Identification of a Novel EF-Loop in the N-terminus of TRPM2 Channel Involved in Calcium Sensitivity.

Yuhuan Luo,Xiafei Yu,Wei Yang,Jianhong Luo,Cheng Ma

doi:10.3389/fphar.2018.00581

Abstract

As an oxidative stress sensor, transient receptor potential melastatin 2 (TRPM2) channel is involved in many physiological and pathological processes including warmth sensing, ischemia injury, inflammatory diseases and diabetes. Intracellular calcium is critical for TRPM2 channel activation and the IQ-like motif in the N-terminus has been shown to be important by mediating calmodulin binding. Sequence analysis predicted two potential EF-loops in the N-terminus of TRPM2. Site-directed mutagenesis combining with functional assay showed that substitution with alanine of several residues, most of which are conserved in the typical EF-loop, including D267, D278, D288, and E298 dramatically reduced TRPM2 channel currents. By further changing the charges or side chain length of these conserved residues, our results indicate that the negative charge of D267 and the side chain length of D278 are critical for calcium-induced TRPM2 channel activation. G272I mutation also dramatically reduced the channel currents, suggesting that this site is critical for calcium-induced TRPM2 channel activation. Furthermore, D267A mutant dramatically reduced the currents induced by calcium alone compared with that by ADPR, indicating that D267 residue in D267–D278 motif is the most important site for calcium sensitivity of TRPM2. In addition, inside-out recordings showed that mutations at D267, G272, D278, and E298 had no effect on single-channel conductance. Taken together, our data indicate that D267–D278 motif in the N-terminus as a novel EF-loop is critical for calcium-induced TRPM2 channel activation.

Highlights

As a member of the mammalian TRP channel family, transient receptor potential melastatin 2 (TRPM2) channel is a calcium-permeable, non-selective cation channel, which is assembled by four subunits surrounding a central ion-permeating pore and each subunit has six transmembrane segments with its N- and C-termini in the cytoplasmic face of the cell (Perraud et al, 2001)
It is well known that adenosine diphosphate-ribose (ADPR) induced the TRPM2 channel activation through interacting with the NUDT9 homology (NUDT9-H) domain in the C-terminus of TRPM2 (Perraud et al, 2001; Kühn and Lückhoff, 2004), and previous studies have demonstrated that calcium has a synergy effect with ADPR to open the TRPM2 channel (Lange et al, 2008; Du et al, 2009)
Our results showed the current density of D267A, G272I, and D278E mutants were much less than that of WT TRPM2 induced by 50 mM Ca2+, supporting that the conserved residues in the first EF-loop is critical for calcium sensitivity of TRPM2

Summary

Introduction

As a member of the mammalian TRP channel family, transient receptor potential melastatin 2 (TRPM2) channel is a calcium-permeable, non-selective cation channel, which is assembled by four subunits surrounding a central ion-permeating pore and each subunit has six transmembrane segments with its N- and C-termini in the cytoplasmic face of the cell (Perraud et al, 2001) This channel is widely expressed in a variety of cells, including neurons (Ye et al, 2014; Ostapchenko et al, 2015; Demirdas et al, 2017), microglia (Haraguchi et al, 2012; Syed Mortadza et al, 2015; Huang et al, 2017), pancreatic β-cells (Yosida et al, 2014; Ito et al, 2017), monocytes and macrophages (Wehrhahn et al, 2010; Zou et al, 2013), endothelial cells (Hecquet et al, 2008; Mittal et al, 2017) and pericytes (Jiang et al, 2017). Many studies show that TRPM2 channel mediates ROS-induced cell death through permeating excessive calcium into cells, which results in many kinds of diseases including ischemia injury, diabetes and inflammatory diseases (Jiang et al, 2010; Syed Mortadza et al, 2015)

Methods

Results

Conclusion