A cell permeable NPE caged ADP-ribose for studying TRPM2

PLoS One. 2012;7(12):e51028. doi: 10.1371/journal.pone.0051028. Epub 2012 Dec 7.

Abstract

Transient potential receptor melastatin-2 (TRPM2) is a non-selective Ca(2+)-permeable cation channel of the TRPM channel subfamily and is mainly activated by intracellular adenosine diphosphate ribose (ADPR). Here we synthesized a 1-(2-nitrophenyl)ethyl caged ADPR (NPE-ADPR) and found that uncaging of NPE-ADPR efficiently stimulated Ca(2+), Mg(2+), and Zn(2+) influx in a concentration-dependent manner in intact human Jurkat T-lymphocytes. The cation influx was inhibited by inhibitors or knockdown of TRPM2. Likewise, uncaging of NPE-ADPR markedly induced cation entry in HEK 293 cells that overexpress TRPM2. As expected, high temperature increased the ability of the photolyzed NPE-ADPR to induce cation entry, whereas acidic pH inhibited. Moreover, the absence of extracellular Ca(2+) significantly inhibited Mg(2+) and Zn(2+) influx after uncaging NPE-ADPR. On the other hand, the absence of extracellular Na(+) or Mg(2+) had no effect on photolyzed NPE-ADPR induced Ca(2+) entry. Taken together, our results indicated that NPE-ADPR is a cell permeable ADPR analogue that is useful for studying TRPM2-mediated cation entry in intact cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcium / metabolism*
  • Calcium Signaling / physiology
  • Cyclic ADP-Ribose / analogs & derivatives*
  • Cyclic ADP-Ribose / chemical synthesis
  • HEK293 Cells
  • Humans
  • Patch-Clamp Techniques
  • TRPM Cation Channels / metabolism*

Substances

  • TRPM Cation Channels
  • TRPM2 protein, human
  • caged cyclic ADP-ribose
  • Cyclic ADP-Ribose
  • Calcium

Grant support

This work was supported by Research Grant Council (RGC) grants (HKU 784710M, HKU 782709M, HKU 785911M) and a National Natural Science Foundation of China (NSFC)/RGC grant from Hong Kong (N_HKU 737/09) and NSFC (20910094), and a Special Fellow Award from the Leukemia and Lymphoma Society of America (J.Y). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.