Ligand recognition and gating mechanism through three ligand-binding sites of human TRPM2 channel

Abstract

TRPM2 is critically involved in diverse physiological processes including core temperature sensing, apoptosis, and immune response. TRPM2’s activation by Ca2+ and ADP ribose (ADPR), an NAD+-metabolite produced under oxidative stress and neurodegenerative conditions, suggests a role in neurological disorders. We provide a central concept between triple-site ligand binding and the channel gating of human TRPM2. We show consecutive structural rearrangements and channel activation of TRPM2 induced by binding of ADPR in two indispensable locations, and the binding of Ca2+ in the transmembrane domain. The 8-Br-cADPR—an antagonist of cADPR—binds only to the MHR1/2 domain and inhibits TRPM2 by stabilizing the channel in an apo-like conformation. We conclude that MHR1/2 acts as a orthostatic ligand-binding site for TRPM2. The NUDT9-H domain binds to a second ADPR to assist channel activation in vertebrates, but not necessary in invertebrates. Our work provides insights into the gating mechanism of human TRPM2 and its pharmacology.

Molecular Regulations and Functions of the Transient Receptor Potential Channels of the Islets of Langerhans and Insulinoma Cells

“…The NUDT9 homology (NUDT9-H) domain of human TRPM2 plays crucial roles in mediating expression of the channel in the plasma membrane and in channel gating. ADPR binds to both the NUDT9-H domain and the TRPM homology regions (MHR) 1 and 2 (MHR1/2) [38]. The NUDT9-H domain of human TRPM2 binds ADPR and promotes channel opening, but does not degrade ADPR.…”

“…The NUDT9-H domain of human TRPM2 binds ADPR and promotes channel opening, but does not degrade ADPR. Channel opening also requires binding of Ca 2+ to the transmembrane domains [38]. 8-Br-cADPR binds only to the MHR1/2 domain and stabilizes the channel at the resting state.…”

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

“…Cryo-EM structures of hTRPM2 have recently improved understanding of the zebrash hTRPM2 apo and ADPR-bound states 30 importantly for hTRPM2 also in the presence of bound ligands. 31,32 However, only recently has their resolution become detailed enough to place ADPR in the binding pocket and has revealed, for the rst time, ADPR binding sites in hTRPM2 that may act synergistically. 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.…”

“…All reactions were performed under an inert atmosphere of argon unless otherwise stated. For NMR experiments; 1 H, 13 31 (1 mL). N,N 0 -dicyclohexylcarbodiimide (20 mg, 0.097 mmol) was added in one portion to the stirred mixture and the mixture was heated at 120 C for 16 h. The reaction mixture was evaporated to dryness and the compound was puried by silica-gel chromatography CH 2 Cl 2 -MeOH-0.1% Et 3 N (10-40%) to afford the title compound 7 as a white solid (18.6 mg, 54%, 1 8 Et 3 N salt).…”

“…This procedure afforded the title compound 5 as colourless glass (0.98 g, 62%) 1. H NMR (400 MHz, d 6 -DMSO) d 8.34 (s, H-8), 8.16 (s, H-2), 7.34 (s, 2H, NH 2 ), 6.19 (d, 1H, J ¼ 2.68 Hz, H-1 0 ), 5.47 (dd, 1H, J ¼ 6.0 Hz, J ¼ 2.96 Hz, H-2 0 ), 5.04 (dd, 1H, J ¼ 6.4 Hz, J ¼ 3.09 Hz, H-3 0 ), 4.39-4.35 (m, 1H, H-4 0 ), 4.31-4.27 ðm; 1H; H À 5 0 a Þ; 4.23-4.18 ðm; 1H; H-5 0 b Þ; 4.04-3.95 (m, 4H, CH 2 CH 3 ), 3.11 (d, 2H, J H-P ¼ 21.36 Hz, CH 2P), 1.54 (s, 3H, C(CH 3 ) 2 ), 1.33 (s, 3H, C(CH 3 ) 2 ), 1.18 (m, 6H, J ¼ 3.16 Hz, CH 2 CH 3 ) 31. P NMR (161 MHz, d 6 -DMSO) d 19.64 (s).…”