Abstract
Zinc ion (Zn(2+)) 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 Zn(2+) on the melastatin transient receptor potential 2 (TRPM2) channel. Zn(2+) 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 Zn(2+) induces channel inactivation. Zn(2+) 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 Zn(2+) inactivation. Alanine substitution scanning mutagenesis of 20 Zn(2+)-interacting candidate residues in the outer pore region of the hTRPM2 channel showed that mutation of Lys(952) in the extracellular end of the fifth transmembrane segment and Asp(1002) in the large turret strongly attenuated or abolished Zn(2+) inactivation, and mutation of several other residues dramatically changed the inactivation kinetics. The mouse (m) TRPM2 channels were also inactivated by Zn(2+), but the kinetics were remarkably slower. Reciprocal mutation of His(995) in the hTRPM2 channel and the equivalent Gln(992) in the mTRPM2 channel completely swapped the kinetics, but no such opposing effects resulted from exchanging another pair of species-specific residues, Arg(961)/Ser(958). We conclude from these results that Zn(2+) inactivates the TRPM2 channels and that residues in the outer pore are critical determinants of the inactivation.
Highlights
Cardiomyocytes, pancreatic -cells, epithelial cells, neutrophils, microglia, monocytes, and macrophages and as lysosomal Ca2ϩ release channels in pancreatic -cells (8, 9)
Unlike recent studies reporting substantial Zn2ϩ permeation of the TRPM3 and TRPM7 channels (26 –28) and potent reversible Zn2ϩ inhibition of the TRPM1 channel (53), here we provide evidence to show that Zn2ϩ induces an irreversible and concentration-independent inhibition or inactivation of the TRPM2 channels and that residues in the outer pore are critical molecular determinants
Effects of Extracellular Zn2ϩ on human TRPM2 (hTRPM2) Channels—We began with experiments to determine the effect of extracellular Zn2ϩ on the ADPR-induced hTRPM2 channel currents, with the hope that Zn2ϩ in mM concentrations would not cause significant inhibition so that we could test whether the channels are permeable to Zn2ϩ by patch clamp recording as shown previously for the TRPM3 and TRPM7 channels (26, 28)
Summary
Cardiomyocytes, pancreatic -cells, epithelial cells, neutrophils, microglia, monocytes, and macrophages and as lysosomal Ca2ϩ release channels in pancreatic -cells (8, 9). They open upon binding of ADP-ribose (ADPR) and structurally related molecules to the TRPM2-specific NUD9-H domain in the distal C-terminal tail (10, 11). TRPM2 channels mediate glucose-evoked Ca2ϩ influx for insulin secretion from pancreatic -cells and facilitation of insulin secretion by incretins (18). These findings raise interests in TRPM2 channels as a therapeutic target to treat inflammatory, neurodegenerative, and cardiovascular disorders including diabetes (8, 9). Unlike recent studies reporting substantial Zn2ϩ permeation of the TRPM3 and TRPM7 channels (26 –28) and potent reversible Zn2ϩ inhibition of the TRPM1 channel (53), here we provide evidence to show that Zn2ϩ induces an irreversible and concentration-independent inhibition or inactivation of the TRPM2 channels and that residues in the outer pore are critical molecular determinants
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