Enzyme activity and selectivity filter stability of ancient TRPM2 channels were simultaneously lost in early vertebrates

Iordanov, I; Tóth, Balázs; Szöllősi, András; Csanády, László

doi:10.7554/elife.44556

Cited by 23 publications

(74 citation statements)

References 40 publications

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“…cDNA was linearized with NheI, transcribed in vitro using T7 polymerase (mMessage mMachine T7 Kit; Thermo Fisher), and cRNA stored at −80°C. For bacterial expression of nvNUDT9-H, the DNA sequence encoding nvTRPM2 residues 1271–1551, with added C-terminal Twin-Strep-tag (General Biosystems), was incorporated into the pJ411 vector as described previously ( Iordanov et al, 2019 ).…”

Section: Methodsmentioning

confidence: 99%

“…Pore opening is triggered by simultaneous binding of cytosolic ADP ribose (ADPR; Perraud et al, 2001 ; Sano et al, 2001 ) and Ca 2+ ( McHugh et al, 2003 ; Csanády and Törocsik, 2009 ), but also requires the presence of phosphatydyl-inositol-4-5-bisphosphate (PIP 2 ) in the inner membrane leaflet ( Tóth and Csanády, 2012 ). These basic functional features of TRPM2 channels have remained unaltered since the first appearance of ancient TRPM2 channels in choanoflagellates ( Iordanov et al, 2019 ). The TRPM2 C-terminal region comprises an ∼270 amino acid domain termed NUDT9-H for its homology to the soluble mitochondrial enzyme NUDT9, a Nudix-family ADPR pyrophosphatase that hydrolyses ADPR into AMP and ribose-5-phosphate (r-5-P; Perraud et al, 2001 ).…”

Section: Introductionmentioning

confidence: 99%

“…The NUDT9-H domain of human TRPM2 (hsTRPM2) is catalytically inactive ( Iordanov et al, 2016 ), but TRPM2 channels from two invertebrate species, the choanoflagellate Salpingoeca rosetta (srTRPM2) and the cnidarian Nematostella vectensis (nvTRPM2), were shown to be active chanzymes that cleave ADPR. Such functional dichotomy is explained by the presence of an intact “Nudix-box” sequence motif in the NUDT9-H domains of invertebrate TRPM2 channels, but by the appearance of mutations in key Nudix-box positions in vertebrates ( Kühn et al, 2016 ; Iordanov et al, 2019 ). Because at the same evolutionary time pore mutations that cause irreversible inactivation also appeared, it was suggested that for invertebrate channels ligand cleavage by the NUDT9-H domain served as an inactivation mechanism which was replaced by pore inactivation in vertebrates ( Iordanov et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

“…Such functional dichotomy is explained by the presence of an intact “Nudix-box” sequence motif in the NUDT9-H domains of invertebrate TRPM2 channels, but by the appearance of mutations in key Nudix-box positions in vertebrates ( Kühn et al, 2016 ; Iordanov et al, 2019 ). Because at the same evolutionary time pore mutations that cause irreversible inactivation also appeared, it was suggested that for invertebrate channels ligand cleavage by the NUDT9-H domain served as an inactivation mechanism which was replaced by pore inactivation in vertebrates ( Iordanov et al, 2019 ). Surprisingly, nvTRPM2 channels were shown to remain activatable by ADPR following deletion of the nvNUDT9-H domain, suggesting the presence of a second ADPR-binding site outside this domain ( Kühn et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

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Selective profiling of N- and C-terminal nucleotide-binding sites in a TRPM2 channel

Tóth

Iordanov

Csanády

2020

Journal of General Physiology

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Transient receptor potential melastatin 2 (TRPM2) is a homotetrameric Ca2+-permeable cation channel important for the immune response, body temperature regulation, and insulin secretion, and is activated by cytosolic Ca2+ and ADP ribose (ADPR). ADPR binds to two distinct locations, formed by large N- and C-terminal cytosolic domains, respectively, of the channel protein. In invertebrate TRPM2 channels, the C-terminal site is not required for channel activity but acts as an active ADPR phosphohydrolase that cleaves the activating ligand. In vertebrate TRPM2 channels, the C-terminal site is catalytically inactive but cooperates with the N-terminal site in channel activation. The precise functional contributions to channel gating and the nucleotide selectivities of the two sites in various species have not yet been deciphered. For TRPM2 of the sea anemone Nematostella vectensis (nvTRPM2), catalytic activity is solely attributable to the C-terminal site. Here, we show that nvTRPM2 channel gating properties remain unaltered upon deletion of the C-terminal domain, indicating that the N-terminal site is single-handedly responsible for channel gating. Exploiting such functional independence of the N- and C-terminal sites, we selectively measure their affinity profiles for a series of ADPR analogues, as reflected by apparent affinities for channel activation and catalysis, respectively. Using site-directed mutagenesis, we confirm that the same N-terminal site observed in vertebrate TRPM2 channels was already present in ancient cnidarians. Finally, by characterizing the functional effects of six amino acid side chain truncations in the N-terminal site, we provide first insights into the mechanistic contributions of those side chains to TRPM2 channel gating.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Selective profiling of N- and C-terminal nucleotide-binding sites in a TRPM2 channel

Tóth

Iordanov

Csanády

2020

Journal of General Physiology

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“…The C-termini of TRPM2, TRPM6, and TRPM7 channels possess enzymatic domains and have been named "chanzymes" [136][137][138][139]. Similar to TRPC4/C5 channels, TRPM channels possess disulfide bonds at their extracellular pore that stabilize the pore domain, but these disulfide-bonds form between cysteines at the pore loop and the pore helix S6 [119].…”

Section: Trpc and Trpm Channelsmentioning

confidence: 99%

TRP ion channels: Proteins with conformational flexibility

et al. 2019

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Ion channels display conformational changes in response to binding of their agonists and antagonists. The study of the relationships between the structure and the function of these proteins has witnessed considerable advances in the last two decades using a combination of techniques, which include electrophysiology, optical approaches (i.e. patch clamp fluorometry, incorporation of non-canonic amino acids, etc.), molecular biology (mutations in different regions of ion channels to determine their role in function) and those that have permitted the resolution of their structures in detail (X-ray crystallography and cryo-electron microscopy). The possibility of making correlations among structural components and functional traits in ion channels has allowed for more refined conclusions on how these proteins work at the molecular level. With the cloning and description of the family of Transient Receptor Potential (TRP) channels, our understanding of several sensory-related processes has also greatly moved forward. The response of these proteins to several agonists, their regulation by signaling pathways as well as by protein-protein and lipid-protein interactions and, in some cases, their biophysical characteristics have been studied thoroughly and, recently, with the resolution of their structures, the field has experienced a new boom. This review article focuses on the conformational changes in the pores, concentrating on some members of the TRP family of ion channels (TRPV and TRPA subfamilies) that result in changes in their single-channel conductances, a phenomenon that may lead to fine-tuning the electrical response to a given agonist in a cell.

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