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.

The nutrient‐sensing repertoires of mouse enterochromaffin cells differ between duodenum and colon

“…This study investigated the nutrient‐sensing capabilities of EC cells from the mouse GI tract. This is the first report of the enrichment of primary EC cells from the duodenum and colon of mice and builds on the capacity for primary EC cell purification in humans and guinea pigs . This approach allows for a paired comparison of gene expression in EC cells obtained from both regions within the same mice.…”

“…This is the first report of the enrichment of primary EC cells from the duodenum and colon of mice and builds on the capacity for primary EC cell purification in humans and guinea pigs. 14,22,33 This approach allows for a paired comparison of gene expression in EC cells obtained from both regions within the same mice. We have identified that EC cells enriched from the mouse duodenum and colon express receptors and transporters capable of sensing luminal sugars and FFAs, which have previously been linked to 5-HT release.…”

Exocytosis in non‐neuronal cells

“…) and that both Vesicular monoamine transporter and the vesicular H + ‐ATPase pump regulate vesicle loading (Raghupathi et al . ). While it was demonstrated that 5‐HT is released from EC cells via exocytosis in a Ca 2+ ‐dependent manner similar to other endocrine cells, EC cells release surprisingly small amounts of 5‐HT given it is contained in large dense core vesicles (Raghupathi et al .…”

“…Some mechanistic insight into the mechanisms governing such small amounts of EC cell 5‐HT release has begun to emerge, and indicate that a smaller diameter fusion pore is able to limit the amount of 5‐HT released per fusion event (Raghupathi et al . ). How fusion pore control differs in these cells compared to other endocrine cells remains unknown.…”

Differential Co-release of Two Neurotransmitters from a Vesicle Fusion Pore in Mammalian Adrenal Chromaffin Cells

“…Kiss-and-Run Fusion Pore Kinetics Are Independent of the Vesicle Matrix By definition, modes of release (sub-quantal versus quantal) are different from modes of vesicle fusion (kiss-and-run versus FFL) because kiss-and-run fusion may produce either quantal (ATP) or sub-quantal (CA) events (Figure 4). To determine whether CgA KD affected SOM-induced fusion pore flickering (or kissand-run) , we assessed fusion pore size during exocytosis-endocytosis using two nano-particle dextrans of different molecular weights and sizes (10 kDa and 40 kDa or 5 nm and 9 nm in diameter) Fulop et al, 2005;Raghupathi et al, 2015), given that SOM-induced endocytosis takes up only 5-nm but not 9-nm dextran in CgA WT cells. CgA KD did not affect the original patterns of SOM-induced uptake of both dextrans, and membrane fusion (kiss-and-run or fusion pore flickers) was not altered by CgA KD (Figure 6).…”