Wu, Z;
Auclair, SM;
Bello, O;
Vennekate, W;
Dudzinski, NR;
Krishnakumar, SS;
Karatekin, E;
(2016)
Nanodisc-cell fusion: control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains.
Scientific Reports
, 6
, Article 27287. 10.1038/srep27287.
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Abstract
The initial, nanometer-sized connection between the plasma membrane and a hormone- or neurotransmitter-filled vesicle –the fusion pore– can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics determine release and vesicle recycling kinetics, but pore properties are poorly known because biochemically defined single-pore assays are lacking. We isolated single flickering pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, “flipped” t-SNAREs. Conductance through single, voltage-clamped fusion pores directly reported sub-millisecond pore dynamics. Pore currents fluctuated, transiently returned to baseline multiple times, and disappeared ~6 s after initial opening, as if the fusion pore fluctuated in size, flickered, and resealed. We found that interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, TMD modifications designed to disrupt v- and t-SNARE TMD zippering prolonged pore lifetimes dramatically. We propose that the post-fusion geometry of the proteins contribute to pore stability.
| Type: | Article |
|---|---|
| Title: | Nanodisc-cell fusion: control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1038/srep27287 |
| Publisher version: | http://doi.org/10.1038/srep27287 |
| Language: | English |
| Additional information: | This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
| Keywords: | Science & Technology, Multidisciplinary Sciences, Science & Technology - Other Topics, ADRENAL CHROMAFFIN CELLS, MEMBRANE-FUSION, TRANSMITTER RELEASE, TRANSIENT PORES, IN-VITRO, EXOCYTOSIS, VESICLES, PLANAR, PATCH, CLAMP |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Clinical and Experimental Epilepsy |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1541643 |
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