Digidata 1322a a d board

Whole cell (current and voltage) patch-clamp recordings were performed using recording pipettes made from thick-wall borosilicate glass with filament (inner diameter: 0.75 mm; Sutter Instruments) pulled on a P-97 Flaming-Brown puller (Sutter). For current-clamp, the internal solution contained the following (in mm): 120 K-gluconate, 20 KCl, 10 HEPES, 2 MgCl₂, 2 Mg2ATP, 0.2 Na3GTP, 0.1 BAPTA, and 0.02% Lucifer yellow, pH 7.3 adjusted with KOH and for voltage-clamp contained the following (in mm): 120 CsMeSO₄, 10 QX-314, 10 HEPES, 1 MgCl₂, 2.5 Mg₂ATP, 0.2 Na₃GTP, 0.1 BAPTA, 10 phosphocreatine, pH 7.3 adjusted with CsOH. Osmolarity for both solutions were in the range 287–295 mOsm. Recordings were discontinued if access resistance was >20 MΩ at the beginning of whole-cell recording with typical access resistances of 10-20 MΩ. Membrane capacitance (C_m) for SACs was 6–10 pF and for PGCs were 5–8 pF. All data were acquired with pCLAMP 9 software using a MultiClamp 700A amplifier, digitized with a Digidata 1322A A/D board (Molecular Devices), low-pass filtered online at 2 kHz (voltage-clamp, sampling rate of 5 kHz) or 10 kHz (current-clamp, sampling rate of 40 kHz).

KCNN3/SK3 channel currents were measured in LGG275 cells transduced with control-YFP or NICD-YFP viruses 3 days after the infection. For electrophysiology, cells were placed in bathing (extracellular) solution composed of 147 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.5 mM MgCl2, 10 mM HEPES, 10 mM glucose, pH 7.4. Recording pipettes filled with a solution containing 135 mM K-methane-sulfonate, 0.1 mM EGTA, 8 mM KCl, 10 mM HEPES, 2 mM MgATP, 0.5 mM NaGTP, pH 7.3 were sealed to the cell membrane. KCNN3/SK3 current was recorded by applying a 600 ms electrical ramp from −80 mV holding potential, gradually increasing up to +60 mV in both control-YFP and NICD-YFP transduced cells. Ionomycin-induced and apamin-sensitive KCNN3/SK3 current densities were measured by adding 10 µM ionomycin and 1 µM apamin to the bathing solution, respectively. All recordings were performed at RT using an Axopatch 200B amplifier and a Digidata 1322A A/D board (Molecular Devices, Berkshire, UK) and acquired at 5 kHz. Current analysis was performed using Clampfit version 10 (Axon Instruments, Molecular Devices).