Resolution of fusion pore formation in a cell-attached patch

Area of Science:

• Neuroscience
• Cell Biology
• Biophysics

Background:

• Exocytosis, the process of neurotransmitter release, begins with fusion pore formation.
• Capacitative current measurements have previously resolved exocytosis of large secretory granules.

Purpose of the Study:

• To extend the capacitative current method for resolving fusion pore formation in single vesicles.
• To investigate the initiation of exocytosis in bovine chromaffin cells.

Main Methods:

• Utilized cell-attached patch recordings to detect capacitative currents.
• Induced exocytosis by increasing intracellular calcium (Ca2+) via ionophore or co-localized Ca2+ channel activation.
• Calculated fusion pore conductance and vesicular membrane potential.

Main Results:

• Successfully resolved fusion pore formation and subsequent exocytosis in single chromaffin cell vesicles.
• Observed capacitative currents evoked by calcium increases.
• Demonstrated that single calcium channel openings trigger fusion pore formation with consistent delay times.

Conclusions:

• The extended capacitative current method accurately resolves fusion pore dynamics in single vesicles.
• This technique is versatile and applicable to various vesicle types and locations, including synaptic vesicles.
• Provides a powerful tool for studying the fundamental mechanisms of exocytosis.