Sites of the NUDT9-H domain critical for ADP-ribose activation of the cation channel TRPM2.
TRPM2 is a cation channel unique within the transient receptor potential family because of its gating by ADP-ribose (ADPR). ADPR gating is enabled by a cytosolic C-terminal Nudix box sequence motif embedded into a region homologous to the NUDT9 ADPR pyrophosphatase. A recently discovered splice variant of TRPM2 (TRPM2-DeltaC) lacks 34 amino acid residues in the NUDT9 domain and is insensitive to ADPR. To analyze in detail which parts of the deleted sequence (DeltaC-stretch) are critical for ADPR gating, we tested mutants that lacked 19, 25, and 29 amino acid residues in the N-terminal part or had amino acid residues substituted in the remaining C-terminal part of the DeltaC-stretch. All of these mutants displayed typical ADPR-induced currents. However, the deletion or substitution of the amino acid residue Asn-1326 immediately downstream of the DeltaC-stretch abrogated ADPR gating. We furthermore analyzed the mutation I1405E/L1406F in the Nudix box of TRPM2, because a considerably decreased AD-PRase activity of the TRPM2 NUDT9-H protein in comparison to the NUDT9 pyrophosphatase has been attributed to the reverse exchange EF --> IL. The I1405E/L1406F variant of TRPM2 failed to respond to ADPR even at concentrations up to 10 mM. We concluded that the DeltaC-stretch contains no individual amino acid residues essential for ADPR gating but may act as a spacer segment stabilizing a conformation necessary for the essential residue Asn-1326 to interact with other channel regions. Enhancing the enzymatic activity of the Nudix box abolishes the ADPR gating of TRPM2, pointing to the requirement of prolonged binding rather than degradation.
Kühn
Frank J P
FJ
Institute of Physiology, Medical Faculty, RWTH Aachen, Pauwelsstr 30, D-52057 Aachen, Germany.
Lückhoff
Andreas
A
D000246:Adenosine Diphosphate Ribose / Q000737:chemistry*; D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D016466:CHO Cells; D002412:Cations; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D017353:Gene Deletion; D007473:Ion Channels / Q000737:chemistry* / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000737:chemistry*; D050053:TRPM Cation Channels; D014162:Transfection
D000596:Amino Acids; D002412:Cations; D017931:DNA Primers; D007473:Ion Channels; D008565:Membrane Proteins; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; D000246:Adenosine Diphosphate Ribose; D001216:Asparagine; C429463:NUDT9; D011755:Pyrophosphatases