#Charged amino acids Patch#
#Charged amino acids free#

Botulinum toxin A cleaves off the 9 C terminal residues of SNAP-25 and impairs release ( Xu et al., 1998).

#Charged amino acids free#

C terminal zippering of the SNARE complex provides the force and free energy to conquer the barriers for fusion ( Gao et al., 2012) under the control of complexin ( Maximov et al., 2009).įusion pore properties depend on multiple factors, including vesicle size ( Klyachko and Jackson, 2002 Flašker et al., 2013), electrostatic interactions ( Han and Jackson, 2005 Calejo et al., 2012), and the energetics of SNARE complex zippering. This stimulus is thought to enable zippering of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex in a vectorial manner from the N terminus toward the C terminus ( Sørensen, 2009) through a mechanism that involves the protein complexin ( Tang et al., 2006 Li et al., 2011). Fusion pore opening and fusion pore expansion are triggered by an increase in intracellular free calcium ions sensed by synaptotagmin ( Fernández-Chacón et al., 2001 Sørensen et al., 2003a Wang et al., 2006). The fusion pore connects the intravesicular lumen to the extracellular space, allowing for release of vesicular contents ( Breckenridge and Almers, 1987). Many types of cells release specific molecules stored in intracellular vesicles by exocytosis via the formation of a fusion pore. 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.

#Charged amino acids Patch#

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.