Zinc Inactivates Melastatin Transient Receptor Potential 2 Channels via the Outer Pore

Abstract

Zinc ion (Zn2+) is an endogenous allosteric modulator that regulates the activity of a wide variety of ion channels in a reversible and concentration-dependent fashion. Here we used patch-clamp recording to study the effects of Zn2+ on the TRPM2 channel. Zn2+ inhibited the human (h) TRPM2 channel currents, and the steady-state inhibition was largely not reversed upon washout and concentration-independent in the range of 30-1000 μM, suggesting that Zn2+ induces channel inactivation. Zn2+ inactivated the channels fully when they conducted inward currents, but only by half when they passed outward currents, indicating profound influence of the permeant ion on Zn2+-inactivation. Alanine substitution scanning mutagenesis of twenty Zn2+-interacting candidate residues in the outer pore region of the hTRPM2 channel showed that mutation of Lys952 in the extracellular end of the fifth transmembrane segment and Asp1002 in the large turret strongly attenuated or abolished Zn2+-inactivation, and mutation of several other residues dramatically changed the inactivation kinetics. The mouse (m) TRPM2 channels were also inactivated by Zn2+ but the kinetics were remarkably slower. Reciprocal mutation of His995 in the hTRPM2 channel and the equivalent Gln992 in the mTRPM2 channel completely swapped the kinetics, but no such opposing effects resulted from exchanging another pair of species-specific residues Arg961/Ser958. We conclude from these results that Zn2+ inactivates the TRPM2 channels and residues in the outer pore are critical determinants of the inactivation.