A new method for isolation and purification of fusion-competent inhibitory synaptic vesicles

Nisha Gopal; Jeremy Leitz; Chuchu Wang; Luis Esquivies; Richard A. Pfuetzner; Axel T. Brunger

Current Research in Physiology (Jan 2024)

A new method for isolation and purification of fusion-competent inhibitory synaptic vesicles

Nisha Gopal,
Jeremy Leitz,
Chuchu Wang,
Luis Esquivies,
Richard A. Pfuetzner,
Axel T. Brunger

Affiliations

Nisha Gopal: Department of Molecular and Cellular Physiology, Stanford University, Stanford, USA; Department of Neurology and Neurological Sciences, Stanford University, Stanford, USA; Department of Structural Biology, Stanford University, Stanford, USA; Department of Photon Science, Stanford University, Stanford, USA; Howard Hughes Medical Institute, Stanford University, Stanford, USA
Jeremy Leitz: Department of Molecular and Cellular Physiology, Stanford University, Stanford, USA; Department of Neurology and Neurological Sciences, Stanford University, Stanford, USA; Department of Structural Biology, Stanford University, Stanford, USA; Department of Photon Science, Stanford University, Stanford, USA; Howard Hughes Medical Institute, Stanford University, Stanford, USA
Chuchu Wang: Department of Molecular and Cellular Physiology, Stanford University, Stanford, USA; Department of Neurology and Neurological Sciences, Stanford University, Stanford, USA; Department of Structural Biology, Stanford University, Stanford, USA; Department of Photon Science, Stanford University, Stanford, USA; Howard Hughes Medical Institute, Stanford University, Stanford, USA
Luis Esquivies: Department of Molecular and Cellular Physiology, Stanford University, Stanford, USA; Department of Neurology and Neurological Sciences, Stanford University, Stanford, USA; Department of Structural Biology, Stanford University, Stanford, USA; Department of Photon Science, Stanford University, Stanford, USA; Howard Hughes Medical Institute, Stanford University, Stanford, USA
Richard A. Pfuetzner: Department of Molecular and Cellular Physiology, Stanford University, Stanford, USA; Department of Neurology and Neurological Sciences, Stanford University, Stanford, USA; Department of Structural Biology, Stanford University, Stanford, USA; Department of Photon Science, Stanford University, Stanford, USA; Howard Hughes Medical Institute, Stanford University, Stanford, USA
Axel T. Brunger: Department of Molecular and Cellular Physiology, Stanford University, Stanford, USA; Department of Neurology and Neurological Sciences, Stanford University, Stanford, USA; Department of Structural Biology, Stanford University, Stanford, USA; Department of Photon Science, Stanford University, Stanford, USA; Howard Hughes Medical Institute, Stanford University, Stanford, USA; Corresponding author. Department of Molecular and Cellular Physiology, Stanford University, Stanford, USA

Journal volume & issue: Vol. 7
p. 100121

Abstract

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Synaptic vesicles specific to inhibitory GABA-releasing neurons are critical for regulating neuronal excitability. To study the specific molecular composition, architecture, and function of inhibitory synaptic vesicles, we have developed a new method to isolate and purify GABA synaptic vesicles from mouse brains. GABA synaptic vesicles were immunoisolated from mouse brain tissue using an engineered fragment antigen-binding region (Fab) against the vesicular GABA transporter (vGAT) and purified. Western blot analysis confirmed that the GABA synaptic vesicles were specifically enriched for vGAT and largely depleted of contaminants from other synaptic vesicle types, such as vesicular glutamate transporter (vGLUT1), and other cellular organelles. This degree of purity was achieved despite the relatively low abundance of vGAT vesicles compared to the total synaptic vesicle pool in mammalian brains. Cryo-electron microscopy images of these isolated GABA synaptic vesicles revealed intact morphology with circular shape and protruding proteinaceous densities. The GABA synaptic vesicles are functional, as assessed by a hybrid (ex vivo/in vitro) vesicle fusion assay, and they undergo synchronized fusion with synthetic plasma membrane mimic vesicles in response to Ca2+-triggering, but, as a negative control, not to Mg2+-triggering. Our immunoisolation method could also be applied to other types of vesicles.

Published in Current Research in Physiology

ISSN: 2665-9441 (Online)
Publisher: Elsevier
Country of publisher: Netherlands
LCC subjects: Science: Physiology; Medicine: Internal medicine: Specialties of internal medicine
Website: https://www.journals.elsevier.com/current-research-in-physiology

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