Figure 1

Effects on activation and inactivation of 2-APB on wild-type nvTRPM2 in patch-clamp experiments. (a) 2-APB does not inhibit ADPR-induced currents. 2-APB (0.1 mM) was added to the standard bath containing 1.2 mM Ca²⁺ just before reaching whole-cell configuration (w.c.). ADPR (0.15 mM) was infused into the cell through the patch pipette together with 1 µM Ca²⁺. As in the control experiment (inset), an immediate current activation was induced by ADPR, followed by a rapid inactivation to background current levels. Substitution of external Na⁺ with the impermeable cation NMDG blocks the remaining currents. (b) 2-APB inhibits current inactivation. Same as in panel a, but the compound (0.1 mM) was pre-incubated for 5 min prior to the current recordings. A plateau-like current was induced by ADPR that was later blocked by NMDG. (c) In the absence of ADPR, 2-APB activates nvTRPM2 only at a high concentration. 2-APB was first applied at a concentration of 0.5 mM and then at 1 mM. Only the highest concentration induced a non-inactivating current that was blocked by NMDG. Statistics of these experiments are shown in the inset. Asterisks indicates significant differences (***P < 0.001; Student’s t-test, n = 4–8). Error bars are s.e. (d) Current activation by 2-APB occurs in a delayed manner but is rapidly reversed after wash-out. Note the delay until the current increases with fast kinetics, resulting in a fairly constant level, whereas removal of 2-APB resulted in an almost immediate abolition of the current. The inset shows voltage ramps taken during various times of the experiment, as indicated. (e) ADPR-induced currents do not inactivate in the presence of 2-APB and the inhibition of current inactivation is rapidly reversed after wash-out. Note that the current decline that results after removal of 2-APB occurs almost immediately and with the kinetics characteristic for ADPR stimulation. (f) 2-APB acts from the outside but not from the inside. 2-APB was present in the pipette solution (1 mM) but the typical currents were induced only after additional application to the bath. All experiments were repeated at least three times confirming the results.