Effects on TRPM2 Channel Gating of Mutations in the NUDT9-H Domain

Abstract

TRPM2 is a Ca2+ permeable cation channel involved in physiological and pathophysiological processes linked to oxidative stress. TRPM2 channels are co-activated by intracellular Ca2+ and ADP ribose (ADPR). ADPR binds to the C-terminal NUDT9-H domain, which shows high sequence similarity to the mitochondrial ADPR hydrolase NUDT9. However, the mechanism by which ADPR binding/hydrolysis regulates TRPM2 channel gating is unclear. To investigate this mechanism, we designed mutations in the NUDT9-H domain expected to alter ADPR binding affinity or ADPRase activity based on a crystal structure of NUDT9. The effects of the mutations on TRPM2 channel gating were tested in inside-out patch recordings.To impair binding of the adenine base, mutations M1286A and N1345A remove side chains which in NUDT9 contact the adenine base. To facilitate binding, S1391M reconstructs such a side chain. By restoring a hydrogen bond to the 2’-OH group Y1349D might facilitate binding of the distal ribose, while H1488A might impair it by removing a hydrogen-bond to the 3’-OH group. Mutants Y1485F and R1433A, respectively, remove a hydrogen bond and a salt bridge to the β-phosphate, expectedly impairing ADPR binding. An opposite effect is expected from L1379R which recreates a salt bridge to the β-phosphate. To enhance catalysis, I1405E recreates the side chain which coordinates Mg2+ in NUDT9. Finally, we tested whether D1468 might act as the catalytic base − if so ADPRase activity should be abolished in D1468A mutant.All mutants formed functional channels. To assay for possible effects on ligand binding and closing rate, we determinted apparent affinities for ADPR and current decay rates upon ADPR removal. Neither was dramatically altered by any of the mutations. We conclude that ADPR hydrolysis is either not required or not rate limiting for channel closure.