Qf100 70 7

HEK cells were maintained and transfected as described above. Cell-attached configuration of the patch clamp technique (Hamill et al., 1981 (link)) was used to record Ba²⁺ currents from single or multiple Ca_V1.2 channels at room temperature (23 ± 2°C) 48–72 h post-transfection. Data acquisition was performed using pCLAMP 9 (Molecular Devices, Sunnyvale, CA, USA) and an Axopatch 200B amplifier (Molecular Devices). Pipettes were pulled from quartz glass capillaries (QF100-70-7.5; Sutter Instrument, Novato, CA, USA) using a Laser-Based Micropipette puller (P-2000, Sutter Instrument, Novato, CA, USA), and filled with 140 mM TEA-Cl, 40 mM BaCl₂, 10 mM HEPES (pH 7.4 adjusted with TEA-OH). To zero membrane potential, the bath solution contained 132 mM K glutamate, 5 mM KCl, 5 mM NaCl, 3 mM MgCl₂, 2 mM EGTA, 10 mM glucose, 20 mM HEPES (pH 7.4 adjusted with KOH) (Dick et al., 2016 (link)). Recordings were low pass filtered with a cutoff frequency of 2 kHz and digitized at 50 ms. Patches were stimulated by a voltage ramp from −80 mV to 50 mV over the duration of 200 ms. The leak for each trace was subtracted using a linear fit added to an exponential fit.