Inside-Out Patch Clamp

Electrophysiological recordings were performed in the inside-out patch-clamp configuration with patch pipettes (1 and 2 μm tip opening) pulled from 1.5 mm borosilicate glass capillaries (Zeitz-Instruments GmbH, München, Germany) using micropipette puller P 97 (Sutter Instruments, Novato, CA, United States). The tips were polished to have a pipette resistance of 2–4 MΩ in the bath solution. The pipette solution contained (in mM) 150 NaCl, 10 HEPES, and 2 CaCl₂ (pH 7.4 with NaOH). The bath solution contained (in mM) 150 NaCl, 10 HEPES, 2 HEDTA (pH 7.4 with NaOH), and no Ca²⁺. Solutions containing 300 μM Ca²⁺ were made by adding the appropriate concentration of CaCl₂ without buffer to a solution containing (in mM) 150 NaCl, 10 HEPES (pH 7.4 with NaOH) as reported previously (Zhang et al., 2005). Bath solution with 0 and 300 μM Ca²⁺ concentrations were applied to cells by a modified rapid solution exchanger (Perfusion Fast-Step SF-77B; Warner Instruments Corp. CT, United States). Membrane currents were recorded with a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale CA, United States) controlled by Clampex 10 via Digidata 1332A (Molecular Devices, Sunnyvale, CA, United States). Data were low-pass filtered at 5 kHz and sampled at 10 kHz. Experiments were performed at room temperature (20–25°C). The holding potential was 0 mV. For measuring steady-state currents, the stimulation protocol consisted of two sweeps with a total duration of 1,000 ms, the first sweep at −100 mV for 500 ms and the second at +100 mV for 500 ms (Figure 2). The effect of the compounds on TRPM4 current has been calculated by averaging the last 100 ms of the second sweep at +100 mV. The I-V relationships (IV curves) have been determined using an I-V protocol from −100 mV to +100 mV for 500 ms with a difference of 20 mV between each sweep (cf. Figure 6). The IV curves have been normalised to the maximum current. No fit was applied.

Validation of cell lines stably expressing TRPM4. (A) Expression of TRPM4 channels in stably transfected cell lines. (A1) Western blot showing TRPM4 expression as detected by anti-TRPM4 antibody from whole-cell extracts prepared from TsA-201 cells. (A2) functional expression of TRPM4 channels in the different stable cell lines (n = 9 to 10 for each condition). **p ≤ 0.01 and ***p ≤ 0.001. (B) Effect of free ATP on TRPM4 current. (B1), representative traces of TRPM4 current activated by 300 μM calcium (black) and inhibited by 10 μM free ATP (blue) currents recorded from excised membrane patches (inside-out) (up and lower panels, respectively, human and mouse TRPM4). (B2), the effect of free ATP on TRPM4 current. The values were fitted with the Hill equation to interpolate IC₅₀ values of free ATP (n = 6 for each concentration).

Effects of CBA on mouse TRPM4 current. Left panel shows raw traces of mouse TRPM4 currents recorded using the step protocol depicted at the top of the figure, with (black) or without (red) 5 μM CBA in the extracellular bath (intracellular application). Right panel shows IV curves, representing the average current amplitude at the end of each voltage step when the TRPM4 current was activated using 300 μM free calcium solution (black) and treated with 5 μM CBA (red) (n = 8). **p ≤ 0.01.