Complexin II facilitates exocytotic release in mast cells by enhancing Ca2+ sensitivity of the fusion process

Satoshi Tadokoro, Mamoru Nakanishi and Naohide Hirashima*

Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan

* Author for correspondence (e-mail: hirashim{at}phar.nagoya-cu.ac.jp)

Accepted 18 February 2005


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 Materials and Methods
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Recent studies have shown that soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in exocytotic release in mast cells as in neurotransmitter release. However, the roles of the proteins that regulate the structure and activity of SNARE proteins are poorly understood. Complexin is one such regulatory protein and is involved in neurotransmitter release, although ideas about its role are still controversial. In this study, we investigated the expression and role of complexin in the regulation of exocytotic release (degranulation) in mast cells. We found that complexin II, but not complexin I, is expressed in mast cells. We obtained RBL-2H3 cells that expressed a low level of complexin II and found that antigen-induced degranulation was suppressed in these cells. No significant changes in the Ca2+ response or expression levels of syntaxins and synaptotagmin were observed in knockdown cells. An immunocytochemical study revealed that complexin II was distributed throughout the cytoplasm before antigen stimulation. However, the distribution of complexin II changed dramatically with stimulation and it became localized on the plasma membrane. This change in the intracellular distribution was observed even in the absence of extracellular Ca2+, while exocytotic release was inhibited almost completely under this condition. The degranulation induced by phorbol 12-myristate 13-acetate and A23187 depended on the extracellular Ca2+ concentration, and its sensitivity to Ca2+ was decreased in knockdown cells. These results suggest that complexin II regulates exocytosis positively by translocating to the plasma membrane and enhancing the Ca2+ sensitivity of fusion machinery, although this translocation to the plasma membrane is not sufficient to trigger exocytotic membrane fusion.

Key words: Mast cell, Complexin, Exocytosis, SNARE, Allergy, Rat


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been shown that soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) proteins play an essential role in exocytotic release in both neuronal cells (Sollner et al., 1993Go; Calakos and Scheller, 1996Go; Brunger, 2001Go) and non-neuronal secretory cells (Wheeler et al., 1996Go; Nagamatsu et al., 1999Go; Reed et al., 1999Go; Flaumenhaft, 1999). In addition to SNARE proteins, several proteins that regulate the conformation and activity of SNARE complexes are involved in exocytosis. Complexin (also called synaphin), a small soluble protein (18-19 kD), is a regulatory protein in the mammalian brain (McMahon et al., 1995Go; Takahashi et al., 1995Go; Ishizuka et al., 1995Go). Complexin interacts with ternary SNARE complex and is thought to stabilize the SNARE complex (Pabst et al., 2000Go; Pabst et al., 2002Go). Based on studies of the three-dimensional structure of the complexin/SNARE complex, it has been suggested that complexin stabilizes the fully assembled SNARE complex (Bracher et al., 2002Go; Chen et al., 2002Go). However, the role of complexin in neurotransmitter release is not yet fully understood. Using a squid giant synapse, Tokumaru et al. (Tokumaru et al., 2001Go) reported that complexin facilitates the association of SNAREs into an intermediate complex that can oligomerize to give a higher order structure that is required for the fusion of synaptic vesicles. They also showed that peptides that prevent complexin from binding to the SNARE complex inhibit evoked transmitter release in a squid giant synapse. Double-knockout mice for complexins I and II show reduced neurotransmitter release (Reim et al., 2001Go). These results suggest that complexin acts as a positive regulator of exocytosis. However, the injection of complexin into Aplysia nerve terminal suppressed transmitter release, while the injection of anti-complexin antibody stimulated neurotransmitter release (Ono et al., 1998Go). The overexpression of complexin in PC12 reduced exocytotic release (Itakura et al., 1999Go). As these findings show, it is still unclear how complexin functions in exocytotic release, even in neuronal cells. Furthermore, it is not clear whether or not complexin is involved in exocytosis in non-neuronal cells. If complexin regulates exocytosis in non-neuronal secretory cells, it would be very interesting to investigate how complexin regulates exocytosis in such cells. As non-neuronal secretory cells are often larger and have bigger secretory granules than nerve terminals, they could provide a useful experimental system for investigating the mechanism by which complexin regulates exocytosis.

In the present study, we examined the expression and role of complexin in mast cells, which are typical non-neuronal secretory cells. In mast cells, cross-linking of high-affinity receptors for IgE (Fc{epsilon}RI) by multivalent antigen activates an intracellular signaling cascade and leads to the exocytotic release of granular contents (degranulation), resulting in allergic responses (Abraham and Malaviya, 1997Go; Swann et al., 1998Go; Turner and Kinet, 1999Go; Kinet, 1999Go). We have previously studied the mechanism of degranulation in mast cells and identified the protein and lipid molecules that regulate this process (Hibi et al., 2000Go; Kato et al., 2002Go; Kato et al., 2003Go). We and other groups have reported that SNARE proteins are involved in degranulation in mast cells (Guo et al., 1998Go; Hibi et al., 2000Go; Paumet et al., 2000Go; Blank et al., 2001Go; Blank and Rivera, 2004Go), although the active isoforms in mast cells are different from those in neuronal cells. However, there is little information available on the proteins that regulate SNARE proteins in mast cells, except for Munc18-2 (Martin-Verdeaux et al., 2003Go).

In this study, we found that complexin II was expressed in mast cells. In experiments using complexin II knockdown cells, we found that complexin II positively regulated exocytotic release in mast cells. Immunocytochemical experiments revealed that complexin II changed its localization from the cytoplasm to the plasma membrane, and this translocation occurred in the absence of extracellular Ca2+, while degranulation was inhibited almost completely. We also found that the degranulation induced by phorbol 12-myristate 13-acetate (PMA) and A23187 depended on extracellular Ca2+ concentration, and its sensitivity to Ca2+ was decreased in knockdown cells. These results suggest that complexin II regulates exocytosis positively by translocating to the plasma membrane and enhancing the Ca2+ sensitivity of fusion machinery, although the association of complexin II with SNARE complex is not sufficient to trigger exocytotic membrane fusion.


    Materials and Methods
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 Materials and Methods
 Results
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Chemicals
PMA, 4-bromo-A23187, (±)-sulfinpyrazone, p-nitrophenyl-N-acetyl-ß-D-glucosaminide and thapsigargin were purchased from Sigma (St Louis, MO). Aprotinin, leupeptin, pepstatin and PMSF were obtained from Wako Pure Chemicals (Tokyo, Japan). All other reagents were of the highest grade available commercially.

Cell culture
A mast cell line, rat basophilic leukemia cell (RBL-2H3), was cultured in Eagle's minimal essential medium from Nissui (Tokyo, Japan) with 10% fetal calf serum (Boehringer Mannheim) at 37°C in an atmosphere of 5% CO2. Mastocytoma P815 was cultured in RPMI1640 (Gibco) supplemented with 10% fetal calf serum at 37°C in an atmosphere of 5% CO2.

RT-PCR
Poly(A)+ RNA was obtained with a QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia Biotech) from 1x 107 cells of RBL-2H3 and cerebrum of Sprague-Dawley rat (6 weeks), and served as a template for cDNA synthesis with SuperScript II RT (Gibco BRL), as reported previously (Hibi et al., 2000Go). The primer pairs used to amplify complexins I and II were 5'-ATGGAGTTCGTGATGAAACAAG-3' (sense)/5'-TTACTTCTTGAACATGTCCTGCA-3' (anti-sense) and 5'-ATGGACTTCGTCATGAAGCA (sense)/5'-TTACTTCTTGAACATGTCCTGCA-3' (anti-sense), respectively. PCR products were extracted from agarose gel with Gene Clean (Bio 101) and subcloned into the TA cloning vector pCRII (Invitrogen). Cloned PCR products were sequenced with a DSQ1000 DNA sequencer (Shimadzu, Kyoto, Japan) using FITC-labeled M13 universal primer.

Western blotting
RBL-2H3 or P815 cells (5x 106) were lysed with lysis buffer (10 mmol/l HEPES, 1% Triton-X100, 1 mmol/l EDTA, 50 mmol/l NaF, 2.5 mmol/l p-nitrophenyl phosphate, 1 mmol/l Na3VO4, 1 mmol/l PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µg/ml aprotinin). After centrifugation at 23,000 g for 20 minutes, supernatant was mixed with an equal volume of Laemmli sample buffer and boiled for 5 minutes. For cellular lysate of rat brain, rat cerebrum lysate was purchased from Transduction Laboratories. Samples were electrophoresed by SDS-PAGE and transferred to a PVDF membrane. After blocking with phosphate buffer containing 5% skimmed milk, blots were probed with primary antibody for 1 hour. As primary antibodies, anti-complexin I antibody (dilution 1:200; Santa Cruz Biotechnology), anti-complexin II antibody (dilution 1:500; Transduction Laboratories), anti-syntaxin-3 antibody (dilution 1:250; Alomone Labs, Israel), anti-syntaxin-4 antibody (dilution 1:500; Santa Cruz Biotechnology), anti-synaptotagmin II antibody (dilution 1:200 Santa Cruz Biotechnology), and ß-actin antibody (dilution 1:4000; Sigma) were used. After being washed with 0.1% Tween 20 in PBS, membrane was treated with anti-mouse IgG conjugated with horseradish peroxidase. Immunoreactivity was detected by enhanced chemiluminescence (ECL, Amersham Pharmacia) with a LAS-1000 (FUJI FILM, Tokyo Japan) and analyzed by Image Gauge (FUJI FILM).

Plasmid construction and transfection
Poly(A)+ RNA was obtained as described above. For the knockdown of complexin II, 5'-GGATCCATGGACTTCGTCATGAAGCA-3' (sense; BamHI site is underlined)/5'-GCGGCCGCTTACTTCTTGAACATGTCCTGCA-3' (anti-sense; NotI site is underlined) was used as a primer pair. For the expression of myc-tagged complexin II, 5'-GGATCCATGGACTTCGTCATGAAGCA-3' (sense; BamHI site is underlined)/5'-GAATTCTTACTTCTTGAACATGTCCTGCA-3' (anti-sense; EcoRI site is underlined) was used as a primer pair. PCR products were extracted from agarose gel with Gene Clean (Bio 101) and subcloned into the TA cloning vector pCRII. Cloned PCR products were sequenced with a DSQ1000 DNA sequencer. Verified cDNA was ligated to pcDNA3 (Invitrogen) in the antisense direction to knockdown the expression of complexin II. For the expression of myc-tagged complexin II, cDNA was ligated to pCMV-Tag5 (Stratagene). RBL-2H3 cells (5x 105 cells/500 µl) were electroporated in cold PBS with 40 µg of plasmid DNA at 250 V and 950 µF using Gene Pulser II (Bio-Rad) (Hibi et al., 2000Go). Stable clones with reduced expression of complexin II were selected by G418 (500 µg/ml) and western blotting with anti-complexin II or anti-myc antibody.

Assay of secreted ß-hexosaminidase
Degranulation of RBL-2H3 cells was monitored by measuring the activity of a granule-stored enzyme, ß-hexosaminidase, secreted in cell supernatant (Amano et al., 2001Go). Cells were sensitized by anti-DNP IgE for 30 minutes and incubated with an average of six DNP groups conjugated with BSA (DNP6-BSA) for 30 minutes at 37°C. Aliquots of supernatant were incubated with substrate solution (2 mmol/l p-nitrophenyl-N-acetyl-ß-D-glucosaminide in 100 mmol/l citrate, pH 4.5) for 1 hour at 37°C. After the reaction was terminated with Na2CO3-NaHCO3 buffer, absorbance at 405 nm was measured. Release activity relative to the total ß-hexosaminidase content of the cells was calculated. Total ß-hexosaminidase content was determined by dissolving cells with 0.1% Triton-X100.

Intracellular Ca2+ measurement
RBL-2H3 cells were loaded with Fura2/AM (Molecular Probes; Eugene, OR) for 30 minutes at 37°C and washed twice with HEPES-buffered saline (140 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l CaCl2, 0.6 mmol/l MgCl2, 0.1% glucose, 0.1% BSA, 0.1 mg/ml sulfinpyrazone and 10 mmol/l HEPES, pH 7.4). For experiments with different extracellular Ca2+ concentration, concentration of CaCl2 in HEPES-buffered saline was varied from 0 to 2 mmol/l. Cells were sensitized with anti-DNP IgE for 30 minutes and stimulated with DNP6-BSA (100 ng/ml). For the stimulation with Ca2+ ionophore and phorbol ester, A23187 (1 µmol/l) and PMA (50 ng/ml) were used instead of DNP6-BSA. The fluorescence intensities with excitation at 340 and 360 nm were measured, and the ratio (F340/F360) and Ca2+ concentration were calculated by a spectrofluorometer linked to a personal computer (RF-5300PC; Shimadzu, Japan), following a procedure described previously (Grynkiewicz et al., 1985Go). During measurement of the intracellular Ca2+ concentration, cells were kept at 37°C.

Immunocytochemistry
Cells (1x 105) were plated in a ZOG-3 glass-bottom chamber (Elekon Science; Chiba, Japan) and incubated for 18 hours. After cells were washed with PBS, they were fixed with PBS containing 4% paraformaldehyde and permeabilized with PBS containing 0.2% Triton-X100. After blocking with 5% BSA in PBS, cells were incubated with anti-complexin II antibody (dilution 1:100; Transduction Laboratories) or anti-myc antibody (9E10, dilution 1:50; Santa Cruz Biotechnology) at 4°C overnight. Cells were treated with fluorescence-labeled secondary antibody (FITC-conjugated anti-mouse IgG) after they were washed three times with PBS for 10 minutes. Fluorescent images were taken with a confocal laser scanning microscope (Zeiss, LSM-510) with a 63x objective lens (Plan-Apochromat 63x/1.4 oil). Samples were excited at 488 nm with an Ar laser and fluorescence was observed with an LP560 filter (Kato et al., 2002Go).


    Results
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 Materials and Methods
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Expression of complexin in mast cells
The expression of complexin in RBL-2H3 cells (a mast cell line) was investigated by RT-PCR. As there are two isoforms of complexin, complexins I and II, we performed PCR with primers specific to complexins I and II. The PCR products amplified with the primer pairs for complexins were electrophoresed, and a clear band at the expected size was detected only in the lane for complexin II (Fig. 1A). The band was excised from agarose gel and the extracted PCR product was sequenced. The sequence was identical to the cDNA sequence of rat complexin II. When RT-PCR was carried out using mRNA from rat brain as a template, expressions of complexin I and II were detected. The expression of complexin was confirmed at the protein expression level by western blotting. As shown in Fig. 1B, the expression of complexin II was detected at about 19 kD (lower panel), while the expression of complexin I was not detected (upper panel). We also investigated the expression of complexin II in mastocytoma, P815. As in the case of RBL-2H3, complexin II but not I was detected in P815 (Fig. 1B).



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Fig. 1. Expression of complexin II in RBL-2H3 cells. (A) RT-PCR products amplified with specific primer pairs for complexins I and II were electrophoresed in agarose gel (1.5%). Far left and far right lanes are for the 100 bp ladder marker (M). PCR products amplified with primer pairs of complexin I (CPX I) and II (CPX II) using templates derived from rat brain (positive control) and RBL-2H3 cells are shown. The expected size of the product is about 400 bp for both isoforms. A single band in the lane for complexin II of RBL-2H3 cells was detected, and the product was identified as complexin II by DNA sequencing. (B) Western blot analysis for complexin. Cell lysates were prepared from RBL-2H3 cells, P815 cells and rat cerebrum and were electrophoresed by SDS-PAGE. After the samples were transferred to a PVDF membrane, blots were probed with primary antigens specific for complexins I and II. Blots were visualized with anti-mouse IgG conjugated with horseradish peroxidase using chemiluminescence methods. Complexin II was detected at about 19 kD (lower panel), but complexin I was not detected (upper panel) in mast cells. (C) Intracellular distribution of complexin II. Complexin II was detected with anti-complexin II antibody and visualized with FITC-conjugated anti mouse IgG. Fluorescent images were collected with a confocal laser scanning microscope. Complexin II was detected in the cytoplasm and the nucleus.

 
To investigate the intracellular distribution of complexin II in mast cells, we carried out immunocytochemical experiments using an anti-complexin II antibody and an FITC-labeled secondary antibody. Complexin II resided throughout the cytoplasm and nucleus, and no localization in a distinct organelle was observed. However, the distribution of fluorescence signal seemed to be punctate rather than uniform (Fig. 1C).

Characterization of complexin II-knockdown cell
To investigate the role of complexin II in exocytotic release in mast cells, we obtained RBL-2H3 cells that expressed a low level of complexin II. After selection with G418, four independent clones with a low expression of complexin II (knockdown cells) were picked up by western blotting analysis. All these clones expressed complexin II at less than 50% of the level in wild-type cells, and they all exhibited similar behaviors in this study. Fig. 2A shows western blotting of cell lysate derived from four knockdown clones. Expression levels of complexin II in knockdown clones (kd-1, 2, 3 and 4) were 42, 46, 40 and 38% of those in wild-type cells, respectively. The expression levels of syntaxin-3 and syntaxin-4, which are SNARE proteins expressed in RBL-2H3 cells and thought to be involved in degranulation, were not significantly changed in knockdown cells (Fig. 2A). In addition, the expression of synaptotagmin II, which is the most abundant isoform of synaptotagmin and regulates exocytotic release in RBL (Baram et al., 1999Go), was not affected (Fig. 2A). The expression of complexin II was attenuated in knockdown cells, but its intracellular distribution was not changed, and complexin II was distributed throughout the cytoplasm and the nucleus, as observed in wild-type cells (Fig. 2B).



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Fig. 2. Characterization of complexin II-knockdown cells. (A) Western blot analysis for complexin II, syntaxins 3, syntaxin 4 and synaptotagmin II in four knockdown clones (kd-1 to kd-4). Expression of complexin II was reduced in knockdown cells, while expression of syntaxins and synaptotagmin II was not affected. (B) Intracellular distribution of complexin II in a kd cell. The fluorescence image was obtained as described in Fig. 1C. (C,D) Timecourse of the intracellular Ca2+ concentration in wild-type (C) and kd cells (D), respectively. Cells were sensitized with IgE and loaded with Fura2-AM, and stimulated with antigen (DNP-BSA) at the time indicated by an arrow. Average fluorescence intensity ratios (F340/F360) were plotted against time. Values are obtained from four independent preparations of wild-type cells and four independent knockdown clones [mean±s.e.m. (n=4)]. No significant differences in the Ca2+ response were detected between wild-type and kd cells.

 
As degranulation is triggered by the elevation of the intracellular Ca2+ concentration due to Ca2+ influx from the extracellular solution into mast cells, we examined the Ca2+ mobilization evoked by antigen in complexin II-knockdown cells. It has been well established that antigen stimulation of mast cells causes an initial Ca2+ increase due to release from intracellular Ca2+ stores followed by a sustained increase due to Ca2+ influx from the extracellular medium. Fig. 2C shows the timecourse of the intracellular Ca2+ concentration after antigen stimulation in wild-type cells. Fig. 2D shows the timecourse of the average Ca2+ concentration in four independent knockdown clones. No significant difference was observed in either phase of the Ca2+ increase between wild-type and knockdown cells. This suggests that the reduction in the expression of complexin II had no effect on events upstream of Ca2+ influx, such as the activation of IgE receptors, tyrosine phosphorylation or opening of Ca2+ channels on the Ca2+ store and plasma membrane.

The exocytotic release of complexin knockdown cells was investigated by quantifying ß-hexosaminidase secreted in medium. Fig. 3A shows the timecourse of degranulation induced by antigen stimulation. A significant inhibition of degranulation was observed in complexin II knockdown cells. There was no difference in the total amount of ß-hexosaminidase between wild-type and knockdown cells.



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Fig. 3. Degranulation in complexin II knockdown cells. (A) Wild-type or knockdown cells were sensitized with IgE and stimulated with antigen (100 ng/ml DNP6-BSA). The quantity of ß-hexosaminidase in the supernatant is expressed as a percentage of total ß-hexosaminidase. Values were obtained from four independent preparations of wild-type cells ({circ}) and four independent knockdown clones ({bullet}) [means.e.m. (n=4)]. (B) Cells were stimulated with A23187 (1 µM) and PMA (50 ng/ml). Values were obtained and plotted as mentioned above.

 
Because the cellular response of RBL-2H3 sometimes varies in individual cells, a selected clone by antibiotics and Western blotting could show impaired degranulation regardless of the expression level of complexin II. Thus, we stimulated cells with a Ca2+ ionophore A23187 and PMA, which mimic the activation of mast cells by bypassing events before Ca2+ influx. In this stimulation condition, degranulation was induced with minimum effects of clonal variance. Furthermore, we can confirm the notion that complexin II regulates processes downstream of Ca2+ influx. As shown in Fig. 3B, degranulation was inhibited in knockdown cells, as observed in the case of antigen stimulation (Fig. 3A). These results suggest that complexin II positively regulates exocytosis in mast cells.

Translocation of complexin II after stimulation
To elucidate the mechanism by which complexin II regulates exocytosis in mast cells, the intracellular localization of complexin II before and after antigen stimulation was observed. Before stimulation, complexin II was distributed throughout the cell as described in Fig. 1. However, upon stimulation, the distribution of complexin II changed dramatically, and it was translocated to the plasma membrane (left images in Fig. 4A,B). This translocation to the plasma membrane was clear at about 3 minutes after stimulation. The distribution of fluorescence signal became more punctate. Similar results were obtained in RBL-2H3 cells expressing myc-tagged complexin II with anti-myc antibody (right images in Fig. 4A,B). Next, we investigated this translocation in the absence of extracellular Ca2+. Translocation to the plasma membrane was observed even in the absence of extracellular Ca2+ (Fig. 4C). Under this condition, exocytotic release was inhibited almost completely (Fig. 4D). Stimulation with thapsigargin in the absence of extracellular Ca2+ gave similar results (Fig. 4E,F). These results suggest that the transient elevation of the intracellular Ca2+ concentration is sufficient for translocation to the plasma membrane, and neither sustained Ca2+ increase due to the influx of extracellular Ca2+ nor the activation of IgE receptors was necessary.



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Fig. 4. Translocation of complexin II after stimulation. Complexin II in RBL-2H3 was visualized with anti-complexin II antibody or anti-myc antibody, using FITC-conjugated anti-mouse IgG, as in Fig. 1C. (A) Distribution of complexin II before antigen stimulation. The left image shows the distribution of complexin II in wild-type cells using anti-complexin II antibody. The right image shows the distribution of myc-tagged complexin II in cells transfected with myc-tagged complexin II using anti-myc antibody. (B) Distribution of complexin II at 5 minutes after antigen stimulation. Complexin II was translocated to the plasma membrane. Left and right images show the distribution of complexin II and myc-tagged complexin II, respectively. (C) Distribution of complexin II at 5 minutes after antigen stimulation in the absence of extracellular Ca2+. Complexin II was translocated to the plasma membrane. (D) Timecourse of antigen-induced degranulation in the absence of extracellular Ca2+. Values were obtained from four independent preparations of wild-type cells [mean±s.e.m. (n=4)]. (E) Distribution of complexin II at 5 minutes after thapsigargin (50 nmol/l) stimulation in the absence of extracellular Ca2+. Complexin II was translocated to the plasma membrane. (F) Timecourse of thapsigargin-induced degranulation in the absence of extracellular Ca2+. Values are obtained from four independent preparations of wild-type cells [mean±s.e.m. (n=4)].

 

Ca-dependence of degranulation on extracellular calcium concentration
Experiments with complexin-knockout mice (Reim et al., 2001Go) suggested that complexin is involved in the calcium-dependence of exocytotic release in neuronal cells. Therefore, we compared the Ca2+-dependence of degranulation in wild-type and knockdown cells. Cells were stimulated with the calcium ionophore A23187 and PMA. The calcium ion concentration in extracellular solution was changed from nominally free to 2 mmol/l. In Fig. 5, the Ca2+-dependent component of degranulation activity, which was calculated by subtracting the value of degranulation at 0 mmol/l, is shown. In both wild-type and knockdown cells, degranulation decreased as the extracellular Ca2+ concentration decreased, but the dose-response curve of knockdown cells was shifted toward a higher concentration of Ca2+ (Fig. 5A). To compare the Ca2+ sensitivity of Ca2+-dependent degranulation between wild-type and knockdown cells, degranulation activity shown in Fig. 5A was re-plotted against intracellular Ca2+ concentration (Fig. 5B). As shown in an inset of Figure 5B, intracellular Ca2+ concentration reached a plateau at about 400 seconds after stimulation. The timecourse of intracellular Ca2+ concentration change induced by PMA and A23187 did not differ between wild-type and knockdown cells. Therefore, we considered an average concentration from 400 seconds to 20 minutes after stimulation as representative of intracellular Ca2+ concentration, and estimated the intracellular Ca2+ concentrations when cells were stimulated in various extracellular Ca2+ concentrations. Using these estimated intracellular Ca2+ concentrations, we re-plotted Ca2+ dependency of degranulation (Fig. 5B). Data were fitted with the Hill equation:

where Rmax is maximum release, [Ca2+] is intracellular concentration of Ca2+, Kd is apparent dissociation constant and n is the Hill coefficient. By nonlinear regression analysis, Rmax, Kd and n were determined. Maximum release (Rmax) of wild-type and knockdown cells predicted by the fit were 37.0 ± 1.4% and 33.6 ± 5.4%, respectively. Kd and n values for wild-type cells were 0.28 ± 0.01 µM and 3.1 ± 0.50. For knockdown cells, the Kd and n were 0.56 ± 0.13 µM and 1.7 ± 0.27, respectively. These values suggest that the Ca2+ sensitivity of degranulation was reduced in knockdown cells.



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Fig. 5. Effects of the extracellular Ca2+ concentration on Ca2+-dependent degranulation. Mast cells were stimulated with PMA and A23187 at various extracellular Ca2+ concentrations. (A) Degranulation activity at 20 minutes after stimulation is plotted against extracellular Ca2+ concentration. Values are expressed as the percentage of total ß-hexosaminidase as shown in Fig. 3, but ß-hexosaminidase at [Ca2+]ex=0 was subtracted. Each point was obtained from four independent preparations of wild-type cells ({circ}) and four independent knockdown clones ({bullet}) [mean±s.e.m. (n=4)]. (B) Degranulation activity is re-plotted against intracellular Ca2+ concentration, which was estimated from Ca2+ concentration at plateau phase after stimulation using Fura-2 as shown in the inset. (Inset) Timecourses of intracellular Ca2+ concentration in wild-type cells induced by PMA and A23187 at various extracellular Ca2+ concentrations, indicated on each line. Cells were stimulated at the time indicated by the arrow. Ca2+ concentration was converted from ratio values and shown as a second ordinate.

 


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We found that complexin II, but not complexin I, was expressed in mast cells. Although these two isoforms are highly homologous (84% amino acid sequence identity) and localized in the brain, they are distributed differently in the brain (Yamada et al., 1999Go). The expression of complexin II in mast cells is consistent with the observation that complexin II is expressed in peripheral tissues, while complexin I is restricted to the brain and spinal cord (Takahashi et al., 1995Go). Recent studies have suggested that complexin II is related to neurological diseases such as Huntington's disease and schizophrenia (Morton et al., 2001Go; Eastwood et al., 2001Go). Morton and Edwardson reported that complexin II was progressively depleted in a mouse model of Huntington's disease, while the expression of complexin I and SNARE proteins remained unchanged (Morton and Edwardson, 2001Go). In addition, the expression of mutant huntingtin blocks exocytosis in PC12 cells through the depletion of complexin II, but not complexin I (Edwardson, 2003). Complexin II knockout mice survive to maturity, but abnormalities in hippocampal long-term potentiation (Takahashi et al., 1999Go) and cognitive function (Glynn et al., 2003Go) have been observed. Therefore, complexin II plays an essential role in neurological function (Glynn et al., 2003Go). In mast cells, complexin II was distributed throughout the cytoplasm and nucleus (Fig. 1C). Because complexin II is a small (18–19 kD) and soluble protein, it is not unusual that it was found in the nucleus, and it might have some function there. No localization of complexin II in a distinct organelle was observed but the distribution was not uniform. The reason for this non-uniform distribution is not known. As shown in Fig. 4B, the punctate distribution became clearer after stimulation and bright spots were observed on the plasma membrane.

In experiments using complexin II knockdown cells, we found that complexin II positively regulated exocytotic release. These inhibitory effects were not due to inhibition of the expression of syntaxin (Fig. 2A) or antigen-induced Ca2+ mobilization (Fig. 2D), which suggests that complexin II plays a role in the process of membrane fusion between secretory granules and the plasma membrane.

To investigate the mechanism of the regulation induced by complexin II, we observed its intracellular distribution after antigen stimulation. Before stimulation, complexin II resided in the cytoplasm; however, it was translocated to the plasma membrane after stimulation (Fig. 4B). This translocation was observed at 3 minutes after stimulation and degranulation also became clear in the same time window, suggesting that this translocation is closely related to degranulation. As complexin selectively associates with the ternary SNARE complex, but not with monomeric SNARE proteins, this translocation might be due to the association of complexin II with SNARE complex on the plasma membrane that is formed by stimulation. The distribution of fluorescence signal became more punctate, and clear spots appeared on the plasma membrane after stimulation. This spot-like structure might reflect the sites of exocytotic membrane fusion. Interestingly, the translocation to the plasma membrane occurred even in the absence of extracellular Ca2+ (Fig. 4C). Furthermore, since stimulation by thapsigargin in the absence of extracellular Ca2+ caused the translocation of complexin II (Fig. 4E), a transient increase in intracellular Ca2+ is sufficient for translocation, and a signal through the IgE receptor is not required. When cells were stimulated without extracellular Ca2+, degranulation was inhibited almost completely (Fig. 4D,F). This suggests that the translocation to the plasma membrane of complexin II is not enough to induce membrane fusion between secretory granules and the plasma membrane, and Ca2+ influx from the extracellular medium triggers membrane fusion. Transient increase in Ca2+ might be necessary to form a ternary SNARE complex, to which complexin II binds. Once the SNARE complex is formed, complexin II can associate with the SNARE complex, and Ca2+ influx from the extracellular medium is not required. This notion is consistent with the model that the Ca2+-independent association of complexin with the SNARE complex puts the complex in a metastable state, which is essential for efficient, fast Ca2+-dependent neurotransmitter release (Chen et al., 2002Go; Marz and Hanson, 2002Go). These observations, that complexin II was translocated to the plasma membrane but translocation itself did not trigger degranulation, were observed for the first time and provide useful information for understanding the mechanism of exocytosis regulation, not only in mast cells but also in neuronal cells.

Interestingly, knockdown of complexin II affected the Ca2+ sensitivity of degranulation (Fig. 5). While maximum release was not significantly changed, Kd and the Hill coefficient suggested that the Ca2+ sensitivity was clearly reduced in knockdown cells. A similar reduction of Ca2+ sensitivity was observed by Reim et al. (Reim et al., 2001Go) in excitatory postsynaptic currents (EPSC) of hippocampal neurons from complexin knockout mice. They investigated the Ca2+ dependence of synchronous evoked transmitter release and estimated the Kd value for extracellular Ca2+ concentration using the Hill equation. They found that the Kd of double knockout mice for complexins I and II increased twice as much as that of single knockout mice for complexin II.

As we could not obtain data at higher concentrations of intracellular Ca2+, due to cellular damage caused by long exposure to high concentrations of Ca2+, the dose-response curve for knockdown clones does not seem to be saturated (Fig. 5B). Nonlinear regression analysis allows us to estimate values related to intracellular Ca2+ dependency, which is extremely important for understanding the mechanism of calcium-dependent secretion, although lack of measured values at saturated phase did not provide an ideal condition for curve-fitting using the Hill equation.

Complexin itself does not have an apparent binding site with Ca2+ and its binding to the SNARE complex is not affected by Ca2+ (Pabst et al., 2000Go). Therefore, it is unlikely that complexin II is the Ca2+ sensor that regulates Ca2+-dependent exocytosis. As complexin II is translocated to the plasma membrane after stimulation, there are at least three steps that may be sensitive to the Ca2+ concentration: the translocation of complexin II to the plasma membrane; the association of complexin II with the SNARE complex on the plasma membrane; and the induction of exocytotic membrane fusion. Considering the finding that exocytotic release requires a sustained increase in the intracellular Ca2+ concentration due to Ca2+ influx, it is probable that the latter two steps have major contributions to the Ca2+-sensitivity of degranulation induced by ionophore and PMA. Change in not only Kd but also the Hill coefficient suggests that complexin II regulates the conformation of Ca2+-binding sites of the Ca2+ sensor. So far, synaptotagmin is the most likely candidate for the Ca2+ sensor. As synaptotagmin has two Ca2+-binding sites, complexin II might regulate the conformation or binding ability of these sites. In mast cells, synaptotagmins II, III, V and IX are expressed (Baram et al., 2001Go), and Baram et al. (Baram et al., 1999Go) reported that synaptotagmin II, which is a major isoform of synaptotagmin, negatively regulates Ca2+-dependent exocytosis in RBL-2H3 cells. As the expression level of synaptotagmin II was not affected in knockdown cells, as shown in Fig. 2A, a study on the interaction of synaptotagmin II with the SNARE complex in the presence of complexin II should shed light on the mechanism of the reduction of Ca2+ sensitivity in complexin II knockdown mast cells.

Pabst et al. (Pabst et al., 2000Go) investigated the association of complexin with the SNARE complex using different isoforms of syntaxin. They showed that complexin binds SNARE complexes comprised of SNAP-25, VAMP 2 and syntaxin 3 as efficiently as it binds with complexes comprised of SNAP-25, VAMP 2 and syntaxin 1. However, complexin did not bind to the SNARE complex containing syntaxin 4. This finding, together with the result that complexin is involved in degranulation, suggests that syntaxin 3 plays a major role as t-SNARE in mast cells. This is also supported by the observation that Munc18-2, which associates with syntaxin 3 but not syntaxin 4, is involved in degranulation in mast cells (Martin-Verdeaux et al., 2003Go).

Very recently, Abderrahmani et al. (Abderrahmani et al., 2004Go) showed that complexin I, but not complexin II, regulates secretion in pancreatic ß cells. They also knocked down the expression of complexin I and showed a reduction in secretion induced by nutrients. As the SNARE complex formed by syntaxin I, VAMP and SNAP25 is required for exocytotic release in pancreatic ß cells (Wheeler et al., 1996Go; Regazzi et al., 1996Go), complexin I serves as a positive regulator of SNARE-like complexin II in mast cells, although different isoforms of SNARE and complexin act in these cells. The involvement of complexin in degranulation provides an insight into the mechanism of allergy and the development of anti-allergy drugs.


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 References
 

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