The proposed channel-enzyme Transient Receptor Potential Melastatin 2 does not possess ADP ribose hydrolase activity

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Abstract

Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca2⁺-permeable cation channel essential for immunocyte activation, insulin secretion, and postischemic cell death. TRPM2 is activated by ADP ribose (ADPR) binding to its C-terminal cytosolic NUDT9-homology (NUDT9H) domain, homologous to the soluble mitochondrial ADPR pyrophosphatase (ADPRase) NUDT9. Reported ADPR hydrolysis classified TRPM2 as a channel-enzyme, but insolubility of isolated NUDT9H hampered further investigations. Here we developed a soluble NUDT9H model using chimeric proteins built from complementary polypeptide fragments of NUDT9H and NUDT9. When expressed in E.coli, chimeras containing up to ~90% NUDT9H sequence remained soluble and were affinity-purified. In ADPRase assays the conserved Nudix-box sequence of NUDT9 proved essential for activity (kcat~4-9s^-1), that of NUDT9H did not support catalysis. Replacing NUDT9H in full-length TRPM2 with soluble chimeras retained ADPR-dependent channel gating (K_1/2~1-5μM), confirming functionality of chimeric domains. Thus, TRPM2 is not a 'chanzyme'. Chimeras provide convenient soluble NUDT9H models for structural/biochemical studies.

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Iordan Iordanov

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-8251-5857
Csaba Mihályi

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-7536-3066
Balázs Tóth

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary

Competing interests
The authors declare that no competing interests exist.
László Csanády

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary

For correspondence
csanady.laszlo@med.semmelweis-univ.hu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-6547-5889

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols of Semmelweis University (22.1/1935/3/2011).

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