Measurements of membrane patch capacitance using a software-based lock-in system.
On-cell patch-clamp capacitance measurements can resolve the fusion of individual vesicles to a membrane patch and the accompanying dilation of the fusion pore. So far, these measurements have used a patch-clamp amplifier in combination with a hardware lock-in amplifier. Usually, solely the capacitance and conductance outputs of hardware lock-in amplifiers were recorded, which needed to be filtered rather heavily to suppress spectral components at the stimulus frequency. Therefore, the temporal resolution was limited, and information carried in the patch current was not utilized. In this paper, we describe an alternative and more versatile approach for measuring patch capacitance and conductance, using a digitally controlled patch-clamp amplifier. The software lock-in system showed better bandwidth and identical signal-to-noise performance needing less instrumentation. High temporal resolution measurements on patches of chromaffin cells showed that vesicle fission can be completed in only tens of microseconds. Capacitance calculation based on the patch current allows for straightforward offline phase correction. Moreover, the close inspection of direct current for the first time revealed small current changes accompanying the fusion and fission of large secretory vesicles, promising new insights into the vesicles' membrane properties. A practical guide to high-resolution on-cell patch-clamp capacitance measurements using the software lock-in is provided.
Neef
Andreas
A
InnerEarLab, Department of Otolaryngology, Goettingen University Medical School, 37099 Goettingen, Germany. aneef@gwdg.de
Heinemann
Christian
C
Moser
Tobias
T
D000465:Algorithms; D000818:Animals; D002138:Calibration; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D002837:Chromaffin Granules / Q000378:metabolism; D025802:Electric Capacitance; D004553:Electric Conductivity; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods; D012984:Software