Am J Physiol Cell Physiol Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 284: C497-C505, 2003. First published October 16, 2002; doi:10.1152/ajpcell.00183.2002
0363-6143/03 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
284/2/C497    most recent
00183.2002v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Su, Z.
Right arrow Articles by Blalock, J. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Su, Z.
Right arrow Articles by Blalock, J. E.
Vol. 284, Issue 2, C497-C505, February 2003

Regulation of Ca2+ release-activated Ca2+ channels by INAD and Ca2+ influx factor

Zhengchang Su1, Douglas S. Barker1, Peter Csutora2, Theresa Chang2, Richard L. Shoemaker1, Richard B. Marchase2, and J. Edwin Blalock1

1 Department of Physiology and Biophysics and 2 Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The coupling mechanism between depletion of Ca2+ stores in the endoplasmic reticulum and plasma membrane store-operated ion channels is fundamental to Ca2+ signaling in many cell types and has yet to be completely elucidated. Using Ca2+ release-activated Ca2+ (CRAC) channels in RBL-2H3 cells as a model system, we have shown that CRAC channels are maintained in the closed state by an inhibitory factor rather than being opened by the inositol 1,4,5-trisphosphate receptor. This inhibitory role can be fulfilled by the Drosophila protein INAD (inactivation-no after potential D). The action of INAD requires Ca2+ and can be reversed by a diffusible Ca2+ influx factor. Thus the coupling between the depletion of Ca2+ stores and the activation of CRAC channels may involve a mammalian homologue of INAD and a low-molecular-weight, diffusible store-depletion signal.

store-operated ion channels; inositol 1,4,5-trisphosphate receptor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AGONIST-RECEPTOR INTERACTIONS at the plasma membrane often lead to the generation of inositol 1,4,5-trisphosphate (IP3), which in turn releases Ca2+ from internal stores in the endoplasmic reticulum (ER). Such depletion of Ca2+ stores gives rise to a signal that activates Ca2+-permeable store-operated channels (SOCs) in the plasma membrane, allowing for the sustained Ca2+ influx termed capacitative or stored-operated Ca2+ entry (6, 36). Four not necessarily distinct models have been proposed to account for the activation mechanism of SOCs (37). First, a physical link between IP3 receptors (IP3R) and SOCs is proposed (15, 17, 22, 35). Changes in the conformation of IP3R upon IP3 binding and/or store depletion activates SOCs. In the second, secretory-like transport vesicles are proposed as a source for the insertion of channels previously not present in the plasma membrane (30, 55). Third, a freely diffusible soluble messenger molecule, termed Ca2+ influx factor (CIF), is formed upon Ca2+ store depletion and activates the SOCs (8, 14, 38, 39, 44, 47), either directly or through interaction with closely associated regulatory proteins. Fourth, SOCs are kept in an inhibited state by an inhibitory mechanism when the Ca2+ stores are full, and discharge of Ca2+ stores removes such an inhibitory mechanism (37).

The Ca2+ release-activated Ca2+ (CRAC) channel is the best characterized of the SOCs (29). Its activation mechanism remains largely unknown (7), although several aspects of this regulation have been established. First, it is clear that depletion of intracellular Ca2+ stores (12, 28) leads to the activation of ICRAC (current conducted by CRAC channels) within tens of seconds. Second, CRAC channels in RBL cells are not likely activated by a secretion-like coupling mechanism (1). Third, CRAC channels in chicken DT40 cells in which all isoforms of IP3R are knocked out are activated normally upon store depletion, suggesting that physical coupling between IP3R and CRAC channels is not a mechanism of CRAC activation (33, 45). Fourth, whole cell patch-clamp experiments have established that achieving this electrophysiological configuration itself leads to ICRAC development, but only after hundreds of seconds and progressively less rapidly as the [Ca2+] in the pipette solution is buffered to levels approaching or exceeding 100 nM (12, 28). Importantly, this activation is seen well before global ER Ca2+ store depletion is observed (19). This finding suggests that low Ca2+ itself is sufficient to activate ICRAC, at least in the context of the large dilution of cytoplasmic constituents resulting from equilibration with the patch pipette solution. One explanation for this result invokes the dissociation of a regulatory factor from the CRAC channel, a dissociation that is facilitated by low levels of Ca2+ and dilution of the cytoplasm. Fifth, we have recently determined (8) that partially purified acid extracts from cells in which Ca2+ stores have been depleted, either with thapsigargin or by genetic disruption of sarcoplasmic endoplasmic reticulum Ca2+ ATPase, will accelerate the activation of ICRAC in whole cell patch-clamp experiments. This provides compelling evidence for the existence of a CIF-like molecule in CRAC channel regulation. However, how CIF might activate CRAC channels is essentially unknown. Here, we begin to reconcile these findings and suggest a unified model for CRAC channel activation.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Rat basophilic leukemia (RBL-2H3) cells were purchased from the American Type Culture Collection (Rockville, MD) and were maintained at 37°C in 5% CO2 in Eagle's minimum essential medium (Sigma, St. Louis, MO) with the following substitutions (Mediatech, Herndon, VA): 10% heat-inactivated fetal calf serum, 10,000 IU/ml penicillin, 10,000 µg/ml streptomycin, 2 mM glutamine, 10 mM sodium pyruvate, 10 mM nonessential amino acid, and 25 mM HEPES. Cells were treated by trypsin, plated on coverslips, and used on days 3 and 4.

Patch-clamp recordings. Whole cell recordings were performed with RBL-2H3 cells at room temperature. Standard external solution contained (in mM) 143 NaCl, 10 CaCl2, 1 MgCl2, 5 D-glucose, 4.5 KCl, 0.5 BaCl2, and 10 HEPES, pH 7.3 adjusted with NaOH. Ca2+-free solution (CFS) was made by replacing CaCl2 in this solution with an equimolar concentration of MgCl2 to abolish ICRAC and to obtain a leak current for subtraction to eliminate possible contamination current from Mg2+-inhibited cation (MIC) channels (34). Divalent cation-free solution (DVF) contained (in mM) 160 Na-methane sulfonate, 5 D-glucose, 2 Na-EGTA, and 10 HEPES, pH 7.2 adjusted with NaOH. Standard internal solution for whole cell recordings contained (in mM) 110 Cs-glutamate, 10 CsCl, 2.9 MgCl2, 0.6 CaCl2, 10 Cs-EGTA, and 30 HEPES, pH 7.2 adjusted with CsOH. The calculated free [Ca2+] and [Mg2+] were 10 nM and 2 mM, respectively. To obtain 100 nM free [Ca2+], the CaCl2 and MgCl2 concentrations in the standard internal solution were changed to 3.7 and 2.6 mM (free [Mg2+] = 2 mM), respectively. In some experiments, intracellular solution contained (in mM) 110 Cs-glutamate, 7 MgCl2, 5 Mg · ATP, 0.5 CaCl2, 10 Cs-EGTA, and 30 HEPES, pH 7.2 adjusted with CsOH. The calculated free [Ca2+] and [Mg2+] were 10 nM and 5 mM, respectively. This solution was used to block MIC channels (18, 26, 34, 40). Whole cell ICRAC was detected by applying a 100- or 320-ms voltage ramp from -100 to 100 mV while the cell was held at -40 mV at a frequency of 1 or 0.2 Hz. The currents measured when the cells were perfused by CFS were used as leak current for subtraction to eliminate possible MIC current contamination (34).

INAD expression and purification. Truncated inad gene produced by NcoI-PstI restriction endonuclease digestion of pCMV4/InaD was ligated into pBADmychisA (Invitrogen) to generate pBADInaDTr. An 890-bp PCR product generated from pCMV4/InaD with the primers Pr.INAD3'END (5'-TCG TCT CTA AAG CTT GGG TGC CTC-3') and Pr.INAD3 (5'-ATG GTC ATC TAT GGC AAG C-3') was digested with HindIII and ligated to PstI-HindIII-digested pBADInaDTr. This linear construct was digested with PstI and self-ligated to produce pBADInaD. Nucleotide sequences of both pBADInaD and pBADInaDTr were confirmed at the Iowa State University DNA Sequencing Facility (Ames, Iowa).

INAD expression from pBADInaD was induced in logarithmically growing Escherichia coli TOP10 cells with 0.2% L-arabinose in Luria-Bertani broth with 100 µg/ml ampicillin for 4 h at 37°C. Cells from 6 liters of induction medium were collected and resuspended in 10 ml of lysis buffer (6 M guanidine · HCl, 20 mM Na · phosphate, pH 7.4, 0.5 M NaCl, 0.1 mM PMSF, 0.1 µg/ml leupeptin, and 0.1 µg/ml Pefabloc) with gentle shaking on ice for 30 min and then disrupted by sonication. Cell debris was pelleted, and soluble proteins were renatured at 4°C by dialysis, first against dialysis buffer (1 M guanidine · HCl, 0.1 mM PMSF, 0.1 µg/ml leupeptin, and 0.1 µg/ml Pefabloc) and twice against water and protease inhibitors (0.1 mM PMSF, 0.1 µg/ml leupeptin, and 0.1 µg/ml Pefabloc). Precipitated proteins were removed by centrifugation, and the supernatant was brought to 30% saturation with ammonium sulfate at 4°C. Precipitated proteins were pelleted, and the supernatant was brought to 40% ammonium sulfate saturation at 4°C. Precipitated protein was again pelleted and then resuspended in loading buffer (20 mM Na · phosphate, pH 5.0, 0.5 M NaCl). Insoluble protein was removed by centrifugation, and the supernatant was applied to a HiTrap chelating column (Pharmacia Biotech) preequilibrated with loading buffer. The column was washed with 10 bed volumes of loading buffer, and INAD was eluted with 5 bed volumes of elution buffer (20 mM Na · phosphate, pH 4.0, 0.5 M NaCl). INAD was then dialyzed twice at 4°C against 1 liter of standard internal solutions for whole cell recordings, with precipitated proteins removed by centrifugation. Final protein concentrations were determined by Bradford assay using a BSA standard curve. The supernatant typically contained a protein concentration of 50-100 µg/ml and was used in the electrophysiological experiments without further dilution.

CIF preparation. We used a previously described procedure to prepare CIF from human platelets (8).

Data analysis. Patch-clamp data were analyzed using pCLAMP 8 (Axon, Foster City, CA). The activation time course of CRAC channels was fitted to equations
I=I<SUB>0</SUB>+I<SUB>1</SUB> exp[−(t − &tgr;<SUB>delay</SUB>)<IT>/&tgr;</IT>)]
where I is current, t is time, I0 and I1 are constants, tau  is the activation time constant, and tau delay is the time when the current develops to 10% of the maximum level after whole cell break-in. Data are shown as means ± SE, and the t-test was used for statistical analysis. The inhibitory effect of 2-aminoethoxydiphenyl borate (2-APB) on CRAC channels in Fig. 2D is defined by
Inhibition = (<IT>I</IT><SUB>before</SUB> − <IT>I</IT><SUB>after</SUB>)/<IT>I</IT><SUB>before</SUB>
where Ibefore is steady-state ICRAC before 2-APB application and Iafter is steady-state ICRAC after 2-APB application. The data are fitted to the Hill equation
Inhibition = Inhibition<SUB>max</SUB>/[1 + (IC<SUB>50</SUB>/[2 − APB]<SUP><IT>n</IT><SUB>H</SUB></SUP>)]
where Inhibitionmax is the maximum inhibition of ICRAC by 2-APB, IC50 is the 2-APB concentration that causes half-maximum inhibition, and nH is the Hill coefficient.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Whole cell ICRAC can be isolated by a leak current subtraction method. Several groups have recently demonstrated that omission of Mg2+ or Mg · ATP in the whole cell pipette can activate a cation current termed Mg2+-nucleotide-regulated metal ion (MagNum) current (11) or Mg2+-inhibited cation (MIC) current (34) in both Jurkat and RBL cells that is presumably encoded by LTRPC7/TRP-PLIK/TRPM7 gene (11, 18, 26, 34). Even though MIC channels can be inhibited intracellularly by Mg2+ alone with an IC50 of 0.5 mM, they still can be sporadically activated during whole cell recordings when intracellular free [Mg2+] is <3 mM (34). Prakriya and Lewis (34) have suggested that pure ICRAC can be isolated from possible MIC-contaminated current by subtracting a leak current taken when cells are exposed to a Ca2+-free but Mg2+-containing solution. To validate this method, we compared the divalent and monovalent currents recorded with two different intracellular solutions after the aforementioned leak current subtraction was performed. As shown in Fig. 1A, when the pipette contained 7 mM MgCl2 and 5 mM Mg · ATP, a typical ICRAC carried by Ca2+ with an inwardly rectifying current-voltage (I-V) relationship and a reversal potential >50 mV (Fig. 1B) was spontaneously activated after whole cell break-in. The channels activated were not permeable to Mg2+ (Fig. 1A), which is a hallmark property of CRAC channels (18, 20, 34). Switching the bath solution from the standard external solution to DVF invoked a larger Na+-carried current with an inwardly rectifying I-V relationship and a reversal potential around 50 mV, which rapidly inactivated in ~20 s to a steady state, another hallmark of ICRAC (2, 18, 20, 34), further suggesting that the current is solely conducted by CRAC channels. These results thus confirmed that high levels of Mg2+/ATP (~5 mM) in the pipette solution could completely block spontaneous activation of MIC channels (18, 20, 34). As shown in Fig. 1, C and D, when the free [Mg2+] level in the pipette was buffered to 2 mM and the aforementioned leak current subtraction method was used, Ca2+ and Na+ currents were recorded that were indistinguishable from those when the pipette contained 7 mM MgCl2 and 5 mM Mg · ATP in terms of Mg2+ impermeability, inwardly rectified I-V relationships for both Ca2+ and Na+ currents, reversal potentials, and inactivation of the monovalent current. These results suggest that the current isolated was solely conducted by CRAC channels and confirms that this leak current subtraction method is sufficient to eliminate possible MIC current contamination (34). We thus used this procedure of leak current subtraction in all the following experiments.


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 1.   Current conducted by Ca2+ release-activated Ca2+ (CRAC) channels (ICRAC) can be isolated by leak current subtraction. A: pipette solution contained 7 mM MgCl2 and 5 mM Mg · ATP. Top trace was measured at 80 mV; bottom trace was measured at -80 mV. Number-labeled arrows indicate time points for display of the current-voltage (I-V) relationship in B. CFS, Ca2+-free solution; DVF, Divalent cation-free solution. B: I-V relationships of current taken at the corresponding time points in A. C: pipette solution contained 2.9 mM MgCl2 (free [Mg2+] = 2 mM). The labels have the same meaning as in A. D: I-V relationships of current taken at the corresponding time points in C.

CRAC channels are not activated by physical interaction between IP3R and CRAC channels. Recent results with an IP3R blocker 2-APB have been used to argue that SOC activation is due to an IP3- and/or depletion-mediated conformational change in the IP3R (22). The relative unimportance of the IP3R as an activator for CRAC channels is suggested by the observation that ICRAC spontaneously and normally developed during whole cell dialysis even in the presence of the IP3 receptor antagonist heparin (0.5 mM) in the pipette solution (Fig. 2A; tau delay = 95 ± 6.1 s for heparin and 100 ± 8.4 s for control, P > 0.1; tau  = 149.4 ± 20.9 s for heparin and 149.5 ± 34.1 s for control, P > 0.25). These results are also consistent with previous reports (1, 5). Similar results were observed with another IP3R inhibitor, xestospongin C (17) (20 µM in pipette solution, data not shown). Thus, if the IP3R is involved in CRAC activation, a conformational change due to IP3 seems not to be required.


View larger version (39K):
[in this window]
[in a new window]
  		Fig. 2.   CRAC channels are not activated by inositol 1,4,5-trisphosphate receptor (IP3R)/channel interaction. A: 0.5 mM heparin in the pipette solution fails to inhibit the activation of ICRAC. tau delay = 95 ± 6.1 and 100 ± 8.4 s for heparin and blank control, respectively (P > 0.1). tau  = 149.4 ± 20.9 and 149.5 ± 34.1 s for heparin and blank control, respectively (P > 0.25; n = 6 for both groups). Data were collected at 0.2 Hz and are presented as means ± SE. B: 75 µM 2-aminoethoxydiphenyl borate (2-APB) in the pipette solution prevents IP3 (10 µM)-induced acceleration of the CRAC channel activation by whole cell dialysis. tau  = 30.0 ± 1.1 s, n = 7; 168.6 ± 8.6 s, n = 7; and 85.2 ± 3.3 s, n = 8, for dialysis with IP3 alone or IP3 plus 2-APB in the pipette solution and for passive dialysis, respectively (P < 0.001 for both IP3 vs. IP3 + 2-APB and IP3 vs. passive dialysis). tau delay = 15.7 ± 0.9 s, n = 7; 41.4 ± 1.4 s, n = 7; and 85.2 ± 3.3 s, n = 8, for dialysis with IP3 alone or IP3 + 2-APB in the pipette solution and for passive dialysis, respectively (P < 0.001 for IP3 vs. IP3 plus 2-APB; P < 0.005 for IP3 vs. passive dialysis). Data were collected at 1 Hz and are presented as means + SE for clarity. C: 75 µM 2-APB in the pipette solution fails to inhibit the activation of ICRAC by whole cell dialysis of the cytosol. However, 0.4 or 2 µM 2-APB from the extracellular side of the patch can partially or completely block ICRAC, respectively. D: dose-response relation of 2-APB inhibition of ICRAC from extracellular side (n = 5). Fitting the data to the Hill equation yielded an IC50 of 0.5 µM and a Hill coefficient of 1.4. Data are presented as means ± SE.

To further explore the possible regulatory role of the IP3R on the CRAC channel, we have also used 2-APB. As previously reported (24), this compound, as shown in Fig. 2B, when included in the pipette solution at 75 µM, blocked IP3 action as evidenced by its delaying the onset of ICRAC development (tau delay = 15.7 ± 0.9 s, n = 7; 41.4 ± 1.4 s, n = 7; and 85.2 ± 3.3 s, n = 8 for IP3 alone, IP3 plus 2-APB, and passive dialysis, respectively; P < 0.001 for both IP3 alone vs. IP3 plus 2-APB and IP3 vs. passive dialysis) as well as by its inhibition of the IP3-mediated acceleration of the development of ICRAC (tau  = 30.0 ± 1.1 s, n = 7; 168.6 ± 8.6 s, n = 7; and 85.2 ± 3.3 s, n = 8 for IP3 alone, IP3 plus 2-APB, and passive dialysis, respectively; P < 0.001 for IP3 alone vs. IP3 plus 2-APB; P < 0.005 for IP3 vs. passive dialysis). However, in the presence of 2-APB in the pipette solution, ICRAC spontaneously developed, although with a smaller amplitude (Fig. 2B; 0.82 ± 0.22 pA/pF for IP3 and 0.52 ± 0.18 pA/pF for IP3 plus 2-APB, P < 0.01). The concentration required to delay ICRAC activation to the time seen with passive dialysis alone was found to be approximately that previously reported for its effects on the IP3R, an IC50 of 42 µM (24). Interestingly, with 75 µM 2-APB in the pipette, ICRAC was completely and reversibly blocked by the application of 2 µM 2-APB to the extracellular side of the patched cell (Fig. 2C). The IC50 for this effect was ~0.5 µM (Fig. 2D). This finding suggests that 2-APB was effective in blocking ICRAC not because of an effect on the IP3R but, rather, by directly affecting the extracellular face of the CRAC channel. The reduced ICRAC in the presence of 75 µM 2-APB in the pipette solution (Fig. 2, B and C) may solely result from its diffusion across the plasma membrane (24) so as to affect the CRAC channel from the extracellular side. Thus, while 2-APB is clearly an IP3R/channel blocker, it appears to be a far more potent CRAC channel blocker. These results cause us to question the possibility that the IP3R is a direct activator of the CRAC channel (22) and strengthen our previous hypothesis that CRAC channels are activated by a CIF-like molecule directly or through a regulatory intermediate (8, 44). The extracellular effects of 2-APB on CRAC channels have also been found by others (1, 4, 33). However, we did not observe a potentiation effect of 2-APB on ICRAC at low concentrations (Fig. 2C) as reported previously (33). This discrepancy might be due to different cells used in the current vs. the previous (Jurkat and DT40 cells) studies.

CRAC channels do not require the continued presence of a diffusible factor once they are activated in whole cell configuration. Although our previous results suggested that CRAC channels are activated by CIF (8, 44), how CIF activates CRAC channels is unknown. If CRAC channels are activated by direct binding of CIF, then ICRAC might be predicted to inactivate after a period of time in whole cell recordings. This might occur because the concentration of CIF would decrease if its rate of synthesis fell behind its rate of removal due to degradation and/or the exchange between the cytosol and pipette solution. In contrast to this prediction, as shown in Fig. 3, after ICRAC was activated by dialysis of the cytosol by pipette solution, it could be recorded more than 120 min after whole cell break-in (n = 4), even though the channels underwent slow inactivation process as previously reported (27, 57). As a result of diffusion (23), this period of time should have washed out a small molecule such as CIF with a molecular weight <1,000 Dalton (38). This might suggest that, once activated, maintaining the CRAC channels in an open state does not require the continued presence of a diffusible molecule such as CIF under this recording condition. In other words, CRAC channels are unlikely activated by direct binding of CIF. Our hypothesis to explain this is that the CRAC channel might be activated by CIF through removal of an inhibitory molecule that otherwise keeps it in a closed state. Krause et al. (19) have recently found that ICRAC could be activated in the absence of global ER store depletion by low cytoplasmic [Ca2+]. One explanation of their results is that a low cytoplasmic [Ca2+] environment promotes the dissociation of the putative inhibitor from the CRAC channel, which then activates. In other words, the inhibitory factor associates with the CRAC channel in a Ca2+-dependent manner. Thus what is the putative inhibitory factor for CRAC channels?


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3.   Activation of CRAC channels does not require the continued presence of a diffusible factor once they are activated. A: ICRAC can be recorded as long as the whole cell configuration is maintained. CRAC channels are activated by whole cell dialysis of the cytosol. ICRAC was detected by a -100- to 100-mV voltage ramp in 320 ms at a 5-s interval, and the current at -80 mV was plotted as a function of time. Horizontal bars indicate the replacement of extracellular Ca2+ by equimolar Mg2+ (CFS; see MATERIALS AND METHODS). B: I-V relationship of current taken at the corresponding number-labeled time points in A. Note the positive reversal potential (>50 mV) and inwardly rectifying I-V relationships of ICRAC.

INAD inhibits CRAC channels in a Ca2+-dependent manner. Detection of light by photoreceptors in the eye of Drosophila requires activation of a cation channel that includes transient receptor potential (TRP) channel subunits (54). Mammalian cells express homologues of TRP, and certain of these can function as SOCs (3). Indeed, there is evidence that mammalian TRPs may be components of CRAC channels (32, 51, 56). In Drosophila, inactivation-no after potential D (INAD), a multivalent PDZ domain-containing protein, functions as a scaffold protein as well as a regulator of the TRP/TRPL channel (21, 41, 43, 48, 49, 53). In particular, it associates with TRP/TRPL through one or two of its PDZ domains with a peptide motif, -S/T-X-V/I, in the carboxyl (COOH) terminal cytoplasmic domain of TRP/TRPL (25, 42), as well as the COOH terminus itself (21). This leads to deactivation of TRP and/or TRPL Ca2+ channel activity (21, 42). A recent study further demonstrated that overexpression of INAD inhibits TRP channels (10). Interestingly, we have found a number of S/T-X-V/I motifs as well as another PDZ domain-binding motif, phi -x-phi , where phi  is a hydrophobic amino acid, in the COOH-terminal domain of TRP1-6 of the mammalian TRP family (data not shown). Indeed, TRP4 and TRP5 can bind to a PDZ-containing scaffold protein, Na2+/H+ exchanger regulatory factor (NHERF) (46). In addition, we also found a putative EF-hand Ca2+-binding motif from G279-I291 in the second PDZ domain of INAD (NH2-GVDPNGALGSVDI-COOH). Consequently, we tested INAD as a surrogate inhibitor of ICRAC.

Figure 4A shows an average whole cell activation profile of ICRAC when the pipette solution is buffered to 10 nM Ca2+ (n = 8). After a 140.0 ± 10.4-s delay, ICRAC begins to activate spontaneously with a time constant tau  of 85.6 ± 3.3 s. In the presence of 100 nM Ca2+ in the pipette solution (Fig. 4B, n = 8), the onset was delayed (tau delay = 320.3 ± 20.1 s, P < 0.05) compared with that when pipette solution contains 10 nM Ca2+, and the activation course was also slower (tau  = 499.3 ± 53.9 s, P < 0.05) compared with that when pipette solution contains 10 nM Ca2+. When electrophoretically pure INAD (73 µg/ml, Fig. 4I) was included in the pipette solution with Ca2+ buffered to 10 nM (Fig. 4C, n = 6), the onset was somewhat delayed (tau delay = 281.1 ± 17.6 s) and more gradual (tau  =160.3 ± 6.6 s) compared with that when INAD was not included (tau delay = 140.0 ± 10.4 s, P < 0.001 and tau  = 85.6 ± 3.3 s, P < 0.001), but full activation was routinely achieved. In contrast, when INAD was included at 100 nM Ca2+, no activation was observed (Fig. 4D, n = 7). This inhibition persisted even after store-depletion with 10 µM ionomycin, suggesting that endogenous CIF, hypothesized to be produced in response to ionomycin-mediated depletion of stores, was not sufficient to activate CRAC in the presence of INAD. No such inhibition was seen in the presence of other proteins, including an extract of E. coli proteins from strains not expressing INAD (Fig. 4E, n = 6, tau delay = 327.7 ± 23.5 s, P > 0.5; tau  = 498.6 ± 67.9 s, P > 0.5). The inability of endogenous CIF to reverse INAD was likely due to the relatively large amount of recombinant INAD and the relatively low concentration of CIF such that only a fraction of INAD is bound to CIF and, hence, CRAC channels stays inactive with INAD bound. To test this hypothesis, we used exogenous CIF at a presumably higher concentration. As shown in Fig. 4F, inclusion of both INAD and platelet CIF (1:50 dilution) in the patch pipette resulted in full activation of ICRAC (tau delay = 128.8 ± 4.6 s, tau  = 44.1 ± 1.4 s, n = 8). In contrast, a small dose of CIF (1:500 dilution) could not reverse the inhibitory effect of INAD on ICRAC activation (Fig. 4G, n = 5), even though this dose of CIF could tremendously accelerate the activation of ICRAC when intracellular [Ca2+] was buffered at 100 nM (Fig. 4H, tau delay = 20.2 ± 2.4 s, tau  = 97.96 ± 3.9 s, n = 5, P < 0.001 compared with data in Fig. 4B).


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 4.   CRAC channels are inhibited by INAD (inactivation-no after potential D) in a Ca2+-dependent manner, and the inhibition is reversed by Ca2+ influx factor (CIF). A: whole cell recording with a pipette solution containing 10 nM free Ca2+ rapidly activates ICRAC: tau delay = 140 ± 10.4 s, tau  = 85.4 ± 3.3 s, n = 8. B: 100 nM free Ca2+ in the pipette solution delays the activation of ICRAC: tau delay = 320.3 ± 20.1 s, n = 8 (P < 0.05 compared with that in A); tau  = 499.3 ± 53.9 s, n = 8 (P < 0.05 compared with that in A). C: INAD (73 µg/ml) only delays the activation of ICRAC when the free Ca2+ level is 10 nM: tau delay = 281.1 ± 17.6 s, tau  = 160.3 ± 6.6 s, n = 6 (P < 0.001 compared with those in A). D: INAD (73 µg/ ml) completely prevents the activation of ICRAC when the free Ca2+ level is 100 nM; even application of 10 µM ionomycin cannot rescue this inhibition (n = 7). E: Escherichia coli extract that does not contain INAD fails to prevent the activation of ICRAC by dialysis in presence of 100 nM free Ca2+: tau delay = 327.7 ± 23.5 s, tau  = 498.6 ± 67.9 s, n = 6 (P > 0.5 compared with those in B). F: CIF (1:50 dilution) in the pipette solution reverses the inhibition of INAD on ICRAC: tau delay = 128.8 ± 4.6 s, tau  = 44.1 ± 1.4 s, n = 8. G: a low dose of CIF (1:500 dilution) cannot reverse the inhibition of ICRAC by INAD (n = 5). H: this low dose of CIF (1:500 dilution), however, accelerates the activation of ICRAC: tau delay = 20.2 ± 2.4 s, tau  = 97.96 ± 3.9 s, n = 5 (P < 0.001 compared with data in B). I: purification of recombinant INAD from E. coli. Protein samples (10 µl each) from various stages of INAD purification were electrophoresed on a 15% SDS-polyacrylamide gel and stained with Coomassie blue. Lane 1, 30% ammonium sulfate precipitate; lane 2, Ni2+ affinity column chromatography of 30% ammonium sulfate precipitate after resolubilization; lane 3, 40% ammonium sulfate precipitate; lane 4, Ni2+ affinity column chromatography of 40% ammonium sulfate precipitate after resolubilization.

Because of CIF's property of overcoming INAD's inhibition of ICRAC, we asked whether a direct association between the two could be established. Affinity-purified INAD was covalently linked to a Sepharose matrix, and a partially purified extract from activated platelets containing CIF was passed over the column. In contrast to a column lacking INAD, the column run-through, although containing nearly all the optical density at 262 nm, was found to be devoid of the CIF activity of the original extract, as assessed by activity in Xenopus oocytes (Fig. 5) (8). Fractions eluting several bed volumes later contained >80% of the activity originally applied to the column. These results suggest that CIF can directly bind to INAD.


View larger version (11K):
[in this window]
[in a new window]
  		Fig. 5.   Elution profile of CIF activity from an INAD-Sepharose column. After HPLC strong anion exchange chromatography, fractions containing CIF activity were dried down and reextracted with methanol. Active fractions were solubilized in 0.1 N acetic acid and buffered to pH 6.0 with NaOH. They were then applied to either an INAD-Sepharose column or to a comparable column without coupled INAD but cyanogen bromide-activated and blocked with Tris buffer. Bed volume (0.5 ml) fractions were collected and assessed for optical density at 262 nm (OD262) and CIF activity using the Xenopus oocyte bioassay. Open bars, activity profile using the uncoupled Sepharose column; filled bars, CIF activity eluted from INAD-Sepharose. Solid line is the OD profile.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several groups have recently demonstrated that low levels of free [Mg2+] (<3 mM) in the pipette solution may activate MIC and ICRAC spontaneously and simultaneously (11, 18, 34) in both Jurkat and RBL cells during whole cell recordings. MIC may be responsible for the previously claimed single-channel current of CRAC channels when divalent cations are taken from both sides of the plasma membrane (4, 9, 13). Single-channel conductance of CRAC channels for Na+ in the absence of extracellular Ca2+ was estimated at 0.2 pS by noise analysis (34), which is beyond the current resolution of patch-clamp recording techniques. Although ICRAC and MIC can be simultaneously and spontaneously activated during whole cell recordings when intracellular free [Mg2+] is low, there are still ways to separate ICRAC and MIC by using the different properties of these currents. In this study we isolated ICRAC from possible MIC-contaminated current by subtracting the leak current recorded while cells were exposed to Ca2+-free but Mg2+-containing solutions after ICRAC was fully activated, as suggested previously by Prakriya and Lewis (34) on the basis that MIC channels are equally permeable to Ca2+ and Mg2+ whereas CRAC channels are only permeable to Ca2+. The success of this method is manifested by the fact that the isolated currents were indistinguishable from currents recorded when MIC was presumably completely blocked by high concentrations of Mg/ATP, in terms of current size, inwardly rectifying I-V relationship, and inactivation of monovalent cation currents (Fig. 1).

It has recently been suggested that the IP3R is involved in the activation of some SOCs (15, 17, 35). However, this appears not to be the case for CRAC channels on the following basis. First, inclusion of the IP3R inhibitor heparin in the whole cell pipette failed to affect the spontaneous activation of ICRAC (Fig. 2A), which is consistent with previous reports (1, 5). Another IP3R inhibitor, xestospongin C, also could not inhibit ICRAC during whole cell recordings when applied from either the intracellular or extracellular side of the plasma membrane (unpublished observation). Second, knockout of all isoforms of IP3R in DT40 cells fails to affect ICRAC activation in these cells (33, 45) (but see Ref. 16).

Although 2-APB can efficiently inhibit CRAC channels, it apparently exerts the effect by directly blocking the CRAC channel extracellularly, instead of disrupting the physical interaction between CRAC channels and IP3R. Some of the previous evidence for SOC/IP3R coupling (22) can now be explained by the finding that 2-APB is a potent and direct CRAC channel blocker. Thus caution needs to be taken when interpreting data with 2-APB. This, of course, does not negate the possibility that the IP3R directly couples to and regulates certain SOCs or mammalian TRP channels such as TRP3 that do not show the precise features of ICRAC (17, 37, 50).

On the basis of the present results with INAD, it is tempting to speculate that mammalian homologues of this protein are key elements in CRAC channel regulation. These data lead to a model of the signaling mechanism that couples ER Ca2+ store depletion in mammalian cells with plasma membrane CRAC channels (Fig. 6). Specifically, there is Ca2+-dependent conformational coupling between a putative INAD homologue (which we termed MINAD, for mammalian INAD) and one or more mammalian TRP subunits comprising the CRAC channel. This occurs through interactions between the TRP and certain PDZ domains on MINAD, which also may serve to anchor the complex to the cytoskeleton (52). In the intact cell, Ca2+ and MINAD function to maintain CRAC channels in the closed state until a diffusible store-depletion signal, CIF, causes the complex to dissociate and CRAC channels to open. After refilling of the stores and the decay of CIF, the complex is reestablished and CRAC channels close. In the absence of CIF, low local [Ca2+] either physiologically or experimentally due to passive dialysis may also result in disassociation of MINAD from the CRAC channel and, thus, ICRAC activation. In this light, it is interesting to note the putative EF-hand Ca2+-binding motif (G279-I291) in the second PDZ domain of INAD. We hypothesize that dissociation of Ca2+ from this site due to low local Ca2+ levels induces a conformational change in MINAD and, thus, dissociation of MINAD from the CRAC channels. This may well explain the finding by Krause et. al (19) that ICRAC can be activated by low levels of cytoplasmic Ca2+ independently of store depletion. Therefore, CIF might serve as a coarse regulator of CRAC channels while the local cytoplasmic Ca2+ serves as a fine tuner.


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 6.   Model for activation of ICRAC. A: a mammalian INAD (MINAD) serves as the organizer of the "signalplex" (21, 48) and as an inhibitor of CRAC channels in an unstimulated cell. PDZ domains within MINAD bind phospholipase C (PLC), the actin-myosin cytoskeleton, and the CRAC channel, while the EF-hand motif of MINAD is occupied by Ca2+. ER, endoplasmic reticulum; PM, plasma membrane; R, receptor; G, G protein. B: when the cell is activated by an agonist binding to its receptor in the plasma membrane, CIF is produced by the Ca2+ store-depleted ER. Binding of CIF to MINAD facilitates dissociation of MINAD from the CRAC channel and allows the channel to open. Alternatively, dissociation of MINAD from the channel can occur when intracellular [Ca2+] is low, such as during whole cell recordings or at some physiological conditions. Dissociation of MINAD from the CRAC channel allows the opening of the channel even in the absence of store depletion.

This model has some very attractive features that add to its credibility. First, TRP mammalian homologues have PDZ domain-binding motifs, as mentioned before. Second, INAD homologues exist in mammalian cells (31). Most interestingly, a PDZ domain-containing mammalian protein, NHERF, which interacts with the cytoskeleton, was recently found to bind to the TRP4 and TRP5 proteins (46). Third, the association/dissociation of CRAC channels and INAD occurs at physiologically relevant [Ca2+] (~100 nM). Fourth, overexpression of INAD inhibits the activation of TRP channels by store depletion (10).

These results also provide further strong support for the existence of CIF. The interaction with INAD may well provide a means to purify and identify this elusive substance. In addition, the "secretory vesicle/kiss and run" model might now be explained by the necessity of delivering vesicles producing CIF to the vicinity of the CRAC channel/INAD-like complex. Finally, results on some SOC with agents that affect the cytoskeleton (30, 55) may well be due to alterations in PDZ domain interactions with cytoskeletal elements, which are known to occur with INAD and other PDZ domain-containing proteins (52).


    ACKNOWLEDGEMENTS

We thank Dr. B. Shieh (Vanderbilt University) for providing the pCmv4/INAD clone and Dr. K. Mikoshiba (Tokyo University) for the generous gift of 2-APB.


    FOOTNOTES

This work was supported in part by National Institutes of Health Grants AI-37670 and HL-68806 (to J. E. Blalock) as well as DK-55647 and HL-68806 (to J. E. Blalock and R. B. Marchase), Juvenile Diabetes Foundation Grant 2000-137 (to R. B. Marchase and J. E. Blalock), and a grant from the C. C. Wu Foundation in Hong Kong (Z. Su).

Address for reprint requests and other correspondence: J. E. Blalock, Dept. of Physiology and Biophysics, Univ. of Alabama at Birmingham, MCLM 898, 1918 Univ. Blvd., Birmingham, AL 35294-0005 (E-mail: Blalock{at}uab.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published October 16, 2002;10.1152/ajpcell.00183.2002

Received 22 April 2002; accepted in final form 8 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bakowski, D, Glitsch MD, and Parekh AB. An examination of the secretion-like coupling model for the activation of the Ca2+ release-activated Ca2+ current ICRAC in RBL-1 cells. J Physiol 532: 55-71, 2001[Abstract/Free Full Text].

2.   Bakowski, D, and Parekh AB. Monovalent cation permeability and Ca2+ block of the store-operated Ca2+ current ICRAC in rat basophilic leukemia cells. Pflügers Arch 443: 892-902, 2002[Web of Science][Medline].

3.   Birnbaumer, L, Zhu X, Jiang M, Boulay G, Peyton M, Vannier B, Brown D, Platano D, Sadeghi H, Stefani E, and Birnbaumer M. On the molecular basis and regulation of cellular capacitative calcium entry: roles for Trp proteins. Proc Natl Acad Sci USA 93: 15195-15202, 1996[Abstract/Free Full Text].

4.   Braun, FJ, Broad LM, Armstrong DL, and Putney JWJ Stable activation of single CRAC-channels in divalent cation-free solutions. J Biol Chem 276: 1063-1070, 2000.

5.   Broad, LM, Braun FJ, Lievremont JP, Bird GS, Kurosaki T, and Putney JWJ 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, 2001[Abstract/Free Full Text].

6.   Clapham, DE. Calcium signaling. Cell 80: 259-268, 1995[Web of Science][Medline].

7.   Clapham, DE. TRP is cracked but is CRAC TRP? Neuron 16: 1069-1072, 1996[Web of Science][Medline].

8.   Csutora, P, Su Z, Kim HY, Bugrim A, Cunningham KW, Nuccitelli R, Keizer JE, Hanley MR, Blalock JE, and Marchase RB. Calcium influx factor is synthesized by yeast and mammalian cells depleted of organellar calcium stores. Proc Natl Acad Sci USA 96: 121-126, 1999[Abstract/Free Full Text].

9.   Fomina, AF, Fanger CM, Kozak JA, and Cahalan MD. Single channel properties and regulated expression of Ca2+ release-activated Ca2+ (CRAC) channels in human T cells. J Cell Biol 150: 1435-1444, 2000[Abstract/Free Full Text].

10.   Harteneck, C, Kuchta SN, Huber A, Paulsen R, and Schultz G. The PDZ-scaffold protein INAD abolishes apparent store-dependent regulation of the light-activated cation channel TRP. FASEB J 16: 1668-1670, 2002[Abstract/Free Full Text].

11.   Hermosura, MC, Monteilh-Zoller MK, Scharenberg AM, Penner R, and Fleig A. Dissociation of the store-operated calcium current ICRAC and the Mg-nucleotide-regulated metal ion current MagNuM. J Physiol 539: 445-458, 2002[Abstract/Free Full Text].

12.   Hoth, M, and Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature 355: 353-356, 1992[Medline].

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

14.   Kim, HY, and Hanley MR. Calcium influx factor (CIF) as a diffusible messenger for the activation of capacitative calcium entry in Xenopus oocytes. Mol Cell 9: 326-332, 1999.

15.   Kiselyov, K, Mignery GA, Zhu MX, and Muallem S. The N-terminal domain of the IP3 receptor gates store-operated hTrp3 channels. Mol Cell 4: 423-429, 1999[Web of Science][Medline].

16.   Kiselyov, K, Shin DM, Shcheynikov N, Kurosaki T, and Muallem S. Regulation of Ca2+-release-activated Ca2+ current (Icrac) by ryanodine receptors in inositol 1,4,5-trisphosphate-receptor-deficient DT40 cells. Biochem J 360: 17-22, 2001[Web of Science][Medline].

17.   Kiselyov, K, Xu X, Mozhayeva G, Kuo T, Pessah I, Mignery G, Zhu X, Birnbaumer L, and Muallem S. Functional interaction between InsP3 receptors and store-operated Htrp3 channels. Nature 396: 478-482, 1998[Medline].

18.   Kozak, JA, Kerschbaum HH, and Cahalan MD. Distinct properties of CRAC and MIC channels in RBL cells. J Gen Physiol 120: 221-235, 2002[Abstract/Free Full Text].

19.   Krause, E, Schmid A, Gonzalez A, and Schulz I. Low cytoplasmic [Ca2+] activates ICRAC independently of global Ca2+ store depletion in RBL-1 cells. J Cell Biol 274: 36957-36962, 1999.

20.   Lepple-Wienhues, A, and Cahalan MD. Conductance and permeation of monovalent cations through depletion-activated Ca2+ channels (ICRAC) in Jurkat T cells. Biophys J 71: 787-794, 1996[Web of Science][Medline].

21.   Li, HS, and Montell C. TRP and the PDZ protein, INAD, form the core complex required for retention of the signalplex in Drosophila photoreceptor cells. J Cell Biol 150: 1411-1422, 2000[Abstract/Free Full Text].

22.   Ma, HT, Patterson RL, van Rossum DB, Birnbaumer L, Mikoshiba K, and Gill DL. Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels. Science 287: 1647-1651, 2000[Abstract/Free Full Text].

23.   Marty, A, and Neher E. Tight-seal whole-cell recording. In: Single-Channel Recording, edited by Sakmann B, and Neher E.. New York: Plenum, 1995, p. 42-44.

24.   Maruyama, T, Kanaji T, Nakade S, Kanno T, and Mikoshiba K. 2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2+ release. J Biochem (Tokyo) 122: 498-505, 1997[Abstract/Free Full Text].

25.   Montell, C. New light on TRP and TRPL. Mol Pharmacol 52: 755-763, 1997[Abstract/Free Full Text].

26.   Nadler, MJ, Hermosura MC, Inabe K, Perraud AL, Zhu Q, Stokes AJ, Kurosaki T, Kinet JP, Penner R, Scharenberg AM, and Fleig A. LTRPC7 is a Mg · ATP-regulated divalent cation channel required for cell viability. Nature 411: 590-595, 2001[Medline].

27.   Parekh, AB. Slow feedback inhibition of calcium release-activated calcium current by calcium entry. J Biol Chem 273: 14925-14932, 1998[Abstract/Free Full Text].

28.   Parekh, AB, Fleig A, and Penner R. The store-operated calcium current ICRAC: nonlinear activation by InsP3 and dissociation from calcium release. Cell 89: 973-980, 1997[Web of Science][Medline].

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

30.   Patterson, RL, van Rossum DB, and Gill DL. Store-operated Ca2+ entry: evidence for a secretion-like coupling model. Cell 98: 487-499, 1999[Web of Science][Medline].

31.   Philipp, S, and Flockerzi V. Molecular characterization of a novel human PDZ domain protein with homology to INAD from Drosophila melanogaster. FEBS Lett 413: 243-248, 1997[Web of Science][Medline].

32.   Philipp, S, Trost C, Warnat J, Rautmann J, Himmerkus N, Schroth G, Kretz O, Nastainczyk W, Cavalie A, Hoth M, and Flockerzi V. TRP4 (CCE1) protein is part of native calcium release-activated Ca2+-like channels in adrenal cells. J Biol Chem 275: 23965-23972, 2000[Abstract/Free Full Text].

33.   Prakriya, M, and Lewis RS. 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, 2001[Abstract/Free Full Text].

34.   Prakriya, M, and Lewis RS. Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels. J Gen Physiol 119: 487-507, 2002[Abstract/Free Full Text].

35.   Putney, JWJ TRP, inositol 1,4,5-trisphosphate receptors, and capacitative calcium entry. Proc Natl Acad Sci USA 96: 14669-14671, 1999[Free Full Text].

36.   Putney, JWJ, and Bird GS. The signal for capacitative calcium entry. Cell 75: 199-201, 1993[Web of Science][Medline].

37.   Putney, JWJ, Broad LM, Braun FJ, Lievremont JP, and Bird GS. Mechanisms of capacitative calcium entry. J Cell Sci 114: 2223-2229, 2001[Web of Science][Medline].

38.   Randriamampita, C, and Tsien RY. Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx. Nature 364: 809-814, 1993[Medline].

39.   Randriamampita, C, and Tsien RY. Degradation of a calcium influx factor (CIF) can be blocked by phosphatase inhibitors or chelation of Ca2+. J Biol Chem 270: 29-32, 1995[Abstract/Free Full Text].

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

41.   Shieh, BH, and Niemeyer B. A novel protein encoded by the InaD gene regulates recovery of visual transduction in Drosophila. Neuron 14: 201-210, 1995[Web of Science][Medline].

42.   Shieh, BH, and Zhu MY. Regulation of the TRP Ca2+ channel by INAD in Drosophila photoreceptors. Neuron 16: 991-998, 1996[Web of Science][Medline].

43.   Shieh, BH, Zhu MY, Lee JK, Kelly IM, and Bahiraei F. Association of INAD with NORPA is essential for controlled activation and deactivation of Drosophila phototransduction in vivo. Proc Natl Acad Sci USA 94: 12682-12687, 1997[Abstract/Free Full Text].

44.   Su, Z, Csutora P, Hunton D, Shoemaker RL, Marchase RB, and Blalock JE. A store-operated nonselective cation channel in lymphocytes is activated directly by Ca2+ influx factor and diacylglycerol. Am J Physiol Cell Physiol 280: C1284-C1292, 2001[Abstract/Free Full Text].

45.   Sugawara, H, Kurosaki M, Takata M, and Kurosaki T. Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor. EMBO J 16: 3078-3088, 1997[Web of Science][Medline].

46.   Tang, Y, Tang J, Chen Z, Trost C, Flockerzi V, Li M, Ramesh V, and Zhu MX. Association of mammalian trp4 and phospholipase C isozymes with a PDZ domain-containing protein, NHERF. J Biol Chem 275: 37559-37564, 2000[Abstract/Free Full Text].

47.   Trepakova, ES, Csutora P, Hunton DL, Marchase RB, Cohen RA, and Bolotina VM. Calcium influx factor directly activates store-operated cation channels in vascular smooth muscle cells. J Biol Chem 275: 26158-26163, 2000[Abstract/Free Full Text].

48.   Tsunoda, S, Sierralta J, Sun Y, Bodner R, Suzuki E, Becker A, Socolich M, and Zuker CS. A multivalent PDZ-domain protein assembles signalling complexes in a G-protein-coupled cascade. Nature 388: 243-249, 1997[Web of Science][Medline].

49.   Tsunoda, S, and Zuker CS. The organization of INAD-signaling complexes by a multivalent PDZ domain protein in Drosophila photoreceptor cells ensures sensitivity and speed of signaling. Cell Calcium 26: 165-171, 1999[Web of Science][Medline].

50.   Vazquez, G, Lievremont JP, St J, Bird G, and Putney JWJ Human Trp3 forms both inositol trisphosphate receptor-dependent and receptor-independent store-operated cation channels in DT40 avian B lymphocytes. Proc Natl Acad Sci USA 98: 11777-11782, 2001[Abstract/Free Full Text].

51.   Warnat, J, Philipp S, Zimmer S, Flockerzi V, and Cavalie A. Phenotype of a recombinant store-operated channel: highly selective permeation of Ca2+. J Physiol 518: 631-638, 1999[Abstract/Free Full Text].

52.   Wes, PD, Xu XZ, Li HS, Chien F, Doberstein SK, and Montell C. Termination of phototransduction requires binding of the NINAC myosin III and the PDZ protein INAD. Nat Neurosci 2: 447-453, 1999[Web of Science][Medline].

53.   Xu, XZ, Choudhury A, Li X, and Montell C. Coordination of an array of signaling proteins through homo- and heteromeric interactions between PDZ domains and target proteins. J Cell Biol 142: 545-555, 1998[Abstract/Free Full Text].

54.   Xu, XZ, Li HS, Guggino WB, and Montell C. Coassembly of TRP and TRPL produces a distinct store-operated conductance. Cell 89: 1155-1164, 1997[Web of Science][Medline].

55.   Yao, Y, Ferrer-Montiel AV, Montal M, and Tsien RY. Activation of store-operated Ca2+ current in Xenopus oocytes requires SNAP-25 but not a diffusible messenger. Cell 98: 475-485, 1999[Web of Science][Medline].

56.   Yue, L, Peng JB, Hediger MA, and Clapham DE. CaT1 manifests the pore properties of the calcium-release-activated calcium channel. Nature 410: 705-709, 2001[Medline].

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

Am J Physiol Cell Physiol 284(2):C497-C505
0363-6143/03 $5.00 Copyright © 2003 the American Physiological Society


This article has been cited by other articles:


Home page
 Physiol. Rev.Home page
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]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
284/2/C497    most recent
00183.2002v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Su, Z.
Right arrow Articles by Blalock, J. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Su, Z.
Right arrow Articles by Blalock, J. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online