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First published online November 23, 2005
doi: 10.1242/10.1242/jcs.02658

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SV2A and SV2C are not vesicular Ca²⁺ transporters but control glucose-evoked granule recruitment

¹ Department of Cell Physiology and Metabolism, University Medical Center, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland
² W.M. Keck Center for Learning and Memory, Department of Neurobiology and Anatomy, University of Texas-Houston Medical School, 6431 Fannin Street, Houston, TX 77030, USA

View larger version (43K): [in a new window]   Fig. 1. Detection of SV2 isoforms in pancreatic endocrine cells. (A) PCR amplification of SV2 cDNAs from rat brain, INS-1E cells and rat islet ß-cells purified by FACS. Control PCR reactions with no reverse transcriptase were performed for each isoform (-). The DNA size markers (bp) are also indicated. (B) Homogenates of rat brain (10 µg), rat islets and INS-1E cells (100 µg each) were separated by SDS-PAGE and subjected to immunoblotting with the monoclonal antibody against SV2 and with rabbit polyclonal antibodies against SV2A, SV2B or SV2C. Positions of molecular size markers are indicated in kDa. (C) Pancreatic endocrine cells were analyzed by confocal microscopy after double immunofluorescence with an antibody against glucagon (a), somatostatin (d) or pancreatic polypeptide (g) (revealed using FITC-coupled antibody) and with the anti-SV2 antibody (b, e and h) (detected using Rhodamine-coupled antibody). The merged images (c, f and i) were obtained after superposition of the green and red channels. Bars, 5 µm.

View larger version (57K): [in a new window]   Fig. 2. Immunolabeling of insulin-secreting cells for insulin, SV2A and SV2C isoforms. INS-1E cells (A,C) and pancreatic ß-cells (B,D) were double-labeled with an antibody against insulin (green) together with anti-SV2A or anti-SV2C (red). The right-hand panels show the overlay of the corresponding green and red channels. Bars, 5 µm.

View larger version (29K): [in a new window]   Fig. 3. Immunofluorescence of insulin-secreting cells for synaptophysin, SV2A and SV2C isoforms. INS-1E cells (A,C) and primary ß-cells (B,D) were double-stained with an antibody against synaptophysin (SVP38) (green) and with SV2A or -C antibodies (red). The right-hand images represent the overlays of the corresponding green and red channels. Bars, 5 µm.

View larger version (39K): [in a new window]   Fig. 4. Subcellular fractionation of SV2 isoforms by sucrose gradient. A postnuclear supernatant of INS-1E cells was loaded on a sucrose density gradient (0.45-2 M sucrose) and centrifuged for 18 hours at 110,000 g. Aliquots of each fraction of the gradient were analyzed by western blotting using antibodies directed against carboxypeptidase H (CPH), synaptophysin (SVP38), SV2, SV2A, SV2B and SV2C. The top panel shows the amount of insulin present in the fractions corresponding to the distribution of insulin granules. Sucrose concentration was calculated from the refractive index of the fractions.

View larger version (31K): [in a new window]   Fig. 5. Effect of SV2A and SV2C overexpression on cytosolic and vesicular [Ca2+]. (A) INS-1E cells were co-infected with AdCAcAq and with adenoviruses expressing the control LacZ, SV2A or SV2C. (a) Immunoblotting analysis of the overexpressed proteins using the SV2 monoclonal antibody. (b) After cytosolic aequorin reconstitution cells were perifused with KRB containing 2.5 mM glucose and stimulated, as indicated, with the same buffer supplement with 30 mM KCl. Insulin secretion was assessed by RIA in the effluent. (c) Peak of [Ca2+]c after KCl or 15 mM glucose (glc) stimulation. Values represent the mean±s.e. of seven independent traces. (B) INS-1E cells were co-infected with AdCAVAMP.Aq and with LacZ, SV2A or SV2C encoding adenoviruses. (a) Western blotting of the overexpressed proteins using anti-SV2 antibody. (b) After Ca2+ depletion and ER.aequorin reconstitution, cells were exposed to Ca2+-free KRB containing 1 mM EGTA. Where indicated, EGTA was replaced with 1.5 mM CaCl2 and the perifusion medium supplemented with 30 mM KCl. (c) Peak of [Ca2+]SG. Data are the mean±s.e. of six independent experiments.

View larger version (26K): [in a new window]   Fig. 6. Effect of SV2A and SV2C overexpression on insulin secretion in INS-1E cells. (A) Western blotting analysis of overexpressed SV2A and -C in INS-1E cells using the antibody against SV2 (top panel). INS-1E cells infected with the control AdCALacZ (LacZ), AdCASV2A (SV2A) or AdCASV2C (SV2C) adenoviruses, were incubated at basal 2.5 mM glucose (glc) and stimulated for 15 minutes with 30 mM KCl, or for 30 minutes with 15 mM glucose. Data are the mean±s.e. of five independent experiments. Asterisks indicate significant differences from levels in the LacZ control at P<0.05. (B) Western blotting of exogenous SV2A and -C in spheroid clusters of INS-1E cells with anti-SV2 antibody (left-hand panel). Spheroids of INS-1E cells infected with LacZ and SV2A, or SV2C encoding adenoviruses, were perifused with 2.5 mM glucose before stimulation with 15 mM glucose for 30 minutes. Values represent the mean±s.e. (n=3 per group) of one out of five independent experiments. Asterisks indicate a significant difference from levels in the LacZ control for corresponding time points at P<0.05.

View larger version (29K): [in a new window]   Fig. 7. Specific reduction of SV2A and SV2C expression by siRNAs. (A) BHK cells were transiently transfected with plasmids encoding SV2A or SV2C. The cells were then collected and the expression of the exogenous proteins was analyzed by western blotting using anti-SV2A (a) or anti-SV2C (b) antibodies. (B) Silencing of the genes was assessed by co-transfecting SV2A or SV2C with either an empty pSilencer vector (control) or a pSilencer vector containing a sequence that directs the synthesis of SV2A siRNA-1, siRNA-2, siRNA-3 (a) or SV2C siRNA-1, siRNA-2, siRNA-3 (b). -, lane with non-transfected cells. After 3 days the cells were homogenized and equal amounts of protein were analyzed by western blotting with anti-SV2A or -SV2C antibodies. (C) INS-1E cells were transiently co-transfected with a plasmid encoding GFP and with an empty vector (control) or with a vector containing either the SV2A siRNA-3 or the SV2C siRNA-2 silencers. Four days later, GFP-expressing cells were enriched by FACS separation. The cells were then homogenized and the same amount of protein was separated by SDS-PAGE and immunoblotted with anti-SV2A (a) or anti-SV2C (c) antibodies. The membranes (a,c) were stripped and then incubated with anti-SV2C (b) and anti-SV2A (d) antibodies, respectively. The lower parts of the membranes were incubated with an antibody against synaptotagmin IX (syt IX). Arrows indicate proteins unrelated to SV2. (D) INS-1E cells were transiently transfected with GFP and with an empty vector (control) or with both SV2A siRNA-3 and SV2C siRNA-2. Half of the homogenates prepared as just described, was loaded on a SDS-PAGE and immunoblotted with anti-SV2A (a) and the other half was immunoblotted with anti-SV2C (b).

View larger version (27K): [in a new window]   Fig. 8. Effect of SV2A and SV2C silencing on cytosolic and vesicular [Ca2+]. (A) INS-1E cells were transiently co-transfected with GFP and an empty vector (Control), or with either the SV2A siRNA-3 (SV2A) or the SV2C siRNA-2 (SV2C). Three days later, the cells were infected with AdCAcAq, further cultured for 24 hours and separated by FACS for enrichment of transfected cells. (i) After cytosolic aequorin reconstitution, the cells were perifused with 2.5 mM glucose and stimulated with 30 mM KCl as indicated. (ii) Peak of [Ca2+]c. Data show the mean±s.e. of four independent traces. (B) INS-1E cells were co-transfected as above. The day after, the cells were infected with AdCAVAMP.Aq, further cultured for 72 hours and separated by FACS. (i) The cells were perifused as detailed in Fig. 5B, panel b. (ii) Peak of [Ca2+]SG. Values represent the mean±s.e. of four independent experiments.

View larger version (23K): [in a new window]   Fig. 9. Impact of SV2A and SV2C suppression on hormone secretion. INS-1E cells were transiently co-transfected with a plasmid encoding hGH together with the empty vector (Control), or the SV2A siRNA-3 (siRNA SV2A), or the SV2C siRNA-2 (siRNA SV2C) or with both silencing vectors (siRNAs SV2A/SV2C). Four days later, the cells were incubated with basal 2.5 mM glucose (A-C, open bars) and stimulated for 15 minutes with 30 mM KCl (A, filled bars) or 30 minutes (B, filled bars) or for the indicated period (C, filled bars) with 15 mM glucose. After 10 minutes, an aliquot of the medium was collected (C, left-hand panel) and the incubation was continued for an additional 20-minute period. Secretion between 10 and 30 minutes was calculated by subtracting the amount of hGH released during the first 10 minutes from that for the entire 30-minute incubation (C, right-hand panel). Data represent the mean±s.e. of three or four independent experiments. The conditions indicated by asterisks are significantly different from levels in the control at P<0.05.

View larger version (20K): [in a new window]   Fig. 10. Effect of SV2A and SV2C depletion on membrane capacitance changes. INS-1E cells were transiently co-transfected with GFP and an empty vector (Control), or with either the SV2A siRNA-3 (siRNA SV2A) or the SV2C siRNA-2 (siRNA SV2C). Four days later, recordings were performed on GFP-expressing cells. (A) Single cell capacitance traces evoked by ten depolarizing pulses to 0 mV from a holding potential of -70 mV (left panels), and the corresponding Ca2+-current traces of the first depolarization in the pulse train (right panels). The pulse train was started 60 seconds after establishing the whole-cell configuration (bottom). Cm, membrane capacitance; fF, femtoFarad. (B) Capacitance changes normalized to the Ca2+ current of the first pulse for every cell, after one and ten pulses. Values are the mean±s.e. of 8 to 12 cells. *P<0.05 vs. levels in the control. pA, picoAmpere; pF, picoFarad. (C) Ca2+-current density measured from the first depolarization in the pulse train. The Ca2+ current was evaluated 3 milliseconds after the onset of depolarization to avoid contamination of fast Na+ current.

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