Direct and Indirect Effectors of the TRPM2 Cation Channel

Abstract

TRPM2 is a Ca2+ permeable cation channel which plays a role in physiological and pathophysiological processes linked to oxidative stress. TRPM2 channels are co-activated by intracellular Ca2+ and ADP ribose (ADPR). In addition, in intact cells, a large number of compounds appear to modulate TRPM2 activity. Superfusion of TRPM2-expressing cells with hydrogen-peroxide (H2O2) activates TRPM2 currents, just as intracellular dialysis of cyclic ADPR (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP). Importantly, H2O2, cADPR, and NAADP enhance ADPR-induced TRPM2 whole-cell currents. Finally, in intact cells AMP acts as a TRPM2 inhibitor. Because in whole-cell recordings the entire cellular machinary involved in nucleotide- and Ca2+-homeostasis is in place, compounds might affect TRPM2 activity either directly, by binding to the TRPM2 protein, or indirectly, by altering the local concentrations of the primary ligands ADPR and Ca2+. To identify direct modulators of TRPM2 activity, we have studied the effects of H2O2, AMP, cADPR, NAADP, and nicotinic acid adenine dinucleotide (NAAD) in inside-out patches excised from Xenopus oocytes expressing human TRPM2, by directly exposing the cytosolic faces of the patches to these compounds. H2O2 (1 mM) and enzymatically purified cADPR (10 μM) failed to activate, while AMP (200 μM) failed to inhibit TRPM2 currents. NAADP acted as a partial agonist (maximal efficacy ∼50%) while NAAD was a full agonist, but both with low affinities (K0.5=104 and 35 μM). Neither of H2O2, cADPR, and NAADP enhanced activation by ADPR. Thus, in a physiological context the above compounds do not directly affect the TRPM2 channel protein. [OTKA grant F68143]