Hum silencer

The I_KACh current in atrial myocytes was measured with standard whole-cell voltage-clamp technique. The Multiclamp 700B (Molecular Devices) amplifier, an A/D converter (Digidata 1550B plus Hum Silencer, Molecular Devices), and the pClamp 10.6 PC software (Molecular Devices) were used for current acquisition. Clampfit 10.6 (pClamp, Molecular Devices) and OriginPro 2018b software packages (OriginLab Corp) were used for data analysis. Whole-cell measurements were performed at room temperature using borosilicate glass microelectrodes with tip resistances of 2.5–3 MΩ filled with pipette solution (in mM): KCl 140, MgCl₂ 1, HEPES 10, EGTA 5, Mg2ATP 5, and GTP 0.1, pH adjusted to 7.2 with KOH. Basal current was recorded by holding the atrial myocyte at −40 mV, followed by 1000 ms steps from −140 mV to +40 mV in 20 mV increments. The extracellular solution contained (in mM): NaCl 100, KCl 50, MgCl₂ 1, HEPES 5, and D-glucose 5.5, adjusted to pH 7.4. To measure the constitutively active I_KACh current in atrial cardiomyocytes, 100 nM Tertiapin-Q (TPQ, Alomone Labs) was applied to the extracellular solution.

Single-barrel recording electrodes (World Precision Instruments, TW150F-3) were pulled (Sutter Instruments, P-87) and flame-polished (ALA Scientific Instruments, CPM-2). The electrode was connected to the head stage (CV-7B, Axon Instruments) of a microelectrode amplifier (Molecular Devices, Axon MultiClamp 700B) with a chlorinated silver wire (Molecular Instruments, 1HLA005). The head stage and suction electrodes were mounted on a motorized micromanipulator (Sutter Instruments, ROE-200 and MPC-200). The antennal nerve was sucked into the recording electrode by manually controlling the strength of external vacuum. The extracellular signal was amplified 100 times at the microelectrode amplifier, digitized (Molecular Devices, Digidata 1550B), and recorded at 10 kHz using the Clampex software (Molecular Devices). The signal was analog-filtered at 10 kHz (Bessel), and 60-Hz main line noise was removed using an adaptive module in the digitizer (HumSilencer, Molecular Devices).