Visualizing structural transitions of ligand-dependent gating of the TRPM2 channel

Abstract

The calcium-permeable transient receptor potential melastatin 2 (TRPM2) channel plays a key role in redox sensation in many cell types 1-3 . Channel activation requires binding of both ADP-ribose (ADPR) 2,4-6 and Ca 2+ 7 . The recently published TRPM2 structures from Danio rerio in the ligand-free and in the ADPR/Ca 2+ -bound conditions represent the channel in closed and open states, which uncover substantial tertiary and quaternary conformational rearrangements 8 .However, it is unclear how these rearrangements occur within the tetrameric channel during channel gating. Here we report two cryo-electron microscopy structures of TRPM2 from the same species in complex with Ca 2+ alone, and with both ADPR and Ca 2+ , determined to an overall resolution of ~3.8 Å and ~4.2 Å respectively. In comparison with the published results, our studies capture TRPM2 in two-fold symmetric intermediate states, offering a glimpse of the structural transitions within the tetramer that bridge the closed and open conformations.3 MainThe transient receptor potential melastatin (TRPM) ion channel family is part of the TRP channel superfamily and comprises eight members (TRPM1 to TRPM8) that carry out diverse functions in a variety of physiological pathways 9 . TRPM2 is a calcium-permeable non-selective cation channel that is widely expressed in the nervous, immune, and endocrine systems, and plays a crucial role in warmth and redox-dependent signaling 1-3 . Studies of TRPM2-deficient mice have shown that TRPM2 channels expressed in sensory and central neurons are responsible for sensation of warm temperatures and body temperature regulation 10,11 . TRPM2 has also been found to be activated by reactive oxygen species (ROS), consequently playing a key role in Ca 2+ signaling involved in chemokine production, insulin production, and cell death under oxidative stress [12][13][14][15][16] .Extensive electrophysiological studies have shown that activation of TRPM2 by ROS is caused by an increase of intracellular ADP-ribose (ADPR), a TRPM2 agonist, and that both ADPR and Ca 2+ are required for channel activation 2,[4][5][6][7]17 . Notably, TRPM2 contains a putative enzyme domain, called the Nudix Hydrolase 9 Homology (NUDT9H) domain located at the C-terminus, which exhibits a high degree of homology to the mitochondrial ADP-ribose pyrophosphatase NUDT9 18,19 . As such, TRPM2 was initially classified as a channel-enzyme in which enzymatic activity was believed to be coupled to channel gating 2,18 . However, subsequent studies have since repudiated this mechanism, as the NUDT9H domain lacks enzymatic activity. Instead it has been suggested that the NUDT9H domain merely serves as a binding site for ADPR 20 .Recently, structures of the zebrafish Danio rerio TRPM2 were reported in the ligand-free closed state and in the ADPR/Ca 2+ -bound open state (hereafter referred to as TRPM2 DR_closed and TRPM2 DR_open respectively) 8 . This study not only revealed the location of the NUDT9H domain, but also showed that the Ca 2+ ion binds in the cavity formed...

Symmetry transitions during gating of the TRPV2 ion channel in lipid membranes

“…Hence, the channel might be utilizing C2 symmetric states as means to achieve full opening in a step-wise manner. Similar C2 symmetric states elicited by ligand binding have been observed in TRPV3 33 and TRPM2 37 channels, which opens up the possibility that C2 symmetry might be widely associated with gating in members of the TRP channel superfamily. Intriguingly, a recent cryo-EM study of the human BK channel reconstituted in liposomes showed that this channel also enters C2 symmetric states 38 , suggesting that two-fold symmetry might also play a role in the molecular mechanisms of other tetrameric ion channels.…”

Symmetry transitions during gating of the TRPV2 ion channel in lipid membranes

“…Hence, the channel might be utilizing C2 symmetric states as means to achieve full opening in a step-wise manner. Similar C2 symmetric states elicited by ligand binding have been observed in TRPV3 (Zubcevic et al, 2018b) and TRPM2 (Yin et al, 2018) channels, which opens up the possibility that C2 symmetry might be widely associated with gating in members of the TRP channel superfamily. Intriguingly, a recent cryo-EM study of the human BK channel reconstituted in liposomes showed that this channel also enters C2 symmetric states (Tonggu and Wang, 2018), suggesting that two-fold symmetry might also play a role in the molecular mechanisms of other tetrameric ion channels.…”

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

“…We favor the former, because the structure of ADPR/Ca 2+ - hs TRPM2 is nearly identical with the non-activated ADPR- hs TRPM2. Moreover, the recently published two-fold symmetric dr TRPM2 adopts a hybrid of alternating non-flipped and flipped conformation of the S4-S5 linkers in TMD (Yin et al, 2019b), the former of which represents an intermediate state after ligand binding but prior to channel opening, which is similar to the TMD conformation of in our ADPR/Ca 2+ -bound hs TRPM2 structure. This further supports that the ADPR/Ca 2+ -bound hs TRPM2 structure represents a pre-open state.…”

“…The hs TRPM2 structures share a four-layer arrangement with dr TRPM2 (Huang et al, 2018; Yin et al, 2019b), having a TMD, MHR3/4, and a ligand-sensing layer that includes the MHR1/2 and NUDT9-H domains, from top to bottom. However, their overall shapes differ, primarily because the NUDT9-H domain is positioned differently.…”