Figure 5

Diminished effects of ADPR and 2-APB in the QLP variant of nvTRPM2, restored by elevated Ca²⁺. (a) ADPR requires extracellular Ca²⁺ in nvTRPM2-QLP in the presence of standard intracellular Ca²⁺ concentrations. With ADPR (0.15 mM) and Ca²⁺ (1 µM) in the pipette solution, current recording was started after extended exposure to DVF. Restoration of standard extracellular Ca²⁺ (1.2 mM) enabled normal currents that were missing without extracellular Ca²⁺. (b) ADPR becomes effective on nvTRPM2-QLP under extracellular divalent-free conditions when intracellular concentrations of Ca²⁺ are strongly increased. Stimulation was performed with ADPR (0.15 mM) and strongly elevated Ca²⁺ (100 µM) in the pipette solution. Note the decelerated inactivation. (c) 2-APB becomes effective in strongly increased concentrations of extracellular Ca²⁺ in nvTRPM2-QLP. Two stimulations were performed with 2-APB (1 mM), first in the presence of a standard extracellular Ca²⁺ (1.2 mM) and then at high Ca²⁺ (10 mM). The pipette solution contained Ca²⁺ (1 µM) but no ADPR. (d) 2-APB becomes effective in standard extracellular Ca²⁺ (1.2 mM) after pre-stimulation with ADPR. The experiment was started with exactly the same protocol as in panel a, followed by a stimulation with 2-APB (1 mM) that this time did induce currents. Note the long lag time as well as the difference in amplitudes between the two stimulations. 2-APB (1 mM) consistently evoked larger currents than ADPR (0.15 mM) alone. (e) Pre-stimulation with low concentrations of ADPR is not sufficient to enable 2-APB effects. With only 15 µM instead of 0.15 mM ADPR in the pipette, small currents were induced but subsequent application of 2-APB (1 mM) failed to evoke the same strong currents as shown in panel c. All experiments were repeated at least three times confirming the results.