Inactivation of TRPM2 Channels by Extracellular Divalent Copper

Cu(2+) 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 Cu(2+). Cu(2+) at concentrations as low as 3 µM inhibited the hTRPM2 channel completely and irreversibly upon washing or using Cu(2+) chelators, suggesting channel inactivation. The Cu(2+)-induced inactivation was similar when the channels conducted inward or outward currents, indicating the permeating ions had little effect on Cu(2+)-induced inactivation. Furthermore, Cu(2+) had no effect on singe channel conductance. Alanine substitution by site-directed mutagenesis of His995 in the pore-forming region strongly attenuated Cu(2+)-induced channel inactivation, and mutation of several other pore residues to alanine altered the kinetics of channel inactivation by Cu(2+). In addition, while introduction of the P1018L mutation is known to result in channel inactivation, exposure to Cu(2+) 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 Cu(2+). Replacement of His995 with glutamine in the hTRPM2 conferred loss of Cu(2+)-induced channel inactivation. Taken together, these results suggest that Cu(2+) 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 Cu(2+) on the human and mouse TRPM2 channels.