Nanodisc-cell fusion: Control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains

• Autores: Wu, Zhenyong; Auclair, Sarah M.; Bello, Oscar Daniel; Vennekate, Wensi; Dudzinski, Natasha R.; Krishnakumar, Shyam S.; Karatekin, Erdem
• Año de publicación: 2016
• Idioma: inglés
• Tipo de recurso: artículo
• Materia: EXOCYTOSIS; MEMBRANE FUSION; NANOSCALE BIOPHYSICS; SNARE; SYNAPTIC VESICLE EXOCYTOSIS
• Institución: Consejo Nacional de Investigaciones Científicas y Técnicas

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.
Fil: Wu, Zhenyong. University of Yale; Estados Unidos. University of Yale. School of Medicine; Estados Unidos
Fil: Auclair, Sarah M.. University of Yale. School of Medicine; Estados Unidos. University of Yale; Estados Unidos
Fil: Bello, Oscar Daniel. University of Yale. School of Medicine; Estados Unidos. University of Yale; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina
Fil: Vennekate, Wensi. University of Yale. School of Medicine; Estados Unidos. University of Yale; Estados Unidos
Fil: Dudzinski, Natasha R.. University of Yale; Estados Unidos. University of Yale. School of Medicine; Estados Unidos
Fil: Krishnakumar, Shyam S.. University of Yale. School of Medicine; Estados Unidos. University of Yale; Estados Unidos
Fil: Karatekin, Erdem. University of Yale; Estados Unidos. University of Yale. School of Medicine; Estados Unidos. Universite Paris Descartes; Francia. Centre National de la Recherche Scientifique; Francia