Elsevier

Cell Calcium

Volume 51, Issue 2, February 2012, Pages 179-185
Cell Calcium

The juvenile myoclonic epilepsy-related protein EFHC1 interacts with the redox-sensitive TRPM2 channel linked to cell death

https://doi.org/10.1016/j.ceca.2011.12.011Get rights and content

Abstract

The transient receptor potential M2 channel (TRPM2) is the Ca2+-permeable cation channel controlled by cellular redox status via β-NAD+ and ADP-ribose (ADPR). TRPM2 activity has been reported to underlie susceptibility to cell death and biological processes such as inflammatory cell migration and insulin secretion. However, little is known about the intracellular mechanisms that regulate oxidative stress-induced cell death via TRPM2. We report here a molecular and functional interaction between the TRPM2 channel and EF-hand motif-containing protein EFHC1, whose mutation causes juvenile myoclonic epilepsy (JME) via mechanisms including neuronal apoptosis. In situ hybridization analysis demonstrates TRPM2 and EFHC1 are coexpressed in hippocampal neurons and ventricle cells, while immunoprecipitation analysis demonstrates physical interaction of the N- and C-terminal cytoplasmic regions of TRPM2 with the EFHC1 protein. Coexpression of EFHC1 significantly potentiates hydrogen peroxide (H2O2)- and ADPR-induced Ca2+ responses and cationic currents via recombinant TRPM2 in HEK293 cells. Furthermore, EFHC1 enhances TRPM2-conferred susceptibility of HEK293 cells to H2O2-induced cell death, which is reversed by JME mutations. These results reveal a positive regulatory action of EFHC1 on TRPM2 activity, suggesting that TRPM2 contributes to the expression of JME phenotypes by mediating disruptive effects of JME mutations of EFHC1 on biological processes including cell death.

Introduction

Mammalian homologues of Drosophila transient receptor potential (TRP) protein form cation channels activated by sensing a variety of physical and chemical stimuli [1], [2]. TRPM2 is a Ca2+-permeable channel activated by ADP-ribose (ADPR) [3], [4] and oxidative stress via β-NAD+[5] and/or ADP-ribose [6], [7], [8], and regulated by various factors [9], [10], [11]. TRPM2 activity has been reported to underlie biological processes such as cell death [5], inflammatory cell migration [8], and insulin secretion [12].

Juvenile myoclonic epilepsy (JME) is the most common form of idiopathic generalized epilepsy, accounting for 10–30% of all epilepsies [13]. The gene encoding EF-hand motif-containing protein, EFHC1, has been identified in the region on chromosome 6p12-p11 associated with JME [14]. EFHC1 is reported to be expressed in brain neurons [14], [15], and in ependymal cells of ventricle walls and other cells that possess motile cilia and flagella [16], [17], [18]. In neuronal cells, EFHC1 increases voltage-dependent R-type Ca2+ channel (CaV2.3) currents and induces neuronal death. However, the previous report [14] suggests that the EFHC1-dependent CaV2.3 Ca2+ current increase is not an exclusive mechanism that underlies EFHC1-induced neuronal death reversed by the JME mutations. EFHC1 may interact with an additional number of proteins in cell death sensitive to JME mutations. With regard to Ca2+ channels linked to cell death, TRPM2 has been reported to elicit hydrogen peroxide (H2O2)- and tumor necrosis factor α-induced cell death [5], [19]. Interestingly, reactive oxygen species (ROS) are reported to be constitutively produced in rat hippocampus [20]. The idea that TRPM2 can be the missing protein linking cell death to EFHC1/JME prompted us to test interaction between EFHC1 and TRPM2. Furthermore, it is important to note that genetic disruption of EFHC1 causes enlargement of ventricles and reduced ciliary beating frequency of ependymal cilia in ventricle [21], and that EFHC1 interacts with microtubules to regulate cell division and cortical development [22]. These recent reports also provide important bases to consider possibilities other than the original scenario focusing on the major role of cell death via R-type Ca2+ channels in the expression of JME phenotypes.

We show here physical association between EFHC1 and TRPM2. EFHC1 enhanced the H2O2 sensitivity of TRPM2 channel activation and cell death. The obtained results may suggest that EFHC1 regulates voltage-independent TRPM2 Ca2+ channels to control Ca2+ homeostasis in neuronal death.

Section snippets

Cell culture and recombinant expression in HEK293 cells

The expression plasmid for human TRPM2 (hTRPM2) was constructed as described previously [5]. HEK293 (ATCC) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 30 units/ml penicillin, and 30 μg/ml streptomycin. HEK293 cells were co-transfected with pEGFP-EFHC1 or pEGFP-C2 vector and one of pCI-neo-hTRPM2 and the vector pCI-neo. Transfection was carried out using SuperFect Transfection Reagent (QIAGEN). Cells were trypsinized and diluted with

EFHC1 physically associates with TRPM2

To assess the localization of TRPM2 and EFHC1 in brain tissues, in situ hybridization analysis was performed. TRPM2 was expressed in hippocampal pyramidal neurons [5], [26], [27] and ependymal cells in ventricle walls, where EFHC1 was coexpressed as previously reported (Fig. 1A). The result suggests that TRPM2 and EFHC1 are co-localized in the same cells in the brain. We next examined whether EFHC1 and TRPM2 coexpressed in the same cells are physically associated. Extracts prepared from HEK293

Discussion

The present investigation reveals that EFHC1, the candidate gene for JME, physically interacts with the TRPM2 channel, and thereby potentiates the TRPM2 channel activity in H2O2-induced cell death. The physical interaction of TRPM2 with EFHC1 is mediated by the putatively cytoplasmic TRPM2 N-terminus and C-terminus. Interestingly, the TRPM2 C-terminus contains the MutT (Nudix) motif that is the action site for TRPM2 activation triggers such as ADPR and β-NAD+ [3], [4], [5]. Ca2+ channels

Conflict of interest statement

There is no conflict of interest.

Acknowledgements

We thank T. Kajimoto for his help in drafting the manuscript and T. Takeuchi for technical assistance.

References (39)

  • T. Okada et al.

    Molecular cloning and functional characterization of a novel receptor-activated TRP Ca2+ channel from mouse brain

    J. Biol. Chem.

    (1998)
  • K. Nagamine et al.

    Molecular cloning of a novel putative Ca2+ channel protein (TRPC7) highly expressed in brain

    Genomics

    (1998)
  • T. Okada et al.

    Molecular and functional characterization of a novel mouse TRP homologue TRP7: Ca2+ permeable cation channel that is constitutively activated and enhanced by stimulation of G-protein-coupled receptor

    J. Biol. Chem.

    (1999)
  • R. Shibata et al.

    A fundamental role for KChIPs in determining the molecular properties and trafficking of Kv4.2 potassium channels

    J. Biol. Chem.

    (2003)
  • D. McHugh et al.

    Critical intracellular Ca2+dependence of transient receptor potential melastatin 2 (TRPM2) cation channel activation

    J. Biol. Chem.

    (2003)
  • D.E. Clapham

    TRP channels as cellular sensors

    Nature

    (2003)
  • A.L. Perraud et al.

    ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology

    Nature

    (2001)
  • Y. Sano et al.

    Immunocyte Ca2+ influx system mediated by LTRPC2

    Science

    (2001)
  • E. Fonfria et al.

    TRPM2 channel opening in response to oxidative stress is dependent on activation of poly(ADP-ribose) polymerase

    Br. J. Pharmacol.

    (2004)
  • Cited by (39)

    • TRPM2 ion channel is involved in the aggravation of cognitive impairment and down regulation of epilepsy threshold in pentylenetetrazole-induced kindling mice

      2020, Brain Research Bulletin
      Citation Excerpt :

      Therefore, we hypothesized that the TRPM2 channel might be involved in the pathogenesis of epilepsy, consistent with previous research indicating that TRPM2 participates in the juvenile myoclonic epilepsy. The juvenile myoclonic epilepsy-related protein EFHC1 facilitates TRPM2-dependent cell death in response to epileptic seizure (Katano et al., 2012). The poly (ADPR) polymerases (ADPRS) can be degraded to ADPR, and the main active form of ADPR is PARP1 (Guse, 2010).

    • EFHC1 variants in juvenile myoclonic epilepsy: Reanalysis according to NHGRI and ACMG guidelines for assigning disease causality

      2017, Genetics in Medicine
      Citation Excerpt :

      Supplementary Table S6a,b online show the published statistical results of 14 experimental measures demonstrating a significant difference between the tested variants and the wild-type protein. Three variants (R159W, R182H, and I619L) classified as benign polymorphism did not produce statistically significant results in almost all of the measures in comparison to the wild type.16,55,56,57 R182H showed a small, but significant difference in apoptotic activity in primary mouse hippocampal neurons in culture.16

    View all citing articles on Scopus
    View full text