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Development and Validation of a Cell-Based High-Throughput Screening Assay for TRPM2 Channel Modulators

Department of Pediatrics and Immunology, University of Washington and Seattle Children's Hospital Research Institute, Seattle, Washington

Ben Buelow

Department of Pediatrics and Immunology, University of Washington and Seattle Children's Hospital Research Institute, Seattle, Washington

Anne-Laure Perraud

Department of Immunology, National Jewish Research Hospital and University of Colorado, Denver

Andrew M. Scharenberg

Department of Pediatrics and Immunology, University of Washington and Seattle Children's Hospital Research Institute, Seattle, Washington, andrewms{at}u.washington.edu

TRPM2 is a member of the transient receptor potential melastatin (TRPM)—related ion channel family. The activation of TRPM2 induced by oxidative/nitrosative stress leads to an increase in intracellular free Ca²⁺. Although further progress in understanding TRPM2's role in cell and organism physiology would be facilitated by isolation of compounds able to specifically modulate its function in primary cells or animal models, no cell-based assays for TRPM2 function well suited for high-throughput screening have yet been described. Here, a novel suspension B lymphocyte cell line stably expressing TRPM2 was used to develop a cell-based assay. The assay uses the Ca²⁺-sensitive fluorescence dye, Fluo-4 NW (no wash), to measure TRPM2-dependent Ca²⁺ transients induced by H₂O₂ and N-methyl-N'-nitrosoguanidine in a 96-well plate format. Assay performance was evaluated by statistical analysis of the Z' factor value and was consistently greater than 0.5 under optimal conditions, suggesting that the assay is very robust. For assay validation, the effects of known inhibitors of TRPM2 and TRPM2 gating secondary messenger production were determined. Overall, the authors have developed a cell-based assay that may be used to identify TRPM2 ion channel modulators from large compound libraries. (Journal of Biomolecular Screening 2008:54-61)

Key Words: TRPM2 ion channels • high-throughput screening • cell-based assay • DT40-TRPM2 cell line • DT40 control cell line • fluorescence Ca2+ probe

This version was published on January 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 1, 54-61 (2008)
DOI: 10.1177/1087057107310986


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