An examination of the secretion-like coupling model for the activation of the Ca2+ release-activated Ca2+ current I(CRAC) in RBL-1 cells

Abstract

One popular model for the activation of store-operated Ca2+ influx is the secretion-like coupling mechanism, in which peripheral endoplasmic reticulum moves to the plasma membrane upon store depletion thereby enabling inositol 1,4,5-trisphosphate (InsP3) receptors on the stores to bind to, and thus activate, store-operated Ca2+ channels. This movement is regulated by the underlying cytoskeleton. We have examined the validity of this mechanism for the activation of I(CRAC), the most widely distributed and best characterised store-operated Ca2+ current, in a model system, the RBL-1 rat basophilic cell line. Stabilisation of the peripheral cytoskeleton, disassembly of actin microfilaments and disaggregation of microtubules all consistently failed to alter the rate or extent of activation of I(CRAC). Rhodamine-phalloidin labelling was used wherever possible, and revealed that the cytoskeleton had been significantly modified by drug treatment. Interference with the cytoskeleton also failed to affect the intracellular calcium signal that occurred when external calcium was re-admitted to cells in which the calcium stores had been previously depleted by exposure to thapsigargin/ionomycin in calcium-free external solution. Application of positive pressure through the patch pipette separated the plasma membrane from underlying structures (cell ballooning). However, I(CRAC) was unaffected irrespective of whether cell ballooning occurred before or after depletion of stores. Pre-treatment with the membrane-permeable InsP3 receptor antagonist 2-APB blocked the activation of I(CRAC). However, intracellular dialysis with 2-APB failed to prevent I(CRAC) from activating, even at higher concentrations than those used extracellularly to achieve full block. Local application of 2-APB, once I(CRAC) had been activated, resulted in a rapid loss of the current at a rate similar to that seen with the rapid channel blocker La3+. Studies with the more conventional InsP3 receptor antagonist heparin revealed that occupation of the intracellular InsP3-sensitive receptors was not necessary for the activation or maintenance of I(CRAC). Similarly, the InsP3 receptor inhibitor caffeine failed to alter the rate or extent of activation of I(CRAC). Exposure to Li+, which reduces InsP3 levels by interfering with inositol monophosphatase, also failed to alter I(CRAC). Caffeine and Li+ did not affect the size of the intracellular Ca2+ signal that arose when external Ca2+ was re-admitted to cells which had been pre-exposed to thapsigargin/ionomycin in Ca2+-free external solution. Our findings demonstrate that the cytoskeleton does not seem to regulate calcium influx and that functional InsP3 receptors are not required for activation of I(CRAC). If the secretion-like coupling model indeed accounts for the activation of I(CRAC) in RBL-1 cells, then it needs to be revised significantly. Possible modifications to the model are discussed.

Figure 2. The cytoskeletal restructuring agent calyculin A does not affect the activation or inactivation of I_CRAC

A, upper panel, development of I_CRAC (measured at -80 mV from the voltage ramps) in control conditions (•) and following pre-incubation with 100 nm calcyculin A for 30 min (○). The I-V relationship for each cell is shown in the middle panel (taken at 76 and 82 s for control and calyculin A, respectively). The lower panel depicts graphs which summarise the relationship between current amplitude and the time constant of activation, and the time to peak of the current. B, intracellular dialysis with 1 μm calyculin A fails to alter I_CRAC. The upper panel shows a typical control recording (InsP₃+ 10 mm EGTA + 2 mm Mg-ATP) and one from a cell dialysed with this solution supplemented with calyculin A. The left and centre plots in the lower panel show that the delay, time to peak and amplitude of I_CRAC were similar for the two conditions (control and calyculin A treated). The graph on the right shows that the extent of inactivation of I_CRAC (measured at 600 s) as well as the time at which half-inactivation had been reached (half-time) were not different between control and calyculin A-treated cells. Hence calyculin A fails to affect the activation of I_CRAC, nor does it reverse activation. C, cells were dialysed with an internal solution containing buffered Ca²⁺ (140 nm) and 2 mm Mg-ATP alone (control) or supplemented with 1 μm calyculin A. After 300 s of dialysis, thapisgargin was applied externally. The histograms compare the extent of activation and the time course of I_CRAC for control versus calyculin A-treated cells. Neither the extent of activation nor the time to peak was significantly different between the two conditions.

Figure 1. Cytoskeletal agents evoke quite marked changes in cell shape

A, a control transillumination image of control (non-treated) RBL-1 cells is shown on the left and one taken after 12 min exposure to 100 nm calyculin A is shown on the right. The cells were from the same coverslip but from different fields of view. Note the rounded, shrivelled appearance following the brief exposure to calyculin A. All cells took on this appearance within 10 min of calyculin A treatment. B, rhodamine-phalloidin labelling of actin in control (left) and calyculin A-treated cells (right). C, a control transillumination image of control cells (left) and of cells exposed to the microfilament-disrupting agent cytochalasin D (1 μm for 45 min). Cells were from the same coverslip but different fields of view. D, distribution of actin in control (left) and cytochalasin D-treated cells using rhodamine-phalloidin labelling. E, transillumination images of control cells and cells exposed to the microfilament stabiliser jasplakinolide (1 μm for 30 min). F, a control transillumination image of cells before (left) and then 18 h after exposure to the microtubule-disaggregating agent nocodazole (5 μm; right). Cells were from different coverlips but from the same preparation. Note the rounded appearance and reduction in cell processes with nocodazole. All the experiments described above were carried out at room temperature and were repeated on at least two different cell preparations. Magnification, × 30.

Figure 3. Pharmacological tools that disaggregate or stabilise the cytoskeleton fail to affect the activation of I_CRAC

A, activation of I_CRAC by passive depletion of stores is not altered by cytochalasin D pre-treatment. The left panel depicts a control recording and one taken after 60 min exposure to 2 μm cytochalasin D. I_CRAC was activated by dialysing the cells with a pipette solution containing 10 mm EGTA. None of the parameters we measured were affected by cytochalasin D (right panels; see text for further details). The filled symbols in the right-hand graph represent control and the open symbols data from cytochalasin D-treated cells. Circles represent the initial slow component of the current and squares represent the secondary large phase (see Fierro & Parekh, 1999, for further details). B, I_CRAC activates normally despite pre-treatment with the microfilament-stabilising peptide jasplakinolide. The left panel shows the time course of development of I_CRAC in a cell pre-exposed to 1 μm jasplakinolide for 34 min. Neither the activation time constant nor time to peak was significantly different between control and jasplakinolide-treated cells (centre plots; •, controls (non-treated); ○, jasplakinolide treated). Increasing the exposure to jasplakinolide to 70 min still did not prevent I_CRAC from activating (histogram in right-hand panel). C, disrupting microtubules by an overnight incubation with 5 μm nocodazole also did not affect I_CRAC activation. The left panel shows a recording following an 18 h exposure to the drug and the I-V relationship (taken at 82 s) is shown. The activation time constant, time to peak and extent of the current were similar between control and nocodazole-treated cells (right-hand graphs; •, control; ○, nocodazole treated).

Figure 4. Store-operated calcium influx is unaffected by interfering with the cytoskeleton

A, a control response. Stores were depleted by applying thapsigargin (Thap, 2 μm) together with ionomycin (Iono, 100 nm) in calcium-free external solution (0.2 mm EGTA) and then 10 mm calcium was applied to the cell. B, the cell was pre-treated with 1 μm jasplakinolide for 45 min and the above protocol repeated. Jasplakinolide was present in all solutions. C-E, pooled data from many cells for the different treatments carried out. C, summary of the amount of Ca²⁺ released from the stores (measured indirectly as the peak of the Ca²⁺ release event). D, the mean amplitude of the Ca²⁺ signal obtained when external Ca²⁺ was re-admitted into the cells. E, the time taken for this Ca²⁺ signal to peak (time to peak), shown for the different treatments. Ca²⁺ influx in cytochalasin D-treated cells tended to develop more slowly than in control, although this was not significant (P = 0.09). In the presence of Li⁺, Ca²⁺ influx tended to be smaller than in control but this again was not quite significant (P = 0.08). For all other conditions, P > 0.15.

Figure 5. Separation of plasma membrane from underlying structures using balloon patching does not affect the activation nor does it induce reversal of activation of I_CRAC

The upper panel shows the development of the ballooned cell and the activation of I_CRAC is shown below. The upper left image shows the cell a few seconds following break-in with a pipette solution containing buffered Ca²⁺ and ATP. The cell was then inflated by applying 3 kPa pressure via a manometer connected to the pipette and the upper middle image shows the cell in the process of ballooning. After around 10 s, the cell reached its maximum inflation (upper right panel) and maintained this shape for the duration of the recording. Note that the intracellular components seem to aggregate at the cell centre and there is a transparent space below the plasma membrane. Following stabilisation, thapsigargin was applied and I_CRAC could still be activated to the same extent and with a similar time course to control (see Fig. 2C). The inset shows I-V relationships 30 s after inflation (i) and then once I_CRAC had peaked (ii). The background current upon break-in has been subtracted. Note that inflation per se did not change membrane conductance. The lower right panel shows the effect of inflation when applied after I_CRAC had been activated (InsP₃+ 10 mm EGTA + thapsigargin). There was a small, reversible further increase in the current, but this was not always seen. Clearly, cell ballooning did not affect I_CRAC significantly in spite of the large structural changes in the vicinity of the plasma membrane.

Figure 6. The InsP₃ receptor antagonist 2-APB appears to block CRAC channels from an external site

A, 2-APB blocks I_CRAC activity when added to the bath but not when included in the recording pipette. Cells were dialysed with 30 μm InsP₃+ 10 mm EGTA + 2 μm thapsigargin. Filled circles show control recordings, taken in the absence of 2-APB. Following incubation of the cells with 20 μm 2-APB for > 15 min, I_CRAC could not be recorded. However, when cells were dialysed with InsP₃+ 10 mm EGTA + thapsigargin and 50 μm 2-APB (i.e. 2-APB was not added to the bath), I_CRAC could be activated. The histogram on the right summarises data from several cells. I_CRAC was reduced by 27 % when 2-APB was included in the pipette and this was significant (P = 0.018). In B, stores were depleted passively by dialysis with 10 mm EGTA alone. Compared with control cells (•), dialysis with 50 μm 2-APB (○) reduced the extent of activation of I_CRAC only slightly (histogram on the right, P > 0.07). In these experiments, around 70 s elapsed following break-in before the current started to develop slowly. The cytosol would therefore have been exposed to appreciable levels of 2-APB prior to the activation of I_CRAC (see text). As with the InsP₃ experiments in A, addition of 2-APB to the bath prevented I_CRAC from being recorded using passive depletion (histogram in B). C, external application of 2-APB rapidly reduces I_CRAC when applied after the current has reached steady state. The left panel shows a typical recording using this protocol and pooled data are summarised in the histogram on the right. The inset of C compares the kinetics of loss of I_CRAC in response to 10 μm La³⁺ (a rapid Ca²⁺ channel blocker) and 20 μm 2-APB using our application system. La³⁺ (10 μm) fully inhibited I_CRAC (3/3 cells) and did this at a rate only slightly faster (around 2-fold) than that seen with 20 μm 2-APB, a concentration that did not fully inhibit the current. In addition to its reported action on the InsP₃ receptor, these results suggest that 2-APB may block CRAC channels directly.

Figure 7. InsP₃ bound to the InsP₃ receptor is not necessary for the activation and maintenance of I_CRAC: studies with the InsP₃ receptor antagonist heparin

A, cells were dialysed with InsP₃, 225 nm Ca²⁺ (buffered with 10 mm EGTA), 2 mm Mg-ATP and 1 mg ml⁻¹ heparin. Following activation of I_CRAC, the current deactivated fully within a further 250 s. This occurred because heparin diffused into the cell relatively slowly but then displaced InsP₃ from the InsP₃ receptors. In the absence of continuous Ca²⁺ release from the stores, SERCA pumps were able to refill the stores in the presence of buffered Ca²⁺ and ATP, thereby deactivating the current. Consistent with this were the findings that the current did not decline much in the presence of heparin when Ca²⁺ and ATP were omitted from the pipette (B), that the decline was only partial when heparin was omitted from the pipette (C) and that the decline was only partial when the SERCA pump blocker thapsigargin (2 μm) was added to the pipette solution (D). The partial loss of I_CRAC reflects Ca²⁺- and phosphorylation-mediated inactivation (see text for references). In E, I_CRAC could be subsequently reactivated by applying the Ca²⁺ ionophore ionomycin to deplete the stores, following full deactivation of the current in the presence of heparin. Because InsP₃ has been displaced from its receptors, this indicates that the current can be activated even though InsP₃ is not bound to the InsP₃ receptor.

Figure 8. Neither Li⁺ nor caffeine interferes with the rate or extent of activation of I_CRAC

A, a control recording from a cell dialysed with a pipette solution containing 10 mm EGTA, to activate I_CRAC passively. The upper panel shows the time course of the current and the lower panel the I-V relationship, taken as the current peaked. The dashed lines in the upper panel represent linear fits to the slow and fast components of development of the current (Fierro & Parekh, 1999). B, a typical recording from a cell that was pre-incubated in 15 mm Li⁺-containing external solution for 105 min and then dialysed with a pipette solution containing 15 mm Li⁺. I_CRAC was evoked passively. C, a representative recording from a cell that was exposed to 10 mm caffeine for 22 min and then dialysed with 10 mm caffeine. I_CRAC was evoked passively. D, summary of the size of the currents for the different conditions (6 control cells, 7 for Li⁺ and 7 for caffeine). E, the delay versus time to peak is plotted for the various conditions. F, the rate of development of I_CRAC for the slow and fast components is plotted against the relative fractional amplitude of I_CRAC. There were no significant differences between any of the parameters shown in D-F for control, Li⁺- or caffeine-treated cells.