Putative chanzyme activity of TRPM2 cation channel is unrelated to pore gating.
Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel expressed in immune cells of phagocytic lineage, pancreatic β cells, and brain neurons and is activated under oxidative stress. TRPM2 activity is required for immune cell activation and insulin secretion and is responsible for postischemic neuronal cell death. TRPM2 is opened by binding of ADP ribose (ADPR) to its C-terminal cytosolic nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain, which, when expressed in isolation, cleaves ADPR into AMP and ribose-5-phosphate. A suggested coupling of this enzymatic activity to channel gating implied a potentially irreversible gating cycle, which is a unique feature of a small group of channel enzymes known to date. The significance of such a coupling lies in the conceptually distinct pharmacologic strategies for modulating the open probability of channels obeying equilibrium versus nonequilibrium gating mechanisms. Here we examine the potential coupling of TRPM2 enzymatic activity to pore gating. Mutation of several residues proposed to enhance or eliminate NUDT9-H catalytic activity all failed to affect channel gating kinetics. An ADPR analog, α-β-methylene-ADPR (AMPCPR), was shown to be entirely resistant to hydrolysis by NUDT9, but nevertheless supported TRPM2 channel gating, albeit with reduced apparent affinity. The rate of channel deactivation was not slowed but, rather, accelerated in AMPCPR. These findings, as well as detailed analyses of steady-state gating kinetics of single channels recorded in the presence of a range of concentrations of ADPR or AMPCPR, identify TRPM2 as a simple ligand-gated channel that obeys an equilibrium gating mechanism uncoupled from its enzymatic activity.
Department of Medical Biochemistry and Magyar Tudományos Akadémia - Semmelweis Egyetem Lendület (MTA-SE) Ion Channel Research Group, Semmelweis University, Budapest H-1094, Hungary.
Balázs
B
Tóth
Department of Medical Biochemistry and Magyar Tudományos Akadémia - Semmelweis Egyetem Lendület (MTA-SE) Ion Channel Research Group, Semmelweis University, Budapest H-1094, Hungary.
Iordan
I
Iordanov
Department of Medical Biochemistry and Magyar Tudományos Akadémia - Semmelweis Egyetem Lendület (MTA-SE) Ion Channel Research Group, Semmelweis University, Budapest H-1094, Hungary csanady.laszlo@med.semmelweis-univ.hu.
László
L
Csanády
D000818:Animals; D006868:Hydrolysis; D015640:Ion Channel Gating; D008024:Ligands; D050053:TRPM Cation Channels / Q000502:physiology*; D014982:Xenopus laevis
D008024:Ligands; D050053:TRPM Cation Channels
ADP ribose analog; Nudix motif; channel enzyme; ligand-gated channel; nonequilibrium