title: Fusion Pore Size Limits 5-HT Release From Single Enterochromaffin Cell Vesicles.
abstract: Enterochromaffin cells are the major site of serotonin (5-HT) synthesis and secretion providing ∼95% of the body's total 5-HT. 5-HT can act as a neurotransmitter or hormone and has several important endocrine and paracrine roles. We have previously demonstrated that EC cells release small amounts of 5-HT per exocytosis event compared to other endocrine cells. We utilized a recently developed method to purify EC cells to demonstrate the mechanisms underlying 5-HT packaging and release. Using the fluorescent probe FFN511, we demonstrate that EC cells express VMAT and that VMAT plays a functional role in 5-HT loading into vesicles. Carbon fiber amperometry studies illustrate that the amount of 5-HT released per exocytosis event from EC cells is dependent on both VMAT and the H(+)-ATPase pump, as demonstrated with reserpine or bafilomycin, respectively. We also demonstrate that increasing the amount of 5-HT loaded into EC cell vesicles does not result in an increase in quantal release. As this indicates that fusion pore size may be a limiting factor involved, we compared pore diameter in EC and chromaffin cells by assessing the vesicle capture of different-sized fluorescent probes to measure the extent of fusion pore dilation. This identified that EC cells have a reduced fusion pore expansion that does not exceed 9 nm in diameter. These results demonstrate that the small amounts of 5-HT released per fusion event in EC cells can be explained by a smaller fusion pore that limits 5-HT release capacity from individual vesicles.
authors.affiliation: Department of Human Physiology and Centre for Neuroscience, Flinders University, Adelaide, Australia.
authors.forename: Ravinarayan
authors.initials: R
authors.lastname: Raghupathi
authors.affiliation: Department of Anatomy and Histology and Centre for Neuroscience, Flinders University, Adelaide, Australia.|Discipline of Medicine, University of Adelaide, Adelaide, Australia.
authors.forename: Claire F
authors.initials: CF
authors.lastname: Jessup
authors.affiliation: Department of Human Physiology and Centre for Neuroscience, Flinders University, Adelaide, Australia.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Lumsden
authors.affiliation: Department of Human Physiology and Centre for Neuroscience, Flinders University, Adelaide, Australia.|South Australian Health and Medical Research Institute, Adelaide, Australia.
authors.forename: Damien J
authors.initials: DJ
authors.lastname: Keating
mesh_terms: D000302:Adrenal Cortex / Q000166:cytology / Q000378:metabolism; D000818:Animals; D004759:Enterochromaffin Cells / Q000378:metabolism*; D005089:Exocytosis / Q000235:genetics; D006168:Guinea Pigs; D008561:Membrane Fusion / Q000235:genetics; D051379:Mice; D022142:Secretory Vesicles / Q000378:metabolism*; D012701:Serotonin / Q000096:biosynthesis* / Q000378:metabolism; D050493:Vesicular Monoamine Transport Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D050493:Vesicular Monoamine Transport Proteins; D012701:Serotonin