Ns510, A High Affinity Fluorescent Catecholamine Sensor For Monitoring Norepinephrine Exocytosis

NS510 is a fluorescent sensor with a high affinity for catecholamines based on a quinolone fluorophore incorporating a boronic acid recognition element and a turn-on response to norepinephrine. The excitation maximum of NS510 is near 488 nm. We are using adrenal chromaffin cells as a model cell to test NS510, and sub-populations of cells enriched in epinephrine versus norepinephrine are isolated using gradient centrifugation. Fluorescence is partially quenched when the sensor interacts with epinephrine and dopamine whereas the fluorescence increases upon binding with norepinephrine. Norepinephrine-enriched chromaffin cells loaded with NS510 exhibit distinct fluorescence punctae using confocal microscopy and total internal reflection fluorescence (TIRF) microscopy, consistent with staining of norepinephrine-containing secretory granules. Amperometry using underlying transparent electrodes in conjunction with TIRF microscopy demonstrates that the sensor can be used to observe destaining of individual chromaffin granules simultaneously with amperometric spikes indicating quantal exocytosis of catecholamine. To further validate the selective staining of norepinephrine-containing vesicles, we labelled fixed cells using an antibody against phenylethanolamine N-methyltransferase (PNMT), an enzyme that converts norepinephrine to epinephrine, and thus is found in epinephrine-enriched, but not norepinephrine-enriched cell populations. NS510 labeling is higher in norepinephrine-enriched cells as validated by PNMT staining. We conclude that NS510 is the highest affinity and most selective fluorescent norepinephrine sensor currently available and can be used for measuring quantal exocytosis of catecholamines in live-cell assays. Supported by NIH R01EB020415.