Goyal, S.; Nguyen, V.; Lyons, S. N.; Dangerfield, T. L.; Yang, W.; Fields, C.; Chan, H.-R.; Mahendravarman, Y.; Cantu, L.; Rubin, N.; Deloney, K.; Johnson, K. A.; Cambronne, X. A.

We have developed a biosensor enabling the dynamic, compartmentalized, and longitudinal measurements of intracellular ADP-ribose (ADPR) in live cells. Free ADPR is a critical signaling metabolite derived from nicotinamide adenine dinucleotide (NAD+). As an agonist for Transient Receptor Potential Melastatin 2 (TRPM2), ADPR levels can regulate immune responses during infection, as well as nociception and adjustment of core body temperature. The study of ADPR signaling has been limited, however, by a lack of methods to measure this metabolite in situ. Using the biosensor and its paired non-responsive control, we determine that intracellular ADPR accumulation was transient and tunable. We found that basal concentrations were in the nanomolar range and could be stimulated ~30-fold to activate TRPM2. We identified that TRPM2 activation, measured by calcium influx, required an intracellular ADPR threshold concentration > 2 M at physiological temperature. We observed that the timing of the ADPR rise coincided with TRPM2 activation, thus providing support for ADPR fluctuations being a critically regulated aspect for channel activation. Notably, transient fluctuations of ADPR were not accurately reflected by measurements of intracellular NAD+ loss or calcium levels.