Transient signaling of free ADP-ribose monitored with an intracellular biosensor

Abstract 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 between 2 – 4 µM 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. Significance Statement We have developed a unique real-time biosensor for free ADP-ribose that is tuned to physiological concentrations and capable of intracellular measurements in individual cells. Using a calibrated system we determined that concentrations and timing of induced intracellular ADPR aligned with the thermosensitive TRPM2 activity. The data support ADPR as a critical component whose intracellular levels are regulated to control TRPM2 channel opening in cells. Competing Interest Statement The University of Texas at Austin has filed a patent application covering the technology described here. Footnotes Updates to this version: - Figure 4 has been updated - Minor additional text edits