Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Abstract Full Text (PDF, 1639K) PPT slides of all figures Correction (v119,p613) Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JGP Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Prakriya, M. Articles by Lewis, R. S. Search for Related Content PubMed PubMed Citation Articles by Prakriya, M. Articles by Lewis, R. S. Social Bookmarking                      What's this?

Article

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305

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

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Although store-operated calcium release–activated Ca²⁺ (CRAC) channels are highly Ca²⁺-selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na⁺ and Cs⁺ with a unitary conductance of 40 pS, and that intracellular Mg²⁺ modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg²⁺-inhibited cation (MIC) channels that open as Mg²⁺ is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8–10 mM intracellular Mg²⁺ to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca²⁺ to divalent-free extracellular solution evokes Na⁺ current through open CRAC channels (Na⁺-I_CRAC) that is initially eightfold larger than the preceding Ca²⁺ current and declines by 80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs⁺ (P_Cs/P_Na = 0.13 vs. 1.2 for MIC). Neither the decline in Na⁺-I_CRAC nor its low Cs⁺ permeability are affected by intracellular Mg²⁺ (90 µM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca²⁺ channels.

Key Words: calcium channel • calcium signaling • ion/membrane channel • TRP-PLIK • LTRPC7

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The depletion of Ca²⁺ from the ER in nonexcitable cells triggers Ca²⁺ influx across the plasma membrane, a process termed store-operated Ca²⁺ entry. Store-operated channels (SOCs)^* are widely, if not ubiquitously, expressed in nonexcitable cells, where they provide a major route for Ca²⁺ entry (for reviews see Parekh and Penner, 1997; Lewis, 1999; Putney and McKay, 1999). One of the best described SOCs, the calcium release–activated Ca²⁺ (CRAC) channel, is present in mast cells, T lymphocytes, and several related cell lines. CRAC channels influence a variety of important physiological processes, including the release of inflammatory mediators from mast cells during allergic reactions, and the generation of [Ca²⁺]_i oscillations leading to gene expression and the differentiation and activation of T cells (for reviews see Parekh and Penner, 1997; Lewis, 2001). The critical role of CRAC channels in human physiology is shown clearly by the devastating immunodeficiencies that arise from the absence of CRAC channel activity in T cells from human patients (Partiseti et al., 1994; Feske et al., 2001).

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 I_CRAC (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 (2–26 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 (P_o > 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 I_CRAC 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 I_MIC, 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.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cells
Jurkat E6-1 human leukemic T cells (American Type Culture Collection) were grown in a medium consisting of RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin. The cells were maintained in log-phase growth at 37°C in 6% CO₂.

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, 3–10 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 I_CRAC 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 22–25°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 2–5 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 1–2 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 I_CRAC) can pose special problems for the isolation of I_CRAC 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 I_CRAC induction) from later currents will not necessarily isolate I_CRAC 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 I_CRAC (Zweifach and Lewis, 1993), but not I_MIC (see Fig. 7 C). Therefore, we used the zero-Ca²⁺ leak subtraction method to isolate I_CRAC in the experiments reported here. For measurements of I_MIC, leak current was collected in 20 mM Ca²⁺ immediately after whole-cell break-in.

View larger version (13K): [in this window] [in a new window]   FIGURE 7. Effects of extracellular divalent cations on MIC current. The cell was treated with 100 µM 2-APB for 15 min before seal formation to irreversibly inhibit ICRAC. Data are not corrected for leak current. Internal solution: MGF. Extracellular solution was alternated between 20 mM Ca2+ and DVF Ringer's as currents were measured in response to voltage ramps from -110 to 90 mV. (A) Parallel activation of inward Na+ current at -110 mV (measured in DVF Ringer's) and outward Cs+ current at 90 mV (measured in 20 mM Ca2+o). (B) Ramp currents collected at the times shown by the open symbols in A. The similar activation time courses of the currents in 20 mM Ca2+ and DVF conditions (shown in A) suggest that the outwardly rectifying currents in 20 mM Ca2+ are due to MIC channels. (C) Removal of extracellular Ca2+ (2 mM Mg2+o) does not alter the inward MIC current.

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Induction of a Large Monovalent Conductance by Washout of Intracellular Mg²⁺
As described in previous studies of Jurkat and human T cells (Kerschbaum and Cahalan, 1998, 1999; Fomina et al., 2000) and RBL cells (Braun et al., 2001), intracellular dialysis with Mg²⁺-free internal solution in the absence of extracellular divalent cations triggers the slow development of a large monovalent conductance. In the experiment shown in Fig. 1, a Jurkat cell was dialysed with a Mg²⁺-free intracellular solution containing HEDTA, and continuous exposure to a DVF Ringer's solution revealed the induction of a large inward current over a period of 400 s (Fig. 1 A). With equimolar extracellular Na⁺ and intracellular Cs⁺, the current reversed at -5 mV (Fig. 1 B), indicating approximately equal permeabilities for Na⁺ and Cs⁺. Substitution of NMDG for Na⁺ selectively suppressed the inward current, demonstrating that the larger cation cannot permeate (Fig. 1, A and B). Early in the induction phase of the whole-cell current, the progressive opening of single channels could be resolved (Fig. 1 C). These channels generally opened in an all-or-none fashion to a very high open probability (P_o = 0.97 at -110 mV; n = 4 cells), such that openings of more than five channels could be clearly resolved under whole-cell conditions as described previously (Kerschbaum and Cahalan, 1999; Fomina et al., 2000). Based on the amplitude and reversal potential of the single-channel current, the average chord conductance was 44 ± 3 pS. We observed similar single-channel and whole-cell currents in human T cells freshly isolated from blood, although their activation following break-in was slower (unpublished data).

View larger version (35K): [in this window] [in a new window]   FIGURE 1. Activation of monovalent current in a Jurkat cell in the absence of extracellular divalent ions and intracellular Mg2+. (A) Time course and selectivity of the current developing in the presence of DVF extracellular solution. The bar indicates sequential changes in the bath solution from 20 mM Ca2+ Ringer's to Na+-DVF to NMDG-DVF (see MATERIALS AND METHODS). Each point represents the mean current during 100-ms steps to -110 mV, after subtraction of the leak current recorded in 20 mM Ca2+ immediately after break-in (time = 0). Internal solution: Cs methanesulfonate/10 HEDTA/0 Mg2+ (MGF). (B) Current-voltage relationship from the cell in A recorded with Na+- or NMDG-based DVF extracellular solution. A 100-ms voltage ramp from -110 to 90 mV was applied. (C) Currents at -110 mV recorded at early times after break-in show progressive activation of single Na+-conducting channels. Channels appear to activate sequentially, opening to very high probabilities in an all-or-none fashion. Numbers on the left indicate time after whole-cell break-in; numbers on the right indicate multiples of -3.9 pA. Same experimental protocol as in A, from another cell. (D) Current-voltage relationship of single channels conducting monovalent ions in an inside-out patch. Same voltage protocol as in B. Bath solution: MGF. Pipette solution: Na+-DVF. (E) Single-channel currents at different potentials in an excised patch. Same conditions as in D. The closed level is indicated by the dashed lines.

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 I_Ca to I_Na 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 I_CRAC.

The Large Monovalent Current Does Not Activate and Deactivate in Parallel With I_CRAC
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 5–10 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 I_CRAC 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 I_CRAC 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.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Depletion of Ca2+ stores does not activate the large monovalent current. (A) Activation of Ca2+ and Na+ currents in a Jurkat cell treated with 1 µM TG for 5 min before seal formation. Current at -110 mV (corrected for leak current collected in 0 Ca2+ Ringer's) is plotted against time after break-in. As indicated by the bar, the extracellular solution was periodically switched between a 20 mM Ca2+ Ringer's and standard DVF solution to measure the Ca2+ and Na+ currents, respectively. (left graph) The Ca2+ current ICRAC is present at the time of break-in, and at these early times only a small transient Na+ current is seen under DVF conditions (arrow). Large currents outside the graph boundaries are omitted for clarity. (Right graph) The same experiment at lower gain shows the slow development of a large Na+ current in DVF solution following break-in. Internal solution: MGF. (B) Plot of the peak Ca2+ () and Na+ () currents measured during applications of 20 mM Ca2+ or DVF solutions, respectively. (C) Monovalent current-voltage relationships recorded under DVF conditions early (41 s; small transient current in A) and late (595 s; large sustained current in A) in the experiment. Note the changes in size, rectification, and reversal potential of the current with time. (D) Current-voltage relations recorded in the presence of 20 mM Ca2+ at early (37 s) and late (565 s) times. A large outwardly rectifying current develops with time. In C and D, the currents from the upper graphs are reproduced as dashed lines in the lower graphs for comparison.

To distinguish between these possibilities, we asked whether the large monovalent conductance deactivates in parallel with I_CRAC 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 I_CRAC (Zweifach and Lewis, 1995). This behavior is illustrated in the experiment of Fig. 3 A (left graph), in which I_CRAC 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 I_CRAC, 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.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. ICRAC and the large monovalent current do not deactivate in parallel. (A) Following store depletion by passive dialysis with 1.2 mM EGTA, exposure to 20 mM Ca2+ evokes ICRAC, which progressively declines due to elevation of intracellular [Ca2+]i and refilling of Ca2+ stores. Current at -110 mV (corrected for leak current collected in 0 Ca2+ Ringer's) is plotted against time after break-in. Periodic exposure to DVF solution shows the development of the large monovalent current, shown at lower gain in the right graph. Note that ICRAC depotentiates during the second exposure to DVF (left), even though the Na+ current during that same period is fairly constant (right). Internal solution: Cs methanesulfonate/1.2 EGTA/0 Mg2+. (B) The Ca2+ () and Na+ currents () measured immediately before and during applications of the DVF solution in A. The monovalent current continues to increase as ICRAC deactivates.

View larger version (17K): [in this window] [in a new window]   FIGURE 4. ICRAC and the large monovalent current exhibit different sensitivities to SKF 96365. All cells were pretreated with 1 µM TG. In B and C, the recordings began after the monovalent current had activated to a steady-state level, as described in Fig. 2. Both currents were measured during steps to -110 mV. (A) Inhibition of ICRAC by SKF 96365 (20 µM). Inhibition and recovery followed exponential time courses with time constants of 17.5 and 19 s, respectively. Internal solution: Cs methanesulfonate/10 BAPTA/8 Mg2+. External: 20 mM Ca2+. (B) The large monovalent current is relatively insensitive to 20 µM SKF 96365. Internal solution: MGF. (C) Even after nearly complete inhibition of ICRAC by SKF 96365 (20 µM), removal of external divalent ions causes the monovalent current to rise rapidly to control levels. Thus, the insensitivity of the monovalent current to SKF 96365 is not explained by a failure to block CRAC channels under DVF conditions. Internal solution: MGF.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. ICRAC and the large monovalent current have differing sensitivities to 2-APB. (A) A low concentration of 2-APB (5 µM) enhances ICRAC after full store depletion by TG. ICRAC is shown at -110 mV. Internal solution: Cs methanesulfonate/10 BAPTA/8 Mg2+. Holding potential: -40 mV (B) A high concentration of 2-APB (50 µM) initially enhances, then produces nearly complete and irreversible inhibition of ICRAC. Experimental conditions as described in A. (C) Effects of 2-APB on the large monovalent current. A high concentration (50 µM) inhibits the current partially and reversibly. In contrast, the irreversible inhibition of ICRAC in the same cell is shown by comparing currents during the first and second applications of Ca2+. 5 µM 2-APB fails to enhance the large monovalent current, although it enhances ICRAC as shown in A. Internal solution: MGF.

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 I_CRAC 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.

View larger version (22K): [in this window] [in a new window]   FIGURE 6. The large monovalent current is inhibited by intracellular Mg2+ and MgATP. (A) High intracellular [Mg2+] prevents activation of the large monovalent current. Mean currents at -110 mV in the presence of DVF Ringer's are shown as a function of time following break-in. Internal solution: Cs methanesulfonate containing either 10 HEDTA/0 Mg2+ (; six cells) or 10 BAPTA/8 mM Mg2+ (; four cells). (B) Dose-response curves for Mg2+-dependent inhibition of the inward Na+ current () recorded at -110 mV in DVF solution and the outward Cs+ current () at 90 mV in 20 mM Ca2+o solution. The two extracellular solutions were periodically alternated as described in Fig. 2. Each point represents the mean ± SEM of 5–7 cells. The solid and dotted lines are fits of the equation I = 1/(1 + ([Mg2+]/K1/2)n) with the following parameter values: K1/2 = 0.6 mM and n = 2.4 for the inward current in DVF; K1/2 = 0.66 mM and n = 2.6 for the outward current in 20 mM Ca2+o. Internal solution: Cs methanesulfonate/10 mM BAPTA containing indicated levels of calculated free Mg2+. (C) The large monovalent current is inhibited by intracellular MgATP. Shown are the mean whole-cell current amplitudes 400 s after break-in with internal solutions containing 4–8 mM MgATP (five cells) or 0 Mg2+/10 HEDTA (six cells), in experiments like the one shown in A. (D) Reversible inhibition of MIC channels by 100 µM Mg2+i in excised patches. Channel activity persisted in this inside-out patch after excision into MGF solution. Application of 100 µM Mg2+ (free concentration, buffered with 10 mM HEDTA) to the cytoplasmic face resulted in reversible closure of two channels. The average current amplitudes during 250-ms steps to -115 mV are shown, with selected sweeps shown at higher time resolution to the right (dashed line indicates 0 current level). Pipette solution: Na-DVF. (E) 2 mM Mg2+ inhibits single-channel monovalent currents irreversibly. Same conditions as in D, except 2 mM Mg2+ was applied with 10 mM BAPTA.

To isolate I_MIC for further characterization, I_CRAC 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, I_MIC (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 I_MIC, the outwardly rectifying current was not observed when 8–10 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 P_Cs/P_Na 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. I_MIC 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 I_CRAC measurements with I_MIC. 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 I_CRAC 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 I_MIC (Fig. 6 C), whereas recordings with 3 mM MgATP exhibited sporadic MIC channel activity. Our combined observations suggest that I_MIC is mediated by TRP-PLIK/LTRPC7 channels, and is probably identical to an outwardly rectifying MgATP-sensitive current (called MagNuM, for Mg²⁺ nucleotide–regulated metal) that has been reported in Jurkat, RBL, and HEK293 cells (Nadler et al., 2001).

Isolating the Monovalent Current through CRAC Channels
Inhibition of I_MIC by intracellular Mg²⁺ offers a convenient strategy for isolating the monovalent current through CRAC channels for characterization. With 8 mM intracellular Mg²⁺ to suppress I_MIC, passive depletion of Ca²⁺ stores by intracellular BAPTA (10 mM) slowly evoked I_CRAC 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 I_CRAC (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 I_CRAC measured just before solution exchange was 7.8 ± 0.4 (n = 17).

View larger version (21K): [in this window] [in a new window]   FIGURE 8. Activation and deactivation of Ca2+ and Na+ currents through CRAC channels. Currents were measured during steps to -110 mV. (A) Activation of ICRAC and the transient Na+ current during passive store depletion with a pipette solution containing 10 mM BAPTA + 8 mM Mg2+. Periodic removal of external divalent ions reveals a transient monovalent current that increases in parallel with ICRAC. (B) The Ca2+ current () and the peak Na+ current () measured immediately before and during each application of DVF solution activate with similar kinetics. (C) Deactivation of ICRAC and the transient monovalent current during intracellular dialysis with 1 mM BAPTA + 8 mM Mg2+. As described in Fig. 3 A, ICRAC deactivates in the presence of low intracellular Ca2+ buffering, presumably due to store refilling. (D) The Ca2+ current () and the peak Na+ current () decline in parallel in the experiment in C. The similar activation and deactivation kinetics for the Na+ and Ca2+ currents support the idea that the transient monovalent current represents Na+ flux through CRAC channels.

The pharmacological profile of the transient Na⁺ current also closely matched that of I_CRAC. As shown in Fig. 9 A, 20 µM SKF 96365 inhibited both currents to similar extents. On average, I_CRAC 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 I_CRAC 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).

View larger version (17K): [in this window] [in a new window]   FIGURE 9. Pharmacological evidence for monovalent currents through CRAC channels. ICRAC was activated by treatment with 1 µM TG for 5 min before seal formation (in A and B) or by passive depletion with 10 mM intracellular BAPTA (in C), with 10 mM intracellular Mg2+ to inhibit IMIC. (A) SKF 96365 (20 µM) inhibits both ICRAC and the transient monovalent current (under DVF conditions). Both currents recover following washout of the drug. (B) A low concentration of 2-APB (5 µM) enhances both ICRAC and the transient monovalent current by severalfold. (C) A high concentration of 2-APB (40 µM) significantly reduces both ICRAC and the transient monovalent current. The inhibition of both currents persists after washout of the drug.

Based on these results as well as the evidence given above that I_CRAC and the transient Na⁺ current activate and deactivate in parallel and are store-dependent, we conclude that 8–10 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⁺-I_CRAC and Ca²⁺-I_CRAC, 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 I_MIC from I_CRAC, 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⁺ (P_Cs/P_Na = 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).

View larger version (19K): [in this window] [in a new window]   FIGURE 10. CRAC channels have a low permeability to Cs+. ICRAC was activated by treatment with 1 µM TG, and measured in response to steps to -110 mV and ramps from -110 to 50 mV. (A) Positive reversal potential of monovalent CRAC current with Cs+ and Na+ as the primary intracellular and extracellular current carriers, respectively. Three ramp currents collected during the depotentiation of Na+-ICRAC (left graph) converge at 50 mV (right). (A and B) Internal solution: Cs methanesulfonate/10 BAPTA/8 Mg2+. (B) Extracellular Cs+ does not conduct significant inward current through CRAC channels under DVF conditions. When the bath solution is changed to a Cs+-based DVF solution, the current at -110 mV drops to 15% of its previous value in 20 mM Ca2+; in contrast, Na+-DVF causes an approximately eightfold increase in the current. Thus, Na+ is 50-fold more conductive than Cs+. (C) Measurement of the CRAC-channel current-voltage relation under DVF conditions in the absence of intracellular Mg2+. A low concentration of 2-APB (5 µM) induces an inward current that sums with IMIC. Ramp currents were averaged before and after 2-APB treatment (bars). Because 2-APB selectively enhances CRAC channel activity (Fig. 5), the difference between the two averaged currents isolates monovalent ICRAC (right). The net current reverses at 40 mV, indicating that the low Cs+ permeability of the CRAC channel is independent of intracellular Mg2+. Internal solution: MGF.

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 10–20 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 I_MIC was likely mistaken for I_CRAC in the absence of intracellular Mg²⁺, we reexamined the role of Mg²⁺ in CRAC channel depotentiation under conditions that prevent activation of I_MIC.

To effectively inhibit I_MIC under conditions of low intracellular Mg²⁺, we applied MgATP in combination with Na₂ATP to reduce free [Mg²⁺]_i. 6–8 mM MgATP + 2–6 mM Na₂ATP in the pipette solution inhibited I_MIC while reducing free [Mg²⁺]_i to calculated values of 92–185 µM (see MATERIALS AND METHODS). Under these conditions, Na⁺-I_CRAC 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 (6–10 mM) or low (131 µM) levels of Mg²⁺_i (Fig. 11, B and C), even though the maximum amplitude of Na⁺-I_CRAC 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.

View larger version (25K): [in this window] [in a new window]   FIGURE 11. Intracellular Mg2+ is not involved in the depotentiation of CRAC channels following removal of extracellular Ca2+. ICRAC was activated by treatment with 1 µM TG, and currents were measured at a step potential of -110 mV. (A) Depotentiation of Na+-ICRAC occurs even in the presence of low cytoplasmic [Mg2+]. The pipette solution contained 8 mM MgATP to inhibit IMIC and 6 mM Na2ATP to reduce free [Mg2+] to 131 µM (calculated). Ramp currents collected at several times during the decline of Na+-ICRAC are shown at the right. (B) The kinetics of Na+-ICRAC decline are not voltage dependent. Transient Na+-ICRAC was evoked by exposure to DVF solution at a holding potential of 40 or -110 mV, with the same intracellular solutions as in A. The peak amplitude of Na+-ICRAC (measured during steps to -110 mV) is larger at the more negative holding potential, presumably due to the voltage dependence of CDP (see Zweifach and Lewis, 1996). However, the extent and rate of depotentiation are unaffected. (C) The rate of depotentiation is unaffected by the level of [Mg2+]i or membrane potential. Exponential curves were fitted to the depotentiation time course with 8 mM Mg2+i at 20 mV (seven cells), 90–131 µM Mg2+i at 20 mV (7 cells), or 131 µM Mg2+ at -110 mV holding potential (three cells). (D) Removal of intracellular Mg2+ does not affect CDP. After exposure to Ca2+-free conditions, readdition of extracellular Ca2+ causes ICRAC to reappear gradually over 10–20 s due to CDP. The extent and time course of CDP were similar in experiments with 8 mM Mg2+i (left) or 0 Mg2+i (right). Holding potential, 20 mV.

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 I_MIC, we were unable to detect any clear single-channel transitions of amplitude >0.4 pA during the induction of Na⁺-I_CRAC by ionomycin in 5/5 cells and during the or depotentiation of Na⁺-I_CRAC. 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 I_CRAC 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.

View larger version (27K): [in this window] [in a new window]   FIGURE 12. Fluctuation analysis of the Na+ current through CRAC channels. ICRAC was induced by treatment with 1 µM TG and was recorded at a constant holding potential of -110 mV. All data are from the same experiment. Internal solution: Cs methanesulfonate/10 BAPTA/8 Mg2+. (A) Mean value and variance of CRAC current in response to 2-APB (5 µM) and removal of divalent cations. Each point represents the values calculated from a 200-ms segment of current data. (B) Sample 200-ms segments of Na+-ICRAC as it depotentiates in the presence of DVF Ringer's. The zero-current level is indicated by the dashed line. (C) Mean-variance analysis of Na+-ICRAC. The plot shows the mean value and variance of 200-ms current sweeps collected as Na+-ICRAC depotentiated in the presence of DVF Ringer's. The data points are well fit by a line with a slope of -31.2 fA. (D) Mean-variance analysis of Ca+-ICRAC. The plot shows the mean value and variance of 200-ms current sweeps collected as Ca+-ICRAC was enhanced by 2-APB in 20 mM Ca2+ Ringer's. The data points are well fit by a line with a slope of -3.9 fA. (E) Spectral analysis of Na+-ICRAC. Spectra were collected and averaged in 0 Ca2+ + 2 µM Ni2+ (dotted trace, "bkgd"), and near the peak of the transient Na+ CRAC current ("peak") and after Na+-ICRAC reached steady-state ("s-s"). Each trace is the average of 3–20 spectra. There is little power above background levels associated with Na+-ICRAC at frequencies >1 kHz.

where ₀² is the variance at the zero-current level. Since I = NiP₀, it follows that

(1)

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 I_CRAC 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⁺-I_CRAC 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 1–2 kHz filtering conditions of the current variance experiments.

Second, noise measurements may underestimate the size of i if P_o is high and the macroscopic current amplitude reflects changes in N, the number of activatable channels, rather than P_o (Jackson and Strange, 1995). In this case, _I² will increase linearly with N:

and the _I²/I ratio underestimates i by a factor of (1 - P_o) (Eq. 1). If this situation applies, a small reduction in the P_o resulting from extracellular blockade of the channels would be expected to produce an increase in channel noise due to an increased number of gating events occurring within each 200-ms sampling period. To examine this possibility, we added 1 µM free Ca²⁺ to the extracellular DVF solution to partially block CRAC channels and reduce channel P_o. The peak I_Na/I_Ca ratio was reduced to 3.1 ± 0.3 (n = 4; versus a ratio of 7.8 ± 0.4 without the blocker), indicating >50% inhibition of the Na⁺-I_CRAC. Under these conditions, the _I²/I ratio was -19 ± 3 fA (n = 4). This ratio is even smaller than the _I²/I ratios obtained in the absence of the blocker.¹ Given these results, it seems unlikely that very high P_o of CRAC channels is causing a gross underestimation of the single channel current. Thus, from the results of noise analysis, we estimate that the monovalent conductance of the CRAC channel is 0.2 pS.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the absence of extracellular divalent cations, removal of intracellular Mg²⁺ during whole-cell recording activates a large monovalent current in T cells and RBL cells. This large Mg²⁺-sensitive current has previously been attributed to monovalent permeation of CRAC channels, and its signature conductance and selectivity have been employed as a biophysical fingerprint to screen candidate CRAC channel genes. In this paper, we show that this large monovalent current, I_MIC, and the 40-pS channels that underlie it, differ in fundamental ways from I_CRAC and therefore represent a distinct ion channel type. These findings necessitate a revision of the biophysical fingerprint of CRAC channels, and have important implications for studies of CRAC channel activation, permeation, and cellular function.

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.

Following removal of extracellular divalents, Na⁺-I_CRAC is transient (Fig. 10 A), whereas I_MIC 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⁺-I_CRAC during store depletion and refilling (Fig. 8), and its sensitivity to SKF 96365 and 2-APB (Fig. 9). Na⁺-I_CRAC 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⁺-I_CRAC and it has the same reversal potential, relative Cs⁺ permeability and sensitivity to SKF 96365 and 2-APB, as the peak current. Although I_MIC 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 (0.5–1 mM; unpublished data). Thus, current decline under DVF conditions cannot be used as a specific indicator for I_CRAC.

Finally, the pharmacological signatures of I_CRAC and I_MIC also differ significantly (Figs. 4 and 9). SKF 96365 at micromolar levels is an effective blocker of I_CRAC (Franzius et al., 1994; Christian et al., 1996a), but not of I_MIC. High concentrations of 2-APB (20–40 µM) inhibit I_CRAC completely and irreversibly (Prakriya and Lewis, 2001), whereas they cause modest and rapidly reversible inhibition of I_MIC. Low concentrations of 2-APB (5 µM) which strongly enhance I_CRAC (Prakriya and Lewis, 2001) fail to affect I_MIC.

The rather large number of properties that distinguish the two conductances strongly suggest that I_CRAC and I_MIC 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 I_CRAC. 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 I_MIC. These issues may have to be revisited using recording conditions explicitly optimized for isolating the monovalent CRAC current. No specific inhibitors of I_MIC are yet known, but high intracellular Mg²⁺ (8–10 mM) or MgATP (>4 mM) effectively blocks I_MIC without affecting I_CRAC. It is important to note that moderate levels of Mg²⁺ (2–3 mM) commonly used in studies of I_CRAC may not be sufficient to completely eliminate MIC activity, as we often found 3–5 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 I_CRAC in the presence of divalent ions, although appropriate subtraction of the "leak" current can alleviate this problem (see MATERIALS AND METHODS).

I_MIC 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 I_CRAC, 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 I_MIC 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 I_MIC 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 250–500 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 I_MIC, which is much larger, more sustained, and less selective than I_CRAC.

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⁺-I_CRAC 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⁺-I_CRAC with I_MIC 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 I_CRAC 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 I_CRAC. In addition, partial inhibition of monovalent I_CRAC with 1 µM extracellular Ca²⁺ did not increase the noise, indicating that whatever the mechanism of activation, the P_o 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⁺-I_CRAC is also about eightfold larger than Ca²⁺-I_CRAC 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 P_o. 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 5,000–10,000. 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⁺-I_CRAC (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⁺ (Ca_V) channels have interesting implications for mechanisms of ion selectivity in CRAC channels. Under physiological conditions, Ca_V channels have an extremely high selectivity for Ca²⁺ over monovalent ions (P_Ca/P_Na > 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⁺-I_CRAC by 50% (see RESULTS).

The most obvious differences between the two channels relates to their unitary conductances and permeability to Cs⁺. Ca_V channels have single-channel conductances for Ca²⁺ in the range of 5–20 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 Ca_V channels. In addition, under DVF conditions Ca_V channels readily pass Cs⁺ (P_Cs/P_Na 0.6; Hess et al., 1986), whereas CRAC channels do not (P_Cs/P_Na = 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 Ca_V channels arises from ion–ion interactions as ions move single-file through the pore (Tsien et al., 1987). Thus, one possibility is that weak ion–ion 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.

	FOOTNOTES

 
^* Abbreviations used in this paper: 2-APB, 2-aminoethyldiphenyl borate; CDP, Ca²⁺-dependent potentiation; CRAC, calcium release–activated Ca²⁺; DVF, divalent-free; MIC, Mg²⁺-inhibited cation; SOC, store-operated channel; TG, thapsigargin.

¹ The explanation for this is not clear, but it is not likely to be due to flickery block occurring at frequencies outside the recording bandwidth, since the fastest unbinding rate for a K_d of 1 µM would be 1,000 s^-1 (assuming a maximum diffusion-limited on rate of 10⁹ M^-1s^-1).

² While this manuscript was under review, Hermosura et al. (2002) published evidence showing that the large sustained monovalent current in RBL cells perfused with Mg²⁺-free intracellular solutions (termed MagNuM) can be separated from I_CRAC. This report and another (Bakowski and Parekh, 2002) also showed that intracellular Mg²⁺ does not significantly affect CRAC channel permeation in RBL cells, in agreement with our results.

	ACKNOWLEDGMENTS

 
The authors would like to thank K. Mikoshiba for the gift of 2-APB. We thank members of the Lewis lab for helpful discussions and Dr. Rick Aldrich, Dr. Adam Zweifach, and Diana Bautista for comments on the manuscript.

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

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

Almers, W., and E.W. McCleskey. 1984. Non-selective conductance in calcium channels of frog muscle: calcium selectivity in a single-file pore. J. Physiol. 353:585–608.[Abstract/Free Full Text]

Bakowski, D., and A.B. Parekh. 2002. Monovalent cation permeability and Ca²⁺ block of the store-operated Ca²⁺ current I_CRAC in rat basophilic leukemia cells. Pflügers Arch. 443:892–902.[CrossRef][Medline]

Braun, F.J., L.M. Broad, D.L. Armstrong, and J.W. Putney, Jr. 2001. Stable activation of single Ca²⁺ release-activated Ca²⁺ channels in divalent cation-free solutions. J. Biol. Chem. 276:1063–1070.[Abstract/Free Full Text]

Broad, L.M., F.J. Braun, J.P. Lievremont, G.S. Bird, T. Kurosaki, and J.W. Putney, Jr. 2001. Role of the phospholipase C-inositol 1,4,5-trisphosphate pathway in calcium release-activated calcium current and capacitative calcium entry. J. Biol. Chem. 276:15945–15952.[Abstract/Free Full Text]

Christian, E.P., K.T. Spence, J.A. Togo, P.G. Dargis, and E. Warawa. 1996a. Extracellular site for econazole-mediated block of Ca²⁺ release-activated Ca²⁺ current (I_crac) in T lymphocytes. Br. J. Pharmacol. 119:647–654.[Medline]

Christian, E.P., K.T. Spence, J.A. Togo, P.G. Dargis, and J. Patel. 1996b. Calcium-dependent enhancement of depletion-activated calcium current in Jurkat T lymphocytes. J. Membr. Biol. 150:63–71.[CrossRef][Medline]

Clapham, D.E., L.W. Runnels, and C. Strubing. 2001. The trp ion channel family. Nat. Rev. Neurosci. 2:387–396.[Medline]

Fanger, C.M., M. Hoth, G.R. Crabtree, and R.S. Lewis. 1995. Characterization of T cell mutants with defects in capacitative calcium entry: genetic evidence for the physiological roles of CRAC channels. J. Cell Biol. 131:655–667.[Abstract/Free Full Text]

Feske, S., J. Giltnane, R. Dolmetsch, L.M. Staudt, and A. Rao. 2001. Gene regulation mediated by calcium signals in T lymphocytes. Nat. Immunol. 2:316–324.[CrossRef][Medline]

Fomina, A.F., C.M. Fanger, J.A. Kozak, and M.D. Cahalan. 2000. Single channel properties and regulated expression of Ca²⁺ release-activated Ca²⁺ (CRAC) channels in human T cells. J. Cell Biol. 150:1435–1444.[Abstract/Free Full Text]

Franzius, D., M. Hoth, and R. Penner. 1994. Non-specific effects of calcium entry antagonists in mast cells. Pflügers Arch. 428:433–438.[CrossRef][Medline]

Hermosura, M.C., M.K. Monteilh-Zoller, A.M. Scharenberg, R. Penner, and A. Fleig. 2002. Dissociation of the store-operated calcium current I_CRAC and the Mg²⁺-nucleotide-regulated metal ion current MagNuM. J. Physiol. 539:445–458.[Abstract/Free Full Text]

Hess, P., and R.W. Tsien. 1984. Mechanism of ion permeation through calcium channels. Nature. 309:453–456.[CrossRef][Medline]

Hess, P., J.B. Lansman, and R.W. Tsien. 1986. Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells. J. Gen. Physiol. 88:293–319.[Abstract/Free Full Text]

Hoth, M. 1995. Calcium and barium permeation through calcium release-activated calcium (CRAC) channels. Pflügers Arch. 430:315–322.[CrossRef][Medline]

Hoth, M., and R. Penner. 1993. Calcium release-activated calcium current in rat mast cells. J. Physiol. 465:359–386.[Abstract/Free Full Text]

Jackson, P.S., and K. Strange. 1995. Single-channel properties of a volume-sensitive anion conductance. Current activation occurs by abrupt switching of closed channels to an open state. J. Gen. Physiol. 105:643–660.[Abstract/Free Full Text]

Kerschbaum, H.H., and M.D. Cahalan. 1998. Monovalent permeability, rectification, and ionic block of store-operated calcium channels in Jurkat T lymphocytes. J. Gen. Physiol. 111:521–537.[Abstract/Free Full Text]

Kerschbaum, H.H., and M.D. Cahalan. 1999. Single-channel recording of a store-operated Ca²⁺ channel in Jurkat T lymphocytes. Science. 283:836–839.[Abstract/Free Full Text]

Lepple-Wienhues, A., and M.D. Cahalan. 1996. Conductance and permeation of monovalent cations through depletion-activated Ca²⁺ channels (I_CRAC) in Jurkat T cells. Biophys. J. 71:787–794.[Medline]

Lewis, R.S. 1999. Store-operated calcium channels. Adv. Second Messenger Phosphoprotein Res. 33:279–307.[Medline]

Lewis, R.S. 2001. Calcium signaling mechanisms in T lymphocytes. Annu. Rev. Immunol. 19:497–521.[CrossRef][Medline]

Ma, H.T., R.L. Patterson, D.B. van Rossum, L. Birnbaumer, K. Mikoshiba, and D.L. Gill. 2000. Requirement of the inositol trisphosphate receptor for activation of store-operated Ca²⁺ channels. Science. 287:1647–1651.[Abstract/Free Full Text]

Nadler, M.J., M.C. Hermosura, K. Inabe, A.L. Perraud, Q. Zhu, A.J. Stokes, T. Kurosaki, J.P. Kinet, R. Penner, A.M. Scharenberg, and A. Fleig. 2001. LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability. Nature. 411:590–595.[CrossRef][Medline]

Parekh, A.B., and R. Penner. 1997. Store depletion and calcium influx. Physiol. Rev. 77:901–930.[Abstract/Free Full Text]

Partiseti, M., F. Le Deist, C. Hivroz, A. Fischer, H. Korn, and D. Choquet. 1994. The calcium current activated by T cell receptor and store depletion in human lymphocytes is absent in a primary immunodeficiency. J. Biol. Chem. 269:32327–32335.[Abstract/Free Full Text]

Prakriya, M., and R.S. Lewis. 2001. Potentiation and inhibition of Ca²⁺ release-activated Ca²⁺ channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP₃ receptors. J. Physiol. 536:3–19.[Abstract/Free Full Text]

Prakriya, M., and R.S. Lewis. 2002. Store-operated calcium channels: properties, functions and the search for a molecular mechanism. In Molecular Insights into Ion Channel Biology in Health and Disease. R. Maue, editor. Elsevier Science, Amsterdam. In press.

Putney, J.W., Jr., and R.R. McKay. 1999. Capacitative calcium entry channels. Bioessays. 21:38–46.[CrossRef][Medline]

Putney, J.W., Jr., L.M. Broad, F.J. Braun, J.P. Lievremont, and G.S. Bird. 2001. Mechanisms of capacitative calcium entry. J. Cell Sci. 114:2223–2229.[Medline]

Romani, A.M., and A. Scarpa. 2000. Regulation of cellular magnesium. Front. Biosci. 5:D720–D734.[Medline]

Runnels, L.W., L. Yue, and D.E. Clapham. 2001. TRP-PLIK, a bifunctional protein with kinase and ion channel activities. Science. 291:1043–1047.[Abstract/Free Full Text]

Rychkov, G., H.M. Brereton, M.L. Harland, and G.J. Barritt. 2001. Plasma membrane Ca²⁺ release-activated Ca²⁺ channels with a high selectivity for Ca²⁺ identified by patch-clamp recording in rat liver cells. Hepatology. 33:938–947.[CrossRef][Medline]

Sigworth, F.J. 1980. The variance of sodium current fluctuations at the node of Ranvier. J. Physiol. 307:97–129.[Abstract/Free Full Text]

Tsien, R.W., P. Hess, E.W. McCleskey, and R.L. Rosenberg. 1987. Calcium channels: mechanisms of selectivity, permeation, and block. Annu. Rev. Biophys. Biophys. Chem. 16:265–290.[CrossRef][Medline]

Voets, T., J.Prenen, A. Fleig, R. Vennekens, H. Watanabe, J.G. Hoenderop, R.J. Bindels, G. Droogmans, R. Penner, and B. Nilius. 2001. CaT1 and the calcium release-activated calcium channel manifest distinct pore properties. J. Biol. Chem. 276:47767–47770.[Abstract/Free Full Text]

Yue, L., J.B. Peng, M.A. Hediger, and D.E. Clapham. 2001. CaT1 manifests the pore properties of the calcium-release-activated calcium channel. Nature. 410:705–709.[CrossRef][Medline]

Zweifach, A., and R.S. Lewis. 1993. Mitogen-regulated Ca²⁺ current of T lymphocytes is activated by depletion of intracellular Ca²⁺ stores. Proc. Natl. Acad. Sci. USA. 90:6295–6299.[Abstract/Free Full Text]

Zweifach, A., and R.S. Lewis. 1995. Slow calcium-dependent inactivation of depletion-activated calcium current. Store-dependent and -independent mechanisms. J. Biol. Chem. 270:14445–14451.[Abstract/Free Full Text]

Zweifach, A., and R.S. Lewis. 1996. Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes. J. Gen. Physiol. 107:597–610.[Abstract/Free Full Text]

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. L. Thompson, O. Mignen, and T. J. Shuttleworth The Orai1 Severe Combined Immune Deficiency Mutation and Calcium Release-activated Ca2+ Channel Function in the Heterozygous Condition J. Biol. Chem., March 13, 2009; 284(11): 6620 - 6626. [Abstract] [Full Text] [PDF]

				I. F. Abdullaev, J. M. Bisaillon, M. Potier, J. C. Gonzalez, R. K. Motiani, and M. Trebak Stim1 and Orai1 Mediate CRAC Currents and Store-Operated Calcium Entry Important for Endothelial Cell Proliferation Circ. Res., November 21, 2008; 103(11): 1289 - 1299. [Abstract] [Full Text] [PDF]

				L. Navarro-Borelly, A. Somasundaram, M. Yamashita, D. Ren, R. J. Miller, and M. Prakriya STIM1-Orai1 interactions and Orai1 conformational changes revealed by live-cell FRET microscopy J. Physiol., November 15, 2008; 586(22): 5383 - 5401. [Abstract] [Full Text] [PDF]

				J. Jin, B. N. Desai, B. Navarro, A. Donovan, N. C. Andrews, and D. E. Clapham Deletion of Trpm7 Disrupts Embryonic Development and Thymopoiesis Without Altering Mg2+ Homeostasis Science, October 31, 2008; 322(5902): 756 - 760. [Abstract] [Full Text] [PDF]

				Y. Gwack, S. Srikanth, M. Oh-hora, P. G. Hogan, E. D. Lamperti, M. Yamashita, C. Gelinas, D. S. Neems, Y. Sasaki, S. Feske, et al. Hair Loss and Defective T- and B-Cell Function in Mice Lacking ORAI1 Mol. Cell. Biol., September 1, 2008; 28(17): 5209 - 5222. [Abstract] [Full Text] [PDF]

				R. L. Schroder, S. Friis, M. Sunesen, C. Mathes, and N. J. Willumsen Automated Patch-Clamp Technique: Increased Throughput in Functional Characterization and in Pharmacological Screening of Small-Conductance Ca2+ Release-Activated Ca2+ Channels J Biomol Screen, August 1, 2008; 13(7): 638 - 647. [Abstract] [PDF]

				S. Thebault, G. Cao, H. Venselaar, Q. Xi, R. J. M. Bindels, and J. G. J. Hoenderop Role of the {alpha}-Kinase Domain in Transient Receptor Potential Melastatin 6 Channel and Regulation by Intracellular ATP J. Biol. Chem., July 18, 2008; 283(29): 19999 - 20007. [Abstract] [Full Text] [PDF]

				S. L. Zhang, J. A. Kozak, W. Jiang, A. V. Yeromin, J. Chen, Y. Yu, A. Penna, W. Shen, V. Chi, and M. D. Cahalan Store-dependent and -independent Modes Regulating Ca2+ Release-activated Ca2+ Channel Activity of Human Orai1 and Orai3 J. Biol. Chem., June 20, 2008; 283(25): 17662 - 17671. [Abstract] [Full Text] [PDF]

				M. A. Spassova, T. Hewavitharana, R. A. Fandino, A. Kaya, J. Tanaka, and D. L. Gill Voltage Gating at the Selectivity Filter of the Ca2+ Release-activated Ca2+ Channel Induced by Mutation of the Orai1 Protein J. Biol. Chem., May 30, 2008; 283(22): 14938 - 14945. [Abstract] [Full Text] [PDF]

				M. Muik, I. Frischauf, I. Derler, M. Fahrner, J. Bergsmann, P. Eder, R. Schindl, C. Hesch, B. Polzinger, R. Fritsch, et al. Dynamic Coupling of the Putative Coiled-coil Domain of ORAI1 with STIM1 Mediates ORAI1 Channel Activation J. Biol. Chem., March 21, 2008; 283(12): 8014 - 8022. [Abstract] [Full Text] [PDF]

				W.-C. Chang, J. Di Capite, K. Singaravelu, C. Nelson, V. Halse, and A. B. Parekh Local Ca2+ Influx through Ca2+ Release-activated Ca2+ (CRAC) Channels Stimulates Production of an Intracellular Messenger and an Intercellular Pro-inflammatory Signal J. Biol. Chem., February 22, 2008; 283(8): 4622 - 4631. [Abstract] [Full Text] [PDF]

				J. Jiang, M.-H. Li, K. Inoue, X.-P. Chu, J. Seeds, and Z.-G. Xiong Transient Receptor Potential Melastatin 7 like Current in Human Head and Neck Carcinoma Cells: Role in Cell Proliferation Cancer Res., November 15, 2007; 67(22): 10929 - 10938. [Abstract] [Full Text] [PDF]

				M. Yamashita, L. Navarro-Borelly, B. A. McNally, and M. Prakriya Orai1 Mutations Alter Ion Permeation and Ca2+-dependent Fast Inactivation of CRAC Channels: Evidence for Coupling of Permeation and Gating J. Gen. Physiol., October 29, 2007; 130(5): 525 - 540. [Abstract] [Full Text] [PDF]

				W.-L. Wei, H.-S. Sun, M. E. Olah, X. Sun, E. Czerwinska, W. Czerwinski, Y. Mori, B. A. Orser, Z.-G. Xiong, M. F. Jackson, et al. TRPM7 channels in hippocampal neurons detect levels of extracellular divalent cations PNAS, October 9, 2007; 104(41): 16323 - 16328. [Abstract] [Full Text] [PDF]

				R. C. E. Wykes, M. Lee, S. M. Duffy, W. Yang, E. P. Seward, and P. Bradding Functional Transient Receptor Potential Melastatin 7 Channels Are Critical for Human Mast Cell Survival J. Immunol., September 15, 2007; 179(6): 4045 - 4052. [Abstract] [Full Text] [PDF]

				B. F. Bessac and A. Fleig TRPM7 channel is sensitive to osmotic gradients in human kidney cells J. Physiol., August 1, 2007; 582(3): 1073 - 1086. [Abstract] [Full Text] [PDF]

				W. I. DeHaven, J. T. Smyth, R. R. Boyles, and J. W. Putney Jr. Calcium Inhibition and Calcium Potentiation of Orai1, Orai2, and Orai3 Calcium Release-activated Calcium Channels J. Biol. Chem., June 15, 2007; 282(24): 17548 - 17556. [Abstract] [Full Text] [PDF]

				R. A. Rose, N. Hatano, S. Ohya, Y. Imaizumi, and W. R. Giles C-type natriuretic peptide activates a non-selective cation current in acutely isolated rat cardiac fibroblasts via natriuretic peptide C receptor-mediated signalling J. Physiol., April 1, 2007; 580(1): 255 - 274. [Abstract] [Full Text] [PDF]

				O. Mignen, J. L. Thompson, and T. J. Shuttleworth STIM1 regulates Ca2+ entry via arachidonate-regulated Ca2+-selective (ARC) channels without store depletion or translocation to the plasma membrane J. Physiol., March 15, 2007; 579(3): 703 - 715. [Abstract] [Full Text] [PDF]

				T. Numata, T. Shimizu, and Y. Okada TRPM7 is a stretch- and swelling-activated cation channel involved in volume regulation in human epithelial cells Am J Physiol Cell Physiol, January 1, 2007; 292(1): C460 - C467. [Abstract] [Full Text] [PDF]

				N. Landman, S. Y. Jeong, S. Y. Shin, S. V. Voronov, G. Serban, M. S. Kang, M. K. Park, G. Di Paolo, S. Chung, and T.-W. Kim Presenilin mutations linked to familial Alzheimer's disease cause an imbalance in phosphatidylinositol 4,5-bisphosphate metabolism PNAS, December 19, 2006; 103(51): 19524 - 19529. [Abstract] [Full Text] [PDF]

				G. B. Montalvo, A. R. Artalejo, and J. A. Gilabert ATP from Subplasmalemmal Mitochondria Controls Ca2+-dependent Inactivation of CRAC Channels J. Biol. Chem., November 24, 2006; 281(47): 35616 - 35623. [Abstract] [Full Text] [PDF]

				R. M. Luik, M. M. Wu, J. Buchanan, and R. S. Lewis The elementary unit of store-operated Ca2+ entry: local activation of CRAC channels by STIM1 at ER-plasma membrane junctions J. Cell Biol., September 11, 2006; 174(6): 815 - 825. [Abstract] [Full Text] [PDF]

				M. Prakriya and R. S. Lewis Regulation of CRAC Channel Activity by Recruitment of Silent Channels to a High Open-probability Gating Mode J. Gen. Physiol., August 28, 2006; 128(3): 373 - 386. [Abstract] [Full Text] [PDF]

				J. Soboloff, M. A. Spassova, X. D. Tang, T. Hewavitharana, W. Xu, and D. L. Gill Orai1 and STIM Reconstitute Store-operated Calcium Channel Function J. Biol. Chem., July 28, 2006; 281(30): 20661 - 20665. [Abstract] [Full Text] [PDF]

				L.-T. Su, M. A. Agapito, M. Li, W. T. N. Simonson, A. Huttenlocher, R. Habas, L. Yue, and L. W. Runnels TRPM7 Regulates Cell Adhesion by Controlling the Calcium-dependent Protease Calpain J. Biol. Chem., April 21, 2006; 281(16): 11260 - 11270. [Abstract] [Full Text] [PDF]

				P. Demeuse, R. Penner, and A. Fleig TRPM7 Channel Is Regulated by Magnesium Nucleotides via its Kinase Domain J. Gen. Physiol., March 27, 2006; 127(4): 421 - 434. [Abstract] [Full Text] [PDF]

				M. A. Spassova, J. Soboloff, L.-P. He, W. Xu, M. A. Dziadek, and D. L. Gill STIM1 has a plasma membrane role in the activation of store-operated Ca2+ channels. PNAS, March 14, 2006; 103(11): 4040 - 4045. [Abstract] [Full Text] [PDF]

				E. Oancea, J. T. Wolfe, and D. E. Clapham Functional TRPM7 Channels Accumulate at the Plasma Membrane in Response to Fluid Flow Circ. Res., February 3, 2006; 98(2): 245 - 253. [Abstract] [Full Text] [PDF]

				Y. Dobrydneva, C. J. Abelt, B. Dovel, C. M. Thadigiri, R. L. Williams, and P. F. Blackmore 2-Aminoethoxydiphenyl Borate as a Prototype Drug for a Group of Structurally Related Calcium Channel Blockers in Human Platelets Mol. Pharmacol., January 1, 2006; 69(1): 247 - 256. [Abstract] [Full Text] [PDF]

				J. Lee, S.-K. Cha, T.-J. Sun, and C.-L. Huang PIP2 Activates TRPV5 and Releases Its Inhibition by Intracellular Mg2+ J. Gen. Physiol., October 31, 2005; 126(5): 439 - 451. [Abstract] [Full Text] [PDF]

				J. A. Kozak, M. Matsushita, A. C. Nairn, and M. D. Cahalan Charge Screening by Internal pH and Polyvalent Cations as a Mechanism for Activation, Inhibition, and Rundown of TRPM7/MIC Channels J. Gen. Physiol., October 31, 2005; 126(5): 499 - 514. [Abstract] [Full Text] [PDF]

				S. Feske, M. Prakriya, A. Rao, and R. S. Lewis A severe defect in CRAC Ca2+ channel activation and altered K+ channel gating in T cells from immunodeficient patients J. Exp. Med., September 6, 2005; 202(5): 651 - 662. [Abstract] [Full Text] [PDF]

				A. Y Estevez and K. Strange Calcium feedback mechanisms regulate oscillatory activity of a TRP-like Ca2+ conductance in C. elegans intestinal cells J. Physiol., August 15, 2005; 567(1): 239 - 251. [Abstract] [Full Text] [PDF]

				J. Jiang, M. Li, and L. Yue Potentiation of TRPM7 Inward Currents by Protons J. Gen. Physiol., July 25, 2005; 126(2): 137 - 150. [Abstract] [Full Text] [PDF]

				M. Matsushita, J. A. Kozak, Y. Shimizu, D. T. McLachlin, H. Yamaguchi, F.-Y. Wei, K. Tomizawa, H. Matsui, B. T. Chait, M. D. Cahalan, et al. Channel Function Is Dissociated from the Intrinsic Kinase Activity and Autophosphorylation of TRPM7/ChaK1 J. Biol. Chem., May 27, 2005; 280(21): 20793 - 20803. [Abstract] [Full Text] [PDF]

				V. Bugaj, V. Alexeenko, A. Zubov, L. Glushankova, A. Nikolaev, Z. Wang, E. Kaznacheyeva, I. Bezprozvanny, and G. N. Mozhayeva Functional Properties of Endogenous Receptor- and Store-operated Calcium Influx Channels in HEK293 Cells J. Biol. Chem., April 29, 2005; 280(17): 16790 - 16797. [Abstract] [Full Text] [PDF]

				A. B. Parekh and J. W. Putney Jr. Store-Operated Calcium Channels Physiol Rev, April 1, 2005; 85(2): 757 - 810. [Abstract] [Full Text] [PDF]

				C. Montell The TRP Superfamily of Cation Channels Sci. Signal., February 22, 2005; 2005(272): re3 - re3. [Abstract] [Full Text] [PDF]

				J. G. J. Hoenderop, B. Nilius, and R. J. M. Bindels Calcium Absorption Across Epithelia Physiol Rev, January 1, 2005; 85(1): 373 - 422. [Abstract] [Full Text] [PDF]

				Q.-H. Liu, X. Liu, Z. Wen, B. Hondowicz, L. King, J. Monroe, and B. D. Freedman Distinct Calcium Channels Regulate Responses of Primary B Lymphocytes to B Cell Receptor Engagement and Mechanical Stimuli J. Immunol., January 1, 2005; 174(1): 68 - 79. [Abstract] [Full Text] [PDF]

				S. I. Zakharov, T. Smani, Y. Dobrydneva, F. Monje, C. Fichandler, P. F. Blackmore, and V. M. Bolotina Diethylstilbestrol Is a Potent Inhibitor of Store-Operated Channels and Capacitative Ca2+ Influx Mol. Pharmacol., September 1, 2004; 66(3): 702 - 707. [Abstract] [Full Text] [PDF]

				H.-Z. Hu, Q. Gu, C. Wang, C. K. Colton, J. Tang, M. Kinoshita-Kawada, L.-Y. Lee, J. D. Wood, and M. X. Zhu 2-Aminoethoxydiphenyl Borate Is a Common Activator of TRPV1, TRPV2, and TRPV3 J. Biol. Chem., August 20, 2004; 279(34): 35741 - 35748. [Abstract] [Full Text] [PDF]

				T. Litjens, M. L. Harland, M. L. Roberts, G. J. Barritt, and G. Y. Rychkov Fast Ca2+-dependent inactivation of the store-operated Ca2+ current (ISOC) in liver cells: a role for calmodulin J. Physiol., July 1, 2004; 558(1): 85 - 97. [Abstract] [Full Text] [PDF]

				H. Kahr, R. Schindl, R. Fritsch, B. Heinze, M. Hofbauer, M. E. Hack, M. A. Mortelmaier, K. Groschner, J.-B. Peng, H. Takanaga, et al. CaT1 knock-down strategies fail to affect CRAC channels in mucosal-type mast cells J. Physiol., May 15, 2004; 557(1): 121 - 132. [Abstract] [Full Text] [PDF]

				D. M. Bautista and R. S. Lewis Modulation of plasma membrane calcium-ATPase activity by local calcium microdomains near CRAC channels in human T cells J. Physiol., May 1, 2004; 556(3): 805 - 817. [Abstract] [Full Text] [PDF]

				C. Zitt, B. Strauss, E. C. Schwarz, N. Spaeth, G. Rast, A. Hatzelmann, and M. Hoth Potent Inhibition of Ca2+ Release-activated Ca2+ Channels and T-lymphocyte Activation by the Pyrazole Derivative BTP2 J. Biol. Chem., March 26, 2004; 279(13): 12427 - 12437. [Abstract] [Full Text] [PDF]

				A. Gore, A. Moran, M. Hershfinkel, and I. Sekler Inhibitory Mechanism of Store-operated Ca2+ Channels by Zinc J. Biol. Chem., March 19, 2004; 279(12): 11106 - 11111. [Abstract] [Full Text] [PDF]

				L. V. Ryazanova, M. V. Dorovkov, A. Ansari, and A. G. Ryazanov Characterization of the Protein Kinase Activity of TRPM7/ChaK1, a Protein Kinase Fused to the Transient Receptor Potential Ion Channel J. Biol. Chem., January 30, 2004; 279(5): 3708 - 3716. [Abstract] [Full Text] [PDF]

				A. V. Yeromin, J. Roos, K. A. Stauderman, and M. D. Cahalan A Store-operated Calcium Channel in Drosophila S2 Cells J. Gen. Physiol., January 26, 2004; 123(2): 167 - 182. [Abstract] [Full Text] [PDF]

				T. Voets, B. Nilius, S. Hoefs, A. W. C. M. van der Kemp, G. Droogmans, R. J. M. Bindels, and J. G. J. Hoenderop TRPM6 Forms the Mg2+ Influx Channel Involved in Intestinal and Renal Mg2+ Absorption J. Biol. Chem., January 2, 2004; 279(1): 19 - 25. [Abstract] [Full Text] [PDF]

				X. Jiang, E. W. Newell, and L. C. Schlichter Regulation of a TRPM7-like Current in Rat Brain Microglia J. Biol. Chem., October 31, 2003; 278(44): 42867 - 42876. [Abstract] [Full Text] [PDF]

				J.-B. Peng, E. M Brown, and M. A Hediger Epithelial Ca2+ entry channels: transcellular Ca2+ transport and beyond J. Physiol., September 15, 2003; 551(3): 729 - 740. [Abstract] [Full Text] [PDF]

				A. Y. Estevez, R. K. Roberts, and K. Strange Identification of Store-independent and Store-operated Ca2+ Conductances in Caenorhabditis elegans Intestinal Epithelial Cells J. Gen. Physiol., July 28, 2003; 122(2): 207 - 223. [Abstract] [Full Text] [PDF]

				K. Gusev, L. Glouchankova, A. Zubov, E. Kaznacheyeva, Z. Wang, I. Bezprozvanny, and G. N. Mozhayeva The Store-operated Calcium Entry Pathways in Human Carcinoma A431 Cells: Functional Properties and Activation Mechanisms J. Gen. Physiol., June 30, 2003; 122(1): 81 - 94. [Abstract] [Full Text] [PDF]

				F.-J. Braun, O. Aziz, and J. W. Putney Jr. 2-Aminoethoxydiphenyl Borane Activates a Novel Calcium-Permeable Cation Channel Mol. Pharmacol., June 1, 2003; 63(6): 1304 - 1311. [Abstract] [Full Text] [PDF]

				F. Vanden Abeele, M. Roudbaraki, Y. Shuba, R. Skryma, and N. Prevarskaya Store-operated Ca2+ Current in Prostate Cancer Epithelial Cells. ROLE OF ENDOGENOUS Ca2+ TRANSPORTER TYPE 1 J. Biol. Chem., April 18, 2003; 278(17): 15381 - 15389. [Abstract] [Full Text] [PDF]

				O. Mignen, J. L. Thompson, and T. J. Shuttleworth Ca2+ Selectivity and Fatty Acid Specificity of the Noncapacitative, Arachidonate-regulated Ca2+ (ARC) Channels J. Biol. Chem., March 14, 2003; 278(12): 10174 - 10181. [Abstract] [Full Text] [PDF]

				A. B Parekh Store-operated Ca2+ entry: dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane J. Physiol., March 1, 2003; 547(2): 333 - 348. [Abstract] [Full Text] [PDF]

				T. Voets, A. Janssens, J. Prenen, G. Droogmans, and B. Nilius Mg2+-dependent Gating and Strong Inward Rectification of the Cation Channel TRPV6 J. Gen. Physiol., February 24, 2003; 121(3): 245 - 260. [Abstract] [Full Text] [PDF]

				Z. Su, D. S. Barker, P. Csutora, T. Chang, R. L. Shoemaker, R. B. Marchase, and J. E. Blalock Regulation of Ca2+ release-activated Ca2+ channels by INAD and Ca2+ influx factor Am J Physiol Cell Physiol, February 1, 2003; 284(2): C497 - C505. [Abstract] [Full Text] [PDF]

				D. E. Clapham Sorting out MIC, TRP, and CRAC Ion Channels J. Gen. Physiol., July 30, 2002; 120(2): 217 - 220. [Full Text] [PDF]

				J. A. Kozak, H. H. Kerschbaum, and M. D. Cahalan Distinct Properties of CRAC and MIC Channels in RBL Cells J. Gen. Physiol., July 30, 2002; 120(2): 221 - 235. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF, 1639K) PPT slides of all figures Correction (v119,p613) Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JGP Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Prakriya, M. Articles by Lewis, R. S. Search for Related Content PubMed PubMed Citation Articles by Prakriya, M. Articles by Lewis, R. S. Social Bookmarking                      What's this?