title: Positively charged amino acids at the SNAP-25 C terminus determine fusion rates, fusion pore properties, and energetics of tight SNARE complex zippering.
abstract: SNAP-25 is a Q-SNARE protein mediating exocytosis of neurosecretory vesicles including chromaffin granules. 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. The deleted fragment contains the positively charged residues R198 and K201, adjacent to layers 7 and 8 of the SNARE complex. 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. Coarse grain molecular dynamics simulations revealed spontaneous transitions between a loose and tightly zippered state at the SNARE complex C terminus. The SNAP-25 K201Q mutant showed no changes compared with SNAP-25 wild-type. 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 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.
authors.affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, and Laboratory for Nanoscale Cell Biology, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Qinghua
authors.identifier: http://orcid.org/0000-0002-3448-5582
authors.initials: Q
authors.lastname: Fang
authors.affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, and Laboratory for Nanoscale Cell Biology, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, and.
authors.forename: Adam Drew
authors.initials: AD
authors.lastname: Herbst
authors.affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, and.
authors.forename: Brian N
authors.initials: BN
authors.lastname: Kim
authors.affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, and Laboratory for Nanoscale Cell Biology, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany mlindau@mpibpc.mpg.de ml95@cornell.edu.
authors.forename: Manfred
authors.initials: M
authors.lastname: Lindau
mesh_terms: D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000704:Analysis of Variance; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D019439:Chromaffin Cells; D003198:Computer Simulation; D025802:Electric Capacitance; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D017711:Nonlinear Dynamics; D018408:Patch-Clamp Techniques; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D050600:SNARE Proteins / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D000596:Amino Acids; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D049452:Green Fluorescent Proteins; D011189:Potassium Chloride; D002118:Calcium
keywords: SNAP-25; amperometry; capacitance; chromaffin cells; exocytosis; fusion pore