30 min after injection of Ca-green dextran, the oocytes were voltage-clamped at 0 mV and stepped to ϩ40 mV every 15 s to monitor I Cl1-S . I Cl1-S (current at the end of the ϩ40 mV pulse, Fig. 1b) is an outward current at depolarizing potentials that is activated quickly (ϳ10 s) after IP 3 injection by Ca 2ϩ released from intracellular stores (16,24,26). In addition, once every 2.25 min, the oocyte was also stepped to Ϫ140 mV to monitor I Cl2 and then to ϩ40 mV to monitor I Cl1-T . I Cl2 (current at the end of the Ϫ140 mV pulse, Fig. 1c) is an inward current that is activated by Ca 2ϩ entry through SOCs driven by the negative membrane potential. I Cl1-T is a transient outward current (peak outward current during the ϩ40 mV pulse, Fig.  1c) that was activated by a depolarizing pulse preceded by a hyperpolarizing pulse to stimulate Ca 2ϩ influx. The Ϫ140 mV pulse was given only once every 2.25 min to minimize Ca 2ϩ influx (and store refilling) during the experiment. For a more detailed discussion of the Cl Ϫ currents see Hartzell and coworkers (24, 27, 28). Fig. 1, a-c, shows the response of Cl Ϫ currents after injection of large amounts of IP 3 (estimated intra-oocyte concentration ϳ20 M). When saturating levels of IP 3 were injected, I Cl1-S (filled squares) was activated immediately. As the stores became depleted of Ca 2ϩ and SOCE developed, I Cl1-T (open triangles) and I Cl2 (open circles) were activated. Injection of IP 3 caused a large increase in Ca 2ϩ fluorescence at all potentials ( Fig. 1d) because of Ca 2ϩ release from stores. Before the peak fluorescence was reached, the fluorescence was the same at all potentials, but afterward the fluorescence during the Ϫ140 mV pulse became greater than the fluorescence during the ϩ40 mV pulse. The difference between the fluorescence at Ϫ140 mV and ϩ40 mV is the voltage-dependent Ca 2ϩ fluorescence, which we have shown is related to Ca 2ϩ entry through SOCs (28).
Ca 2ϩ Waves Stimulated by AdA Are Very Slow-Injection of large amounts of AdA (estimated intra-oocyte concentration ϳ2 M; note that AdA is 10 -100 times more potent than IP 3 (13)) produced rather similar effects on the Cl Ϫ currents to those produced by IP 3 (Fig. 2, a-c). There was a striking difference, however, in the kinetics of the Ca 2ϩ fluorescence change produced by IP 3 and by AdA. The Ca 2ϩ fluorescence did not begin to increase for several min and peaked ϳ8 min after injection of AdA (Fig. 2d). By comparison, after IP 3 injection, the Ca 2ϩ fluorescence peaked in less than 2 min (Fig. 1d).
It may seem surprising in Fig. 2 that the Ca 2ϩ wave peaked so much more slowly than I Cl1-S . It should be noted that the 1-mm diameter oocyte is on the stage of an inverted microscope and that the injection takes place at the top, whereas the confocal image plane is Ͻ30 m from the bottom. Cl Ϫ currents, At the end of the experiment the oocyte was exposed to 14 M ionomycin in calcium-free Ringer (Ionom./0 Ca 2ϩ ) to release any residual Ca 2ϩ from stores (see Fig. 6). Note that I Cl-1T in this cell does not completely inactivate when the cell is switched to Ca 2ϩ -free Ringer, because not all of the Ca 2ϩ in the bath had been removed by the time the pulse that stimulated I Cl1-T occurred. Typically, when the cell is switched to Ca 2ϩ -free Ringer after AdA injection, I Cl1-T and I Cl2 completely inactivated as after IP 3 injection (Fig. 1a). This cell is representative of six cells. which are measured from the entire surface of the oocyte, increase as soon as Ca 2ϩ is released from stores near the injection site. However, the slow increase in Ca 2ϩ fluorescence partly reflects the very slow transit time of the Ca 2ϩ wave from the injection site to the confocal image plane ϳ1 mm away. There is some variability in the lag period between AdA injection and the increase in Ca 2ϩ fluorescence. This variability is most likely related to the depth and position of the injection pipette in the oocyte.
The Ca 2ϩ waves induced by injection of smaller amounts of AdA moved even more slowly. In Fig. 3a, typical traces of Ca 2ϩ fluorescence at ϩ40 mV in response to injection of large amounts of IP 3 (ϳ20 M, filled squares), large amounts of AdA  (ϳ2 M, open circles), and small amounts of AdA (ϳ5 nM, open triangles) are superimposed. In the case of low concentrations of adenophostin, the time-to-peak of the Ca 2ϩ fluorescence was ϳ20 min. Fig. 3b shows averages of the time-to-peak of the Ca 2ϩ fluorescence to these injections. The time-to-peak for large concentrations of AdA was Ͼ2 times slower than for large concentrations of IP 3 , and the time-to-peak for small concentrations of AdA was Ͼ7 times slower than for large concentrations of IP 3 . It was not possible to measure the time-to-peak for small IP 3 concentrations because small IP 3 concentrations produced oscillating Ca 2ϩ waves that exhibited no clear peak. The slowness of the Ca 2ϩ wave is illustrated in a different way in the images in Fig. 3c. After injection of AdA, the spread of the Ca 2ϩ fluorescence is very slow relative to the spread of the IP 3 -induced wave of Ca 2ϩ release.
These data confirm our earlier suggestion that AdA causes release of Ca 2ϩ from stores much more slowly than IP 3 does. These findings support the idea that small concentrations of AdA do not stimulate I Cl1-S because slow release of Ca 2ϩ from stores does not elevate Ca 2ϩ in the vicinity of the Cl Ϫ channels to an activating level. This could occur if efflux and/or local Ca 2ϩ buffering removes free Ca 2ϩ as rapidly as it is released, so that an effective Ca 2ϩ concentration is not attained. Fig. 3 shows that low concentrations of AdA release Ca 2ϩ from stores, the question remains whether the stimulation of Ca 2ϩ entry by low concentrations of AdA is because of depletion of stores. For example, the AdA-stimulated Ca 2ϩ release might be so slow that the stores refill. To examine this question, we measured the effects of low concentrations of AdA (ϳ5 nM) that did not activate I Cl1-S on Ca 2ϩ store depletion. Fig. 4 shows the results of a typical experiment. Injection of 10 nl of 0.5 M AdA did not detectably stimulate I Cl1-S (Fig. 4, a-c), but both I Cl1-T and I Cl2 developed robustly. I Cl1-T and I Cl2 were dependent on extracellular Ca 2ϩ , and their activation corresponded to the activation of SOCE (16). Ca 2ϩ fluorescence began to increase about 5 min after AdA injection and continued to increase for 20 min (Fig. 4d). Voltage-dependent Ca 2ϩ fluorescence (open circles), which reflects SOCE, developed shortly after Ca 2ϩ release and remained at a high level for the duration of the experiment. To test whether stores were depleted of Ca 2ϩ , ionomycin in Ca 2ϩfree Ringer was applied to release Ca 2ϩ from any remaining stores. Ionomycin had only a very small effect on I Cl1-S and had no effect on the Ca 2ϩ fluorescence at ϩ40 mV. This showed that the stores had been virtually completely depleted of Ca 2ϩ by AdA.

Small Concentrations of AdA Completely Deplete Ca 2ϩ Stores-Although
This result contrasts to that observed when small amounts of IP 3 were injected (Fig. 5). Concentrations of IP 3 that stimulated Ca 2ϩ influx, as determined by the presence of voltage-dependent Ca 2ϩ fluorescence and activation of I Cl1-T and I Cl2 , inevitably stimulated I Cl1-S . In some cells, as in Fig. 5, the increase in I Cl1-S was not accompanied by a significant increase in Ca 2ϩ fluorescence, because the IP 3 effect was local and did not propagate into the region of the oocyte that was imaged. Both voltage-dependent Ca 2ϩ fluorescence and I Cl1-T and I Cl2 eventually declined to base line. Application of ionomycin at the end of the experiment evoked a large increase in Ca 2ϩ fluorescence and in I Cl1-S , showing that the stores were not completely depleted of Ca 2ϩ .
To obtain a more quantitative measure of the extent of store  , n ϭ 7). The speed of the Ca 2ϩ release wave after IP 3 or AdA injection was estimated by calculating the time required for the Ca 2ϩdependent fluorescence to reach its maximal value. It was not possible to perform the same analysis on cells injected with low levels of IP 3 , because in many instances, such IP 3 injections lead to repetitive Ca 2ϩ waves that oscillate and not a single wave that sweeps through the entire oocytes as observed when the oocyte is injected with high IP 3 levels or AdA. The speed of the wave was significantly slower (p Ͻ 0.006) between high IP 3 and AdA injections and between high and low AdA injections. c, images of Ca-green-1 fluorescence in response to injection of 23 nl of 1 mM IP 3 (High IP 3 ) and 10 nl of 0.5 M AdA (Low AdA). The times in min at which the confocal images were taken are indicated in the top left corner of each image. IP 3 or AdA were injected at time 0. depletion after injection of IP 3 or AdA, we calculated the ratio of ionomycin-induced Ca 2ϩ release to IP 3 -or AdA-induced Ca 2ϩ release. This ratio gives a measure of the level of residual Ca 2ϩ in intracellular stores after IP 3 R agonist injection. The results from these experiments are shown in Fig. 6. Injections of high IP 3 , high AdA, or low AdA all left the stores largely depleted of Ca 2ϩ . In contrast, low IP 3 concentrations were less effective in depleting the stores. These data show that concentrations of AdA that did not noticeably activate I Cl1-S were capable of depleting intracellular Ca 2ϩ stores to similar levels as high concentrations of IP 3 .
Thus, we conclude that AdA stimulates SOCE as a consequence of depletion of internal Ca 2ϩ stores and not by some direct effect on SOCs. Furthermore, previous conclusions, based on Ca 2ϩ -activated Cl Ϫ current activation, which suggested that low concentrations of AdA stimulate SOCE without releasing Ca 2ϩ from stores (17), can be explained by the observation that slow release of Ca 2ϩ from stores is often insufficient to activate I Cl1-S .
Effect of AdA on SOCE Requires Active IP 3 R-If this conclusion is correct, the effects of AdA on SOCE should depend on the ability of the IP 3 R to release Ca 2ϩ . Thus, treatments that suppress IP 3 R function should inhibit the effects of AdA injections. We suppressed IP 3 R function either by injecting the competitive inhibitor heparin (Fig. 7) or by reducing IP 3 R expression by injection of antisense oligonucleotides to the Xenopus IP 3 R (Fig. 8).
Injection of heparin to block the IP 3 R significantly reduced I Cl2 and I Cl1-T currents induced by small AdA injections (Fig. 7,  a-b). In a similar fashion, heparin blocked the Cl Ϫ current response induced by IP 3 (Fig. 7, c-d). Reducing IP 3 R levels by antisense oligonucleotides as described previously by others (25,29) also reduced the effects of IP 3 and AdA treatments on I Cl1-T and I Cl2 (Fig. 8). The effects of antisense treatment were less pronounced than the effects of heparin, but it was clear that antisense had a significant effect. Note that although antisense treatment inhibited I Cl2 and I Cl1-T in response to IP 3 injection, there was no decrease in levels of I Cl1-S (Fig. 8d). Actually, I Cl1-S was slightly potentiated as compared with sense-injected cells (Fig. 8c). This observation could be explained if one assumes there are two distinct subpopulations of IP 3 receptors with differential turnover rates of the IP 3 R. If we postulate the existence of subsets of stores, one close to the I Cl1-S Cl Ϫ channels containing IP 3 Rs with a very slow turnover rate and a second located further from the Cl Ϫ channels containing an IP 3 R population that turns over rapidly, then injection of antisense IP 3 oligonucleotides will reduce the levels of IP 3 Rs in the latter subset faster, resulting in sufficient Ca 2ϩ release after IP 3 injection to activate I Cl1-S but insufficient release from most of the stores to induce significant SOCE.

DISCUSSION
In many cell types, release of Ca 2ϩ from endoplasmic reticulum stores stimulates Ca 2ϩ influx into the cytosol from the extracellular space through SOCs by a process termed SOCE. The mechanisms by which release of Ca 2ϩ from stores stimu-  1 and 2 in a. c, current traces corresponding to the Ϫ140 mV/ϩ40 mV pulse combination labeled 3 and 4 in a and d. Ca 2ϩ fluorescence during the ϩ40 mV pulses from the 0-mV holding potential (F Caϩ40 (filled squares)) and at -140 mV (F Ca-140 (open circles)) every 10th pulse. At the end of the experiment, the oocyte was exposed to 14 M ionomycin in calcium-free Ringer (Ionom./0 Ca 2ϩ ) to release any residual Ca 2ϩ from stores (see Fig. 6). This cell is representative of eight cells.
lates SOCE is unknown, but one hypothesis states that the endoplasmic reticulum releases a diffusible chemical messenger that opens SOCs. The search for such a calcium influx factor has so far not been very fruitful, and the putative calcium influx factors that have been discovered have not found universal acceptance (9,10). When it was suggested that AdA could stimulate Ca 2ϩ influx without stimulating Ca 2ϩ release from stores (17), some hope was raised that clues to the structure of calcium influx factors would be learned from AdA. The suggestion that AdA could stimulate Ca 2ϩ entry without depleting Ca 2ϩ from stores was based on the observation that low concentrations of AdA did not stimulate Ca 2ϩ -activated Cl Ϫ currents in the absence of extracellular Ca 2ϩ and therefore did not release Ca 2ϩ from stores but did stimulate Ca 2ϩ -activated Cl Ϫ currents in the presence of Ca 2ϩ influx. The present studies using Ca 2ϩ imaging demonstrate, however, that even very low amounts of AdA (calculated oocyte concentration ϳ5 nM) released Ca 2ϩ from stores. Under these conditions, even though I Cl1-S was not activated, the stores were completely depleted of Ca 2ϩ as demonstrated by the inability of ionomycin to increase Ca 2ϩ fluorescence. We believe that the Ca 2ϩ released from stores by low concentrations of AdA is unable to activate I Cl1-S because of its significantly slower rate of Ca 2ϩ release (ϳ7 times slower than IP 3 ).
How do the kinetics of Ca 2ϩ release from stores determine the response of the Cl Ϫ channels? It has been suggested that I Cl1-S responds to the rate-of-change of cytosolic Ca 2ϩ (20) because the peak activation of I Cl1-S corresponds to the maximum rate of change of cytosolic Ca 2ϩ and because the amplitude of I Cl1-S does not correlate with the steady-state levels of cytosolic Ca 2ϩ . However, we have shown (27) that the turn-off of I Cl1-S is not explained by inactivation of the current as previously sug-  1, 2, and 5 in a. c, current traces corresponding to the Ϫ140 mV/ϩ40 mV pulse combination labeled 3 and 4 in a. d, Ca 2ϩ fluorescence during the ϩ40 mV pulses from the 0-mV holding potential (F Caϩ40 (filled squares)) and at -140 mV (F Ca-140 (open circles)) every 10th pulse. At the end of the experiment, the oocyte was exposed to 14 M ionomycin in calcium-free Ringer (Ionom./0 Ca 2ϩ ) to release any residual Ca 2ϩ from stores (see Fig. 6). This cell is representative of 14 cells. gested (20). Furthermore, we have found that the Ca 2ϩ concentration measured by cytosolic Ca 2ϩ dyes (such as Ca 2ϩ -green dextran) does not reflect the concentration of Ca 2ϩ just below the plasma membrane (measured by lipophilic Ca 2ϩ dyes such as Ca-green C18) (28). We have presented evidence that the subplasmalemmal Ca 2ϩ concentration changes much more FIG. 7. Heparin blocks both IP 3 -and AdA-dependent store depletion. a-b,cells were injected with 10 nl of 0.5 M AdA alone (a; n ϭ 5) or preinjected with 92 nl of 100 mg/ml heparin before AdA injection (b; n ϭ 6). c-d, cells were injected with 10 nl of 50 M IP 3 alone (c; n ϭ 5) or preinjected with 92 nl of 100 mg/ml heparin before IP 3 injection (d; n ϭ 3). The site of injection is indicated by the arrow. Cl Ϫ currents are measured as described in Fig. 1. quickly than does the Ca 2ϩ concentration deeper in the cytosol because plasma membrane Ca 2ϩ efflux systems can rapidly clear Ca 2ϩ from the subplasmalemmal space. Consequently, we would predict that the subplasmalemmal Ca 2ϩ concentration would depend on the relative rates of Ca 2ϩ release from stores and cytosolic Ca 2ϩ buffering and Ca 2ϩ efflux from the oocyte. If Ca 2ϩ release is slow, the concentration of Ca 2ϩ in the subplasmalemmal space may not rise sufficiently to activate Ca 2ϩactivated Cl Ϫ channels.
The different kinetics of Ca 2ϩ release produced by AdA and IP 3 are probably related to differences between AdA and IP 3 activation of IP 3 Rs. First, the apparent diffusion coefficient of AdA or IP 3 in the cytosol will depend on the fraction of molecules () that are bound to the IP 3 R at any one time (D obs ϭ D/). Because AdA has a 100-fold higher affinity for the IP 3 R than IP 3 does, AdA diffusion will be slower because a larger fraction of the total AdA (compared with IP 3 ) will be bound to IP 3 Rs. Second, AdA exhibits a higher cooperativity in activating IP 3 Rs than IP 3 does. Hirota et al. 1995 (13) have shown that IP 3 has a Hill coefficient of 1.8 for Ca 2ϩ release by the type 1 IP 3 R, whereas the Hill coefficient for AdA was 3.9. This implies that at least 2 molecules of IP 3 and 4 molecules of AdA are needed to open an IP 3 R. This factor will also contribute to the slow movement of the Ca 2ϩ release wave in response to small amounts of AdA. Accordingly, the elementary Ca 2ϩ release events ("Ca 2ϩ puffs") induced by AdA have been shown by Marchant and Parker (30) to be smaller and faster than those induced by IP 3 . Because Ca 2ϩ waves are initiated by the summation of Ca 2ϩ puffs, the smaller and faster puffs induced by AdA may contribute to the slower propagation of the AdA wave. However, the mechanisms by which AdA releases Ca 2ϩ from stores remains to be fully elucidated.
Although low concentrations of AdA evoke a slow release of Ca 2ϩ from stores and little or no I Cl1-S , high concentrations release Ca 2ϩ only about 2-fold more slowly than IP 3 and also evoke significant I Cl1-S . This finding that AdA activates different I Cl1-S responses depending upon the kinetics of Ca 2ϩ release from intracellular stores is interesting because it provides another example of how the temporal features of a Ca 2ϩ signal contribute to its physiological consequences. Different receptors can induce different Ca 2ϩ release kinetics depending on factors including spatial localization of the receptor and/or IP 3 -sensitive stores or the activation of different PLC isoforms (31)(32)(33)(34)(35)(36). These different release kinetics can then play an important role in determining which effectors are activated.