Insights into the Functions of Munc18-1 in Neurotransmitter Release
| dc.contributor.advisor | Rizo-Rey, José | en |
| dc.contributor.committeeMember | Luo, Xuelian | en |
| dc.contributor.committeeMember | Kavalali, Ege T. | en |
| dc.contributor.committeeMember | Liu, Yi | en |
| dc.creator | Su, Lijing | en |
| dc.date.accessioned | 2015-06-01T21:38:13Z | |
| dc.date.available | 2015-06-01T21:38:13Z | |
| dc.date.created | 2013-05 | |
| dc.date.issued | 2013-03-07 | |
| dc.date.submitted | May 2013 | |
| dc.date.updated | 2015-06-01T21:37:45Z | |
| dc.description.abstract | Neurotransmitter release is an exquisitely regulated process that transmits signals between neurons. The release process includes: docking of synaptic vesicles at the active zone of the pre-synaptic plasma membrane, priming to a release ready state, and then membrane fusion and release of neurotransmitters triggered by Ca2+. Several conserved proteins are involved in regulating the entire process. The central membrane fusion machinery in neurons includes Munc18-1 and the SNARE proteins syntaxin-1, SNAP-25 and synaptobrevin. The SNAREs form tight SNARE complexes that bring the vesicle membrane and plasma membrane into close proximity and provide forces to induce membrane fusion. Munc18-1 is essential because deletion of Munc18-1 in mice leads to a complete loss of neurotransmitter secretion. However, its molecular mechanism of action is still unclear. This work is aimed to unravel the critical roles of Munc18-1 in regulating neurotransmitter release. The functions of Munc18-1 discovered so far are related to the SNAREs. Recently we found that Munc18-1 interacts with synaptobrevin and the SNARE four-helix bundle with week affinity, which have been shown to stimulate in vitro SNARE-dependent liposome fusion. Biophysical characterization of these two interactions could provide important information to uncover the roles of Munc18-1 in membrane fusion. Cross-linking and NMR spectroscopy experiments showed that Munc18-1 interacts with the C-terminus of the synaptobrevin SNARE motif through some positively charged residues located in its domain 3a. NMR spectroscopy and ITC experiments revealed that the Munc18-1 N-terminal domain interacts with the C-terminal part of synaptobrevin and syntaxin-1 on the SNARE four-helix bundle, and that the affinity is higher than full length Munc18-1. In our in vitro reconstitution experiments that try to establish the vital functions of Munc18-1 and Munc13 in neurotransmitter release, I found that Munc18-1 displaces SNAP-25 from syntaxin-1/SNAP-25 complex to form Munc18-1/syntaxin-1 complex on liposomes in the presence of NSF/α-SNAP/ATP. When NSF/α-SNAP were incorporated in the lipid mixing assays between synaptobrevin-liposmes and syntaxin-1/SNAP-25-liposomes, Munc18-1 together with Munc13 activate lipid mixing that is inhibited by NSF/α-SNAP. These results suggest that Munc18-1 functions with Munc13 to promote SNARE complex formation in an NSF/α-SNAP resistant manner and to guide the synaptic vesicle exocytosis through a tightly regulated pathway. | en |
| dc.format.mimetype | application/pdf | en |
| dc.identifier.oclc | 910559212 | |
| dc.identifier.uri | https://hdl.handle.net/2152.5/ETD-UTSWMED-2013-05-104 | |
| dc.identifier.uri | https://hdl.handle.net/2152.5/1592 | |
| dc.language.iso | en | en |
| dc.subject | Membrane Fusion | en |
| dc.subject | Munc18 Proteins | en |
| dc.subject | Nerve Tissue Proteins | en |
| dc.subject | Neurotransmitter Agents | en |
| dc.subject | Synaptic Vesicles | en |
| dc.title | Insights into the Functions of Munc18-1 in Neurotransmitter Release | en |
| dc.type | Thesis | en |
| dc.type.material | Text | en |
| thesis.degree.department | Graduate School of Biomedical Sciences | en |
| thesis.degree.discipline | Integrative Biology | en |
| thesis.degree.grantor | UT Southwestern Medical Center | en |
| thesis.degree.level | Doctoral | en |
| thesis.degree.name | Doctor of Philosophy | en |