Inactivation of TRPM2 channels by extracellular divalent copper.

Wenyue Yu,Yang Zheng,Xupang Hu,Lin-Hua Jiang,Wei Yang,Jianhong Luo,Shang-Zhong Xu

doi:10.1371/journal.pone.0112071

Wenyue Yu, Yang Zheng + Show 5 more

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https://doi.org/10.1371/journal.pone.0112071

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Abstract

Cu2+ is an essential metal ion that plays a critical role in the regulation of a number of ion channels and receptors in addition to acting as a cofactor in a variety of enzymes. Here, we showed that human melastatin transient receptor potential 2 (hTRPM2) channel is sensitive to inhibition by extracellular Cu2+. Cu2+ at concentrations as low as 3 µM inhibited the hTRPM2 channel completely and irreversibly upon washing or using Cu2+ chelators, suggesting channel inactivation. The Cu2+-induced inactivation was similar when the channels conducted inward or outward currents, indicating the permeating ions had little effect on Cu2+-induced inactivation. Furthermore, Cu2+ had no effect on singe channel conductance. Alanine substitution by site-directed mutagenesis of His995 in the pore-forming region strongly attenuated Cu2+-induced channel inactivation, and mutation of several other pore residues to alanine altered the kinetics of channel inactivation by Cu2+. In addition, while introduction of the P1018L mutation is known to result in channel inactivation, exposure to Cu2+ accelerated the inactivation of this mutant channel. In contrast with the hTRPM2, the mouse TRPM2 (mTRPM2) channel, which contains glutamine at the position equivalent to His995, was insensitive to Cu2+. Replacement of His995 with glutamine in the hTRPM2 conferred loss of Cu2+-induced channel inactivation. Taken together, these results suggest that Cu2+ inactivates the hTRPM2 channel by interacting with the outer pore region. Our results also indicate that the amino acid residue difference in this region gives rise to species-dependent effect by Cu2+ on the human and mouse TRPM2 channels.

Highlights

The TRPM2 channel belongs to the melastatin subfamily of the mammalian transient receptor potential (TRP) channels, which share several conserved domains with other TRPM channels, such as the TRPM homology domains (MHD domains) in the Nterminus and the TRP box and coiled-coil domain in the Cterminus [1,2,3,4]
While the hTRPM2 channels were completely inhibited by Cu2+ independently of concentrations from 3 mM to 1 mM, the time required for 90% inhibition (t90%) was concentration-dependent from 98.4616.7 s (n = 4) at 3 mM to 7.161.5 s (n = 5) at 1 mM (Fig. 1B)
We found that extracellular Cu2+ inactivates the human but not the mouse TRPM2 channel, and revealed a striking species-dependent effect

Summary

Introduction

The TRPM2 channel belongs to the melastatin subfamily of the mammalian transient receptor potential (TRP) channels, which share several conserved domains with other TRPM channels, such as the TRPM homology domains (MHD domains) in the Nterminus and the TRP box and coiled-coil domain in the Cterminus [1,2,3,4]. Previous studies showed that the TRPM2 channel can undergo rapid inactivation upon exposure to extracellular proton and Zn2+ that interact selective residues in the pore region [9,16,17]. Mutation of the residues in the pore region can strongly alter the channel inactivation. Cu2+ is the third abundant trace metal in the human body, and plays a critical role in a variety of physiological and pathological conditions. Several studies suggest Cu2+ and Zn2+ regulate cell functions via altering the activity of a variety of ion channels [26,27]. Elucidating the mechanisms regulating ion channels by Cu2+ is critical for a better understanding of its physiological and pathological roles in humans.

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