Article ; Online: Development and optimization of a high-throughput bioassay for TRPM7 ion channel inhibitors.
Journal of biomolecular screening
2010 Volume 15, Issue 5, Page(s) 498–507
Abstract: ... no reports of selective inhibitors of TRPM7. The authors developed and optimized a fluorescent dye ... TRPM7 is a ubiquitously expressed and constitutively active divalent cation channel essential ... that stably overexpress TRPM7. The following bioassay conditions were evaluated: (a) cell density, (b) dye ...
| Abstract | TRPM7 is a ubiquitously expressed and constitutively active divalent cation channel essential for cell survival and proliferation because it provides a mechanism for Mg2+ entry. This makes the channel an attractive target for proliferative diseases. In keeping with its role in Mg2+ homeostasis, TRPM7 is inhibited by intracellular Mg2+ and Mg-ATP. TRPM7 has been implicated in anoxia-mediated cell death following brain ischemia. Despite its critical role in ischemic cell death and cell proliferation, there are no reports of selective inhibitors of TRPM7. The authors developed and optimized a fluorescent dye-based bioassay measuring the fluorescence quench of fura-2 by TRPM7-mediated Mn2+ influx in HEK293 cells that stably overexpress TRPM7. The following bioassay conditions were evaluated: (a) cell density, (b) dye loading conditions, (c) bioassay temperature, (d) concentration of the fura-2 quenching agent Mn2+, and (e) concentration of vehicle solvent. The bioassay was validated by measuring the effects of the known (nonselective) inhibitor 2-APB and La3+ on Mn2+ influx, and furthermore, the performance of the assay was evaluated by screening a subset of a marine bacteria-derived extract library. The quality of the bioassay window is excellent based on an established statistical parameter used to evaluate high-throughput screening window quality (Z and Z' factors > or =0.5). |
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| MeSH term(s) | Animals ; Cell Line ; Fluorescent Dyes/metabolism ; Fura-2/analogs & derivatives ; Fura-2/metabolism ; High-Throughput Screening Assays/methods ; High-Throughput Screening Assays/standards ; Humans ; Magnesium/metabolism ; Manganese/metabolism ; Patch-Clamp Techniques ; Poloxamer/metabolism ; Probenecid/metabolism ; Protein-Serine-Threonine Kinases ; Reproducibility of Results ; Surface-Active Agents/metabolism ; TRPM Cation Channels/antagonists & inhibitors ; Uricosuric Agents/metabolism | |||||
| Chemical Substances | Fluorescent Dyes ; Surface-Active Agents ; TRPM Cation Channels ; Uricosuric Agents ; fura-2-am (105344-37-4) ; Poloxamer (106392-12-5) ; Manganese (42Z2K6ZL8P) ; Protein-Serine-Threonine Kinases (EC 2.7.11.1) ; TRPM7 protein, human (EC 2.7.11.1) ; Magnesium (I38ZP9992A) ; Probenecid (PO572Z7917) ; Fura-2 (TSN3DL106G) | |||||
| Language | English | |||||
| Publishing date | 2010-04-22 | |||||
| Publishing country | United States | |||||
| Document type | Journal Article ; Research Support, N.I.H., Extramural | |||||
| ZDB-ID | 1433680-7 | |||||
| ISSN | 1552-454X ; 1087-0571 | |||||
| ISSN (online) | 1552-454X | |||||
| ISSN | 1087-0571 | |||||
| DOI | 10.1177/1087057110368294 | |||||
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| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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