The Mechanism Of Extracellular Divalent Copper Induced TRPM2Channel Inactivation

Background Cu2+is a critical trace mental element in organisms especially in mammals. Many studies have showed that Cu2+can regulate the activity of a number of ion channels and receptors. TRPM2channel is a unselective cation channel which can be activated by ADPR. Accumulative evidences have implied that TRPM2is correlated to oxidative stress in physiological process in organisms. Recently, several researches have shown that Zn2+and H+can lead to inactivation of TRPM2channels. Cu2+which works as a similar cation is proved to have the same result, but the mechanism is still unclear. Objectives To explore the influence and the molecular mechanism of TRPM2channels treated with Cu2+. Methods Using whole-cell recording to explore the influence of Cu2+on TRPM2; constructing mutants of TRPM2by molecular methods to find the mechanism of interaction between Cu2+and TRPM2channels; using outside-out patch to explore the influence of Cu2+on single channel conductance of TRPM2channels. Results In the experiences, we observed that Cu2+inactivated the human TRPM2channel even down to3μM, besides, the decay time of inactivation is in a dose-dependent way. Cu2+treatment after TRPM2channel open resulted in completely inactivation in both inward and outward current, indicating the ion conducting is not effected by Cu2+. And the same result can be got from the outside-out patch which shows no significant change by Cu2+treatment. By monitoring the change of mutants of the charge residues in pore region, we identified the His995residue is the key position for Cu2+induced human TRPM2inactivation, and several mutants strongly changed the TRPM2inactivation kinetics by Cu2+. Unlike human TRPM2, mouse TRPM2channel whose histidine is replaced by glutamine is insensitive to Cu2+. So we concluded that Cu2+inactivated human TRPM2channel instead of mouse TRPM2channel by interacting with several residues in the outer pore region, which implies that the function of Cu2+in pathological or physiological process is species dependent.