ABSTRACT
Detection of Released Molecules................................................................................................. 316
Patch Amperometry...................................................................................................................... 317
Patch Amperometry Requires Reversed Patch Clamp Electrode Configuration .................... 317
Equipment and Setup ............................................................................................................... 317
Data Acquisition ................................................................................................................... 317
Capacitance Measurements .................................................................................................. 318
Amperomety ......................................................................................................................... 322
Microscopy ........................................................................................................................... 322
Design of the Electrode Holders .............................................................................................. 322
Manually Adjustable Electrode Holder ............................................................................... 323
Motorized Electrode Holder ................................................................................................. 324
Patch Pipettes ........................................................................................................................... 326
Carbon Fiber Electrode Fabrication ......................................................................................... 326
Carbon Fiber Fabrication Setup ........................................................................................... 326
Carbon Fiber Pulling ............................................................................................................ 327
Testing the CFE in the Patch Amperometry Configuration ................................................ 328
Recording Chamber Design ..................................................................................................... 329
Patching of Chromaffin Cells ................................................................................................... 329
Capacitance Calibration ........................................................................................................... 330
Analysis of Exocytotic Events ..................................................................................................... 331
Vesicle Capactiance and Fusion Pore Conductance................................................................ 331
Release of Molecules ............................................................................................................... 333
Quantal Size, Vesicle Size, and the Fusion Pore .................................................................... 334
Summary and Discussion ............................................................................................................. 334
Acknowledgments ........................................................................................................................ 334
References .................................................................................................................................... 335
Exocytosis of vesicles as small as 60 nm in diameter can be detected by cell membrane admittance
measurements with the patch clamp technique in the cell attached configuration [2-4], and by
amperometry with a carbon fiber electrode (CFE) [5]. The admittance measurement provides the
membrane capacitance that reveals changes in surface area due to the incorporation of the vesicular
membrane into the plasma membrane. Each fusing vesicle causes a stepwise increase in capaci-
tance [2]. The admittance measurement also provides the membrane conductance and the fusion
pore conductance during an exocytotic event [4]. With amperometry, released molecules that are
readily oxidizable are electrochemically detected at the surface of the CFE. This technique is able
to resolve release events down to a few ten-thousands of molecules [6].
