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Function and modulation of TRPM2 channels

Zou, Jie (2013) Function and modulation of TRPM2 channels. PhD thesis, University of Leeds.

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Abstract

Melastatin-related transient receptor potential 2 (TRPM2) channel is a Ca2+-permeable cation channel that is gated by ADP-ribose (ADPR) and also activated by reactive oxygen species (ROS) such as H2O2. TRPM2 channel are shown to be critically involved in several physiological and pathological cell processes. Previous studies have reported inhibition of the human TRPM2 channel by extracellular acidic pH. However, the underlying mechanism is not fully understood. In the present study, I performed patch-clamp recordings to examine the effect of extracellular acidic pH on ADPR-induced currents in HEK293 cells heterogeneously expressing human TRPM2 (hTRPM2) or mouse TRPM2 (mTRPM2) channels. The results showed that the inhibition was substantially reversible upon brief exposure to acidic pH but became irreversible after prolonged exposure, supporting the mechanism in which protons bind to and inhibit the open TRPM2 channel and the proton-binding induces further conformational changes leading to channel inactivation. Furthermore, the mTRPM2 channel exhibited a lower sensitivity to, and slower kinetics of, inhibition, than the hTRPM2 channel. A residue in the pore region (His-995 in hTRPM2 and Gln-992 in mTRPM2) had a crucial role in determining such species differences. The pharmacology of the TRPM2 channel is poor, with no specific inhibitor. Here, I examined the effects of 48 hit compounds identified from screening chemical libraries on hTRPM2 channels expressed in HEK293 cells. Four compounds inhibited H2O2-induced Ca2+-response with a micromolar to submicromolar potency and abolished ADPR-induced currents at 10 μM, indicating that they act as TRPM2 channel inhibitors. The TRPM2 channel was reported to be functionally expressed in macrophage cells, but its role in mediating ROS-induced Ca2+ signalling and cell death is largely unclear. This study examined the contribution and mechanism of the TRPM2 channel in H2O2-induced Ca2+-responses and cell death in RAW264.7 and differentiated THP-1 macrophage cells and peritoneal macrophage cells isolated from TRPM2+/+ and TRPM2-/- mice. The results showed that TRPM2 channels operated as cell surface Ca2+-permeable channels and constituted the principal Ca2+ signalling mechanism, but played a limited role in cell death. In summary, the results from my study provided useful information to advance the understanding of the pharmacology and functional roles of the TRPM2 channels.

Item Type: Thesis (PhD)
ISBN: 978-0-85731-642-4
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds)
Identification Number/EthosID: uk.bl.ethos.605310
Depositing User: Repository Administrator
Date Deposited: 07 May 2014 09:47
Last Modified: 25 Jul 2018 09:49
URI: http://etheses.whiterose.ac.uk/id/eprint/5902

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