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* Department of Physiology and Biophysics and Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
Correspondence: Address reprint requests to J. Edwin Blalock, PhD, University of Alabama at Birmingham, Dept. of Physiology and Biophysics, MCLM 898, 1918 University Blvd., Birmingham, AL 35294-0005. Tel.: 205-934-6439; Fax: 205-934-1446; E-mail: blalock{at}uab.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). In lymphocytes, mast cells and rat basophilic leukemia (RBL) cells, depletion of internal Ca2+ stores by either antigen/agonist binding or sarcoplasmic/endoplasmic reticulum Ca2+ (SERCA) pump inhibitors (e.g., thapsigagin), or dialysis of the cytosol by a whole-cell pipette solution during patch-clamp recordings activate a highly Ca2+-selective ion channel termed the Ca2+ release-activated Ca2+ (CRAC) channel (Lewis and Cahalan, 1989; Zweifach and Lewis, 1993; Hoth and Penner, 1992, 1993; Premack et al., 1994), which has an extremely low unitary conductance for Ca2+ (24 fs) (Zweifach and Lewis, 1993). Even though the CRAC channel is the most widely accepted, and well-characterized SOCs (Parekh and Penner, 1997), its molecular identity, activation mechanism, and regulation are yet to be fully determined.
One intriguing property of the CRAC channel is its high Ca2+ selectivity under normal physiological conditions. Its selectivity for Ca2+ is >1000 times higher than for Na+, which is even higher than that of voltage-gated Ca2+ channels (Hoth and Penner, 1993). However, in the absence of any extracellular divalent cations, it becomes permeable to monovalent cations (Hoth and Penner, 1993; Premack et al., 1994) with a larger single-channel conductance (unitary conductance = 2 pS for Na+ (Prakriya and Lewis, 2002)). A similar phenomenon has been observed for voltage-gated Ca2+ channels (Almers et al., 1984; Fukushima and Hagiwara, 1985; Almers and McCleskey, 1984; Hess and Tsien, 1984; Hess et al., 1986). However, in contrast to voltage-gated Ca2+ channels where Na+ and Ca2+ currents show similar kinetics (Almers et al., 1984; Fukushima and Hagiwara, 1985; Almers and McCleskey, 1984; Hess and Tsien, 1984; Hess et al., 1986), the Na+ current through CRAC channels (Na+-ICRAC) inactivates rapidly by an unknown mechanism. The rapid inactivation of Na+-ICRAC is interesting for two reasons: first, this type of inactivation has not been observed in any other well-known ion channels to our knowledge, and therefore might represent a new form of ion channel inactivation. Second, understanding the mechanism underling the inactivation of Na+-ICRAC may reveal a novel regulatory mode for CRAC channels. In this study we have shown that the inactivation of Na+-ICRAC is due to the dissociation of Ca2+ ions from a site on the outside of the CRAC channel, and Ca2+ binding to this site otherwise potentiates the current. Moreover, Ca2+ occupancy is necessary for the normal functioning of CRAC channels.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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)
Patch clamp recordings
Whole-cell recordings were performed with RBL-2H3 cells at room temperature with an Axonpatch 200B amplifier (Axon Instruments, Fox City, CA). Standard external solution (SES) contained (in mM) 143 NaCl, 10 CaCl2, 1 MgCl2, 5 D-glucose, 4.5 KCl, 0.5 BaCl2, and 10 HEPES, pH 7.3 adjusted with NaOH. Nominal Ca2+-free solution was made by replacing Ca2+ in SES with equal-molar Mg2+. The currents measured in this solution were used as leak currents for subtraction to eliminate possible contamination currents from Mg2+-inhibited cation channels (Prakriya and Lewis, 2002; Su et al., 2002). In some experiments as labeled in the figures (Figs. 3 A and 6 C), the external solution contained (in mM) 149 N-methyl-D-glutamine (NMDG) chloride, 10 CaCl2 or MgCl2 or BaCl2 or MnCl2 or NiCl2 or ZnCl2, 5 D-glucose, and 10 HEPES, pH 7.3 adjusted with NMDG. External solutions containing 50 or 1 mM Ca2+ used in Figs. 4 and 6 were made by replacing 10 mM Ca2+ in this solution with the corresponding molarity of Ca2+ and changing NMDG-Cl level to 89 or 162.5 mM to maintain the total osmolarity. External solutions containing from 10 nM to 100 µM free [Ca2+] used in Fig. 4 were made by adding an appropriate amount of CaCl2 to the following metal-ions-free solution (MIF) containing (in mM) 158 NMDG-Cl, 10 HEPES, 5 glucose, and 2 NMDG-EGTA, pH 7.3 adjusted by NMDG. The divalent cation free solution (DVF) contained (in mM) 160 Na-methane sulphonate, 5 D-glucose, 2 Na-EGTA, and 10 HEPES, pH 7.2 adjusted with NaOH. External solutions containing from 10 nM to 100 µM free Ca2+ used in Fig. 5 A were made by adding appropriate amounts of CaCl2 to DVF. Standard internal solution for whole-cell recordings contained (in mM) 110 Cs-glutamate, 10 CsCl, 2.9 MgCl2, 0.6 CaCl2, 10 Cs-EGTA, and 30 HEPES, pH 7.2 adjusted with CsOH. The calculated free [Ca2+] and [Mg2+] are 10 nM and 2 mM, respectively. The whole-cell currents shown in Figs. 1 A, 3 A, 5 A, and 6 C were detected by applying a 100-ms voltage ramp from -100 to 100 mV at a frequency of 1 Hz while cells were held at -40 mV, and currents at -80 mV are displayed. The whole-cell currents shown in Fig. 7 were detected by applying an 80-ms voltage step of -80 mV at a frequency of 2 Hz while cells were held at +60, 0, or -60 mV. The signal was filtered at 1 kHz with a built-in lowpass Bessel filter of the amplifier and digitized at 5 kHz. In all other experiments to detect the fast transition of the currents, the currents were continuously measured at -60 mV, filtered at 1 kHz with the built-in lowpass Bessel filter of the amplifier, and digitized at 5 kHz. Fast solution exchange was achieved using a mobile linear array of wide-tipped puffer pipettes. Only experiments with a solution exchange time <150 ms were included in the analysis.
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, 1, and 2 are the activation or inactivation time constants. The dose-response data shown in Figs. 4 E and 5 D were fitted to the Hill equation:
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). Notably, although Na+-ICRAC was initially larger, it inactivated rapidly (Fig. 1 A). A similar phenomenon was seen when Na+ was replaced by Li+ or K+ (data not shown). This confirms the previous similar observations in Jurkat T (Prakriya and Lewis, 2002), rat mast (Hoth and Penner, 1992, 1993), and RBL cells (Kozak et al., 2002; Hermosura et al., 2002). To reveal the details of the transition of the current during the solution exchange, we continuously recorded the current from other cells with a higher signal-sampling rate under a holding potential of -60 mV (see Materials and Methods). As shown in Fig. 1, C and D, such recordings revealed that the kinetics of the current induced by solution exchange consists of three phases (Fig. 1 D). In phase I, the current first slightly decreased when the Ca2+-containing solution SES was changed to DVF (Fig. 1 D). This phase can be fitted to a single-component exponential equation with a time constant of 74.0 ± 16.3 ms (n = 20), which reflects the rate of solution exchange to remove the Ca2+ blockage of Na+-ICRAC (Fig. 1 D). We therefore termed this phase the preactivation phase of Na+-ICRAC. In phase II, the current rapidly increased to a maximum (peak) in 779.1 ± 87.2 ms (n = 17), reflecting the activation process of Na+-ICRAC, and we thus termed it the activation phase of Na+-ICRAC. It can be fitted to a single-component exponential equation with a time constant of 367.2 ± 11.0 ms (n = 17) (Fig. 1 D). The current then rapidly inactivated to a steady state in phase III, so we termed this phase the inactivation phase of Na+-ICRAC. It can be fitted to a two-component exponential equation with time constants 1 = 912.0 ± 269.0 ms and 2 = 10421.4 ± 1270.0 ms (n = 17) (Fig. 1 D). To quantitatively analyze the inactivation of the Na+-ICRAC, we defined an inactivation index as the ratio of the peak Na+-ICRAC and the Na+-ICRAC measured at 20 s after the peak (Fig. 1 C):
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The 20-s measurement was chosen to reflect the fact that the Na+-ICRAC inactivates to a steady state in 20 s after reaching the peak (Fig. 1 C). The inactivation index of Na+-ICRAC is 2.9 ± 0.14 (n = 16) when the extracellular solution was switched from SES to DVF (Fig. 1 C inset). As shown in Fig. 1 E, the inactivation index remained relatively stable during the activation and slow inactivation of CRAC channels (Zweifach and Lewis, 1995b; Parekh, 1998), suggesting that it is a useful parameter with which to investigate effects of the other factors on the rapid inactivation of Na+-ICRAC.
Preexposing cells to metal ions confers the three-phase kinetics of ICRAC during the solution exchange
Because the complex three-phase kinetics of the ICRAC occurred when all the metal ions were replaced by Na+, we tested the effects of preexposure of cells to cations on the kinetics of Na+-ICRAC. As shown in Fig. 2 A, preexposing a cell to the MIF solution (see Materials and Methods) before switching to DVF abolished the three-phase kinetics of the ICRAC during solution exchange and greatly reduced the size of peak Na+-ICRAC. However, preexposing the cell to the SES before switching to DVF restored the three-phase kinetics of the ICRAC (Fig. 2 A) and to a large extent the size of the peak Na+-ICRAC. The smaller size of the restored peak Na+-ICRAC is due to the unavoidable slow inactivation of the CRAC channel (Zweifach and Lewis, 1995b; Parekh, 1998). As shown in Fig. 2 B, superimposing the two segments (6-s long) of current as indicated by the horizontal bars labeled "6 s" in Fig. 2 A over time revealed some marked contrasts in kinetics of the currents. First, when the cells were preexposed to MIF, the Na+-ICRAC activated almost immediately after switching to DVF, whereas when the cells were preexposed to SES, the current first underwent the preactivation phase of the Na+-ICRAC, i.e., the current first decreased before the Na+-ICRAC developed (Fig. 2 B). Second, when the cells were preexposed to MIF, the time for Na+-ICRAC to develop to its peak (time to peak) was longer (3.73 ± 0.34 s, n = 7) compared to that when the cells were preexposed to SES (0.78 ± 0.09 s, n = 18) (Fig. 2, B and C). The activation time course, when the cells were preexposed to MIF, can be fitted to a two-component exponential equation with time constants 1 = 70.1 ± 5.5 ms and 2 = 516.1 ± 53.8 ms (n = 7) (Fig. 2 B), whereas that when the cells were exposed to SES can be fitted to a single-component exponential equation with a time constant of 308.2 ± 12.0 (n = 18). (Fig. 2 B). Third, the magnitude of the peak Na+-ICRAC when the cells were preexposed to MIF is only about the same size as the steady-state Na+-ICRAC when the cells were preexposed to SES (Fig. 2, A and D). Fourth, the inactivation index of Na+-ICRAC when cells were preexposed to MIF is close to 1 (0.99 ± 0.08, n = 7) (Fig. 2 E), indicating that there is no further inactivation of Na+-ICRAC after preexposing to MIF. These results strongly suggest that preexposure of the cell to MIF has already inactivated the current to a steady state, and therefore it cannot be further inactivated. In other words, preexposure of cells to the SES potentiates Na+-ICRAC, whereas preexposure of cells to MIF inactivates it. We therefore hypothesized that association of a metal ion to a site on the CRAC channel might potentiate Na+-ICRAC, whereas its dissociation from this site might result in the inactivation of the Na+-ICRAC.
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; Christian et al., 1996), we tested the effect of various divalent cations on the inactivation of the Na+-ICRAC. As shown in Fig. 3 A, preexposing a cell to 10 mM Ca2+ (Ca2+ is the only metal cation in the solution; see Materials and Methods) greatly potentiated Na+-ICRAC that inactivated rapidly. Both its peak value (-7.85 ± 0.73 pA/pF, n = 7; Fig. 3 B) and inactivation index (2.84 ± 0.15, n = 7; Fig. 3 C) are similar to those seen when the cells were preexposed to SES (Fig. 2, D and E). However, preexposing the cell to the CRAC channel blockers Ni2+ or Zn2+ at 10 mM, failed to potentiate Na+-ICRAC as evidenced by a small peak Na+-ICRAC (-1.55 ± 0.12 pA/pF, n = 6 for Ni2+ and -1.6 ± 0.14 pA/pF, n = 6 for Zn2+) and an inactivation index of 
1 (-1.01 ± 0.01, n = 6 for Ni2+ and 1.02 ± 0.03, n = 6 for Zn2+). Preexposing the cell to 10 mM Ba2+, Mn2+, or Mg2+ only slightly potentiated Na+-ICRAC, because the current had a smaller peak Na+-ICRAC (-2.57 ± 0.44 pA/pF, n = 7, for Ba2+; -2.63 ± 0.55 pA/pF, n = 6 for Mn2+; and -2.50 ± 0.57 pA/pF, n = 7 for Mg2+; Fig. 3 B) and a smaller inactivation index (1.34 ± 0.11, n = 7 for Ba2+; 1.32 ± 0.09, n = 6 for Mn2+; and 1.25 ± 0.09, n = 7 for Mg2+; Fig. 3 C). Reexposing the cell to 10 mM Ca2+ restored the potentiation and inactivation index of the Na+-ICRAC (Fig. 3 A). These results strongly suggest that Ca2+ is the most potent cation for potentiating Na+-ICRAC. Surprisingly, though Ca2+-ICRAC in Jurkat T cells is potentiated by Ni2+ (Zweifach and Lewis, 1996; Christian et al., 1996), Na2+-ICRAC in RBL-2H3 cells is not by either Ni2+ or Zn2+.
By preexposing cells to solutions containing various levels of free Ca2+ with NMDG as the only other cation (see Materials and Methods) followed by exposure to DVF, we further showed that extracellular Ca2+ can dose-dependently potentiate Na+-ICRAC (Fig. 4, A and B). To eliminate the effects of the slow inactivation of ICRAC, we cycled through the preexposing Ca2+ levels in both increasing and decreasing orders. Similar results were obtained in both sets of experiments (Fig. 4, C and D). Fig. 4, B and D are examples of two superimposed 10-s segments of current taken from Fig. 4, A and C, as indicated by the horizontal bars labeled as "10 s" in Fig. 4, A and C, respectively. The inactivation indices for the same preexposing Ca2+ levels (1 nM and 10 mM) are similar, no matter whether preexposing Ca2+ levels were applied increasingly or decreasingly, suggesting that this ordering has little effect on the inactivation of Na+-ICRAC, even though the current undergoes unavoidable slow inactivation. This again suggests that an inactivation index is a good measure of Na+-ICRAC inactivation. Fig. 4 E shows the pooled data from both types of experiments. Fitting the data to a Hill equation yielded a Kd of 20.4 µM. However, this value of Kd is likely to be an overestimate of the real value for the Ca2+ binding site, because the preexposing Ca2+ levels are used in the calculation. This concentration is rapidly diluted by DVF, and hence the actual Ca2+ levels around the channel are likely to be lower than the preexposing levels (a more accurate estimation of the Kd is given below). Nevertheless, these results strongly suggested that Ca2+ binding to a specific site on the CRAC channel potentiates Na+-ICRAC and that dissociation of Ca2+ from it inactivates Na+-ICRAC. We thus termed this site a Ca2+-dependent potentiation site (or a potentiation site for simplicity). A Ca2+-binding site on the extracellular side of the CRAC channel has been suggested to account for the potentiation of the Ca2+ current in Jurkat T cells (Zweifach and Lewis, 1996; Christian et al., 1996).
The affinity of the potentiation site for Ca2+ is higher than that of the permeation site
The sequential rapid activation and inactivation of the Na+-ICRAC might reflect a relief of Ca2+ blockage of Na+-ICRAC and subsequent dissociation of Ca2+ from the potentiation site when the extracellular solution was rapidly switched from a Ca2+-containing one to DVF. In other words, Ca2+ ions may be removed from the site in the channel that would otherwise prevent Na+ from entering the pore before they dissociate from the potentiation site. We term this site a permeation site, and presume that it is located in the selectivity filter, as has been suggested for the voltage-gated Ca2+ channels (Ellinor et al., 1995b; Yang et al., 1993b). To test this, we preexposed cells to SES followed by exposure to solutions containing various levels of free Ca2+, with Na+ as the only other cation (see Material and Methods). As shown in Fig. 5 A, higher levels (>1 µM) of Ca2+ inhibited Na2+-ICRAC. Similar results were obtained when the Ca2+ levels were applied in a reverse order (data not shown). Fig. 5 B shows the pooled relationship of the steady-state Na+-ICRAC (measured 20 s after peak; see Fig. 1 C) and the perfusing Ca2+ levels (
1 µM) from both types of experiments. Fitting the data to a Hill equation yielded a Kd of 13.7 µM (Fig. 5 B, n = 5). This Kd reflects the affinity of the permeation site for Ca2+ to block Na+-ICRAC. Interestingly, superimposing the activation and inactivation processes of normalized Na+-ICRAC recorded in various levels of free Ca2+ (
10 µM) reveals that Ca2+ dose-dependently delayed the inactivation of Na+-ICRAC (Fig. 5 C), which is consistent with the idea that dissociation of Ca2+ from the potentiation site inactivates the Na+-ICRAC. Fig. 5 D is a plot of the pooled inactivation index of Na+-ICRAC as a function of [Ca2+] of the perfusion solutions. Fitting these data to a Hill equation yielded a Kd of 186 nM (Fig. 5 D, n = 5). This Kd seems to be a more accurate estimate of the dissociation constant of the potentiation site, because when Na+-ICRAC reaches the peak value used to compute the inactivation index (Fig.1 C), the preexposing solution has been fully washed out. Thus, it has little effect on the Ca2+ levels in the subsequent perfusion solutions. Therefore, the Ca2+ levels in this calculation are now likely to reflect the actual Ca2+ levels around the channel. Hence, this Kd reflects the affinity of the potentiation site for Ca2+, which is lower than the Kd for the Ca2+ permeation site. These results demonstrate that the affinity of the potentiation site for Ca2+ is higher than that of the permeation site, and therefore, a level of [Ca2+] can be reached such that the potentiation site is occupied by Ca2+ ions whereas the permeation site is not. Taken together, these results strongly suggest that Ca2+ ions are removed from the permeation site before they dissociate from the potentiation site during solution exchange.
Ca2+ occupancy of the potentiation site is required for the normal functioning of CRAC channels
In RBL-2H3 cells, Parekh and Penner (1997) have reported that they failed to see a similar potentiation of ICRAC by Ca2+ as reported for the Jurkat T cells (Zweifach and Lewis, 1996; Christian et al., 1996). As a result of our finding with Na+-ICRAC, we revisited the effect of the Ca2+ binding on potentiation of the Ca2+-ICRAC channels in RBL-2H3 cells. As shown in Fig. 6, A and B, when a cell was preexposed to 10 mM Ca2+, and thus the potentiation sites were presumably fully occupied by Ca2+, increasing the extracellular Ca2+ level to 50 mM rapidly increased the ICRAC due to a larger driving force. The time course of the current increase can be fitted to a single-component exponential equation with a time constant of 49.1 ± 12.5 ms (n = 8) (Fig. 6 B). In contrast, after occupying Ca2+ ions were fully washed away by DVF, an increase in the extracellular Ca2+ level to 50 mM first inhibited Na+-ICRAC and later only slowly increased the Ca2+ current (Fig. 6, A and B). The time course of this Ca2+-ICRAC increase phase could also be fitted to a single-component exponential equation but with a much longer time constant of 509.6 ± 60.9 ms (n = 6) (Fig. 6 B). These results strongly suggest that Ca2+ binding to the potentiation site also potentiates the Ca2+-ICRAC. The reason that Parekh and Penner (1997) failed to see the Ca2+ dependent potentiation of Ca2+-ICRAC is probably due to a different protocol. For example, if the rate of solution exchange is slow, such potentiation is masked (Z. Su and J. E. Blalock, unpublished observation). However, in contrast to the previous finding (Zweifach and Lewis, 1996; Christian et al., 1996) in Jurkat T cells, the CRAC channel blockers Ni2+ and Zn2+ failed to potentiate Ca2+-ICRAC in RBL-2H3 cells. The time course of Ca2+-ICRAC increase after the cells were preexposed to 10 mM Zn2+ or Ni2+ could be fitted to a two-component exponential equation with time constants 1 = 15.5 ± 2.9 s, 2 = 91.8 ± 11.5 s, n = 6 for Zn2+, and 1 = 3.6 ± 0.6 s and 2 = 69.5 ± 3.4 s, n =6 for Ni2+ (Fig. 6, C and E). These are significantly slower than the time constant when cells were preexposed to 10 mM Ca2+ ( = 49.1 ± 12.5 ms; Fig. 6 B, P < 0.001). Ba2+, Mn2+, and Mg2+ may partially potentiate Ca2+-ICRAC, because the time course of the current increase after the cell was preexposed to these ions could be fitted to a single-component exponential equation with a time constant = 3.4 ± 0.27 s, n = 6; 15.0 ± 3.2 s; n = 6; and 4.2 ± 0.4 s, n = 6, respectively. These are slower than when the cell was preexposed to Ca2+, but significantly faster than those when the cell was preexposed to Ni2+ (P < 0.001) and Zn2+ (P < 0.001). This is consistent with the finding that Ni2+ and Zn2+ could not potentiate Na+-ICRAC in RBL cells (Fig. 3) but Ba2+, Mn2+, and Mg2+ were partially effective. The rapid increase in Ca2+-ICRAC due to increased extracellular Ca2+ level when the potentiation site is fully occupied by Ca2+ suggests that Ca2+ binding to this site is necessary for the normal functioning of CRAC channels, as previously suggested for the CRAC channels in Jurkat T cells (Zweifach and Lewis, 1996; Christian et al., 1996), though slight differences clearly exist between these two cell types.
Ca2+ binding to the potentiation site is voltage dependent
We next tested the effect of transmembrane potential on Ca2+-dependent potentiation of CRAC channels by measuring the current evoked by applying an 80-ms voltage step of -80 mV from a holding potential (Vp) of 60, 0, or -60 mV at a frequency of 2 Hz. Fig. 7 A shows one of such experiments (the current displayed was taken at 70 ms after the initiation of the -80-mV voltage steps). As shown in Fig. 7, B–D, when the cell was exposed to SES, the increases in Ca2+-ICRAC evoked by the -80-mV voltage steps from the indicated holding potential could be fitted to a single-exponential equation. However, the time constants of the current increase for preholding potentials -60 and 0 are significantly faster (0.203 ± 0.013 ms, n = 10 for -60 mV; 0.263 ± 0.009 ms, n = 12 for 0 mV) than for a preholding potential of 60 mV (0.738 ± 0.035 ms, n = 14, P < 0.001). These results suggest that positive transmembrane potential do not facilitate Ca2+-ICRAC. In addition, as shown in Fig. 7, A, E, and F, under the preholding potential of 60 mV, Ca2+ only slightly potentiated Na+-ICRAC, as its inactivation index (1.4 ± 0.1, n = 5) as well as its peak value (8.2 ± 1.2 pA/pF, n = 5) was significantly smaller than the respective values for preholding potentials of 0 (2.3 ± 0.2; 10.6 ± 0.7 pA/pF, n = 5) and -60 mV (3.1 ± 0.3; 14.1 ± 1.0 pA/pF, n = 5). Data shown in Fig. 7, E and F, are pooled from experiments with different orders of voltage application to minimize the influence of the slow inactivation of ICRAC. These results suggest that at positive transmembrane potentials, the binding affinity of Ca2+ to the potentiation site is lower, and hence, little potentiation of ICRAC is seen. This result is consistent with the previous findings in Jurkat T cells by Zweifach and co-workers (Zweifach and Lewis, 1996).
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DISCUSSION |
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; Christian et al., 1996). Ba2+, Mn2+, and Mg2+ can also slightly potentiate the channel presumably by binding to the same site. That Mn2+ and Mg2+ can potentiate the CRAC channel suggests that the potentiation site is not located inside of the conducting pore, because CRAC channels do not permeate Mn2+ and Mg2+ (Figs. 1 A, 3 A, 6 C; also Hoth and Penner (1993) and Prakriya and Lewis (2002)). The potentiation site is likely located outside or at the outer mouth of the pore as suggested before for Jurkat T cells (Zweifach and Lewis, 1996; Christian et al., 1996). Several results presented here strongly support the idea that Ca2+ ions dissociate from the permeation sites (e.g., Ca2+ sites in the selectivity filter) well before they dissociate from the potentiation sites when the extracellular solution is rapidly exchanged from Ca2+-containing ones to DVF. First, the average Kd for the inhibition site (13.7 µM) is 74-fold higher than that for the potentiation site (186 nM). Second, when the Ca2+ level was not high enough (<100 µM) to completely block the CRAC channels, increase in [Ca2+] delayed the inactivation of Na+-ICRAC (Fig. 5, A and C), suggesting that the potentiation sites can be occupied by Ca2+ ions whereas the inhibition sites are not. And third, CRAC channels lose their selectivity well before undergoing a full inactivation (Fig. 1, C and D) during solution switch, suggesting that the relief of the inhibition of Na+-ICRAC by Ca2+ happens well before the loss of potentiation of Na+-ICRAC by Ca2+.
In fact, modulation of ion channels by extracellular Ca2+ is not unique to the CRAC channel. It has been reported that Ca2+ potentiates nicotinic acetylcholine receptors (nAChRs) by binding to a site on the extracellular domain of the 
-subunit of the nAChRs (Galzi et al., 1996; Le et al., 2002). Such binding greatly increases the open probability of nAChR channels and thus enhances the ensemble current, even if the unitary conductance decreases slightly (Mulle et al., 1992; Vernino et al., 1992). Armstrong and colleagues (Armstrong, 1999; Armstrong and Cota, 1991, 1999; Horn, 1999) have also demonstrated that Ca2+ occupancy of a site on the voltage-gated Na+ channels in squid giant axon is required for channel closing and that nonoccupied channels fold reversibly into a nonfunctional conformation. Ca2+ occupancy of the potentiation site of CRAC channels also seems to be required for the channels to remain in a functional conformation, and nonoccupied channels appear to fold into a less functional conformation, because without Ca2+ binding to this site the channels are not fully permeable to Ca2+ even though they are in the fully active state (Fig.6, A and B). In other words, without Ca2+ occupancy at the potentiation site, only a small fraction of CRAC channels are permeable to either Ca2+ or Na+, and/or CRAC channels have a decreased single-channel conductance or open probability. However, the extremely small single-channel conductance of CRAC channels (Zweifach and Lewis, 1993; Prakriya and Lewis, 2002) prevents a detailed experimental analysis to provide further clarity.
Although CRAC channels in Jurkat T cells and RBL cells have many similar biophysical properties including inwardly rectifying I-V relationships, high Ca2+ selectivity, fast and slow inactivations, and rapid inactivation of monovalent cation currents (Zweifach and Lewis, 1995a,b; Parekh, 1998; Fierro and Parekh, 1999), they differ in some other properties. These include pore size, permeability to other cations in the absence of Ca2+ (Bakowski and Parekh, 2002), and responses to blockers (Su et al., 2002), suggesting that the molecular identities of CRAC channels in these two cell types are different. In this study, we found that the CRAC channel blockers Zn2+ and Ni2+ failed to potentiate ICRAC in RBL-2H3 cells when either Ca2+ or Na+ were the permeating ions. This result is in contrast to findings with CRAC channels in Jurkat T cells, in which CRAC channels can be potentiated by Ni2+ as effectively as by Ca2+. This provides additional evidence to support the idea that CRAC channels in Jurkat T and RBL cells are distinct subtypes within a CRAC channel family. Currently, there is intensive interest in the molecular cloning of CRAC channels (Clapham, 1996, 2002). This analysis may need to keep in mind the possibility of several subtypes of CRAC channels in different cell types or organisms.
The potentiation of CRAC channels by Ca2+ as well as the complex three-phase kinetics of ICRAC during the solution exchange can be easily explained considering the roles of the potentiation as well as the permeation sites on CRAC channels and their different affinities for Ca2+ (Fig. 8). Under normal physiological conditions, both types of the Ca2+ sites are occupied by Ca2+ ions. The Ca2+ occupancy of the permeation site, presumably located in or near the selectivity filter as suggested for voltage-gated Ca2+ channel (Yang et al., 1993a; Ellinor et al., 1995a), prevents Na+ from entering the pore even though [Na+] is 80 times higher than [Ca2+] in the extracellular space. Ca2+ binding to the potentiation site insures the normal functioning of the channels (higher open probability or larger single-channel conductance). In the preactivation phase (phase I) of the Na+-ICRAC, the transient decrease in the current when the Ca2+-containing solution is switched to the DVF, results from the anomalous mole fraction phenomena due to the decreased driving force of the Ca2+ current, and the concomitant blockage of the Na+ current by Ca2+ binding to the permeation site. In the activation phase (phase II) when the [Ca2+] further decreases, Ca2+ falls off the permeation site due to its lower affinity whereas the potentiation site is still occupied by Ca2+ because of its higher affinity, thus a robust Na+ influx occurs, resulting in a larger current due at least to a larger unitary conductance for Na+ (Prakriya and Lewis, 2002). As time elapses, Ca2+ begins to dissociate from the potentiation site because [Ca2+] decreases further, resulting in the inactivation of current due to decreased open probability or single-channel conductance. Na+-ICRAC thus enters the inactivation phase (phase III).
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institutes of Health (grants AI37670 to J.E.B. and grants DK55647 and HL 68806 to J.E.B. and R.B.M.), Juvenile Diabetes Foundation (grant 2000-137 to R.B.M. and J.E.B), and a grant from C.C. Wu Foundation in Hong Kong (to Z.S.).
Submitted on April 11, 2003; accepted for publication October 14, 2003.
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REFERENCES |
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