Multiclamp commander

Electrophysiological recordings were digitized, using a Digidata 1322 A A/D interface (Molecular Devices), at a sampling rate of 20 kHz (low-pass filtered at 10 kHz) and recorded with the pClamp 10 software (Molecular Devices). The amplifier settings of the Multiclamp 700B were controlled through Multiclamp Commander (Molecular Devices).
After 50 ms baseline recording, 1 s current steps of –350 to +350 pA were applied to the patched cells in increments of 25 pA, after which an additional 1 s of post-step membrane potential was recorded. The trigger time between these episodes was 3 s. Voltage deflections in response to current steps of –50 to +50 pA were used to calculate the input resistance. Initial RMP was obtained immediately upon breaking into the cell. Voltage sag in response to negative current steps was calculated by dividing the steady-state voltage deflection during the late phase of the –100 pA current step by the peak of the voltage deflection during the same step. AP threshold was determined as the membrane voltage at which the derivative of the voltage trace exceeded 40 mV/ms. AP width describes the width of the AP at 50% of its amplitude.
Data was analyzed using Axograph X software (Axograph Scientific, Australia), MATLAB (MathWorks), as well as Microsoft Excel (Microsoft Corp., WA) and Prism 8 (GraphPad, CA) and visualized in Acrobat Illustrator (Adobe, CA).

For Ca_V2.1, HEK293 cells were transfected with 0.5 µg Ca_V2.1-WT or 0.5 µg Ca_V2.1-V1686M and co-transfected with 0.5 µg Ca_Vβ2, 0.5 µg Ca_Vα2δ1 and 0.2 µg EGFP. All transfections were done using siLentFect™ Lipid (Bio-Rad, Copenhagen, Denmark) according to manufacturer’s instructions. Patch-clamp experiments were performed at room temperature. Currents were measured 72 h after transfection from single fluorescent CHO or HEK cells using a MultiClamp 700B amplifier and MultiClamp Commander (Molecular Devices, Axon Instruments, Sunnyvale, California, United States). The cells were superfused with an extracellular solution containing the following (in mM): 140 TEA-Cl, 3 CsCl, 2.5 CaCl₂, 1.2 MgCl₂, 10 HEPES and 10 Glucose, pH adjusted to 7.4 with NaOH. Pipettes were pulled from borosilicate glass capillaries (Harvard Apparatus, Holliston, United States) using a DMZ Universal Puller (Zeitz Instruments, Martinsried, Germany) and had a resistance of 4.0-6.0 MΩ when filled with intracellular solution containing the following (in mmol/L): 140 CsCl, 1 EGTA, 4 Na₂ATP, 0.1 Na₃GTP and 10 HEPES, pH adjusted to 7.2 with CsOH. Data were acquired using a Digidata 1,440 Converter and the software pClamp 10.4 Commander (Molecular Devices).