Elsevier

Neuropharmacology

Volume 47, Issue 3, September 2004, Pages 450-460
Neuropharmacology

Flufenamic acid is a pH-dependent antagonist of TRPM2 channels

https://doi.org/10.1016/j.neuropharm.2004.04.014Get rights and content

Abstract

Like a number of other TRP channels, TRPM2 is a Ca2+-permeable non-selective cation channel, the activity of which is regulated by intracellular and extracellular Ca2+. A unique feature of TRPM2 is its activation by ADP-ribose and chemical species that arise during oxidative stress, for example, NAD+ and H2O2. These properties have lead to proposals that this channel may play a role in the cell death produced by pathological redox states. The lack of known antagonists of this channel have made these hypotheses difficult to test. Here, we demonstrate, using patch clamp electrophysiology, that the non-steroidal anti-inflammatory compound flufenamic acid (FFA) inhibits recombinant human TRPM2 (hTRPM2) as well as currents activated by intracellular ADP-ribose in the CRI-G1 rat insulinoma cell line. All concentrations tested in a range from 50 to 1000 μM produced complete inhibition of the TRPM2-mediated current. Following FFA removal, a small (typically 10–15%) component of current was rapidly recovered (time constant ~3 s), considerably longer periods in the absence of FFA produced no further current recovery. Reapplication of FFA re-antagonised the recovered current and subsequent FFA washout produced recovery of only a small percentage of the reblocked current. Decreasing extracellular pH accelerated FFA inhibition of TRPM2. Additional experiments indicated hTRPM2 activation was required for FFA antagonism to occur and that the generation of irreversible antagonism was preceded by a reversible component of block. FFA inhibition could not be induced by intracellular application of FFA. ADP-ribose activated currents in the rat insulinoma cell line CRI-G1 were also antagonised by FFA with concentration- and pH-dependent kinetics. In contrast to the observations made with hTRPM2, antagonism of ADP-ribose activated currents in CRI-G1 cells could be fully reversed following FFA removal. These experiments suggest that FFA may be a useful tool antagonist for studies of TRPM2 function.

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)

  • A.L Perraud et al.

    TRPM2 Ca(2+) permeable cation channels: from gene to biological function

    Cell Calcium

    (2003)
  • H.L Wilson et al.

    ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase act as a redox sensor

    J. Biol. Chem.

    (2001)
  • W Zhang et al.

    A novel TRPM2 isoform inhibits calcium influx and susceptibility to cell death

    J. Biol. Chem.

    (2003)
  • S Bevan et al.

    Sensory neuron-specific actions of capsaicin: mechanisms and applications

    Trends Pharmacol. Sci.

    (1990)
  • L.M Boland et al.

    Omega-conotoxin block of N-type calcium channels in frog and rat sympathetic neurons

    J. Neurosci.

    (1994)
  • Cited by (0)

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