Fluorescent Labeling and Quantification of Vesicular ATP Release Using Live Cell Imaging

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• Adenosine triphosphate (ATP) is a key signaling molecule in purinergic neurotransmission.
• Vesicular nucleotide transporter (VNUT), encoded by SLC17A9, facilitates ATP transport into vesicles.
• Understanding vesicular ATP release mechanisms is crucial for neuroscience research.

Purpose of the Study:

• To describe methods for fluorescent labeling of VNUT-positive cells.
• To quantify vesicular ATP release using live cell imaging techniques.
• To investigate the mechanism of ATP release in neurons.

Main Methods:

• Preparation of viable dissociated neurons.
• Cellular labeling with anti-VNUT antibodies and fluorescent ATP markers (quinacrine or MANT-ATP).
• Confocal live cell imaging to observe VNUT-positive cells and vesicular ATP markers, and quantify fluorescence changes upon stimulation.

Main Results:

• VNUT-positive cells showed colocalization with fluorescent ATP markers near the cell membrane.
• Depolarization with high potassium solution induced a reduction in membrane fluorescence, indicating ATP release.
• Cadmium pretreatment blocked fluorescence changes, suggesting calcium-dependent exocytosis.

Conclusions:

• A novel technique for quantifying vesicular ATP release from VNUT-positive neurons was established.
• The results support a calcium-dependent exocytotic mechanism for ATP release in purinergic neurotransmission.
• This method can be applied to study ATP release in both the central and peripheral nervous systems.