Inactivation of TRPM2 channels by extracellular divalent copper.
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.
Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Zhejiang Province Key Laboratory of Neurobiology, Department of Neurobiology, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China.
Wenyue
W
Yu
Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang, Henan Province, China; School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.
Lin-Hua
LH
Jiang
Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Zhejiang Province Key Laboratory of Neurobiology, Department of Neurobiology, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China.
Yang
Y
Zheng
Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Zhejiang Province Key Laboratory of Neurobiology, Department of Neurobiology, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China.
Xupang
X
Hu
Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Zhejiang Province Key Laboratory of Neurobiology, Department of Neurobiology, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China.
Jianhong
J
Luo
Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Zhejiang Province Key Laboratory of Neurobiology, Department of Neurobiology, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China.
Wei
W
Yang
D019943:Amino Acid Substitution; D000818:Animals; D003300:Copper / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D050053:TRPM Cation Channels / Q000235:genetics / Q000378:metabolism*
D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; D003300:Copper