Ligand-induced activation of human TRPM2 requires the terminal ribose of ADPR and involves Arg1433 and Tyr1349

Abstract

TRPM2 (transient receptor potential channel, subfamily melastatin, member 2) is a Ca2+-permeable non-selective cation channel activated by the binding of adenosine 5′-diphosphoribose (ADPR) to its cytoplasmic NUDT9H domain (NUDT9 homology domain). Activation of TRPM2 by ADPR downstream of oxidative stress has been implicated in the pathogenesis of many human diseases, rendering TRPM2 an attractive novel target for pharmacological intervention. However, the structural basis underlying this activation is largely unknown. Since ADP (adenosine 5′-diphosphate) alone did not activate or antagonize the channel, we used a chemical biology approach employing synthetic analogues to focus on the role of the ADPR terminal ribose. All novel ADPR derivatives modified in the terminal ribose, including that with the seemingly minor change of methylating the anomeric-OH, abolished agonist activity at TRPM2. Antagonist activity improved as the terminal substituent increasingly resembled the natural ribose, indicating that gating by ADPR might require specific interactions between hydroxyl groups of the terminal ribose and the NUDT9H domain. By mutating amino acid residues of the NUDT9H domain, predicted by modelling and docking to interact with the terminal ribose, we demonstrate that abrogating hydrogen bonding of the amino acids Arg1433 and Tyr1349 interferes with activation of the channel by ADPR. Taken together, using the complementary experimental approaches of chemical modification of the ligand and site-directed mutagenesis of TRPM2, we demonstrate that channel activation critically depends on hydrogen bonding of Arg1433 and Tyr1349 with the terminal ribose. Our findings allow for a more rational design of novel TRPM2 antagonists that may ultimately lead to compounds of therapeutic potential.

TRPM2 channel: A novel target for alleviating ischaemia‐reperfusion, chronic cerebral hypo‐perfusion and neonatal hypoxic‐ischaemic brain damage

“…The TRPM2 channel belongs to the superfamily of transient receptor potential (TRP) channels [15][16][17][18] and is a tetrameric Ca 2+ -permeable non-selective cation channel that is gated by intracellular ADP-ribose (ADPR) and cyclic ADPR. [19][20][21][22][23][24][25] Intracellular Ca 2+ can bind to and activate the TRPM2 channels, 26 and warm temperature (≥35°C) can also induce the TRPM2 channel opening independently of, and more often in synergy with, ADPR or cyclic ADPR. 27,28 The TRPM2 channel can be potently activated after exposure to pathologically relevant concentrations of ROS, which is thought to stimulate ADPR generation via poly(ADPR) polymerase (PARP), particularly PARP-1, and poly(ADRP) glycohydrolase (PARG) in the nucleus, and also via NADase in the mitochondria.…”

Roles of NAD+ and Its Metabolites Regulated Calcium Channels in Cancer

“…has made brilliant contributions to the identification of these NAD + metabolites as these ion channels activators/regulators, which have been cited above. More importantly, based on the fact that these ion channels have been regarded as new cancer therapeutic targets, Professor Potter has designed and synthesized a series of ligands/inhibitors of these calcium channels [ 72 , 154 , 155 , 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 , 168 ], which certainly play critical roles in the development of anti-cancer drugs targeting these ion channels. In addition, some potassium and sodium channels can also be activated by NAD + and its metabolites, and there are also some calcium channels whose activity can be indirectly affected by NAD + and its metabolites.…”

Synthesis of phosphonoacetate analogues of the second messenger adenosine 5′-diphosphate ribose (ADPR)

“…32 The requirement for an ADPR mimic to bind to both sites, which possess very distinct shapes, 30 may explain why very few synthetic ADPR analogues have activated the channel. 11,15 Despite this recent advance, the structure activity relationship for hTRPM2 is not fully understood, and ADPR analogues remain essential for ligand-based drug design. The crystal structure of the macro domain of thermophilic protein Af1521 bound to ADPR was solved in high resolution (1.5Å, PDB 2BFQ).…”

“…9,10 Small changes to the structure of ADPR have demonstrated profound effects on activity and, as a result, synthetic analogues closely based on the ADPR structure have been very informative. [11][12][13] Analogues with modications in the adenosine 11,13 and terminal ribose [14][15][16][17] motifs have been prepared via coupling of a modied 5 0 -O-phosphoryl adenosine or ribose to its 5 0 -Ophosphoryl counterpart using morpholidate, diphenylphosphonate or imidazolide methods to activate one phosphate towards nucleophilic attack and thus generate the pyrophosphate. Notwithstanding their utility for studying ADPR processes using single cell techniques such as patch clamping, further studies even with many of these designed analogues are limited by the negative charge and potential biological instability of the pyrophosphate moiety.…”