Elsevier

Neuropharmacology

Volume 50, Issue 1, January 2006, Pages 89-97
Neuropharmacology

Characterisation of recombinant rat TRPM2 and a TRPM2-like conductance in cultured rat striatal neurones

https://doi.org/10.1016/j.neuropharm.2005.08.021Get rights and content

Abstract

TRPM2, a member of the TRP ion channel family, is expressed both in the brain and immune cells of the monocyte lineage. Functionally, it is unique in its activation by intracellular ADP-ribose and both oxidative and nitrosative stress. To date studies of this channel have concentrated on human recombinant channels and rodent native preparations. This provides the potential for cross-species complications in the interpretation of native tissue observations based on recombinant channel phenotype. Consequently, we have cloned and heterologously expressed rat TRPM2 (rTRPM2) in HEK293 cells. We find that, like hTRPM2, it responds to intracellular ADP-ribose in a manner dependent on extracellular Ca2+. At the single channel level rTRPM2 is a slow gating, large conductance (84 pS) channel that rapidly runs down in isolated membrane patches. Pharmacologically, rTRPM2 is rapidly and irreversibly blocked by clotrimazole (10 μM), thus resembling hTRPM2 but not the TRPM2-like current of the rat-derived insulinoma CRI-G1, which exhibits reversible inhibition by this agent. We show that cultured rat striatal neurones exhibit an ADP-ribose-activated conductance at both the whole cell and single channel level. Pharmacologically this neuronal current can be irreversibly inhibited by clotrimazole. It is also sensitive to removal of extracellular Ca2+, suggesting that it is mediated by TRPM2-containing channels. These data provide a functional characterisation of heterologously expressed rTRPM2 and demonstrate that, in addition to the previous descriptions in immune cells, microglia and insulinomas, a TRPM2-like conductance can be found in neurones derived from the rodent CNS.

Introduction

More than 20 different TRP channels have been identified in mammals (Clapham et al., 2003). The various members of this large family are activated by a diverse range of stimuli. For example, a host of molecular agonists as well as a number of these channels can be activated by thermal and/or physical (i.e. mechanical) stimuli.

The large majority of the TRP channels are non-selective cation channels and often exhibits substantial permeability to the important second messenger ion Ca2+. One such channel is TRPM2. Uniquely, this member of the melastatin subfamily of TRP channels is activated by intracellular ADP-ribose at concentrations in the micromolar range (Perraud et al., 2001, Sano et al., 2001, Inamura et al., 2003). TRPM2 is also activated in response to oxidative and nitrosative stress; the former is typically elicited in vitro by the application of H2O2 (Hara et al., 2002, Wehage et al., 2002, Zhang et al., 2003). The electrophysiological properties of human TRPM2 (hTRPM2) have been described in detail in a number of recent papers. Pharmacologically, TRPM2 has been shown to be inhibited by 2 different classes of compounds, namely the fenamates, such as flufenamic acid, and anti-fungal imidazoles such as clotrimazole (Hill et al., 2004a, Hill et al., 2004b). The antagonism of hTRPM2 produced by these compound classes of molecule exhibits certain defining features, most strikingly a dependence on channel activation and a seemingly irreversible block.

Insulinoma cell-lines, such as the rat-derived CRI-G1 line, exhibit robust currents in response to intracellular application of NAD+ or ADP-ribose (Inamura et al., 2003, Herson et al., 1997). At both the macroscopic and microscopic levels these ADP-ribose-induced responses are very similar to those produced by ADP-ribose stimulation of heterologously expressed recombinant TRPM2 (Perraud et al., 2001). This has led to the suggestion that these responses in insulinoma cells are mediated by TRPM2 channels. One pharmacological difference is clearly apparent, however, when recombinant hTRPM2 and responses in CRI-G1 cells are compared. This is in the nature of the inhibition by both fenamates and imidazoles, which is very freely reversible in the insulinoma cells. This led us to question whether this reversibility of antagonism in the insulin-secreting cells simply reflects the pharmacology of rat TRPM2 or some other factor, for example the formation of heteromeric TRPM2-containing channels in these cells derived from a pancreatic tumour. To address this we cloned rat TRPM2 and characterised its electrophysiological and pharmacological properties when heterologously expressed in HEK293 cells. In parallel, we have characterised another TRPM2-like endogenous conductance from the rat, an ADP-ribose-activated current in cultured rat striatal neurones.

Section snippets

Molecular cloning of rat TRPM2 (rTRPM2)

The full-length DNA sequence of the rat TRPM2 gene was not available in the public domain. Partial sequences representing the rat TRPM2 gene were identified by performing a homology search with public and proprietary sequence databases using the mouse TRPM2 protein (AJ344343) and TBALSTN2 (Altschul et al., 1990). Rat sequence fragments were assembled and the predictions were made using the mouse protein in the GENEWISE gene prediction program (http://www.sanger.ac.uk/Software/Wise2). As a

Results

The deduced amino acid sequence of the rTRPM2 monomer has 1508 amino acids and a calculated unglycosylated molecular weight of 172 kDa. The sequences of rat (AY749166) and human (AJ878416) TRPM2 share 83 and 85% identity at the nucleotide and protein levels, respectively. One notable region of difference between rat and human TRPM2 is within a 25 amino acid region of the core nudix hydrolase domain. Here the identity between the rodent and human proteins falls to 75–78%. In contrast, the

Discussion

In this paper we have provided the first description of the cloning and functional properties of rat TRPM2. We have also described the properties of TRPM2-like ADP-ribose activated currents in cultured rat striatal neurones, which are very likely to be the same channels described as NSNAD by Smith et al. (2003). To date, all published data on the physiology and pharmacology of recombinant TRPM2 have concerned the human form initially cloned by Nagamine et al. (1998). In contrast, most

Acknowledgements

K.H. was in receipt of a Marie Curie EU Framework V Postdoctoral Fellowship.

References (22)

  • S.E. Browne et al.

    Oxidative stress in Huntington's disease

    Brain Pathol.

    (1999)
  • Cited by (0)

    View full text