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. 2010 Aug 11;30(32):10683-91.
doi: 10.1523/JNEUROSCI.5210-09.2010.

The association of dynamin with synaptophysin regulates quantal size and duration of exocytotic events in chromaffin cells

Arlek M González-Jamett  1 , Ximena Báez-Matus, Montserrat A Hevia, María José Guerra, María José Olivares, Agustín D Martínez, Alan Neely, Ana M Cárdenas
Affiliations

The association of dynamin with synaptophysin regulates quantal size and duration of exocytotic events in chromaffin cells

Arlek M González-Jamett et al. J Neurosci. .

Abstract

Although synaptophysin is one of the most abundant integral proteins of synaptic vesicle membranes, its contribution to neurotransmitter release remains unclear. One possibility is that through its association with dynamin it controls the fine tuning of transmitter release. To test this hypothesis, we took advantage of amperometric measurements of quantal catecholamine release from chromaffin cells. First, we showed that synaptophysin and dynamin interact in chromaffin granule-rich fractions and that this interaction relies on the C terminal of synaptophysin. Experimental maneuvers that are predicted to disrupt the association between these two proteins, such as injection of antibodies against dynamin or synaptophysin, or peptides homologous to the C terminal of synaptophysin, increased the quantal size and duration of amperometric spikes. In contrast, the amperometric current that precedes the spike remained unchanged, indicating that synaptophysin/dynamin association does not regulate the initial fusion pore, but it appears to target a later step of exocytosis to control the amount of catecholamines released during a single vesicle fusion event.

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Figures

Figure 1.
Figure 1.
Synaptophysin binds to dynamin in bovine chromaffin cells. A, Protein extracts from cultured bovine chromaffin cells were incubated in the presence of Ca2+/Mg2+ or EGTA for 30 min, and then subjected to immunoprecipitation with a monoclonal anti-dynamin antibody. Bound proteins were analyzed by immunoblotting with anti-Dyn and anti-Syn antibodies. B, Protein extracts from non-depolarized chromaffin cells, or cells depolarized with 100 mm KCl (5 min) in the presence of 2.5 mm Ca2+, 5 mm EGTA, or 10 mm Co2+ were immunoprecipitated with a monoclonal anti-dynamin antibody. EGTA and Co2+ were present 15 min before and during the depolarizing pulse. Bound proteins were analyzed by immunoblotting with antibodies against Syn and Dyn. C, GST alone or GST-Cterm was immobilized on glutathione-agarose beads and incubated with protein extracts from chromaffin cells. The bound material was subjected to immunobloting with anti-Dyn and anti-GST antibodies.
Figure 2.
Figure 2.
Immunodetection of dynamin and synaptophysin in chromaffin granules. A, Fractions 2–16 collected from a continuous sucrose density gradient (1.0–2.2 m sucrose) layered with protein extracts from chromaffin cells were subjected to gel electrophoresis and immunodetected with anti-Dyn, anti-DBH, and anti-Syn. B, Purified chromaffin granule membranes were subjected to immunoblotting with anti-Dyn, anti-DBH, and anti-Syn. C, GST alone or GST-Cterm were immobilized on glutathione-agarose beads and incubated with protein extracts from chromaffin granules. The bound material was subjected to immunobloting with anti-Dyn and anti-GST antibodies. D, Chromaffin granule extracts were subjected to immunoprecipitation with anti-Syn. Bound proteins were analyzed by immunoblotting with anti-Dyn and anti-Syn. E, Supernatants from pull-down assays were subjected to immunoprecipitation with anti-Syn, and then bound proteins were analyzed by immunoblotting with anti-Dyn and anti-Syn.
Figure 3.
Figure 3.
Microinjections of antibodies against synaptophysin or dynamin increase duration and quantal size of depolarization-induced exocytotic events. Chromaffin cells were injected with anti-Syn, anti-Dyn or anti-His. The exocytosis response was evoked by high K+ and measured 30–45 min after injections. A, Representative amperometric traces from noninjected cells (Control) or cells injected with anti-Syn. B, Cumulative histograms of the number of amperometric events from control (white circles), anti-Syn (black squares) and anti-Dyn injected cells (gray triangles). C, Representative amperometric spikes from noninjected cells (Control), anti-Syn and anti-Dyn injected cells. D, Representative amperometric spike with its parameters: Q and t1/2. E, Averaged values for Q and t1/2. Data are means ± SEM of averages per cell (14–24 cells from 3 to 5 different cultures). *p < 0.05 compared with control cells.
Figure 4.
Figure 4.
Microinjections of synaptophysin C-terminal derivatives increase duration and quantal size of depolarization-induced exocytotic events. Chromaffin cells were injected with GST (40 μm), GST-Cterm (10 and 40 μm), GST-Cterm plus anti-Syn antibody (10 and 40 μm), C-term240-290 (10 μm), or C-term240-290 plus anti-Syn antibody (10 μm). Thirty to forty-five minutes after injections, exocytosis responses evoked by high K+ were measured. A, Amino acid sequence of the C-terminal domain of rat synaptophysin (top, gray) and the synthetic peptide C-term240-290 (bottom, black). Tyrosine residues are shown in bold. Underlined amino acids indicate the epitope recognized by anti-Syn. B, Protein extracts from chromaffin cells were immunoprecipitated with a polyclonal anti-synaptophysin antibody, in the presence of 10 μm C-term240-290, GST, or GST-Cterm. Bound proteins were analyzed by immunoblotting with antibodies against Syn, Dyn, and VAMP-2. C, Cumulative histograms of the number of amperometric events from control cells (white circles), cells injected with 40 μm GST-Cterm (black square) or 10 μm C-term240-290 (gray triangles). D, Representative amperometric traces from cells injected with GST, GST-Cterm or C-term240-290. E, Averaged values for Q and t1/2. Data are means ± SEM of averages per cell (12–24 cells from 3 to 5 different cultures). *p < 0.05 compared with control cells. Insets: Immunoblotting showing that GST-Cterm and C-term240-290 are recognized by the anti-Syn antibody.
Figure 5.
Figure 5.
Microinjections of either derivatives of the C terminal of synaptophysin or anti-Syn do not affect the fusion pore dynamics. A, Example traces with a long-lasting foot event (shaded area) showing foot amplitude and foot duration. B, Examples of spikes with foot signals from different experimental conditions. C, Averaged values for frequency (percentage of spikes with foot per cell), foot amplitude, and foot duration of amperometric spikes of cells noninjected (Control) or injected with GST (40 μm), GST-Cterm (40 μm), C-term240-290 (10 μm), or anti-Syn (10 μm). Data are means ± SEM average per cell (14–22 cells from 3 to 5 different cultures).
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