# Structure and dynamics of exocytotic fusion pores

> **NIH NIH R35** · UNIVERSITY OF WISCONSIN-MADISON · 2020 · $522,780

## Abstract

PROJECT SUMMARY/ABSTRACT
 During exocytosis, fusion pores form the first aqueous connection that allows escape of neurotransmitters
and hormones from secretory vesicles. Although it is well established that SNARE complexes catalyze fusion,
the structure and composition of fusion pores remain unknown. This is the central question in the field of
membrane fusion, as the mechanism of fusion cannot be solved until the structure of the first key intermediate
in this pathway, the fusion pore, has been elucidated. The main objective of this proposal is to gain new
insights into fusion pore composition, structure, and dynamics, using both reconstitution and cell-based
approaches. A major limitation in the biochemical study of fusion pores in cells concerns their low abundance
and ephemeral nature. For example, in neuroendocrine cells, the duration of the initial open state of the fusion
pore is of the order of msec; the pore then either closes (kiss-and-run exocytosis), or dilates to yield full fusion.
To overcome this limitation, we have begun to study fusion pore structure, in vitro, by exploiting the rigid
framework of nanodiscs. SNARE-bearing proteoliposomes dock and fuse with nanodiscs that harbor cognate
SNAREs. Since nanodiscs are bounded by membrane scaffolding proteins, the pores cannot dilate, and hence
can be studied biochemically. Using this system, we have begun to interrogate the properties of reconstituted
fusion pores. Our preliminary data indicate that, contrary to the common view that fusion pores are purely
lipidic, they are in fact hybrid structures, composed of both lipids and proteins. We will use a simulation
approach to derive a new model for fusion pore structure, and conduct cryo-electron microscopy studies to
visualize this structure. We will also use a variety of cargos of varying diameter, in conjunction with optical
sensors that report their release during fusion, to determine the size of the pore, and to determine whether
pore diameter is `plastic' and varies with the number of SNARE proteins. We will also probe for interactions
between cargo and SNARE transmembrane domains by exploiting electrostatic interactions between them.
The nanodisc system will be adapted to single molecule studies, to monitor pore opening and closing of
individual pores in real-time, and to directly assess the impact of regulatory factors on pore stability. These in
vitro experiments will be complimented by our ongoing direct measurements of fusion pores in chromaffin cells,
using carbon fiber amperometry, by comparing the effects of SNARE mutations in these two systems. We will
draw parallels between these systems so that we can arrive at unified, physiologically relevant models for
pores. Finally, we will also design novel optical probes, based on pH sensitive dyes conjugated to quantum
dots, to study fusion pores in cultured neurons. These latter studies will address the highly controversial topic
of kiss-and-run exocytosis versus full fusion. T...

## Key facts

- **NIH application ID:** 9823896
- **Project number:** 5R35NS097362-04
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Edwin R Chapman
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $522,780
- **Award type:** 5
- **Project period:** 2016-12-01 → 2024-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9823896

## Citation

> US National Institutes of Health, RePORTER application 9823896, Structure and dynamics of exocytotic fusion pores (5R35NS097362-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9823896. Licensed CC0.

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