Neurosensor 521: A Fluorescent Sensor for Selective Labeling of Norepinephrine-Containing Vesicles

Abstract

We have developed a novel fluorescence-based turn-on molecular sensor (NeuroSensor 521) that selectively binds to primary amines via iminium ion formation. The fluorescence increases > 5-fold upon binding norepinephrine and is accompanied by a red shift in the absorption maximum from 448 to 488 nm. Norepinephrine and dopamine bind with a ∼10-fold higher affinity (∼10 mM) than other alkyl amines such as glycine, but secondary amines such as epinephrine do not have a measurable binding affinity. We used confocal microscopy to image chromaffin cells labeled with NS521. Chromaffin cells were separated into norepinephrine-enriched and epinephrine-enriched fractions by centrifugation on a Percoll gradient. The norepinephrine-enriched cell population shows strong, punctate fluorescence compared to the epinephrine-enriched cell population, which exhibits only marginal fluorescence. To further validate the selective labeling of norepinephrine-containing vesicles, we labeled fixed cells using an antibody against phenylethanolamine N-methyltransferase (PNMT). The NE-enriched cell population stains brightly with NS521 and weakly with the fluorescent PNMT secondary antibody while the opposite is true for the EP-enriched cell population. Next, we used Total Internal Reflection Fluorescence Microscopy (TIRFM) to image the bottom surface of chromaffin cells loaded with NS521. Clear punctate fluorescence consistent with labeling of individual norepinephrine-containing granules was observed. We conclude that NeuroSensor 521 represents a convenient method to selectively stain norepinephrine and dopamine in neurosecretory vesicles. Supported by the NSF (CHE-1112194) and the NIH (R43 MH096650).