We used National Instruments board PCI-6052E to generate the command potential and collect signals, and we used an Axopatch-1D (Molecular Devices) for patch clamp. Electrode tips were dipped in molten hard dental wax (Kerr Corporation) before cutting and polishing to reduce stray capacitance. For excised patches, electrodes with ∼15 μm inner diameters were employed. The giant patch was excised by essentially aspirating the cell into a second pipette with a sharp, unpolished edge (Hilgemann and Lu, 1998 (link)). The patches were positioned in front of a temperature controlled (∼30°C) solution outlet immediately after excision. Membrane fusion was triggered by moving the patch to a solution outlet containing 0.2 mM free Ca. Capacitance and conductance were measured using the Lindau-Neher method (Lindau and Neher, 1988 (link)). Sine waves generated by Capmeter 6 with 20 mV peak-to-peak amplitude at 2 kHz were applied to the cell. The current output from the patch clamp was low-pass filtered at 10 kHz. When sine wave perturbation was employed, the optimal phase angle was determined as described above. When patch amperometry was employed, a hardware lock-in amplifier (SR830; Stanford Research Systems) was employed, as it allowed a higher signal-to-noise ratio at oscillation frequencies >3 kHz. Sine waves with Vrms of 20 mV at 10 kHz were usually employed. The signals were recorded by Capmeter 1.
For whole-cell recording, with ∼5 μm inner diameter pipette tips, membrane fusion was initiated via perfusion of Ca-containing (nitrilotriacetic acid-bufferd) solution through a quartz capillary with a 40 μm outlet, manipulated within the patch pipette to a distance of 50∼100 μm from the cell opening (Hilgemann and Lu, 1998 (link)). Square wave 20 mV (peak-to-peak) perturbation at 0.5 kHz was employed in all experiments presented in this article for whole-cell capacitance recording, with cell parameters determined by Capmeter 6 as described above.
For whole-cell recording, with ∼5 μm inner diameter pipette tips, membrane fusion was initiated via perfusion of Ca-containing (nitrilotriacetic acid-bufferd) solution through a quartz capillary with a 40 μm outlet, manipulated within the patch pipette to a distance of 50∼100 μm from the cell opening (Hilgemann and Lu, 1998 (link)). Square wave 20 mV (peak-to-peak) perturbation at 0.5 kHz was employed in all experiments presented in this article for whole-cell capacitance recording, with cell parameters determined by Capmeter 6 as described above.
