CaT1 and the Calcium Release-activated Calcium Channel Manifest Distinct Pore Properties*

The calcium release-activated calcium channel (CRAC) is a highly Ca2+-selective ion channel that is activated on depletion of inositol triphosphate (IP3)-sensitive intracellular Ca2+ stores. It was recently reported that CaT1, a member of the TRP family of cation channels, exhibits the unique biophysical properties of CRAC, which led to the conclusion that CaT1 comprises all or part of the CRAC pore (Yue, L., Peng, J. B., Hediger, M. A., and Clapham, D. E. (2001) Nature 410, 705–709). Here, we directly compare endogenous CRAC with heterologously expressed CaT1 and show that they manifest several clearly distinct properties. CaT1 can be distinguished from CRAC in the following features: sensitivity to store-depleting agents; inward rectification in the absence of divalent cations; relative permeability to Na+ and Cs+; effect of 2-aminoethoxydiphenyl borate (2-APB). Moreover, CaT1 displays a mode of voltage-dependent gating that is fully absent in CRAC and originates from the voltage-dependent binding/unbinding of Mg2+ inside the channel pore. Our results imply that the pores of CaT1 and CRAC are not identical and indicate that CaT1 is a Mg2+-gated channel not directly related to CRAC.

In non-excitable cells, calcium transients upon receptor stimulation generally consist of two phases: a rapid rise due to Ca 2ϩ release from IP 3 -sensitive 1 intracellular Ca 2ϩ stores, which is followed by sustained Ca 2ϩ entry through store-operated plasma membrane channels (1). Of all store-operated channels, CRAC is functionally the best described, but its molecular identity and the mechanism of its store-dependent activation remain elusive (2). Several members of the TRP family, a group of cation channels related to the Drosophila transient receptor potential (trp) gene product (3,4), have been implicated in store-dependent Ca 2ϩ influx (5)(6)(7)(8)(9), but until recently none of them was found to exhibit the unique biophysical properties of CRAC (3).
In a recent study, Yue et al. (10) reported that CaT1 (11), a TRP family member homologous to Caenorhabditis elegans OSM-9, manifests the pore properties and store dependence of CRAC, and it has been proposed that CaT1 comprises all or part of the CRAC pore (10,12,13). To further investigate this intriguing possibility, we made a direct comparison of CaT1, heterologously expressed in human embryonic kidney (HEK-293) cells, and endogenous CRAC in rat basophilic leukemia (RBL) cells. RBL cells were chosen because they were the source of the CaT1 clone used by Yue et al. (10) and have been widely used as a model system to study CRAC (2). We confirm the resemblance of currents through CRAC and CaT1 but additionally present a number of novel features that are incompatible with the proposed equality of the CaT1 and CRAC pores.

EXPERIMENTAL PROCEDURES
Cell Culture and Transfection-HEK-293 cells and RBL-2H3 cells were grown in Dulbecco's modified Eagle's medium containing 10% (v/v) human serum, 2 mM L-glutamine, 2 units/ml penicillin, and 2 mg/ml streptomycin at 37°C in a humidity-controlled incubator with 10% CO 2 . HEK-293 cells were transiently transfected with the pCINeo/IRES-GFP/mCaT1 vector using previously described methods (14), and electrophysiological recordings were performed between 8 and 24 h after transfection.
Unless indicated differently, currents were measured in response to voltage ramps (Ϫ100 to ϩ100 mV in 100 ms) applied at a frequency of 0.5 Hz from a holding potential of 0 mV. Series resistances were between 3 and 10 megaohms and were compensated for 50 -80%. Voltage errors were generally Ͻ10 mV. Currents were sampled at 10 kHz and filtered at 2 kHz using an eight-pole Bessel filter. For the accurate determination of the currents through CRAC, background currents measured under identical ionic conditions but before store depletion were subtracted. Averaged data are expressed as the mean Ϯ S.E. from n cells. The significance of differences between means was evaluated using Student's unpaired t test.

RESULTS
Immediately after obtaining the whole-cell configuration, CaT1-transfected HEK-293 cells exposed to 10 mM extracellular Ca 2ϩ and with intracellular Ca 2ϩ buffered at 100 nM (to maintain full Ca 2ϩ stores) displayed a large membrane current (Fig. 1, A and B). This current subsequently decayed to reach a lower, but still robust, steady state level (Fig. 1A). The CaT1mediated current was strongly inwardly rectifying ( Fig. 1B) and reversed at positive potentials (39 Ϯ 7 mV; n ϭ 18) in agreement with previous work (10). Depletion of intracellular Ca 2ϩ stores with the Ca 2ϩ ionophore ionomycin (2 M) did not induce any store-dependent activation of CaT1 but instead caused a transient inhibition of the CaT1-current by 26 Ϯ 4% (n ϭ 8) (Fig. 1A). A similar inhibition by 30 Ϯ 6% (n ϭ 7) was observed when Ca 2ϩ stores were passively depleted by application of the sarcoendoplasmic reticulum Ca 2ϩ -ATPase inhibitor, 2,5-di-t-butyl-1,4-benzohydroquinone (20 M). We attribute the inhibitory effect of store-depleting agents to an increase of intracellular Ca 2ϩ as has been previously described for epithelial calcium channel (ECaC1; see Refs. 15 and 16), a close homologue of CaT1. Under identical intra-and extracellular ionic conditions, RBL-2H3 cells displayed only a very small background current that reversed at negative potentials (Ϫ41 Ϯ 2 mV; n ϭ 26). Depletion of Ca 2ϩ stores by extracellular application of 2 M ionomycin ( Fig. 1A) or intracellular perfusion with IP 3 (20 M; data not shown) activated CRAC, as indicated by the appearance of a Ca 2ϩ -selective, inwardly rectifying current that reversed at positive potentials (42 Ϯ 7 mV; n ϭ 26) (Fig. 1B).
The permeability of both CaT1 and CRAC in the presence of extracellular Ca 2ϩ is at least 100 times higher for Ca 2ϩ than for Na ϩ , but removal of all divalent cations from the extracellular medium renders both channel pores highly permeable for monovalent cations (2,10). This loss of selectivity is presumably because of the removal of divalents from negatively charged residues (aspartates or glutamates) lining the Ca 2ϩselective pore (17), as has been previously shown for voltagegated Ca 2ϩ channels and for ECaC1 (18,19). When switching from the normal extracellular solution containing 10 mM Ca 2ϩ to a DVF solution, the CaT1-mediated current gradually increased following an exponential time constant of 9 Ϯ 4 s (n ϭ 14) (Fig. 1C). We interpret this time-dependent increase of the current as the slow removal of the Ca 2ϩ -induced inactivation of CaT1, as has been previously demonstrated for its close homologue, ECaC1 (15,16). After reaching its maximal amplitude, the monovalent CaT1 current remained stable for more than 15 min ( Fig. 1C and data not shown). In contrast, the current through CRAC, when preactivated by ionomycin in the normal extracellular solution containing 10 mM Ca 2ϩ , reached its maximal amplitude immediately after switching to DVF solution and decayed subsequently with a time constant of 12 Ϯ 4 s (n ϭ 17) (Fig. 1C).
Current-voltage relationships obtained during linear voltage ramps in DVF solutions revealed several significant differences between CaT1 and CRAC (Fig. 1D). First, the reversal potential for CaT1 (18 Ϯ 2 mV; n ϭ 8) was significantly less positive than for CRAC (45 Ϯ 6 mV; n ϭ 8). We examined whether this difference reflected different monovalent cation permeability profiles of the CaT1 and CRAC channel pores and found that, although both channels display a Na ϩ Ϸ Li ϩ Ͼ K ϩ Ͼ Cs ϩ Ͼ NMDG permeability sequence, CRAC has a much lower relative Cs ϩ permeability (P Cs /P Na ϭ 0.48 Ϯ 0.03 for CaT1 versus 0.12 Ϯ 0.04 for CRAC; p Ͻ 0.001). Second, inward rectification was much more pronounced for CaT1 than for CRAC. The rectification score (defined as the ratio of the current amplitudes at potentials 40 mV negative and positive to the reversal potential) was 9.9 Ϯ 1.1 (n ϭ 15) for CaT1 compared with 1.5 Ϯ 0.1 for CRAC (n ϭ 19; p Ͻ 0.001). Third, the current-voltage relationship for CaT1 currents measured using the linear voltage ramp protocol displayed a negative slope at potentials below Ϫ80 mV, which was not observed for CRAC. As shown below, this negative slope originates from the time-dependent removal of intracellular Mg 2ϩ block.
Monovalent cation currents through CaT1 activated during membrane hyperpolarization from ϩ20 mV to Ϫ100 mV and deactivated during subsequent steps to less hyperpolarized . Currents were measured during the final step to Ϫ100 mV and normalized to the steady state current at this potential. potentials ( Fig. 2A). The fraction of open channels at the end of each voltage step was assessed from the initial current amplitude during a final step to Ϫ100 mV and exhibited a Boltzmann dependence on membrane potential (20), with a slope factor of 8 mV and half-maximal deactivation at Ϫ24 mV (Fig. 2D). This voltage dependence was abolished when Mg 2ϩ was omitted from the intracellular solution (Fig. 2, C and D), indicating that the gating mechanism of CaT1 is a voltage-dependent block/unblock of the channel pore by intracellular Mg 2ϩ . Using pipette solutions containing different concentrations of Mg 2ϩ , we found that half-maximal block at 0 mV was achieved with ϳ200 M intracellular Mg 2ϩ . In contrast, monovalent cation currents through CRAC did not exhibit such voltage dependence (Fig. 2, B and D), not even with 10 mM intracellular Mg 2ϩ (n ϭ 5; data not shown).
It should be noted that CaT1 currents remained strongly inwardly rectifying in symmetric monovalent cation solutions and in the absence of intracellular Mg 2ϩ , indicating that rectification represents an intrinsic pore property of CaT1. Moreover, the strong inward rectification and voltage-dependent Mg 2ϩ block of CaT1 were preserved in cell-free inside-out patches (data not shown).
2-aminoethoxydiphenyl borate (2-APB), an inhibitor of IP 3 receptors, is known to inhibit store-dependent Ca 2ϩ influx pathways in a variety of cells (21)(22)(23)(24)(25). We confirmed that 50 M 2-APB caused a rapid and complete block of CRAC in RBL cells (Fig. 3A), which was voltage-independent ( Fig. 3B and data not shown) and slowly reversible upon washout (Fig. 3A). The inhibitory effect of 2-APB on CRAC was dose-dependent with half-maximal inhibition at 5.5 M (Fig. 3D). In contrast to a recent study (25), we did not observe a consistent initial increase in CRAC activity upon application of 2-APB. However, we cannot exclude the possibility that some 2-APB-dependent potentiation was masked by the rapid blocking effect of the drug. Although 2-APB has been shown to be membrane-permeable, two observations indicate that the target for 2-APB block of CRAC is exposed to the extracellular medium. First, the onset of block occurred with at time constant of ϳ2 s (Fig. 3A), which is almost as fast as block of CRAC by extracellular Zn 2ϩ , Cd 2ϩ , or La 3ϩ (data not shown) and much faster than what has been reported for IP 3 receptor-mediated effects of 2-APB in intact cells (22). Second, dialysis of the cytoplasm with a pipette solution supplemented with 50 M 2-APB did not markedly prevent activation of CRAC by ionomycin (data not shown; see also Ref. 23). From this we conclude that the interaction site for 2-APB is either on the extracellular part of the CRAC channel itself or at least closely associated therewith. In remarkable contrast with its inhibitory effects on CRAC, 50 M 2-APB potentiated CaT1-mediated currents by ϳ25% (Fig. 3, A  and D). The potentiating effect of 2-APB did not show marked voltage dependence (Fig. 3C), was fully reversible upon washout (Fig. 3A), and could be repeated several times in the same cell. A similar but less pronounced potentiation was obtained with 5 M 2-APB (Fig. 3D). Application of 250 M 2-APB also caused a potentiation of CaT1, which was mostly followed by a slow and moderate inhibition of the currents by ϳ20% and/or activation of linear leak currents. Thus, CRAC is at least 100 times more sensitive for 2-APB block than CaT1.
According to Yue et al. (10), the properties of CaT1, notably its activation by active store depletion, were dependent on the expression level of the channel protein. Therefore, we wanted to make sure that the differences we observed between CaT1 and CRAC were independent on the number of CaT1 channels per cell. Like Yue et al. (10), we measured CaT1 currents within 12 h after transfection and selected for cells that had current densities below 100 pA/pF at Ϫ100 mV with 10 mM extracellular Ca 2ϩ (mean peak amplitude, 78 Ϯ 9 pA/pF; n ϭ 8). Despite the fact that the average CaT1 current density in this group of cells was ϳ5 times lower than the overall average (356 Ϯ 44 pA/pF; n ϭ 25), they displayed all of the above  Channel Pores of CaT1 and CRAC Are Not Identical 47769 described CaT1 features: inhibition of 24% by 2 M ionomycin (n ϭ 2); strong inward rectification in DVF solution (rectification score, 12.7 Ϯ 3.2; n ϭ 4); relative Cs ϩ permeability (P Cs / P Na ) of 0.52 Ϯ 0.06 (n ϭ 5); voltage-dependent gating in the presence of intracellular Mg 2ϩ with a slope factor of 8 mV and half-maximal deactivation at Ϫ25 mV (n ϭ 4); potentiation to 121% of control by 50 M 2-APB (n ϭ 2). Thus, the biophysical and pharmacological properties that distinguish heterologously expressed CaT1 from endogenous CRAC in RBL cells are not influenced by the expression level of the CaT1 channel protein. Moreover, virtually identical results were obtained in CaT1-expressing Chinese hamster ovary cells (data not shown), indicating that the expression system does not significantly affect the properties of the CaT1 channel.

DISCUSSION
To evaluate the hypothesis that CaT1, a member of the TRP family of cation channels, constitutes all or part of CRAC, the calcium release-activated Ca 2ϩ channel, we made a direct comparison of the biophysical and pharmacological properties of both channels under identical experimental conditions. At first sight, the similarities between CRAC and CaT1 (Table I) are indeed striking. However, it should be noted that many of the common characteristics of the CRAC and CaT1 pores (high Ca 2ϩ selectivity; anomalous mole fraction effect; divalent and monovalent cation permeability sequence; block by inorganic cations; loss of selectivity in the absence of divalents; singlechannel conductance to Na ϩ in the range of 10 -100 picosiemens) are not only shared by ECaC1 (15,26,27), which has a pore region virtually identical to the pore region of CaT1, but also shared to a large extent by voltage-operated Ca 2ϩ channels (VOCCs) (17, 28 -30), which have pore regions that are only weakly homologous to CaT1/ECaC1. In VOCCs and ECaC1, high Ca 2ϩ selectivity depends on negatively charged amino acids (glutamates and/or aspartates) in the pore region (18,19). It is believed that high affinity binding of Ca 2ϩ ions to these sites prevents, by electrostatic repulsion, monovalent cations from passing through the pore while simultaneously allowing high rates of Ca 2ϩ flux (17). Such a configuration implies that the pore becomes readily permeable to monovalent cations when the extracellular concentration of Ca 2ϩ and other divalent cations is so low that the Ca 2ϩ -binding sites are no longer occupied (20). Intermediate Ca 2ϩ concentrations block monovalent permeation but yield only small Ca 2ϩ fluxes. Thus, the conductance of these channels as a function of the extracellular Ca 2ϩ concentration goes through a minimum, a phenomenon termed anomalous mole fraction behavior, which has been observed in VOCCs (17), ECaC1/CaT1 (10,27), and CRAC (31). Moreover, because negatively charged Ca 2ϩ -binding sites generally display a high affinity for transition metals such as Cd 2ϩ , Zn 2ϩ , or La 3ϩ (20), it is not unexpected that these cations exert an inhibitory effect on the different Ca 2ϩ selective channels. Thus, it appears that most of the pore properties common for CRAC and CaT1 are features inherent to all highly Ca 2ϩselective pores in biological membranes.
As summarized in Table I, CaT1 differs from CRAC in RBL cells in the following salient features: effect of ionomycin-induced store depletion; current decay in DVF solution; relative permeability for Cs ϩ versus Na ϩ ; inward rectification; voltagedependent block by intracellular Mg 2ϩ ; and effect of extracellular 2-APB. Obviously, some of these differences might be explained by cell-specific modulation of channel function. However, monovalent permeability, channel rectification, and voltage-dependent block represent biophysical properties that are directly related to the channel pore (20). Thus, our data demonstrate that the pore properties of CRAC and CaT1 are clearly distinct, thereby ruling out the possibility that CaT1 encompasses the full CRAC pore. We cannot definitely exclude the possibility that the CRAC pore consists of CaT1 plus an additional subunit, but this additional channel subunit would then have to abolish the intracellular Mg 2ϩ -sensitivity of CaT1, provide for the 2-APB inhibition, and significantly alter the ion selectivity and inward rectification of the putative heteromultimeric channel. An alternative and presumably more likely explanation is that CRAC and CaT1 are two distinct channels with a number of overlapping properties but nevertheless encoded by different genes. In that case, we have provided a number of useful tools to discriminate between both channels in native tissue.