Effects of external and internal Ca²⁺ on protons' inhibition of TRPM2. (A) Concentration-dependent effects of external acidic pH on TRPM2 in the solutions containing 200 µM and 2 mM [Ca²⁺]_o, respectively. Whole cell currents were recorded with pipette solutions containing minimal [Ca²⁺]_i buffered by 10 mM EGTA and 500 µM ADPR. Ramp protocol (−100 to +100 mV) was used to elicit TRPM2 currents. Current amplitude at +100 mV was measured and normalized to the maximal current amplitude at pH 7.4. The proton dose–response curve was rightward shifted at 200 µM of external [Ca²⁺]_o, and the IC₅₀ was changed from 5.4 ± 0.05 pH units (2 mM [Ca²⁺]_o; n = 8) to 7.5 ± 0.1 pH units (200 µM [Ca²⁺]_o; n = 8). (B) Effects of internal [Ca²⁺]_i on concentration-dependent inhibition of TRPM2 by external protons. Whole cell currents of TRPM2 were recorded in the 2 mM of external Ca²⁺ with pipette solutions containing 100 nM or 100 µM [Ca²⁺]_i in the presence of 200 µM [ADPR]_i. Protons produced similar inhibitory effects on TRPM2 at 100 nM (n = 9) and 100 µM (n = 6) of internal [Ca²⁺]_i (with 200 µM ADPR), respectively.