1 TRPM2 (melastatin-like transient receptor potential 2 channel) is a nonselective cation channel that is activated under conditions of oxidative stress leading to an increase in intracellular free Ca 27; 6.3570.18; 5.2970.12, respectively). The order of potency of the PARP inhibitors in these assays (SB7501394PJ344DPQ) was the same as for inhibition of isolated PARP enzyme. 4 SB750139-B, PJ34 and DPQ had no effect on inward currents elicited by intracellular ADP-ribose in tetracycline-induced TRPM2-HEK293 cells, suggesting that PARP inhibitors are not interacting directly with the channel. 5 SB750139-B, PJ34 and DPQ inhibited increases in [Ca 2 þ ] i in a rat insulinoma cell line (CRI-G1 cells) endogenously expressing TRPM2 (pIC 50 vs 100 mM H 2 O 2 : 7.6470.38; 6.6870.28; 4.7870.05, respectively). 6 These data suggest that oxidative stress causes TRPM2 channel opening in both recombinant and endogenously expressing cell systems via activation of PARP enzymes.

Eight members of the TRP-melastatin (TRPM) subfamily have been identified, whose physiological functions and distribution are poorly characterized. Although tissue expression and distribution patterns have been reported for individual TRPM channels, comparisons between individual studies are not possible because of variations in analysis techniques and tissue selection. We report here a comparative analysis of the expression patterns of all of the human TRPM channels in selected peripheral tissues and the central nervous system (CNS) using two distinct but complimentary approaches: TaqMan and SYBR Green real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). These techniques generated comparative distribution profiles and demonstrated tissue-specific co-expression of TRPM mRNA species, indicating significant potential for the formation of heteromeric channels. TRPM channels 2, 4, 5, 6, and 7 in contrast to 1, 3, and 8 are widely distributed in the CNS and periphery. The tissues demonstrating highest expression for individual family members were brain (TRPM1), brain and bone marrow (TRPM2), brain and pituitary (TRPM3), intestine and prostate (TRPM4), intestine, pancreas, and prostate (TRPM5), intestine and brain (TRPM6), heart, pituitary, bone, and adipose tissue (TRPM7), and prostate and liver (TRPM8). The data reported here will guide the elucidation of TRPM channel physiological functions.Key Words: Ion channel; TRPM; Expression; TaqMan; SYBR Green. INTRODUCTIONThe mammalian transient receptor potential (TRP) channels form a superfamily consisting of diverse nonselective cation channels that both sense and Address correspondence to Shaun McNulty, Neurology and GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Research and Development Limited, Harlow, UK. E-mail: shaun.mcnulty@ntworld.com 159 160 E. Fonfria et al. respond to changes in the cellular environment [for reviews see (1-3)]. These channels are structurally similar to the first nonmammalian TRP channels identified and characterized from Drosophila (4,5). Together with voltagegated Ca 2+ channels, they likely provide the major route of Ca 2+ entry in numerous cell types. Mammalian TRP channels possess six putative transmembrane domains that are thought to assemble in vivo as tetramers. They have a wide tissue distribution and most channels demonstrate the potential for alternative splicing. The existence of TRP heterodimers and splice variants increases significantly the potential for functional diversity. To date, the in vivo physiological functions of many TRP channels remain poorly characterized.The TRPM subfamily was named after the first member identified (Melastatin) and is also referred to as the long TRP channel family due to the large N-and C-termini regions that these channels contain. This family is composed of eight channels that possess distinct biophysical and physiological properties [for review see (6,7)]. A number of these channels (including TRPM2) are permeable to extracellular calcium on activati...

The ATP-gated receptor P2X is expressed in multiple malignant tumours including neuroblastoma, melanoma, prostate, lung and breast. P2X has a significant role in mediating diverse cell responses, which upon dysregulation are associated with tumour initiation and development. The rapid, ATP-mediated activation of P2X induces a fast-inward cation current in cells. However, prolonged ATP-mediated activation of P2X leads to formation of a pore that increases membrane permeability and eventually causes cell death. This presents a potential paradox, as the tumour microenvironment contains extracellular ATP at levels sufficient to activate the P2X pore and trigger cell death. However, P2X expression is associated with enhanced cancer cell survival, proliferation and metastatic potential. At least one distinct conformational form of P2X, termed non-pore functional P2X (nfP2X), has been described, which is not able to form a functional pore. We demonstrate for the first time in this study that exposure to a high ATP concentration, equivalent to those measured in the tumour microenvironment, drives nfP2X expression and also that nfP2X is essential for tumour cell survival. We show that monoclonal antibodies raised against a P2X amino acid sequence (200-216), whose conformation is distinct from that of wild-type (WT) P2X, bind specifically to nfP2X expressed on the surface of tumour cells. We also show that nfP2X is broadly expressed in patient-derived tumour sections from a wide range of cancers. Therefore, antibodies raised against E200 provide tools that can differentiate between forms of the P2X receptor that have a key role in cancer.

Amyloid b-peptide (Ab) is the main component of senile plaques which characterize Alzheimer's disease and may induce neuronal death through mechanisms which include oxidative stress. To date, the signalling pathways linking oxidant stress, a component of several neurodegenerative diseases, to cell death in the CNS are poorly understood. Melastatin-like transient receptor potential 2 (TRPM2) is a Ca 2+ -permeant non-selective cation channel, which responds to increases in oxidative stress levels in the cell and is activated by oxidants such as hydrogen peroxide. We demonstrate here that Ab and hydrogen peroxide both induce death in cultured rat striatal cells which express TRPM2 endogenously. Transfection with a splice variant that acts as a dominant negative blocker of TRPM2 function (TRPM2-S) inhibited both hydrogen perox-