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Depletion of intracellular calcium stores activates a calcium conducting nonselective cation current in mouse pancreatic acinar cells.

Receptor-mediated Ca2+ release from inositol (1,4,5)-trisphosphate (IP3)-sensitive Ca2+ stores causes “capacitative calcium entry” in many cell types (Putney, J. W., Jr. (1986) Cell Calcium 7, 1-12; Putney, J. W., Jr. (1990) Cell Calcium 11, 611-624). We used patch-clamp and fluorescence techniques in isolated mouse pancreatic acinar cells to identify ion currents and cytosolic calcium concentrations under conditions in which intracellular Ca2+ stores were emptied. We found that depletion of Ca2+ stores activated a calcium-release-activated nonselective cation current (ICRANC) which did not discriminate between monovalent cations. ICRANC possessed a significant conductance for Ca2+ and Ba2+. It was not inhibited by La3+, Gd3+, Co2+, or Cd2+ but was completely abolished by flufenamic acid or genistein. In whole cell and cell-attached recordings, a 40-45 pS nonselective cation channel was identified which was activated by Ca2+ store depletion. Calcium entry as detected by single cell fluorescence measurements with fluo-3 or fura-2, showed the same pharmacological properties as ICRANC. We conclude that in mouse pancreatic acinar cells 40-45 pS nonselective cation channels serve as a pathway for capacitative Ca2+ entry. This entry pathway differs from the previously described ICRAC (Hoth, M., and Penner, R. (1992) Nature 355, 353-356) in its ion-selectivity, pharmacological profile, and single-channel conductance.

In several nonexcitable cell types activation of cell membrane receptors by hormones or neurotransmitters results in a biphasic calcium signal. An initial Ca 2ϩ peak produced by calcium release from intracellular inositol (1,4,5)-trisphosphate (IP 3 ) 1 -sensitive Ca 2ϩ stores is followed by a sustained Ca 2ϩ plateau due to Ca 2ϩ entry from the extracellular space (1)(2)(3)(4)(5). The hypothesis that it is the decrease in the Ca 2ϩ concentration in the internal store which causes Ca 2ϩ influx into the cell was first proposed in 1986 by Putney and has been termed "capacitative calcium influx" (1,2). Accordingly, not only hormonal stimulation, but also Ca 2ϩ pool depletion following treatment with inhibitors of the Ca 2ϩ -ATPase such as di-tert-butyl-hydroquinone (t-BHQ) or thapsigargin (6) or with Ca 2ϩ ionophores (7) leads to activation of Ca 2ϩ entry.
In mast cells (7), RBL cells (8), Xenopus oocytes (9), and Jurkat T-cells (10), capacitative calcium influx is mediated by ion channels (I CRAC ) which have a high selectivity for calcium and is inhibited by La 3ϩ . At present it is unclear, however, if Ca 2ϩ influx through I CRAC is a common mechanism or if there exist different Ca 2ϩ influx channels in different nonexcitable cell types. In pancreatic acinar cells capacitative calcium influx has been described by means of fluorescence measurements (11,12) and we have recently shown that genistein blocks Ca 2ϩ entry in mouse pancreatic acinar cells (13).
We describe here that calcium store depletion by the agonist acetylcholine (ACh), IP 3 , the Ca 2ϩ -ATPase inhibitor t-BHQ, or the Ca 2ϩ ionophore ionomycin activates a calcium conducting nonselective cation channel which can also be blocked by genistein but not by La 3ϩ . Capacitative Ca 2ϩ entry as measured by fluorescence methods was also completely inhibited by genistein, whereas La 3ϩ had no effect on Ca 2ϩ influx in mouse but completely inhibited Ca 2ϩ influx in rat pancreatic acini. This indicates the presence of different Ca 2ϩ influx pathways in different animal species. We conclude from our data that in mouse pancreatic acinar cells the "calcium release-activated nonselective cation current" (I CRANC ) is responsible for capacitative calcium entry.

EXPERIMENTAL PROCEDURES
Cell Preparation-Mouse and rat pancreatic acinar cells were prepared from male CD-1 mice and male Wistar rats, respectively, as described previously (14). Acinar cells from male Wistar rats were prepared in the same way (14).
Electrophysiology-Patch-clamp experiments were performed in the tight-seal, whole cell, and cell-attached configuration (15) at room temperature (24 Ϯ 2°C) in a standard bath solution containing in mM: 140 NaCl, 4.7 KCl, 1.3 CaCl 2 , 1 MgCl 2 , 10 HEPES, 10 glucose, pH 7.4. Patch pipettes were manufactured from borosilicate glass capillaries and had resistances of 2 to 4 M⍀ when filled with a standard buffer containing in mM: 125 K ϩ -Asp, 15.5 NaCl, 1 MgCl 2 , 10 HEPES, 10 EGTA, 30 KOH, 2.5 mM Mg-ATP, 70 nM free Ca 2ϩ , pH 7.2. In some experiments BAPTA (10 mM) was used instead of EGTA, which did not influence the results. Patch-clamp experiments were recorded with a computer controlled EPC9 patch-clamp amplifier (HEKA; Lambrecht, Germany). Cell capacitance and series resistance were calculated with the softwaresupported internal routines of the EPC9 and compensated before each experiment. Data were sampled at 1 kH on the computer hard disk after low pass filtering at 400 Hz. In whole cell experiments voltage ramps were applied every 2 s to the cells (Ϫ140 to 100 mV, slope 1 V/s) near the reversal. Either the resulting I/V curve is shown or current values at Ϫ60 mV (reversal potential of Cl Ϫ currents) were extracted from every ramp and presented as time courses. Single-channel measurements were done in the whole cell mode with the same pipette solution as described above. In the cell-attached configuration pipettes were filled with the standard bath solution. Single-channel data were collected on a video tape by use of a modified pulse code modulator (Sony, PCM 501), * This work was supported by grants from the Deutsche Forschungsgesellschaft (Sonderforschungsbereich 246/A9 and B11) and from the Bundesministerium fü r Bildung, Wissenschaft, Forschung und Technologie (BOE/21/10246A). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The permeability ratios of I CRANC were calculated as, (E rev ϭ reversal potential (V), T ϭ temperature (K), P ca ϭ relative permeability for calcium, P cat ϭ relative permeability for cations, [Ca] o ϭ calcium concentration outside the cell, [cat] i ϭ concentration of monovalent cations inside the cell) E rev was measured, P Ca was set to 1, and P cat was calculated.
Fluorescence Techniques-Isolated single cells and acini were loaded with 3-4 M fluo-3/AM or fura-2/AM for 30 min at room temperature. After dye loading the cell suspension was stored at 4°C and used for experiments within 5 h. Measurements with fluo-3 were done with a confocal laser scanning microscope (Zeiss Axiovert 35 microscope equipped with the confocal laser scanning and imaging system Bio-Rad MRC-600) on single cells from multiple cell clusters (up to 5 cells) as described in greater detail elsewhere (13). The internal [Ca 2ϩ ] is given as the mean fluorescence of fluo-3.
Fura-2 ratiometric measurements were made with an imaging system (Zeiss Axiovert 135 equipped with an apparatus from T.I.L.L. Photonics, Munich) on single cells from multiple cell clusters. Cells were excited alternately at 345 and 380 nm wavelength and the emission was collected for 50 -200 ms above 510 nm wavelength. The results are given as the ratio of the fluorescence intensities at the different wavelengths (345/380).

Depletion of Intracellular Ca 2ϩ Pools Activates a Nonselective
Cation Current-In order to activate capacitative Ca 2ϩ entry we depleted intracellular Ca 2ϩ stores of isolated mouse pancreatic acinar cells by extracellular addition of Ca 2ϩ -ATPase inhibitor t-BHQ (1 M), ACh (1 M), ionomycin (10 M) or by intracellular addition of IP 3 (10 M). Cells were dialyzed in the whole cell mode with the Ca 2ϩ chelators EGTA (10 mM) or BAPTA (10 mM) through the patch pipette (free Ca 2ϩ clamped to 70 nM) to prevent increases in free [Ca 2ϩ ] leading to activation of previously described Ca 2ϩ -dependent Cl Ϫ and nonselective cation currents (16).
Under activating conditions, currents began to develop, but with different time courses depending on the test substances used for store depletion ( Fig. 1, a and b). A Ca 2ϩ -free pipette solution induced a slow response (t 90 ϭ 484 Ϯ 137 s), most likely by depletion of stores through Ca 2ϩ leaks. ACh, t-BHQ, or intracellular application of IP 3 accelerated the current development significantly (t 90 ϭ 240 Ϯ 90 s, mean of the pooled data from all three conditions) and ionomycin induced the fastest response (t 90 ϭ 90 Ϯ 17 s). To obtain current-voltage curves (I/V curves) we applied voltage ramps (Ϫ140 to 100 mV, 1 V/s). The linearity of the I/V curve (Fig. 1c) indicates voltage independence. The maximal conductance of the current was variable from cell to cell. The mean was 253 Ϯ 211 pS/pF (n ϭ 17) in the case of t-BHQ activation. The conductance did not differ significantly when currents were activated by ACh (220 Ϯ 99 pS/pF, n ϭ 5), IP 3 (228 Ϯ 111 pS/pF, n ϭ 6), or ionomycin (195 Ϯ 109 pS/pF, n ϭ 13).
Ion Selectivity of I CRANC -Ion exchange experiments revealed a permeability sequence for the current of Rb ϩ Ͼ K ϩ ϭ Na ϩ Ͼ Ͼ N-methyl-D-glucamine (NMDG ϩ ) as determined by changes in the reversal potentials (Fig. 1d, n ϭ 5). A Cl Ϫ component could be excluded since no changes in current were observed at the reversal potential of monovalent cations (0 mV, Fig. 1c). Furthermore, substitution of bath NMDG-Cl with NMDG-aspartate had no effect on outward currents (Fig. 1e, FIG. 1. Activation of a nonselective cation current by depletion of intracellular Ca 2؉ pools. a, time course of activation of nonselective cation currents (I CRANC ) in response to Ca 2ϩ store depletion. Control: standard pipette solution contained 2.5 mM ATP and 70 nM free Ca 2ϩ to maintain filling of the Ca 2ϩ pool. No current was activated (n ϭ 5) under these conditions. Ca 2ϩ free, 0 ATP: no Ca 2ϩ and no ATP were added to the pipette solution (n ϭ 5). IP 3 : 10 M IP 3 was added to the standard pipette solution (n ϭ 6). ACh: ACh (1 M) was added to the bath solution (n ϭ 5). t-BHQ: t-BHQ (1 M) was added to the bath solution (n ϭ 9). Ionomycin: ionomycin (10 M) was added to the cells from the bath side by a wide-tipped glass capillary (n ϭ 13). t 0 (arrow) indicates the time of addition of test substances to the bath medium (t-BHQ, ACh, and ionomycin). In the case of IP 3 activation t 0 is the beginning of the whole cell configuration. Current traces were generated by applying voltage ramps (Ϫ140 to 100 mV, 1V/s) every 2 s. The actual current values at V hold ϭ Ϫ60 mV (reversal potential of Cl Ϫ ions) were extracted and plotted against the time. The data shown are representative of a total given in b. b, columns present the latencies between addition of test substances and 90% activation of the depletionactivated current (t 90 ). Averaged data are presented as the mean Ϯ S.D. Student's t test was used to determine significant differences between the t 90 values of currents activated with different substances. The latencies of t-BHQ, ACh, IP 3 (p Ͻ 0.005), and ionomycin (p Ͻ 0.001) were significantly shorter in comparison to the 0 Ca 2ϩ /0 ATP condition. While the effects for t-BHQ, ACh, and IP 3 did not differ significantly from each other (p Ͼ 0.05) the ionomycin effect was significantly faster (p Ͻ 0.005). n gives the number of independent experiments with a single cell each. c, representative I/V curves taken before and following activation of I CRANC with t-BHQ (1 M). d, permeabilities for different monovalent cations. Currents were activated by t-BHQ (1 M). The standard bath solution was exchanged for solutions with equimolar concentrations of NMDG ϩ , Rb ϩ , or K ϩ instead of Na ϩ as indicated. The order of the reversal potentials were taken as indicators for the relative permeabilities (one representative out of five similar experiment is shown; for clarity the I/V curve for KCl is not shown, since it is identical to the NaCl curve). The determined permeability sequence is RbϾNa ϭ KϾNMDG. e, permeability for Cl Ϫ and aspartate Ϫ . Currents were activated by t-BHQ (1 M). First Na ϩ was exchanged for NMDG ϩ thereafter in the same experiment Cl Ϫ was exchanged for aspartate (Asp) as indicated (one representative out of six similar experiments is shown). n ϭ 4). The whole cell current was also able to carry Ca 2ϩ and Ba 2ϩ ions (Fig. 2a). Referring to the more negative reversal potentials measured after Na ϩ substitution by Ca 2ϩ (⌬V rev(Na/ Ca) ϭ Ϫ23 Ϯ 6.5 mV, n ϭ 7), an apparent permeability ratio for the cations inside versus Ca 2ϩ outside the cell of 13:1 was calculated. Because I CRANC does not discriminate between Na ϩ and K ϩ , this permeability ratio equals the ratio for both Na ϩ : Ca 2ϩ and K ϩ :Ca 2ϩ .
Inhibitors of I CRANC and Ca 2ϩ Influx-To test if the described current is responsible for capacitative calcium influx in pancreatic acinar cells we compared pharmacological properties of I CRANC and Ca 2ϩ influx measured as fluo-3 fluores-cence with a confocal microscope. In fluorescence experiments the plateau phase of the calcium signal is exclusively produced by extracellular calcium entering the cell (13,17). Both I CRANC and calcium influx were inhibited by genistein (Figs. 2, b and c, and 3, a and c). Therefore it seemed likely that Ca 2ϩ influx occurred through the same genistein-inhibitable calcium pathway. Other tyrosine kinase inhibitors such as tyrphostin 25 (100 M, n ϭ 4, data not shown) showed 50% inhibition of both I CRANC and Ca 2ϩ -influx measured with fluorescence while lavendustin A and tyrphostin B56 (n ϭ 5, 100 M each) had no effect. The dihydroxy analog of genistein (daidzein, 50 M, n ϭ 10, data not shown) which lacks the ability to regulate tyrosin kinase was also ineffective on capacitative Ca 2ϩ influx. The assumption that Ca 2ϩ influx as measured with fluo-3 occurred through I CRANC was further substantiated by the finding that flufenamic acid (100 M) (Fig. 3b) , n ϭ 3), and Mn 2ϩ (1 mM, n ϭ 3) (data not shown) had no effect on both Ca 2ϩ influx and current. In particular the lack of effect of La 3ϩ on I CRANC measured in the presence of sodium ( fig. 3c, left) or in the presence of calcium (Fig. 2c), and on the capacitative calcium entry measured with fluorescence techniques (Figs. 3c and 4a) is remarkable because in other systems La 3ϩ is known as an inhibitor of capacitative Ca 2ϩ influx and in higher concentration also of Ca 2ϩ efflux due to Ca 2ϩ pump inhibition (18). We therefore tested the effect of La 3ϩ in more detail. We took into consideration that the maintenance of a Ca 2ϩ plateau, which is usually interpreted to show Ca 2ϩ influx, should also occur if both Ca 2ϩ influx and Ca 2ϩ extrusion was inhibited by La 3ϩ . In this case the effect of La 3ϩ could not be taken as indication for Ca 2ϩ influx following Ca 2ϩ release. We therefore tested a wide range of La 3ϩ concentrations (100 nM, 1 M, 10 M, 100 M, and 250 M) to inhibit agonist-stimulated Ca 2ϩ entry without Ca 2ϩ extrusion at low concentrations and to inhibit also Ca 2ϩ extrusion at higher concentrations (5,17,18). In no case did La 3ϩ reduce fluo-3 fluorescence whereas subsequent application of genistein in the presence of La 3ϩ ([La 3ϩ ], 100 or 250 M) always abolished the calcium plateau (Fig. 3c). It therefore appears to be unlikely that La 3ϩ inhibited Ca 2ϩ entry in this concentration range. To further test the effect of La 3ϩ on Ca 2ϩ influx we performed the experiments shown in Fig. 4a, indicating that following hormonal depletion of Ca 2ϩ stores in the absence of Ca 2ϩ , readdition of Ca 2ϩ caused Ca 2ϩ influx which was not inhibited by La 3ϩ .
Comparison of Ca 2ϩ Influx in Mouse and Rat Pancreatic Acinar Cells-Since La 3ϩ had been described to inhibit capacitative Ca 2ϩ influx in rat pancreatic acinar cells using fura-2 (17), we repeated the experiments with La 3ϩ in rat to compare the results with mouse pancreatic acinar cells. In mouse pancreatic acinar cells La 3ϩ had no effect on calcium entry as measured with fluo-3 (see Fig. 4a, left) or fura-2 (see Fig. 4a, right). However, in rat pancreatic acinar cells capacitative Ca 2ϩ entry was inhibited by 100 M La 3ϩ (Fig. 4b). Our results confirm experiments described previously by Tsunoda et al. (17) on rat pancreatic acinar cells and indicate different capac-itative Ca 2ϩ influx pathways in mouse and rat. In contrast, genistein (50 M) completely inhibited capacitative Ca 2ϩ influx in both mouse (see Fig. 3, a, right, and c, right) and rat as measured by fluorescence (data not shown).
Resolution of Single Channel Events in I CRANC -In some experiments (n ϭ 6 out of n ϭ 32 and 7 out of 52 cells) in which store depletion resulted in the activation of only a small current it was possible to recognize single channel events in the whole cell mode of the patch-clamp technique. The I/V curves of these channels were linear and reversed at 0 mV (Fig. 5a). The conductance in the whole cell mode measured with standard solutions was in the range of 40 -45 pS (43 Ϯ 3.3 pS, n ϭ 7). In . A smaller nonselective cation channel (26 Ϯ 3.2 pS, n ϭ 4, right I/V curve) marked in the trace by filled circles was seen in around 95% of experiments. In 10% of experiments a bigger channel (41 Ϯ 3.3 pS, n ϭ 4, left I/V curve) marked by triangles in the trace was recognized in addition to the smaller one. The currents shown were measured at V hold ϭ Ϫ40 mV. c, similar results were obtained by depleting intracellular calcium pools with ACh (500 nM, n ϭ 3). The ACh activation of single channels was reversible (back control). All single channel traces were taken from representative experiments. In all traces "C3" denotes the closed-channel current level. the cell-attached mode the 40 -45 pS channel could be irreversibly activated with t-BHQ (Fig. 5b) or reversibly with ACh (Fig.  5c). Under these conditions the channel always coexisted with the previously described 27 pS channel (Fig. 5b) which is Ca 2ϩactivated and therefore is observed under conditions at which cytoplasmic Ca 2ϩ rises (19). Because the 27 pS channel has a high density (about 500 channels/cell (20)) compared to the 45 pS channel (about 70 channels/cell, calculated as the mean single cell conductance/single channel conductance assuming a P O of 0.6 for the single channel) it is not surprising that the 40 -45 pS channel was seen in the cell-attached mode in only Ϸ10% of the cells. The possibility that the two conductance levels which we found in cell-attached experiments are sublevels of one channel type can be ruled out by the finding that in all whole cell experiments only a single conductance level of 40 -45 pS was observed. Excising patches with the 40 -45 pS channel into a standard bath solution resulted in an immediate run-down of this channel type leaving only the 27 pS channel active (n ϭ 4).

DISCUSSION
The results presented here indicate that capacitative Ca 2ϩ entry in mouse pancreatic acinar cells is produced by a calcium release-activated Ca 2ϩ conducting nonselective cation channel (I CRANC ). The main characteristic of I CRANC is its insensitivity to La 3ϩ which argues against the presence of the previously described I CRAC (7) in mouse pancreatic acinar cells. Flufenamic acid, an inhibitor of nonselective cation channels (21), and genistein, a tyrosine kinase inhibitor, also inhibited both I CRANC and capacitative Ca 2ϩ entry. The genistein effect should be emphasized because it was shown before (13) that it inhibits calcium entry without affecting calcium release from IP 3 -sensitive pools. We believe that these pharmacological similarities of I CRANC and capacitative calcium entry are consistent with the possibility that I CRANC is responsible for capacitative Ca 2ϩ influx in mouse pancreatic acinar cells. The mechanism for the effect of genistein on calcium influx is not yet clear because genistein effects are diverse (22). The lack of effect of other tyrosine kinase inhibitors like lavendustin A or tyrphostin B56 on mouse pancreatic acinar cells led us to conclude that a more direct interaction between channel and genistein rather than the proposed inhibition of a tyrosine kinase (23,24) is responsible for inhibition.
Comparison of I CRANC to Other Capacitative Ca 2ϩ Influx Currents-The nonselective cation current, which is activated by Ca 2ϩ store depletion (I CRANC ) in mouse pancreatic acinar cells, differs from capacitative Ca 2ϩ influx described in other systems. In mast cells and other cell types I CRAC seems to be the dominant influx pathway for Ca 2ϩ (7)(8)(9)(10). It is highly Ca 2ϩ selective and can be inhibited over 90% with low concentrations of La 3ϩ (10 M) and in part by Cd 2ϩ and Co 2ϩ (25). Single channels producing I CRAC could not be identified so far and noise analysis of I CRAC made it likely that the single channel conductance is much below the resolution threshold of the patch-clamp technique (25). Comparing the characteristics of I CRANC as described here with those of I CRAC it appears that both currents are different although they have the same activation mechanism.
Single channels activated by Ca 2ϩ store depletion were identified in vascular endothelium (26). These channels were relatively Ca 2ϩ selective (permeability ratio Ca/Na ϭ 10/1) and had a conductance of 11 pS (with 10 mM Ca 2ϩ ). Also in A431 cells single channels activated by store depletion were found which had a relative low conductance in the presence of high Ca 2ϩ or Ba 2ϩ concentrations (200 mM Ca 2ϩ , conductance 2 pS or 160 mM Ba 2ϩ , conductance 16 pS, respectively) (27). These channels clearly differ from the I CRANC channel in mouse described here.
Nonselective cation channels have been discussed as candidates for mediating Ca 2ϩ influx in nonexcitable cells (28). While it had been described for several systems that agonist activated nonselective cation channels allow calcium influx into cells (29 -31) our study demonstrates that depletion of Ca 2ϩ stores can directly activate those channels.
Comparison of I CRANC and I CRAC -Whereas I CRAC is inhibited by several di-and trivalent ions such as Co 2ϩ , Cd 2ϩ , and most effectively by La 3ϩ (25), these ions are ineffective in mouse pancreatic acinar cells. In particular the lack of effect of La 3ϩ on I CRANC measured as electrical current and as calcium fluorescence with both fluo-3 and fura-2 should be emphasized. La 3ϩ at low concentrations (micromolar) has been reported to inhibit capacitative calcium entry, while at higher concentrations (millimolar) it inhibited the calcium extrusion mechanism in lacrimal acinar cells (18). Moreover it was shown in rat pancreatic acinar cells (17) and guinea pig pancreatic acinar cells (5) that La 3ϩ (25-250 M) inhibited capacitative calcium entry. In mouse pancreatic acinar cell 1 mM La 3ϩ inhibited Ca 2ϩ extrusion and the authors assumed that Ca 2ϩ influx was inhibited, too (32). Direct evidence for this assumption was not given, however. In contrast to these results we did not find any inhibition of Ca 2ϩ entry by La 3ϩ up to 250 M in mouse pancreatic acinar cells with different experimental protocols including different depletion methods (ACh and t-BHQ) and different detection methods (measurements of electrical current and of fluorescence with fluo-3 or fura-2). The reason for the discrepancy between the data in the literature and our data are most likely due to species differences which can also be concluded from our own results which compare the effects of 100 M La 3ϩ in rat and mouse (Fig. 4). These differences in the La 3ϩ sensitivity between rat and mouse make it likely that rat but not mouse pancreatic acinar cells use I CRAC for capacitative Ca 2ϩ influx as it had been assumed already by Bahnson et al. (33).
If in addition to I CRANC mouse pancreatic acinar cells would also contain I CRAC we would have expected at least, in part, inhibition of capacitative Ca 2ϩ influx by La 3ϩ . It therefore appears that in mouse pancreatic acinar cells I CRAC is not present or in such small amounts that it is undetectable by our methods. In conclusion our data indicate that a Ca 2ϩ conducting nonselective cation channel is activated following depletion of intracellular IP 3 -sensitive Ca 2ϩ stores in mouse pancreatic acinar cells.
The characteristics of I CRANC are different from I CRAC previously described in mast cells and other cell types (34) in that the latter is highly Ca 2ϩ selective, completely inhibited by La 3ϩ , and in part by Cd 2ϩ and Co 2ϩ . Evidence suggests that mammalian Ca 2ϩ influx channels are homologues of insect trp and trpl channels (35)(36)(37). Whereas trp is selective for Ca 2ϩ , has a high La 3ϩ sensitivity, and is activated by Ca 2ϩ store depletion (and therefore seems to have similarities with I CRAC , 38), trpl is a nonselective cation channel conducting also Ca 2ϩ and Ba 2ϩ ions. It has a lower La 3ϩ sensitivity compared to trp but does not seem to be activated by Ca 2ϩ store depletion (35,38,39). Whether I CRANC in pancreatic acinar cells shares genetic homologies with these insect channels remains to be determined in future studies.