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First published online 3 February 2004
doi: 10.1242/jcs.00941

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Evidence for structural and functional diversity among SDS-resistant SNARE complexes in neuroendocrine cells

Department of Pharmacology, University of Vienna, Waehringerstrasse 13a, 1090 Vienna, Austria

View larger version (29K): [in a new window]   Fig. 1. Identification of SDS-resistant SNARE complexes in PC12 cell membrane extracts. SDS-membrane extracts were separated on SDS-PAGE, transferred to nitrocellulose membranes and were immunoblotted with polyclonal and monoclonal antibodies against SNARE. (A) Immunoblot of unboiled samples and samples that had been boiled before electrophoresis from a single membrane preparation using MAB333 (SB1), MAB331 (S25) and MAB336 (STX1). The insert at the bottom shows the anti-SNARE bands that correspond to the monomeric antigens from a shorter film-exposure of the nitrocellulose membrane. Of the three antibodies only MAB331 (antibody against SNAP-25) detects heat-sensitive SDS-resistant SNARE complexes. However, the amount of monomeric syntaxin and synaptobrevin is increased in boiled samples, suggesting that in the unboiled samples a fraction of these proteins is engaged in protein complexes where epitopes are inaccessible for the respective antibody. Immunoblots shown in B to E were performed with unboiled SDS-samples. (B) Syntaxin antibody clone HPC-1 (STX2) detects, besides the monomeric antigen at about 36 kDa, two high-molecular weight bands at positions, which correspond to the complex bands identified with MAB331 and shown in A (at 230 and 100 kDa). (C) Syntaxin-antibody clone 78.3 (STX3) recognizes only the 230 kDa protein band (with low staining intensity) which is also detected by MAB331 (S25), whereas synaptobrevin antibody clone 69.1 (SB2) fails to detect any slow migrating bands. (D) After heat-treatment of the nitrocellulose membrane, STX3 recognizes both high-molecular weight bands that are also detected by MAB331 (S25). (E) On heat-treated nitrocellulose membranes, synaptobrevin antibody clone 69.1 (SB2) detects the 230 kDa band, whereas synaptobrevin antibody AB5856 (SB3) recognizes only the 100 kDa protein band. Note that the samples separated in lanes 1-3 and lanes 4 and 5 (from left to right), were from the same membrane preparation. The preparation analysed in lanes 4 and 5 contained a third SDS-resistant band at about 55 kDa (asterisk). The recognition patterns of the antibodies were verified in at least three independent experiments.

View larger version (24K): [in a new window]   Fig. 2. SNARE-immunoreactive protein bands are identical in immunoblots of total PC12 cell extracts and in extracts of isolated PC12 cell membranes. (A) Increasing amounts (left to right) of total membrane protein extracted from PC12 cell membranes were loaded as indicated, separated on SDS-PAGE and transferred to a nitrocellulose membrane. SNAP-25-immunoreactive material was identified by anti-SNAP-25 (MAB331) antibody at a position characteristic for monomeric SNAP-25 and at 100 and 230 kDa, respectively. (B) Similar blot as shown in A for protein samples from whole PC12 cells directly lysed in SDS-sample buffer.

View larger version (23K): [in a new window]   Fig. 3. Temperature dependence and variation of relative amounts of SDS-resistant SNARE complexes from PC12 cell membranes. (A) An anti-SNAP-25-immunoblot was performed on PC12 cell SDS-membrane-extracts that had been exposed to different temperatures (as indicated) for 5 minutes before separation by SDS-PAGE. (B) SNAP-25-immunoreactive bands of membrane extracts from cells lysates kept at –20°C (lanes 1, 2) or +24°C (lanes 3, 4) for 16 hours before protein preparation. (C) Comparison of 100 kDa and 230 kDa SNAP-25-immunoreactive bands from two independent preparations (lanes 1, 2) of PC12 cells.

View larger version (25K): [in a new window]   Fig. 4. Effect of K+-stimulation on SNARE complexes. 230 kDa, 100 kDa and 25 kDa SNAP-25-immunoreactive bands are shown from samples extracted from unstimulated (control) cells (C) and from cells that were stimulated for 5 minutes with 57 mM K+ or 110 mM K+ (57K, 110K, respectively).

View larger version (31K): [in a new window]   Fig. 5. Membrane-depolarization-induced effects on SNARE complexes in PC12 cells that were stimulated with high levels of K+ require elevations of intracellular Ca2+. (A) Effects of membrane depolarization and repolarization on SNARE complexes in cells at different extracellular Ca2+ concentrations or in the presence of 1 mM Cd2+. 230 kDa and 100 kDa SNAP-25-immunoreactive bands from PC12 cell membrane extracts from cells treated for 5 minutes in the following way: (lane C) incubation in physiological buffer containing 1.5 mM Ca2+ (control), (lane D) stimulation with 80 mM K+ buffer containing 1.5 mM Ca2+, (lane D/R) stimulation with 80 mM K+ buffer containing 1.5 mM Ca2+, followed by a 45 minute incubation at 37°C in physiological buffer containing 0.1 mM Ca2+, (lane D 0.1) stimulation with 80 mM K+ buffer containing 0.1 mM Ca2+, (lane D Cd2+) stimulation with 80 mM K+ buffer containing 1.5 mM Ca2+ and 1 mM Cd2+. (B) The effect of membrane depolarization in PC12 cells with 110 mM K+ on syntaxin (clone 78.3)-immunoreactive protein bands (intact cells, left panel) is shown together with a comparison of the intensities of protein bands recognized by the same antibody in PC12 cell membrane extracts from permeabilized cells exposed to 0.1 µM free Ca2+ and 250 µM free Ca2+ (right panel). Immunoblots were performed on heat-treated nitrocellulose membrane. Stimulation of intact cells with 110 mM K+ for 10 minutes led to a decrease in the intensity of the 230 kDa band (62% of the band intensity obtained from an extract of non-depolarized control cells, left column) and to an increase in the intensity of the 100 kDa band (157% of control). Exposure of permeabilized cells to 250 µM free Ca2+ showed a reduced intensity of the 230 kDa band (to 63%) when compared with the intensity of the 230 kDa band when 100 nM free Ca2+ were present. The intensities of the 100 kDa band after stimulation of cells exposed to 0.1 µM and 250 µM free Ca2+ showed no difference (intensity in 250 µM Ca2+ was 99.6% of intensity in 0.1 µM Ca2+). Notice that the 100 kDa and 230 kDa bands are from films exposed to the blotting membrane for different durations. (C) Effect of depolarization, repolarization and digitonin (digi)-permeabilization of PC12 cells. SNAP-25-immunoreactive protein bands obtained from membrane extracts from PC12 cells exposed for 10 minutes to the indicated conditions are compared with those of the control cells (4 mM K+ and 10 mM Ca2+) (bands on left side). Repolarization was for 45 minutes at 37°C (bands on right side).

View larger version (17K): [in a new window]   Fig. 8. Comparison of Ca2+ elevations evoked by membrane depolarization with 57 mM and 110 mM K+. (A) Averaged fura-2 fluorescence ratios (F340/F380) measured in single PC12 cells stimulated with 57 mM K+ (white triangles) or with 110 mM K+ (black triangles). Fluorescence images were acquired every 15 seconds. Superfusion was switched from physiological buffer (containing 4 mM K+) to high K+ depolarization buffer at 1.85 minutes. The symbols indicate the mean fluorescence ratio of 16 (57 mM K+) and 14 cells (110 mM K+). (B) Analysis of Ca2+ responses induced by 57 mM K+ and 110 mM K+ from experiments such as the one shown in A. The bars show the average response (± s.d.) of 4 (57 mM K+) and 3 (110 mM K+) experiments (7-16 cells per experiment).

View larger version (30K): [in a new window]   Fig. 6. Effect of submicromolar free Ca2+ on SNARE complexes in permeabilized PC12 cells. (A) SNAP-25-immunoreactive bands from samples extracted from PC12 cells that had been exposed to intracellular buffer containing 100, 250, 500 or 1000 nM free Ca2+ in the presence of 10 µM digitonin for 20 minutes before membrane preparation. (B) Comparison of the levels of SNAP-25-immunoreactive proteins in membrane extracts of cells permeabilized in the presence of 100 nM and 1000 nM free Ca2+. Data are from three independent experiments. Levels of the 230 kDa band (light grey bar) and the 100 kDa band (dark grey bar) observed in the presence of 1000 nM free Ca2+ are shown as percentage (± s.d.) of the signals obtained with 100 nM free Ca2+.

View larger version (16K): [in a new window]   Fig. 7. Comparison of [3H]noradrenaline (3H-NA) release evoked by 57 mM and 110 mM K+-depolarization. (A) 3H-NA release evoked within 10 minutes of stimulation with 57 mM K+ or 110 mM K+. The bars show the average response (± s.d.) of three measurements in % of total radioactivity. (B) 3H-NA release induced within 20 minutes (black horizontal bar) of stimulation with 57 mM K+ and 110 mM K+ in superfused PC12 cells. Symbols show the average radioactivity (± s.d.) in superfusates collected every 4 minutes. Values were corrected for basal release, which was determined 12 minutes before membrane depolarization with K+.

View larger version (16K): [in a new window]   Fig. 9. Comparison of the effects of short- and long-lasting K+-depolarizations on SNARE complexes. (A) SNAP-25-immunoreactive bands from membrane extracts of PC12 cells that were exposed to normal extracellular buffer (control, C) or to 57 mM K+ buffer for 5 or 10 minutes before membrane preparation. (B) Densitometric analysis of western blots with samples obtained after 5 or 10 minute depolarization with K+ (top graph, 57 mM K+; bottom graph, 110 mM K+). Averaged values (± s.d.) for the 100 kDa and 230 kDa bands from three to four experiments are shown as percentage of control.

View larger version (28K): [in a new window]   Fig. 10. The model describes how moderate and intense stimulation of exocytosis might affect SDS-resistant SNARE complexes in neuroendocrine cells. (Left) Ca2+ elevations to submicromolar concentrations (e.g. by 57 mM K+) stimulate vesicle priming and support low rates of vesicle exocytosis (left drawing: (1) unprimed vesicle, (2) primed vesicle). As a consequence, the amount of fusion-competent SDS-resistant SNARE complexes is increased. This is illustrated in the lower panel showing cells permeabilized in the presence of 100 nM (1) and 1000 nM (2) free Ca2+. (Right) Ca2+ elevations to micromolar concentrations (e.g. by 110 mM K+) support vesicle priming and high rates of vesicle exocytosis [right drawing, (3)]. The immediate fusion of primed vesicles leads to a reduction of fusion-competent SDS-resistant SNARE complexes via a post-fusion action of NSF [right drawing, (4)] [shown in the lower panel for a 5 minute stimulation of intact cells with 110 mM K+ (4) compared with SNAP-25-immunoreactive protein bands obtained from non-depolarized control cells (3)].

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