Figure 3

Dependence on Ca²⁺ of currents induced by ADPR or 2-APB in nvTRPM2. (a) For the stimulation with ADPR, intracellular Ca²⁺ is not required. The experiment was performed in standard bath (1.2 mM Ca²⁺) but with a pipette solution in which Ca²⁺ was buffered with EGTA to below 10 nM as indicated. (b) Extracellular Ca²⁺ is not required for the ADPR-dependent stimulation, but for the current inactivation. The current recording was performed with a pipette solution containing 1 µM Ca²⁺ but in the extended absence of extracellular Ca²⁺, achieved by multiple superfusion of the cells with a divalent-free solution. Note that the inactivation takes place considerably slower than under control conditions (inset Fig. 1a), reaching about 50% of the maximal current after 40 s. (c) Intracellular Ca²⁺ is required for the stimulation with 2-APB. Under the same Ca²⁺ conditions as in panel a (no ADPR), 2-APB (1 mM) induced only miniscule currents. (d) Extracellular Ca²⁺ is required for the stimulation with 2-APB. After extended exposure to DVF, 2-APB (1 mM) was without effect but did induce characteristic currents when applied a second time in the presence of a standard extracellular Ca²⁺ (1.2 mM). As in panel b, the pipette solution contains 1 µM Ca²⁺. (e) Strongly elevated intracellular Ca²⁺ cannot substitute extracellular Ca²⁺. Same experimental conditions as in panel d, but the intracellular solution contained 100 µM Ca²⁺. All experiments were repeated at least three times confirming the results.