Digidata 1320a

Kv1.3 currents were recorded from CD8⁺ T cells in whole-cell voltage clamp configuration using an AxoPatch 200B Amplifier (Molecular devices). Pipettes were formed from Borosilicate glass (TW150F-4, World Precision Instruments) with a P-97 horizontal puller (Sutter Instruments) and had a resistance between 4 and 7 MΩ when filled with intracellular pipette solution containing (in mmol/L) 140 KF, 55 EGTA, 5 CaCl₂, 10 MgCl₂, and 10 HEPES [pH 7.22; (19 (link))]. The external solution contained (in mmol/L) 145 NaCl, 5 KCl, 1 MgCl₂, 2.5 CaCl₂, 5.5 glucose, and 10 HEPES (pH 7.4,all reagents from Millipore-Sigma). Kv1.3 currents were elicited by 800-ms step pulse depolarization from -80mV to +50 mV. Data were acquired using pCLAMP 8.0 software (Molecular Devices) through a 16-bit A-D/D-A interface (Digidata1320A, Molecular Devices). Data were low pass filtered frequency at 2 kHz and digitalized at 100 kHz. The amplitude of the peak current was determined at +50 mV. The verity of Kv1.3 currents was confirmed by blocking the channels by ShK-Dap22 (Bachem), a specific Kv1.3 channel blocker (28 (link)).

mIPSCs were recorded in ACSF that, along with DNQX (10 μM) and D-AP-5 (50 μM), included tetrodotoxin (TTX; 1 μM) to block APs, sIPSCs were recorded without TTX. Whole-cell voltage-clamp recording of mIPSCs was conducted using a high-chloride internal pipette solution, which resulted in an inward chloride current with cells clamped at −76 mV (corrected for liquid junction potential in Clampfit). The pipette solution consisted of the following (in mM): 100 CsCH₃O₃S, 50 CsCl, 3 KCl, 0.2 BAPTA, 10 HEPES, 1 MgCl₂, 2.5 Phosphcreatine-2Na, 2 Mg-ATP, and 0.25 GTP-Tris, titrated to pH 7.2–7.3 with 3 M CsOH (osmolarity 280–290 mOsm). In all experiments, lidocaine N-ethylbromide (QX-314; 5 mM) was added to the pipette solution on the day of the experiment. Synaptic currents were recorded using an Axopatch 700B amplifier (Molecular Devices), filtered at 2 kHz, sampled at 20 kHz, digitized (Digidata 1320 A; Molecular Devices), and stored for off- line analysis (using Minianalysis software written in IGOR Pro; Wavemetrics, Lake Oswego, OR) (Hwang and Copenhagen, 1999). Access resistance stability (10–18 MΩ; 80% compensation) was monitored using a 2 mV voltage step applied every 120 s, and data from cells were discarded when >15% change occurred.

For patch-clamp recording, the recording culture dish was mounted on the stage of an inverted microscope (TMD300; Nikon, Tokyo). The indifferent electrode was an Ag-AgCl wire connected to the culture dish. Membrane voltages and currents were recorded in the whole-cell configuration using a patch-clamp amplifier (Axopatch 200B; Molecular Devices, San Jose, CA, USA) linked to a computer [35 (link),36 (link),37 (link)]. The voltage-clamp and current-clamp procedures were controlled by pCLAMP software (Molecular Devices). The data were low-pass filtered with a cut-off frequency of 5 kHz and then digitized at 10 kHz by an analog-to-digital interface (Digidata 1320A; Molecular Devices). Cultured cells were perfused at 1 mL/min with Ringer solution bubbled with 100% O₂. The composition of HEPES-buffered Ringer solution (in mM) was 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (the pH was adjusted to 7.4 with KOH). The recording pipette was filled with pseudo-intracellular solution with the following composition (in mM): 140 mM KCl, 1 mM CaCl₂, 2 mM MgCl₂, 5 mM BAPTA, 10 mM HEPES. The solution was adjusted with KOH to pH 7.4. The pipette resistance was 6–8 MΩ. Tetrodotoxin (1 µM TTX, a voltage-gated sodium channel blocker) was applied through the bath.

BK currents at single-channel resolution were recorded from excised, inside-out (I/O) membrane patches. Both bath and electrode solutions contained (mM) 130 KCl, 5 EGTA, 1.6 HEDTA, 10 HEPES, 5.59 CaCl₂ ([Ca²⁺]_free≈30 μM), 2 MgCl₂, pH 7.35. For experiments with [Ca²⁺]_free≈3 μM, both bath and electrode solutions contained (mM) 130 KCl, 5 EGTA, 1.6 HEDTA, 10 HEPES, 4.49 CaCl₂, 2.44 MgCl₂, pH 7.35. Nominal free Ca²⁺ was calculated with MaxChelator Sliders. Patch-recording glass electrodes were fire-polished on a microforge WPI MF-200 (World Precision Instruments) to give resistances of 5–9 MΩ when filled with electrode solution. An agar bridge with Cl^- as the main anion was used as ground electrode. When required by experimental design, excised patches were exposed to a stream of CLR-containing or CLR-free bath solution that was applied onto the patches by using a computerized and pressurized OctaFlow system (ALA Scientific Instruments) via a micropipette tip with an internal diameter of 100 μm. Ionic current was recorded with an EPC8 amplifier (HEKA) at 1 kHz using a low-pass, eight-pole Bessel filter (model 902LPF; Frequency Devices). Data were digitized at 5 kHz using Digidata 1320A and pCLAMP 8.0 (Molecular Devices). For a proper comparison with data previously obtained by us and others, all studies were conducted at room temperature (20–22º C).