Store depletion fails to activate CRAC channels in SCID T cells
In principle, several mechanisms could abrogate Ca²⁺ influx subsequent to store depletion. These include defective expression or function of store-operated CRAC channels, as well as less direct mechanisms involving depolarization of the cell's membrane potential and consequent reduction of the driving force for Ca²⁺ entry. To distinguish between these possibilities, we directly measured CRAC channel function in SCID patients and control T cells using the whole-cell patch-clamp technique.

Control and SCID T cells were pretreated with 1 µM TG for 15 min to deplete Ca²⁺ stores completely. Subsequent exposure of control T cells to 20 mM Ca²⁺ produced an inward Ca²⁺ current in 5/5 cells (Fig. 2 A), which was identified as CRAC current (I_CRAC) based on several key characteristics. First, the response to rapid voltage ramps showed an inwardly rectifying current-voltage relationship and lack of an obvious reversal potential, both of which are typical of I_CRAC (Fig. 2 B). Second, as previously shown for I_CRAC in Jurkat T cells (23), the Ca²⁺ current in normal human T cells was potentiated strongly by 5 µM 2-APB (240 ± 45% enhancement; n = 4 cells; Fig. 2 C). Third, removal of all extracellular Ca²⁺ and Mg²⁺ by application of a divalent-free (DVF) solution produced an inward Na⁺ current that initially was approximately eightfold larger than the preceding Ca²⁺ current, and slowly declined by 90% (Fig. 2 A). It is well known that removal of divalent cations allows Na⁺ to permeate CRAC channels but does not sustain their activity, leading to a slow rundown of the current in Jurkat T cells (24, 25) and RBL cells (26).

View larger version (25K): [in this window] [in a new window]   Figure 2. Functional CRAC channels are undetectable in whole-cell patch-clamp recordings from SCID T cells. (A) Ca2+ and Na+ currents through CRAC channels in a control T cell. Peak currents recorded every 1.5 s during brief steps to –110 mV are shown. Exposure of this TG-pretreated cell to 20 mM Ca2+ evoked an inward CRAC Ca2+ current, whereas subsequent exposure to DVF solution allowed Na+ to carry an approximately eightfold larger inward current that declined slowly due to depotentiation. (B) Current-voltage relationship of the CRAC Ca2+ current. The graph shows the average of 10 leak-corrected responses to voltage ramps recorded from 78–92 s shown by the gray bar in A. (C) 2-APB strongly enhanced ICRAC. After development of ICRAC, 5 µM 2-APB was applied as indicated. Peak currents at –110 mV plotted as in A. (D) CRAC Ca2+ and Na+ currents were absent in SCID T cells. Same protocol as in A. (E) Current-voltage relationship of the current recorded in D. (F) Application of 5 µM 2-APB failed to elicit a response in the SCID cells. (G) Summary of the Ca2+ (ICRAC) and Na+ (IDVF) current densities in control (Ca2+ current: n = 5 cells; Na+ current: n = 4 cells) and SCID (Ca2+ current: n = 6 cells; Na+ current: n = 4 cells) T cells normalized to the capacitance of each cell (a measure of surface area).

Residual Ca²⁺ influx in SCID T cells: evidence for a defect in CRAC channel activation
The absence of CRAC channel activity in SCID T cells could arise from two potential sources: aberrant expression of CRAC channels (e.g., a null mutation in the structural gene for the channel or faulty trafficking to the plasma membrane) or defective activation of CRAC channels in response to store depletion. Clues that distinguish between these mechanisms came from observations of residual Ca²⁺ influx in the presence of high extracellular Ca²⁺ levels. Although store-depleted SCID cells fail to display detectable I_CRAC and lack Ca²⁺ influx in 2 mM Ca²⁺_o, they do exhibit a small degree of Ca²⁺ influx in the presence of 20 mM Ca²⁺_o. This influx occurred in all SCID cells, and evoked a transient increase in [Ca²⁺]_i (Fig. 3 A). The initial slope of the [Ca²⁺]_i increase was measured as an indicator of the apparent Ca²⁺ influx rate, which in SCID cells was 1–2% of the rate observed in control T cells under similar conditions (Fig. S2 A, available at http://www.jem.org/cgi/content/full/jem.20050687/DC1). The transient time course of the response is similar to that observed in TG-treated normal Jurkat T cells after initiating influx with low concentration of Ca²⁺_o (27); most likely this is due to a slow increase in Ca²⁺ extrusion by the plasma membrane Ca²⁺-ATPase (PMCA), because delayed modulation of PMCA has been shown to create a transient [Ca²⁺]_i increase in response to constant Ca²⁺ influx through CRAC channels (28).

View larger version (19K): [in this window] [in a new window]   Figure 3. Residual Ca2+ influx in SCID T cells is due to CRAC channels with an altered sensitivity to 2-APB. (A) Residual Ca2+ influx in SCID cells is store-dependent. Exposure to 20 mM Ca2+ before treatment with TG elicits a small [Ca2+]i increase in control (black trace) but not SCID T cells (red trace). The small influx in control cells may reflect a low level of CRAC channel activity induced by previous incubation in 0 Ca2+. Following store depletion with TG, readdition of 20 mM Ca2+ caused a small transient [Ca2+]i increase in SCID T cells; the initial rising phase indicates an influx rate 1.5% of that in control T cells (also see Fig. S2 A). (B) Residual Ca2+ influx is sensitive to inhibition by a high concentration of 2-APB. Addition of 2-APB (75 µM; blue trace) blocked Ca2+ entry and caused [Ca2+]i to decline rapidly in contrast to untreated SCID T cells (red trace). (C) Residual Ca2+ influx was inhibited by Gd3+ (green trace) or La3+ (blue trace; 2 µM). (D) Inhibition of residual Ca2+ influx by SKF 96365. Application of SKF 96365 (30 µM) before readdition of Ca2+ completely prevented the increase in [Ca2+]i (blue trace). (E, F) A low concentration of 2-APB failed to enhance residual Ca2+ influx in SCID T cells. (E) 3 µM 2-APB enhanced Ca2+ influx in control T cell (black trace), but did not affect the Ca2+ transient in SCID cells (red trace). (F) 2-APB (5 µM; blue trace) added with 20 mM Ca2+ did not increase the Ca2+ influx rate, as indicated by the initial slope (d[Ca2+]i/dt), or the peak Ca2+ response beyond that seen in untreated SCID T cells (red trace). Plots in A–F show the average and SEM values from 30 cells.

A distinctive property of CRAC channels is that their activity is enhanced by low concentrations (1–5 µM) of 2-APB (23). Accordingly, 2-APB (3 µM) added to control T cells following Ca²⁺ readdition caused a distinct further increase in [Ca²⁺]_i (Fig. 3 E). Given that the residual Ca²⁺ influx in SCID T cells displays several key properties of the CRAC channel, it was surprising that 2-APB at low concentrations (5 µM) failed to enhance residual influx when added near the peak of the Ca²⁺ transient (Fig. 3 E). A similar lack of effect was noted when 2-APB was added at a longer time (240 s) following Ca²⁺ readdition (unpublished data). To maximize the detectability of a change in influx rate, we also added 2-APB simultaneously with Ca²⁺; under these conditions, 2-APB also failed to enhance the rate of Ca²⁺ influx in SCID T cells (Fig. 3 F; 0.90 nM/s versus 0.87 nM/s with or without 5 µM 2-APB, respectively). The inability of low concentrations of 2-APB to enhance residual Ca²⁺ influx in SCID cells cannot be explained by a defect that simply reduces the number of CRAC channels in the plasma membrane, because a small number of normal channels in the membrane would be expected to show normal enhancement by 2-APB (e.g., see Fig. 2 C). Enhancement of I_CRAC by 2-APB is believed to involve augmentation of the activation process (32). Thus, an alternative explanation of our findings is that the SCID cells have a primary defect in the CRAC channel activation process, such that overall activity and enhancement are only 1–2% of the control level. Such a defect in SCID cells could arise from abnormal generation or response to the activation signal (see Discussion).

A primary defect in the activation, rather than the expression or targeting, of CRAC channels also is consistent with a molecular analysis of potential CRAC channel genes in the SCID cells. A variety of genes in the trp family, including trpc1 (9), trpc3 (10), trpc4 (33), and trpv6 (11, 12) have been proposed to form parts of store-operated Ca²⁺ channels, in general, or the CRAC channel, in particular. We found that transcripts for trpc1, c3, c4, c5, c7, trpv5, and v6, are present in control T cells and at similar levels in SCID T cells (Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20050687/DC1). In addition, no mutations were detected in transcripts of trpc1, c3, m2, v5, or v6 amplified by RT-PCR or in gene loci, including all known exons and splice junctions for trpc3, c5, c6, m5, v5, and v6 using DNA from both patients, their parents, one healthy brother, and one independent control (unpublished data). Furthermore, patients were heterozygous by haplotype mapping for the region containing trpv5 and v6 genes on chromosome 7q31, incompatible with the autosomal recessive mode of inheritance in this disease (unpublished data). Finally, retrovirus-based overexpression of myc-tagged TRPV6 failed to restore TG- or TCR-triggered Ca²⁺ influx in SCID T cells or TG-triggered influx in fibroblasts (unpublished data). Collectively, these observations lend further support to the notion that the locus of the SCID defect involves the activation, rather than the expression, of store-operated Ca²⁺ channels.

Given the evidence for a regulatory defect in CRAC channel activation, we examined the expression of stromal interaction molecule (STIM)1, a conserved ER protein recently shown to be essential for CRAC channel activation in multiple cell types (34). Endogenous STIM1 protein levels in T cells from SCID patients were comparable to those of control T cells and Jurkat cells (Fig. 4 A). In addition, no mutations in STIM1 were apparent from sequencing genomic DNA from the SCID patients. To test whether STIM1 overexpression could rescue the Ca²⁺ influx defect in cells from the SCID patients, their fibroblasts were cotransfected with pSTIM1 and GFP. Virtually all GFP⁺ cells also expressed STIM1 as determined by immunocytochemistry (Fig. 4 B). However, GFP⁺ cells did not show increased store-dependent Ca²⁺ influx compared with GFP^– cells, which—like untransfected SCID fibroblasts and T cells—are Ca²⁺ influx deficient (8). Despite the failure to rescue normal function, STIM1 localization seemed to be similar in transfected SCID and control fibroblasts (Fig. 4 C). Together, these results suggest that the Ca²⁺ influx defect is not due to inadequate expression of STIM1.

View larger version (40K): [in this window] [in a new window]   Figure 4. The lack of store-operated Ca2+ influx in SCID cells is independent of STIM1 expression. (A) STIM1 protein expression in SCID T cells. Whole-cell lysates from control and SCID patients' T cells, Jurkat, and TE671 cells were immunoblotted with STIM1 mAb, detecting a dominant band at 80–85 kD. (B) Exogenous expression of STIM1 fails to rescue Ca2+ influx in SCID fibroblasts. STIM1 was coexpressed with eGFP in fibroblasts from SCID patients (top panels). Virtually all cells expressing eGFP (green) showed a strong STIM1 signal (red) by immunocytochemistry. 48 h after transfection, cotransfected SCID fibroblasts were stimulated with TG (1 µM) in the absence of Ca2+ followed by readdition of 2 mM Ca2+ (bottom right). Mean Ca2+ responses for GFP+ (green) and GFP– (black) cells from the same experiment are shown. The Ca2+ response in untransfected control fibroblasts (bottom left) is shown for comparison. Results shown are representative of five experiments. (C) STIM1 shows a similar distribution in fibroblasts from SCID patients and controls. STIM1 was expressed in fibroblasts from one control and one SCID patient and protein localization detected by immunocytochemistry 48 h later. Merged images show substantial overlap of STIM1 and calreticulin indicating predominant localization of STIM1 in the ER in control and SCID fibroblasts. Images shown were obtained by deconvolution of an image z-stack.

TRPM7 is a widely expressed Ca²⁺- and Mg²⁺-permeable ion channel (35, 36). Native TRPM7 channels in T cells, called Mg²⁺-inhibited cation (MIC) channels or Mg²⁺ nucleotide-regulated metal channels typically are activated by the washout of intracellular Mg²⁺ and ATP during whole-cell recording (25, 35). Because of their slow induction kinetics, MIC channels initially were confused with CRAC channels; however, a variety of evidence argues that they are molecularly distinct (25, 26, 37). In SCID and control T cells, introduction of intracellular Mg²⁺-free solutions evoked the slow activation of currents with the properties of MIC. As expected for I_MIC, the currents showed outward rectification in the presence of extracellular Ca²⁺ and a linear current-voltage relationship reversing around 0 mV in the absence of external divalent ions (Fig. 5 A). The amplitude and activation kinetics of MIC current in SCID T cells also were similar to that of control T cells, demonstrating normal MIC channel expression and properties (Fig. 5 B). The nearly complete absence of I_CRAC in these cells provides strong genetic evidence that MIC and CRAC currents arise from different gene products.

View larger version (15K): [in this window] [in a new window]   Figure 5. MIC/TRPM7 channels are normal in SCID T cells. (A) Current-voltage relationships showing the typical permeation properties of MIC channels in control (left) and SCID T cells (right). Each graph show currents from one cell after 400 s of whole-cell recording in the presence of 20 mM Ca2+ and shortly after removal of divalent ions (DVF solution). (B) Activation kinetics and magnitude of IMIC in SCID T cells are normal. The plot shows peak currents at +100 mV in the presence of 2 mM Ca2+o in control (closed circles; n = 4 cells) and SCID T cells (open circles; n = 5 cells) as a function of time after break-in with Mg2+-free pipette solution.

IKCa1 currents were evoked in SCID and control T cells by intracellular dialysis with a buffered solution of 2 µM Ca²⁺ in the presence of equal concentrations of intracellular and extracellular K⁺. IKCa1 current was identified in response to voltage ramps from –100 to +100 mV by the increased inward current at membrane potentials negative to –50 mV; at more positive potentials, time- and voltage-dependent activation of Kv1.3 sums with IKCa1 to produce an "N"-shaped current-voltage relation (Fig. 6 A). The current below –50 mV was blocked by 50 nM charybdotoxin (CTX), as expected for IKCa1 channels (39, 40). With 2 µM Ca²⁺ in the pipette solution, the current densities at –100 mV were similar in the control and SCID T cells (Fig. 6 A, right panel), indicating normal IKCa1 expression and function in SCID cells.

View larger version (31K): [in this window] [in a new window]   Figure 6. Expression of Ca2+-activated and voltage-gated K+ channels in SCID T cells. (A) Amplitude of IKCa1 is normal in SCID cells internally dialyzed with 2 µM Ca2+. IKCa1 current density measured from CTX-inhibited currents at –100 mV is plotted for control (n = 5 cells) and SCID (n = 7 cells) T cells (right). (B) Voltage-dependent K+ currents in control (top) and SCID T cells (bottom), elicited by depolarizations from –70 to +70 mV in steps of 10 mV. The inset compares peak K+ current densities at +70 mV in control (n = 5 cells) and SCID (n = 8 cells) T cells. (C) Cumulative inactivation of Kv1.3 current, elicited by steps to +60 mV applied every 1 s. Top: the first 10 responses to this protocol in a SCID cell. Bottom: average peak currents during successive pulses in control (closed circles; n = 4 cells) and SCID cells (open circles: n = 5 cells), relative to the first pulse. (D) Inhibition by ShK-Dap22. K+ current in response to a pulse to +60 mV was blocked by 200 pM ShK-Dap22 in a SCID T cell (top). The degree of block is compared for control (n = 4) and SCID (n = 5) cells (bottom).

Consistent with these results, Kv1.3 transcript levels did not differ significantly between patient and control T cells (Fig. S4 A, available at http://www.jem.org/cgi/content/full/jem.20050687/DC1). Kv1.3 protein was detected readily in patient and control T cells by immunoblotting (Fig. 4S B) and at the cell surface by immunocytochemistry using Kv1.3-specific antibodies (Fig. 4S C, top panels). Flow cytometric measurements of surface binding of fluorescently labeled ShK-F6CA (42) confirmed equivalent surface expression of Kv1.3 in SCID and control T cells (Fig. 4S C, lower panel). ß subunits associated with Kv1.3, including Kvß1, Kvß2, and Kvß3, also were expressed normally at the mRNA and protein level (Fig. 4S, A and B).

Kv1.3 channel activation is altered in SCID T cells
Although the expression, inactivation, and pharmacologic profile of Kv1.3 channels was normal in the SCID patient cells, further examination revealed abnormal voltage dependence and kinetics of channel activation. Kv1.3 current in SCID cells showed a normal threshold for activation (approximately –50 mV), but the voltage dependence of channel activation was reduced markedly, as indicated by the diminished slope of the conductance-voltage relation (Fig. 7 A). The K⁺ conductance increases e-fold per 11 mV in the SCID cells relative to e-fold per 6 mV in controls, effectively shifting the voltage for half-maximal activation by 12 mV. Altered voltage sensitivity of Kv1.3 channels in the patient cells was accompanied by slower than normal activation and deactivation kinetics (Fig. 7, B and C). These changes in gating seem to be restricted to activation and deactivation; the rate of C-type inactivation of Kv1.3 currents in SCID T cells was normal (Fig. 7 D), consistent with their normal cumulative inactivation characteristics (Fig. 6 C).

View larger version (29K): [in this window] [in a new window]   Figure 7. Voltage dependence and kinetics of Kv1.3 activation and deactivation are altered in SCID T cells. (A) The voltage sensitivity of Kv1.3 conductance in SCID cells is reduced. Kv1.3 conductance was measured from tail currents as described (see Materials and methods). Boltzmann curves were fitted and normalized to maximal conductance using the parameter values Gmax = 8.85 nS, V1/2 = –43.9 mV, and k = 6 mV (control) or Gmax = 8.67 nS, V1/2 = –31.6 mV, and k = 11 mV (SCID). (B) Activation kinetics of Kv1.3 current are slowed in SCID T cells. Currents evoked by pulses from –50 to +30 mV in control (black) and SCID T cells (red) are normalized to peak current at each voltage and superimposed (left). Activation kinetics were quantified by fitting single exponential functions to the final part of the rising phase of each current (right). (C) Deactivation kinetics are slowed in SCID cells. Normalized tail currents from a control (black) and a SCID T cell (red) upon repolarization to voltages of –100 to –50 mV (excluding –90 mV) after a 200-ms step to +60 mV are shown (left). Deactivation kinetics were measured from single exponential fits to each tail current (right). (D) Slow C-type inactivation of Kv1.3 current is not altered in SCID T cells. Currents from control (top) and SCID T cells (bottom) in response to 500-ms steps from +10 mV to +80 mV are shown (left). The extent of inactivation at the end of each pulse (right) is similar for control (black; n = 4 cells) and SCID cells (red; n = 5 cells).

	DISCUSSION

Top Abstract RESULTS DISCUSSION MATERIALS AND METHODS References

Importantly, these results also confirm the essential role of the CRAC channel in mediating Ca²⁺ influx in response to TCR stimulation. A recent study proposed that Ca_V channels underlie TCR-mediated Ca²⁺ entry in murine T cells (13), based on the endogenous expression of and ß subunits of L-type Ca²⁺ channels and depolarization-activated L-type Ca²⁺ currents in normal T cells, as well as abnormally small Ca²⁺ signals in response to TCR stimulation in mutant mice lacking the ß4 subunit of the Ca²⁺ channel. These results are surprising considering that voltage-gated Ca²⁺ currents in T cells have not been detectable in a large number of electrophysiological studies, and membrane depolarization has never been shown to elevate [Ca²⁺]_i in T cells. In addition, it is not clear whether TCR cross-linking activates Ca_V channels. In the study by Badou et al. (13), anti-TCR antibodies evoked current fluctuations after an artificially imposed membrane depolarization but not at the cell's natural membrane potential. Finally, although normal and SCID human T cells express several isoforms of the Ca_V 1 subunit, thorough tests failed to reveal any Ca²⁺ current in response to depolarization before or after TCR stimulation (unpublished data). Further work is needed to understand the role of Ca_V channel proteins in human T cells, and to explore additional functions of the ß subunits. In contrast, the absence of I_CRAC in SCID T cells clearly supports the idea that voltage-independent CRAC channels are the major, if not the sole, pathway for Ca²⁺ entry in response to stimulation through the TCR (43). Such a role also is consistent with previous studies of human SCID T cells (6, 7) and CRAC-deficient mutant Jurkat T cells (27, 44), in which a lack of CRAC channel activity was associated with the absence of TCR-mediated Ca²⁺ influx and deficient activation of transcriptional pathways, cytokine secretion, and T cell activation.

The molecular basis of store-operated Ca²⁺ entry is one of the enduring mysteries of cell biology. Despite more than a decade of work by many laboratories focused on this question, little is known about the nature of the CRAC channel or its activation mechanism at a molecular level. Many genes and mechanisms have been proposed, but supporting evidence for many of these has been inconsistent. Of the TRP channels that have been suggested as candidates for CRAC channel subunits (TRPC1, TRPC3, TRPV6), we found that all were expressed in the SCID cells and did not display mutations, and that overexpression of TRPV6 was unable to rescue store-operated Ca²⁺ entry. The possible role of TRPV6 in CRAC channel formation is controversial. Early work indicated that TRPV6 shares several characteristic properties with CRAC, such as divalent cation selectivity, inactivation, and sensitivity to La³⁺ (11). In addition, overexpression of a dominant negative TRPV6 pore mutant was able to suppress endogenous I_CRAC in Jurkat cells (12); together, these studies supported the view that TRPV6 may encode at least part of the CRAC channel pore. However, other studies have shown differences with CRAC in terms of ion selectivity and rectification, effects of intracellular Mg²⁺ and 2-APB (15), and unitary conductance (25); furthermore, knockdown of TRPV6 with antisense or RNAi methods fails to reduce I_CRAC in RBL cells (45). Given the evidence that the SCID cells may bear a defect in the CRAC channel activation mechanism, our experiments do not exclude a role for TRPV6 in CRAC-mediated Ca²⁺ influx in normal lymphocytes; however, we reasonably can exclude their causative involvement in the CRAC deficiency in the SCID patients.

Which aspect of the CRAC channel activation process is deficient in the SCID T cells? The cells display a small amount of CRAC channel activity, identified by its store dependence and sensitivity to block by 2-APB, Gd³⁺, La³⁺, and SKF 96365; the properties of this residual Ca²⁺ influx can offer clues about the underlying defect. One possibility is that the CRAC channel is not expressed at the cell surface, because of a mutation affecting ion conduction, a defective promoter, or mutations interfering with transport and trafficking to the plasma membrane. If this were the case, the residual influx would represent a small number of normal channels, or a normal number of channels with greatly reduced ion conduction activity. One would predict that the activity of these channels would be enhanced by a low concentration of 2-APB (Fig. 3, E and F). This was not observed, arguing against the presence of a small number of otherwise normal CRAC channels in the plasma membrane.

An alternative explanation for this result is that the SCID cells harbor a defect in the CRAC channel activation process which impairs channel activity as well as the ability of 2-APB to enhance it. Recent evidence suggests that 2-APB inhibits several members of the TRP channel family by interfering with channel activation rather than blocking channel conduction itself. For example, 2-APB inhibits currents through Drosophila TRP channels activated by light or through mammalian TRPC3 channels activated by PLC-coupled receptors, but does not affect the same channels after they are activated directly by metabolic stress or diacylglycerol, respectively (46, 47). A defect in the activation machinery also may explain why store-operated Ca²⁺ influx was not observed in SCID T cells transfected with TRPV6. A defect in CRAC channel activation could occur at many levels between store depletion and channel opening, including the ER Ca²⁺ sensor, the proteins that communicate store depletion to the channel, and the ability of the CRAC channel itself to respond to the activation signal. Therefore, successful complementation of the SCID cells may reveal crucial information about the nature of the link between store depletion and channel activation, as well as the molecular mechanism of CRAC channel potentiation by 2-APB.

The expression and gross properties of several other channels (TRPM7, Kv1.3, and IKCa1) were normal in the SCID cells, suggesting that the mutation in the SCID cells specifically targets CRAC channel activation rather than channel expression, in general. However, we were surprised to find that the activation kinetics and voltage sensitivity of Kv1.3 are altered; the voltage sensitivity is reduced from a normal value of 6 mV per e-fold change in conductance to 11 mV, with a slowing of channel activation and deactivation. There are several ways in which Kv1.3 channels are known to be modulated in vivo. Association with ß subunits promotes cell surface expression of the Kv1.3 subunit, and thereby, increases current amplitude without altering the channel's voltage dependence or activation kinetics (48). Given that Kv1.3 current amplitude, cell surface expression of Kv1.3 (Fig. S4 C), and Kvß1 and Kvß2 expression are normal in the SCID T cells, a ß subunit defect seems unlikely. Phorbol ester shifts the voltage dependence of Kv1.3 in T cells toward positive voltages but without the large decrease in voltage sensitivity (as indicated by the slope of the conductance-voltage curve) we have observed in SCID cells (49). Likewise, expression of v-src is known to cause a similar shift in voltage dependence without reducing voltage sensitivity, and also greatly slows the rate of inactivation, which also differs from the behavior of Kv1.3 in SCID cells (50). Thus, it seems unlikely that the abnormal K⁺ channel properties observed in SCID T cells are simply due to altered phosphorylation by protein kinase C or a tyrosine kinase.

The association of altered K⁺ channel kinetics with the lack of CRAC channel activity in SCID T cells suggests a functional link between CRAC and Kv channels. Although further work is needed to establish the nature of this link, one intriguing possibility is that Kv1.3 and CRAC channels are regulated by a common protein which is targeted by the SCID mutation. This protein target could be involved in posttranslational modification of both channels or could be a common subunit, adaptor, or scaffolding protein, which could influence clustering or activation of CRAC and Kv1.3. The PMCA and Kv1.3 bind to the scaffold MAGUK protein SAP97 (hDlg) present in T cells (51, 52), and the sensitivity of PMCA in T cells to local gradients of Ca²⁺ near CRAC channels suggests that CRAC and PMCA are in close proximity to each other (53). These results raise the possibility that SAP97 may bring CRAC, PMCA, and Kv1.3 together in a multiprotein complex. Preliminary results suggest that the expression and localization of SAP97 are normal in SCID T cells and fibroblasts (unpublished data); thus, the SCID phenotype does not seem to involve a loss or gross mistargeting of SAP97. Further studies to examine the physical association of Kv1.3 with CRAC channels and other proteins in T cells may offer a new strategy for understanding the regulation of store-operated Ca²⁺ entry.

	MATERIALS AND METHODS

Top Abstract RESULTS DISCUSSION MATERIALS AND METHODS References

 
Cells.
Continuously growing T cell lines were derived from the peripheral blood lymphocytes of two patients and four healthy donors (22). Foreskin fibroblasts from newborn SCID patient 2 and a healthy newborn (Hs27 cell line, American Type Culture Collection) were immortalized by retroviral transduction with a telomerase expression plasmid (hTERT, gift of S. Lessnick, Dana-Farber Cancer Institute, Boston, MA). Patients in this study were included in a human subjects protocol which was reviewed and approved by the CBR Institute's internal review board. The rhabdomyosarcoma cell line TE671 was obtained from American Type Culture Collection.

Solutions and chemicals.
Standard extracellular Ringer's solution contained (in mM): 155 NaCl, 4.5 KCl, 2 CaCl₂, 1 MgCl₂, 10 D-glucose, and 5 Na-Hepes (pH 7.4). In Ca²⁺-free Ringer's, 1 mM EGTA + 2 mM MgCl₂ was substituted for CaCl₂. For measurements of I_CRAC, 20 mM CaCl₂ was used. DVF Ringer's solution contained (in mM): 155 Na methanesulfonate, 10 HEDTA, 1 EDTA, and 10 Hepes (pH 7.4 with NaOH). For measurements of K_Ca currents, NaCl in Ringer's solution was replaced with KCl. Internal solutions for recordings of I_CRAC contained (in mM): 150 Cs methanesulfonate, 8 MgCl₂, 10 BAPTA, and 10 Cs Hepes (pH 7.2). For recordings of I_MIC, internal solution contained (in mM): 150 Cs methanesulfonate, 10 HEDTA, 0.5 CaCl₂ and 10 Cs-Hepes (pH 7.2). For recordings of K_Ca current, internal solution contained (in mM): 150 K aspartate, 2 MgCl₂, 10 K Hepes, 10 EGTA, and added CaCl₂ to yield a free [Ca²⁺] of 2 µM. For recordings of Kv1.3 current, internal solution contained (in mM): 150 K aspartate 4 MgCl₂, 10 EGTA, and 10 K Hepes. pH of all internal solutions was 7.2. Solutions were applied to the cells with a rapid multi-barrel local perfusion pipette (25). 2-APB was purchased from Sigma-Aldrich and TG was purchased from LC Biochemicals.

Cell extracts, immunoblotting, and immunocytochemistry.
Preparation of total cellular extracts, immunoblotting, and immunocytochemistry was done as described (19) (see supplemental Materials and methods, available at http://www.jem.org/cgi/content/full/jem.20050687/DC1). Antibodies against STIM1 were obtained from BD Biosciences and as a gift of G. Velicelebi (Torrey Pines Therapeutics, La Jolla, CA). Antibody against calreticulin was purchased from Abcam (Cambridge, MA). For overexpression of STIM1, full-length STIM1 (NM_003156) cDNA (Openbiosystems) was subcloned into pENTR11 (Gateway system, Invitrogen) and transferred into MSCV-containing retroviral destination vector GFP-KV-DV for stable transduction of fibroblasts essentially as described (54). Immunocytochemical analysis was performed on an Axiovert S200 epifluorescence microscope (Carl Zeiss Microimaging, Inc.) with Openlab digital imaging software (Improvision); deconvolution of z-sections was performed to subtract out-of-focus signals.

Single-cell Ca²⁺ imaging.
T cells were loaded with 1 µM fura-2/AM (Molecular Probes) in RPMI 1640 + 10% FBS for 30 min at 22–25°C, and fibroblasts grown directly on UV-sterilized cover slips were loaded with 3 µM fura-2/AM for 45 min at 22–25°C. Ca²⁺ imaging was done with a Zeiss Axiovert S200 epifluorescence microscope and OpenLab imaging software (Improvision) as described (8). To actively deplete Ca²⁺ stores, T cells were incubated with 5 µg/ml biotinylated anti-CD3 antibody (UCHT1, BD Biosciences) for 10 min at 22–25°C followed by cross-linking with 5 µg/ml streptavidin (Pierce Chemical Co.).

Patch-clamp electrophysiology.
Whole-cell recording was conducted at 22–25°C using standard methods (25). Currents were filtered at 1 or 2 kHz with a 4-pole Bessel filter and sampled at 5 kHz. For measurements of CRAC and MIC currents, a voltage protocol consisting of a 100-ms step to –110 mV followed by a ramp from –110 to +90 mV lasting 100 ms was applied. Voltage pulses were delivered at 1-s intervals from a holding potential of +20 mV. Unless noted otherwise, all data were corrected for liquid junction potential of the pipette solution and for leak currents collected in Ca²⁺-free Ringer's solution; averaged results are presented as the mean value ± SEM.

IKCa1 currents were measured by stepping the voltage to –100 mV for 100 ms followed by a ramp from –100 to +100 mV for 100 ms. For measurements of Kv1.3 currents, voltage pulses were delivered every 15–20 s from a holding potential of –80 mV, and currents were leak subtracted using the P/8 method with an offset of –20 mV. For conductance-voltage curves, tail currents were measured 200 µs after membrane repolarization. Curves were fit with a Boltzmann function: G(V) = G_max/{1 + exp[(V-V_1/2/k)]}, where G_max is the maximal conductance, V_1/2 is the voltage for half-maximal activation, and k is the voltage sensitivity in mV.

Online supplemental material.
Fig. S1 shows evidence that expression levels of IP₃ receptors and PLC are similar in control and SCID T cells. Fig. S2 quantitates store-operated Ca²⁺ influx in individual SCID T cells and shows that it is inhibited by 2-APB. Fig. S3 shows that expression levels of mRNA for TRPC1-5, TRPC7, TRPV5, and TRPV6 in SCID T cells are all within normal range. Fig. S4 shows evidence that protein expression of Kv1.3 and it's ß subunits are normal in SCID calls, as are mRNA levels for Kv1.3, IKCa1, Kv3.1, and Kvß1, ß2, and ß3. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20050687/DC1.