Miz-2, A New Catecholamine-Selective Fluorescent Sensor for Visualizing Norepinephrine

Abstract

We have developed a fluorescence-based turn-on molecular sensor (NeuroSensor 521) based on a coumarin aldehyde scaffold that allows for the selective recognition and sensing of norepinephrine in live and fixed chromaffin cells. Based on NeuroSensor 521, we have now developed Miz-2: a promising member of a new series of catecholamine-selective sensors, which has both a functional aldehyde group to bind to primary amines, and other recognition elements to improve selectivity for binding catecholamine over other amines. The excitation maximum of Miz-2 is near 488 nm. Fluorescence is quenched when the sensor interacts with epinephrine and dopamine whereas the fluorescence increases upon binding with norepinephrine. We are using adrenal chromaffin cells as a model cell to test fluorescent catecholamine sensors and sub-populations of cells enriched in epinephrine versus norepinephrine can be isolated upon gradient centrifugation. Norepinephrine-enriched chromaffin cells exhibit distinct fluorescence punctae by total internal reflection fluorescence (TIRF) microscopy when loaded with Miz-2 that are consistent with labeling of granules. Upon stimulation with a high-K+ solution, punctae abruptly disappear during TIRF imaging, consistent with granule fusion and loss of dye via exocytosis. Using underlying transparent electrodes, we recorded amperometric spikes consistent with quantal exocytosis of catecholamine in cells loaded with the sensor. Experiments are in progress to combine TIRF imaging with amperometric measurements in underlying transparent electrodes to resolve features of norepinephrine release from individual granule. Supported by NIH R01EB020415.