Flufenamic acid is a pH-dependent antagonist of TRPM2 channels
Introduction
In recent years, molecular cloning has identified greater than 20 members of the mammalian transient receptor potential (TRP) family. These are subdivided into three major subgroups, namely, the vanilloid receptor family (TRPVs), the short TRP channels (TRPCs) and the melastatin-related (or long) TRP channels (TRPMs) (Clapham et al., 2003). We have become particularly interested in one member of the latter family, known as TRPM2, for a number of reasons including a potential role in cell death resulting from oxidative stress. For an extensive review of the properties of TRPM2, see Perraud et al. (2003).
TRPM2 is reported to be most abundantly expressed in the brain, within which it exhibits a widespread expression pattern (Smith et al., 2003, Kraft et al., 2004). In addition, TRPM2 is also expressed by neutrophil granulocytes (Heiner et al., 2003) and insulinoma cell lines such as the CRI-G1 cells (Herson et al., 1999, Inamura et al., 2003). Heterologous expression of TRPM2 yields a voltage-independent, Ca2+-permeable, non-selective cation conductance (NSCC). This conductance can be activated by molecular species generated under conditions of oxidant stress, for example, hydrogen peroxide and β-NAD+. In addition, TRPM2 is potently activated by intracellular ADP-ribose a molecule closely related to β-NAD+ (Perraud et al., 2001, Inamura et al., 2003). ADP-ribose levels may additionally be modulated by cellular redox status via the regulation of enzyme pathways that synthesise and metabolise NADH, NAD+, ADP-ribose and its cyclical analogue cyclic ADP-ribose (see Wilson et al., 2001). Similar H2O2 and ADP-ribose activated currents can be observed in CRI-G1 and other TRPM2 expressing cells.
Like many of the TRP channels, with the exception of TRPV1, the pharmacology of TRPM2 is poorly understood. As described above, ADP-ribose and β-NAD+ activate TRPM2 through binding to an intracellular site. Furthermore, activity of TRPM2 depends strictly on both intra- and extracellular Ca2+ (McHugh et al., 2003), with Ca2+ being permissive for gating in both cases. Indeed, without the convenience of known extracellular agonists (or voltage-dependent gating), the most commonly used experimental method to switch between activated and deactivated TRPM2 is to greatly reduce the extracellular Ca2+ concentration, having first activated the channel with intracellular ADP-ribose (see Fig. 1A for an example). Although it has been shown that a hydrogen peroxide-mediated depolarisation of striatal neurones, which might be due to activation of TRPM2, can be blocked by the free radical scavengers DMTU (Smith et al., 2003), no antagonists acting directly on TRPM2 have been identified to date.
The arylaminobenzoate flufenamic acid (N-[3-(trifluoromethyl)-phenyl]anthranilic acid, FFA) is a member of the pharmacological family of fenamates. These analgesic molecules are non-steroidal anti-inflammatory agents that are capable of producing anti-inflammatory effects in the central nervous system and elsewhere (Chen et al., 1998). Additionally, fenamates, including FFA, produce inhibition of a variety of ion channel responses in a range of tissues. These actions are capable of encompassing both anion and cation channels. Examples of actions at the former channel class include inhibition of Ca2+-activated chloride currents (Kim et al., 2003) and interactions with GABAA receptors in the brain (Maksay et al., 1998, Sinkkonen et al., 2003). FFA-mediated inhibition of cation channels includes block of voltage-gated Na+ and K+ channels (Lee et al., 1996, Lee and Wang, 1999, Lee et al., 2003). Furthermore, FFA blocks a range of Ca2+-permeable NSCCs (Cho et al., 2003). In physiological systems, the activity of these NSCCs is triggered by increases in intracellular Ca2+ concentration that result from G-protein coupled receptor or Ca2+-permeable ion channel activation (Yamashita et al., 2003, Tozzi et al., 2003). In addition, FFA has been shown to modulate gap junction activity in a pH-dependent manner (Srinivas and Spray, 2003).
In this study, we describe inhibition of human recombinant TRPM2 by FFA and additionally present evidence for inhibition of TRPM2-like currents in CRI-G1 cells.
Section snippets
Cell culture
Human embryonic kidney (HEK293) cells expressing tetracycline-inducible flag-tagged TRPM2 (TRPM2-HEK293 cells) (Perraud et al., 2001) were a kind gift from A.M. Scharenberg (University of Washington). They were grown in minimum essential medium (MEM) supplemented with non-essential amino acids, 10% foetal calf serum and 0.2 mM l-glutamine under 95% air and 5% CO2 at 37 °C. TRPM2 expression was induced by incubating cells for 24 h with 1 μg/ml tetracycline. Cells were used for patch clamp
Effect of FFA on recombinant human TRPM2
Induced expression of TRPM2 in HEK293 cells lead to the observation of a large non-selective, non-desensitising, cation current that was present only when ADP-ribose was included in the patch pipette. This current had a mean amplitude of 5.4±0.6 nA at −50 mV and was not observed in untransfected HEK293 cells. This ADP-ribose-activated TRPM2-mediated current was rapidly eliminated by replacement of extracellular Ca2+ with Ba2+ (Fig. 1A) or removal of extracellular Ca2+ (data not shown). The
Discussion
As a result of its activation by species produced by oxidative stress (β-NAD+, H2O2 and possibly ADP-ribose, see Wilson et al., 2001) and its substantial Ca2+ permeability, TRPM2 is a good candidate for a channel that triggers cellular responses to changes in oxidative load. An extension of this is the consideration that TRPM2 is a “suicide channel” causing cells to overload with Ca2+ and become irreversibly compromised under conditions of oxidative stress. A consequence of this latter
Acknowledgements
The authors thank Dr. A. Scharenberg (University of Washington) for the kind gift of the HEK293-TRPM2 cell line. K.H. is in receipt of EU Framework V Postdoctoral Fellowship.
References (30)
- et al.
Fenamates protect neurons against ischemic and excitotoxic injury in chick embryo retina
Neurosci. Lett.
(1998) - et al.
Molecular and functional characterization of the melastatin-related cation channel TRPM3
J. Biol. Chem.
(2003) - et al.
LTRPC2 Ca2+-permeable channel activated by changes in redox status confers susceptibility to cell death
Mol. Cell
(2002) - et al.
Hydrogen peroxide induces intracellular calcium overload by activation of a non-selective cation channel in an insulin-secreting cell line
J. Biol. Chem.
(1999) - et al.
Inhibition of hKv2.1, a major human neuronal voltage-gated K+ channel, by meclofenamic acid
Eur. J. Pharmacol.
(1999) - et al.
Mechanism of action of the non-steroidal anti-inflammatory drug flufenamate on [Ca2+]i and Ca(2+)-activated currents in neurons
Cell Calcium
(1996) - et al.
Diclofenac inhibition of sodium currents in rat dorsal root ganglion neurons
Brain Res.
(2003) - et al.
Bimodal action of furosemide on convulsant [3H]EBOB binding to cerebellar and cortical GABA(A) receptors
Neurochem. Int.
(1998) - et al.
Critical intracellular Ca2+ dependence of transient receptor potential melastatin 2 (TRPM2) cation channel activation
J. Biol. Chem.
(2003) - et al.
Functional characterisation of human TASK-3, an acid-sensitive two-pore domain potassium channel
Neuropharmacology
(2001)