TRPM2 is not permeable to protons. (A) Inward currents of TRPM2 recorded by holding the TRPM2-transfected cell at −100 mV. No current was activated in NMDH-Cl solutions at pH_o 4.5 or 7.4, where Tyrode's solution generated large inward current. (B) In mock-transfected cells, NMDH-Cl solutions at pH_o 4.5 or 7.4 could not induce any current. (C) TRPM2 current elicited by the ramp protocol (−100 to +100 mV) in Tyrode's solution (red). NMDG-Glu at pH_o 4.5 did not induce any inward proton current (green). The pipette solution for this experiment contained NMDG-Glu to favor proton permeation. (D) Inward current of TRPM2 recorded at various external solutions obtained in C. Currents were measured at −100 mV and plotted as a function of time. (E) Effects of holding potentials on the inhibition of TRPM2 by external protons. Dose–response curves were constructed by normalizing the inward currents (−100 mV) at each pH_o to the maximal current amplitude (−100 mV) obtained at pH_o 7.4. The IC₅₀s were 5.3 ± 0.05 (n = 9) at HP = 0 mV and 5.5 ± 0.14 (n = 9; P > 0.05) at HP = −100 mV. (F) Inhibition of TRPM2 by acidic pH_o (pH_o = 4.0) could not be reversed by high pH_i. Inward currents (−100 mV) and outward currents (+100 mV) plotted against time under the indicated conditions. TRPM2 currents were elicited by voltage ramps with the CsSO₃CH₃ pipette solution containing 100 nM Ca²⁺ and 200 µM ADPR. Note that TRPM2 was completely and irreversibly blocked by external solutions at pH_o 4.0. 30 mM NH₄Cl, used to increase intracellular pH, could not reverse the effects of pH_o 4. Similar results were obtained in five separate experiments.