Similar articles

Abstract: As a crucial signaling molecule, calcium plays a critical role in many physiological and pathological processes by regulating ion channel activity. Recently, one study resolved the structure of the transient receptor potential melastatin 2 (TRPM2) channel from Nematostella vectensis (nvTRPM2). This identified a calcium-binding site in the s2–;s3 loop, while its effect on channel gating remains unclear. Here, we investigated the role of this calcium-binding site in both nvTRPM2 and human TRPM2 (hTRPM2) by mutagenesis and patch-clamp recording. Unlike hTRPM2, nvTRPM2 cannot be activated by calcium alone. Moreover, the inactivation rate of nvTRPM2 was decreased as intracellular calcium concentration was increased. In addition, our results showed that the four key residues in the calcium-binding site of s2–;s3 loop have similar effects on the gating processes of nvTRPM2 and hTRPM2. Among them, the mutations at negatively charged residues (glutamate and aspartate) substantially decreased the currents of nvTRPM2 and hTRPM2. This suggests that these sites are essential for calcium-dependent channel gating. For the charge-neutralizing residues (glutamine and asparagine) in the calcium-binding site, our data showed that glutamine mutating to alanine or glutamate did not affect the channel activity, but glutamine mutating to lysine caused loss of function. Asparagine mutating to aspartate still remained functional, while asparagine mutating to alanine or lysine led to little channel activity. These results suggest that the side chain of glutamine has a less contribution to channel gating than does asparagine. However, our data indicated that both glutamine mutating to alanine or glutamate and asparagine mutating to aspartate accelerated the channel inactivation rate, suggesting that the calcium-binding site in the s2–;s3 loop is important for calcium-dependent channel inactivation. Taken together, our results uncovered the effect of four key residues in the s2–;s3 loop of TRPM2 on the TRPM2 gating process.

S2-S3 loop中钙离子结合位点对人类及海葵来源TRPM2通道门控过程的影响研究

[1]Autzen HE, Myasnikov AG, Campbell MG, et al., 2018. Structure of the human TRPM4 ion channel in a lipid nanodisc. Science, 359(6372):228-232.

[2]Csanády L, Törőcsik B, 2009. Four Ca²⁺ ions activate TRPM2 channels by binding in deep crevices near the pore but intracellularly of the gate. J Gen Physiol, 133(2):189-203.

[3]Du JY, Xie J, Yue LX, 2009. Intracellular calcium activates TRPM2 and its alternative spliced isoforms. Proc Natl Acad Sci USA, 106(17):7239-7244.

[4]Gao GF, Wang WW, Tadagavadi RK, et al., 2014. TRPM2 mediates ischemic kidney injury and oxidant stress through RAC1. J Clin Invest, 124(11):4989-5001.

[5]Hermosura MC, Cui AM, Go RCV, et al., 2008. Altered functional properties of a TRPM2 variant in Guamanian ALS and PD. Proc Natl Acad Sci USA, 105(46):18029-18034.

[6]Huang S, Turlova E, Li FY, et al., 2017. Transient receptor potential melastatin 2 channels (TRPM2) mediate neonatal hypoxic-ischemic brain injury in mice. Exp Neurol, 296:32-40.

[7]Huang YH, Winkler PA, Sun WN, et al., 2018. Architecture of the TRPM2 channel and its activation mechanism by ADP-ribose and calcium. Nature, 562(7725):145-149.

[8]Jiang LH, Yang W, Zou J, et al., 2010. TRPM2 channel properties, functions and therapeutic potentials. Expert Opin Ther Targets, 14(9):973-988.

[9]Kheradpezhouh E, Ma LL, Morphett A, et al., 2014. TRPM2 channels mediate acetaminophen-induced liver damage. Proc Natl Acad Sci USA, 111(8):3176-3181.

[10]Kühn F, Kühn C, Lückhoff A, 2015. Functional characterisation of a TRPM2 orthologue from the sea anemone Nematostella vectensis in human cells. Sci Rep, 5:8032.

[11]Kühn F, Kühn C, Lückhoff A, 2017. Different principles of ADP-ribose-mediated activation and opposite roles of the NUDT9 homology domain in the TRPM2 orthologs of man and sea anemone. Front Physiol, 8:879.

[12]Lange I, Penner R, Fleig A, et al., 2008. Synergistic regulation of endogenous TRPM2 channels by adenine dinucleotides in primary human neutrophils. Cell Calcium, 44(6):604-615.

[13]Li X, Jiang LH, 2018. Multiple molecular mechanisms form a positive feedback loop driving amyloid β42 peptide-induced neurotoxicity via activation of the TRPM2 channel in hippocampal neurons. Cell Death Dis, 9(2):195.

[14]Luo YH, Yu XF, Ma C, et al., 2018. Identification of a novel EF-Loop in the N-terminus of TRPM2 channel involved in calcium sensitivity. Front Pharmacol, 9:581.

[15]Miller BA, Hoffman NE, Merali S, et al., 2014. TRPM2 channels protect against cardiac ischemia-reperfusion injury: role of mitochondria. J Biol Chem, 289(11):7615-7629.

[16]Nagamine K, Kudoh J, Minoshima S, et al., 1998. Molecular cloning of a novel putative Ca²⁺ channel protein (TRPC7) highly expressed in brain. Genomics, 54(1):124-131.

[17]Perraud AL, Fleig A, Dunn CA, et al., 2001. ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology. Nature, 411(6837):595-599.

[18]Tóth B, Csanády L, 2010. Identification of direct and indirect effectors of the transient receptor potential melastatin 2 (TRPM2) cation channel. J Biol Chem, 285(39):30091-30102.

[19]Wang LF, Fu TM, Zhou YM, et al., 2018. Structures and gating mechanism of human TRPM2. Science, 362(6421):eaav4809.

[20]Yang W, Zou J, Xia R, et al., 2010. State-dependent inhibition of TRPM2 channel by acidic pH. J Biol Chem, 285(40):30411-30418.

[21]Yang W, Manna PT, Zou J, et al., 2011. Zinc inactivates melastatin transient receptor potential 2 channels via the outer pore. J Biol Chem, 286(27):23789-23798.

[22]Yonezawa R, Yamamoto S, Takenaka M, et al., 2016. TRPM2 channels in alveolar epithelial cells mediate bleomycin-induced lung inflammation. Free Radic Biol Med, 90: 101-113.

[23]Yu PL, Xue XW, Zhang JM, et al., 2017. Identification of the ADPR binding pocket in the NUDT9 homology domain of TRPM2. J Gen Physiol, 149(2):219-235.

[24]Yu WY, Jiang LH, Zheng Y, et al., 2014. Inactivation of TRPM2 channels by extracellular divalent copper. PLoS ONE, 9(11):e112071.

[25]Zhang Z, Tóth B, Szollosi A, et al., 2018. Structure of a TRPM2 channel in complex with Ca²⁺ explains unique gating regulation. Elife, 7:e36409.