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Type I, Continuous expansion type II, expansion with plateau (duration of the plateau is longer than 10 ms) type III, expansion with flicker(s) ( G p decreases >50%).
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Real part ( R e, black) and imaginary part of admittance change ( I m, red), fusion pore conductance ( G p, blue), and fusion pore capacitance ( C v, green) are shown. A, Three types of fusion pore expansion modes. * p < 0.05 (one-way ANOVA test).įusion pore conductance analysis for SNAP-25 R198 mutants. The dotted lines show fits of the decay with double exponential fit. G, Survival curves of foot duration for SNAP-25 wt (black n = 407 events) and SNAP-25 mutants R198Q (green, n = 244 events), R198E (red, n = 133 events), K201Q (orange, n = 467 events), and K201E (blue, n = 193 events). B– F, Statistical analysis of quantal size ( B), spike half-width ( C), spike amplitude ( D), foot duration ( E), and foot amplitude ( F) obtained from cells overexpressing SNAP-25 wt ( n = 16 cells, 904 events) or R198Q ( n = 15 cells, 595 events), R198E ( n = 15 cells, 331 events), K201Q ( n = 18 cells, 1038 events), or K201E ( n = 16 cells, 434 events) mutants. A, A single amperometric event from the SNAP-25 wt trace on expanded scale illustrating the parameters analyzed for amperometric spikes (foot amplitude, foot duration, half-width, amplitude, and quantal size). Positively charged amino acids at the SNAP-25 C terminus accelerate the transmitter release from single vesicles. SNAP-25 amperometry capacitance chromaffin cells exocytosis fusion pore.Ĭopyright © 2015 the authors 0270-6430-10$15.00/0. The results show that the positively charged amino acids at the SNAP-25 C terminus promote tight SNARE complex zippering and are required for high release frequency and rapid release in individual fusion events. However, K201E, R198Q, and R198E displayed reduced release frequencies, slower release kinetics, and prolonged fusion pore duration that were correlated with reduced probability to engage in the tightly zippered state. The SNAP-25 K201Q mutant showed no changes compared with SNAP-25 wild-type. Coarse grain molecular dynamics simulations revealed spontaneous transitions between a loose and tightly zippered state at the SNARE complex C terminus.
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To determine how fusion pore conductance and dynamics depend on these residues, single exocytotic events in bovine chromaffin cells expressing R198Q, R198E, K201Q, or K201E mutants were investigated by carbon fiber amperometry and cell-attached patch capacitance measurements. The deleted fragment contains the positively charged residues R198 and K201, adjacent to layers 7 and 8 of the SNARE complex. Previous results with a SNAP-25 construct lacking the nine C terminal residues (SNAP-25Δ9) showed changed fusion pore properties (Fang et al., 2008), suggesting a model for fusion pore mechanics that couple C terminal zipping of the SNARE complex to the opening of the fusion pore. SNAP-25 is a Q-SNARE protein mediating exocytosis of neurosecretory vesicles including chromaffin granules.