Electrophysiological Characterization of TMEM16F

TMEM16F currents were recorded in whole-cell configuration using an Axopatch 200B amplifier (Molecular Devices) and the pClamp software package (Molecular Devices). Glass pipettes were pulled from borosilicate capillaries (Sutter Instruments) and fire-polished using a microforge (Narishge) to reach a resistance of 2–3 MΩ. The pipette solution (internal) contained (in mM) 140 CsCl, 1 MgCl₂, and 10 HEPES, plus 1 CaCl₂ to avoid the long-delay activation of TMEM16F(Liang and Yang, 2021 ; Zhang et al., 2022 (link)). The bath solution contained 140 CsCl, 10 HEPES, and 5 EGTA. Adjusted to pH 7.3 for both sides with CsOH. Currents were recorded about 2 minutes after whole-cell formation to ensure the activation of TMEM16F(Liang and Yang, 2021 ; Zhang et al., 2022 (link)).
Currents were normalized to cell capacitance as current density. $I-V$ curves were constructed from steady-state currents after depolarization. Individual $I-V$ curves were fitted with a Boltzmann function,
$$I(V)=\frac{I_{max}}{1+e^{\frac{-Z(V-V0.5)}{RT}}}$$
where $I_{max}$ denotes the maximal current, $V_{0.5}$ denotes the voltage of half-maximal activation of conductance, $z$ denotes the net charge moved across the membrane during the transition from the closed to the open state, and $F$ denotes the Faraday constant.