Patch Clamp Electrophysiology

TRPM7 currents (I_TRPM7) were measured in whole cell configuration as illustrated (Fig. S1C). The standard external solution contained (in mM): 135 Na-methanesulfonate, 5 Cs-gluconate, 2.5 CaCl₂, 10 HEPES, pH 7.3 (adjusted with NaOH), and osmolality 280–290 mOsm/Kg. The standard pipette solution contained (in mM): 110 Cs-gluconate, 0.5 NaCl, 0.75 CaCl₂, 10 HEPES, 10 HEDTA, 1.8 Cs₄-BAPTA, 2 Na₂ATP, pH 7.3 (adjusted with CsOH), and osmolality 273 mOsm/Kg. Free [Ca²⁺] = ~100nM and was calculated via the Maxchelator algorithm (http://maxchelator.stanford.edu/webmaxc/webmaxcS.htm). Peritoneal macrophages were freshly-isolated prior to analysis by peritoneal lavage. MgCl₂ (10 mM) was added to the external solution to inhibit TRPM7 currents. The recording protocol used 400 ms ramps from −100 mV to +100 mV and a holding potential (HP) of 0 mV. Signals were low-pass filtered at 5 kHz and sampled at 10 kHz. All electrophysiology experiments were conducted at RT (~23°C) using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA).