A melastatin-like transient receptor potential 2 (TRPM2) channel is activated in concert with Ca(2+) by intracellular adenosine diphosphoribose (ADPR) binding to the channel's enzyme Nudix domain. Channel activity is also seen with nicotinamide dinucleotide (NAD(+)) and hydrogen peroxide (H(2)O(2)) although the mechanisms remain unknown. Hence, we tested the effects of ADPR, NAD(+) and H(2)O(2) on the activation of TRPM2 currents in transfected Chinese hamster ovary (CHO) cells. The CHO cells were transfected with cDNA coding for TRPM2. The intracellular solution used EDTA (10 mM) as a chelator for Ca(2+) and heavy metal ions. Moreover, we balanced the intracellular Ca(2+) concentration at 1 mu M. H(2)O(2) (10 mM) in the bath chamber was extracellularly added although ADPR (0.3 mM) and NAD(+) (1 mM) in pipette solution were intracellularly added. Using these conditions, the channel currents were evoked by the three stimulators. The time course of ADPR, NAD(+) and H(2)O(2) effects was characterized by a delay of 0.6, 3.0 min and 2-5 min, respectively and a slow current induction reached a clear plateau with ADPR and NAD(+) although H(2)O(2) currents continued to gain in amplitude over at least 15 min and it did not reach a clear plateau in many experiments. Furthermore, H(2)O(2)-induced a single-channel conductance in the current study; the first time that this has been resolved in CHO. The conductance of ADPR and H(2)O(2) was 48.80 pS and 39.14 pS, respectively and the cells seem to be separately activated by ADPR and H(2)O(2). In conclusion, we observed further support for a calcium influx pathway regulated separately by oxidative stress and ADPR in TRPM2 channels in transfected cells. A second novel result of the present study was that the TRPM2 channels were constitutionally activated by H(2)O(2).