Role of the Inositol 1,4,5-Trisphosphate Receptor in Ca2+ Feedback Inhibition of Calcium Release-activated Calcium Current (I crac)
1999; Elsevier BV; Volume: 274; Issue: 46 Linguagem: Inglês
10.1074/jbc.274.46.32881
ISSN1083-351X
AutoresLisa M. Broad, David L. Armstrong, James W. Putney,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoWe examined the activation and regulation of calcium release-activated calcium current (I crac) in RBL-1 cells in response to various Ca2+ store-depleting agents. With [Ca2+] i strongly buffered to 100 nm,I crac was activated by ionomycin, thapsigargin, inositol 1,4,5-trisphosphate (IP3), and two metabolically stable IP3 receptor agonists, adenophostin A andl-α-glycerophospho-d-myoinositol-4,5-bisphosphate (GPIP2). With minimal [Ca2+] i buffering, with [Ca2+] i free to fluctuateI crac was activated by ionomycin, thapsigargin, and by the potent IP3 receptor agonist, adenophostin A, but not by GPIP2 or IP3 itself. Likewise, when [Ca2+] i was strongly buffered to 500 nm, ionomycin, thapsigargin, and adenophostin A did and GPIP2 and IP3 did not activate detectableI crac. However, with minimal [Ca2+] i buffering, or with [Ca2+] i buffered to 500 nm, GPIP2 was able to fully activate detectableI crac if uptake of Ca2+intracellular stores was first inhibited. Our findings suggest that when IP3 activates the IP3 receptor, the resulting influx of Ca2+ quickly inactivates the receptor, and Ca2+ is re-accumulated at sites that regulateI crac. Adenophostin A, by virtue of its high receptor affinity, is resistant to this inactivation. Comparison of thapsigargin-releasable Ca2+ pools following activation by different IP3 receptor agonists indicates that the critical regulatory pool of Ca2+ may be very small in comparison to the total IP3-sensitive component of the endoplasmic reticulum. These findings reveal new and important roles for IP3 receptors located on discrete IP3-sensitive Ca2+ pools in calcium feedback regulation ofI crac and capacitative calcium entry. We examined the activation and regulation of calcium release-activated calcium current (I crac) in RBL-1 cells in response to various Ca2+ store-depleting agents. With [Ca2+] i strongly buffered to 100 nm,I crac was activated by ionomycin, thapsigargin, inositol 1,4,5-trisphosphate (IP3), and two metabolically stable IP3 receptor agonists, adenophostin A andl-α-glycerophospho-d-myoinositol-4,5-bisphosphate (GPIP2). With minimal [Ca2+] i buffering, with [Ca2+] i free to fluctuateI crac was activated by ionomycin, thapsigargin, and by the potent IP3 receptor agonist, adenophostin A, but not by GPIP2 or IP3 itself. Likewise, when [Ca2+] i was strongly buffered to 500 nm, ionomycin, thapsigargin, and adenophostin A did and GPIP2 and IP3 did not activate detectableI crac. However, with minimal [Ca2+] i buffering, or with [Ca2+] i buffered to 500 nm, GPIP2 was able to fully activate detectableI crac if uptake of Ca2+intracellular stores was first inhibited. Our findings suggest that when IP3 activates the IP3 receptor, the resulting influx of Ca2+ quickly inactivates the receptor, and Ca2+ is re-accumulated at sites that regulateI crac. Adenophostin A, by virtue of its high receptor affinity, is resistant to this inactivation. Comparison of thapsigargin-releasable Ca2+ pools following activation by different IP3 receptor agonists indicates that the critical regulatory pool of Ca2+ may be very small in comparison to the total IP3-sensitive component of the endoplasmic reticulum. These findings reveal new and important roles for IP3 receptors located on discrete IP3-sensitive Ca2+ pools in calcium feedback regulation ofI crac and capacitative calcium entry. inositol 1,4,5-trisphosphate Ca2+ release-activated Ca2+ current 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid l-α-glycerophospho-d-myo-inositol-4,5-bisphosphate HEPES-buffered saline solution Many extracellular stimuli act through cell surface receptors to promote generation of intracellular inositol 1,4,5-trisphosphate (IP3)1 and consequently release intracellular Ca2+ stores (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6174) Google Scholar). The release of stored Ca2+ is commonly accompanied by influx of Ca2+ from the extracellular space, through the "capacitative Ca2+ entry" pathway (2Putney Jr., J.W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2109) Google Scholar, 3Putney Jr., J.W. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1261) Google Scholar, 4Putney Jr., J.W. Capacitative Calcium Entry. Landes Biomedical Publishing, Austin, TX1997Crossref Google Scholar). Although depletion of intracellular stores and activation of capacitative calcium entry are inextricably linked, the underlying mechanism remains poorly defined (4Putney Jr., J.W. Capacitative Calcium Entry. Landes Biomedical Publishing, Austin, TX1997Crossref Google Scholar). Store-operated currents, which are proposed to mediate capacitative calcium entry, have been measured in various cell types (5Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1291) Google Scholar). To date, "Ca2+ release-activated Ca2+ current" (I crac) is the best defined member of the store-operated current family (6Hoth M. Penner R. Nature. 1992; 355: 353-355Crossref PubMed Scopus (1491) Google Scholar). I crac, measured in mast cells, T-lymphocytes, and rat basophilic leukemia (RBL-1) cells is both highly selective for Ca2+ as the permeant ion and strongly inhibited by Ca2+ feedback (5Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1291) Google Scholar,7Zweifach A. Lewis R.S. J. Gen. Physiol. 1995; 105: 209-226Crossref PubMed Scopus (335) Google Scholar, 8Zweifach A. Lewis R.S. J. Biol. Chem. 1995; 270: 14445-14451Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 9Hoth M. Penner R. J. Physiol. (Lond.). 1993; 465: 359-386Crossref Scopus (661) Google Scholar). At least two forms of Ca2+-dependent inactivation of I crac have been reported, termed fast and slow inactivation. Fast inactivation occurs on a sub-second time scale and is caused by increases in sub-plasmalemmal Ca2+ concentration in the vicinity of the Ca2+channel (7Zweifach A. Lewis R.S. J. Gen. Physiol. 1995; 105: 209-226Crossref PubMed Scopus (335) Google Scholar, 9Hoth M. Penner R. J. Physiol. (Lond.). 1993; 465: 359-386Crossref Scopus (661) Google Scholar). Slow inactivation, on the time scale of tens to hundreds of seconds, is caused by increases in the bulk cytosolic [Ca2+] and is partly dependent on refilling of intracellular Ca2+ stores (8Zweifach A. Lewis R.S. J. Biol. Chem. 1995; 270: 14445-14451Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). In order to maximize, or indeed detect, the typically small whole-cell CRAC currents, it is necessary to minimize these forms of Ca2+ feedback. First, the [Ca2+] i is tightly buffered to basal levels with high concentrations of Ca2+ chelators such as BAPTA or EGTA. Second, the cell membrane potential is held at depolarized levels to decrease the driving force for Ca2+ entry between current measurements. A major disadvantage of these conditions is the inability to measure simultaneous changes in [Ca2+] i . Recently, simultaneous measurements of [Ca2+] i and I crac have been reported in RBL-1 cells, an immortalized mast cell line (10Huang Y. Takahashi M. Tanzawa K. Putney Jr., J.W. J. Biol. Chem. 1998; 273: 31815-31821Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In this study, the degree of Ca2+ buffering was reduced from a level which effectively clamps [Ca2+] i to basal concentrations, to a minimal level, which permitted fluctuations of [Ca2+] i. Under these conditions, adenophostin A, an IP3 receptor agonist with 100-fold higher affinity for the IP3 receptor compared with the native ligand IP3 (11Takahashi M. Tanzawa K. Takahashi S. J. Biol. Chem. 1994; 269: 369-372Abstract Full Text PDF PubMed Google Scholar) induced Ca2+ release and activation ofI crac (10Huang Y. Takahashi M. Tanzawa K. Putney Jr., J.W. J. Biol. Chem. 1998; 273: 31815-31821Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Several other agents that would be expected to deplete intracellular stores, including analogues of IP3 ((2,4,5)IP3, 3-deoxy-3-fluoro-IP3), thapsigargin (a SERCA inhibitor), and ionomycin (a Ca2+ ionophore), did not activateI crac under these conditions. It was suggested that the unique ability of adenophostin A to activateI crac was perhaps due to action at the level of Ca2+-dependent inactivation of the CRAC channel. In the present study we investigated activation ofI crac in RBL-1 cells under conditions of weak Ca2+ buffering, while simultaneously monitoring [Ca2+] i. We compared the ability of various store depletion agents and combinations of these agents to induce a rise in [Ca2+] i and to activate detectableI crac. We now find that adenophostin A, thapsigargin, and ionomycin all activate I cracunder low buffering conditions; however, IP3 and a stable analogue of IP3,l-α-glycerophospho-d-myoinositol-4,5-bisphosphate (GPIP2), do not. This failure was not the result of Ca2+ feedback on the CRAC channel. Rather, deactivation of the current appears due to inactivation of IP3 receptors and rapid refilling of critical Ca2+ stores thereby diminishing the signal for activation of I crac. This work reveals a new mechanism that involves the IP3receptor for Ca2+ feedback on I crac. These IP3 receptors are likely located in spatially restricted regions within the endoplasmic reticulum and are closely coupled to activation and regulation ofI crac. Rat basophilic leukemia cells (RBL-1) were purchased from the ATCC (1378-CRL). Cells were cultured in Earle's minimal essential medium with Earle's salts, 10% fetal bovine serum, 2 mml-glutamine, 50 units/ml penicillin, and 50 mg/ml streptomycin (37 °C, 5% CO2). For experiments, cells were passaged onto glass coverslips (number 11/2) and used 12 -36 h after plating. Coverslips with attached cells were mounted in a Teflon chamber and incubated at room temperature for 25 min in HEPES-buffered saline solution (HBSS; in mm, 140 NaCl, 4.7 KCl, 10 CsCl, 1.8 CaCl2, 1.13 MgCl2, 10 glucose, and 10 HEPES, pH 7.2) containing 1 μm Fura-2 AM (Molecular Probes). Cells were then washed and bathed in HBSS for at least 10 min before Ca2+measurements were made. Fluorescence was monitored by placing the Teflon chamber with the coverslip of Fura-2-loaded cells onto the stage of a Nikon Diaphot microscope (40× Neofluor objective). Cells were excited by light (340 and 380 nm) from a Deltascan D101 (Photon Technology International Ltd.) light source equipped with a light path chopper and dual excitation monochromators. Emitted fluorescence (510 nm) was collected by a photomultiplier tube (Omega). All experiments were conducted at room temperature (22 °C). Calibration of [Ca2+] i was performed by reference to a look-up table created from Ca2+ standards supplied by Molecular Probes. Patch clamp experiments were conducted in the standard whole-cell recording configuration (12Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15145) Google Scholar). Patch pipette (2–4 megaohm, Corning glass, 7052) solutions contained (in mm) 140 cesium aspartate, 2 MgCl2, 10 HEPES, 1 MgATP, and either 10 BAPTA-Cs4 (with free Ca2+set to 100 or 500 nm, calculated using MaxChelator software (version 6.60) or 0.1 BAPTA-Cs4 (with no Ca2+added), pH 7.2. Fura-2 free acid (50 μm) was included in the pipette as indicated. Bath solution (HBSS) was as described above, except CaCl2 was increased to 10 mm for Ca2+-HBSS or omitted for nominally Ca2+-free HBSS (10 mm MgCl2 was included in nominally Ca2+-free HBSS). 0.2 mm EGTA was included where indicated. In all experiments, upon forming the whole-cell configuration the cell membrane potential was held at +30 mV (to minimize Ca2+entry and Ca2+-dependent inactivation of CRAC channels). Periodically (once every 5 s) the membrane potential was stepped to −100 mV (for 20 ms to assessI crac), and then a voltage ramp to +60 mV, over a period of 160 ms, was applied. Currents are normalized to cell capacitance. All voltages are corrected for a 10 mV liquid-junction potential. Membrane currents were amplified with an Axopatch-1C amplifier (Axon Instruments, Burlingham, CA). Voltage clamp protocols were implemented and data acquisition performed with PCLAMP 6.1 software (Axon Instruments). Currents were filtered at 1 kHz and digitized at 200-μs intervals. Adenophostin A was a gift from Drs. M. Takahashi and K. Tanzawa (Sankyo Co., Ltd., Tokyo, Japan). IP3 and ionomycin were from Calbiochem. Thapsigargin was from LC Laboratories, and GPIP2 was from Roche Molecular Biochemicals. Cs4BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) and Fura-2 were from Molecular Probes (Eugene, OR). With pipette solutions strongly buffered (with 10 mm BAPTA) to 100 nmfree [Ca2+], and following the voltage protocol described under "Experimental Procedures," I crac was activated by a variety of store depletion agents in agreement with previous studies (6Hoth M. Penner R. Nature. 1992; 355: 353-355Crossref PubMed Scopus (1491) Google Scholar, 9Hoth M. Penner R. J. Physiol. (Lond.). 1993; 465: 359-386Crossref Scopus (661) Google Scholar, 10Huang Y. Takahashi M. Tanzawa K. Putney Jr., J.W. J. Biol. Chem. 1998; 273: 31815-31821Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 13Huang Y. Putney Jr., J.W. J. Biol. Chem. 1998; 273: 19554-19559Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) (Fig. 1). Intracellular delivery of IP3 (20 or 40 μm), GPIP2 (100 or 200 μm), a non-metabolizable analogue of IP3, or adenophostin A (2 μm), or extracellular addition of thapsigargin (1 μm) or ionomycin (500 nm) activated an inward current with properties characteristic of I crac. In each case, the current decreased rapidly upon removal of extracellular Ca2+ (Fig. 1 B), showed inward rectification, and reversed direction at a potential positive of +30 mV (Fig.1 A). There were no significant differences among the maximum amplitudes of the currents activated by these agents (Fig.1 C). The development times were also similar, although slightly prolonged for thapsigargin (Fig. 1 C; complete time course of I crac activation with 10 mm BAPTA, 100 nm [Ca2+] i is shown by the open circles in Fig. 3).Figure 3Adenophostin A, thapsigargin, and ionomycin, but not IP3 and GPIP2 ,activate I crac when [Ca2+] i is strongly buffered to activated levels (500 nm). A–E, cells were patched with pipette solutions containing 10 mm BAPTA and free Ca2+ set to either 500 nm (filled circles) or 100 nm (open circles).A, 2 μm adenophostin A (AdA);B, 20 (in this figure) or 40 μm (not shown) IP3; C, 100 (in this figure) or 200 μm (not shown) GPIP2 was also included in the pipette solution; D, 1 μm thapsigargin (TG); E, 500 nm ionomycin (IONO) was applied to the outside of the cell for the period shown. Cells were held at 30 mV, and the voltage protocol described in Fig. 1 was used. Traces are means ± S.E. (n ≥ 5).View Large Image Figure ViewerDownload Hi-res image Download (PPT) With pipette solutions buffered with 0.1 mm BAPTA, [Ca2+] i was weakly buffered and free to fluctuate. Under these conditions, each of the store-depleting agents caused an increase in [Ca2+] i in Fura-2-loaded cells (Fig. 2, A–E upper traces). Adenophostin A (Fig. 2 A), IP3 (Fig. 2 B), and GPIP2 (Fig.2 C) each produced a biphasic increase in [Ca2+] i as follows: first a peak then a lower plateau, sustained slightly but significantly above basal levels. Thapsigargin (Fig. 2 D) and ionomycin (Fig. 2 E), agents that bypass the IP3 receptor, produced sustained and monophasic increases in [Ca2+] i. Importantly, all the sustained increases in Ca2+ were reversed by removal of extracellular Ca2+ and therefore reflected Ca2+entry. Ca2+ entry presumably occurs mostly during the brief hyperpolarizing pulses. In support of this idea, expansion of the Ca2+ traces revealed episodic Ca2+ peaks, with a 5-s periodicity, that correspond to membrane hyperpolarizations. These Ca2+ peaks were absent when extracellular Ca2+ was removed (data now shown). Simultaneous measurements of membrane currents in the same Fura-2-loaded cells revealed that adenophostin A, thapsigargin, and ionomycin, but neither IP3 nor GPIP2, induced detectable activation of I crac (Fig. 2,A–E, lower traces). The currents activated by adenophostin A (Fig. 2 A), thapsigargin (Fig. 2 D), and ionomycin (Fig. 2 E) in weakly Ca2+-buffered cells displayed properties characteristic ofI crac. In each case, the current decreased rapidly upon removal of extracellular Ca2+. The currents also showed inward rectification and reversed direction at a potential positive of +30 mV (not shown). Under these conditions adenophostin A (1.08 ± 0.12 pA/pF), thapsigargin (1.05 ± 0.14 pA/pF), and ionomycin (1.29 ± 0.13 pA/pF) caused similar increases in I crac(Fig. 2 F), but all of the values were significantly less than those measured with strong Ca2+ buffering (Fig.1 C), being 45 ± 5, 43 ± 6, and 46 ± 3%, respectively. Following break in with pipettes containing adenophostin A, or following application of ionomycin, activation ofI crac occurred after short delays of 49 ± 11 and 88 ± 9 s, respectively (Fig. 2, A andE). However, there was a considerably longer delay between the delivery of thapsigargin and subsequent activation ofI crac (181 ± 19 s) (Fig.2 D). I crac was activated by thapsigargin with a mean development time (time from the initial increase in current to development of current; see Ref. 13Huang Y. Putney Jr., J.W. J. Biol. Chem. 1998; 273: 19554-19559Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar)) of 375 ± 35 s, considerably slower than that for adenophostin A (132 ± 25 s) or ionomycin (145 ± 22 s) (Fig.2 F, open bars). The initial rise in Ca2+ induced by thapsigargin indicates blockade of endoplasmic reticulum Ca2+-ATPases and the subsequent leak of Ca2+ from intracellular stores. The delay before detectable I crac activation and the slow time course of development presumably reflect the time it takes to empty, to the necessary degree, the stores linked to I cracactivation. It is because of this delayed and slow activation ofI crac with thapsigargin that our earlier study failed to detect activation of I crac by thapsigargin in RBL-1 cells (10Huang Y. Takahashi M. Tanzawa K. Putney Jr., J.W. J. Biol. Chem. 1998; 273: 31815-31821Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Also, in our earlier study (10Huang Y. Takahashi M. Tanzawa K. Putney Jr., J.W. J. Biol. Chem. 1998; 273: 31815-31821Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) ionomycin failed to activate I crac. However, a substantially higher concentration of ionomycin was used (5 μm, as opposed to 500 nm in the current study), and at this concentration ionomycin raises [Ca2+] i to extremely high levels, in excess of 1 μm, which likely caused a strong Ca2+-dependent inactivation ofI crac. We next considered how minimal Ca2+ buffering might prevent IP3 and GPIP2 from activating detectableI crac. Direct effects of global [Ca2+] i on CRAC channels is unlikely to be responsible, because the agents that activatedI crac actually raised steady-state [Ca2+] i to somewhat higher levels than did IP3 or GPIP2. Thus, either IP3 and GPIP2 are capable of raising Ca2+ to higher levels than other agents in small, discrete regions close to the CRAC channels or, alternatively, I crac activated by IP3 and GPIP2 is for some reason more sensitive to Ca2+ inhibition. Thus, we studied the effect of increased cytosolic Ca2+ on I cracunder conditions whereby both sub-plasmalemmal and global [Ca2+] i were strongly buffered with 10 mm BAPTA. We buffered [Ca2+] i to either a basal value ([Ca2+] i ∼100 nm) or a value ([Ca2+] i ∼500 nm) similar to that recorded in weakly buffered cells after activation with store depletion agents (Fig. 2 E). Under these conditions (10 mm BAPTA, [Ca2+] i ∼500 nm), adenophostin A (Fig. 3 A), thapsigargin (Fig.3 D), and ionomycin (Fig. 3 E) activatedI crac; IP3 (Fig. 3 B) and GPIP2 (Fig. 3 C) did not. Thus, IP3and GPIP2 apparently fail to activateI crac because with these agents the signaling mechanism is more sensitive to inhibition by elevated [Ca2+] i . Adenophostin A (2.18 ± 0.03 pA/pF), thapsigargin (2.57 ± 0.65 pA/pF), and ionomycin (2.95 ± 0.45 pA/pF) caused similar increases in I crac. These values were also similar to measurements made with free Ca2+ buffered to 100 nm (Fig. 1 C), being 90 ± 3, 104 ± 26, and 105 ± 20%, respectively. It is worth noting, however, that although the peak magnitude of I cracmeasured with free Ca2+ set to 100 or 500 nmwas similar, a slow inactivation of the current was prominent at the higher cytosolic [Ca2+]. I cracactivated by adenophostin A fell to 90 ± 12 and 56 ± 9% of the peak after 300 s, at 100 and 500 nm free Ca2+, respectively. Over the same period,I crac activated by ionomycin also declined more at 500 nm, falling to 62 ± 9% of the peak compared with 87 ± 11% with 100 nm free Ca2+. The mean development time of I crac for thapsigargin at 500 nm [Ca2+] i (415 ± 27 s) was again much slower than for adenophostin A (94 ± 8 s) and ionomycin (68 ± 5 s). There was also a considerable delay before thapsigargin induced detectable activation of I crac (295 ± 34 s) compared to when free [Ca2+] i was set to 100 nm (91 ± 14 s) (Fig. 3 D). For both adenophostin A and ionomycin, activation ofI crac was seen after only a short delay (42 ± 12 and 88 ± 9 s, respectively). Buffering sub-plasmalemmal [Ca2+] gradients (10 mm BAPTA), and raising bulk cytosolic [Ca2+] (500 nm), mimicked some of the effects of low Ca2+ buffering, namely failure of GPIP2and IP3 to activate I crac and slow activation of I crac by thapsigargin. Given that adenophostin A, ionomycin, and thapsigargin all activateI crac under conditions where IP3 and GPIP2 are ineffective, direct Ca2+-dependent inactivation of the CRAC channel seems unlikely to be the cause. Rather, an involvement of the IP3 receptor itself would be indicated. Thus,I crac could be inactivated if, when IP3 and GPIP2 are used, the IP3receptors were desensitized, and as a result the stores signalingI crac activation were refilled. Increases in [Ca2+] i are known to promote dissociation of IP3 from the IP3 receptor leading to faster IP3 receptor inactivation (14Hannaert-Merah Z. Coquil J.-F. Combettes L. Claret M. Mauger J.-P. Champeil P. J. Biol. Chem. 1994; 269: 29642-29649Abstract Full Text PDF PubMed Google Scholar). Adenophostin A, by virtue of its high affinity for the IP3 receptor, may be less susceptible to this increased rate of dissociation. Adenophostin A would therefore prolong IP3 receptor activation, relative to the lower affinity agonists (GPIP2 and IP3), and maintain depleted Ca2+ stores. If indeed IP3 receptor inactivation does lead to rapid store refilling and turns off detectable I crac, then prevention of Ca2+ re-uptake (with thapsigargin) should prevent the re-uptake of Ca2+ that occurs with IP3 and GPIP2, and thus these agents would act more like adenophostin A. To test this hypothesis, thapsigargin was applied to cells loaded with Fura-2 and patch-clamped in the cell-attached mode (Fig. 4 A). Patch pipettes contained 100 μm GPIP2 and 0.1 mm BAPTA. After 50–70 s, a slight increase in [Ca2+] i was detected indicating SERCA inhibition (Fig. 4 A, upper trace). The whole-cell mode was then established in order to deliver GPIP2 and measureI crac (Fig. 4 A, lower trace). GPIP2 caused a further, more substantial increase in [Ca2+] i which was sustained (compare with GPIP2 alone, Fig. 2 C). Importantly, the increase in [Ca2+] i that occurred upon delivery of GPIP2 was accompanied by rapid activation ofI crac. I crac was activated after only a short delay (33 ± 8 s) and with a time course of 86 ± 14 s. Had the current been activated by thapsigargin alone, the delay from break-in would have been in excess of 3 min and the development time in excess of 5 min (inferred from Fig. 2 D). Development of I crac in the presence of GPIP2 is therefore triggered by the rapid release of Ca2+ through activated IP3 receptors and independent of the slow leak of Ca2+ induced by thapsigargin alone. The peak amplitude of I cracwas not significantly altered by the presence (1.53 ± 0.23 pA/pF) or absence of GPIP2 (1.05 ± 0.14 pA/pF, Fig.2 F). 2Although the mean value for the current in the presence of GPIP2 and thapsigargin was almost 50% larger than that for thapsigargin alone, the difference was not statistically significant by either t test or analysis of variance, owing to the variability in values of I crac from one preparation to another. However, we note that the value for GPIP2 plus thapsigargin (1.53 ± 0.23 pA/pF) was similar to the value for ionomycin under the same conditions (1.29 ± 0.13 pA/pF). This suggests that if GPIP2 plus thapsigargin does induce a current larger than for thapsigargin alone, it is likely due to increased release of Ca2+ rather than a specific effect of GPIP2 on the channel activation mechanism. This is to be expected if GPIP2 increases the rate of release, through opening IP3 receptors, but not the overall extent. The preceding data suggest that when Ca2+ is free to increase, re-uptake of Ca2+ prevents detectable activation of I crac by GPIP2. If this assumption is correct, then delivery of thapsigargin before GPIP2, with Ca2+ clamped to 500 nmwith 10 mm BAPTA, should also allow rapid activation ofI crac (Fig. 4 B). As predicted, in cells pretreated with thapsigargin for 50–70 s, delivery of GPIP2 led to a rapid activation ofI crac. Activation was detected after only a short delay (34 ± 11 s) and had a development time of 81 ± 13 s, compared with 415 ± 27 s for thapsigargin alone. The amplitude of the current induced by GPIP2 and thapsigargin combined (3.16 ± 0.28 pA/pF) was once again not significantly greater than that that seen with thapsigargin alone (2.57 ± 0.65 pA/pF). These results reveal that SERCA pumps need to be blocked if GPIP2 is to activate detectableI crac when [Ca2+] i is weakly buffered. Refilling of stores by SERCA pumps must occur very quickly, and therefore, a decrease in IP3 receptor activity must also occur quickly to explain the complete lack of detectableI crac activation by GPIP2 or IP3. GPIP2 is successful in activatingI crac after thapsigargin addition because despite rapid IP3 receptor desensitization, Ca2+ cannot be re-accumulated into the critical stores. This predicts that if the addition of the SERCA inhibitor is delayed for even a short interval following addition of GPIP2,I crac activation will still fail. As shown in Fig. 4 C, this is indeed the case. When thapsigargin was added 50–70 s after break-in with GPIP2, no rapid activation of I crac was observed; ratherI crac activation occurred slowly (311 ± 7 s) and after a latency of 222 ± 21 s, similar to activation of I crac by thapsigargin alone (see Fig. 2 D). Collectively these results indicate that IP3 and GPIP2 activate IP3 receptors and allow a rapid release of Ca2+ from intracellular stores, but this receptor activity and enhanced Ca2+ release are transient. Hence, when re-uptake of Ca2+ is blocked prior to GPIP2 exposure, the release induced by GPIP2 is sufficiently sustained to activate I crac rapidly (Fig. 4 A). However, if Ca2+ re-uptake is allowed to proceed for even a minute after GPIP2 delivery, blockade of SERCA pumps at this stage does not rapidly activateI crac, because the IP3 receptors have already desensitized and Ca2+ has been re-accumulated (Fig. 4 C). Finally, therefore, we attempted experiments designed to demonstrate more directly the re-uptake of Ca2+into pools regulating I crac and to determine their size relative to the presumably larger IP3- and thapsigargin-sensitive stores. To this end, we activated signaling in cells with either GPIP2 or adenophostin A, and we examined the size of the [Ca2+] i signal on application of thapsigargin in Ca2+-free media (Fig.5, lower panels). On delivery of adenophostin A or GPIP2, cells were exposed to either Ca2+-free or Ca2+-containing media before assessing the thapsigargin-sensitive store content in order to determine the contribution of Ca2+ influx to the content of the stores. A residual, thapsigargin-sensitive, Ca2+ store was detected in cells after exposure to adenophostin A (Fig.5 A) or GPIP2 (Fig. 5 B) in the absence of Ca2+ influx (dotted lines). When Ca2+ influx was allowed before store assessment (dashed lines), some refilling of stores occurred after both agents, although slightly more refilling appeared to occur in the presence of GPIP2. In each case, however, Ca2+stores were not completely refilled, because control cells exposed only to thapsigargin showed the largest release of Ca2+(solid lines). Assessment of the Ca2+ stores by inhibition of SERCA pumps revealed Ca2+ release kinetics for adenophostin A (Fig.5 A, dashed lines) which were distinct from that in control cells (Fig. 5, solid line) or GPIP2-treated cells (Fig. 5 B, dashed lines). For adenophostin A-treated cells, the rate of Ca2+ release was faster than in control cells, with [Ca2+] i returning to base line within 514 ± 31 s, compared with 1058 ± 60 and 966 ± 92 s for control or GPIP2-treated cells, respectively. This finding fits with our prediction that, in the presence of adenophostin A, IP3 receptors remain more active and this maintains critical stores depleted andI crac-detectable. GPIP2 did not significantly increase the kinetics of Ca2+ release over that in control cells, suggesting minimal residual activation of IP3 receptors. Although the residual Ca2+ store was similar in cells treated with adenophostin A or GPIP2, simultaneous current measurements revealed I crac activation in response to adenophostin A only (Fig. 5, upper panels). This indicates that the pool of Ca2+ involved in controllingI
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