Address correspondence to Richard S. Lewis, Beckman Center B-111A, Stanford University School of Medicine, Stanford, CA 94305. Fax: (650) 725-8021; E-mail: rslewis{at}stanford.edu
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ABSTRACT |
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Key Words: calcium channel calcium signaling ion/membrane channel TRP-PLIK LTRPC7
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INTRODUCTION |
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In view of their critical physiological functions, considerable effort has been focused on isolating the gene(s) encoding the CRAC channel and on understanding how the channel's gating is regulated. Although several genes of the TRP family have been proposed to encode the CRAC channel, it is unclear as to whether the currents resulting from heterologous expression of these genes are identical to native ICRAC (Putney and McKay, 1999; Clapham et al., 2001
; Prakriya and Lewis, 2002
). Several classes of activation mechanisms are currently being studied, including a diffusible messenger released from the ER, depletion-triggered insertion of CRAC channels into the plasma membrane, and physical coupling between the channels and IP3 receptors in the ER. However, there is as yet no consensus on which if any of these mechanisms may be correct (Putney et al., 2001
). Progress in resolving these issues has been hampered by several factors that are unique to this class of channels, including a lack of selective inhibitors, very small whole cell currents (on the order of a few pA per cell), and an extremely small single-channel Ca2+ conductance (226 fS estimated from noise analysis) (Zweifach and Lewis, 1993
), which has precluded single-channel recording in membrane patches.
The study of currents carried by monovalent cations through CRAC channels could, in principle, bypass some of these problems. Like voltage-gated Ca2+ channels (Almers and McCleskey, 1984; Hess et al., 1986
), CRAC channels under physiological conditions are exquisitely selective for Ca2+, conducting Ca2+
1,000 times better than the more prevalent Na+ (Hoth and Penner, 1993
; Hoth, 1995
). However, as with voltage-gated Ca2+ channels (Almers and McCleskey, 1984
), the removal of extracellular divalent cations renders CRAC channels permeable to Na+ (Hoth and Penner, 1993
; Lepple-Wienhues and Cahalan, 1996
). Initially, upon removal of extracellular divalents, this Na+ current is approximately six times larger than the preceding Ca2+ current, but it declines by >90% over tens of seconds. The slow loss of channel activity is reversed following readdition of extracellular Ca2+, a process referred to as Ca2+-dependent potentiation (CDP) (Christian et al., 1996b
; Zweifach and Lewis, 1996
). Kerschbaum and Cahalan (1998)
found that removal of intracellular Mg2+ made the Na+ current larger and sustained, leading to the idea that intracellular Mg2+ is required for CRAC channel depotentiation. Furthermore, in the absence of intracellular Mg2+ the whole-cell current was seen to arise from the progressive, all-or-none activation of single 40-pS channels having a high open probability (Po > 0.9) (Kerschbaum and Cahalan, 1999
). Similar results were later found in human T cells (Fomina et al., 2000
) and RBL cells (Braun et al., 2001
). The Na+ currents were considered to arise from CRAC channels based on their slow time course of activation and inhibition by extracellular Ca2+, Mg2+, Ni2+, and Gd3+ (Kerschbaum and Cahalan, 1999
; Fomina et al., 2000
; Braun et al., 2001
). These results are significant because resolution of CRAC currents at the single-channel level is expected to greatly facilitate studies of the molecular mechanism of store-operated Ca2+ entry. In fact, the conductance, selectivity, and high open probability of the Na+-conducting channels was exploited in studies of the CRAC channel's activation mechanism (Braun et al., 2001
; Rychkov et al., 2001
), changes in CRAC channel expression during T cell activation (Fomina et al., 2000
), and for the identification of genes that may encode the CRAC channel pore region (Yue et al., 2001
).
Although the discovery of the large, sustained mono-valent current in the absence of intracellular Mg2+ has offered new opportunities for molecular characterization of CRAC channels, we noted several discrepancies in our own studies which led us to examine its identity in greater detail. We have found that the large sustained monovalent current seen with Mg2+-free intracellular solutions arises from a store-independent channel that differs from ICRAC in its ion selectivity, pharmacology, and regulation. Because its activity is suppressed by intracellular Mg2+, we refer to this channel as the Mg2+-inhibited cation (MIC) channel. By using conditions that prevent the activation of IMIC, we have been able to characterize monovalent fluxes through CRAC channels. Several key properties of monovalent CRAC channels, including ion selectivity, unitary conductance, and regulation by intracellular Mg2+, differ significantly from those described previously. These new results reveal similarities and differences in the ionic selectivity mechanisms of store-operated and voltage-gated Ca2+ channels, and force a revision of the biophysical fingerprint of CRAC channels that will have important implications for the identification of CRAC channel genes.
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MATERIALS AND METHODS |
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Solutions and Chemicals
The standard extracellular Ringer's solution contained (in mM): 155 NaCl, 4.5 KCl, 2 or 20 CaCl2, 1 MgCl2, 10 D-glucose, and 5 Na-HEPES (pH 7.4). Ca2+-free Ringer's was prepared by substituting 1 mM EGTA + 2 mM MgCl2 for CaCl2. The divalent-free (DVF) Ringer's solutions contained (in mM): 155 Na, Cs, or NMDG methanesulfonate, and 10 HEDTA, 1 EDTA and 10 Hepes (pH 7.4 with NaOH, CsOH, or HCl, respectively). The standard internal solution contained (in mM): 150 Cs methanesulfonate, 310 mM MgCl2, 10 BAPTA, and 10 Cs-Hepes (pH 7.2). The Mg2+-free (MGF) intracellular solution contained (in mM): 150 Cs methanesulfonate, 10 HEDTA, 0.5 CaCl2 (calculated [Ca2+]i = 10 nM) and 10 Cs-Hepes (pH 7.2). Where noted, 10 mM BAPTA was substituted for HEDTA. In the experiments on ICRAC deactivation, the intracellular solution contained (in mM): 150 Cs methanesulfonate, 2 mM CsCl, 1.2 EGTA or 1 BAPTA, and 10 Cs-HEPES (pH 7.2). In the excised patch experiments, the pipette solution contained an Na-based DVF solution (composition listed above), and the cytoplasmic face of the patch was exposed to the MGF solution (listed above, but with 0 CaCl2) to which an appropriate quantity of MgCl2 added to yield the free [Mg2+] indicated in the figure legends. MgATP and Na2ATP (Sigma-Aldrich) were added to the intracellular solution in some experiments, and free [Mg2+]i and [Ca2+]i were calculated using MaxChelator software (WEBMAXC 2.10, available at http://www.stanford.edu/~cpatton/webmaxc2.htm).
2-aminoethyldiphenyl borate (2-APB) was provided by Dr. K. Mikoshiba (Tokyo University, Japan). In some experiments, 2-APB obtained from Sigma-Aldrich was used; no difference was found between the drugs from the different sources. Stock solutions of 2-APB and thapsigargin (Sigma-Aldrich) were prepared in DMSO at concentrations of 20 mM and 1 mM, respectively; SKF 96365 (Sigma-Aldrich) was dissolved in deionized water at a concentration of 10 mM. The drugs were diluted to the concentrations indicated in the legends and applied to the cells using a multi-barrel local perfusion pipette with a common delivery port. The time for 90% solution exchange was measured to be <1 s, based on the rate at which the K+ current reversal potential changed when the external [K+] was switched from 2 to 150 mM.
Patch-Clamp Measurements
Patch-clamp experiments were conducted in the standard whole-cell recording configuration at 2225°C using an Axopatch 200 amplifier (Axon Instruments, Inc.), an ITC-16 interface (Instrutech) and a Macintosh G3 computer. Recording electrodes were pulled from 100-µl pipettes coated with Sylgard and fire-polished to a final resistance of 25 M. Stimulation and data acquisition and analysis were performed using in-house routines developed on the Igor Pro platform (Wavemetrics). The holding potential was 20 mV unless otherwise indicated. Voltage stimuli usually consisted of a 100-ms step to -110 mV, immediately followed by a 100-ms ramp from -110 to 90 mV applied every 12 s. Currents were filtered at 1 kHz with a 4-pole Bessel filter and sampled at 5 kHz without series resistance compensation. Data are corrected for the liquid junction potential of the pipette solution relative to Ringer's in the bath (-10 mV) and of the bath DVF solution relative to Ringer's in the bath-ground agar bridge (5 mV). The averaged results are presented as the mean value ± SEM. Curve fitting was done by least-squares methods using built-in functions in Igor Pro 4.0. For the analysis of MIC channel kinetics, excised patches showing the activity of only one MIC channel were selected and processed using TAC (Bruxton Corporation). Channel transitions were idealized by setting a discriminator at 50% of the current between the open and closed levels, and the channel kinetics were obtained from the idealized traces.
Leak Current Subtraction
The activity of MIC channels when [Mg2+]i is 3 mM (conditions of the majority of published papers on ICRAC) can pose special problems for the isolation of ICRAC in Jurkat cells. In our experience, MIC activity often changes significantly during the course of whole-cell recording (depending, among other things, on the free [Mg2+] in the pipette). Thus, the commonly employed practice of subtracting the current present at the start of whole-cell recording (before ICRAC induction) from later currents will not necessarily isolate ICRAC cleanly. A more consistent leak subtraction method is to expose the cell to a Ca2+-free extracellular solution (0 Ca2+/3 Mg2+) shortly before or after measurement of the Ca2+ current, and to use this current as the "leak," because these ionic conditions eliminate ICRAC (Zweifach and Lewis, 1993
), but not IMIC (see Fig. 7 C). Therefore, we used the zero-Ca2+ leak subtraction method to isolate ICRAC in the experiments reported here. For measurements of IMIC, leak current was collected in 20 mM Ca2+ immediately after whole-cell break-in.
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RESULTS |
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In previous studies, the large, sustained monovalent current and the underlying 40-pS single-channel currents were ascribed to the activity of CRAC channels (Kerschbaum and Cahalan, 1998, 1999
; Fomina et al., 2000
; Braun et al., 2001
). However, we noted several discrepancies in our own experiments that led us to question this conclusion. First, monovalent inward current under Mg2+i-free conditions activated much more slowly than the Ca2+ current in the presence of 20 mM extracellular Ca2+, with half times of 210 ± 27 s (n = 5) and 109 ± 16 s (n = 6), respectively. Second, the ratio of ICa to INa varied widely among cells (from 13 in Fig. 1 A to more than 100), even to the point where monovalent currents in the nA range occurred in some cells lacking a measurable Ca2+ current. Finally, using a DVF pipette solution we observed 40-pS channels in the majority of cell-attached patches from resting cells in which stores were not deliberately depleted, suggesting that the large monovalent current may be store-independent. To determine whether it does in fact reflect CRAC channel activity, we compared its dependence on store depletion and its pharmacological profile with that of ICRAC.
The Large Monovalent Current Does Not Activate and Deactivate in Parallel With ICRAC
If the large monovalent current flows through CRAC channels, then it should be active immediately upon establishing the whole-cell recording configuration in cells with empty Ca2+ stores. To test this, 1 µM thapsigargin (TG) was applied for 510 min before seal formation to fully deplete stores and maximally activate CRAC channels. Soon after break-in to the whole-cell configuration, application of 20 mM Ca2+ to the cell causes an inward current to develop over 10 s (Fig. 2 A). This inward current is ICRAC as judged from its current-voltage relationship (Fig. 2 D, top graph), Ca2+o- and store-dependence, sensitivity to various pharmacological agents (unpublished data), and characteristic delayed appearance following each application of Ca2+. This latter process, termed CDP, has been described previously (Zweifach and Lewis, 1996
). Following Ca2+ readdition, the first application of DVF solution revealed only a small, transient inward Na+ current at -110 mV that declined >60% within 20 s (arrow, Fig. 2 A). Subsequent short applications of DVF solution revealed the slow development of a large Na+ current over the next 400 s, shown more clearly at lower gain (Fig. 2 A, right graph). Two observations suggest that the large Na+ current and ICRAC are not related. First, the amplitudes of the inward Ca2+ and Na+ currents did not increase in parallel (Fig. 2 B), causing the ratio of the Na+ to the Ca2+ current to change from
2:1 at the beginning of the experiment (t = 37 s) to
50:1 at later times (t = 570 s). Thus, the amplitudes of the Ca2+ current and the large Na+ current are not well correlated in time. Second, during each application of DVF, the large Na+ current was roughly constant, even though CRAC channels appeared to be closing. This is shown by the small initial Ca2+ current seen immediately after each Ca2+ readdition, followed by a prominent increase due to CDP. Similar results were obtained in five cells.
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To distinguish between these possibilities, we asked whether the large monovalent conductance deactivates in parallel with ICRAC in response to store refilling. We have shown previously that in the presence of weak intracellular Ca2+ buffering (1 mM EGTA in the pipette solution), prolonged Ca2+ influx through CRAC channels can overwhelm the buffer, causing a global rise of [Ca2+]i, store refilling, and deactivation of ICRAC (Zweifach and Lewis, 1995). This behavior is illustrated in the experiment of Fig. 3 A (left graph), in which ICRAC measured in the presence of 20 mM Ca2+ activated slowly in response to passive store depletion, rose to a peak, then declined over
150 s back to baseline. DVF solution was applied periodically in this experiment to monitor the large monovalent current. The monovalent current did not decline in parallel with ICRAC, but instead grew progressively larger over 400 s (Fig. 3 A, right). A plot of the time courses of the Ca2+ current and the Na+ current in this cell shows clearly that the amplitude of the large Na+ current increases even as CRAC channels close (Fig. 3 B). Similar results were observed in 5/5 cells. Together with the changes in ion selectivity and kinetic behavior noted above, these results argue strongly that the large monovalent current arises from store-independent channels distinct from the CRAC channel.
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In sum, multiple independent lines of evidence demonstrate that the large monovalent current evoked in the absence of extracellular divalent ions and intracellular Mg2+ does not flow through CRAC channels. The large monovalent current is not store-dependent, its time course is independent of changes in CRAC channel activity, it is pharmacologically distinct from ICRAC and its expression is regulated independently of CRAC in mutant cells. In the following section we examine the properties of the large monovalent conductance and describe methods for inhibiting it so that monovalent currents through CRAC channels can be measured in isolation.
The Large Monovalent Current Is Inhibited by Intracellular Mg2+
The induction of the large monovalent current by intracellular pipette solutions lacking Mg2+ suggests that it may be activated as intracellular Mg2+ is dialysed out of the cell. We obtained direct support for this idea at both the whole-cell and single-channel levels. Addition of 8 mM MgCl2 to the whole-cell pipette solution completely suppressed the activation of the large monovalent current under DVF conditions (Fig. 6 A) with an IC50 of 0.6 mM (Fig. 6 B). Intracellular Mg2+ also inhibited the development of the outwardly rectifying current seen in 20 mM Ca2+o (Fig. 2 D) with about the same efficacy (Fig. 6 B), suggesting that both currents arise from the same channel.
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To isolate IMIC for further characterization, ICRAC was blocked irreversibly by pretreatment with 100 µM 2-APB for 15 min. Following break-in with a Mg2+-free internal solution, ramp currents were recorded while switching between 20 mM Ca2+ and DVF Ringer's solutions. Under these conditions, IMIC (observed during brief applications of DVF Ringer's) developed in parallel with an outwardly rectifying current (observed during applications of 20 Ca2+ Ringer's) (Fig. 7, A and B). Similar behavior was seen in 5/5 cells. As with IMIC, the outwardly rectifying current was not observed when 810 mM Mg2+ was included in the pipette solution (unpublished data), supporting the idea that this current is conducted by MIC channels.
Under DVF conditions, MIC currents reverse at -6 ± 2 mV (n = 5), indicating a PCs/PNa ratio of 1.2. In the presence of extracellular divalent ions, however, the permeability of MIC channels is complex. Addition of divalent ions transforms the linear I-V relation in DVF conditions to a outwardly rectifying one, suggesting that external divalent ions block monovalent current flow in a voltage-dependent manner. IMIC reverses around 0 mV under these conditions also (Fig. 7), consistent with a lack of selectivity among cations. Our preliminary results from ion substitution experiments indicate that Na+, Ca2+, and Mg2+ all carry current through MIC channels at negative potentials; however, Mg2+ and Ca2+ also block the passage of Na+, making it difficult on the basis of ion substitution experiments to determine the exact proportion of current carried by each ionic species. For this reason, we did not characterize the permeation properties of MIC channels further, but instead focused on ways to prevent contamination of ICRAC measurements with IMIC. Importantly, the inward MIC current is unaffected by switching from 20 mM Ca2+o to a Ca2+-free Ringer's solution containing 2 mM Mg2+ (Fig. 7 C). Therefore, for purposes of isolating ICRAC at negative potentials, leak subtraction using currents obtained in a Ca2+-free solution effectively removes the contaminating current arising from MIC channels (see MATERIALS AND METHODS).
A recently cloned member of the TRP family of ion channels, called TRP-PLIK (Runnels et al., 2001) or LTRPC7 (Nadler et al., 2001
), has an ionic selectivity and current-voltage relation quite similar to that of MIC. LTRPC7 is expressed in Jurkat cells and is inhibited by intracellular Mg2+ as well as by intracellular MgATP (Nadler et al., 2001
). Likewise, we found that MgATP at concentrations of 4 mM and higher in the intracellular pipette solution completely inhibited the development of IMIC (Fig. 6 C), whereas recordings with 3 mM MgATP exhibited sporadic MIC channel activity. Our combined observations suggest that IMIC is mediated by TRP-PLIK/LTRPC7 channels, and is probably identical to an outwardly rectifying MgATP-sensitive current (called MagNuM, for Mg2+ nucleotideregulated metal) that has been reported in Jurkat, RBL, and HEK293 cells (Nadler et al., 2001
).
Isolating the Monovalent Current through CRAC Channels
Inhibition of IMIC by intracellular Mg2+ offers a convenient strategy for isolating the monovalent current through CRAC channels for characterization. With 8 mM intracellular Mg2+ to suppress IMIC, passive depletion of Ca2+ stores by intracellular BAPTA (10 mM) slowly evoked ICRAC in the presence of 20 mM Ca2+ (Fig. 8 A). Periodic exposure to DVF solution revealed a monovalent current which developed with the same time course as ICRAC (Fig. 8 B). Similar results were seen in 7/7 cells. The monovalent current was transient during each DVF episode, peaking immediately after solution exchange and subsequently declining with a time constant of 10 s. The ratio of the peak amplitude of the Na+ current to ICRAC measured just before solution exchange was 7.8 ± 0.4 (n = 17).
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The pharmacological profile of the transient Na+ current also closely matched that of ICRAC. As shown in Fig. 9 A, 20 µM SKF 96365 inhibited both currents to similar extents. On average, ICRAC was reduced by 84 ± 3% (n = 4), whereas the inactivating Na+ current was reduced by 74 ± 3% in the same cells. Moreover, 5 µM 2-APB potentiated both currents, enhancing ICRAC by 241 ± 41% (n = 7) and the peak amplitude of the transient Na+ current in the same cells by 185 ± 31% (Fig. 9 B). 2-APB also enhanced the steady-state component of the Na+ current by a similar amount (224 ± 26%; n = 6). These results suggest that the residual current remaining after the Na+ current has declined is also due to CRAC channels, a conclusion that is consistent with the observation that the peak and the residual currents reverse at the same potential (see below).
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Based on these results as well as the evidence given above that ICRAC and the transient Na+ current activate and deactivate in parallel and are store-dependent, we conclude that 810 mM intracellular Mg2+ suffices to isolate the monovalent current through CRAC channels in the absence of extracellular divalent ions. For convenience, the inward Na+ and Ca2+ currents through CRAC channels will hereafter be referred to as Na+-ICRAC and Ca2+-ICRAC, respectively.
The Monovalent Selectivity of CRAC Channels Is Not Influenced by Intracellular Mg2+
The identification of a Mg2+-sensitive monovalent current raises new questions about the possible role of Mg2+ in shaping the properties of CRAC channels. Previous studies have concluded that intracellular Mg2+ blocks permeation of Cs+ through CRAC channels under DVF conditions (Kerschbaum and Cahalan, 1998). Using the methods described above for separating IMIC from ICRAC, we reassessed the role of Mg2+ in CRAC channel selectivity.
Fig. 10 A shows the transient monovalent CRAC current with 8 mM Mg2+i and with equimolar Cs+ and Na+ as the principal intracellular and extracellular cations, respectively. The I-V relations recorded as the current depotentiated show pronounced inward rectification and intersect at a single potential. This supports the idea that the whole-cell current decreases due to the closure of a single channel type, i.e., CRAC channels, and that the residual steady-state current is also due to CRAC channels as discussed above. The crossover point in eight cells was 52 ± 2 mV, implying that CRAC channels under DVF conditions are only weakly permeable to Cs+ (PCs/PNa = 0.13 based on the Goldman-Hodgkin-Katz equation). A similar reversal potential was obtained from single ramp currents after subtraction of leak current recorded in 0-Ca Ringer's (unpublished data). Thus, unlike MIC channels, CRAC channels in the absence of divalent cations select strongly against Cs+ (compare Fig. 7 B). Consistent with this conclusion, replacement of extracellular Na+ with Cs+ eliminates nearly all of the inward monovalent current through CRAC channels (Fig. 10 B).
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Depotentiation of CRAC Channels Is Independent of Intracellular Mg2+
The activity of CRAC channels slowly declines by up to 80% after removal of extracellular Ca2+, and recovers over 1020 s after its reapplication by a process referred to as Ca2+-dependent potentiation, or CDP (Christian et al., 1996b; Zweifach and Lewis, 1996
). Thus, the current's decline following the removal of extracellular divalents reflects depotentiation. Previous findings that transient monovalent currents (thought to be through CRAC channels) became large and sustained in the absence of intracellular Mg2+ led to the suggestion that internal Mg2+ is required for the depotentiation process, and that CDP may arise from the expulsion of Mg2+ ions from the pore of CRAC channels by permeant Ca2+ ions (Kerschbaum and Cahalan, 1998
). Because IMIC was likely mistaken for ICRAC in the absence of intracellular Mg2+, we reexamined the role of Mg2+ in CRAC channel depotentiation under conditions that prevent activation of IMIC.
To effectively inhibit IMIC under conditions of low intracellular Mg2+, we applied MgATP in combination with Na2ATP to reduce free [Mg2+]i. 68 mM MgATP + 26 mM Na2ATP in the pipette solution inhibited IMIC while reducing free [Mg2+]i to calculated values of 92185 µM (see MATERIALS AND METHODS). Under these conditions, Na+-ICRAC still depotentiated after removal of extracellular divalents (Fig. 11 A), and the I-V relationship of the current was similar to that recorded with 8 mM Mg2+i. Neither the time course (Fig. 11 C) nor the amplitude of depotentiation were altered relative to cells with 8 mM Mg2+i; the fraction of current remaining after 50 s of DVF application was 20 ± 4% (n = 7) with 131 µM Mg2+i vs. 18 ± 2% (n = 8) with 8 mM Mg2+i. Finally, if depotentiation were to arise from Mg2+ binding in the pore of the CRAC channel and within the membrane field, hyperpolarization would be expected to inhibit it by reducing Mg2+ binding. However, shifting the holding potential from 40 mV to -110 mV did not significantly alter the rate of depotentiation at either high (610 mM) or low (131 µM) levels of Mg2+i (Fig. 11, B and C), even though the maximum amplitude of Na+-ICRAC was increased, consistent with the voltage dependence of CDP (Zweifach and Lewis, 1996
). Together, these results argue against a necessary role for intracellular Mg2+ in the depotentiation of CRAC channels.
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Estimating the Unitary Na+ Current through CRAC Channels by Noise Analysis
The finding that the 40-pS monovalent channels seen under DVF conditions are MIC channels raises new questions about the unitary monovalent conductance of CRAC channels. Under DVF conditions with 10 mM intracellular Mg2+ to inhibit IMIC, we were unable to detect any clear single-channel transitions of amplitude >0.4 pA during the induction of Na+-ICRAC by ionomycin in 5/5 cells and during the or depotentiation of Na+-ICRAC. This contrasts with the obvious appearance of single-channel MIC currents when intracellular Mg2+ is washed out during whole-cell recording (Fig. 1). Therefore, we applied stationary fluctuation analysis to estimate the single-channel monovalent CRAC conductance.
Cells were treated with 1 µM TG to activate CRAC channels, and after establishing whole-cell recording the mean macroscopic current (I) and corresponding variance (I2) were measured at a holding potential of -110 mV. Application of 5 µM 2-APB enhanced ICRAC in the presence of 20 mM Ca2+ and caused a barely detectable increase in current noise (Fig. 12 A). Subsequent removal of divalent cations (DVF conditions) evoked a transient Na+ current through CRAC channels in parallel with a more robust increase in noise. Several 200-ms current sweeps used to compute the current mean and variance for the DVF condition are shown in Fig. 12 B, and indicate the lack of any clear single-channel transitions as the current declines. Plots of the current variance against mean current amplitude were well fitted by straight lines (Fig. 12, C and D). The average slope (
I2/I) was -3.8 ± 0.6 fA (n = 5) in 20 mM Ca2+, and -31 ± 2 fA (n = 10) under DVF conditions.
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Given these assumptions, the linear shape of the I2/I relation implies that the open probability of the channel is very low (<0.5) and that the unitary current amplitude is given by the slope (Eq. 1). Thus, the results would suggest that the unitary amplitude of monovalent ICRAC at -110 mV is -31 fA; given the reversal potential of 52 mV, this corresponds to a single-channel chord conductance of
0.2 pS.
There are several ways in which fluctuation analysis may seriously underestimate the unitary conductance. First, the bandwidth of the recording may not extend to high enough frequencies to capture the fluctuations due to very brief gating events. To test this, we analyzed the power spectrum of the current noise under DVF conditions. Fig. 12 E shows the background spectrum obtained in 0 Ca2+ + 2 mM Ni2+, together with the spectrum of Na+-ICRAC at its peak and after it depotentiated to a steady level. In both cases, the noise approaches the background level at frequencies above 1 kHz, suggesting that no large component of high-frequency noise was missed under the 12 kHz filtering conditions of the current variance experiments.
Second, noise measurements may underestimate the size of i if Po is high and the macroscopic current amplitude reflects changes in N, the number of activatable channels, rather than Po (Jackson and Strange, 1995). In this case,
I2 will increase linearly with N:
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DISCUSSION |
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MIC and CRAC Currents Arise from Distinct Channels
A number of characteristics distinguish MIC channels from CRAC channels. These include the mode of activation, inhibition by Mg2+ or MgATP, kinetic properties, selectivity, pharmacology, and unitary conductance. These differences are summarized in Table I and are discussed below.
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Following removal of extracellular divalents, Na+-ICRAC is transient (Fig. 10 A), whereas IMIC is relatively sustained (Fig. 5 C). The peak phase of the Na+ current is linked to CRAC channels, based on its close correlation with Ca2+-ICRAC during store depletion and refilling (Fig. 8), and its sensitivity to SKF 96365 and 2-APB (Fig. 9). Na+-ICRAC depotentiated in DVF solutions to an average level of 20%. We believe the steady-state activity is also from CRAC channels because its amplitude varies in parallel with the peak Na+-ICRAC and it has the same reversal potential, relative Cs+ permeability and sensitivity to SKF 96365 and 2-APB, as the peak current. Although IMIC is generally sustained under Mg2+i-free conditions after application of DVF solutions, it declines slowly in the presence of low levels of Mg2+i (0.51 mM; unpublished data). Thus, current decline under DVF conditions cannot be used as a specific indicator for ICRAC.
Finally, the pharmacological signatures of ICRAC and IMIC also differ significantly (Figs. 4 and 9). SKF 96365 at micromolar levels is an effective blocker of ICRAC (Franzius et al., 1994; Christian et al., 1996a
), but not of IMIC. High concentrations of 2-APB (2040 µM) inhibit ICRAC completely and irreversibly (Prakriya and Lewis, 2001
), whereas they cause modest and rapidly reversible inhibition of IMIC. Low concentrations of 2-APB (
5 µM) which strongly enhance ICRAC (Prakriya and Lewis, 2001
) fail to affect IMIC.
The rather large number of properties that distinguish the two conductances strongly suggest that ICRAC and IMIC arise from two different channels. However, an alternative explanation might be that removal of intracellular Mg2+ merely alters the properties of CRAC channels, making them acquire the characteristics of what we now call MIC channels (Kerschbaum and Cahalan, 1998). We do not believe that the MIC channel is an altered state of the CRAC channel for several key reasons. First, it seems unlikely that removal of Mg2+ alone would be able to alter such a diverse set of properties, including store-dependence, depotentiation, ion selectivity, pharmacological profile, and unitary conductance. Second, we find that removal of intracellular Mg2+ does not in fact reduce the selectivity of CRAC for Na+ over Cs+ (Fig. 10 C), nor does it prevent depotentiation of CRAC channels (i.e., make CRAC activity sustained) under 0-Ca2+o conditions (Figs. 2 A, 3 A, and 11 D). Finally, MIC channel activity is normal in mutant Jurkat cells that have very low levels of ICRAC. Thus, the most parsimonious explanation for these data is that MIC and CRAC channels are separate and distinct proteins.2
In light of our results, various conclusions made in previous reports regarding the regulation of CRAC channels probably apply more to MIC channels than to CRAC channels. For example, the voltage-dependent block of monovalent CRAC currents by extracellular Ca2+ (Kerschbaum and Cahalan, 1998; Fomina et al., 2000
), the upregulation of CRAC channels in activated T cells (Fomina et al., 2000
), and regulation by cytoplasmic Mg2+ and Ca2+ (Braun et al., 2001
) were all measured under conditions optimized for activation of IMIC. These issues may have to be revisited using recording conditions explicitly optimized for isolating the monovalent CRAC current. No specific inhibitors of IMIC are yet known, but high intracellular Mg2+ (810 mM) or MgATP (>4 mM) effectively blocks IMIC without affecting ICRAC. It is important to note that moderate levels of Mg2+ (23 mM) commonly used in studies of ICRAC may not be sufficient to completely eliminate MIC activity, as we often found 35 active MIC channels in Jurkat cells under these conditions. Although this represents a small fraction of the total MIC conductance in the cell, the unitary current of MIC channels is so much larger than that of CRAC channels that it can seriously contaminate noise measurements under DVF conditions (see below). Activity of MIC channels can also lead to inadvertent contamination of ICRAC in the presence of divalent ions, although appropriate subtraction of the "leak" current can alleviate this problem (see MATERIALS AND METHODS).
IMIC May Arise from TRP-PLIK/LTRPC7 Channels
Given that MIC channels are distinct from CRAC, what is their molecular identity? Recently, a novel member of the TRP family of ion channels has been cloned which has been named TRP-PLIK (Runnels et al., 2001) or LTRPC7 (Nadler et al., 2001
). Several characteristics of TRP-PLIK/LTRPC7 are similar to MIC in Jurkat cells. LTRPC7 is expressed in cell lines commonly employed for studies of ICRAC, such as Jurkat T cells and RBL cells. Activation of LTRPC7 in transfected cells is elicited by whole-cell dialysis with chelators of Mg2+, and introduction of Mg2+ and/or Mg-ATP inhibits channel activity (Nadler et al., 2001
). TRP-PLIK/LTRPC7 produces a nonselective cation channel with strong outward rectification in the presence of extracellular divalent ions (Nadler et al., 2001
; Runnels et al., 2001
), much like the outward rectification of IMIC we observe under similar conditions (Fig. 7). Elimination of extracellular Ca2+ or Na+ alone does not affect the inward current through LTRPC7 (Nadler et al., 2001
) or MIC channels (Fig. 7 C and unpublished data). Together, these similarities to IMIC in Jurkat cells suggest that TRP-PLIK/LTRPC7 encodes the MIC channel.
The activation mechanism and physiological roles of MIC channels are not well understood at this point. A small number of MIC channels appear to be active in resting Jurkat cells, based on the current seen in DVF conditions immediately after break-in with 0-Mg2+ pipette solution (Fig. 6 A). Although depletion of cytoplasmic Mg2+ can further activate MIC channels, it seems unlikely that this is the physiological stimulus (see Nadler et al., 2001), as cytosolic Mg2+ in many cells is held relatively constant between 0.5 and 1 mM (Romani and Scarpa, 2000
). Its sensitivity to inhibition by Mg2+-nucleotides has led to the hypothesis that it opens in response to declining ATP levels (Nadler et al., 2001
), but there is also evidence that channel activation requires kinase activity of the COOH-terminal PLIK domain (Runnels et al., 2001
). The physiological consequences of MIC activation are also unclear. Nadler et al. (2001)
have proposed that LTRPC7 provides a conduit for Ca2+ entry that regulates mitochondrial activity and ATP homeostasis (Nadler et al., 2001
). However, the precise Ca2+ permeability of this channel has not been measured. Although it has been stated that the block of Na+ flux by external divalent ions renders LTRPC7 solely permeable to divalent ions at negative potentials (Nadler et al., 2001
), our preliminary experiments on MIC channels in Jurkat cells suggest that Na+ can carry inward current in the presence of Mg2+o (unpublished data). Thus, while MIC channels may provide a pathway for Ca2+ entry, they may also depolarize the cell by conducting monovalent ions. From the relative amplitudes of the single-channel and whole-cell currents we estimate that Jurkat cells express
250500 MIC channels per cell.
Intracellular Mg2+ and CRAC Channel Function
In previous studies, the removal of intracellular Mg2+ under DVF conditions appeared to change a small transient and Na+-selective current through CRAC channels into a much larger, sustained, and nonselective current with equal permeability to Cs+ and Na+ (Lepple-Wienhues and Cahalan, 1996; Kerschbaum and Cahalan, 1998
). These observations were interpreted to mean that Mg2+i removal prevents depotentiation of CRAC and alters their selectivity. We have found that intracellular Mg2+ does not affect Ca2+-dependent potentiation of CRAC channels, or the reverse process of depotentiation (Fig. 11), nor is it required to achieve selectivity for Na+ over Cs+ (Fig. 10 C). This discrepancy with the earlier results can now be explained by the fact that removal of Mg2+i activates IMIC, which is much larger, more sustained, and less selective than ICRAC.
The Unitary Conductance of CRAC Channels
Our results indicate that the monovalent conductance of CRAC channels is too small to be resolvable in whole-cell recordings. Noise analysis of whole-cell Na+-ICRAC suggests a conductance in the range of 0.2 pS. This is dramatically different from recent reports in T cells and RBL cells, where a conductance of
40 pS was reported for monovalent currents through CRAC channels (Kerschbaum and Cahalan, 1999
; Fomina et al., 2000
; Braun et al., 2001
). This discrepancy is explained by the fact that the earlier experiments were performed in the absence of cytoplasmic Mg2+, which as we have shown here activates store-independent MIC channels. Our estimate of the unitary conductance is also smaller than an earlier estimate of 2.6 pS based on noise analysis (Lepple-Wienhues and Cahalan, 1996
). Using the ionic conditions of this earlier study (3 mM intracellular Mg2+ + 1 µM extracellular Mg2+), we also detect a significantly larger noise, which appears to arise from flickery block of several active MIC channels in the cell. Thus, even slight contamination of Na+-ICRAC with IMIC may lead to large differences in the estimated unitary currents.
We considered several possible factors that could lead us to underestimate the unitary currents. Current fluctuations could be missed if they occur at frequencies outside the recording bandwidth. However, spectral analysis showed that most of the power of the current noise occurred at frequencies <1 kHz, well within the recording bandwidth (Fig. 12 E). A more difficult problem could arise if activation of ICRAC occurs through an increase in N, the number of activatable channels, each of which has a high open probability and therefore contributes little noise. However, we were unable to detect discrete jumps in current during the activation or depotentiation of monovalent ICRAC. In addition, partial inhibition of monovalent ICRAC with 1 µM extracellular Ca2+ did not increase the noise, indicating that whatever the mechanism of activation, the Po of active CRAC channels conducting Na+ is quite small. Together with our estimates of the single channel conductance, these observations set limits on the maximum size of the single-channel conductance to <1 pS.
Our estimate of -3.8 fA at -110 mV for the unitary Ca2+ current compares well with a previous estimate in 110 mM Ca2+o at -80 mV (-3.7 fA) (Zweifach and Lewis, 1993). Note that this estimate is roughly eightfold smaller than the single-channel estimates of the Na+ current given above (-31 fA). The fact that the whole-cell Na+-ICRAC is also about eightfold larger than Ca2+-ICRAC suggests that the increase in whole-cell current seen upon exchanging the Ca2+o solution for the DVF solution can entirely be accounted for by the increase in conductance of the CRAC channels without a significant change in channel Po. From the ratio of the unitary and whole-cell peak Na+ currents, we estimate that the number of CRAC channels per cell is at least 5,00010,000. More would be expected if the open probability is low, as is suggested by the linear relation of variance to mean current. Finally, it should be noted that our estimate for the unitary conductance of Na+-ICRAC (0.2 pS) is significantly smaller than the 42-pS conductance reported for the CaT1 channel under similar DVF conditions (Yue et al., 2001
), consistent with recent suggestions that CaT1 may not comprise the complete CRAC-channel pore (Voets et al., 2001
).
Selectivity of CRAC Channels
Similarities and differences between the permeation properties of CRAC channels and voltage-gated Ca2+ (CaV) channels have interesting implications for mechanisms of ion selectivity in CRAC channels. Under physiological conditions, CaV channels have an extremely high selectivity for Ca2+ over monovalent ions (PCa/PNa > 1,000). The high Ca2+ selectivity is thought to arise from the high-affinity binding of Ca2+ within the pore, which prevents Na+ from conducting. In fact, the removal of extracellular divalent ions allows Na+ to conduct freely (Almers and McCleskey, 1984; Hess and Tsien, 1984
). CRAC channels show a comparably high selectivity for Ca2+ over monovalent ions (Hoth and Penner, 1993
), and similarly become freely permeable to Na+ in the absence of extracellular divalents. Thus, it is likely that the Ca2+ selectivity of CRAC channels also arises from high affinity Ca2+ binding within the pore, and this is consistent with the ability of 1 µM Ca2+ to block Na+-ICRAC by
50% (see RESULTS).
The most obvious differences between the two channels relates to their unitary conductances and permeability to Cs+. CaV channels have single-channel conductances for Ca2+ in the range of 520 pS and 85 pS for Na+ under DVF conditions (Hess et al., 1986
). By contrast, the unitary conductances of CRAC channels is estimated to be
21 fS for Ca2+ and 0.2 pS for Na+, or
500-fold smaller than CaV channels. In addition, under DVF conditions CaV channels readily pass Cs+ (PCs/PNa
0.6; Hess et al., 1986
), whereas CRAC channels do not (PCs/PNa = 0.13; Fig. 10; see also Lepple-Wienhues and Cahalan, 1996
). These observations suggest that although CRAC channels and voltage-gated Ca2+ channels achieve selectivity for Ca2+ over other ions by high affinity Ca2+ binding within the pore, many structural features may be significantly different between these two channel types.
What unique structural features of CRAC channels could give rise to such a low conductance? A popular model proposes that the high throughput of CaV channels arises from ionion interactions as ions move single-file through the pore (Tsien et al., 1987). Thus, one possibility is that weak ionion interactions underlie the low flux rate of Ca2+ through CRAC channels. However, this idea cannot adequately explain why CRAC channels would have an equally low conductance for monovalent ions. We hypothesize that CRAC channels possess a nonselective rate-limiting barrier to ion permeation in series with the selectivity filter. Such a barrier may explain not only the uniformly low permeability to various ions, but also the lack of permeability to Cs+. Identification of CRAC channel genes will provide much needed tools for elucidating the mechanism of CRAC channel permeation. In the interim, systematic examination of permeation of various ions through native CRAC channels will improve our understanding of the properties of the channel pore.
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FOOTNOTES |
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ACKNOWLEDGMENTS |
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This work was supported by a postdoctoral fellowship from the Irvington Foundation for Immunological Research to M. Prakriya and National Institutes of Health grant GM45374 to R.S. Lewis.
Submitted: 27 December 2001
Revised: 1 April 2002
Accepted: 3 April 2002
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REFERENCES |
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