The Transient Receptor Potential Melastatin 2 (TRPM2) protein is a recently identified transmembrane cation channel permeable to Ca2+. It is abundantly expressed in the brain, pancreas, and in phagocytic cells. Ca2+ influx through the TRPM2 pore is a key step in immunocyte activation, insulin secretion, and post-ischemic neuronal cell death. Thus, TRPM2 is an emerging target in the treatment of multiple diseases, including neurological and immunological conditions and diabetes. While under certain conditions inhibition of TRPM2 activity would seem beneficial (e.g., stroke, myocardial infarction, Alzheimer’s disease, chronic inflammatory diseases, hyperinsulinism), in other cases hopes for a clinical benefit are set on TRPM2 stimulation (e.g., diabetes, and diseases associated with TRPM2 loss-of-function mutations like Amyotrophic Lateral Sclerosis, Parkinsonism Dementia, and certain forms of bipolar disorder). Understanding TRPM2 structure and mechanism will be vital for designing drugs that specifically target this protein.
TRPM2 channels are co-activated by intracellular ADP-ribose (ADPR) and Ca2+: the channels open only in the combined presence of both ligands. In recent work we have clarified the mechanism of Ca2+-activation and the location of the activating sites. In the presence of ADPR, activation by intracellular Ca2+ follows the Monod-Wyman-Changeux mechanism; binding of each of four Ca2+ ions incrementally increases the open-closed equilibrium constant ~33-fold. The Ca2+ binding sites are located intracellularly from the gate, but in a shielded crevice near the pore entrance (Fig. 3; Ref. 3). Because of the positive feedback by Ca2+ entering through the pore, a single brief intracellular Ca2+ signal seems sufficient to trigger prolonged, self-sustained TRPM2 activity in intact cells provided that ADPR is available. Thus, while Ca2+ is the trigger, ADPR controls the timing of TRPM2 channel activity in living cells.
ADPR is produced under conditions of oxidative stress, such as the respiratory burst in immune cells or ischemia in the brain. ADPR binds to the carboxy-terminal NUDT9-H domain of the channel which functions as an enzyme that cleaves ADPR (Fig. 4). This enzymatic activity is rather surprising, since ion channels are passive devices which facilitate the flux of ions down their electrochemical gradients and therefore, unlike active transporters, do not require energy input to operate their gates. Thus, just as CFTR, TRPM2 ranks among the very few known ion channels whose gating is linked to an enzymatic activity (“chanzymes”).
What is the best strategy for manipulating TRPM2 activity? Because TRP-family channels are involved in diverse physiological processes, any useful TRPM2 agonists/antagonists will need to be highly selective. This singles out the NUDT9-H domain, the only protein segment unique to TRPM2, as the most attractive pharmacological target. And yet, at the moment, our understanding of the role of this chanzyme domain in TRPM2 gating is extremely limited. Thus, the goal of our research is to understand the mechanism by which ADPR binding to the NUDT9-H domain drives TRPM2 gating. Specifically, we would like to identify how strictly enzymatic activity and gating are coupled, which step of the catalytic cycle is linked to opening and closure of the ion pore, and which are the key amino acid residues responsible for ADPR binding/hydrolysis. Using this information, we hope to be able to design ADPR analogs that specifically activate or inhibit TRPM2.
Fig. 3. TRPM2 regulation by Ca2+.
Left, Upon removal of internal Ca2+TRPM2 channels close fast if extracellular [Ca2+] is low, but ~20-fold slower if extracellular [Ca2+] is high.
Right, Cartoon explanation of the effect of extracellular Ca2+. Top: Without extracellular Ca2+, the activating sites rapidly loose Ca2+ and quickly close.
Bottom: In high extracellular Ca2+, the activating sites, located in a deep vestibule of the channel, remain liganded longer due to Ca2+ entry through the open pore.
Fig. 4. TRPM2 membrane topology and enzymatic function.
TRPM channels are homotetramers with a membrane topology resembling that of voltage-gated cation channels. TRPM2 is activated by binding of ADPR to a unique C-terminal domain called NUDT9-H for homology with the mitochondrial enzyme NUDT9 (see structure). The NUDT9-H domain was shown not only to bind, but also to hydrolyze ADPR into AMP and ribose-5-phosphate (Perraud et al. 2001. Nature 411:595-99).