TRPM2, a member of the melastin related TRP (transient receptor potential) channel family, is widely and most abundantely expressed in the brain (Kraft et al. 2003) but is also found in pancreatic beta cell-lines (Herson et al. 1999). TRPM2 forms a Ca²⁺-permeable, non-selective, cation channel activated by species generated by oxidant stress, including intracellular ADP-ribose, hydrogen peroxide, and β-NAD⁺.To date no potent antagonists of this channel have been described.HEK293 cells expressing tetracycline-inducible Flag-tagged TRPM2 (TRPM2-HEK293 cells) and CRI-G1 rat insulinoma cells were grown under standard tissue culture conditions. TRPM2 expression was induced by incubating cells for 24h with 1µg/ml tetracycline. All experiments were performed at room temperature using the whole cell variant of the patch clamp technique. Data are presented as mean ± S.E.M.When 100µM ADP-ribose was included in the patch pipette solution TRPM2-HEK293 exhibited a large inward current at −70 mV. This current was not present in parental HEK-293 cells and, characteristically of TRPM2, could be eliminated by removal of extracelluar Ca²⁺.Extracellular application of FFA (50-1000 µM) produced complete block of this TRPM2-mediated current. The rate of development of this block was dependent on FFA concentration (time to half-maximal block (t_1/2) 91.6±1.6s (50µM, n=3), 40.4±4.1s (100µM, n=7), 13.1±2.3s (200µM, n=4), 2.6±0.4s (500µM, n=3), and 1.5±0.5s (1mM, n=3), and appeared voltage-independent. Unlike the current inhibition produced by removal of extracellular Ca²⁺, the majority of FFA-mediated block was irreversible. The kinetics of antagonism by 100µM FFA were pH-dependent (e.g. pH 6 t_1/2=3.8±0.4s; pH 7.4 t_1/2=40.4±4.1s, n=7; pH8 t_1/2=135.9±27.4s, n=3). Furthermore antagonism seemed to require the channel to be activated. In corresponding experiments performed on CRI-G1 cells, FFA (50-1000 µM) produced a similar antagonism of ADP-ribose induced currents. The time to half maximal block was somewhat faster than for HEK293-TRPM2 cells (t_1/2=21.8±1.0s (50µM, n=4), 14.6±3.8s (100µM, n=5), 5.3±1.9s (200µM, n=4), 3.9±0.6s (500µM, n=4), 1.5±0.2s (1 mM, n=3), and could be speeded further by lowering the external pH to 6.0 (t_1/2=3.8±0.6s (n=4), 100 µM FFA). Unlike TRPM2-HEK293 cells the majority of the block observed in CRI-G1 cells was readily reversed on FFA removal.